978-981-19-8714-4

Lecture Notes in Electrical Engineering 989

Koushik Guha
Gorachand Dutta
Arindam Biswas
K. Srinivasa Rao Editors

MEMS and
Microfluidics
in Healthcare
Devices and Applications Perspectives
Lecture Notes in Electrical Engineering

Volume 989
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Koushik Guha · Gorachand Dutta ·
Arindam Biswas · K. Srinivasa Rao
Editors
MEMS and Microfluidics

in Healthcare
Devices and Applications Perspectives
Editors
Koushik Guha Gorachand Dutta
Department of Electronics School of Medical Science and Technology
and Communication Engineering Indian Institute of Technology Kharagpur
National Institute of Technology Silchar Kharagpur, West Bengal, India
Silchar, Assam, India
K. Srinivasa Rao
Arindam Biswas Department of Electronics
School of Mines and Metallurgy and Communication Engineering
Kazi Nazrul University KL University
Asansol, West Bengal, India Guntur, Andhra Pradesh, India
ISSN 1876-1100 ISSN 1876-1119 (electronic)

Lecture Notes in Electrical Engineering
ISBN 978-981-19-8713-7 ISBN 978-981-19-8714-4 (eBook)
https://doi.org/10.1007/978-981-19-8714-4
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Preface
The significance of medical MEMS is increasing exponentially from past two

decades. There exist numerous MEMS diagnostic devices for detection of blood pres-
sure, glucose, TB, Hepatitis, etc. This is possible due to the multi-disciplinary fields
fusing together to make a potent solution for the existing problems. MEMS has proven
to be capable of paving bridge between medicine and engineering field, which helps
in developing medical instruments. The controllability of sample volume, response
time, and size of these devices is satisfactory. The research on the biomaterials to
realize such medical devices has a huge scope in future. Furthermore, the book is
aimed to study different microfluidic devices. The microfluidics technology has vast
number of medical applications ranging from µTAS, drug delivery, DNA sequencing,
cancer and COVID-19 detection, and organ on chip. Presently, organ-on-chip tech-
nology is prevailing in the scientific community, it can be expected that within a
decade there will be marketed microphysiological devices. Microfluidics is capable
of regenerating any organ functional physiology and pathophysiology which can be
useful in disease diagnosis and organ replacement as well. The present book focuses
on the past developments and future research directions in MEMS and microflu-
idic medical device field, the research available currently on these instruments is
intriguing. Though there were plenty of marketed devices, MEMS technology has
not yet been explored to its potential. This book primarily focuses on the reader’s
enlightenment on MEMS medical devices by introducing all the diagnostic devices
and treatment tools at one place. The book covers in-depth technical works and
general introductions to the devices such that the book can reach technical and
general audience as well. In addition, the fabrication techniques of bio-MEMS and
bio-microfluidic devices will also be elaborated, which makes the book a complete
guide from device development to fabrication stages.
v
vi Preface
Therefore, in this text, all the above mentioned are covered through chap-
ters spanning from 1 to 12. The first chapter “N/MEMS Biosensors: An Intro-
duction” covering Introduction to N/MEMS Biosensors and the second chapter
“Lab-On-A-Chip Technology in Health Care” explains Lab-on-a-Chip Technology
in Health Care. The third chapter “A Review on Recent Trends in the Segregation
of Red Blood Cells Using Microfluidic Devices” presents recent trends in the segrega-
tion of red blood cells using microfluidic devices. The fourth chapter “A Road Map
to Paper-Based Microfluidics Towards Affordable Disease Detection” deals with
paper-based microfluidics and its journey toward affordable disease diagnosis and
the fifth chapter “Critical Review and Exploration on Micro-pumps for Microfluidic
Delivery” presents a critical review and exploration on micro-pumps for microfluidic
delivery. The sixth chapter “Fabrication Techniques and Materials for Bio-MEMS”
introduces fabrication techniques and materials for bio-MEMS. The seventh chapter
“In-silico Analysis of Expandable Radiofrequency Electrode for Ablation of Hepatic
Tumors” proposes an in-silico analysis of expandable radiofrequency electrode for
ablation of hepatic tumors and the eighth chapter “Lab-On-Chip Electrochemical
Biosensor for Rheumatoid Arthritis” describes a work on lab-on-chip electrochem-
ical biosensor for rheumatoid arthritis. The ninth chapter “Transdermal Injection
with Microneedle Devices in Healthcare Sector: Materials, Challenging Fabrica-
tion Methodologies, and its Limitations” indulges with transdermal injection with
microneedle devices in healthcare sector: materials, challenging fabrication method-
ologies, and its limitations. The work on an economical and efficient method for the
fabrication of spiral micromixer is presented in the tenth chapter “An Economical
and Efficient Method for the Fabrication of Spiral Micromixer”. The eleventh chapter
“Damping Estimation and Analysis for High Performance Inertial MEMS for Early
Detection of Neurological Disorders During Pregnancy” is all about damping estima-
tion and analysis for high-performance inertial MEMS for early detection of neuro-
logical disorders during pregnancy. Finally, the last chapter “Affinity Biosensing:
Modeling of Adsorption Kinetics and Fluctuation Dynamics” presents the concept
of affinity biosensing: modeling of adsorption kinetics and fluctuation dynamics.
Silchar, India Dr. Koushik Guha

Kharagpur, India Gorachand Dutta
Asansol, India Arindam Biswas
Guntur, India K. Srinivasa Rao
Contents
N/MEMS Biosensors: An Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Vinayak Pachkawade
Lab-On-A-Chip Technology in Health Care . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Neha Mishra
A Review on Recent Trends in the Segregation of Red Blood Cells
Using Microfluidic Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Subhash Turaka, Valanukonda Bhavya Bhargavi,
Naga Nikhila Nandam, Shahed Baba Syed, Nanda Sai Donepudi,
Dhanya Yalamanchili, Koushik Guha, and Jasti Sateesh
A Road Map to Paper-Based Microfluidics Towards Affordable
Disease Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Mareedu Nagavalli, Tatineni Sharmila Swaroopa,
Pannangi Sri Vidya Gayathri, Vuyyuru Dinesh Kumar Reddy,
Nanda Sai Donepudi, Dhanya Yalamanchili, Koushik Guha,
and Jasti Sateesh
Critical Review and Exploration on Micro-pumps for Microfluidic
Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
J. Prithvi, B. S. Sreeja, S. Radha, C. Joshitha, and A. Gowthami
Fabrication Techniques and Materials for Bio-MEMS . . . . . . . . . . . . . . . . 101
Sudhanshu Dwivedi
In-silico Analysis of Expandable Radiofrequency Electrode
for Ablation of Hepatic Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Shaik Sadikbasha and Ashish. B. Deoghare
Lab-On-Chip Electrochemical Biosensor for Rheumatoid Arthritis . . . . 157
Rahul Kumar Ram, Nirmita Dutta, Jai Shukla, and Gorachand Dutta
vii
viii Contents
Transdermal Injection with Microneedle Devices in Healthcare

Sector: Materials, Challenging Fabrication Methodologies, and its
Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
A. Gowthami, B. S. Sreeja, and S. Radha
An Economical and Efficient Method for the Fabrication of Spiral
Micromixer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Ekta Tripathi, Pallab Sarmah, Promod Kumar Patowari,
and Sukumar Pati
Damping Estimation and Analysis for High Performance Inertial
MEMS for Early Detection of Neurological Disorders During
Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Sonali Biswas, Anup Kumar Gogoi, and Moushumi Biswas
Affinity Biosensing: Modeling of Adsorption Kinetics
and Fluctuation Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Olga Jakšić
Editors and Contributors
About the Editors
Koushik Guha is awarded Distinguished Faculty Award 2021 by NIT Silchar for his
outstanding performance in teaching and research. Recently, he has been awarded
IETE R S Khandpur award 2022 for his achievements and outstanding contribu-
tion at national and international level in the field of ‘Medical Instrumentation’
covering education, research, design, development, and production. Dr. Guha is also
awarded Institute of Smart Structures and Systems Young Scientist award (ISSS,
IISc Bangalore) 2021 for his contribution in MEMS/NEMS sensors and actuators.
He is Optimistic Researcher and Associate Professor in the Department of Elec-
tronics and Communication Engineering, National Institute of Technology Silchar,
India. He is now Associate Dean of Academic Affairs and Former Assoc. Dean of
Students Welfare in NIT Silchar. Dr. Guha is awarded five international patents and
one national patent. He has been nominated Fellow of Institution of Electronics and
Telecommunication Engineers (IETE) and Institute of Scholars (InSc) Government
of India. He has been Active and Regular Reviewer of (SCI/SCIE journals) and
various IEEE hosted/sponsored conferences.
Gorachand Dutta Ph.D., is Assistant Professor with the School of Medical Science
and Technology, Indian Institute of Technology Kharagpur. He received his Ph.D.
in Biosensor and Electrochemistry from Pusan National University, South Korea,
Postdoctoral fellowships in the Department of Mechanical Engineering, Michigan
State University, USA, and Department of Electronic and Electrical Engineering at
University of Bath, UK. He has expertise on electrochemical biosensors.
Arindam Biswas received M.Tech. degree in Radio Physics and Electronics from
University of Calcutta, India, in 2010 and Ph.D. from NIT Durgapur in 2013. He
has worked as Postdoctoral Researcher at Pusan National University, South Korea.
Dr. Biswas has received DST-JSPS Invitation Fellow at RIE, Japan, DST-ASEAN
Invitation Fellow at Duy Tan University, Vietnam and Taylors University, Malaysia,
ix
x Editors and Contributors
and Visiting Fellow to Department of Electrical and Computer Engineering, National

University Singapore. He has worked as Specially Appointed Associate Professor
(Visiting) at Research Institute of Electronics, Shizouka University, Japan. He has
been selected for IE (I) Young Engineer Award: 2019–2020; KNU Best Researcher
Award (Engineering and Technology): 2021; and KNU Best Faculty Award-2022
(Faculty of Science and Technology). Presently, Dr. Biswas is working as Associate
Professor in School of Mines and Metallurgy at Kazi Nazrul University, West Bengal,
India, where he has been actively engaged in teaching, research, and administration.
He has 53 technical papers in different journals and 55 conference proceedings and
8 authored books, 18 edited volumes, and 10 chapters with international repute.
Dr. Biswas has produced six Ph.D. students till date. His research interest is in
carrier transport in low-dimensional system and electronic device, nonlinear optical
communication, THz semiconductor source, IoT, and optimization.
K. Srinivasa Rao was born in Andhra Pradesh, India. He received Master’s and Ph.D.
degree from Central University. He is presently working as Professor and Head of
Microelectronics Research Group, Department of Electronics and Communication
Engineering in Koneru Lakshmaiah Education Foundation (Deemed to be Univer-
sity), Guntur, Andhra Pradesh, India. His current research areas are MEMS-based
Reconfigurable Antenna’s actuators, Bio-MEMS, RF MEMS Switches, RF MEMS
Filters, MOSFET, FinFETs, etc. He received Young Scientist Award from Depart-
ment of Science and Technology, Government of India, in 2011. He also received
UGC Major Research Project in 2012. He received early career research Award from
SERB, Government of India, in 2016. Presently, he is working on MEMS project
worth of 40 Lakhs funded by SERB, Government of India. He has published more
than 140+ international research publications and presented more than 55 conference
technical papers around the world. He is Member of IETE, ISTE, and IEEE.
Contributors
Valanukonda Bhavya Bhargavi Department of Electronics and Communication

Engineering, Velagapudi Ramakrishna Siddhartha Engineering College, Kanuru,
Vijayawada, Andhra Pradesh, India
Moushumi Biswas Silchar Medical College and Hospital, Silchar, India
Sonali Biswas Jorhat Engineering College, Assam Science and Technology Univer-
sity, Jorhat, India
Ashish. B. Deoghare Department of Mechanical Engineering, National Institute of
Technology Silchar, Assam, India
Nanda Sai Donepudi Siddhartha Government College and Hospital, Vijayawada,
India
Editors and Contributors xi
Gorachand Dutta NanoBiosensors and Biodevices Lab, School of Medical

Sciences and Technology, Indian Institute of Technology, Kharagpur, West Bengal,
India
Nirmita Dutta NanoBiosensors and Biodevices Lab, School of Medical Sciences
and Technology, Indian Institute of Technology, Kharagpur, West Bengal, India
Sudhanshu Dwivedi S.S. Jain Subodh P.G. (Autonomous) College, Jaipur, India
Anup Kumar Gogoi Indian Institute of Technology Guwahati, Amingaon, Guwa-
hati, India
A. Gowthami Materials and MEMS Laboratory, Department of Electronics and
Communication Engineering, Sri Sivasubramaniya Nadar College of Engineering,
Chennai, Tamilnadu, India
Koushik Guha Department of Electronics and Communication Engineering,
National Institute of Technology Silchar, Assam, India
Olga Jakšić Center of Microelectronic Technologies, Institute of Chemistry, Tech-
nology and Metallurgy, National Institute of the Republic of Serbia, University of
Belgrade, Beograd, Serbia
C. Joshitha Department of Electronics and Communiction Engineering, Koneru
Lakshmaiah Education Foundation, Hyderabad, Telangana, India
Neha Mishra Indian Institute of Technology, Bombay, India
Mareedu Nagavalli Department of Electronics and Communication Engineering,
Velagapudi Ramakrishna Siddhartha Engineering College, Kanuru, Vijayawada,
Andhra Pradesh, India
Naga Nikhila Nandam Department of Electronics and Communication Engi-
neering, Velagapudi Ramakrishna Siddhartha Engineering College, Kanuru,
Vinayak Pachkawade VPACHKAWADE Research Center, Nagpur, India
Sukumar Pati Department of Mechanical Engineering, National Institute of Tech-
nology Silchar, Silchar, Assam, India
Promod Kumar Patowari Department of Mechanical Engineering, National Insti-
tute of Technology Silchar, Silchar, Assam, India
J. Prithvi Department of Electronics and Communication Engineering, Sri Siva-
subramaniya Nadar College of Engineering, Chennai, Tamil Nadu, India
S. Radha Materials and MEMS Laboratory, Department of Electronics and
Communication Engineering, Sri Sivasubramaniya Nadar College of Engineering,
Chennai, Tamilnadu, India
xii Editors and Contributors
Rahul Kumar Ram NanoBiosensors and Biodevices Lab, School of Medical

Sciences and Technology, Indian Institute of Technology, Kharagpur, West Bengal,
India
Vuyyuru Dinesh Kumar Reddy Department of Electronics and Communication
Shaik Sadikbasha Department of Mechanical Engineering, National Institute of
Technology Silchar, Assam, India
Pallab Sarmah Department of Mechanical Engineering, National Institute of
Technology Silchar, Silchar, Assam, India
Jasti Sateesh Department of Electronics and Communication Engineering, Vela-
gapudi Ramakrishna Siddhartha Engineering College, Kanuru, Vijayawada, Andhra
Pradesh, India
Tatineni Sharmila Swaroopa Department of Electronics and Communication
Jai Shukla NanoBiosensors and Biodevices Lab, School of Medical Sciences and
Technology, Indian Institute of Technology, Kharagpur, West Bengal, India
B. S. Sreeja Department of Electronics and Communication Engineering, Sri Siva-
subramaniya Nadar College of Engineering, Chennai, Tamil Nadu, India;
Materials and MEMS Laboratory, Department of Electronics and Communica-
tion Engineering, Sri Sivasubramaniya Nadar College of Engineering, Chennai,
Tamilnadu, India
Pannangi Sri Vidya Gayathri Department of Electronics and Communication
Shahed Baba Syed Department of Electronics and Communication Engineering,
Ekta Tripathi Department of Mechanical Engineering, National Institute of Tech-
nology Silchar, Silchar, Assam, India
Subhash Turaka Department of Electronics and Communication Engineering,
Dhanya Yalamanchili Siddhartha Government College and Hospital, Vijayawada,
India
N/MEMS Biosensors: An Introduction
Vinayak Pachkawade
Abstract In the twenty-first century, biosensors have gathered much wider attention
than ever before, irrespective of the technology that promises to bring them forward.
With the recent COVID-19 outbreak, the concern and efforts to restore global health
and well-being are rising at an unprecedented rate. A requirement to develop precise,
fast, point-of-care, reliable, easily disposable/reproducible and low-cost diagnostic
tools has ascended. Biosensors form a primary element of hand-held medical kits,
tools, products, and/or instruments. They have a very wide range of applications
such as nearby environmental checks, detecting the onset of a disease, food quality,
drug discovery, medicine dose control, and many more. This chapter explains how
Nano/Micro-Electro-Mechanical Systems (N/MEMS) can be enabling technology
toward a sustainable, scalable, ultra-miniaturized, easy-to-use, energy-efficient, and
integrated bio/chemical sensing system. This study provides a deeper insight into the
fundamentals, recent advances, and potential end applications of N/MEMS sensors
and integrated systems to detect and measure the concentration of biological and/or
chemical analytes. Transduction principle/s, materials, efficient designs, including
readout technique, and sensor performance are explained. This is followed by a
discussion on how N/MEMS biosensors continue to evolve. The challenges and
possible opportunities are also discussed.
Keywords Biosensors · N/MEMS · Health and well-being · BioMEMS ·

Applications
1 Introduction
Biosensors are playing a crucial role in today’s biomedical science and technology
[1]. Biosensors allow the sensitive and precise detection of a range of biological or
chemical contaminants [1–5]. It is a device that measures biological or chemical
reactions and generates an output signal in proportion to the concentration of a target
V. Pachkawade (B)
VPACHKAWADE Research Center, 440032, Nagpur, India
e-mail: [email protected]; [email protected]
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 1
K. Guha et al. (eds.), MEMS and Microfluidics in Healthcare, Lecture Notes
in Electrical Engineering 989, https://doi.org/10.1007/978-981-19-8714-4_1
2 V. Pachkawade
analyte in the reaction. Typical elements of a biosensor are shown in Fig. 1. It consists
of the following elements: (1) a small inlet/channel (μfluidic) to collect/navigate the
sample/solution to the sensing system. A sample may be in the form of a solid, liquid,
gas, or combination thereof. A sample in solid form may contain dust, dirt, soot, or
smoke particles. Samples in the gaseous form include air with a mixture of oxygen,
nitrogen, carbon dioxide, or other hazardous gas elements. A liquid sample may
include bodily fluids, for example, nasal swabs, saliva, blood, semen, sweat, urine,
etc. Analyte/s in the samples are a target substance to be detected. For example,
particulate matter (PM) is an analyte whose concentration in the surrounding air
is to be measured. Triglyceride is an analyte whose concentration in the blood is
to be measured. In clinical applications, analytes of interest are glucose, vitamins,
hemoglobin, molecules, proteins, amino acids, urea, bodily gases, toxins, specific
biomarkers (to detect chronic disease), living cells, and pathogens (viruses and
bacteria). (2) Next element is called bioreceptors (see Fig. 1). Bioreceptors are a group
of molecules that are deposited/immobilized (as a thin film/layer) onto the surface
of a transducer. Bioreceptors interact with the sample/solution to precisely recog-
nize the target analyte/antigen. Such interaction between the bioreceptor molecules
and target analyte is termed biorecognition. The output of a biorecognition event
may result in a change in mass, light, color, temperature, pH level, etc. (3) A sensor
converts this form of energy into a measurable signal in either optical or electrical
form (conductance/impedance, charge/current, potential/voltage, frequency/phase,
etc.). Here, we use the words transducer and sensor interchangeably. (4) A sustaining
electronics in the biodevice/module can process the sensor output by further ampli-
fication, filtering, analog-to-digital (A/D) conversion, and microprocessor/memory
storage. Eventually, the module shows the reading on the computer or an embedded
display in real time. A wireless data transmission may also be added to the unit.
Figure 2 shows the information on the potential areas of applications of biosensors.
Biosensors are widely being deployed for environmental check (to detect hazardous
gas elements), to detect the onset of the existing/future disease, monitoring the quality
of food and beverages at places where these are stored, on farms to inspect soil quality,
in medicine dose control, and many other.
2 Characteristics of a Biosensor
In the biosensing development platform, the following parameters are of the most
importance. Researchers are continuously finding ways to improve the following
features (see Fig. 3).
Sensitivity/Resolution: It is defined as the smallest possible change in the phys-
ical/biological/chemical properties of a transducer that can be resolved after the
biomass/target analyte/s are adsorbed/absorbed by the sensor surface (i.e., after the
reaction occurs). Sensitivity (also expressed in %) can be given as the ratio of change
in the sensor output per unit change in the input. It is also called the detection limit.
N/MEMS Biosensors: An Introduction 3
Fig. 1 A typical bio/chemical sensing platform. Bioreceptors enable the selective detection of
a target analyte in the sample. A transducer’s job is to convert the biorecognition process into
measurable output. Such measurable output is then correlated to the detection and concentration of
a target analyte
Fig. 2 A diagram showing

potential application areas
where biosensors are used.
There are many other areas
where biosensors are
primarily used
4 V. Pachkawade
Fig. 3 Performance parameters in biosensor development
A mass sensor, for example, can show resolution up to fg/ml to record the concentra-
tion of analyte traces in a liquid sample. Measuring a lowest possible concentration
of a specific analyte/s (for example, antigen) can be associated with an underlying
medical condition for which doctors can prescribe a test.
Precision: Precision is an important feature of a biosensor. Preci-
sion/selectivity/specificity indicates the ability of a bioreceptor thin film molecule
to detect/recognize a specific analyte within a mixture of other contaminants in a
sample under test (see Fig. 1). An example of precision is the contact of an antigen
with the antibody. Typically, antibodies act as bioreceptors and are immobilized on
the surface of the transducer. A solution/sample that contains the antigen is exposed
to the transducer, where antibodies should interact specifically with the antigens. To
develop a biosensor, precision is the key characteristic.
Stability: The stability of a sensor is the characteristic to avoid responding to changes
in the environmental conditions (i.e., temperature, humidity, vibrations, pressure,
etc.), thereby preventing a false output. To address this issue, a provision is made in
the embedded electronics to compensate for environmental drifts in the sensor output.
Such common-mode signals can be canceled using a differential sensor readout.
Reproducibility: Reproducibility is the capability of the biosensor module to

produce the same output when the experiment/test is repeated. It implies that the
sensor provides an average value close to the correct value when a sample is
measured several times. Reproducible sensor output indicates the high consistency
and robustness of a biosensor.
Noise floor: This parameter provides the baseline above which a sensor output can
reliably be detected/measured. Noise in the sensing system stems from i) the intrinsic
noise in the sensing element and ii) electronics. Therefore, a sensor is designed to
reduce the overall noise. Lower the noise floor, better the sensor signal-to-noise ratio
(S/N), or say sensitivity/resolution.
Linearity and Dynamic range: A sensor can show linear changes in the output per
unit change in the input. The output of a sensor should produce a linear response to
different concentrations being measured. This characteristic is also often represented
by the % nonlinearity in the sensor output. Linear dynamic range (expressed in dB)
is the ratio of the maximum to the minimum sensor output.
3 N/MEMS Biosensor (BioMEMS)
N/MEMS features scalability, ultra-high sensitivity, energy efficiency, and compat-

ibility to easily integrate with microelectronics[6–8]. N/MEMS offers outstanding
precision in the detection and measurement of ultra-small elements. This advantage
comes with the miniaturization N/MEMS offers. For example, a nominal mass of
N/MEMS can scale down to nanograms or even smaller. It is therefore possible to
detect and measure things at 10–15 , 10–18 , or even lower scale. Miniaturization also
means a small footprint, weight, energy, and Internet of sensors (IoS), all at reduced
system-level cost. With N/MEMS, it is possible to precisely detect the target analyte
and measure its concentration. This is achieved through precisely recording even the
minutest shift in the sensor output [4, 9, 10]. Transduction principles such as piezo-
electricity, piezo-resistivity, electro-static, electro-magnetic, electro-thermal, optical,
etc. are popularly used in the development of the N/MEMS-enabled bio/chemical
sensors.
3.1 Mass Accumulation
N/MEMS devices are operated by the principle of gravimetric sensing [7, 11]. Mass
accumulation is characterized by the adsorption/absorption (biorecognition) of the
mass of a particle or target element in a biological/chemical sample. N/MEMS trans-
ducer is a mechanical cantilever/tuning-fork/plate/disk/bar/membrane. This trans-
ducer is set in a static or a dynamic mode (vibrate at a particular frequency called a
natural resonant frequency). Figure 4 shows a schematic illustration of an N/MEMS
6 V. Pachkawade
Fig. 4 A static N/MEMS transducer that can be used to sense and quantify target bio/chemical
contaminants in a sample. Part a shows a stress profile of a structure that can be used to use piezo-
resistive transduction. Part b shows a displacement profile of a structure that can be used to use
capacitive transduction
transducer that can be used as a primary element in a typical N/MEMS biosensing

platform. As seen in Fig. 4, a transducer (cantilever in this case) in a static form is
used for sensing purposes. A transducer surface is functionalized to attract specific
molecules. The functionalization takes place by depositing/loading nanoparticles
(for example, gold nanoparticles) onto the surface of a cantilever. After this, target
molecules are attached to the nanoparticles. Such mass accumulation leads to the
gradient in (i) the surface stress (σ) (see Fig. 4a), (ii) surface strain, and (iii) a posi-
tion/displacement of a cantilever (see Fig. 4b). In other words, the transducer is
actuated by the component of the force exerted by the bio/chemical reaction that
occurs at the surface of a transducer.
For the sensing part, a set of resistors (forming a Wheatstone bridge electronic
circuit) is embedded at the other end of a cantilever, where it is fixed/anchored (see
Fig. 4). At this end, the stress is maximum. Another viable method of sensing is to
monitor a change in the parallel plate capacitance at the free end of a cantilever. At
this end, displacement is maximum. A bottom electrode placed beneath the free end
of the cantilever can be used to detect the capacitive gradient when the cantilever
moves.
3.2 Materials
Usually, silicon/polysilicon is used to construct such a transducer. In recent years,

piezoelectric materials (for example, aluminum nitride (AlN) and quartz) have
become a popular choice in realizing several potential applications in BioMEMS [4].
However, materials such as silicon, piezo, etc. are suited only for non-invasive appli-
cations, a procedure that does not require inserting an instrument through the skin
or into a body. Such materials can be a part of N/MEMS-enabled biosensors. These

sensors can then be used for monitoring environmental parameters, lab-on-chip,
laboratory tests, wearable gadgets, etc. Given the biocompatibility issue, materials
such as Polydimethylsiloxane (PDMS) and other polymers are also widely used for
biosensing [12]. Another application of PDMS is that a transducer can be embedded
into the μ channels made by PDMS for lab-on-chip fluid testing applications.
3.3 N/MEMS Resonators for Ultra-Precise Bio/Chemical

Sensing
N/MEMS resonators are one of the most promising candidates to develop a wide
range of applications in medical diagnostic and instrumentation [1, 4, 8, 13–16].
N/MEMS resonators offer extremely high-quality factors (Q) and high frequency.
These two parameters result in exceptionally high parametric sensitivity. Addition-
ally, the N/MEMS resonant sensing platform provides good output stability. Due to
their highly precise output, N/MEMS resonators are extremely useful to sense envi-
ronmental, physical, biological, and chemical quantities. They can detect multiple
analytes in parallel, and offer immunity toward a false output. Being a primary
component in many commercial applications, N/MEMS resonators are today a
popular choice to develop low-cost, miniaturized, reliable, reproducible, and precise
sensing solutions. Possible areas of applications are point-of-care/remote monitoring
of target parameters in the environment, agriculture, medicine, health, and well-
being, thus democratizing sensing across the globe. N/MEMS resonators operate on
the principle of gravimetric sensing. The N/MEMS mass sensor is reported to detect
particle concentration as low as attogram (ag) or even zeptogram (zg). Figure 5 shows
a representative example of a N/MEMS resonator that can be used to detect and quan-
tify the concentration of a target contaminant in a biological/chemical sample. Upon
adsorption/absorption of a target trace/particle, a shift in the resonant frequency of a
transducer can be accurately recorded (in real time). These shifts can be correlated
to the concentration of the contaminant/s. The essential assumption is that adsorp-
tion/absorption of a mass, Δm is much smaller than the nominal mass, M of the
resonant transducer (Δm << M).
3.4 Multiple Analyte Detection Using N/MEMS
Figure 6 shows a platform parallel processing using N/MEMS sensors. As seen,

C1 −Cn are the cantilevers arranged as an array. These sensors are attached to a
common base. Each of the cantilevers is coated/immobilized/functionalized with a
specific bioreceptor thin film to attract the target molecules/particles. Such a sensing
platform is highly useful for the fast testing of multiple parameters in bio/chemical
8 V. Pachkawade
Fig. 5 A representative example schematic of a resonant mass sensor for particle detection and
concentration measurement of a target analyte. Δf indicates the shift in the resonant frequency, i.e.,
transducer output
samples. Due to simultaneous testing and processing, the overall cost is low. Such
a platform is compact and efficient. However, a specific coating of n number of
sensing units in an array is required. Such a requirement may be a trade-off with the
advantages in terms of cost and time.
4 Summary, Outlook, and Conclusion
This chapter introduced the basics of biosensors and how N/MEMS sensors can
be used in several bio/chemical applications. The chapter started by providing
an overview of a typical N/MEMS-enabled bio/chemical sensing platform. The
N/MEMS sensors can be used to detect and precisely quantify several biolog-
ical and chemical contaminants. This scientific area requires expertise, experience,
and collaborations across the multi-domains (mechanics, optics, biology, chemistry,
physics, and microelectronics). A static mode N/MEMS can be used to monitor
Fig. 6 An array of N/MEMS sensors to specifically detect and measure the concentration of a target
analyte in a sample. A parallel test and processing of multiple analytes in the sample are useful in
terms of cost and time
the surface stress, strain, or displacement when the mass of a target particle/analyte
is attached to the sensor. An electro/optical sensing mechanism or a combination
thereof can then be used to read and correlate the sensor output to the presence
and concentration of the target element/s in a sample. A dynamic (resonant) mode
N/NEMS is also used as a high-sensitivity transducer in biological and chemical
applications. Here, a transducer element is set to vibrate at the designed frequency.
When the mass of a target particle/analyte is adsorbed/absorbed on the chemically
functionalized surface of a transducer, a change in the effective mass of the resonant
transducer results in a change in the frequency.
N/MEMS-enabled hand-held biosensor modules can be employed for many appli-
cations. These applications are health care, surgery, drug discovery/dose control,
recognition of pollutants, detection of micro-organisms responsible for causing
a disease, and markers to indicate disease in bodily liquids/fluids (blood, urine,
saliva, semen, sweat, etc.). Typical applications of BioMEMS also include sensors
measuring intravascular blood pressure, therapeutics applications (e.g., drug delivery
actuators, disease monitors), pacemakers, and defibrillators. Biosensors can be
implantable or wearable. These are also part of sensing systems for real-time moni-
toring of several body parameters such as pulse rate, blood pressure, oxygen, and
neurological activities.
10 V. Pachkawade
References
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field: A brief review
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for healthcare and biomedical applications: A review. Meas J Int Meas Confed 167. https://doi.
org/10.1016/j.measurement.2020.108293
3. Choi JR (2020) Development of point-of-care biosensors for COVID-19
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Lab-On-A-Chip Technology in Health
Care
Neha Mishra
Abstract In the current era when early disease diagnosis is a need of the hour for
the proper cure, LOC devices are the most reliable and promising solution. Lab-
on-a-chip (LoC) devices are used for point-of-care diagnostics as they are compact,
cost-effective, integrable, and multiple diagnostics can be performed on a single
chip. For this reason, there are different diagnostic techniques used for LOC devices
such as optical detection, PCR, qPCR, paper-based assays, and lab-on-a-chip using
microfluidic platforms. The main focus of the aforesaid technique is the simplification
of device design with multiplexed processes and the availability of automation to
make LOC devices user-friendly. In this chapter, different techniques and processes
are discussed which are used in diagnostic and healthcare applications.
Keywords Microfabrication · Biosensor · Microfludic channel · Optical detection
1 Introduction
A microfluidic platform consists of a group of precisely defined, fabricated microflu-

idic operating components. Miniaturization, automating, and parallelizing chemical
processes [1, 2] are made possible by lab-on-a-chip (LOC) technologies. Because
they employ less chemical reagent in modularly constructed, miniaturized devices,
LOC devices have a lower cost advantage. The ability to operate quickly in a small
space is another significant benefit of LOC devices; this is made possible by the
parallelization of response chambers. The throughput and automation of analyt-
ical systems are also increased by LOC technology. To regulate fluidics at the
micro- and nanoscales, LOC devices can be created (Fig. 1), and depending on
the scale difference, these devices are sometimes referred to as microfluidics and
nano-fluidics. Micro-channels or nano-channels in LOC technology offer control
of fluids in minuscule quantities to enable biological processes in incredibly small
volumes.
N. Mishra (B)
Indian Institute of Technology, Bombay, India
e-mail: [email protected]
12 N. Mishra
Sample Sample Sample Sample

purification mixing separation Diagnostic
Fig. 1 Basic component of lab-on-a-chip technology
Utilizing the photolithography process, integrated circuits and microchannels can

have fluid control components and routes created. For LOC to become a comprehen-
sive system, it also needs the integration of micro-pumps, valves, micro-electrodes,
electrical fields, and micro-electronics [3].
Smaller and faster electronic devices are produced as a result of advances in
integrated circuit technology and wafer fabrication facilities. Silicon or glass and
polymers are the foundation of microfluidic manufacturing. Despite having well-
controlled mechanical and chemical qualities, silicon and glass require more expen-
sive manufacturing processes. One mould can be used as a master for fabricating
multiple devices in the case of polymers, however, using soft lithography or hot
embossing. This makes it possible to produce disposable goods in large quantities.
In contrast, polymers’ mechanical and chemical properties have reliability issues,
which necessitate surface modification for reliable device functionality [4]. In many
cases, PoC diagnostics involve lateral flow assays. These assays include moving a
liquid sample that contains the target analyte through various zones of polymeric
strips that have immobilized capture probes that can interact with the analyte as
shown in Fig. 2 [5, 6]. Commercially available pregnancy tests for the detection
of human chorionic gonadotropin hormone present in bio-analyte (urine) are widely
used for lateral flow assays. These tests use a sandwich-based immunoassay to detect
the target protein, which is realized by a colour shift that can be seen with the unaided
eye [5–8]. The key benefits of lateral flow tests include their affordability, conve-
nience of use, simplicity, and extended shelf life. However, multiplexing and flow
rate control are difficult with lateral flow assays since they call for a lot of chemicals
and relatively significant volumes of material [5–8]. By offering fine flow control
through various microfluidic channel geometry, microfluidic technology has been
used to overcome these restrictions [8, 9]. Figure 2 illustrates the basic schematic
diagram of the lateral flow assay, which explains how the process works. A small
amount (generally in microliters) of the analyte is placed into the sample pad and then
the analyte will go slowly to the probe pad through capillary action. The target analyte
is beholden by tagged detection probes. It will take to the detection membrane, and
then be captured on a line where capture probes are immobilized [5]. For proof-
of-concept diagnostics of several analytes, capillary-driven microfluidic chips have
been employed [6, 10–13]. For instance, a triage system is commercially available,
which consists of a disposable protein chip and a portable analyzing device, that
attempts to identify a wide range of medical disorders [14, 15] (Table 1).
Lab-On-A-Chip Technology in Health Care 13
Fig. 2 Schematic diagram of lateral flow assay strip. Reproduced with permission from
Anfossi et al., Biosensors 9(1):2 (2018)[5]
Table 1 LOC Technologies in healthcare [2]

LOC area LOC application Products References
Diagnostics PoC, PCR, qPCR, DNA Fluidigm, biomark HD, Agilent [16–25]
isolation, electrophoresis, lab chip, Elvesys system
sequencing, Colorimetric
detection, Optical absorbance,
Amperometric sensor
Genomics Microarray for next-generation Nanopore, Illumina hiseq, [26–28]
sequencing PasificBio, Ion torrent
Biochemical Immunological assays, Dexcom G5, Preganews [29–31]
Assays Pregnancy kit,
glucose Monitoring
Proteomics MS, SDS-PAGE 908 Devices zip chip [32, 33]
Biosensors Optical biosensors-based IST AG IV4 biosensor [34–36]
devices, Bio-affinity and
bio-catalytic devices,
Antigen–antibody-based
devices
Cell research Flow cytometers, cell culturing Fluidigm helios [37]
and monitoring
One of the major fields of application of lab-on-a-chip is molecular biology;

it includes biochemical, research related to cell and proteomic, biosensors, and is
further used for drug design and development. Diagnostic and genomic analyses are
the primary uses for LOC. There are numerous molecular biology studies present
which include PCR, DNA isolation, qPCR, sequencing, and electrophoresis by using
LOC. It has enormous scope to develop PCR microfluidics devices that can be used
for speeding up the amplification of DNA and RNA. LOC PCR devices provide quick
viral and bacterial identification during any pandemic to prevent infection. Different
genetic diagnostic mechanisms are laboratory-independent and there are used for
the detection of HIV and HBV infection. These methodologies are made possible
because LOC technologies have reduced the size of diagnostic devices.
These emerging LOC devices can also be utilized to identify the biomarkers of
diseases with a genetic base. LOC devices are also used in the food industry for the
14 N. Mishra
detection of pathogens present in food. Earlier for this purpose DNA sequencing was
used which was a time-consuming and costly process.
It took 15 years to complete the first human genome project. Today, a human
genome can be sequenced using LOC devices in a matter of hours, which is faster
than traditional methods. Next-generation sequencing devices are mostly used in
laboratories for reading millions of DNA fragments on a single chip using LOC
technologies. Nanopore Technologies is one of these companies and has produced
the smallest and fastest platform that is now awaiting commercialization. Mass spec-
trometry (MS) analysis, cell extraction, electrophoresis, blotting, and digestion are
all components of protein analysis. LOC combines every phase of protein analysis
onto a single chip [2]. The integrating steps speed up the analysis by several minutes.
In LOC systems, the crystallization process can be parallelized and accelerated to
better understand its structure. Immunoassays are another LOC use that can decrease
the reaction time in comparison to the standard method. The best platform for cell
research is LOC microchannels; microfluidics allows for the control of cell flow,
tagged antibody staining and imaging, cell differentiation, cell sorting, and cytometry
in cell biology experiments [2].
1.1 Diagnostics
LOCs are used in a variety of chemical and molecular diagnostic procedures,

including heavy metal ion detection [18, 20, 35], biological analyte present in biolog-
ical fluid [15, 19, 21, 22, 24] antigen–antibody interaction, DNA extraction, PCR,
qPCR, electrophoresis, DNA purification, molecular detection, and others.
1.1.1 Optical Detection Techniques for the Detection of the Analyte

Using a Microfluidic Channel
There are various methods currently used for the sensing and detection of relevant
biological and chemical analytes present in biological as well as water samples. For
this purpose, optical absorbances, as well as evanescent-based optical techniques,
are used [18, 19, 21, 22, 35].
1.1.2 LOC Devices Used for the Extraction and Purification of DNA
For diagnostic tests, nucleic acid from biological cells must be purified. The two main
processes in the extraction of DNA are cell lysis (disruption of the cell membrane and
nucleus membrane) and DNA purification (removal of undesirable RNA, membrane
lipids, and proteins). Devices for lysing LOC cells can use a variety of lysis tech-
niques, including chemical, thermal, ultrasonic, electrical, mechanical, etc. Most
macroscale DNA purification methods employed in extraction columns, which rely
on DNA adsorption on silica beads in particular buffer conditions, are portable to

microfluidic techniques. Therefore, silica beads can either be inhibited during the
washing and elution phases of LOC devices by mechanical barriers or by silica-coated
magnetic beads which can be easily stopped by a magnet [17].
1.1.3 Molecular, qPCR, and PCR Detection Using LOC Devices
PCR is the next most popular analysis following DNA extraction. Numerous PCR
uses are similar to sequencing techniques both directly and indirectly. Undoubtedly,
one of the applications of direct PCR is the amplification of DNA sequences that aids
in the observation of minuscule amounts of DNA (e.g., for the detection of different
pathogen such as viruses, fungi, and bacteria). The creation of multiple LOC PCR
machines is impacted by the significance of PCR in genetic analysis. Rapid thermal
transfer for quick and coupled PCR is caused by the miniaturization of volume and
increased surface-to-volume ratio. To merge PCR and electrophoresis on-chip, PCR
additionally needs a post-analysis to allow for the size identification of amplicons
by electrophoresis [38].
Originally, gels comprised primarily of agarose (for longer DNA) and polyacry-
lamides were used for electrophoresis (for shorter DNA). DNA electrophoresis was
one of the first molecular processes that could be incorporated into a chip with the
development of LOCs [39]. This miniaturization made it possible to speed up the
electrophoresis process, even more, use fewer reagents, and assemble other previ-
ously described DNA manufacturing steps on a chip. Another method that has been
tailored for LOC devices is qPCR, which has the advantages of being quicker (auto-
matic detection during PCR), more sensitive, and long-lasting. The below Fig. 3
shows the basic process flow of the micro-PCR system.
A device is being developed by Elvesys, which is an ultra-fast qPCR microfluidic
system for the molecular analysis of numerous diseases, including ebola and anthrax.
Using an ultra-fast pressure controller, fluorescence reader, and depending on ultra-
fast temperature control, it takes only a fraction of a minute and also has detection
efficiency comparable to the commercial system [40].
Another newly-emerging field is digital microfluidics which deals with emulsions
and droplets inside LOC devices. Droplets can collect extremely small amounts of
DNA, and limits can be raised by detecting one copy numbers using LOC droplet
qPCR as shown in Fig. 3 [25].
1.2 Genomic Application
Due to the results of sequencing initiatives like the Human Genome Project, the
discipline of genomics has experienced a remarkable expansion in recent years.
Sequencing-related projects help advance the field’s technology. Analyzing DNA
sequences to determine their nucleotide order is known as sequencing. The Sanger
16 N. Mishra
Sample
collection
RNA
Result
Extraction
Micro PCR
system
RNA
Microflui
Amplificat
dic chip
ion and
input
detection
Fig. 3 Microfluidic RT-PCR assay
method, on which the initial sequencing was based, produces DNA fragments of
various lengths with fluorophores that correspond to various nucleotide ends. By
substituting a tiny capillary filled with gel for the polyacrylamide gel slab used in
conventional DNA sequencing, miniaturization of the process was made possible.
The volume of the necessary DNA sample was reduced by the capillaries’ modest
size [41].
The next-generation sequencing technologies for ultra-fast DNA sequencing
currently developed are Sequencing-by-hybridization (SBH), nano-pore sequencing,
and sequencing-by-synthesis (SBS). High-throughput sequencing’s cost and duration
can be significantly decreased by using the LOC method for these technologies [27].
1.3 Microarray
Multiple biosensors must be integrated in conjunction with DNA microarrays for

the investigation of complicated DNA sample expression. Miniaturization, speed,
and precision are the key characteristics of lab-on-a-chip devices. Rapid multiplex
analysis of nucleic acid samples using this technology has enormous potential for
applications such as forensic investigation, drug screening, infectious agent identifi-
cation, differential gene expression measures, and the diagnosis of genetic illnesses.
Numerous elements of genetic study are being revolutionized by this usage of DNA
microarrays [28].
1.4 Biochemical Applications
The LOC technology can be coupled with immunological and enzymatic assays
on a single microfluidic platform, as in the case of monitoring glucose and insulin
simultaneously. The availability of a LOC that can track both glucose and insulin
simultaneously holds enormous promise for better diabetic management.
The incorporation of pertinent sample pre-treatment/clean-up processes necessary
for whole-blood analysis is critical for the proper and effective realization of such
glucose monitoring. The assembling of additional tests and analyte handling steps can
be done using the new LOC technique, which is desirable for developing miniature
clinical devices [31].
1.5 Proteomics
One of the serious challenges affecting science in the post-genome era is proteomics.
Proteome profiling, the most fundamental form of proteomics, is a difficult task that
involves identifying all the proteins identified in each sample. Owing to its broad
range of concentrations, diversity of molecules, and capacity to bind to solid surfaces,
the proteome presents special analytical challenges. LOC devices help create creative
solutions to challenging analytical challenges like proteome profiling. The four areas
of this application where LOC devices are advanced are chemical processing, sample
pre-concentration and clean-up, chemical separations, and interfaces with mass spec-
trometry [42]. LOC is a small device for protein separation and detection that takes
minimal reagents is simple to operate and completes tests relatively quickly. LOC
protein devices can manufacture a wide range of data points, allowing for the extrac-
tion of the most information from each sample. Another benefit is that it is simple to
compare information from LOC devices to data collected using 2D-PAGE [32].
LOC proteins sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE) isolation with UV spectrophotometry is the most frequently used approach
for sorting and quantifying protein combinations. SDS-PAGE frequently takes a lot
of work and time. LOC devices hasten separation and pave the way for protein
sizing automation [33]. LOC MS protein profiling is one technique for measuring
proteins. These devices are utilized for indirect infusion into the mass spectrometer
during Capillary Electrophoresis (CE) segregation, on-chip processing of the sample,
and infusion of CE before MS analysis, it was claimed. The goal of the project is
to use electrospray ionization MS to provide an interconnection between both the
spectrometer and the LOC [33].
18 N. Mishra
Recognition
Analyte Transducer Electric Signal
Element
Fig. 4 Basic element of biosensors
1.6 Biosensors
Lab-on-a-chip biosensors, which are small and biologically active devices, are used to
find the target analytes. Such systems directly couple a biorecognition element (direct
interaction with the intended analyte) with a physical transducer, which transforms
the biorecognition process into electric signals as illustrated in Fig. 4. Bio-catalytic
and bio-affinity devices are the two categories of LOC biosensors. Devices that use
bio-affinity are based on the target analyte binding selectively to a partner ligand that
is encapsulated on the surface (e.g., antibody, oligonucleotide).
Single-stranded (ss) DNA probes, for instance, are fixed on the transducer surface
in hybridization biosensors. The attachment of an adequate hybridization indica-
tion after binding allows for the detection of duplex formation. But in bio-catalytic
devices, the target substrate is recognized by an immobilized enzyme. For instance,
glucose oxidase immobilized sensor strips for diabetes monitoring [36]. The biosen-
sors are also used for the detection of certain biomarkers for the detection of chronic
diseases like cancer. Monitoring of cancer cell biomarkers is a need of the hour. For
this purpose SPR and LSPR-based biosensors are prominently used [43].
1.7 Cell Research
A study into a single cell may be carried out on a very small scale thanks to the employ-
ment of LOC devices. Single-cell handling and manipulation are typically necessary
because, after being positioned inside the microchannel, the single cell must also
be put or trapped at a certain location. It should be possible for the microsystem to
carry out minimal analyte present in the sample fluid. LOC systems also made it
possible to control the surrounding environment of a cell using different methodolo-
gies such as controlling the physical or chemical nature of the surface to which the cell
attaches, surrounding temperature, and pH. It is possible to use several, frequently
intricate fluid-handling components when the cell is to be subjected to diverse kinds
of stimuli. These concepts suggest that cell location and chemical stimulation must
be combined. To prevent sample wastage, which might happen if multiple devices
will be used, a single platform is used for cell lysis and analysis [44, 45].
Flow Cytometry is one of the most commonly and successfully developed and
used lab-on-a-chip devices. LOC flow cytometer systems have advanced quickly
over the past ten years as a result of the growing need for data on cellular analysis
that is of higher quality and broader scope as well as the complexity involved and
availability of microfabrication technologies. As a result of increasing technological
capabilities and expanding needs, microfluidics in the flow cytometric examination
of cells and particles has experienced several exciting advancements [37].
1.8 Organ-On-A-Chip
The term “organ-on-a-chip” (OOC) refers to a microfluidic cell culture system that
has constantly perfused chambers colonized by a variety of cell types to mimic the
physiology of different tissue and organ types. OOC has the potential to be a valuable
tool in the application of drug research and development by accurately recapturing
levels of tissue and organ functionality. The different components of organ-on-a-chip
are shown in Fig. 5 [46].
Fig. 5 Basic components of an organ on a chip. Reproduced with permission from Peng Zhang
Elsevier, (181–198), 2022
20 N. Mishra
1.9 Drug Design and Development
To help guide the emergence of new pharmaceuticals, the industry needs new tools,
especially ones that can predict how potential new treatments might behave in humans
based on how well they operate in cells and animals [34]. Even though some analyt-
ical applications of Lab-on-a-chip systems in the innovation and use of biopharma-
ceuticals seem simple (like analytical techniques to measure and enhance the yield
of protein drugs like therapeutic antibodies), others, like assays based on primary
human cells, are technically more challenging but are still possible, at least in some
circumstances. In any setting, the LOC platform could be used repeatedly in a highly
flexible and reproducible way [44].
2 Conclusion and Future Directions
A recent field termed LOC deals with the miniaturization of laboratory devices
into micro dimension devices. The major advantage of LOC devices is that they
are cost-effective, user-friendly, multiple processes can be performed at the same
time sensitive and speedy device. Aforesaid devices conclude that they are used
for advanced diagnostic purposes shortly by taking a look at commercialized LOC
equipment like RT-PCR, pregnancy kit, DNA sequencing, and glucose monitoring. In
future, more goods created with LOC technology will be available in the market. Day
by day, the industry will produce LOC devices more affordably. LOC development
has a direct impact on molecular-based inventions that are employed in daily life.
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A Review on Recent Trends
in the Segregation of Red Blood Cells
Using Microfluidic Devices
Subhash Turaka, Valanukonda Bhavya Bhargavi, Naga Nikhila Nandam,

Shahed Baba Syed, Nanda Sai Donepudi, Dhanya Yalamanchili,
Koushik Guha, and Jasti Sateesh
Abstract The isolation of Red Blood Cells (RBCs) has become a broad area of
research in recent times. The early segregation of RBCs from the blood prevents
them from lysis. The segregation of RBCs using traditional techniques like centrifu-
gation has become outdated due to the usage of bulky equipment. This paper reviews
the functions of RBCs, the age-old techniques that were practically used to distin-
guish RBCs, and their drawbacks. The assessment of microfluidic devices which are
prevalently used in present-day diagnostics that are promised to replace the bottle-
necks posed by the traditional methods is also presented. This review aims to project
the recent advancements in microfluidics, their applications, and the segregation of
microfluidic particles using them. The modern approaches that can separate RBCs
virtually using electroosmotic phenomena like di-electrophoresis are also reviewed.
The present scenarios for the separation of RBCs with a FEM tool computer-aided
design for virtual analysis are also discussed.
Keywords Isolation · Red Blood Cells · Centrifugation · Microfluidics ·

Di-electrophoresis
1 Introduction
The human body resembles a very complex and technological machine. It functions
as a single entity, although it is made up of numerous interconnected systems. Each
system is linked to a certain function that is generally required for an individual’s
S. Turaka · V. B. Bhargavi · N. N. Nandam · S. B. Syed · J. Sateesh (B)

Department of Electronics and Communication Engineering, Velagapudi Ramakrishna Siddhartha
Engineering College, Kanuru, Vijayawada, Andhra Pradesh 520007, India
N. S. Donepudi · D. Yalamanchili
Siddhartha Government College and Hospital, Vijayawada 520008, India
K. Guha
Department of Electronics and Communication Engineering, National Institute of Technology
Silchar, Assam 780010, India
26 S. Turaka et al.
Fig. 1 The figure illustrates the classification of blood in the human body
well-being. The ability of the body systems to function together assures survival.
As a result, both the composition and working of the human body are obscured [1].
Among all the body systems, transport systems that consist of the blood, cardiovas-
cular, and lymphatic systems guarantee that all body cells have insight into different
nutrients necessary to sustain them and offer a way of eliminating waste. The blood
exports substances throughout the body using a network of blood vessels. The main
functions of blood are respiratory, transportation, temperature regulation, excretory
functions, immunity, acid–base balance, etc [2]. Blood can be categorized into two
parts: plasma and blood cells. Plasma consists of 95% water with a wide range
of substances and proteins like albumins, globulins, electrolytes, nutrients, waste
products, hormones, and gases as shown in Fig. 1. Blood cells can be classified into
erythrocytes, leukocytes, and thrombocytes as shown in Fig. 1 to perform functions of
gaseous exchange, protection, and blood clotting. Red Blood Cells (RBCs) constitute
99% of blood cells [1].
2 Red Blood Cells and Their Functions
Erythrocytes are hollowed disks without nuclei which are around 7 μm in circumfer-
ence [1]. Their primitive function is to transport gases, primarily oxygen, although
they also transport carbon dioxide. Their distinctive form is well adapted to their
function; the concavity improves the surface area available for gas exchange, while
the core portion’s thinness allows for quick gas entrance and escape. The cells are
elastic, allowing them to pass through tiny holes, and they lack intramolecular struc-
tures, enabling hemoglobin, a large colored protein involved in gas transport, to
A Review on Recent Trends in the Segregation of Red Blood Cells … 27
occupy a wider area. In clinical practice, measurements of red cell counts, volume,
and hemoglobin concentration are helpful measures.
RBCs do not split as they lack nuclei, so they should be restored regularly by
parent cells from the red bone marrow, which may be observed at the extremities of
bones. Before being absorbed into circulation, RBCs experience a number of stages
during their development. In circulation, erythrocytes have a lifespan of roughly
120 days in adults and 70 days in infants [3]. In the typical healthy body, there are
roughly 30 trillion RBCs, about 25% of the overall cell count, and 1% of these,
mostly older cells, are removed and destroyed every day [1]. Since the bone marrow
generates erythrocytes at the same rate as they are destroyed, red cell levels stay
relatively constant. A homeostatic negative feedback system is responsible for the
regeneration of RBCs [4]. Erythropoietin, a hormone generated mostly by the kidney,
controls red blood cell formation [5]. When erythropoietin concentrations are low,
red cell production is inhibited, resulting in anemia.
Phagocytic reticuloendothelial cells do hemolysis or blood degradation [6]. These
cells can be found in different organs, but the spleen, bone marrow, and liver are the
most common sites of hemolysis. The cell membranes of erythrocytes grow weaker as
they age, making them more prone to hemolysis. The iron produced by hemolysis
is stored in the body and utilized to make new hemoglobin molecules in the bone
marrow. The hem component of hemoglobin is used to make biliverdin [7]. It is
nearly completely converted to the yellow color bilirubin before being linked to
plasma globulin and transmitted to the liver. It is transformed in the liver from a
fat-soluble to a water-soluble state and then excreted as component bile. As a result,
RBCs must be separated from the blood to be protected against hemolysis.
3 Laboratory Techniques for the Segregation of RBCs
The conventional laboratory approach for the separation of RBCs is centrifugation

which is based on the density and volume of particles [8]. The other techniques that are
practiced for the isolation of RBCs are density gradient centrifugation, agglutination,
and phase partitioning as shown in Fig. 2 [9–15]. The principle involved in every
technique has been discussed further.
3.1 Centrifugation
Centrifugation is a methodology that utilizes centrifugal force to remove blends or

particulates from a solution. The major components required for centrifugation are
centrifuge and centrifuge rotors. When rotating swiftly, the centrifuge rotor forces
the heavier molecules to the narrow end and the milder particles to the upper end.
The centrifuge rotors play a crucial role in the segregation process. The centrifuge
rotor can be categorized into three types: swinging-bucket rotors, vertical rotors, and
28 S. Turaka et al.
Fig. 2 The figure illustrates different laboratory techniques that are in practice for the segregation
of RBCs
fixed-angle rotors which are used to determine the type, speed, and range of the
volume. Depending on the type of rotor used, centrifugation can be classified into
two types: differential and density gradient as shown in Fig. 2. Density gradient can
further be classified into rate-zonal and isopycnic.
As the rotor rotates in a centrifuge under the influence of centrifugal force, the
blood particles in the solution residues at a rate that is in correlation with the applied
centrifugal force. Buoyant and frictional forces oppose the centrifugal force operating
on blood particles. As a result, the blood particles migrate from the point of rotation.
When the induced centrifugal force is greater than the two opposing forces the blood
particles precipitate at a constant rate as shown in Fig. 3. In Fig. 3, RBCs are at the
lower end as they have low density, whereas plasma is at the upper end due to its
high density. The middle layer in Fig. 2 is a buffy coat which is a combination of
platelets and white blood cells (WBCs).
Fig. 3 The figure depicts the isolation of blood particles under the influence of centrifugal force
The factors that affect the centrifugation process are the density of the sample and
solution, viscosity, temperature, and rotation speed. However, the significant draw-
backs of this process are its bulky equipment, the transformation of cell structure
when subjected to high gravity, salts present in the blood that can cause corro-
sion to the equipment, requires costly infrastructure for continuous centrifuges [16].
Moreover, the centrifugation technique is based on the size of the blood particles
which alters as they age in circulation. To overcome this effect, density gradient
centrifugation came into the picture which is based on the density of particles.
3.2 Density Gradient Centrifugation (DSC)
In hematology, difficulties such as partitioning erythrocytes into age-dependent frac-

tions and preparing blood samples rich in reticulocytes are of major interest. The
frequently utilized approach for the separation of RBCs is density gradient centrifu-
gation [10–12]. Density gradient centrifugation is almost similar to that of the conven-
tional centrifugation process but is based on density rather than size. In DSC, each
particle has a unique collection of physical traits or biological features, that may
be utilized to separate and isolate it. The focus of density gradient centrifugation
is on two factors: size and density. The amount of time needed for this treatment
is determined by the particle size. Tiny particles take longer to cross through the
bigger particulate region and accept the position deeper in the gradient, whereas
larger particles reach their point of stability sooner.
DSC uses subsidiary materials like bovine serum albumin, gum acacia, etc., which
the reader may refer to [9, 11, 17–21]. The reagent in DSC is a substance that aids
in the isolation or separation of cells. These items can not only speed up the process,
but they can also improve purity and throughput. The reagents can considerably
enhance the accuracy of DSC by preventing particles from aggregating, generating
a set divider, or removing remaining red blood cells. But these reagents can cause
various types of cancer which are not desirable. Hence, there is a need for another
separation technique that does not require reagents. Differential centrifugation (DC)
is a technique that does not use reagents but segregates particles based on their mass.
DC is sometimes seen to be a more straightforward method of centrifugation. Cells
and organelles are separated using it, while molecules and particles are separated
using density gradient centrifugation. The sort of physical qualities on which the
two centrifugation processes are based is the fundamental distinction between them.
Although differential centrifugation is simpler, density gradient centrifugation may
separate considerably smaller particles. But the physical properties of blood alter
as they age in circulation. A comparative study has been drafted about the aging of
blood by researchers using agglutination.
30 S. Turaka et al.
3.3 Agglutination
Antibodies can cluster cells or particles in a process termed agglutination, in addi-

tion to precipitating soluble molecules and flocculating molecules in suspension.
The existence of immunoglobulin against microbial species or erythrocytes can be
detected through agglutination. On a glass plate or a microtiter plate, agglutination
experiments are typically rapid and simple to perform. The process of agglutina-
tion can be performed by direct and indirect Coomb’s test. In the direct Coomb’s
test, first, a sample of blood is extracted with hemolytic anemia and antibodies
attached to RBCs. Coomb’s reagent is blended with the collected blood sample then
clumping (agglutination) is observed after the cross-linkage of antibodies. In the indi-
rect method, patient serum containing antibodies is drawn and donor blood is mixed
with the collected serum. As a result, the patient’s antibodies bind to donor RBCs.
After adding a few drops of Coomb’s reagent agglutination reaction is observed.
When RBCs are subjected to agglutination it is observed that the younger RBCs
have more negative charge when compared to older RBCs [22].
3.4 Phase Partitioning
The topology of microvascular networks is complicated, with many bifurcating

vessels. At diverging bifurcations, phase partitioning of RBCs (which contains
various enzymes) occurs, resulting in a diverse RBC distribution that impacts the
oxygen supply to living tissues [23]. T-butanol and ammonium sulphate are used
in three-phase partitioning (TPP) to extract important nucleoids from aqueous solu-
tions. The approach may be used upstream as well as downstream with basic steps
that can be scaled up. Various laboratories have isolated around 25 lipids and proteins
using TPP–t-butanol [24]. When enough salts, like ammonium sulphate, are added to
tertiary butanol, the blood distinguishes into two stages: a higher t-butanol stage and
a lower hydrated stage. If RBCs are observed in the initial hydrated stage, they may
divide into a third stage, midway between the lower hydrated and higher t-butanol
stages, based on the quantity of ammonium sulphate added. This is the foundation of
the technical term “three-phase partitioning,” or TPP, for isolating and concentrating
proteins.
The above-mentioned approaches for the separation of RBCs depend on the phys-
ical and chemical properties of blood. The properties of blood alter as they age in
circulation. Furthermore, these approaches require reagents like polyethylene glycol
and phthalates which can cause life-threatening diseases. Moreover, the laboratory
techniques require bulky equipment, huge laboratory, and human intervention. To
overcome these, microfluidic devices have been developed for the separation of
RBCs.
4 Microfluidics and Applications
Microfluidics has the potential to impact a large assortment of fields, which includes
biological analysis, optics, and information technology [25]. As a variety of innova-
tive components and techniques for injecting, blending, regulating, and preserving
fluids in microfluidic channels have been developed and applied, the range of applica-
tions of microfluidics in genetics and numerical biochemistry has risen [26]. Despite
fast advancement, some issues remain addressed, including sample preparation and
introduction, connecting microlevel channels with human intervention, dealing with
a variety of testable quantities, and mobility.
Microfluidics can change how biology is currently handled [27]. Microfluidic
systems enable the isolation, manipulation, and examination of tiny groups of cells,
or even single cells [28–30]. The capabilities of microfluidic devices relevant to
chemurgy are miniaturization, small volume, large surface area, scaling out, automa-
tion, etc. For personalized care, microfluidic devices can gather factors such as
proteomic, metagenomic, and genetic data [31, 32].
Microfluidic systems are emerging in a large assortment of applications, making
them impossible to assess all within a given amount of time. Some of the applica-
tions are arrays (interaction of a large number of molecules), gradients, microfilters,
droplets, etc.
5 Physics of Microfluidics
To comprehend and deal with microfluidics, it is necessary to first comprehend the

physics that prevails at the micro range. Other microfluidic reviews are available [33–
37], but none of them includes a thorough examination of the quantum mechanics of
the microlevel and how it enables particular devices. The physics of microfluidics is
addressed in this section, with pointers to more comprehensive studies.
At the microscale, factors that are not present in ordinary life become prominent
[38]. Because of scaling, it’s generally detrimental to downsize existing big devices
and expect them to work well at the microscale [39]. Designs must be developed to
take benefit of microscale forces. Laminar flow, diffusion, Reynolds number, surface
area to volume ratio, and surface tension are some of the factors which are prevalent
in microfluidics.
5.1 Laminar Flow
A phenomenon in which the motion of particulate in a liquid medium is not a

stochastic phenomenon of time is known as laminar flow. The microchannel stream
is usually laminar because of its limited size [40]. The major consequence of laminar
32 S. Turaka et al.
flow is that two or more streams running nearby will not mix unless they do so
through diffusion. For conducting experiments and sorting particles by size, diffusion
across laminar flows in a microfluidic device has been employed [41, 42]. Another
approach made possible by the laminar stream is the formation of fluid particles,
barring eccentric influences on both sides, which remain reasonably well-formed.
These particulates may be transported about in an organized fashion, allowing a wide
range of cellular investigation options.
5.2 Diffusion
Diffusion is the phenomenon by which a dense concentration of molecules in an area

spreads radially outward owing to random motion, resulting in a constant average
particle concentration across the volume. D2 = 2dt may be used to accomplish one-
dimensional diffusion, where D is the particle’s distance traveled in time period t and
d is the diffusion parameter. Because displacement alters from the square power on
the microlevel, diffusion evolves even more essential.
5.3 Reynolds Number
A fluid flow’s Reynolds number (Re) indicates whether the flow is laminar or turbu-
lent. Below is a detailed description of the laminar flow. Turbulent flow is erratic
as well as uncertain. The Reynolds number may be determined using the following
formula:
ρvdh
Re = (1)
μ
where ρ is the density of the fluid, v is the fluid velocity, dh is the circumference of
the hydraulic, and μ is the viscosity of the fluid.
The value of Re 2300, as computed by the formula, implies a laminar flow. The
liquid starts to exhibit indications of turbulence when Re approaches 2300, and when
Re exceeds 2300, the flow is deemed turbulent [27].
5.4 Surface Area to Volume Ratio
The component which is essential in the micro range is the surface area to volume
ratio (SAV). Capillary electrophoresis (CE) in microchannels is more efficient when
the SAV ratio is high because surplus heat is removed more quickly. Unfortunately,
when utilizing electrokinetic flow to transfer fluids, the high SAV ratio permits macro-
particles to readily absorb and bind to channel surfaces, lowering the performance.
5.5 Surface Tension
Adhesion among molecules of a solution at the fluid/gas interface causes surface

tension. The solvent surface-free energy is the amount that shows how much tension
is present on its surface. The height at which liquid can pass through a capillary is
proportional to the liquid surface energy and conversely to the capillary radius. The
distances that solvents will migrate solely on capillary pressure are substantial when
microchannels with sizes in the range of microns are employed.
The Young–Laplace equation may be used to compute the pressure created by a
surface of the liquid with orthogonal radii of curvature R1 and R2 and is given by

1 1
P = γ + (2)
R1 R2
where γ is the solution surface energy, The height of the wall is defined by R, which
extends to infinity, and the equation is reduced to
γ
P = (3)
R
It calculates the pressure at the liquid border linking two cosmic long plates
isolated by 2R. If R1 = R2 and the region is spherical, the equation becomes
2γ
P = (4)
R
6 Modeling of Microfluidic Devices for Medical

Applications
Microfluidics, a branch of biomedical research, has shown to be a profitable sector for

replacing regular assessments and detection procedures, conducting basic botanical
investigations in cells and disorders. Microfluidics investigations have enabled the
development of a variety of assuring biomedical applications, including biosensing
elements, point-of-care medical diagnostics, drug tests, content providers, innate
medical gadgets, unique biomimetics, artificial tissues, and unicellular studies,
among others [43]. Different micro- or nanotechnology methods are employed and
can be chosen based on the eventual use of the microdevice.
34 S. Turaka et al.
In the application, material selection is also critical. The typical materials used
are silicon, glass, thermoplastics, and elastomers [44]. Out of these elastomers like
Polydimethylsiloxane (PDMS) has moderate protein crystallization, porosity, and
droplet formation. It also provides good permeability, bio-culture, and disposable
device use.
The connection and/or merging of components for the applications for which the
device is being created is another key part of microfluidic device construction. Micro
heaters, injectors, detectors, electroosmotic liquid taps, and readout electronics, may
be integrated to create full microfluidic systems with impressive capabilities [45].
7 Microfluidic Cell Separation and Sorting Techniques
Despite product evolution into microdevices, the downsizing of lab-on-a-chip

(LOC) devices persists as a hedge. The mixing, pumping, isolation, and manage-
ment of liquids at the microscopic level are constrained by minimal testing quan-
tities and stream velocity requisite for biomimetic assessment, as well as the
microscale features of the systems. At the microscale, the primary physical and chem-
ical processes differ from those at the macroscale, resulting in greater complexity
in the glide and migration process. To counter these restrictions, many experiments
have been conducted to improve the modeling of microfluidic devices and isolation
equipment which may be implemented on LOC systems while tackling the quasi
behavior of bulky corporeal fluids [46–50].
Microfluidic equipment may incorporate several cell sorting methods based on
physical factors, offering a precise interface for manipulating cellular components
and extracting forces in a range of methods, as well as allowing for the entire sovereign
assessment of phenomenal components [51]. In particular, cell sorting approaches
have evolved specifically for cell absorption; claret endowment; blood fractiona-
tion; cell categorizing (separation of cells by type); and cell eliminator, which can
act as cell segregator [52]. Active technologies, such as di-electrophoresis, magne-
tophoresis, acoustophoresis, and optical tweezers, are based on microelectromechan-
ical systems and increase fluid control by employing mobile components or external
mechanical forces [53]. The recent electrokinetic phenomenon used for the segre-
gation of RBCs is di-electrophoresis [8, 54]. When polarizable particles floating in
a liquid are exposed to a quasi-electric field, di-electrophoresis is induced, and it is
often employed to manipulate bio-particles, including cells in microfluidic devices
[55–59].
Electrical charges are stimulated on the cell/medium junction when a polarisable
particle, such as a cell, is introduced to an electric field. When dipoles are oriented
parallelly, electrical stress is formed by the induced charges. When a homogeneous
electric field is provided, the coulombic forces experienced by either side of the dipole
are equal, resulting in equivalent and neutralizing forces exerted on the cell. In a quasi-
electric field, the imposed coulombic forces are uneven, providing a resultant force
on the cell. Based on its comparable polarizability about the surrounding medium,
the cell can be lured to or driven from regions of the intense electric field. If cells
have a spherical form, they are subjected to a DEP force, which may be stated as
follows:

ε∗p (ω) − εm∗ (ω)
FDEP = 4π εm r Re ∗
3
· ∇E2 (5)
ε p (ω) + 2εm∗ (ω)
where εm and εp are the permittivities of the medium and particle, r is the radius of
the sphere, and E is the induced electric field.
Because red blood cells have a more intricate structure, comprising a cytosolic core
encased inside a plasma membrane, the single-shell spherical model is frequently
used to compute the Real part of Clausius Mossotti’s (CM) factor. The CM factor
obtained for RBCs is negative, and hence RBCs follow negative di-electrophoresis
as shown in Fig. 4. Whereas the negative DEP response of cells is somewhat lower
at 100 MHz than at 100 kHz, the chosen frequency of 100 MHz guarantees that the
thermal stress delivered to the blood owing to the Joule heating effect is kept to a
minimum.
Bharat et al. [54] constructed a microfluidic device to separate RBCs using di-
electrophoresis. The platelet is split by the induced electric field (n-DEP), which
results in separation. Using di-electrophoresis field flow fractionation, a modified
solution is used to isolate thrombocytes from blood. The microfluidic system is used
to pre-focus and fractionation features to capture cells in contrast to regions relying
on their area. By establishing a nonuniform electric field and placing alternating
polarity electrodes, the particle trajectories are also varied. This sorting mechanism
might be used with a system that sorts cells according to their “opacity.“ Variations of
the design specifications, such as the applied separating voltage, frequency, pressure,
and flow inlet rates, were tested using the 2D finite element model as shown in Fig. 5.
An electric potential of 5 V is applied and the materials used are silicon, germanium,
and copper.
Fig. 4 The figure illustrates the mechanism of negative di-electrophoresis

36 S. Turaka et al.
Fig. 5 The microfluidic device with applied voltage, outlet velocity, pressure and outlet velocity
[56]
The lower inlet’s velocity is substantially higher (853 m/s) than the top inlet’s
(134 m/s) to concentrate all of the infused particulates toward the upper output, as
seen in Fig. 5. The velocity differential between the separation (highest velocity)
and collection (lowest velocity) regions allows particles to quickly approach the
collecting zone when an electric field is supplied with an initial frequency of 100 kHz.
The maximum potential is determined to be 1.45 × 10–3 mV.
Mitra et al. [8] designed a microfluidic device that uses di-electrophoresis for the
isolation of RBCs with dimensions 3, 5, and 8 μm diameters as shown in Fig. 6. The
dimensions of the design are 820 × 320 μm with two inlets and two outlets. Blood is
first injected into the inlet, then into the microscale, which encounters a quasi-electric
field, causing blood cells to separate. During the filtering process, Newton’s law of
motion is employed as the primitive expression to define the particle orientation,
which incorporates drag force, Brownian force, and DEP force. The finite element
approach is used to solve the governing equation (FEM). Up to 3 s of transient analysis
have been performed. Three different voltages (3, 5, and 7 V) were employed, with
red blood cell diameters ranging from 3, 5, and 8 μm. The work concluded that the
RBCs with a 5 μm diameter and an applied voltage of 5 V produced an efficient
segregation process.
Fig. 6 Particle trajectory

during filtering for various
RBC diameters at t = 3 s
with a 5 V applied voltage.
(The red hue denotes RBCs,
whereas the blue tint denotes
Platelets) [8]
Sahin et al. utilized microfluidic technology to obtain RBCs from B-lymphocytes

(B-Cells). Di-electrophoresis is a method to handle and isolate micron-scaled
substances. RBCs and B-Cells with circumferences of 8.8 m and 10.3 m are subjected
to dielectrophoretic manipulation. A microdevice with a side electrode is simulated
and the results are exhibited. RBCs and B-Cells could be separated with a magnitude
of 10 kHz and a stream rate of 1.5 L/s. RBCs and B-Cells could be separated with
98 percent efficiency using an induced electric potential of 0.06 V [60].
Salahi et al. showed the use of RBCs as heterogeneous particles with care-
fully controlled subcellular electrodiagnosis with related fluorescence intensity is
described in this paper. Each customized RBC form could recognize unicellular
sensitivity relying on phenomenological resistance indicators which are equipped
with electrical designs to quantify rheological statistics employing glutaraldehyde
fusion to alter membrane capability and membrane resealing beyond electrolyte inva-
sion to transform inner cytosolic conductivity and fluorescence in a congruent fashion
as shown in Fig. 7. Mono cell resistance data from unfamiliar erythrocyte variants
can be mapped against these models, allowing for the quick assessment of subcellular
physical and biological data and DEP sorting circumstances unaccompanied by the
use of tedious algorithms with unfamiliar parameters [61].
The computer-aided design modeling of a microfluidic channel capable of
isolating thrombocytes and erythrocytes from other blood cells is presented by
Praveen et al. [62] as shown in Fig. 8. The use of negative dielectrophoretic (n-
DEP) force in combination with drag force allows for separation based on their
sizes. Within the microchannel, a 38° angled electrode array separated by 70 μm is
developed and studied for quasi-electric field distribution. The projectile trajectories
module simulates particle motion inside the microchannel under an induced electric
field to show separation. The CM factor, n-DEP force, and drag forces are calculated
using numerical research. The work concluded that the electrode with an applied
electric potential of 5 V, electrode array angle of 38°, and a displacement of 70 μm
can be used for the efficient separation of RBCs from blood platelets.
Fig. 7 Subcellular electrical prediction of RBCs with relevance to fluorescence intensity [61]
38 S. Turaka et al.
Fig. 8 The figure depicts the behavior of distinct blood molecules under a 5 V applied voltage of
a di-electrophoretic-based microfluidic channel
Shirmohammadli et al. constructed a microfluidic device for the analysis of diver-

gent electrodes in a DEP-based microfluidic device for cell segregation as shown
in Fig. 9 [63]. This study eliminates the need for initial RBC lysis by separating
MCF-7 tumor cells from leukocytes and erythrocytes. The differential electrodes
not only successfully suppressed the requirement for high applied voltages via dc
di-electrophoresis but also reduced the joule heating effects, according to numerical
simulation findings. Aside from that, the configuration of the divergent electrodes
was shown to have a significant impact on separation performance. Designing the
outputs, as well as altering the electrode voltage, has made it possible for the gadget
to be tunable for a variety of cancer cells with varying radii. More importantly,
the device’s capacity to deflect waste cells (WBCs and RBCs) out of the porous
channel before cancer cells has been shown to improve separation efficiency and
purity. The suggested system was capable of discerning distinct cells with grab
purity and accuracy of more than 83 percent and 100 percent, respectively, using
varied diameters.
Amir et al. designed a trapezoidal microfluidic device for cell segregation of blood
by acoustic force [64]. Unlike prior research in which the stream was rectangular, the
channel in this article is trapezoidal to improve the dissolution rate. WBC’s position
from the axis will change when the trapezoidal leg tilt changes. A Bio-MEMS channel
with two inlets for sheath flow and blood particulates, a trapezoidal main conduit,
and three exits was created as shown in Fig. 10. On the LiNbO3 substrate, there are
two aluminum interdigital transducers. We used alternating voltage on transducers
to generate the acoustic force. Because of the positive acoustic distinction factor,
blood particles will be pushed toward the acoustic nodes. Because acoustic radiation
force is proportional to particle size and compressibility, cells may be sorted by
diameter and compressibility, terminating in particle separation. The work modified
the trapezoidal channel tilt, the input signal, and the fluid velocity to optimize the
device.
Zhao et al. demonstrated how to separate nano/microparticles using a high-
performance, detachable acoustofluidic platform as shown in Fig. 11. The
Fig. 9 The figure illustrates the proposed device for cell separation with two inlets for buffer and
cell suspension. The WBCs and RBCs will flow to outlets 1 and 2 with MCF-7 cells flowing directly
to the CTC outlet [63]
Fig. 10 Top view of the device with tilt angle = π/20 [66]
proposed expendable acoustofluidic devices accomplish acoustic radiation forces

similar to those engendered by emerging permanently bound, non-disposable devices
by utilizing unidirectional interdental transducers (IDTs), a blended path design with
hard/soft materials, and tilted-angle standing SAWs (taSSAWs). Not only micropar-
ticles, but even nanoparticles may be separated using our disposable instruments.
Furthermore, they can distinguish pathogens from human erythrocytes with up to
96 percent purity. Overall, we created a disposable acoustofluidic platform based
40 S. Turaka et al.
Fig. 11 Proposed design of

detachable acoustofluidic
isolation chip utilizing a
single direction IDT-based
design [65]
on unidirectional IDT for micro/nanoparticle separation that has good separation

efficiency, adaptability, and biocompatibility [65].
Mantegazza et al. proved that in a sophisticated in vitro network with homoge-
neous bifurcations and 176 microchannels, quantifiable data for phase separation was
acquired. The investigations revealed that hematocrit is heterogeneously distributed,
confirming the traditional finding that the branch with the highest blood fraction
also had the highest RBC fraction (classical partitioning). In the event of a distorted
hematocrit pattern in the parental vessels of bifurcations, a reversal of this traditional
phase transition (reverse partitioning) was found [23]. They constructed a microflu-
idic device with an inlet and outlet that comprises 49 hexagon components as shown
in Fig. 12. The flow was generally going from left to right. The microchannels were
10 μm wide, 8 μm in height, and 85 μm long. A conventional microscopic field of
view of 512 × 512 pixels is represented by the picture at the bottom right. The work
concluded that the RBC flux fractions were frequently over proportional in branches
with larger blood flow fractions.
Zhang et al. proposed a microfluidic device for the segregation of RBCs from
platelets. The study offers a particle separation di-electrophoresis microfluidic device
that exploits dielectric characteristics to size-based fractionate red blood cells and
platelets. Using COMSOL Multiphysics, the propagation of the electron beam in the
device and the trajectory of the particulates in the capillary are estimated for various
electrode geometries, voltages, and chip exit configurations based on the control
variables. Negative di-electrophoresis, which uses an AC signal of 100 kHz, affects
both erythrocytes and platelets. The platelets flow out of the left nozzle under the
joint effect of fluid force and DEP force, whereas the bigger RBCs are subjected to a
higher DEP force and are skewed toward the right outlet as shown in Fig. 13. On this
premise, a more optimized microfluidic chip capable of properly separating particles
is eventually selected via statistical analysis [66].
Eugen et al. designed a microfluidic device for the isolation of CTCs (Circulating
tumor cells) from RBCs using DEP. The device is modeled in a way that it has one inlet
and three outlets. The interdigital electrodes provide a quasi-alternate electric field
Fig. 12 The figure depicts the top view of an enlarged microfluidic device with a mono inlet and
outlet
Fig. 13 The figure encapsulates the separation of RBCs from platelets using DEP force [66]
that isolates the biological particles within the device from the appropriate terminal
as shown in Fig. 14. Simulations were carried out with COMSOL Multiphysics. In
the simulation, the CTCs and RBCs are modeled as spheres with afferent attributes.
The work concluded that the RBCs move through the first zone of strong electric
field contours and toward the outlet with negative electric potential due to its initial
momentum. While the CTC particle attaches directly to the electrodes and proceeds
over the high electric field gradient zone, propelled by its initial velocity. The particle,
however, remains connected to the electrodes in this situation and does not meet the
predicted exit, as in Fig. 15 [67]. This research might be expanded to include testing
numerous CTC lines at different flow rates and electric field frequencies.
42 S. Turaka et al.
Fig. 14 The figure illustrates the proposed DEP device for the segregation of CTCs from RBCs
Fig. 15 Figure depicts particle trajectories at t = 300 s, CTC: blue, RBCs: red, and electric potential:
greyscale
8 Conclusion
The review focused on the microfluidic devices and old approaches that were utilized
to separate RBCs and their limitations. Red blood cell separation is used to identify
sickle cells. The RBCs in the blood have a big volume and can be easily separated
by centrifugation. However, centrifugation necessitates the use of bulky equipment
and large samples. The bottleneck of the traditional approaches can be overcome by
microfluidic devices. Microfluidics is a well-known technology with unique char-
acteristics that have the potential to revolutionize illness detection and treatment.
It has been well established for micron-sized particle sorting because it provides
a comparatively simple, low-cost, and continuous particle separation technique.
Microfluidic devices are subtle in the areas of biomedical applications. Moreover, this
technique demonstrates a new concept for particle-sorting applications in medical
biology for its simplicity and efficiency. Microfluidic devices use techniques like
di-electrophoresis for cell sorting. In the fields of biomedical and biosensors, the
DEP force has numerous uses which are used to separate RBCs utilizing microflu-
idics have been discussed in this review. RBC separation using DEP demonstrates
how RBCs can be selectively filtered from a blood sample. RBCs, which seem to be
larger than other blood cells, are exposed to a greater force and are therefore deflected
more. But the short come of DEP force is that it fails for large-size RBC particles [8].
Furthermore, the recent microfluidic devices that are used to isolate RBCs occupy a
large area and have high power consumption [8, 23, 54, 60–67]. Hence, future works
will focus on novel approaches for RBC particle isolation that is independent of size,
optimization of the device, affordability, and low power consumption.
Acknowledgements The authors would like to thank the Department of Electronics and Commu-
nication Engineering, Velagapudi Ramakrishna Siddhartha Engineering College for providing the
necessary infrastructure.
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A Road Map to Paper-Based
Microfluidics Towards Affordable
Disease Detection
Mareedu Nagavalli, Tatineni Sharmila Swaroopa,

Pannangi Sri Vidya Gayathri, Vuyyuru Dinesh Kumar Reddy,
Nanda Sai Donepudi, Dhanya Yalamanchili, Koushik Guha,
and Jasti Sateesh
Abstract Microfluidic technology (μF) is an approach in managing very small

volumes of fluids through patterned tiny channels. The present chapter focuses on
the importance of designing microfluidic systems, their capabilities, and their appli-
cations. As microfluidic diagnostics are complex and unaffordable, paper-based
microfluidic technology has become viable for low-cost disease diagnosis. The
branch of microfluidics that manages very small amounts of fluids through capil-
lary action and are made of paper and porous materials are termed paper-based
microfluidics (PBM). This review chapter mainly illustrates the significance of PBM
devices and their capabilities, applications, and advantages over microfluidics. Diag-
nostic applications in the detection of chronic diseases, cancer, dengue, glucose, and
tuberculosis with μF and μPAD are also reviewed to the finest level in this review. In
the current scenario, Artificial Intelligence (AI) and Internet of Things have become
an integral part of every technology. Integration of μF and μPAD with IoT and AI
aids to produce better design considerations, which are also covered in this review.
Keywords Microfluidics · Paper-based microfluidics · Disease diagnosis · And

affordable diagnostics
M. Nagavalli · T. Sharmila Swaroopa · P. Sri Vidya Gayathri · V. D. K. Reddy · J. Sateesh (B)

Department of Electronics and Communication Engineering, Velagapudi Ramakrishna Siddhartha
Engineering College, Kanuru, Vijayawada, Andhra Pradesh 520007, India
N. S. Donepudi · D. Yalamanchili
Siddhartha Government College and Hospital, Vijayawada 520008, India
K. Guha
Deparment of Electronics and Communication Engineering, National Institute of Technology
Silchar, Assam 780010, India
48 M. Nagavalli et al.
1 Microfluidics
1.1 Introduction to Microfluidics
Life-threatening diseases such as cancer, dengue, and TB cannot be rapidly diagnosed

using various microscopy techniques. Microfluidics plays a vital role in such micro-
level disease detection by utilizing channels with dimensions of 0.1 times micro
meters [1]. The main objective of microfluidic technology is to provide a single
platform from the input of samples to the disclosure of diagnostic results. Other
technologies used for the diagnosis of a disease cannot produce rapid response, so
microfluidic systems are also denominated as μTAS (micro-total analysis systems),
LOC (Lab-On-Chip), and biochip devices [2]. In macroscopic systems, performing
single-cell assays and parallelized experiments is very difficult. Miniaturization of
microfluidics became the only solution for all those drawbacks and has numerous
advantages which include reduced utilization of sample and reagents, minimized cost
of reagent, narrowing the risk of contamination, decreasing the cost of analysis, and
increasing throughput [1]. With numerous proven and pertinent applications within
the areas of biological sciences, engineering sciences, genomics, chemical sciences,
proteomics, and biodefence, microfluidics evolved as the dominant technology [3].
During the early days of microfluidics, most of the devices are fabricated using silicon
material. Later, silicon is replaced with polymer materials like Polymethylmethacry-
late (PMMA) [4], Polystyrene (PS) [5], Polycarbonate (PC) [6], and Polydimethyl-
siloxane (PDMS) [7]. Among all these polymer materials, PDMS is currently used for
fabricating microfluidic devices in laboratories due to its easy cloning, transparency,
biocompatibility, and low cost [5]. Microfluidic devices fabricated with PDMS have
various biomedical applications, namely DNA analysis, immunochemical assays,
and cell-based assays [8–19].
1.2 Applications of Microfluidics
Microfluidics has expanded applications in the areas of molecular and clinical diag-
nostics [21], neuronal studies [22], biological sciences such as chemical biology [23,
24], quantitative biology [25], stem cell biology [26], and fluid mechanics. One of
the widely used applications is the LOC devices. A LOC device integrates one or
more operations on a single integrated circuit as shown in Fig. 1.
This device performs various operations and processing steps that include sample
separation viz. electrophoresis, molecular exclusion, and liquid chromatography.
Applications of microfluidic devices in biological systems include the delivery of
drugs, microscopic sensors, namely cellular-level quantum dots, and nano-scaled
electrodes [29–36]. Furthermore, there exist a few other applications of microfluidics
including C. elegans immobilization [37], pH control [37], drug administering [37],
A Road Map to Paper-Based Microfluidics Towards Affordable Disease … 49
Fig. 1 A microfabricated
system for aligning
miniature molecules with
intracellular resolution [24].
Reproduced with permission
from Weibel, Douglas B.,
and George M. Whitesides.
Current opinion in chemical
biology 10.6 (2006):
584–591
gradients generation [37], point-of-care devices [37], and cell analysis [37]. Applica-
tions of microfluidics also stretch in chemical biology which includes microdilution,
gel structures, droplets, cell painting, and the study of single cells [24]. Stem cell
biology using microfluidics has extended its wings widely for a decade that includes
high throughput screening and regeneration of stem cell physiological environment
such as differentiation and isolation [26]. In microfluidic devices, molecular diag-
nostics are extensively used in pathogen detection. This technique also helps in the
detection of cancer and diagnosis of diseases caused by bacteria and viruses such as
HIV, Cholera, and Syphilis [21].
1.3 Capabilities of Microfluidics
All the microfluidic devices designed for diagnosis are aimed at the detection of a
single disease. However, diagnostic devices have to be capable of detecting multiple
diseases to satisfy market needs [21]. In today’s world, 95% of mortality is due to
inadequate diagnostics and lack of treatment-providing centres for many infectious
diseases like HIV, COVID-19, AIDS, TB, etc. [27]. To date, epidemics like the 2009
H1N1 influenza and 2019 COVID have highlighted the necessity of rapid diagnostic
tools to restrain transmittable diseases [28]. The critical analysis of the physiolog-
ical and pharmacological responses at one-cell level can be achieved by microfluidic
platforms [22]. Microfluidics can minimize monotonous operations; increase sensi-
tivity, specificity, and reliability, and reduce the time of analysis [1]. They can also
diagnose and segregate the molecular components when analytical devices require
high resolution and smaller footprints. Microfluidics has the capability of regulating
the concentration of molecules in analytical devices.
2 Paper-Based Microfluidics
2.1 Introduction to Paper-Based Microfluidics
Paper-based microfluidics is the sub-division of microfluidic technology, where the

devices are made using paper to manage a small quantity of fluids via capillary
forces [39]. PBM is also termed lab-on-paper or μPAD [38]. For decades, analytical
chemistry uses paper as the basic platform, and various forms of highly engineered
paper with various distinct properties are also used [44]. Paper became an attrac-
tive substrate because of the following reasons: (1) easy availability, (2) very cheap
cellulose material, (3) transports tiny amounts of fluids with capillary forces, and (4)
biocompatibility [38]. Capillary pressure generates the flow that results in capillary
formation in the material by cellulose fibre wetting. In paper devices, capillary flow
rate, thickness, size, and permeability (porosity) are some of the factors determining
fluidic flow. There are microchannels that are built on the paper substrate so that
fluid flows in a controlled manner [38] as shown in Fig. 2. In Fig. 2 below is a paper-
based microfluidic (PBM) device fabricated by photolithography; urine is given as a
sample to that device. Fabrication of μPAD involves the patterning of hydrophobic
barriers on paper to generate hydrophilic microchannels. In Fig. 3, using a wax pencil
a microchannel is patterned on laboratory paper and a kitchen towel. Microchannel
is patterned on one side of the laboratory paper in one case and in the next case,
it is patterned on either side, and in the other it is patterned on the kitchen towel.
On single-sided laboratory paper, fluid leakage can be observed and on double-sided
laboratory paper smooth flow can be observed whereas on kitchen towel incomplete
wax diffusion occurs so fluid leakage can be observed [41].
Paper-based microfluidics has various applications that include testing of food
quality, environment monitoring, and health diagnostics and have potential advan-
tages like minimal cost, speed detection, user friendliness, biocompatibility, sensi-
tivity, specificity, etc.
Fig. 2 Paper-based
analytical microfluidic
device schematics that can
detect glucose and protein in
urine simultaneously [40].
Reproduced with permission
from Journal of biological
engineering 5.1 (2011): 1–22
Fig. 3 To design a
paper-based microfluidic
device in a Nitro Cellulose
(NC) membrane using wax
printing [41]. Reproduced
with permission from Lu, Y.,
Shi, W., Qin, J., & Lin, B.
(2010). Analytical
Chemistry, 82(1), 329–335
2.2 Advantages of Microfluidics
In comparison with other microfluidic devices, paper-based devices are sensitive

and affordable and fit themselves into point-of-care (POC) testing because of natural
disposability, situational flexibility, and capability to store and analyse the target
at the site of requirement. The main reason for choosing paper as starting material
for fabricating micro-analytical devices is that it wicks aqueous fluids. The fabri-
cated paper-based device is used for numerous purposes such as recognition of dyes
and pigments, and urine tests [43]. The wicking of liquid makes passive transport
of fluids without active pumping on μPADs. In addition, paper has several other
crucial advantages such as high bioavailability, being inexpensive, biodegradable,
and biocompatible representing an alternate material for making diagnostic devices.
2.3 Capabilities
PBM is used for robust diagnostics which require less volume of fluid for testing. The
paper which is made up of cellulose or polymers of cellulose controls the movement
of fluids by slow capillary actions via pores without the help of any external actuation
or pumps [45]. PBM can detect nucleic acid which helps in disease detection such
as cancer [48]. Open channel microfluidics can be created with the help of paper
microfluidics which enables water–oil emulsification by controlling the speed of
fluids. It also creates a preferable size of the emulsion particle such that it can be
integrated into a drug or tablet [49]. In the delivery of the drug to the target site at the
correct times, paper-based microfluidics plays a crucial role [51]. PBM also helps to
manage the rate of flow of drugs and studies the effectiveness of drug and their side
effects at different concentrations [50, 51]. Long channels can be created through a
micropatterning technique by wax printing in PBM [53]. Micropatterning leads to
the creation of POC testing which diagnoses the condition of patients at their location
[54]. To improve the diagnosis of disease at an earlier stage of the development, an
amplification strategy under isothermal conditions is required such as Loop Mediated
Isothermal Amplification (LAMP) which is highly compatible with POC analysis.
The use of LAMP-based technology along with paper-based microfluidics results in
the development of portable POC nucleic acid assays that can amplify the DNA/RNA
which enables in the detection of diseases like malaria, pneumonia, Ziko virus, and
food pathogens [57].
3 Paper and Its Materials
Paper is a thin sheet obtained by chemical and mechanical treatment of cellulose

fibres. Several sources of cellulose fibres such as cotton, bamboo, grass, and wood
have been used for obtaining μPADs [58]. The finest quality of cotton which contains
98% of α-cellulose is used for the manufacturing of chromatography and filter papers
[59]. The chromatography paper helps to identify the amino acids, bases, proteins,
and low concentrations of DNA/RNA [46]. In Fig. 4, a PBM device is fabricated to
measure the least functioning hydrophilic channel that has Grade 1 Chromatography
paper as substrate [53].
Paper which is made from pure cotton is widely used for diagnostic purposes.
Whatman chromatography paper, nitrocellulose membrane/paper, Whatman filter
paper, and Whatman 105 lens paper are the most commonly used papers for the
Fig. 4 Examples of wax-printed Micro Paper-based Analytical Devices (µPADs) [53]. Repro-
duced with permission from Carrilho, E., Martinez, A. W., & Whitesides, G. M. (2009). Analytical
Chemistry, 81(16), 7091–7095
fabrication of PBM [38, 39, 60–65]. Nitrocellulose membrane/paper is a 3D porous

structure that is ideal for protein immobilization and is made out of pure cellulose
nitrate [61]. It has a smooth surface, homogeneous pore size (0.45 μm), and stable
flow of liquid [38]. Hence, nitrocellulose membrane is used in numerous applications
like western blotting, test strips based on gold nanoparticles, dot ELISA, DNA immo-
bilization, protein immobilization, and cell immobilization [62, 65–70]. Whatman
chromatography paper and Whatman filter paper belong to the family of cellulose-
based paper [38]. Despite its hydrophilic nature, cellulose paper is widely used due
to its affordability and rapid penetration of liquid [71]. Whatman filter is the most
famous cellulose paper due to particle withholding capability, well-categorized rate
of flow, and permeability. In 2007, the Whiteside group patterned Whatman filter
paper was fabricated using the photolithography technique for colourimetric anal-
yses of urine and protein pH [69]. Whatman filter and chromatography papers are
categorized into four grades (Grades 1–4) depending on the pore size. Cellulose
paper containing the maximum percentage of alpha-cellulose is used for making
Whatman chromatography paper. Whatman filter paper is incompatible with normal
filter paper in many ways. In comparison with normal filter paper, the Whatman filter
paper is worthy and best for conducting qualitative analysis. Qualitative filter papers
like Whatman filter paper are majorly based on diverse properties which include
thickness, retention, and weight. In everyday applications, Grade 1 filter paper is the
most commonly used filter paper for clarifying liquids. With an analogous increase in
filtration time, Grade 2 filter paper is slightly more retentive than Grade 1. As Grade
3 filter paper is double the thickness of Grade 1, it can hold more precipitate without
clogging and has improved wet strength that is well suited for sample transport after
collection. For the retention of coarse particles and gelatinous precipitates, such as
aluminium hydroxide, a fast filter material is used known as Grade 4 filter paper.
Grade 5 filter paper provides the equivalent slow rate of flow. Whatman filter No 1 is
a kind of filter paper whose pore size is 11 μm and allows medium flow rates [62].
Whatman filter No 4 also belongs to the Whatman filter paper family; it has a greater
pore size (22–25 μm) and is ideal for etch printing fabrication techniques [65]. White-
side patterned the Whatman chromatography paper with SU8 photoresist through
photolithography for the detection of glucose and proteins simultaneously [69].
4 Applications
4.1 Chronic Diseases
Nowadays, diagnosis and monitoring of chronic diseases is a major challenge that

requires established laboratory infrastructure and specialized instrumentation. The
Most Common Chronic Diseases (CD) are chronic respiratory diseases (CRD),
diabetes, and chronic kidney diseases (CKD) [72]. The diseases of the airways and
other structures of the lung lead to chronic respiratory diseases (CRD). Some of the
most common CRDs are pulmonary hypertension, chronic obstructive pulmonary
disease (COPD), minor respiratory infections frequently, and asthma. For a person
affected with CKD, their kidney gradually loses the filtering capacity of blood to
eliminate wastes and also has several adverse effects like premature birth, kidney
failure, cardiovascular disease, and, ultimately, death. The main causes of the most
common chronic disease diabetes are insufficient insulin produced by the pancreas
and the body’s inefficiency to properly utilize the produced insulin. Levels of blood
sugar are regulated by the insulin hormone. Diabetes or diabetes mellitus (DM) is a
metabolic disorder that has very high levels of blood sugar over a long period [73].
Nowadays, monitoring and diagnosis of chronic disease (CD) is a major challenge
that requires established laboratory infrastructure and specialized instrumentation.
The moratorium on CD diagnosis leads to a significantly increased economic
burden [74]. Among the existing laboratory clinical approaches, microfluidic tech-
nology gained increased engrossment over the past few decades due to the miniatur-
ization of bioanalytical assays for improving diagnostic performance. In microfluidic
technology, the prognosis, diagnosis, and monitoring of chronic disease require the
measurement of biomarkers effectively [75–82]. Hepatitis C virus (HCV) belongs to
the family of Flaviviridae, which is an RNA virus that accounts for acute infection.
HCV is the primary basis of hepatocellular carcinoma and liver cirrhosis, causing a
large number of deaths worldwide. The immunoassays of HCV are classified into 3
Fig. 5 For diagnosis and detection of anti-HCV using paper-based immunoassay [83]. Reproduced
with permission from Mu, Xuan, et al. Analytical chemistry 86.11 (2014): 5338–5344
sub-divisions, targeting core antigen detection of HCV, antibody detection of anti-

HCV, and multiple HCV antigen detection. Biomarker detection uses some conven-
tional methods like mass spectrometry (MS), enzyme-linked immunosorbent assay
(ELISA), polymerase chain reaction (PCR) chromatography, and gel electrophoresis
[74–82]. It is expected that these contemporary approaches will be soon embraced
and deployed in point-of-care (POC) settings leading to betterment in the patient’s
life quality.
In Fig. 5, ELISA Indirect is performed on patterned NC (nitro-cellulose) for
detecting quantitative analysis of IgG. ELISA Indirect is considered as a two-step
binding process that contains 1st antibody (primary) and enzyme-linked 2nd antibody
(secondary) that is complementary to the primary. Incubation of 1st antibody with
antigen is followed by 2nd antibody. Horseradish peroxidase (HRP) could catalyse
substrate and produce a signal like colour precipitate or chemiluminescence as shown
in Fig. 6.
4.2 Dengue
Dengue virus (DENV) is the most commonly encountered infection worldwide.

Approximately 200 million people are infected every year due to DENV [84, 85].
DENV belongs to the family of flavivirus genus and people are exposed to dengue by
Aedes mosquitoes. Four serotypes of DENV, i.e., DENV 1–4 are known. Dengvaxia
and CYD-TDV are the WHO recommended Dengue vaccines for all 4 serotypes.
Fever, rashes, and musculoskeletal pain are commonly experienced symptoms of
DENV [86]. Numerous laboratory diagnostic tests exist for DENV detection which
include hemagglutination-inhibition test, 50% plaque reduction neutralization test,
viral isolation, enzyme-linked immunosorbent assay (ELISA), and molecular assays
[87–93]. IgG (Immunoglobulin G), IgM (Immunoglobulin M), and NS1 (Non-
structural protein 1) are the DENV markers; this simplifies the Paper Analytical
Devices (PADs). IgG/IgM and NS1 diagnosis using lateral flow assays produce
Fig. 6 Procedure of diagnosis of dengue in paediatric serum using paper-based microfluidics [86].
Reproduced with permission from Choi, Jane Ru, et al. Biosensors and Bioelectronics 74 (2015):
427–439
qualitative results [93–95]. Paper-based analytical microfluidic devices for diag-

nosis of DENV markers are fabricated using sandwich immunoassay on Grade 2
chromatography paper patterned with wax activated with antibodies of anti-dengue
NS 1 monoclonal for the POC testing of NS1 dengue marker [86].
DEN-NS1-PAD (Paper Analytical Devices): Fig. 7 consists of four major areas
of detection of DENV; those are sample, detection, conjugate, and absorbent areas.
These areas are allied by a single channel which is bounded by a hydrophobic barrier
patterned with wax. The sample given to the sample area absorbs over the channel. In
the conjugate area, sample interacts with Gold Nanoparticle–Antibody Conjugates
(AuNPs-Ab) and in the detection area sample interacts with anti-NS1antibody that
shows dengue NS1 positive results; meanwhile, at the control spot AuNPs-Ab inter-
acts with the captured antibody. Red colour generated at the test and control spots
indicates that the given sample is dengue positive and that is observed by a scanner,
Fig. 7 Modification and fabrication procedure of electrochemical paper-based aptasensors [98].

Reproduced with permission from Wang, Yang. Biosensors and Bioelectronics 136 (2019): 84–90
smartphone, and even the naked eye, Fig. 6. Early detection of dengue is not geared
up to a crowing point; the researchers may investigate it.
4.3 Cancer
Cancer is a frightening disease or set of diseases which is the preeminent cause of

death worldwide [96]. Cancer is stated as the cells of uncontrolled growth. These
cells might be formed as abnormal cells. The mutilation cells do not die and become
cancer cells owing to uncontrolled growth. These destruction cells are transferable
easily through the blood into several body organs. The indexes of cancer are elicited
from the genre of cancer, and certain patients don’t show indexes or markers until it
reaches distant advanced stages.
The cancer disease affects and progresses in three steps: (1) Initiation, (2) Promo-
tion, and (3) Progression. Currently, we have several sorts of treatments for cancer,
i.e., (1) Surgery, the surgeon cuts the cancer tumour; (2) Radiation therapy, which
uses immense shots of radiation to eradicate tumour cells; (3) Chemotherapy, used
to eliminate cancer cells using drugs, etc. Through wax and screen printing, PBM
was fabricated and it enables sample filtration and injection functionalities. The
authors opted for the label-free electrochemical method which enables rapid testing
and designed a simple PBM that involves an inlet hole sample, filter hole, counter
electrode screen printing, reference electrode screen printing, diagnosis zone, and
working electrode screen printing as shown in Fig. 7. This PBM can diagnose two
biomarkers from a single sample that are integrated onto a single device.
4.4 Glucose
Glucose is an important medical analyte and also an important metabolic interme-

diator [99, 100]. It acts as an indicator for several diseases which includes glucose
metabolism disorders and islet cell carcinoma [99–102]. Detection of concentration
levels of glucose helps to regulate insulin levels in order to avoid diabetes compli-
cations. Colourimetric and electrochemical μPAD are familiar in the detection of
glucose levels [101].
Figure 8 shows the procedure for fabricating dumbbell-shaped Electrochemical
Paper-Based Analytical Devices (ePADs) using the wax dipping technique: (a) Iron
mould designing, (b) Alignment of Whatman No 1 paper and blood separation paper,
(c) ePADs assembly construction steps, and (d) Blood separation ePADs [105].
Noiphung, Julaluk, et al. designed μPAD to measure the glucose level from a blood
sample using plasma isolation methods [105]. In this, electrochemical dumbbell-
shaped paper-based analytical devices (ePADs) were used to obtain uniform plasma
isolation. The blood sample was given to the separation zone of ePADs, and in 4 min
plasma separation takes place. Isolated plasma from the separation zone transmits to
the detection zone. Glucose oxidase immobilized in the middle of the paper device
in the detection zone detects the glucose in isolated plasma. Hydrogen peroxide is
generated when the reaction between glucose and enzyme is passed through Prussian
blue modified screen-printed electrodes (PB-SPEs). Chronoamperometry is used to
measure the currents at the optimal detecting potential for H2 O2 (Hydrogen peroxide)
where glucose concentrations in whole blood are proportional [105].
Fig. 8 Wax dipping technique for identification of Glucose [105]. Reproduced with permission
from Noiphung, Julaluk, Analyticachimicaacta 788 (2013): 39–45
Fig. 9 Represents label-free

electrochemical μPAD using
carbon/ferrocene nanotubes
for detection of
mycobacterium tuberculosis
DNA [104]. Reproduced
with permission from Wu,
Jiandong, NPJ digital
medicine 1.1 (2018): 1–11
4.5 Tuberculosis
Tuberculosis is a very serious infective disease which mainly deteriorates lungs’

health. In 2020, an estimated 10 million people developed active TB, resulting in
1.5 million deaths, making it the second leading cause of death from an infectious
disease after COVID-19 [106]. Mycobacterium tuberculosis (MTB) bacteria causes
Tuberculosis (TB). Generally, TB not only affects the lungs but also affects other
parts of the body [106]. Chronic cough with blood-containing mucus, fever, night
sweats, and weight loss are typical symptoms of active TB. TB is a communicable
disease; the spreading happens through air when the infected person coughs, spits,
speaks, or sneezes [106]. The current routine diagnostic tests for TB are chest X-ray,
tissue culture, tuberculin skin test (TST), and acid-fast staining. These diagnostic tests
are time-consuming, and require laboratory equipment and well-trained technician
(Fig. 9).
5 Integration of MF and PµF with AI and IoT
Electronic devices, sensors, and software collectively form a network that can
communicate with each other. This network is termed Internet of Things (IoT). Large
volumes of data can be generated by the networks, sensors, and users which assists
in gaining knowledge and developing applications with the help of Artificial Intelli-
gence (AI) approaches. The combination of AI and IoT facilitates the advancement
of public safety, education, health care, transportation, and various such domains
offering valuable services. Over the last decade, the phenomenon of μF and Paper-
Based Microfluidics are coupled with Artificial Intelligence (AI) and Internet of
Things (IoT). It utilizes nano-functionalized materials that have been identified as the
economical and sustainable tool for point-of-care-testing (POCT) for the diagnosis of
various diseases. The aim of designing these devices is to provide cost-effective, eco-
friendly, rapid, and accurate results [107]. Traditional hospital management systems
contain problems like work overload for doctors and nurses, loads of paperwork,
and many more. Integration of IoT into the hospital environment can avoid all these
drawbacks and replace the voluminous paperwork with a centralized and automated
database. Artificial Intelligence (AI) and Machine Learning (ML) are proven as better
tools to predict the result through analysis of available data in a database.
6 Conclusion
An accurate disease diagnosis is crucial for successful treatment. The diagnostic

equipment available are costly, bulky, and time-consuming, which necessitates the
need for new and innovative techniques. The field of microfluidics and its branch
paper-based microfluidics have great potential in revolutionizing diagnostic equip-
ment. Their applications are becoming more apposite in molecular diagnostics and
life sciences. The success of microfluidics and paper-based microfluidics is attributed
to the distinct chemical and physical features of fluids at micron size. The advan-
tages of μF and μPAD are cost-effective, easy to use, biocompatible, and easily
disposable. The μF and μPAD are capable of implementing many functions within
them that include flow control components such as valves and pumps. The COVID-
19 pandemic has highlighted the spotlight on the necessity of μF and μPAD for
rapid diagnostics. The paper-based diagnostics are relatively simple, fast, and cost-
effective; the reason for such advantages is clearly described in the paper. Future
researchers should focus on improving the reliability and accuracy of the device. It
would be a major breakthrough if a single paper-based device for multiple disease
detection. Further, the integration of μF and μPAD with IoT and AI will allow the
device to quickly reach the hands of users.
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208
Critical Review and Exploration
on Micro-pumps for Microfluidic
Delivery
J. Prithvi, B. S. Sreeja, S. Radha, C. Joshitha, and A. Gowthami
Abstract This review covers various types of micro-pumps designs, and the design
aspects of an IPMC actuator-based micro-pump are much focused on flow rate as the
primary objective while maintaining a low power consumption. A detailed compar-
ison of various diaphragm micro-pump types and pump structures is discussed. The
need for durability led to the choice of valve-less micro-pump over their active coun-
terparts. Choice of PEGDA (Polyethyleneglycoldiacrylate) as the membrane material
for actuation has been proposed due to the ease of curing and biocompatibility aspect.
In addition to the review, a 2D simulation of IPMC membrane has been performed
for obtaining corresponding values of membrane displacement in COMSOL Mutli-
physics. The valve-less nozzle/diffuser configuration with a conical angle of 4–6˚
allows the pump to provide the desired flow rate. The simulation results show the
relationship between applied voltage, frequency, dimensions and displacement of
actuator and the flow rate and accumulated flow volume for various cone angles of
valve. Further, the backflow rates for conical and tesla valves have been observed at
various pressures. Tesla valve exhibits a much lesser backflow rate of 240 μl/s at an
inlet pressure of 130 Pa.
Keywords Micro pumps · Low power · IPMC membrane · Flowrate
1 Introduction
Microfluidics deals with the study of fluid flow in extremely small amounts typically
in the nano-liter to typically a few microliters range in a miniaturized system of
the milli-order. The main functions performed are sample preparation, purification,
transport, biosensing, and detection. The physics of fluid flow is no longer in terms of
J. Prithvi · B. S. Sreeja (B) · S. Radha · A. Gowthami

Department of Electronics and Communication Engineering, Sri Sivasubramaniya Nadar College
of Engineering, Chennai, Tamil Nadu 603110, India
C. Joshitha
Department of Electronics and Communiction Engineering, Koneru Lakshmaiah Education
Foundation, Hyderabad, Telangana 500075, India
66 J. Prithvi et al.
conventional models and negligent parameters such as surface tension become domi-
nant characteristics. Keeping these difficulties in mind, the research into MEMS and
NEMS has been rapidly increasing in recent years for biomedical applications. Due
to availability of technologies with the potential to revolutionize the current manu-
facturing processes, such as laser micromachining and 3D printing, the prototyping
phase has been substantially reduced. In recent years, the most important advance-
ment of MEMS and NEMS in biomedicine is microfluidic transdermal drug delivery
(TDD) systems. TDD systems are involved in the movement of pharmaceutical drugs
through the skin for subsequent distribution in the human body. TDD system consists
of micro-pumps, micro-needles, reservoir, micro-flow sensor, blood pressure sensor,
and required electronic circuit for necessary operations. A schematic of a sample
TDD system is shown in Fig. 1.
The micro-pump is the main component of the TDD system that performs the
actuation process required to transport the drugs from a reservoir at specific volumes.
The main performance metrics are the flow rate desired in the system, the power
consumption, and the efficiency of electrical energy conversion to mechanical energy.
They can be of two types—Mechanical and Non-mechanical based on the type of
actuation. Mechanical micro-pumps need moving parts to perform the actuation like
diaphragm and check valves, whereas the non-mechanical counterparts use electrical,
chemical, or magnetic energy to produce kinetic momentum.
Some types of mechanical micro-pumps are Electrostatic [1, 2], Piezoelectric [3–
9], Thermo-pneumatic [10–13], Bimetallic, Shape Memory Alloy (SMA) [14], Ion
Conductive Polymer Film (ICPF) [15–22], MEMS-based pumps [47, 48, 50], Elec-
tromagnetic, and Phase-Change Type pumps. Some types of non-mechanical pumps
Fig. 1 Schematic of sample TDD system

Critical Review and Exploration on Micro-pumps for Microfluidic Delivery 67
Fig. 2 Schematic of
mechanical diaphragm type
micro-pump
are Magnetohydrodynamic (MHD), Electrohydrodynamic (EHD), Electro-osmotic,

Electrowetting, Bubble-type, Flexural Planar Wave (FPW), Electrochemical, and
Evaporation type pumps. Since insulin is the primary focus of this work and it does
not exhibit any significant magnetic or chemical properties that can be utilized for
actuation, and to reduce design complexity, mechanical pumps have been chosen for
micro-pump design (Fig. 2).
A typical diaphragm type mechanical micro-pump consists of a membrane that
takes energy in the form of electricity, heat, liquid pressure, air pressure and converts it
into some form of mechanical energy. Fluid flow is achieved by oscillatory movement
of the diaphragm which creates a difference in pressure. When low pressure is created,
fluid flows into the chamber through the inlet valve and during high pressure, fluid is
pushed out through the outlet valve. The pressure generated inside the chamber is a
function of stroke volume (∆V) produced by the actuator which must contend with
dead volume of the chamber (V0).
Pumps can either consist of single chamber or multiple chambers placed sequen-
tially in series or parallel. Such pumps are called peristaltic micro-pumps that transfer
fluid through the alternating oscillation of the diaphragms in different chambers. This
could lead to better control of volume transfer as well as better pump efficiency.
Micro-valves are used to regulate fluid flow at inlets and outlets basing on pumping
motion. They can be of two types—active or passive. Active micro-valves use an
actuating force similar to the main diaphragm and improve efficiency but are limited
by design complexity and fabrication cost. Electrostatic, Thermo-pneumatic, and
Piezoelectric are some of the mechanisms that have been reported for active valves.
Passive valves, on the other hand, obtain the valving effect as a difference of pressure
between the inlet and outlet valve.
A third type are valve-less micro-pumps which don’t use check valves but rely
on nozzle/diffuser action for flow rectification. Figure 3 shows the schematic repre-
sentation of the setup. The flow is directed in such a way that more fluid enters the
chamber through the inlet than exits the outlet in supply mode. The reverse occurs
during pump mode. Predominant research has been conducted in cone-shaped valve-
less nozzle/diffuser setup but there exists another specially shaped valve called the
tesla valve as shown in Fig. 4. Like conical valves, they offer minimum resistance
to flow of fluid in one direction indicated by the blue lines, whereas resistance is
increased in the opposite direction by looping back the fluid on itself as indicated
by the red lines thus arresting the fluid flow. The ease of fabrication of tesla valves
makes it an important alternative to conical valves.
Biocompatibility of MEMS-based micro-pumps is becoming increasingly impor-
tant and is being regarded as a key requirement for drug delivery systems. As there is
the possibility of implanting micro-pumps inside the human body, the materials
used for fabrication are required to fulfill rigorous biocompatibility and biosta-
bility constraints. Further, the implanted pump should be able to withstand long-
term physiological exposure of surrounding tissues. In line with these conditions,
there is a recent trend in usage of polymers as a substrate material since they are
widely used in medicine and are suitable for human implantation. Polymer mate-
rials such as Polymethylmethacrylate (PMMA) [49], Polydimethylsiloxane (PDMS),
and Polyethyleneglycoldiacrylate (PEGDA) are being used in fabrication of MEMS
micro-pumps.
Fig. 3 Schematic of valve-less micro-pump
Fig. 4 Schematic of tesla

valve. a Flow in the blocking
direction, part of the fluid is
turned around and interferes
with the forward flow. b
Flow in the unhindered
direction [U.S. Patent
1,329,559 “Valvular
Conduit”]
2 Background Study
Piezoelectric micro-pumps function by the application of electrical energy to a piezo-

electric crystal whose structure is altered due to net polarization at the surface, leading
to mechanical stress and thereby actuation. These actuators have large actuation and
fast response time but are difficult to fabricate. They also exhibit small stroke volumes
at high voltages. Figure 5 exhibits the representation of piezoelectric micro-pump.
Liu et al. have proposed a disposable, high-performance piezoelectric micro-
pump with four chambers in serial connection for closed-loop insulin therapy system
[23]. Outflow resolution of 6.23 × 10-5 ml/pulse was observed. The maximum
backpressure of 22 kPa was reported at applied voltage 36 Vpp and 200 Hz frequency.
Electrostatic micro-pumps involve actuation by electrostatic form of attraction
and repulsion between two plates separated by a small distance. The plate attached
to the membrane performs supply and pumping respectively is depicted in Fig. 6.
The fabrication is easy, power consumption is low and response times are fast, but
it provides very low stroke volume. Lee et al. fabricated and tested a peristaltic
electrostatic gas micro-pump that employed fluidic resonance for high flow rate
[24]. The micro-pump presented pressure ranges from 3.3 to 7.3 kPa and flow rates
from 0.07 to 0.29 sccm.
Fig. 5 Piezoelectric
micro-pump
Fig. 6 Electrostatic
micro-pump
Thermo-pneumatic micro-pumps function on thermal expansion for actuation

where a chamber above the membrane is filled with air which is then heated and
cooled to provide expansion and compression using a heater and cooler respectively
is shown in Fig. 7. This type of pump generates strong pressure and displacement of
membrane; however, the driving power is constantly held high.
Kim et al. proposed a thermo-pneumatic micro-pump with a glass layer, indium tin
oxide heater, polydimethylsiloxane (PDMS) chamber, PDMS membrane, and PDMS
cavity [25]. The flow rate of 0.078 μL/min was achieved at applied voltage of 55 V
with frequency of 6 Hz. Chia et al. proposed a novel thermo-pneumatic peristaltic
micro-pump comprised of two separate zones for air heating and fluid squeezing [10].
The temperature elevation of 2.0 K was reported on the fluid pumping area. Tan et al.
fabricated a peristaltic micro-pump by bonding a PDMS part with micro-channels to
the PDMS/PMMA (polymethylmethacrylate) part where PDMS/adhesive membrane
worked like a pneumatic actuator [27]. The maximum flow rate of 96 l μL/min was
achieved.
Electromagnetic actuation is large and covers a longer distance as compared to
electrostatic actuation. It needs low voltage, but an external source is required for
actuation such as a permanent magnet is depicted in Fig. 8. On small scale, this type
Fig. 7 Thermo-pneumatic
micro-pump
Fig. 8 Electromagnetic
micro-pump
of actuation has no benefit because it is reduced by the cube of scaling factor. The
driving coils or permanent magnets bond directly with the membrane and provide
a magnetic field. However, at the same time, the size is compromised. Usually,
electromagnetic micro-pumps have high power consumption and heat dissipation.
Yamahata et al. fabricated and characterized a reciprocating PMMA ball valve
micro-pump with electromagnetic actuation. The micro-pump showed a backpres-
sure of 35 kPa and flow rate of 6 mL/min at 2 W electromagnetic actuation power
with 20 Hz resonant frequency [28]. Halhouli et al. worked on the design of a novel
electromagnetic pump that is based on the rotation of two hard magnets kept in
channel, with opposing polarity [29]. The maximum flow rate of 13.7 mL/min at
200 rpm and a pressure of 785 Pa at 136 rpm were observed.
Bimetal refers to an object that is composed of two different metals jointed together
as in Fig. 9. The thermal expansion coefficients of these metals are different. The
deflection of a diaphragm made of bimetallic materials is induced against thermal
alternation if the two chosen materials possess adequately discriminative thermal
expansion factors.
Zou et al. [30] designed a micro-pump that operated on both bimetallic thermal
actuation and thermal pneumatic actuation mechanisms. When the bimetallic actu-
ator made of Al/Si membrane was heated, the membrane deformed in downward
direction. At the same time, the gas in the air chamber expended due to the heat to
support bimetallic actuation. The flow rate of 336 μL/min was achieved when the
open pressure was 0.5 kPa.
ICPF actuator shows high speed response. However, the positioning control is
difficult. Physically it looks like a—sandwich diaphragm between two thin films of
metallic or electro-active polymers that are placed on both sides of the polymer. These
two films have high electrical conductivity. Both ends of the diaphragm are fixed in
case of rectangular diaphragm or the outer edge in case of circular diaphragm, and
the ICPF diaphragm can be controlled by bending in the direction of either upside
or downside if an appropriate pair of voltages is applied at the electrodes as shown
in Fig. 10. The ICPF actuator is commonly called an artificial muscle because of the
large bending displacement, low actuation voltage, and biocompatibility. Fang and
Tan [26] proposed a control-oriented model to envisage the deformation of diaphragm
and the flow rate. Experimental results of the polypyrrole (PPy) actuated micro-pump
Fig. 9 Bimetallic
micro-pump
Fig. 10 ICPF micro-pump
showed that the maximum flow rate of 1260 μL/min was observed at the voltage of
4 V.
The basic principle used in phase-change type actuators and micro-pumps is the
vaporization and condensation phenomenon. In vaporization, the phase transition
occurs from liquid phase to vapor phase. While in condensation, the change of the
physical state occurs from gaseous phase to liquid phase. The phase-change type
micro-pump consists of a heater, diaphragm, and working fluid chamber.
Sim et al. [31] proposed a phase-change type micro-pump consisting of a pair of
Al flap valves and a phase-change type actuator. The actuator comprised of a heater,
working fluid chamber, and silicone rubber diaphragm as shown in Fig. 11. The
diaphragm was actuated by the vaporization and the condensation of the working
fluid in the chamber of the pump. The maximum flow rate of 6.1 μL/min was achieved
at applied voltage of 10 V with 0.5 Hz frequency and 60% duty ratio for zero pressure
difference.
SMA are the metals which exhibit two unique properties such as pseudo elasticity
and the shape memory (SM) effect. They have the capability of changing their shapes
Fig. 11 Phase-Change
micro-pump
upon application of an external stimulus. The SM effect involves a phase transfor-

mation between two solid phases. At high temperature the phase is called austenite
and at low temperature the phase is called martensite. SMA starts in martensite
phase and transforms into austenite phase after being heated. This property of mate-
rials is useful to make SMA micro-pump. SMA have many attractive properties like
high force to volume ratio, ability to recover large transformation stress and strain
upon heating and cooling processes, high damping capacity, chemical resistance, and
biocompatibility. Usually, the deformation of SMA cannot be precisely controlled
and investigated due to temperature sensitivity.
Zhang and Qiu [32] reported a Ti/Ni/Copper (Cu) shape memory thin film micro-
pump comprised of a TiNiCu/Si driving membrane, pump chamber and two inlet
and outlet check valves. The hysteresis width ∆T of 9 °C was observed. Setiawan
[14] reported the performance assessment of SMA spring as actuator for gripping
manipulation. The SMA actuator was a TiNi tensile spring with diameter of 50 mm
wire and 350 g hanging mass.
From the available options, ICPF, Electromagnetic, and Thermo-pneumatic have
desirable characteristics but however owing to size and voltage supplied, ICPF
have the necessary trade-off required to satisfy the key parameters expected in a
TDD system micro-pump. Literature has been studied to cover the various design
configurations adapted for ICPF micro-pumps.
Pak et al. [33] presented a micro-pump which combined Poly Dimethylsiloxane
(PDMS) micro-channel and ICPF actuator. In the micro-channel, there is a nozzle,
diffuser structure which controls fluid flow from inlet to outlet. The recorded flow
rate was 9.97 μl/min in response to 8Vp-p and 0.5 Hz input voltage. The membrane
was fabricated using commercial Nafion 112 conductive polymer polypyrrole as
electrode where in order to have best performance, and it needed to have 17–19 μm
thick electrode.
Lee and Kim [34] tried to model ICPF disk as a micro-pump diaphragm. Low cost
and ease of manufacturing were the key factors considered when trying this method
as opposed to other technologies. A finite element analysis (FEA) was used to find
the shape of IPMC diaphragm and its electrodes and the estimation of its stroke
volumes (∆V). Based on the optimum designed nozzle/diffuser structure and the
estimated stroke volume of the IPMC diaphragm, the flow rate of the IPMC-based
micro-pump was estimated at a low Reynolds number (about 50) and it means that this
IPMC-based micro-pump is an appropriate choice for microfluidic devices because in
microfluidic devices we face with low Reynolds number fluids. The assumed IPMC
for this model had a Nafion membrane with Pt electrodes and based on the calculated
results for the IPMC diaphragm, the optimum center displacement (the best result to
have maximum pumping rate) was 0.966 mm, in which the radius of the electrode
was 8.5 mm. Based on the numerical result of this study, in response to a 2 V and
0.1 Hz input voltage, the flow rate of this micro-pump is 8.2 μl/s (492 μl/min).
Nguyen et al. [35, 36] presented a flap valve-based IPMC micro-pump. In this
device, a multilayered IPMC has been fabricated with a consecutive recasting
method, which consists of a Nafion/modified silica layer as a membrane sandwiched
between two thin layers containing Nafion, layered silicate, and conductive mate-
rial particles (Ag Nanopowder) as electrodes. The developed modified multilayered
IPMC has better blocking force without making any constraint and problem for its
bending displacement. The main material using in the flap valve was PDMS (due
to its flexibility and solving the sealing problem), but the whole of micro-pump is a
composition of several PMMA (Poly(methyl methacrylate)) layers that is equipped
with IPMC diaphragm and PDMS flap valve. Its flow rate is 760 μl/min in response
to a 3 V and 3 Hz input voltage.
Santos et al. [37] presented a micro-pump with two different types of diaphragms
proposed by the way in which voltage was applied to them. As shown in Fig. 12,
the second diaphragm has four additional branches in comparison with the first
diaphragm where we can apply a voltage to the IPMC by two thin copper rings
attached to these branches as shown in Fig. 13 while in diaphragm 1, two copper
wires are soldered to the center of the Pt electrodes in both sides as shown in Fig. 14.
The IPMC diaphragm of second diaphragm is more practical and durable and hence
the micro-pump has been fabricated by this diaphragm. The proposed micro-pump
is a four layers nozzle/diffuser pump as depicted in Fig. 15 with two acrylic layers
on top and down, with a layer of IPMC diaphragm covered by a layer of Teflon.
This pump was fabricated and the stroke volume of both IPMC diaphragms was
tested with different values of supplied electric current. The results showed an asym-
metric behavior of upward and downward displacements for both diaphragms. Stroke
volumes up to 80 μl were measured and in response to a 0.1 Hz input voltage (ampli-
tude was not reported), the flow rate of the fabricated micro-pump with second
diaphragm is reported to be about 8.02 μl/s (481.2 μl/min).
Guo and Wei [38] presented a micro-pump with an improved diaphragm was
presented to overcome the limitation of a single disk shaped diaphragm. This kind of
ICPF disk is clamped at all edges, which makes an obstacle for ICPF deformation,
Fig. 12 a First diaphragm. b Second diaphragm [37]

Fig. 13 Voltage application corresponding to second diaphragm [37]
Fig. 14 Voltage application corresponding to first diaphragm [37]
i.e., it decreases the ICPF bending, and consequently, the pumping ability of ICPF-
based micro-pump will be decreased significantly. In order to solve this problem and
increasing the deformation and pumping ability of the micro-pump, Guo presented a
new form of ICPF diaphragm using four pieces petal-shaped ICPF actuator instead
of a disk-shaped one. This petal-shaped structure has been depicted in Fig. 16 where
four quarter-disk-shaped ICPF actuators are attached on a thin PDMS diaphragm. In
this structure, the ICPFs are only clamped on one edge, and they are freer to bend and
make an up-down movement in the PDMS diaphragm so that the pumping operation
occurs. The 3D schematic of micro-pump consists of a petal-shaped ICPF actuator in
a sealed actuating chamber, and two check valves with a PDMS diaphragm attached
to the ICPF actuator. Under applying voltage, the ICPF and then PDMS diaphragm
will be moved up and down. Thus, this movement makes the pumping pressure,
Fig. 15 Exploded view of the micro-pump structure [37]
and the fluid will be pumped from the inlet to the outlet. The functionality of the
micro-valves of this micro-pump is keeping the directional flow during the pumping
process. The micro-valves are fabricated using PDMS diaphragms. The working
principle and duties of these valves are as follows:
1. When pressure is applied from the bottom, the outlet diaphragm will be lifted to
allow the fluid flow out through the outlet.
2. When pressure is applied from the top, the inlet diaphragm will be pushed down
and allows the fluid flow into the pump through the inlet.
The proposed micro-pump has been made in the form of a cube with the size of
30 mm × 30 mm and 27 mm (height). A petal-shaped IPMC actuator is used as the
active element of the pump with a PDMS diaphragm and under experimental tests,
this IPMC-based micro-pump was able to pump the fluids by 202 μl/min flow rate
in response to a 5-V 2-Hz applied voltage.
In all the previous ICPF-based micro-pumps, a disk-shaped or petal-shaped IPMC
was used as diaphragm or active element of the micro-pump, but McDaid et al.
[39] presented a new version of IPMC-based micro-pump using the ICPF in its
standard cantilever form. The authors believe the major issue in integrating ICPF in
microfluidic devices is its control in order to have a reliable and durable actuation
over a long period and many cycles. In the proposed structure, there is no diaphragm,
and the pumping actuator is an IPMC in its cantilever beam form. The device has
Fig. 16 Petal-shaped diaphragm before (left) and after (right) application of voltage
been fabricated by four layers of Perspex (Poly methyl methacrylate) for making
the inlet channel, the outlet channel, the pump chamber where the ICPF actuator
is embedded in it, and the bottom layer as the tank that allows the IPMC remains
hydrated throughout all experiments and hence it can work durable over a long time.
A thin layer of latex also has been attached to the bottom of the pump chamber, so
when the IPMC is deforming, the volume of the pump chamber also will be changed.
Hence, pressure is produced, and pumping of the fluid is obtained. In order to have
a continuous pumping actuation, a sinusoid IPMC displacement is needed. By an
open loop test, the optimum frequency to have the highest pumping rate was obtained
around 0.1 Hz. Of course, the high pumping rate is not the desire of all applications.
For example, in some drug delivery microfluidic chips, the lower pumping rates
are needed. Hence the proposed controller can work by lower frequencies too, for
example, 0.05 Hz.
Rajapaksha et al. [40] demonstrated a high-performance actuator based on
cross-linked poly(ethylene glycol) diacrylate (PEGDA) modified with Thiosiloxane
(TS) and ionic liquid (1-hexyl-3-methylimidazolium hexafluorophosphate) placed
between 2 μm thick poly(3,4-etylenedioxythiophene) polystyrene sulfonate
(PEDOT: PSS) electrodes. The maximum observed displacement was 2.5 mm at
a sinusoidal voltage of 3 V at 0.5 Hz. The displacement reduces exponentially
with increasing frequency for a polymer formed using 50% ionic liquid and 50%
PEGDA/TS composite. At the same composition, the ICPF shows fastest switching
time of 2 s which enables faster actuation speeds if required [43, 44].
Lastly, the literature relevant to conical valves has been explored to identify a
suitable design that can be incorporated in the fabrication process. Schabmueller
et al. [41] developed a valve-less piezoelectric pump with cone-shaped tubes. The
tubes had a moderate cone angle of 35.3˚ etched in the 111 plane on a single crystal
Table 1 Summary of ICPF-based micro-pumps

Reference Voltage/frequency Device Electrode Shape Main measurement
material material index
Pak et al. 8 V/0.5 Hz PDMS Ppy Square FR: 9.97 μl/min
Lee and 2 V/0.1 Hz Unknown Platinum Disk FR: 492 μl/min
Kim
Ngugen 3 V/3 Hz PDMS, Silver Disk FR: 760 μl/min
et al. PMMA nanopowder
Santos Unknown/0.1 Hz Acrylic, Platinum Disk FR: 481.2 μl/min
et al. Teflon,
Copper
Guo and 5 V/2 Hz Unknown Gold Petal FR: 202 μl/min
Wei
Mc Daid Unknown/0.1 Hz Persperex, Platinum Cantilever Diaphragm
et al. Latex Displacement ~300 μm
silicon. A liquid pump rate of 1500 μl/min and a gas pumping rate of 690 μl/min
were achieved using PZT actuator (Table 1).
Pun et al. [30] developed cone-shaped tubes laterally placed using Hot Embossing
Technique as opposed to DRIE or Advanced Silicon Etching (ASE). This resulted in
a preformed structure within the mold and internal stress was avoided. In addition,
inserting sacrificial layer in order to obtain the fluidic cavity wasn’t necessary, thus
simplifying the fabrication process. The final structure with a cone angle of 7˚ is
obtained.
Huang et al. [42] designed a single nozzle valve-less micro-pump using layers
of PMMA and adhesives. Carbon dioxide laser etching is used to create the pump
chamber, which consists of a circular chamber and an exponentially curved nozzle
as shown in Fig. 17. A peak flow rate of 0.4 ml/min was achieved at 20 V voltage
at a frequency of 70 Hz. A PZT piezoelectric disk attached to brass sheets in direct
contact with the chamber was used for actuation.
He et al. [5] developed conical valve micro-pump with a cone angle of 7˚. The
paper shows extensive relationship between frequency of operation of a PZT-based
piezoelectric actuator and cone angles. For frequencies lesser than or equal to 100 Hz,
the efficiency is independent of frequency, between 100 and 1000 Hz the efficiency
increases and beyond that efficiency decreases for a cone angle less than 20˚. For
cone angles greater than 40˚, efficiency increases beyond 1000 Hz. The optimal angle
for lesser than 1000 Hz is ten degrees. The pump design is shown in Fig. 18. All the
Fig. 17 Schematic of single

nozzle rain drop shaped
micro-pump [42]
Fig. 18 Schematic of 7°
conical valve micro-pump
[5]
above micro-pumps are much useful for biomedical applications and drug delivery
systems [51–59].
3 Modeling IPMC Membrane and Nozzle/Diffusor Valve

in COMSOL Multiphysics
There are three basic phenomena which define the characteristics of the physical
model of IPCF actuators: (1) electric field change inside the polymer due to distri-
bution of mobile cations and the applied electric potential at the electrodes (2) Mass
transfer using diffusion and migration transport (3) Occurrence of stress and strain
due to redistribution of ions and water inside the polymer membrane. The IPMC is
made of ICPF sandwiched between two metal electrodes [45, 46]. When voltage is
applied to the electrodes, cation flux or ionic current is induced by the imposed elec-
tric field. In case of water-based IPMC, migrating cations drag the water molecules
along, causing osmotic pressure changes and therefore swelling of the polymer near
the cathode and contraction near the anode. This in turn results in bending of material
towards the anode.
3.1 Governing Equations
The ionic current in the polymer is calculated with the Nernst-Planck equation:
∂C
∂t
+ ∇.(−D∇C − zμFC∇ϕ − μC∆V ∇ P) = 0 (1)
where C is the cation concentration, μ is the mobility of cations, D is the diffusion

constant, F is the Faraday constant, z is the charge number, ∆V is molar volume
that quantifies the cation hydrophilicity, P is solvent pressure, and ϕ is the electric
potential in the polymer. Mobility can be expressed as
Fig. 19 Conceptual model

of IPMC with gradients [45]
μ= D
RT
(2)
where R is gas constant and T is absolute temperature. Equation (1) is the main
governing equation for describing the transduction phenomena in IPMC materials.
There are three gradients present namely the potential gradient ∇ϕ, the concentration
gradient ∇C, and solvent pressure gradient ∇V. Conceptual model of IPMC along
with gradients is shown in Fig. 19.
The potential ϕ is defined by Poisson’s equation:
ρ
−∇ 2 ϕ = ε
(3)
where ρ is the charge density and is defined as
ρ = F(C − Ca ) (4)
where Ca is the major anion concentration. The variable ε is the effective dielectric
permittivity that can be explicitly written as
ε = εo εr (5)
where ε0 is the dielectric permittivity in vacuum. While cation concentration is

governed by Eq. (1), anion concentration is related to the local volumetric strain as
dV = ∇.u (6)
where u is the local displacement vector. A positive value of the volumetric strain
means an increase in local volume and vice versa. The volume changes affect the
local polymer anion concentration as they are part of the polymer backbone. Hence
the anion concentration is expressed as:
Ca = Co (1 − dV) (7)
where C0 is the initial anion concentration. It must be noted that for most practical
calculations it is reasonable to approximate anion concentration to C0 . The solvent
pressure is caused by local strain in the polymer matrix, forcing the solvent from
the concave side to the convex side of IPMC. Effective cation transport due to this
term is governed by the pressure gradient ∇P and molar volume constant ∆V in the
ionic flux term in Eq. (1). Pressure P is the solvent pressure caused by the strain in
the polymer. According to the momentum conservation, the solvent pressure and the
pressure of the polymer p are related as follows:
∇(P + p) = 0 (8)
It has been shown that
p(dV) = E(1−v)
(1+v)(1−2v)
dV (9)
where E is Young’s modulus of the material and v is Poisson’s ratio. By knowing

these constants, Navier’s equations can be constructed for displacements:
−∇.σ = F (10)
where F is the force per unit volume. Body force F is defined as a function of charge
density ρ:
F = f (ρ) (11)
3.2 Boundary Conditions
This section presents the boundary conditions that are used to solve the governing
equations described in the previous section. Figure 20 represents the polymer domain,
electrode domains, and boundaries of the actuator model. Equations (1) and (3) are
solved in the polymer domain δ and the domains ψ1 and ψ2 are used for electrodes
which are used to apply electric potential.
For Eq. (1) for domain δ at the boundaries ∂δ1-4 :
−D ∂C
∂n
− zμFC ∂ϕ
∂n
=0 (12)
For Eq. (3) which is used to solve potential inside the polymer:
At boundary ∂ δ2 and ∂δ4 :
ϕ∂δ4 = ϕ∂δ2 = V (13)

Fig. 20 2D model of actuator with all boundaries and domains
At boundary ∂δ1 and ∂δ3:

∂ϕ∂δ3 ∂ϕ∂δ1
∂n
= ∂n
=0 (14)
For Ohm’s law in electrode domain ψ1 and ψ2:
V∂Ψ 1 = Vpos , VΨ 2 = Vneg (15)
where Vpos and Vneg are applied electric potentials. The input applied signal is
a sinusoidal wave set at a specific amplitude and specific frequency for positive
terminal and ground at negative terminal.
3.3 Nozzle/Diffuser
In most pumps, mechanical check valves were used for the inlet and outlet ports, either
with membranes or flaps. The process of designing and fabricating this type of valve
is very complex. The pump itself may suffer from problems such as high pressure
drop across the valves. Some crucial properties like backward flow and switching
speed have to be controlled precisely in order to achieve a working miniature pump.
Moreover, wear and fatigue can be critical issues, especially in polymer-fabricated
devices. This may result in reduced lifetimes and reliabilities. There is also the risk
of valve blocking by even small particles, which instantly degrade the pumping
performance or cause damage to sensitive fluids. This limits the application range
of most valve-based miniature pumps to filtered media. Therefore, there was a need
for miniature pumps with no movable parts.
Valve-less miniature pumps can eliminate these problems. The diffuser/nozzle
setup provides flow-rectifying properties like a check valve. Literature shows a
maximum achievable forward–backward flow ratio of 2.23 for this type of valve
which is sufficient for a pump. A diffuser is a diverging duct, and nozzle is a
converging duct. These ducts are designed to have lower pressure loss in the diffuser
direction than in the nozzle direction for the same flow velocity. When chamber
volume increases (the supply mode), the inlet element acts as a diffuser with a lower
flow restriction than the outlet element, which acts as a nozzle. Therefore, a larger
volume is transported through the inlet into the chamber than through the outlet.
On the other hand, during decreasing chamber volume (the pump mode), the outlet
element acts as a diffuser with a lower flow restriction than the inlet element, which
acts as a nozzle. This means that a larger volume is transported through the outlet out
of the chamber than through the input. As a result, a net volume is transported (i.e.,
pumped) from the inlet side to the outlet side. Therefore, a complete pump cycle
is achieved, even though the fluid traveled in both directions of the diffuse/nozzle
element.
The dimensions that decide the flow rate of the ducts are the smaller diameter,
larger diameter, height of the cone, and the angle of inclination of cone with side
wall which is an optimum of 5˚ when dynamic fluid resistance is minimum. The inlet
pressure and fluid density are other deciding factors for the pressure distribution flow
velocity across the ducts.
3.4 Pump Designs for Fabrication
The following pump designs were proposed for fabrication of micro-pump: single
chamber as shown in Fig. 21 with left-hand side inlet and right-hand side outlet, dual
chamber with 180˚ out of phase pumping using conical valves as shown in Fig. 22
with right-hand side inlet and left-hand side outlet and single chamber pump with
tesla valve as shown in Fig. 24 with right-hand side inlet and left-hand side outlet.
Rectangular inlet and outlet chambers have been provided in double chamber design
for increased pumping efficiency by promoting fluid pumping in alternate cham-
bers whereas in single chamber design, cylindrical chambers have been provided to
prevent excess backflow (Fig. 23).
Fig. 21 Single chamber

conical valve-less
micro-pump
Fig. 22 Dual chamber

conical valve-less
micro-pump
Fig. 23 Single chamber

tesla valve-less micro-pump
The pumps were being tested with the existing, popular piezoelectric buzzer-based
actuation as well as the proposed PEGDA-based ICPF membrane-based actuation.
The pump chamber was proposed to be fabricated using impression molding using
PDMS polymer (Tables 2 and 3).
Table 2 Specifications of
Part Dimensions
single chamber conical
valve-less pump Main block 30 × 60 × 6 mm
Diaphragm holder radius 21 mm
Pumping chamber radius 19 mm
Nozzle/diffuser base diameter 1.8 mm
Nozzle/diffuser base height 10 mm
Nozzle/diffuser angle 5˚
Fluid receiving chamber diameter 4 mm
Tube fitting pipe diameter 4.8 mm
Part Dimensions
dual chamber conical
valve-less micro-pump Main block 60 × 60 × 6 mm
Nozzle/diffuser base diameter 1.8 mm
Nozzle/diffuser base height 10 mm
Nozzle/diffuser angle 5˚
Fluid receiving chamber diameter 35 × 5 × 3 mm
Fig. 24 Inverse 3D print of

double chamber conical
valve micro-pump and tesla
valve
4 Simulation Results
4.1 IPMC Actuator
The model is designed as shown in Fig. 25. The modules used in COMSOL Multi-
physics to implement the IPMC model in 2D are Transport of Diluted Species (tds)
which is responsible for Eq. (1) and boundary condition Eq. (12), Electric currents
(ec) which is used to provide potential and boundary conditions corresponding to
Eqs. (13), (14) and (15), Solid mechanics (solid) to implement Eq. (9) and boundary
condition is implemented using Dirichlet boundary for Eq. (10), General Form PDE
(g) to implement Eqs. (3), (4), (5) and the various constants needed to solve the
system of equations is provided in Fig. 26. The applied voltage at the top is Vpos
= 0.5*sin(2*pi*t) V at a frequency of 1 Hz and at the bottom is Vneg = 0 V. The
width of the IPMC is modeled as coming out of the plane and approximated to 2D
calculations. The deformation plot is shown in Fig. 27 where the scale shows the
minimum and maximum value of stress across the 2D domain. The displacement
plot of the mid-point of the actuator where the displacement is highest at any given
point of time is shown in Fig. 28.
Parametric studies were performed by varying the length of the IPMC actuator
and the frequency of the sine voltage provided to perform the actuation. It can be
observed that the displacement steadily increases with increase in length however
at a length of 71.07 mm, the displacement drops to a really low value as seen in
Fig. 29. This can be attributed to poor charge distribution and low control of the
actuator when the actuator is excessively long. The frequency of input sine wave
voltage was varied from 0 to 3 Hz with step increments of 0.5 Hz is displayed in
Fig. 30. The maximum displacement which is nearly 20 mm is observed at 0.5 Hz
which drops sharply with increasing frequency. This observation can be attributed to
poor response time of ionic current flow within the polymer with higher frequencies.
Fig. 25 IPMC model
Fig. 26 Parametric constants for IPMC model

Fig. 27 Surface stress (Pa) with length (mm) of IPMC along x-axis and displacement (mm) along
y-axis
Fig. 28 Displacement (mm) versus time (s) of IPMC actuator of length 51.07 mm, thickness
0.57 mm actuated at 0.5 V, 1 Hz
Fig. 29 Displacement (mm) versus time (s) of varying IPMC actuator lengths at 0.5 V, 1 Hz
Fig. 30 Displacement (mm) versus time (s) of varying voltage frequencies at 0.5 V
4.2 Nozzle/Diffuser
In order to analyze the effectiveness of a passive valve, an existing model of a

piezoelectric pump was used so that the results generated could be attributed to
solely the study of the nozzle/diffuser setup. The modules used in COMSOL Multi-
physics to achieve this study are Solid mechanics (solid), Laminar flow. The para-
metric constants required are shown in Fig. 31. Due to computational difficulty of
IPMC actuator in 3D, passive valve characterization has been performed using a
piezoelectric pump with a 12 V, 60 Hz supply voltage.
The volume of pump chamber used is 4.5 ml with dimensions 30 mm × 30 mm
× 10 mm. The valves are symmetrically placed equidistant from the edge of the
pump at the bottom. The height of valve is 10 mm, the larger diameter of inlet and
Fig. 31 Parametric constants for valve simulations

outlet is 3 mm while the smaller diameter is controlled by cone angle which is at 5˚

corresponding to roughly 1.06 mm radius. The volume of the valves is approximately
52 μl. The model designed is shown in Fig. 32. The model has been bisected along
the x–z plane to reduce computational complexity since the pump is symmetric in
nature. The flow rate is calculated as the spatial integral of the volume of liquid
passing through the inlet and outlet. The flow rate was found as shown in Fig. 33
which is 2.4 ml/min at steady state. The flows below 0 represent backward flow
which is less than 10 μl/min.
The flow rate for different cone angles was plotted as shown in Fig. 34 and as
expected with shrinking diameters of the smaller end the flow rate changes accord-
ingly. However, there is no major change in variation of flow rate between various
angles. The maximum outflow at every angle is shown in Fig. 35 which is a better
representation of the variation.
The above results show that valve-less model for micro-pumps is a suitable alter-
native to their active counterparts. Not only do they save on power, but also the manu-
facturing difficulty is overcome, and wear and tear associated is reduced. Figure 33
illustrates acceptable backflow values that can be fine-tuned by varying the cone
angle as observed in Figs. 31 and 35. Since the effective height of valve was kept
constant, the flow rate doesn’t seem to improve with different angles and could have
been limited by the smaller diameter of the valve. Additionally, the overall volumetric
out flow varies nearly linearly with time which implies that fluid is transported effi-
ciently with minimum delay as shown in Fig. 36. The response times of active valves
could be prone to slow response due to the actuation mechanism in case of dual valve
type micro pump. Additional study has to be conducted to verify if usage of multiple
valves with greater backflow resistance can improve efficiency and flow control and
if variation of sidewall curvature has any effect on flow rate control.
Fig. 32 Pump model with nozzle/diffusor valves

Fig. 33 Inlet flow rate (ml/s) and Outlet flow rate (ml/s) versus time
Fig. 34 Flow rate (ml/s) for different conical angles of valve
Fig. 35 Maximum outflow rate (ml/s) versus cone angle

Fig. 36 Accumulated flow volume versus time for different cone angles
Figures 37, 38, 39, 40, 41 and 42 show the flow rate and pressure contours of a 10˚
angle conical valve in the direction opposite to the flow of regular valve operation. The
negative pressure shown is due to the formation of eddies that create low-pressure
areas and drastically reduce flow rates. There is a 30% increase in flow rate with
increasing inlet pressure which settles to a nearly constant value at the inlet pressure
of 130 Pa. The outlet pressure on the other hand shows a steady decrease with
increasing inlet pressure with a 5% decrease across three different pressures. The
maximum backflow rate observed is 3 ml/s.
Tesla valve under similar conditions exhibits a much lesser backflow rate as shown
in Figs. 43, 44, 45, 46, 47 and 48. There is a maximum backflow rate of 240 μl/s at an
inlet pressure of 130 Pa. The observed rise in back flow rate in 60% for every 60 Pa
Fig. 37 Flow rate at 10 Pa inlet pressure for conical valve

raise in inlet pressure. The reduction in backflow rate can be attributed to the observed
lack pressure variation at the inlet and outlet thus restricting backflow. The difference
in pressure is hardly 1% as shown in the figures. The direction of flow is from right
to left where it offers greater resistance to flow. The flow rate is calculated across a
cross-sectional area of 1 mm cube as shown in proposed fabrication dimensions in
Table 4.
Fig. 40 Conical valve-Outlet pressure contours for 10 Pa inlet pressure

Fig. 43 Flow rate at 10 Pa inlet pressure for tesla valve


Fig. 46 Outlet pressure contours at 10 Pa inlet pressure for tesla valve

Part Dimensions
single chamber tesla
valve-less micro-pump Main block 200 × 30 × 6 mm
Tesla valve cross section 1 × 1 mm
Tesla valve length 60 mm
Angle of islands 53˚
Fluid receiving chamber diameter 4 mm
5 Conclusion
This review article shows the comparison of various types of micro-pumps and
majorly focused on IPMC-based micro-pumps. The IPMC micro-actuators consume
less power, and hence it is possible to obtain low power operation. This is due to
low voltage operation of IPMC actuator. The conical angles were analyzed for opti-
mizing the performance with respect to flow rate. Using valve-less nozzle/diffuser
setup with conical angle of 4–6˚, the pump provides desired flow rate. However,
the computational effort for the simulation of IPMC actuator is exceptionally large.
Furthermore, there exists no concise methodology for modeling IPMC-based actu-
ators. The simulation performed uses a white box model which is solved using
approximation methods targeted at error reduction over several segregated itera-

tions. The current model does not converge for 3D simulations in the electric current
domain hence, direct characterization of pump in relation to actuator performance
needs a different approach. Although the effectiveness of valve should be indepen-
dent of type of diaphragm actuated micro-pump, a suitable methodology for relation
of IPMC membrane displacement and flow rate across nozzle/diffuser type passive
valves is required.
Fabrication designs were proposed for single chamber conical valve micro-pump,
double chamber conical valve micro-pump functioning at 180˚ out of phase with each
other and single chamber tesla valve micro-pump according to the specifications. The
simulation results show the expected flow rates achievable for different fluid pressures
that may arise giving rise to the desired flow rate. Future work would involve a suitable
fabrication method that doesn’t involve the difficulty faced in impression molding
technique.
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Fabrication Techniques and Materials
for Bio-MEMS
Sudhanshu Dwivedi
Abstract Microelectromechanical systems (MEMS) based advanced point-of-care

rapid diagnostic solutions with high degree of sensitivity along with high accuracy
are required that can remain efficient even after a decrement in the form factor of
the sensor. MEMS technology is at the forefront of development of miniaturized
biosensor devices in bulk batches that offer a highly efficient, sensitive, accurate,
precise and commercial platform. Arranged arrays of MEMS devices can be spatially
covered in more than an area of 1000 mm2 . Bio-MEMS devices are fabricated by
the conventional micromachining techniques employing oxidation, thin film depo-
sition by sputtering, e-beam & thermal evaporation, and chemical vapor deposition
(CVD). Photolithography is applied for patterning of the micrometre sized geomet-
rical shapes, while electron beam lithography (EBL) patterns nanostructures down to
few nanometres scale in reference to the nano-electromechanical-systems (NEMS)
for advanced bio-detection applications including lab-on-a-chip technology. Bulk
micromachining offers high commercially viable technology involving selective
removal by etching of the bulk substrate materials that develop MEMS components,
such as, cantilevers and beams. Wet or dry etching methods can be employed for
the bulk substrate material removal by selective elimination of unmasked areas for
the patterning of geometrical shapes and patterns. Faster etch rates are obtained by
chemical wet etching while dry etching technique offers fabrication of anisotropic
geometrical patterns with high aspect ratio. Lift-off is also a conventional commercial
technique that develops patterns on the surface of the material. Stereo-lithography
is an advanced 3D fabrication technology that has a commercial orientation focused
on ultraviolet (UV) radiation-based curing of the polymer solution for fabrication
of high aspect ratio structures in a layer-by-layer approach. Bio-MEMS/NEMS
relies on different types of biomaterials, such as, DNA and RNA, that are used as
biomimetic materials. The other categories are inorganic (Si, GaAs, Ge, SiC, Si3 N4 ,
SiO2 and glass/quartz for biomedical applications) and organic materials (PMMA,
SU-8, PDMS,) for use in MEMS/NEMS. Polymers and plastic substrates are more
favourable because of ease of micromachining, faster prototyping along with higher
mechanical bending characteristics, and low costs. Moreover, optically transparent
S. Dwivedi (B)
S.S. Jain Subodh P.G. (Autonomous) College, Jaipur, India
102 S. Dwivedi
substrates can be used in optical detection techniques along with being biologi-
cally compatible. Paper microfluidics is another special variant of bio-MEMS due
to its features of low-cost technology, biodegradable nature along with the normal
wicking action. Paper microfluidics has been employed in paper immunoassays
and electrophoresis. Microfluidic approaches in reference to bio-MEMS manipu-
lates very small quantities of fluid over the microfabricated substrates and combines
electronics for rapid testing device prototypes. There have also been efforts for the
fabrication of microchannels in glass and/or fused quartz and other similar types
of substrates using ultrafast lasers for microfluidics that combine optical detection
techniques as well. This chapter is a concise effort to present an overview of various
micro- and nanofabrication technologies for bio-MEM/NEMS device fabrication for
sensing platforms. Technical nitty-gritties of photolithography and EBL have been
presented in a critical manner along with different materials used in the advanced
bio-MEMS/NEMS micro- and nanofabrication. A small portion is also devoted to
the description of cleanroom technology including its classifications with a note on
international standardization protocols. Apart from this, deposition techniques have
also been discoursed in special reference to bio-MEMS/NEMS. Superhydrophobic
feature or wetness property and its importance in the bio-MEMS/NEMS based
sensing platforms have also been discussed.
1 Introduction
Microelectromechanical systems or MEMS entails the fabrication of micron-sized

devices by the use of a host of fabrication techniques of thin film deposition,
photolithography and wet- or mostly dry-etching techniques [1, 2]. Fabrication of
nano-sized devices by the above techniques including electron beam lithography
(EBL) for nano-patterning is termed as nanoelectromechanical systems or NEMS
[1, 2]. This technology is a combination of microelectronics and μm- or nm-sized
mechanical structures obtained by micromachining. This technology is referred to
as microsystems technology (MST) in Europe while as micro-machines in Japan.
MEMS is a word that pertains to a relevant technological platform stating that elec-
trical circuits and correlated components are combined to form a mechanical device
in form of a sensor or actuator. Microelectronics and micromachined structures can
be integrated during or after the process of fabrication and for this a standard design
process is extensively tested before the actual production of the device structure.
MEMS is an established commercially viable technology that ensures production
of micromachined architectural systems with enhanced performances. MEMS is
decades old technology now widely used for the fabrication of accelerometers as
navigational sensors, radiofrequency (RF) MEMS for automotive radar sensors in the
operational frequency range of 76–81 GHz, MEMS based fabrication of 3D architec-
tures, robust MEMS devices for ground aero-propulsion wind tunnels, MEMS based
inertial measurement units (IMUs) for missile applications including applications
in high speed re-entry spacecraft guidance, navigation as well as control (GNC)
Fabrication Techniques and Materials for Bio-MEMS 103
systems, fuel cell applications, applications in integrated circuitry (IC), geotech-

nical underground sensor including monitoring applications and also inclusive of
bio-MEMS or even bio-NEMS based implantable devices [2, 3].
Bio-MEMS or bio-NEMS is a sub-branch of MEMS/NEMS that makes use of
prevalent techniques in MEMS/NEMS for the fabrication of bio- or medical-devices
[4–7]. In relation to bio-MEMS, the important ingredients are inclusive of microfab-
rication of devices on silicon (Si), glass and polymer substrates, microfluidics and
the associated electrokinetics, physics of devices that include sensors as well as actu-
ators, micro-total-analysis systems (μTAS), lab-on-a-chip or LOC device, detection
as well as measuring and quantification systems, packaging of bio-MEMS systems,
RF safety measures, power systems & effective transportation of data packages
as well as a host of biological systems that include clinical medicines and corre-
lated systems, deoxy-ribose nucleic acid (DNA), proteomics, genomics and protein
microarrays.
Fabrication of bio-MEMS devices involves four different strategies that include
photolithography-based patterning of the material, (ii) deposition of thin films over
the substrates followed by patterning, (iii) selective etching of material to form
uniform or shape-anisotropic structures and (iv) bonding in which two substrates
are bonded with each other.
2 Fabrication Technology
The first and the foremost step in fabrication methodology on a substrate involves the
meticulous cleaning process. Different types of substrates are silicon (Si), germa-
nium (Ge), gallium arsenide (GaAs), sapphire (Al2 O3 ) or even glass substrates,
such as, fused silica. Very often Si is used in the fabrication of MEMS, switching
and memory devices for logic & memory operations. Si fabrication technology is
highly developed device processing technology because of low cost of the raw mate-
rial of SiO2 for Si wafer manufacturing along with being readily available. This is
despite the fact that Si is an indirect bandgap semiconductor material [8], while Ge
is a direct bandgap semiconductor material [9] and possesses much reduced elec-
tron mobilities in comparison to Ge. Lin et al. has specifically worked on indirect
to direct bandgap engineering in ultrathin Si semiconductor films [8]. Fadaly et al.
reported on highly efficient light emission from direct bandgap Ge semiconductor
materials [9]. Cleaning of Si substrates is performed by the RCA (Radio Corpora-
tion of America) method, Piranha cleaning methods, dipping in HF solution and/or
even cleaning with acetone, methanol & isopropyl alcohol subsequently followed by
cleaning with doubly deionized water (resistivity, ρ = 18.2 MΩ-cm at T = 25 °C).
A dehydration bake is performed before application of photoresist (PR) over Si
substrates for removal of residual H2 O molecules or water vapor molecules attached
to the substrate. Wafer priming is the process of enhancement of adhesion by use of
adhesion promoter, hexamethyldisiliazane [linear formula: (CH3 )3 SiNHSi(CH)3 ],
or application of plasma based etching of the substrate surface to coarsen it. After
104 S. Dwivedi
cleaning of the substrate, an oxide layer of SiO2 is very often required. This SiO2
layer not only acts as an insulating layer but can also be used as a protective mask in the
subsequent processing steps of etching, for example. Oxidation of Si wafers can be
done both by wet and dry oxidation methods in a tube furnace at higher temperatures
for dry oxidation (900–1150 °C) and at relatively temperatures in case of wet oxida-
tion (700 °C). The first step in the photolithography process involves the application
of a thin photosensitive organic polymer layer, i.e., of liquid PR over the oxidized
substrate surfaces. Si substrate is held in a spin-coater with the help of a vacuum chuck
so that it does not get thrown away during the spinning process, and to ensure forma-
tion of uniform layers of PR over the substrate surface. Spinning of Si substrate is
performed at highly controlled speeds in one or even more steps. Usual spin speeds are
in the range of 1500–8000 rpm causing liquid PR material to drive towards the edges
of the substrate due to centrifugal forces. The material keeps on compiling at the edges
where surface tension keeps on holding the material and is thrown out on exceeding
this force. The generated PR thickness is the function of molecular weight, solution
concentration and the spin speeds. Optimization of spinning process is highly desir-
able for effective transfer of the patterns in addition to the control of defect density
in the spun polymer. Subsequently, the substrate is soft baked to remove remaining
solvents in the spun PR to ensure that no built-in stresses are generated as a result of
residual liquid. Prebaking or soft-baking is performed at T = 75–100 °C for removal
of residual solvent and subsequent promotion of adhesion of PR to the substrate
surface. After soft-baking, the pattern is transferred onto the PR by light exposure
through a photomask consisting of a pattern to be replicated with desired geometrical
features. Light sources can be a mercury lamp, ultraviolet (UV) light and even laser
sources. Light radiation from mercury lamp can be of wavelengths 435 nm (g-line) or
even 365 nm (i-line). UV light has the wavelength of 285 nm, while laser sources have
typical wavelengths of 193 and 248 nm. The smallest feature that can be lithographed
is almost proportional to the wavelength of the source that is implemented for expo-
sure. The exposed PR becomes either soluble or insoluble in the developer solution
depending on itself being a positive or negative PR. Profiling of side-walls of PR is
of critical importance that can be better adjusted by appropriate control of exposure
time and dosage, strength of the developer as well as the time of development. Post-
exposure development and treatment is required for cessation of reactions initiated
during the exposure and even inducing newer reactions. Post-exposure treatments are
inclusive of post-exposure baking, vacuum reactions, interaction in an environment of
a reactive gas and even flood exposure from different radiation sources. Aqueous alka-
line solutions are generally used for the development of positive PRs while organic
solvents are used for the development of negative PRs. After the development proce-
dure, undesired residual PR is eliminated before further processing. This process is
known as descumming that removes undesired PR with the help of mild plasma-based
procedure typically known as plasma ashing. Highly energetic O2− ions are generated
in the plasma by use of O2 gas that strikes the undesired PR in a controlled manner
to burn it out. Hard-baking or post-baking is performed to enhance adhesion qualities
of PR over the substrate that has been rendered to be weak as a result of penetration
of the developer solution into the PR-substrate interface or swelling mainly in case
(b) (c)
(a)
Si Si
Si substrate with negative PR
Si
Photomask Alignment & Light Exposure
(e)
(d)
(f)
Si Si Si
Removal of PR
Sputter Deposition of Metal Side-Walls : Lift-Off
Fig. 1 Microfabrication technology process steps are shown with the help of a process flow
diagram. As shown below (a) RCA cleaned wafer is taken, which is, (b) spun-coated with a nega-
tive PR followed by (c) proper photomask alignment and light exposure to develop PR for pattern
formation. Lift-off generates non-uniform sidewalls as shown in (d) followed by (e) metal sputtering
and (f) subsequent removal of PR, more specifically by plasma-ashing in case of a negative PR [10]
of negative PRs. Post-baking removes residual solvents from PR along with a proper
heat-treatment or annealing to fill-up gaps between PR and the substrate. Post-baking
is performed at higher temperatures of 120 °C to induce hardness in the thin PR layer
to enhance tolerance level against etching as well as deposition steps. Sacrificial PR
mask is used as a patterned covering layer in further processing steps involving
etching and/or deposition. In the etching process, the masked PR layer is the protec-
tive layer against the etching solution that can be in a liquid form, gas or even plasma.
After printing of the pattern, PR is ashed out for subsequent processing. In certain
types of MEMS device architectures, PR is an integral part and it may not be essential
to ash it out. Microfabrication process steps are shown schematically in Fig. 1.
2.1 Photolithography
Semiconductor technology relies on photolithography for the development of

microstructures for the production of architectural systems for MEMS. Photolithog-
raphy is an optical lithography technology employing mostly UV light for generation
of the user-defined pattern by application of the photomask [10–13]. Photolithog-
raphy technique has been discussed in a critical manner by Mahalik in his book on
Micromanufacturing and Nanotechnology [10]. Hubenthal has given technical details
on photolithography in the work titled as “Noble Metal Nanoparticles: Synthesis and
106 S. Dwivedi
Optical Properties” [11]. Cirelli, Watson & Nalamasu have provided excellent tech-
nical knowhow about optical lithography in “Encyclopedia of Materials: Science and
Technology” [12]. Similarly, May and Sze have discussed about principles of optical
lithography in their book on “Fundamentals of Semiconductor Fabrication” [13].
Operative principle of optical lithography is based on the application of a photo-
sensitive mask to fabricate the geometrical patterns by using a light-sensitive PR
which has already been blanket-deposited over a substrate. The photosensitive mask
is fabricated over special glass plates in the locality of a clean room that is typically
designed with all the desired geometrical boundaries, sizes and shapes. The photore-
sist or PR is an organic liquid material with special photosensitive properties that is
spun onto a substrate using a spin-coater followed by light exposure and baking [14–
16]. Photoresist materials have been discussed in good amount by K. S. Gandhi [14]
and Turner and Daly [15]. An important note is that photolithography is performed
in a yellow or red-green lightened clean room to prevent initiation of photo-induced
reactions in the photosensitive resist material. The technology is based on the fact
that PRs are sensitive only to light radiations that have wavelengths shorter than
those of yellow light. The wavelength of the yellow light usually lies in the range
of 560–590 nm towards which the PR is not sensitive so that a photoreaction can
never be induced, and bonds in the photosensitive PR material are not broken. Only
high frequency or short wavelength light radiation can impart sufficient energy to
cause photo-induced bond-breaking in PRs. However, with advancing technology
day-by-day, there are efforts to develop highly efficient PRs along with processing
of PRs in rather miniaturized machines with reduced processing steps to have lesser
dependency on yellow light environment inside the clean room.
There are two types of PR, the positive PR and the negative PR. If the PR becomes
soft bonded or bond-breaking happens in the material as a result of light exposure,
and it becomes soluble in the PR developer solution in contrast to the unexposed
part of the same PR, it is termed as a positive PR. On the other hand, if the PR gets
hardened with bonds becoming stronger due to enhanced crosslinking on exposure
to the light so that it becomes insoluble into the PR developer solution in comparison
to the unexposed part, it is called as negative PR. Both positive and negative PRs
have merits and demerits, and are applied according to the requirements. Negative
PRs possess excellent adhesion over Si substrates in comparison to the positive PRs,
are less expensive, have usually organic base, while positive PR have aqueous base,
minimum feature size obtainable from positive PR is 0.5 μm while it is 2 μm from
negative PR, and step coverage is better with a positive PR than with that obtained
from a negative PR. Patterning with photolithography has been shown schematically
in Fig. 2 given below which shows the use of a photomask-based pattern-generation
procedure by the application of a positive PR.
As pointed out in the above schematic to generate patterns with a positive PR,
initially a thin metal layer is deposited over the substrate. A host of techniques
can be employed for the purpose that include chemical vapor deposition (CVD),
thermal evaporation, e-beam evaporation, sputtering, molecular beam epitaxy (MBE)
and/or pulsed laser deposition (PLD), and may be other clean-room physical vapor
deposition (PVD) techniques, such as, atomic layer deposition (ALD). Licari and
Fig. 2 Photolithographic (d) Soft-Baked Patterned PR

(a)
process with positive PR for Metal Layer
pattern generation over a Metal Layer
metal film in the form of a
Substrate
schematic Substrate
(b)
Positive Photoresist (PR)
Metal Layer (e) Etching of Metal Layer
Substrate
Substrate
(c) UV Light
(f) Patterned Metallic Layer

Photomask
Photoresist (PR)
Metal Layer
Substrate
Substrate
coworkers have given details of different thin film processes in “Hybrid Microcircuit
Technology Handbook (Materials, Processes, Design, Testing and Production)” [16].
The choice of the deposition technique depends on the specific requirements of high
through-put, congruent thin films, uniform with well-connected grain morphology,
epitaxial or polycrystalline films, and single or multilayer deposition including the
area of deposition for production of devices over large-sized wafers. For example,
CVD can deposit large-area thin films over large-sized substrates (8 inches or 12
inches sizes), while PLD usually deposits uniform and congruent thin films in small
areas of 1 × 1 cm2 over the substrates. For commercial purposes, state-of-the-art
highly automated and sophisticated large area deposition techniques are required. In
the research domain, state-of-the-art techniques, such as, PLD are also useful. Now,
for geometrical patterns generation on the deposited metallic layer, positive PR is
spun over the deposited metallic film using a spin-coater that is subsequently exposed
to UV-light. Geometrical patterns are transferred with the help of a photomask that
needs to be produced as shown in Fig. 2(c). Before exposure to light, spun PR-coated
substrate is soft-baked in a temperature range of 60–90 °C to evaporate any liquid
phase and help facilitate normal bonding in the spun PR layer. Light exposure with
the help of a photomask causes geometrical pattern generation over the positive PR
as per its photosensitivity mechanism discussed above. Subsequently, positive PR
is developed in the PR developer solution followed by hard-baking in the usual
temperature range of 120–180 °C. The uncovered or open metal areas (those lying
108 S. Dwivedi
outside the coverage of PR) are etched away with the help of an etchant solution by
wet or dry etching methods selectively as shown in Fig. 2(e) followed by removal of
PR to expose the protected parts to get the finally patterned structures of the metallic
film as shown in Fig. 2(f).
Patterns can also be generated over metallic films using a negative PR [17] as
shown in the process flow given below in Fig. 3. In this approach, a negative PR is
spun over the substrate using a spin-coater that is subsequently exposed to light with
the help of a photomask after the process of soft-baking as shown in steps (a), (b) and
(c) of Fig. 3. In the next steps, negative PR is subjected to the negative PR developer
solution followed by hard-baking. Now, metal films are deposited over the structures
obtained in step (c) of Fig. 3 to fill the voids and empty areas as shown in Fig. 3(d)
followed by etching to get patterned films without PR as shown in Fig. 3(e). Ceyssens
and Puers have discussed the technology of SU8 negative PRs in the “Encyclopedia
of Nanotechnology” [17].
Photolithography is a parallel process technique that is applied for commercial
level production of MEMS devices in addition to the device research. The main
drawback associated with photolithography is that it is a diffraction-limited technique
defined by the Abbe-Rayleigh criterion as follows [10–13],
Fig. 3 Photolithographic (d) Metal Deposition

(a)
process with negative PR for
Negative Photoresist (PR)
pattern generation over a
metal film in the form of a
schematic Substrate
Substrate
(b) UV Light
(e) Selective Etching To Remove PR

Photomask
Negative Photoresist (PR)

Substrate
Substrate
(c) Soft-Baked Patterned PR
Substrate
λ λ
d mi n = 0.61 = 0.61 (1)
nsi nα NA
Here, λ is the wavelength of the point source, α is the angle of aperture, NA is
numerical aperture, n is the refractive index of the surrounding medium and d min
entails the minimum size of structure that is illuminated with the point source of
light. As the above criterion entails in Eq. (1), shorter wavelengths and high NAs
are required to produce small-sized structures. A high-pressure mercury lamp is
good-enough to write (word “litho” means to write) structures with features above
250 nm. Shorter wavelengths should be used for writing structures in the range of
150–250 nm. An excimer laser is the best one to produce wavelengths in the specified
range. Different excimer laser sources produce lights of different wavelengths that
includes ArF and KrF sources with wavelengths of 193 and 248 nm, respectively.
Herve et al. have produced an effective comparison of the use of ArF and KrF laser
sources-based microfabrication process [18]. For the production of structures with
features below 100 nm, extreme ultraviolet (EUV) lithography uses much shorter
wavelengths down to 13.4 nm. Endert et al. talked about a novel laser cavity combined
with a line narrowing module for the deep ultraviolet (DUV) optical lithographic
tool that utilized excimer laser steppers [19]. An important task is the reduction of
minimum feature sizes and increment of aspect ratio of the geometrical features
to be fabricated. In the contact exposure technique, the resolution limit for UV
photolithography with wavelength of 365 nm is counted as follows in the manner
expressed by Maalouf et al. [20] and Kumar et al. [21].
√
Resolution Limit ∼ k(Constant) x (λh) (2)
√
Here, h is the thickness of the photoresist, k usually has a value of 2 and λ
is the wavelength of light that is exposed onto the sample. The value of constant k
expresses the diffraction limitation that can be reduced by optimization of exposure
to the UV light with desired intensity level. Several parameters have been tuned for
reduction of the k-value that include reducing the thickness of the PR and applica-
tion of low wavelength light for improvement of the resolution. Bourdillon et al. was
successful in generating features down to 16 nm with the help of X-ray lithography
that reduced constant k to the value of 0.65 to improve the resolution [22]. DUV
photolithography is a technique that can be performed with excimer laser sources of
wavelengths 193 nm (ArF) or 248 nm (KrF) that fall on the lower side as demon-
strated by Jain et al. [23]. Mojarad et al. successfully generated spatial line structures
possessing periodicity of 14 nm along with a resolution of 7 nm [24]. In the next-
generation lithographic methods, immersion lithography is extensively used in the
latest times to produce feature sizes down to 25 nm [25]. In this lithography tech-
nology, the air-gap between the lens and the PR-spun substrate is filled with a liquid
possessing refractive index greater than 1 (μ > 1). Owa and Nagasaka pointed out
improvement in resolution as a result of increment in refractive index of the medium
(perfluoropolyether or PFPE, μ = 1.37) and also in NA as defined by Eq. (1) [26].
110 S. Dwivedi
Photolithography involves a chain of controlled processes that is performed

strictly in a clean room environment. A cleanroom entails a highly cleaned room with
controlled environment in which minute dust particles, aerosol particles, airborne
microbes and even other much smaller contaminants not visible to the eye are filtered
out using special types of high-class filters. Other important factors that need to be
controlled in a cleanroom are inclusive of humidity inside the chamber, air-flow and
temperature. Different types of cleanrooms are possible that depend on the quantity
of contaminants present inside the room after filtration. High efficiency particulate
air or HEPA filter is used for contamination removal to the tune of 99.97% that
effectively removes minute dust particles, bacteria or other smaller particulates not
generally visible to the eye. Another type of filter is ultra-low particulate air or ULPA
that is used in commercial filters for the removal of particulate contaminants that are
extremely smaller in size. Both types of filters generate efficiency of 99.97% of
filtration with HEPA possessing the capability of removal of smaller particles of
sizes 0.3 μm, while ULPA can filter particles as small as 0.12 μm. T. Sandle has
discussed in detail about the cleanrooms, isolators and cleanroom technology [27].
International Organization For Standardization (ISO) 14,644–1 states the following
formulae for the cleanroom classification possessing maximum concentration of
particles per class and per particle size [28],
( )2.08
0.1
C N = 10 N
(3)
D
Here, CN is the maximum concentration of particles present per unit volume

(1 m3 ) of airborne particles. These are considered to be equal to or higher in size
in comparison to the particle size under consideration that has been approximated
by rounding-off to the closest whole number upto three significant digits. Here, N
expresses the ISO class number, D represents the particle size in microns and 0.1
is a constant that has been expressed in μm. As per ISO 3, class 1 consists of 1000
particles per m3 of sizes equal to or less than 0.1 μm, 237 particles per m3 of sizes
equal to or less than 0.2 μm, and 102 particles per m3 of sizes equal to or less
than 0.3 μm. Similarly, class 10 is defined by ISO 4 and all the above counts are
multiplied by 10 to get the number of particles per m3 for different sizes of particles.
Similarly, class 100 is defined by ISO 5 and all the above counts in case of class
1 are multiplied by 100 to get the number of particles per m3 for different sizes of
particles. Similarly, class 1000 is defined by ISO 5 and all the above counts in case of
class 1 are multiplied by 1000 to get the number of particles per m3 for different sizes
of particles. Usually, before processing of substrates, they are cleaned by acetone,
methanol or preferably with non-reacting isopropyl alcohol followed by cleaning
with deionized water (DI).
Exposure is the process of production of an imprint on the PR with the help of a
photomask that restricts light in certain parts but letting it through in other parts. The
different modes of exposures are inclusive of contact and proximity, and the projec-
tion types. In the contact mode of exposure, the photosensitive PR spun substrate is
placed and pressed in “contact” mode below the photomask that consists of opaque
chromium patterns, after which the sample is exposed followed by the developing
steps. An important phenomenon is the near-field diffraction after passing through
the interface of PR-photomask deep into the PR so that it causes diminishing contrast
with increasing depth. This is owed to the speedy decline of the highest-order evanes-
cent waves as a function of the increasing distance from the interface. This rapid
decline of the highest-order evanescent waves can be shed-off in parts by applica-
tion of thinner PR. There are technologies of plasmon resonances and lensing films
that are applied for contrast enhancements. The advantages of contact photolithog-
raphy include refraining away from the requirement of complicated projection optics
between the object and its image. Further, it does not suffer from limitations as expe-
rienced by projection optics of capturing only a limited spatially confined spectrum
originating out of the object, and restriction of the resolution limit originating from
the finitely sized final imaging lens and its distance from the plane of the image.
McNab et al. reported the resolution of the contact photolithography to be way ahead
λ/20 periodicity [29]. Drawback associated with contact photolithography is due to
the much smaller dust particles or Si specks entering forcefully into the mask and
causing defects into the substrates as well. In order to avoid these pitfalls, proximity
lithography can be adopted in which a small gap of typically 10–50 μm is introduced,
which in turn, causes diffraction-limited phenomenon on the edges of the features so
that the resolution gets typically limited in the range of 2–5 μm. Contact or proximity
printing is also known as shadow printing and the minimum printable line-width (l m )
is given as follows,
l m = (λg)1/2 (4)
Here, g is the spacing that is constituted after the photomask and substrate are
brought closer to each other including thickness of the PR, and λ is the wavelength of
light radiation that is applied for exposure. Like before, the same conclusion is drawn
from Eq. (3) that spatial separation of l m can be minimized significantly by reduction
of g and λ. Figure 4 given below shows the optics involved in the photolithography
process.
Photolithography can be performed in different modes of (a) contact, (b) proximity
and (c) projection optics as shown below in Fig. 5(a–c).
2.2 E-Beam Lithography (EBL)
In electron beam lithography (EBL), an electron beam scans the thin photosensitive
material layer over a substrate for patterning of very small and precise feature sizes
down to atomic scale [31–33]. E-beam is advantageous for patterning of very small
feature sizes because of much smaller wavelengths of electron beam in contrast to
the wavelength of light used in photolithography. EBL forms a potential alternate
technique for UV photolithography on account of capabilities of higher resolution
112 S. Dwivedi
MASK SUBSTRATE
Space linewidth
Plane of Image
Plane of Object
θ
Optical Axis
Δz
Fig. 4 Schematic of the optical phenomena happening during photolithography process where θ
is the conic half-angle that converges in form of an image at the side of the substrate [30]
enabling smaller feature sizes and the costings involved. Photolithography systems
are diffraction limited due to maximum resolution of the involved optics limited by
the type of wavelength being used for patterning. Resolution enhancement method-
ologies, such as, immersion and phase shifting lithographies, result in increment
of processing times of photomasks, pricing as well as increasing complicacy. In
case of commercial production, the costs involved rose significantly even to few
million dollars according to the node of technology applied for fabrication. In case
of electrons, the de Broglie wavelength can be expressed as,
h 1.23
λ= √ = √ nm (5)
2m E K i net i c E K i net i c
Here, λ is the wavelength of electrons used and EKinetic is the kinetic energy of
electrons forming the beam which is expressed in terms of electron volts (eV).
From λ = hp = m.v h
, the famous de Broglie equation, wavelengths are typically very
small usually of the order of few angstroms with mass of electrons being, m = 9.1 ×
10–31 kg, h is Planck’s constant, and v is the speed. Here, it is assumed that the elec-
trons are moving effectively below the speed of light that is in negation of the special
relativity effects. Electron wavelengths are typically a fraction of the unit angstrom for
a few electron volts of energy imparted. This actually reduces the diffraction-limited
patterning effects very significantly that happens in case of UV photolithography. EBL
systems are highly advanced, such that, they do not require the use of photomasks
and apply a projection system for patterning. Hence, resolutions are limited by the
beam column system and not by the electron wavelengths. Diffraction-limitation by
wavelength of electrons become effective only when very high-resolution systems
possessing very low throughput are required. W.F.R Pease has reported a nice work on
EBL [31]. On the other hand, Pala [32] and Wilson [33] have presented nice theoretical
explanations on EBL.
An EBL system consists of different components of electron sources, electron
lenses, apertures, beam blankers, stigmators, and stage. Electrons are ejected from
(a) Contact Printing (b) Proximity Printing
UV Lamp UV Lamp
Lens
Lens
Mask
Mask
PR PR
Substrate Substrate
(c) Projection Printing

UV Lamp
Lens
Mask
PR
Substrate
Fig. 5 Schematics of different modes of photolithography process that include a contact printing,
b proximity printing and c projection printing. Projection lithography is diffraction limited and is
taken care of by adjustment of the refractive index of the medium, thickness of the PR and the
corresponding gap
the source by either thermionic emission or field emission techniques which rely
on taking out electrons from the cathode either by thermal induced emission or by
application of a large amount of electric field, respectively. Thermionic emission is
generally attained by the use of tungsten (W) or tungsten thoriated filaments and
lanthanum hexaboride (LaB6 ) which is a better source for thermionic emission of
electrons. However, thermionic emission sources are limited by crossover diameter
114 S. Dwivedi
or finite source size causing broader beam energy distribution profiles. Field emission
relies on application of high potentials to surpass the surface potential acting as a
barrier in case of emitter sources. Electron source generates the electrons that should
be possessing brightness or high intensity, small electron beam spot sizes, higher
levels of uniformities & stabilities. Higher brightness or intensity of the electron
beam leads to faster rate of reaction with the organic photosensitive layer. This even-
tually results in shorter times of exposure as well as enhanced throughputs. Reduced
brightness or intensities of the electron beam are needed in case of requirement of
higher resolution or smaller feature sizes. Smaller virtual size of the electron source
generates smaller spot sizes ensuring minimized application of lenses suitable for
attainment of higher resolution and demagnification of the beam column as well.
Electron beams with narrow energy distribution is another important feature in order
to minimize chromatic aberration effects.
Electromagnetic lenses are used in EBL similar to that of the transmission electron
microscope (TEM). Classical optics laws are applied in modulation of the electron
motion under the effect of electromagnetic (EM) waves. Electrons in the form of clas-
sically charged particles are modulated through an external electric field or magnetic
fields. The electric field force is denoted by FE = −eE and the magnetic field force
through FM = −ev × B. Magnetic lenses are usually suitable for application in beam
column because they produce highly reduced aberrations in comparison to electro-
static lenses. Application of a radial magnetic field generates the rotation of electrons
about the optical axis of the electromagnetic lens causing components of velocity in
a tangential direction with reference to the optical axis. The velocity component of
electrons now begins to have an interaction with the magnetic field along the axis.
Electrons start experiencing a force along the direction of optical axis that brings them
in vicinity of the axis. Beam deflectors perform the task of scanning the electron beam
over an area called as scan field over target surfaces. The mechanism of beam deflec-
tion must be highly predictable for fabrication of high precision very small feature
sizes, and hence is required to be perfectly linear along with minimized disruption in
the beam spot size and showing non-hysteretic features. Magnetic deflectors are more
effective in the generation of precise and accurate beam deflection in comparison to
electrostatic beam deflectors. Two types of apertures are blanking and beam-splitting
apertures that cause deflection of the beam at a distance away from the hole of the
aperture, and by setting the beam convergence angle (α) that guides the lens aberra-
tion effects, resolution along with limiting the beam current, respectively. Stigmators
are used to remove astigmatism that generates focusing conditions of different orders
over spatially different positions.
2.3 Lift-Off Process
Lift-off is a typical patterning technique in MEMS used in case of noble metals

or materials that are difficult to be processed by dry etching techniques based on
plasma [34, 35]. It is a technique of micro- or nanostructuring to create patterns of
a specific material or noble metals over the substrate surface by employing a sacri-
ficial layer. García et al. used lift-off and etching technologies to fabricate magnetic
microsensors and compared the two techniques in a critical manner in the exper-
imental framework [34]. Negative PR was applied in case of the lift-off method
followed by patterning before depositing the material. Lift-off of the unwanted parts
of deposited films were removed effectively and highly efficient magnetoimpedance
responses were recorded for the micron-sized geometrical structures. Positive PR
was applied for patterning by the wet-etching method and the magnetoimpedance
response was not even distant comparable to the architectures fabricated by lift-off
technology [34]. Cheung et al. experimentally performed the polydimethylsiloxane
(PDMS)-stamp-supported monolayers in bimetallic configuration by the chemical
lift-off lithography (CLL) technology [35]. This technology makes patterns of self-
assembled monolayers of functional molecules by the application of PDMS stamps.
A schematic of the lift-off process has been shown below in Fig. 6.
Step coverage in the lift-off technique is poor as it involves the application of an
intermediate layer along with a deposition process. Lift-off technique involves (a)
deposition of an intermediate thick layer of oxide (SiO2 ) or PR possessing a mild
reentrant profile followed by patterning, (b) deposition of material that is to be in a
specific patterned structure along with thickness to be only a part of the thickness
(a) Inverse structure formation by patterning

of sacrificial layer
(d) Target material deposition
(b) Intermediate or sacrificial layer deposition
(e) Patterned target material on substrate surface by

washing of the sacrificial layer along with target material
(c) Substrate Cleaning deposited top on of it.
Fig. 6 Schematic representation of lift-off process in MEMS technology with the help of a sacrifi-
cial oxide or photoresist layer. First process, a involves usual RCA cleaning of the substrate surface
so that no oxides remain on the surface, b second part involves the deposition of sacrificial or inter-
mediate layer, c patterning of the sacrificial layer to create inverse structure patterns, d, deposition
of the target material, and e creation of typically shaped & micro- or nanoengineered structures by
washing-off the sacrificial layer followed by deposition of target material
116 S. Dwivedi
of the intermediate layer, any technique that possess poor step-coverage offers an
effective alternate e.g., evaporation, (c) final step is the lift-off of the intermediate
oxide layer or PR that will be possible due to the higher degree of stress formation
in portions possessing poor step-coverages made possible eventually by fracture of
the metallic layer or material layer to generate patterns of the deposited metals or
materials.
Generally, lift-off technique is applied to generate patterns for the formation of
metal interconnects. Multiple sacrificial layers of different types of photosensitive
materials can also be applied that possess the capability to produce architectural
features to protect side-walls of the photosensitive material from getting covered
with the metal during the metal deposition process. Lift-off presents an effective
alternative technique in case of materials that cannot be etched-out directly and may
lead to undesirable consequences on the material layer underneath. Disadvantages
of the lift-off technique are retention, ears formation and redeposition. Retention is a
problem in lift-off so that undesirable metal layer sections adhere to the substrate so
that complete lift-off of the layer is prevented. Another reason is the non-dissolution
of the photoresist in the dissolution solvent so that it becomes difficult to perform
lift-off properly. “Ear” formation takes place when the depositing metal covers the
side-walls of the photoresist and points upwards in the standing instruction. The
possibility of these “ear structures” falling over the substrate surface equally exist
forming undesirable structures over substrate surfaces and may cause undesirable
connections. The third one is redeposition that entails re-attachment of the metal
particles in a random manner at undesirable locations during the process of lift-off.
2.4 Etching Technology: Wet and Dry Etching Techniques
2.4.1 Wet or Chemical Etching
Wet or chemical etching is applied on a large scale in the semiconductor and micro-
electronics manufacturing industry [13, 14, 36]. Chemical etching is a high potential
technique for the removal of blanket depositions of oxides, nitrides, metals and even
III-V compound semiconductors. Here, blanket deposition means the depositions of
materials that are performed over the whole substrate surface area. The substrates
are immersed in the etchant solution under application of suitable temperature and
mechanically controlled agitation to ensure uniform and faster etching rates. Etching
rate denotes the amount of film peeled-off in a single unit of time. For optimization of
the etching recipe, substrates consisting of layers of desired materials are immersed
in the etchant solution and time is measured with a stop watch. The amount of
minimum time consumed to peel-off a particular thickness of the deposited film
attests to the etching rate, i.e., thickness etched per minute. In the wet chemical
etching process, diffusion of the reactants takes place in a direction pointing towards
the substrate surface followed by chemical reactions, and eventually removal of the
substrate-based materials happens, such as, oxides or the deposited thin film material
gets peeled-off by the process of diffusion. Chemical or wet etching produces etch
rates that are generally isotropic which means that the etch rates along the plane,
lateral etch rates as well as the etch rates perpendicular to the plane or vertical etch
rates remain the same. In the microelectronics manufacturing production lines, an
essential requirement is the uniformity of the etching rates to ensure consistency of
removal across a series of wafers and in successive automated runs as well. Etch rate
uniformity can be expressed as follows,
Maximum Etch Rate − Minimum Ech Rate

Etch Rate Uniformity (%) = × 100%
Maximum Etch Rate + Minimum Etch Rate
Orientation-dependent etching can be performed using chemical etchants in which

case specifically oriented crystal planes of single crystal Si can be etched preferably
or comfortably. For example, the number of available bonds in a single unit of area
for (110) and (100) crystal planes of Si are larger than in the case of (111) oriented
single crystal Si. Hence, etch rate is faster for (110) and (100) oriented crystal planes
of singly crystalline Si than for (111) oriented crystal planes based single crystals
of Si. The lowest bond density happens in case of (111) oriented crystal planes and
hence the slowest etch rate is obtained in this case. Etch rates for Si usually depend
on the bond densities that are in the order of 1: 0.707: 0.557 for orientations of
(100):(110):(111) of crystal planes. Such etching reactions do not produce sufficient
amount of heat on account of being slow and, hence, are reaction-rate limited. The
slowest etch rates for (111) orientations are owed to the faster oxidizing capabilities
of (111) orientation at reduced temperatures that are generally encountered during
the wet chemical etching process typically in the temperature range of 0–100 °C.
Covering of surfaces with oxide layers facilitates hindering of the dissolution process
in the etchant solution, thereby generating slower etch rates. Si etching is usually
done by oxidation with HNO3 to form SiO2 followed by etching with hydroflu-
oric or HF acid so that it dissolves SiO2 to form H2 SiF6 . For etching of SiO2 ,
generally HF solution is mixed with ammonium fluoride (NH4 F) that alters the pH
value to form a buffer HF solution or BHF. This has the effect of restoration of the
depleting fluoride (F− ) ions to ensure an overall highly stable etching mechanism.
Important parameters are etchant concentration, etchant solution, temperature, agita-
tion including microstructure, porous nature, density and impurities and, in addition,
defects in oxide that have a bearing on the etch rate. In case of polysilicon, much
faster etching rates can be achieved because of the occurrence of grain-boundaries
in the polycrystalline materials. In case of Si3 N4 layers, a thin SiO2 layer is usually
deposited over it followed by PR coating for effective patterning by etching in the
boiling H3 PO4 solution.
Chemical etching suffers from the major drawback of undercut formation in the
layer just below the masking layer that generates loss of resolution in geometrical
patterns to be produced as a result of etching. Ideally, for better patterns having
resolution much smaller in comparison to the thickness of the film, dry plasma-based
etching must be applied to ensure the anisotropic etching pattern. Isotropic etching
is favourable in cases when the thickness of the film is lesser than the resolution of
118 S. Dwivedi
the patterns. This can be expressed more precisely with the following condition as
given below in the form of a mathematical equation,
l
Af ≡ 1 − (6)
hf
Here, Af is the degree of anisotropy, l is the lateral length covered as a result of

etching below the PR mask layer and hf denotes the thickness of the layer material.
For anisotropic etching, the conditional equality of 1 ≥ Af > 0 holds well.
2.4.2 Dry Etching Technique
Dry etching techniques are basically plasma-assisted etching techniques that are
actually very important in ultra-large-scale integration (ULSI) chip technology
for transfer of patterns over the substrate surfaces with high-fidelity [13, 14, 36].
Different variants of dry etching techniques include reactive ion etching (RIE),
plasma-assisted etching, reactive ion beam etching, magnetically enhanced reaction
ion etching or magnetically RIE, and high-density plasma etching or HDP etching.
In the dry etching technique, material removal is performed by physical bombard-
ment of the substrate surfaces with chemically inert mobile projectile atoms or ions
that are highly mobilized by gas discharges. This results in the transfer of momentum
to the atoms of the deposited layer or of substrate surfaces in elastic collisions so that
the imparted energy starts exceeding the binding energy of atoms of the surfaces.
The maximum transferrable energy (Em ) from the projectile atoms or ions of mass
M1 and energy E0 to the substrate surface or surface layers of mass M2 is expressed
as follows,
[ ]
4M 1 M 2
Em = E0 (7)
(M 1 + M 2 )2
The above equation shows that it is advisable to use heavy inert gases, such as,
Ar as projectiles for the sputtering process to knock out the material atoms that are
to be etched out. The process is performed at relatively high energies (usually below
~2 keV) so that atoms or ions are able to cause considerable damage in the surface
layers as a result of penetration quite deeply inside the surfaces. At rather higher
energies, all projectiles have the tendency to escape and are not able to reach the
surfaces appropriately causing considerable losses in energy underneath the surface.
Consequently, the yield of sputtering is not dependent on the energy imparted to
the ions for most of the materials. Angle of incidence of the projectiles or atoms
and ions onto the surfaces is also of paramount importance. For smaller angles of
projections, penetration depth increases that generates poor dislodgement of atoms
from the surfaces. Sputtering yield is found to increase with increment in the angle
of incidence in case of polycrystalline targets with the maximum obtainable yield
happening at an angle of incidence of 60°. Dry etching techniques are particularly
useful for multi-layered thin film stacks ensuring minimized undercut formation in
all the subsequent layers to be etched. A usual drawback is the extremely difficult
removal of photoresist layers by chemical methods after ion-based etching.
Plasma etching relies on plasma formation by arcing the inert gas molecules kept
at low pressures in a vacuum chamber. Two variants of plasma etching methodolo-
gies are physical etch and chemical etches. Physical etching methodology is typified
by sputter etching in which the positive ions formed out of inert gas molecules strike
the surfaces with high kinetic energies to knock out the material to be etched. Small
amounts of negative ions are also formed in the plasma that do not possess suffi-
cient kinetic energy to sputter out the material effectively and do not even reach
closer to the surfaces. Thus, the physical method completely relies on sputtering the
material out for etching purposes by heavy energetic bombardment of ions on the
surfaces. The second variant of plasma etching relies on the generation of neutrally
reactive species in the plasma that undergo chemical reaction with the materials on
surfaces to be etched out and the formation of volatile products takes place. An advan-
tage of this technique is the reduced damage as a result of heavy ion-bombardment
caused damages. However, it yields isotropic etch profiles of the structures. A justi-
fied combination of physical and chemical etching methods produces anisotropi-
cally etched structures, significantly controlled damages due to ion-bombardment
over surfaces along with high selectivity. Reactive ion etching or RIE is a variant
of plasma-based etching in which reactive ions are generated and etching of the
materials takes place which is a chemical process. In RIE, the wafer is placed on a
substrate holder that forms the RF capacitively coupled electrode in the configuration
of a parallel-plate integrated diode system. In this way, the substrate placed on the
grounded bottom electrode experiences a negative self-bias at a lower operating pres-
sure of typically less than 500 mTorr leading to heavy energetic ion-bombardments.
Etch selectivity in RIE is usually improved by inducting an appropriate etch chem-
istry. Triode-configuration RIE systems are also possible in which ionic transport is
differentiated from the plasma production process. Etch selectivity in such systems
is maintained by control of the ion energy by application of a bias applied separately
to the electrode holding the substrates.
Dry chemical etching processes involves the use of gaseous chemical reagents
for the purpose of etching. Gaseous reagents SF6 and HCl have been employed
for etching of Si, while AsCl3 and HCl gases have been used for dry chemical
etching of GaAs. Highly selective gaseous etching of SiO2 can be performed with
interhalogenic compounds of BrF3 , ClF3 , BrF5 and IF5 with an etch rate of 0.5–
5 μm.min−1 in an isotropic pattern. A combination of UV radiation and ozone
(O3 ) gas has been used for the stripping of PR layers with a high etching rate of
1400 Å.min−1 at the processing temperature of 300 °C. Usually reactive gaseous
reagents are fed into the chamber having pressures in the range of 0.001 to 10 Torr
under a glow discharge for dry chemical etching. Free electrons receive energy from
the glow discharge that is lost in successive collisions with gas molecules by both
elastic and inelastic collisions. Inelastic collisions dominate the mechanism of energy
transfer involving much larger magnitudes that is accompanied in the process of
excitation of gaseous species. As a result, a plasma chemistry is involved that guides
120 S. Dwivedi
the reactions taking place in partially-ionized gases that consist of electrons, ions
and free radicals. House et al. has nicely pointed-out about anisotropic etching in the
article on dry etching [37]. Morozov et al. reported about the fabrication of shape-
anisotropic vertically aligned Si nanostructures by the dry etching technique [38].
Ganjian et al. patterned biofunctional titanium nanostructures by the reactive ion
etching technique [39].
Exposed photoresist removal is usually performed by plasma-ashing technique
which is a dry chemical etching technique. In this technique, substrates consisting of
patterned and exposed photoresist layers are placed inside the chamber in presence
of a plasma formed out of arcing the oxygen (O2 ) gaseous molecules.
e + O 2 → 2 O − + e−
→ O + O + e−
Free radicals induced oxidation or burning of the PR molecules takes place at

temperatures of 40–50 °C. Different parameters controlling the process include
pressure of the O2 gas, RF power, temperature of the exposed substrate, number
of substrates and flow rate of the gas inside the chamber. Several closely spaced
substrates can be cleaned off from the exposed PRs in a single run. The process is
diffusion-controlled so that the etching rate depends on the diameter of the substrates,
spacing between them, in addition to the etching rate being slower in the middle
while faster at the perimeters of the substrates. O2 plasma has actually no or very
little effect on the surfaces holding the exposed PRs, such as, Si substrate, GaAs
substrates, Au etc. This is the most positive factor of this technique allowing to use
it till complete removal of PR happens without any considerations of substrate or
underlayer damages. In MEMS/NEMS, plasma-ashing is generally required very
often for the removal of the exposed PR [36].
2.5 Stereolithography
Stereolithography is a versatile tool for additive manufacturing or 3D printing of

highly precise complex structures with reasonable expenditure. Stereolithography
has been utilized for the fabrication of a wide variety of applications including
microfluidic devices for biodevices, medical implants, sensors as well as energy
storage devices. Stereolithographic process takes place in a container that consists
of the oligomers, liquid monomers and photoinitiators so that a photopolymeriza-
tion reaction induced by UV photons takes place. Epoxy-, acrylate- and vinyl-based
polymers are mostly used as photocurable resins in stereolithography. Epoxy-based
polymer materials have the tendency to get cured even when light radiation from the
laser does not hit the material. Acrylates, on the other hand, tend to stop curing on
cessation of UV light onto the material. In the stereolithography process, first of all,
a 3D image of the desired structure is designed on a computer. This is transformed

into a set of sliced hundreds or thousands of layers of very thin cross-sections. A
large container or bath has an attachment for moving upwards along with photocur-
able resins. A movable attachment further supports the portion under patterning by
a thickness of the order that is normally ~0.1 mm per unit layer. Laser beam scans
the unit layer of liquid solution contained into the bath as shown in Fig. 7. UV light
induced photopolymerization takes place at the place where it hits the surface so that
the photopolymer becomes hardened. The construction of the system consists of a
platform that works as an attachment situated beneath the surface of the bath. UV
laser source of low power orientation strikes the layer solidifying it from the middle
cross-section but does not have an effect on other part that is in excess. UV laser
is guided to move in x–y directions with the help of a galvanometer scanner that is
subsequently followed by the elevator bringing down the platform to the liquid solu-
tion. Laser then starts scanning from the left side and the solidified layer is coated
with the liquid followed by tracing of the second layer by the laser just upside next
to this layer. This process keeps on repeating till the final prototype is fabricated.
The solidified part is then eliminated from the bath followed by its cleaning to take
out the excess liquid solution. In the final step, the model is kept in an ultraviolet
oven for curing it completely. All these steps involved in stereolithography based
fabrication have been shown below in Fig. 7 in the form of a schematic. Huang et al.
has reviewed the stereolithography process with full technical knowhow in a detailed
manner [40].
3 Paper Micro- and Nanofluidics
Paper microfluidics is the technology of fabrication of micro- and nanochannels on

papers, paper-like materials or porous membranes that can absorb or drop-off liquid
by capillary action [42–45]. Nishat et al. reviewed the technology and utility of
paper microfluidics for the fabrication of devices in an easier manner [42]. Reboud
et al. demonstrated the paper microfluidics technology that integrates vertical flow
specimen-processing stages inculcating paper-folding for the preparation of whole-
blood specimen [43]. This was combined with an identical thermal amplification
along with a lateral flow detection. The technology provided for the quick and
efficient malaria detection that combined origami for enabling sensitive and multi-
plexed assays. Noviana et al. has critically reviewed microfluidic paper-based analyt-
ical devices (μPADs) [44]. Ghosh et al. reports on the fabrication of laser-printed
microfluidic paper-based analytical devices or LP-μPADs for point-of-care applica-
tions [45]. Paper microfluidics can be employed for many applications out of which
the most important is the development of point-of-care health diagnostic devices.
This technology has helped to do away with cumbersome, time-consuming and costly
disease diagnosis analytical procedures. In this technology, hydrophobic boundaries
are defined over paper-like substrates for fabrication of hydrophilic channels and
122 S. Dwivedi
Solidified Resin
Laser
Laser
Resin
(a) Resin layer being solidified (b) Initial layer tracing by (c) Post-initial layer tracing by UV
on a platform UV laser selectivity laser tracing from the left side
Supportive Layer
Laser
Resin
(d) Repeated UV laser scans to (e) Repeated UV laser scans to (f) Finally obtained patterns after
build the bulk layer build the bulk layer removal of support structures
Fig. 7 Schematic of the stereolithography process [41]
regions. Very small volumes of fluids in the usual range of 10–6 –10–9 L are trans-
ported across these hydrophilic channels constituted by patterning of hydrophobic
barriers. In addition, these devices are portable, possess the ability to carry out multi-
plexed assays, are generally cost-effective and do not require cleanroom facilities for
fabrication. Cellulose fibres get pressed in the form of an interlaced network to form
the paper and the corresponding capillary action. Some of the common materials for
paper microfluidics is the cellulose-based paper, litmus paper, filter paper, nitrocel-
lulose membranes and chromatography paper. Cellulose fibres possess a structural
configuration in which large density of −OH groups and some of the −COOH
functional groups are arranged to form the hydrophilic paper. Nitrating the cellulose,
meaning replacement of −OH groups by −NO3 groups, produces the nitrocellu-
lose polymer which is then casted in the form of membranes possessing controlled
porosity. Such nitrated cellulose membranes have high degree of affinity to bind
proteins finding sound applications in western blots and lateral-flow assays. Cellu-
lose based papers are used mostly in the fabrication of microPADs and dipsticks.
Lateral-flow assays consist of multiple porous membranes that are utilized for home
pregnancy tests, detection of diseases, pollutants, toxins, pathogens, and are used
even for the detection of Covid-19.
4 Thin Film Deposition Techniques
Thin film is the first and the foremost requirement in the state-of-the-art device fabri-
cation technology, be it the CMOS scaling process or MEMS/NEMS. Thin films are
usually defined as the layers having thickness less than 1 μm in a 2D geometry. In
the modern times, state-of-the-art deposition techniques are available that possess
the capability to deposit layers down to near atomic level thicknesses. The deposited
thin films may be epitaxial, polycrystalline or even amorphous depending on the
deposition parameters and the nucleating surfaces present over the substrates or the
substrate surfaces itself. Mostly employed deposition techniques in the clean room
fabrication are the chemical vapor deposition (CVD) and physical vapor deposi-
tion (PVD) techniques that include sputtering, and possibly other techniques. For
the simplistic MEMS/NEMS devices, metal sputter deposition, e-beam evaporation
and thermal evaporation are sufficient to deposit normal metal layers. However, in
case of complex material deposition at the commercial scale, CVD and sputtering
are preferred over other techniques because of certain advantages attached to these
techniques. PVD is a collective term used to define the techniques that involve vapor-
ization of the solid-state material and subsequent condensation over top surfaces of
the substrates held under the atmosphere of vacuum conditions. PVD can be more
specifically defined as an atomistic procedure of deposition in which a wafer is held
in the substrate-holder under usually high vacuum conditions or inert gases are flown
at low rates to maintain low pressures in the vacuum chamber or even generation of a
plasma or plasma-enhanced atmosphere [46–48]. Subsequently, the source material
is broken down to eject atoms, molecules or even generation of plasma & plume
can happen followed by cooling down of these energetically forced species over the
substrate surfaces, and hence the nucleation. Reactive deposition happens when a
reactive gas is introduced into the vacuum chamber for deposition. Abegunde et al.
reviewed the different thin film deposition techniques in a critical manner [46]. Ross-
nagel et al. discussed about the various PVD based thin film fabrication techniques in
detail [47]. In a previous work on fabrication of solar cells, I have presented a detailed
account on sputtering and thermal evaporation techniques for thin film deposition in
detail [48]. In a the previous work pertaining to spintronics, deposition techniques
have been discussed in a special context to this topic [49]. In my previous works,
pulsed laser deposition (PLD) has been used for the deposition of stable ferromag-
netic chromium oxide thin films for enhanced room temperature magnetoresistance
(MR) and for insulating TiO2 thin films [50–53].
Different major steps involved in the PVD process are outlined below,
(i) All the target solid-state materials in the form of a solid disk of desired thickness
are converted to vapor phase by evaporation as a result of temperature or e-
beam exposure or ejection of materials by striking of the target surfaces by
plasma in sputtering. Thus, the first step involves the formation of vapor phase
of the target material that is to be deposited.
(ii) Vapor-phase species are transported towards the substrate under streamlined
molecular flow environment. Temperature inside the chamber has a specific
124 S. Dwivedi
bearing over these produced vapor-phase species, and thermal scattering events
can also happen. During the streamlined molecular transport of the vapor-
phase material to the substrate, there will happen huge number of collisions
if the partial pressure of gases or material vapours is high enough. Thus, the
molecular transport from the source material towards the substrate surface
forms the second step.
(iii) Molecular transport towards the substrate enforces nucleation over the surfaces
in a manner controlled by distance between source and the substrate, tempera-
ture, reactive environment and surface modified substrates. Specific crystalline
substrate surfaces can nucleate epitaxial, polycrystalline or even amorphous
phase films that can eventually be controlled by changed thermodynamics by
changes in temperature and possibly other parameters. Less mobile vapor-phase
species may form an amorphous phase even over crystalline substrates. Simi-
larly, substrate temperature can change the thermodynamics of nucleation of
crystallites that alters surface mobilities of the depositing atoms and molecules.
Substrate temperature is of paramount importance in the film formation physics.
Thus, nucleation and thin film growth is the third and final step of the thin film
growth.
In the current chapter, the focus is to shed light on sputtering, thermal and e-beam
evaporation PVD processes in short.
4.1 Sputtering
Sputtering involves ejection of atoms and molecules from the target surfaces by
atomic level collision induced cascades through ions present in the plasma formed by
high electric discharge of inert gases [46, 49]. This methodology of ejection of atoms
and molecules by atomic level collisions is known as “sputtering” or knocking out of
the material atoms. Highly energetic gaseous ions from the plasma are accelerated
towards the substrate surface that knock out the material by transfer of momentum.
This is a completely different process from thermal evaporation whereby atoms
and molecules are knocked out due to heavy strike of ions on the surfaces along
with shorter target-to-substrate distance. Different variants of sputtering include
magnetron sputtering, reactive sputtering, radiofrequency (RF) or diode or cathode
sputtering and bias sputtering. DC sputtering is based on the twin planar electrodes
whereby the source disk forms the cathode and substrate forms the anode. An inert
gas is flown inside the vacuum chamber to form the plasma of energetic ions to
initiate atomic level collisions at the substrate surface. Usually, argon (Ar, at. wt. =
39.95) is implemented for the purpose because of its higher mass as compared to
other inert gases of neon (Ne, at. wt. = 20.18) and helium (He, at. wt. = 2). The
reason is that Ar can impact the substrate surface more energetically in comparison
to Ne and He (Fig. 8).
(a) Substrate [Anode]

(b) Vacuum Chamber
Ar Inlet Vacuum Pump
Plasma Plasma
Molecular Transport
Cathode Shield
Target [Cathode]
Ar+
Substrates [Anode]
Heater
Ar Gas Inlet
Target [Cathode]
High Voltage To Vacuum Pump
Fig. 8 a & b Schematics of a sputtering system portraying the substrate placed on an anode and
the target placed over the cathode, formation of plasma ions (Ar+ ) inside the vacuum chamber that
strikes the target surface constituting the molecular transport towards the substrate
A direct current or DC voltage maintains the glow discharge that is applied

between the source and the substrate. Due to sustained glow discharges or plasma
formation, atoms and molecules are sputtered out of the substrate surface and start
condensing on the substrate surface to enforce nucleation and growth of a thin film.
Usually, the source disk material is a metal or a conducting material that possesses
features to sustain glow discharge or current flow between the metallic electrodes.
Reactive sputtering involves a process in which a reactive gas, e.g., O2 or N2 is
flown into the vacuum chamber in a controlled manner so that they react with the
atoms or molecules ejecting out of the target material that leads to the formation of
a compound film over the substrate surface. An enhancement in the amount of the
reactive gas transforms the upper layer of the target surface from a single phase to
the compound type of phase formation that may cause alterations in the property of
the material. Only Ar gas can be used for the purpose or in combination with the
ionized non-inert gas or reactive gas or introduction of only the ionized non-reactive
or reactive gas in the deposition chamber can also be performed. Stoichiometry of the
compound film can be controlled by the regulation of percentage of the reactive gas to
be introduced into the deposition chamber so that the produced thin film is different
from the target material. In radiofrequency (RF) sputtering, current is applied at
radio frequencies with alternating potentials to avoid piling of charges. Alternating
current or periodically varying current thus mitigates the effect of accumulation of
charges in each phase of the cycle when it is made to flow continuously along a
single direction. Electrons are driven towards the target placed on the cathode during
the positive cycle of the applied voltage resulting in generation of negative bias. On
the other hand, during the negative cycles, knocking of target surfaces by plasma
ions continues to eject atoms & molecules at an operational frequency of 13.56 MHz
of RF. Accumulation of charges over specific target surfaces is a serious issue that
can generate sparkling in the plasma resulting in droplets formation over produced
thin films, and hence degrading the quality. Further, this can cease the sputtering of
126 S. Dwivedi
atoms & molecules from the target surfaces that eventually terminates the process.
Thus, target surfaces are cleaned-up in each cycle in the RF sputtering so that charge
build-up is protected that diminishes the unwanted arcing or sparkling. An advan-
tage of RF sputtering is that it facilitates stable and sustained plasma formation at
lower pressures of 1 mTorr to 15 mTorr resulting in highly reduced collisions of
Ar gas ions so that a highly streamlined molecular transport of the target material
happens towards the substrate. RF sputtering is also helpful in minimization of the
formation of “race-track erosion” over the target surfaces. Due to the ac character of
the RF discharge, height of depth along with the width of the race-track happens to
be highly reduced in dimensions in addition to the minimized confinement of elec-
trons in the applied magnetic field. This results in larger spread-outs of the plasma
confined material so that shallower but larger & wider plasma spreads happen to
produce better and highly uniform coatings.
Magnetron sputtering makes use of the magnetic field to accelerate the rate of
ionization delivered by the current to the target material that eventually results in
highly improved deposition rates. Another advantage of magnetron sputtering is that
it restricts secondary electrons generally ejected from the target surface due to heavy
ion-striking in the neighbourhood of the target. There is also another benefit attached
to this specific technique of magnetron sputtering that it allows sputtering at a reduced
pressure and voltages of 100 Pa and −500 V, respectively, in comparison to 10 Pa
and 2–3 kV, respectively for the normal sputtering procedure. The two variants of
magnetron sputtering are balanced and unbalanced magnetron sputtering. In the first
variant, balanced magnetron sputtering, the plasma largely remains confined to the
target region. On the other hand, in unbalanced magnetron sputtering, magnetic field
lines are directed in parts towards the substrate in addition to the closed field lines.
Kelly et al. has presented a nice review on magnetron sputtering and has discussed
the details of the same [54]. Figure 9 given below shows the schematic of a DC
magnetron sputtering mechanism and subsequent plasma-based knock-out of atoms
or molecules from the target material to form thin deposition over substrates [55].
Pulsed magnetron sputtering or PMS is another variant of magnetron sputtering
that is a short-pulse of magnetron discharge tuned to the orders of microseconds (μs)
in a cycle of low duty operating in the medium frequency range of 10–200 kHz as
shown in Fig. 10 [56]. In PMS, short or intermittent pulsing is used rather than the
continuous mode of magnetic field applied in conventional magnetron sputtering.
Collision of positive ions develops positive charges over the target surfaces resulting
in a reduced drift of positive ions in a direction pointing towards the target. The two
sub-variants of PMS are the unipolar and bipolar PMS.
In the unipolar PMS, pulsed voltage is applied to the target that lies between the
standard operating voltage and the ground. Power waveform experiences positive
voltages at a particular operational frequency in the unipolar PMS to clean target
surfaces including the removal of charges accumulated over dielectric surfaces.
In the bipolar PMS, the voltage applied to the target is switched to positive voltages
during the off-cycle of the intermittent pulse duration reflecting a reverse nature. Two
pulses that are 180° out-of-phase are applied so that the magnetron can be swapped
from the cathode to the anode and vice-a-versa for the mitigating effect of charge
DC Voltage
Source
Magnetron Cathode
Array of Magnets
S N S
Target For Magnetic Field ( H )
Sputtering
Plasma Zone
Ionized Ar Gas
Plasma Zone
Plasma Zone
Thin Film Substrate
Ar Gas Inlet
Anode
Vacuum Pump
Fig. 9 Schematic of a DC magnetron sputtering system portraying substrate placed on an anode

and target at the cathode [55]. A magnetic assembly is at the core of magnetron cathode providing
acceleration to the charged ions. A high-density plasma of Ar gas (marked with crossed circles)
takes place between the magnetron cathode and anode so that atoms and the molecules (shown in
form of yellowish cubes) are ejected at a faster pace from the target surface at the cathode. Molecular
transport towards the substrate takes place instantly so that nucleation starts happening and thin
film growth is initiated
accumulation over dielectric surfaces. Super-dense plasma is created due to this

specific short-pulsing process that results in the deposition of highly uniform thin
films. Advantages associated with PMS as compared to conventional magnetron
sputtering are the deposition of smoother layers even over irregular and complicated
substrate surfaces, removal arc event attached to the reactive magnetron sputtering
and exclusion of low rates of deposition as obtained in RF sputtering in addition to the
target poisoning. Duplex sputtering is yet another variant of sputtering that combines
two or more deposition techniques for the production of functional materials of high
grading.
Power, pulse frequency, pulse duration including pressure are the common param-
eters that determine plasma formation process, time period and sustainability of the
plasma as well as its stabilization. The PMS system can be operated in voltage &
current modes.
128 S. Dwivedi
Discharging Dielectric Thin Films
Sputter Deposition of Dielectric Materials Thin Films

Time
0
Off-Cycle or Reverse Cycle

Voltage
τrev τOn τCycle

On-Cycle
Charge Accumulation Over Dielectric Surfaces
Fig. 10 Schematic of voltage as a function of time (τ ) in a pulsed magnetron sputtering or PMS

system for deposition of dielectric thin films showing off- and on-cycles during the process [56].
It reflects accumulation of charges over sputtered dielectric surfaces during the on-cycle and
discharging the surfaces during off-cycle. Off-cycle is also called as the “reverse” cycle (τ rev )
during which polarity of the applied voltage inverts from negative to positive (typically 20 V) with
smaller amplitudes. A negative voltage of a few hundred orders is applied to the target surface when
a power is applied and this constitutes the “on-time (τ On )” of the process. During the on-cycle,
dielectric surfaces are charged and are discharged during the off-cycle. The involved physics is that
the on-cycle must be short in timescales so that the accumulated charges over dielectric surfaces
do not result in a luminous discharge in this time period. On the contrary, off-cycle should be of
sufficient timescales so that the collected charges can be cordoned-off completely. This helps in the
prevention of accrual of charges in the subsequent repetitive on- and off-cycles
Shorter pulse durations but higher frequencies favour plasma accumulation to

operate PMS in the constant voltage mode. In contrast, at longer pulse durations
but lower frequencies, PMS operates in the constant current mode so that a high
voltage accelerates the plasma ions highly energetically towards the target surfaces
to reconfigure the current as well as the rate of deposition.
E-beam and thermal evaporation are other conventional techniques that are
frequently used in MEMS/NEMS for metal deposition of nanometres thickness [46,
49]. E-beam deposited thin films are more uniform, congruent, continuous in a well-
connected morphology in contrast to the thin films deposited by thermal evaporation.
E-beam evaporation is a deposition technique in which an e-beam scans the surface
of the material to impart a huge amount of energy onto the material’s surface of which
thin films are to be deposited so that it sublimates without undergoing melting. The
solid material is placed in a crucible which is maintained at a lower temperature using
a water-cooling circuit. This protects the diffusion of impurities from the crucible
into the material that is eventually evaporated in the crucible for thin film formation.
Crucibles should be restricted for the sublimation of one type of material only to
avoid contamination. For example, if an e-beam system is used for gold (Au) depo-
sition for metallization in MEMS/NEMS, the crucible and hence the system will
be Au-contaminated for the deposition of other materials. Hence, Au-contaminated
e-beam system can be used only for the Au-interconnects, contact pads or thin films
in MEMS/NEMS or in CMOS technology. Contamination-free crucibles are highly
costly and they can be used in a specific range of temperature only. For example,
graphite-made crucibles can be used for e-beam sublimation at very high tempera-
tures, but however, carbon may enter the thin films in ppm or ppb or even in few
thousands range of concentration of the depositing material. An added advantage
of e-beam evaporation is that the different source materials can be introduced into
the path of the scanning electrons inside the vacuum chamber to generate multiple
thin films in a single point of time. The disadvantages associated with e-beam evap-
oration include difficulty in controlled cleaning of source surfaces, potential X-ray
based damages and rather difficult improvement of the step-coverage. In contrast to
a thermal evaporator utilizing temperature based melting and subsequent evapora-
tion for thin film deposition, e-beam evaporator can induct higher temperatures for
material sublimation so as to ensure significantly higher rates of deposition, refrac-
tory materials (W, Ta etc.) and high temperature materials. Thermal evaporation
is rather an old technique with lots of literature already available and hence I will
restrict myself to e-beam evaporation only. Details about the thermal evaporation are
available in my another chapter on “Spintronics: The Realm of Nanotechnology”
[49]. In other report, I have outlined the importance of an important variant of PVD,
the pulsed laser deposition (PLD) technique, to deposit even phases as metastable
as half-metallic ferromagnetic CrO2 for room temperature spintronic applications
[50–53].
4.2 Chemical Vapour Deposition (CVD)
Chemical vapor deposition or CVD is a technique for the development of solid thin
films or coating over various substrates in semiconductor materials process tech-
nology. Creighton & Ho have pointed out that, mostly, it is used for the fabrication of
high quality, congruous thin films of well-connected granular configuration over large
area silicon (Si) substrates [55]. It is preferably not used for metallization purposes
for the fabrication of interconnects and metal contact pads for which sputtering, and
even e-beam & thermal evaporation techniques are used widely. In a previous work on
nanoelectronics, field effect transistors including metal oxide field effect transistors
(MOSFETs) have been discussed including metallization of contact pads and inter-
connects [57]. CVD consists of a process flow chamber in which precursor gases are
made to flow over materials placed at a desired temperature so that chemical reactions
take place to deposit thin films over the substrate surfaces. The resulting chemical
by-products are vented out of the chamber in addition to the process gas left unreacted
after the deposition. Control of various parameters including tailoring of pressures
from sub-torr level total pressures to pressures higher than the atmospheric pressures,
130 S. Dwivedi
change of temperatures and cold-wall & hot-wall reactors that provide wider func-
tionality to the CVD process. Various types of CVD processes are thermal CVD,
hot-wire CVD, atmospheric CVD, metal–organic CVD (MOCVD), organo-metallic
CVD, organometallic vapour phase epitaxy (OMVPE) and metalorganic vapour
phase epitaxy (MOVPE) [46]. In the enhanced CVD processes, plasmas, photons,
ions, combustion reactions, lasers and even hot filaments are applied for enhance-
ment of the deposition rates and or lowering temperatures of deposition. Advantages
of CVD are inclusive of deposition of thin films that are usually conformal to higher
orders. Conformal means the deposited films have a thickness on the sidewalls that
is quite comparable to the film thickness over the top side. A specific advantage
of CVD is the deposition of thin films of very high purity that emerge out of the
filtration techniques to remove impurities including elimination of a high vacuum
environment and line-of-sight between the source and the substrate similar to sput-
tering, & evaporation techniques, and eventually the possibility of deposition of a
wide variety of materials. Disadvantages of CVD are inclusive of non-volatility of
precursors at or near-room temperatures typically, toxicity of chemical precursors
including by-products, deposition of thin films at higher temperatures that produce
stress in thin films leading to mechanical instabilities, and higher costs of precur-
sors specially in case of metal–organic precursor materials. CVD plays a crucial
role in the fabrication of MEMS devices since most of such devices are fabricated
from polysilicon films deposited over Si substrates consisting of intermediate sacri-
ficial SiO2 layers or masks that are subsequently removed by the etching process to
expose the devices [58]. Polycrystalline Si and oxide layers of high quality can be
fabricated using CVD or plasma-enhanced CVD (PECVD) so as to establish the
device structure in a direction perpendicular to Si substrate [59]. On the other hand,
various other steps involving lithographic & etching processes define device struc-
ture in rest of the two directions (X and Y). Current fabrication technology has aims
to integrate microelectronics manufacturing with MEMS processing on a single unit
of the substrate for enhanced multifunctional devices at the micro- and nanometre
scale. Epitaxial heterostructures consist of 2D quantum wells and superlattices that
are made-up of 1–10 nm thick strained epitaxial layers. Additionally, vertical cavity
surface emitting laser (VCSEL) consists of mirror stacks that are made-up of several
alternating semiconductor layers of typical thickness in the range of 50–100 nm with
a precision of more than 1 nm. OMVPE and atomic layer epitaxy (ALE) are the
techniques that perform the task efficiently to the orders of subnanometer scale.
Hot wall reactors-based CVD consists of a chamber consisting of an enclosure of
a furnace that induces temperature-based heating effects inside the chamber. After
setting of temperatures to the desired levels, reactive gases are flown inside the
chamber at lower pressures of the orders of torrs. Reactors can be of large sizes for
large-area coating or may consist of shelves for coating of several parts in a single
point of time. These reactors have the capacity to process large-batches of closely
spaced Si substrates, generally over hundred, for semiconductor processing simulta-
neously. Usually, uniform substrate temperatures are generated without any thermal
gradient that drives the formation of uniformly thick coatings. Hot wall reactors start
possessing thicker coatings from inside on successive depositions requiring extensive
cleaning procedures. In the cold wall reactors, walls are made to cool and substrate
is kept at a suitable temperature. The walls are made-up of a quartz material with
a chiller attached for water-cooling. Si or compound semiconductor substrates are
placed on a rotating holder possessing a system for resistive heating from the bottom
side. Relatively higher pressures are used in a cold-wall reactor along with a carrier
gas flown into the reactor consisting of diluted precursors. In contrast to hot wall
reactors, cold wall reactors do not suffer from the disadvantage of higher levels of
cleaning due to lesser deposition on its walls and operational power of highly reduced
energies including imposition of lesser thermal stresses due to substrates undergoing
quicker heating and cooling cycles. Drawbacks attached to this technique are inclu-
sive of non-uniformly deposited thin films because of generation of thermal gradients
on the substrate, thermal stress imposition because of quicker heating and cooling
cycles along with batch-production sizes that are not larger in number similar to hot
wall reactors-based CVD.
Plasma enhanced CVD or PECVD is another variant of CVD in which an inert
gas is ionized by the application of electric currents to form ionized gas environ-
ment to constitute plasma for initiating the reactions [60]. This is helpful in lowering
down the higher temperatures of deposition generally used in a CVD process. A great
advantage is the significant lowering of deposition temperatures in PECVD. Depo-
sition can be performed even at room temperature so as to avoid temperature-based
effects, and thus, it becomes possible to coat the organic polymer including other
types of materials possessing low melting temperatures (M.P.). Hence, PECVD can
be used for the deposition of both inorganic and organic materials based on highly
reactive precursors. A herd of ionized inert gas particles, innumerable electrons and
neutral atoms constitute the highly energetic plasma state that can be partially or
completely ionized. However, it must be noted that plasma is a neutral state of matter
so that the net charge is always zero. Two methods of plasma production are the
heating of the gas to produce temperature-based ionization for which higher temper-
atures are required, and the other one is based on the application of an electrical
current for ionizing the gas. The electrical energy can be imparted to the gas in
various frequency ranges of audio frequency of 10–20 kHz, RF of 13.56 MHz and
microwave frequency of 2.45 GHz that actually produce different types of plasmas.
Plasma causes disintegration of the material to be deposited that generates free radi-
cals, ions and sub-monomers. Further, the highly energetic plasma matter causes the
monomers to undergo plasma or radical polymerization. Chemically reactive species
are obtained when neutral molecules interact with the plasma species in addition to the
generation of radicals, polymerization reactions, etching reactions or even generation
of implantation reactions. In PECVD, monomers are generally taken in the liquid or
gaseous states for comfortable vapour formation. In case of solid monomers, subli-
mation system is required to evaporate the material that allows the deposition of a
range of materials. In the gaseous or liquid state with high vapour pressures, disso-
ciation of the precursor material takes place followed by high mobilization of the
dissociated species that makes the deposition happen at highly reduced temperatures.
In the case of a polymer substrate, etching, deposition and cross-linking & function-
alization take place that modify polymer substrate surfaces. Plasma polymerization
132 S. Dwivedi
adopts a random radical recombination approach in which a radical kick starts the
polymerization reaction inducing radicalization that results in the generation of the
polymer at the end of the reaction. Radicals are present in each of the series of the
polymer produced to induce cross-linking of the polymer to produce a polymer of
the cross-linked structure. Plasma produced polymer thin films do not resemble a
traditional polymer because of highly branched and cross-linked structures along
with “sub-units” of the plasma produced polymers not remaining to be same after
plasma-processing. For preservation of “sub-units” of the plasma produced polymers,
optimization of the internal and external plasma parameters is required.
5 Materials for Bio-MEMS
Both inorganic and organic materials are employed in the bio-MEMS/NEMS tech-
nology. Si and glass are widely used in the MEMS technology because of high devel-
opments in this field. Si is an indirect low bandgap semiconductor material, while
glass is dielectric in nature and falls in the category of wide bandgap materials. Si and
glass possess sound mechanical properties, while glass can be used for optical device
fabrication as well. Si is transparent to the infrared radiation while remaining opaque
to other wavelengths. Glass offers uniform surfaces for the fabrication of microflu-
idic devices, highly sensitive optical sensing platform in addition to the formation of
hermetically sealed devices owing to the creation of covalent bond of high strengths.
Glass based device processing is too costly and complicated. Etching is an integral
part of the device fabrication technology. As is well known, glass is amorphous in
nature and does not possess any crystalline character. Drawbacks associated with Si
and glass include high brittleness of both the materials, costly and limited disposable
bio-MEMS devices are possible only because of higher costs involved in the fabrica-
tion process [7]. Polymers offer an effective alternate as substrates for microfluidics
and bio-MEMS device fabrication. In addition, polymer substrates are less brittle,
dielectric in nature, possess sound thermal properties & high chemical stability, are
glassy, optically transparent, existing in soft or hard states, offer elastic platform & the
possibility of fabrication of disposable devices on account of being lesser expensive,
and are applicable in-vivo drug delivery platforms. An added advantage is the ease of
micromachining of these polymeric substrates by the routine etching techniques of
VLSI technology or even by laser-based microfabrication to pattern microchannels.
Ultrafast laser-based micro-patterning of thin films is possible for advanced MEMS
and for even deposition processes. In a work on microfabrication, micro-patterning
of indium thin films was performed for the generation of micron and submicron
particles using femtosecond laser-induced forward transfer (LIFT) [61].
Laser based microfabricated channels can also act as optical waveguides
for the fabrication of optical circuitry for quantum computers, and even light-
sensitive microfluidic and bio-MEMS devices. Laser based microfabrication requires
processing of optically transparent polymeric substrates by scanning of laser beam
across the surface to create spatially separated microchannels at equal distances. Light
can also be coupled from each of the ends of the microchannels to act as waveguides
in the refractive index modified areas inside the microchannels. Basically, laser beam
is focused at a specific distance deep inside the substrate to fabricate refractive index
modified regions that act as a sort of optical cables. Such microfabricated channels
can be used both in the form of microchannels and as optical waveguides as well. The
drawback associated with laser-based microfabrication is the fabrication of uneven
sidewalls of the deeply buried microstructures. Controlled energy should be imparted
by the laser pulses otherwise cracks due to heated zones may happen on polymeric
or glass substrates. Pulsed femtosecond oscillator lasers that can produce nanoen-
ergy pulses are suitable for this type of fabrication that can modify the refractive
index in a highly controlled manner. A train of pulses of the laser beam strikes deep
inside the substrate focused by means of suitable optics. This modifies the refractive
index inside the substrate in a highly controlled manner that is achieved by scanning
the substrate placed over a 3D translation stage possessing the capability to make
movements in three dimensions.
5.1 Inorganic Materials
Si is the most abundant material existing in compound form with other materials.
Single crystal Si is highly suitable for microelectronics and bio-MEMS devices as
it forms the platform for device fabrication and integration in the form of wafers
or substrates. Pure single crystal Si wafers can be fabricated using the Czochralski
(CZ) method. Si is mechanically stable, can be integrated easily onto substrates of
the same materials, possesses Young’s modulus of ~2 × 105 MPa similar to steel,
has a density of ~2.3 gcm−3 which makes it as light as aluminium, an elevated
melting point (M.P.) of 1400 °C makes it stable for use at higher temperatures,
does not show any mechanical hysteresis making it ideal for actuators and sensors,
possesses highly flat surfaces for coatings and subsequent multi-layered coatings, and
overall availability of its raw material of SiO2 has made the processing technology
advanced and even cost-effective. Single crystal Si structure is “face centered cubic
or fcc” in which eight atoms are placed at the corner while six atoms happen to
occur on the faces. However, single crystal Si has four extra atoms in its interior
that makes the total number of available atoms in a single crystal Si structure to be
eighteen. Unsymmetrical or uneven distribution of atoms inside the single crystal
Si develops anisotropic or directional mechanical properties in this semiconductor
structure, but however, Si is normally treated as an isotropic material overall. Miller
indices of the three distinct planes of a cubic crystal are inclusive of (001), (010),
(100) for the top, right and front faces, (110) for the diagonal face and (111) for the
inclined face. These crystal plane orientations possess their own intrinsic energies
depending on the number of atoms forming that orientation. Hence, (100) offers
least resistance and is the easiest to work with on account of the minimum number
of atoms forming this specific crystal orientation. Similarly, (110) crystal orientation
offers the ultraclean surfaces for microfabrication purposes. On the contrary, (111)
134 S. Dwivedi
consists of the shortest bonds formed between the atoms to form the strongest plane
and hence the most difficult to work with. Other Si based materials used in bio-
MEMS are silicon-di-oxide (SiO2 ), silicon carbide (SiC), silicon nitride (Si3 N4 ) and
polysilicon or polycrystalline silicon as well. Polysilicon or polycrystalline Si can be
deposited by various techniques that consist of Si crystals of different orientations
and sizes that form grain boundaries and also induce resistance related to them.
Polysilicon is usually highly doped and is deposited for the production of gates for
transistors and localized resistors. Polysilicon crystals are stronger than the single
crystal Si.
SiO2 is the most readily available material that offers good thermal as well as
electrical insulation protective layers, is also used in the form of oxide masks as
replacement of PRs for device patterning, deep buried oxide layers, and the initial
use in the form of device isolation layers decades ago. It forms a significant and
cost-effective use as “oxidic masking layer” for deposition, diffusion and etching
processes in microfabrication. It is also used as a “sacrificial layer” in surface micro-
machining [13, 14]. It can be produced by both dry and wet oxidation methods in a
furnace. In wet oxidation, steam is made to flow at a specific temperature to oxidize
the Si substrate surface, while in dry oxidation, O2 is flown over the high temperature
heated substrates. Generally, SiO2 has the resistivity of ≥1016 Ω-cm and a dielectric
constant of 3.9. SiC is used as a masking layer in microfabrication technology owed
to its high M.P. and the capability to resist chemical reactions, along with a higher
dimensional stability.
Silicon nitride (Si3 N4 ) possesses superior electrical insulation properties, and is
used as a masking layer in the deep etching process owing to its highly powerful
resistive nature against oxidation including several etchants [13, 14]. It is prepared
by the following reaction,
3SiCl2 H2 + 4NH3 → Si3 N4 + 6HCl + 6H2
High quality Si3 N4 layers are grown by LPCVD and PECVD techniques.
LPCVD grown Si3 N4 layers have a dielectric constant of 6–7 & resistivity of 1016
Ω-cm, while PECVD grown layers possess a dielectric constant of typically 6–9 &
resistivity of typically ~1016 Ω-cm. The etch rates of LPCVD grown Si3 N4 layers in
concentrated HF acid are 200 Å per minute, while the etch rate is 5–10 Å per minute
in boiling HF.
Quartz is silicon-di-oxide or SiO2 in which the unit cell acquires the shape of a
tetrahedron. It is highly applicable in microfluidics for biomedical purposes. Further,
it offers a high degree of insulation in microsystems or MEMS. This material is rather
difficult to etch out and is normally etched with a combined recipe of HF-NH4 F in
the required geometries.
5.2 Organic Materials
Organic materials are inclusive of polymers that consist of plastics, plexiglass, lucite
and adhesives. Polymer substrates can also be doped with suitable agents that can
possess electrical conductivities as well. In MEMS and microsystems fabrication
technology, organic substrates made-up of conductive polymers are frequently used
[7, 60]. B. Bhushan has presented an account of bioMEMS and bioNEMS that
gives details about biomimetics, and correlated materials & devices. Ferroelectric
polymers are another interesting material behaving similar to piezoelectric crystals
that can be manipulated as the actuation controller in a range of MEMS devices,
such as, micro pumping. Polymers are also used in microfluidics for facilitation
of effective electro-osmotic flow by coating polymers with specific properties over
the capillary tubes. Maheshwari and Ramgopal Rao have pointed out various mate-
rials involved in the fabrication of bio-MEMS [7]. In this article, electronic mate-
rials, such as, SU8 photopolymer, poly-dimethylsiloxane (PDMS) and poly-methyl
meth acrylate (PMMA) have been discussed in special reference to bio-MEMS.
SU8 is an epoxy polymer which is negatively toned so that photo-induced cross-
linking of 8-epoxy groups happens on exposure to UV-light [17]. SU8 is made-up
of Bisphenol A Novolac epoxy usually made to dissolve in gamma-butyrolactone
(GBL) or cyclopentanone in which triarylsulfonium/hexafluoroantimonate is added
in about 10 wt% of proportion to act as a photoacid generator. This photoacid gener-
ator is responsible for polymerization along with a unit photon triggering many poly-
merization reactions at a single time. In the next step, it is post-baked at a suitably
high temperature for effective cross-linking of the epoxy groups in advanced config-
urations as a result of curing. SU8 is a highly used negatively toned PR in MEMS
applications owing to its compatibility in micromachining process, bio-compatibility
features, higher degree of resistivity to chemicals, and generation of high aspect ratio
micro- and nanostructures with high mechanical stabilities. However, surface chem-
istry reactions need to be performed on SU8 before performing bio-applications
of the cell sorter or microchannels. Poly-dimethyl-siloxane (PDMS) is a specific
class of materials of silicones that belongs to the category of viscoelastic polymer
of molecular formula of [-(C2 H6 OSi)n ] [15, 21]. PDMS is widely used in microflu-
idic devices for biological sensing applications owing to capabilities to polymerize at
rather low temperatures, capabilities to deform reversibly, optically transparent nature
for wavelengths of 280 nm and higher, bio-compatible nature along with the capacity
to regenerate micrometre-sized features certifying high degree of reproducibility.
PDMS is highly compatible for the development of cell growth systems owing to
its permeable characteristics towards carbon-di-oxide (CO2 ) and oxygen (O2 ). Pure
PDMS possess hydrophobic surfaces along with highly reduced surface energies
that can enforce the adsorption of hydrophobic compounds and bio-molecules over
its surfaces subsequently lower the sensing capabilities of devices. Hence, specific
surface chemistry led modifications are required for enhancing the functionality. An
associated drawback is the huge difference between PDMS and metals pertaining to
Young’s modulus that causes stress generation including buckling of metallic bonds
136 S. Dwivedi
along with crack formation. This results in difficulty in the formation of electrical
interconnects and contact pads in the form of thin films over the PDMS surfaces.
Poly-methyl-meth-acrylate (PMMA) is a widely used, low-cost polymer with trans-
parent features that can be applied in the form of a masking material in the fabrica-
tion technology along with use as a structural material [15, 21]. It is widely used in
bio-MEMS applications in the form of microfluidic devices.
Polymer materials must undergo specific surface chemistry reactions for modifi-
cation of polymer surfaces. Polymers used for optical sensing applications or devices
must not suffer from auto-fluorescence in addition to coating with bio-fouling mate-
rial for doing away with the adsorption of bio-molecule hydrophobic organic mate-
rials. Polymers selected for bio-MEMS should have temperature-tolerance character-
istics along with a sense of wearability towards organic solvents. Different techniques
for polymer materials processing for patterning of devices include hot embossing,
photo- and electron beam lithographies (EBL) laser ablation and X-ray lithographies.
MEMS devices length scales are smaller than 1 mm but greater than 100 nm,
while NEMS length scales are smaller than 100 nm. In that sense, quantum-dot tran-
sistor is usually at the scale of 300 nm. Biomaterials, such as, DNA molecules are
typically ~2.5 nm wide in scales, biological cells are thousands of nanometres in
diameter and molecular gears are typically ~10 nm in length scales. Hence, it can
be realized that nature has already provided us with well structured, designed and
patterned biomaterials that can eventually be utilized for bio-MEMS/NEMS devices.
This will be actually helpful in the fabrication of devices that can be exploited at
the human-interface consisting of highly advanced electronics at the micro- and
nanoelectronics regime in form of an integrated circuitry. This is further coupled
with advanced approaches of artificial intelligence (AI), machine learning and data
analytics for next-generation commercial production of advanced devices for bio-,
safety-, medical- and defence-applications. Machine learning is already integrated
in research domain these days for the development of advanced, well-planned, struc-
tured and well-designed biomaterials. Microelectronics manufacturing technology is
usually based on Si fabrication technology exploiting conventional surface and bulk
micromachining techniques. Si as a substrate can have detrimental effects on the
human body on application in form of microimplants. For elimination of this anomaly,
Si substrate surfaces are passivated with protein coatings for mimicking a biolog-
ical surface. In this way, Si based microimplants & devices are made compatible
with the human body. Nanoscale 3D structural conformation of the protein provides
information about the function imparted by the protein as a biomaterial. Bio-MEMS
exploits functional specificity of the polymers along with large-scale self-assembly of
arranged molecules at the nanoscale for a rich variety of operations. Bio-MEMS can
be applied for the development of biosensors consisting of specific protein antigens
that bind to the specific protein antibodies. This is the particular bio-sensing mech-
anism driven by affinity towards specific moieties that is applicable in therapeutics.
Proteins used in bio-MEMS should display higher degree of wear resistance in case of
direct contact with the circulatory blood flow without getting eliminated and tissues
as well. Adhesion of the mobile parts in polymer based micro-nanofluidic biodevices
should be minimized. Biomolecules in the fluidic form get adhered to the walls of
the microchannels restricting transport along the length of the channel, and hence,
microchannel surfaces must be mobilized accordingly. Hence, hydrophobicity or
wettability of a surface is of critical importance in bio-MEMS/NEMS as well [62, 63].
It is measured by the static contact angle made between the hydrophilic spherically
shaped water droplet and the corresponding surface under study. The contact angle
range is defined to be 90° < θ ≤ 180° for non-wetting of the surfaces by the liquid.
Superhydrophobic surfaces are defined to be surfaces with contact angle in the range
of 150–180°. Superhydrophobic surfaces must also possess very low hysteresis (θ H )
of the water contact angle for self-cleaning purposes. Superhydrophobic surfaces
have applications in energy conversion in addition to energy conservation, are good
for fuel economies, while reversible superhydrophobicity provides novel routes for
energy conversion, such as, microscale capillary engine. Self-cleaning capability
is acquired by the specific feature of water droplets rolling off over the superhy-
drophobic surfaces with some degree of slip and taking away minute contaminants
with them, hence the “lotus effect” [60, 61]. Contact angle hysteresis is a measure of
the heterogenous character of the surfaces and roughness and portrays the difference
occurring between forwarding and back-geared contact angles that are realistically
two separate values being of highly stable nature. The removal of contaminant parti-
cles happens on the lower values of contact angle hysteresis so that rolling happens
in addition to sliding. Self-cleaning surfaces can have a wide variety of applications
that include solar panels, self-cleaning windows, textiles, roof tiles, paints, and in
micro- and nanochannels in micro- and nanofluidics.
6 Future Scope
Bio-MEMS is at the forefront in development of biomedical sensing devices for

various types of disease detection, point-of-care health applications. Bio-NEMS is
all set to create a revolution in biomedical sensing devices in health monitoring with
significantly enhanced efficiencies for non-invasive patient health monitoring. State-
of-the-art diagnostic tools can be developed by applying micro- and nanofabrication
technologies. Recently, outbreak of Covid-19 created a high degree of turbulence
worldwide. However, with modern scientific advancements, rapid testing equip-
ments were developed that had high sensing capabilities towards the novel coro-
navirus. MEMS/NEMS based biomedical sensors and diagnostic tools is a huge
commercial technology that is expected have a global market of around 15 billion
US dollars by the end of 2022. Bio-MEMS/NEMS based sensing devices can be used
for detection of diseases, such as, malaria, cholera, Ebola, leprosy, schistosomiasis,
and many other diseases. Diagnosis methodologies include culture techniques for
microorganism detection, serological detection procedures in case of pathogenic
markers, such as, proteins & antigens, and many other different ones. With the
advent of bioweapons methodologies for modern warfare without directly hitting on
a country, bio-MEMS/NEMS has become increasingly important for state-of-the-
art rapid detection and sensing platforms. Modern viruses are continuously striking
138 S. Dwivedi
the society across the world. For example, Covid-19 proved to be highly fatal for
humans across the world. Similarly, a new virus, named as lumpi skin disease virus,
is striking the cows in a damaging manner. For agriculture dependent countries like
India, it is extremely important to develop bio-MEMS/NEMS based rapid detection
and sensing technologies so that administration of drugs, medicines & injections can
be performed at an early stage. Hence, bio-MEMS/NEMS is the need of the hour
not only to protect humans but animals as well, and all societies and countries must
focus on it in an out-of-box manner.
7 Conclusion
Bio-MEMS/NEMS is currently at the forefront of technology for point-of-care

health applications, fabrication of advanced biosensors, biodevices, drug-delivery
systems, lab-on-a-chip technology for early diagnosis, and advanced microimplants
that are biocompatible. Photolithography combined with wet- or dry-etching is
a rather conventional method for the MEMS/NEMS based production of struc-
tures. The technology is well-established and commercial production of devices
is already in place. Dry etching process can produce even anisotropic structures for
advanced MEMS/NEMS fabrication. Superhydrophobic and self-cleaning surfaces
with specific surface energies are important for micro- and nanofluidic systems.
Stereolithography is a state-of-the-art additive manufacturing technique for the
production of 3D printed structures with many applications in consumer electronics,
medical & surgical equipments, dental applications, and even defence applications.
Stereolithography is a very stable, fully automatic process producing structures
with high accuracy and precision. Once started, the process advances automatically
without requirement of any attention. A drawback associated with stereolithography
is the generation of warping and curling due to the inclusion of water in the resins
over a period of time in thinner areas. Another is the non-curing of certain parts even
after the exposure of laser due to energy imparted in form of conical shapes by the
laser spot. Moreover, stereolithography is a costly system with costs of laser guns
being very high along with costs of the photocurable resins also being high.
Micro- and nanofluidic are also in use for bio-MEMS/NEMS device fabrication
by conventional lithographic technologies. Electron beam lithography (EBL) can be
employed for device patterning at the nanometric level for NEMS fabrication. Both
positively- and negatively toned photosensitive polymer solutions are used in NEMS
as well. Nanochannels can be fabricated by both conventional lithography as well
as by laser-based microfabrication in glasses and/or polymer substrates. Glass or
polymer substrates are optically transparent to light offering the possibility of micro-
and nanochannels fabrication for light-sensitive detection of biomolecules.
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00003-3
In-silico Analysis of Expandable
Radiofrequency Electrode for Ablation
of Hepatic Tumors
Shaik Sadikbasha and Ashish. B. Deoghare
Abstract The current study evaluates radiofrequency (RF) ablation of hepatic

tumors with an expandable multi-prong RF applicator. To assess the localized cooling
effect of blood flow on necrosis volume, a three-dimensional computational model
of hepatic tissue, cancer tumor, blood vessel, and RF applicator was created. The
finite element method is used to calculate the effect of blood vessel location, diam-
eter, change in input voltage, and tumor property uncertainty on ablation volume. An
increase in blood vessel diameter causes a 6.52% reduction in ablation volume, and
a 10 mm diameter blood vessel located next to a tumor causes a 12.19% reduction
in ablation volume.
Keywords Hepatic tumors · Bio-heat equation · Hepatocellular Carcinoma ·

Ablation volume
1 Introduction
Cancer kills millions of people worldwide each year, with Hepatocellular Carcinoma
(HCC) being the third leading cause of death in Asia and Africa [1]. Surgical resection
is the most commonly used treatment method for cancer tumors. However, open
surgery removes part of tissue permanently which shortens the organ and tumor
location restricts the use of surgical resection in some cases. To overcome these
limitations, researchers were drawn to minimally invasive percutaneous techniques
such as RF ablation, Laser, Microwave, and Cryoablation as an alternative to surgical
resection. RF ablation is a clinically well-accepted hyperthermia technique that uses
heat energy to effectively treat multifocal and surgically non-resectable tumors. Its
ability to ablate targeted tissue while causing minimal normal tissue invasion and
low cost made it an attractive alternative to open surgery. When compared to surgical
resection, RF ablation as an initial treatment for HCC has better long-term outcomes
and survival rates [2].
S. Sadikbasha · Ashish. B. Deoghare (B)

Department of Mechanical Engineering, National Institute of Technology Silchar, Assam 788010,
India
144 S. Sadikbasha and Ashish. B. Deoghare
Large blood vessels cause localized cooling and reduce the efficacy of hyper-
thermia treatment, according to Roemer et al. [3]. Kolios et al. [4] developed a numer-
ical model to investigate the cooling effect of large blood vessels in heated tissue.
Chinn et al. [5] investigated the effect of vascular occlusion on hepatic tumor RF
ablation. Jain and Wolf [6] presented a novel three-dimensional model to investigate
the effect of blood flow on lesion volume in RF ablation using a single-tip electrode.
Supan et al. [7], Haemmerich et al. [8], Suarez et al. [9], and Huang et al. [10] inves-
tigated the effect of a large blood vessel on ablation volume using isothermal and
convective boundary conditions with an expandable electrode. However, assuming
isothermal and convective boundary conditions at the tissue-blood vessel interface
overestimate ablation volume, and results obtained under these conditions may not
accurately predict temperature profiles. As a result, combining blood flow analysis
with electrothermal analysis yields a more accurate estimate of ablation volume.
Haemmerich et al. [11] studied the variation of electrical conductivity in rat tumors
in vivo and discovered that it was significantly different from normal tissue conduc-
tivity. Liu et al. [12] investigated the relationship between temperature and perfusion
rate and discovered that increased blood flow decreases coagulation volume and time
required to achieve thermal equilibrium. Schutt et al. [13] compared various perfu-
sion models, their effect on temperature profiles, and confirmed that multi-tined
electrodes are more susceptible to perfusion change. Hall et al. [14] validated the
effect of perfusion, thermal, electrical conductivity, and cell death models on abla-
tion volume, establishing that perfusion and cell death models are critical parameters
for accurate ablation volume prediction. Various protocols for increasing ablation
volume have been developed, including internally cooled electrodes [15], multiple
electrodes [16], two-compartment models [17], expandable electrodes [7, 8, 10], and
bipolar RF ablation [18]. These studies, however, show that taking into account the
same properties for both tissue and tumor may not provide an accurate prediction of
ablation volume and temperature profiles. As a result, a variation in tumor properties
with temperature, as well as uncertainty in tumor properties, require more attention
for accurate analysis. The purpose of this study is to determine the effect of blood
flow velocity, blood vessel diameter, and the relative location of a blood vessel with a
tumor on ablation volume. The impact of input voltage, tumor density, specific heat,
thermal conductivity, electrical conductivity, and perfusion rate on ablation volume
and temperature distribution is also investigated. This study aids the therapist in the
effective pre-planning of RF ablation treatment for hepatic tumors.
2 Materials and Methods
A schematic diagram used for computational analysis is shown in Fig. 1. It shows liver
tissue, the spherical tumor is located at the center, a blood vessel, and an RF applicator
inserted in the tumor. A 15 gauge RITA Medical Systems Model30 umbrella-shaped
expandable four-tined RF applicator [7] is used to destroy tumor by heating as shown
in Fig. 1. The liver is a complex organ and its physical properties are inhomogeneous.
In-silico Analysis of Expandable Radiofrequency Electrode … 145
Fig. 1 Schematic of the computational domain for RF ablation
Therefore, investigations are carried out for tumor cells by varying density, specific
heat, thermal conductivity, electrical conductivity, and perfusion rate to study the
influence on temperature distribution and ablation volume. Blood vessels of diameter
more than 6 mm cause localized cooling in heated tissue, hence a blood vessel is
modeled in hepatic tissue and its diameter has been varied from 6 to 13 mm to
evaluate the effect on ablation volume and temperature distribution. The influence
of relative distance between tumor and blood vessel on temperature change and an
ablation volume is studied by locating blood vessel at 21 and 26 mm from the tumor
center. Analysis has been carried out at 30, 70, and 90 mm/s velocity of blood flow
to evaluate the effect on ablation volume and temperature profiles [6].
In RF ablation procedure electrical potential is applied to RF applicator at 500 kHz
frequency for the period of 8 min to destroy the targeted tissue. The electrical potential
applied generates a flow of alternating current in conductive electrodes which causes
ion agitation and produces localized heating. This results in resistive heating of liver
and tumor tissue in the proximity of electrodes. Intercellular protein denaturation,
cell membrane destruction occurs at the temperatures beyond 45 °C [7] and produces
coagulative necrosis.
2.1 Governing Equations
The heat generated from the electrodes propagates to surrounding tissue through
conduction mode. The maximum temperature reduces the fourth power as the
distance from the electrode increases in tissue [4]. Tumor cells near to large blood
vessels are more difficult to ablate because blood flow causes localized cooling effect
and influences heat deposition in tissue. Hence, theoretical formulation of RF ablation
procedure is necessary to evaluate the localized cooling effect for accurate prediction
of ablation volume and to plan the treatment effectively.
Table 1 Properties of materials used for computational domains [7, 19, 20]
( ) ( ) ( W ) ( )
Domain Material ρ mkg3 J
c kg.K k m.K σ mS Other properties
Insulation Polyurethane 70 1045 0.026 1 × 10−5

Trocar Stainless steel 21,500 132 71 4 × 106
Electrodes Ni–Ti alloy 6450 840 18 1 × 108
(/ )
Tissue Liver 1060 3600 0.512 0.333 0.0064 ω 1 s
(/ )
Tumor Liver tumor 999 4200 0.552 0.117 0.0156 ω 1 s
Blood Blood 1050 4180 0.543 0.667 0.0035 μ[Pa.s]
Modeling of heat transfer and temperature distribution in biological tissues is

expressed using bioheat Eq. (1) [7].
∂T
ρC = ∇.k∇T + ρb Cb ωb (Tb − T ) + Q e + Q m (1)
∂t
where ρ is the density of the medium(kg/m3 ), C is the specific heat (J/kg.K), T is the
temperature(K), t is the time(s), k is the thermal conductivity(W/m.K), subscript b
denotes blood, and ω is the blood perfusion(1/s). The properties of materials used
for computational domains are given in Table1. ( / )
The equation involves the metabolic heat energy Q m W m 3 generated in the
body
( which is negligible when compared to other terms in Eq. (1), source energy
W / )
Qe m generated by electrodes, ρb Cb ωb (Tb − T ) is the heat carried away due
3
to the blood perfusion, ∇.k∇T is the heat energy propagated in tissue through
conduction mode and ρC ∂∂tT is the energy deposited in the tissue over the period
of time.
The source energy Q e is evaluated from the distribution of current density and
electrical field intensity in the computational domain. The distribution is expressed
by generalised Laplace Eq. (2), from this equation voltage distribution is evaluated.
∇.[σ ∇V ] = 0 (2)
where V is the voltage (V) and σ is the electrical conductivity (S/m).

The electrical field intensity and the current density are obtained from Eqs. (3)
and (4) from the voltage distribution.
E = −∇V (3)
J = σ.E (4)
where E is the electric field intensity(V/m) and J is the current density(A/m2 ). The
resistive heat generated by current density is expressed by Eq. (5)
Q e = J.E (5)
The thermo-electrical system further needs to be coupled with a fluid flow field
to determine the localized cooling effect caused by blood flow. The continuity (6),
Navier Stokes (7), and energy Eqs. (8) are solved over the computational domains.
→ · V→ = 0
∇ (6)
−
→
∂V → −
− → 1 −→ μb 2 −→
+ V .∇ V = − ∇ P + ρb −
→g + ∇ V (7)
∂t ρb ρb
( )
∂T − →
ρC + V .∇T = ∇.k∇T + ρb Cb ωb (Tb − T ) + Q e (8)
∂t
−
→ −
→
where V is the velocity vector, P is the pressure vector, − →g is the gravitational
acceleration vector, and μ is the viscosity of blood.
The sensitivity of electrical, thermal conductivity, and blood perfusion rate to the
temperature [21] is evaluated using Eqs. (9), (10), and (11)
σ (T ) = σ0 (1 + 0.02(T − Tb ) (9)
where σ0 is the baseline conductivity, Eq. (9) considers 2% per °C variation in

electrical conductivity for both tumor and tissue [21].
k(T ) = k0 (1 + 0.013(T − 310.13) (10)
where k0 is the baseline thermal conductivity, Eq. (10) considers 1.3% per K variation
in thermal conductivity for both tumour and tissue [21].
⎧ ⎫
⎨ ωb0 [ ] Ω(t) ≤ 0 ⎬
ωb = ωb0 1 + 25Ω(t) − 260Ω(t)2 0 < Ω(t) ≤ 0.1 (11)
⎩ ⎭
ωb0 e[−Ω(t)] Ω(t) > 0.1
where ωb0 is the baseline perfusion rate, Ω(t) is the thermal induced tissue damage.
The destruction of cancer cells in RFA occurs due to direct and indirect heating
mechanisms. At the end of the procedure of RFA, three zones of necroses tissue,
sub-lethal damage tissue, and normal tissue can be observed. The tissue subjected to
temperature more than 50 °C is considered necroses tissue because denaturation of
protein, cell membrane collapses, metabolism, and enzymatic function halt occurs
at 50 °C. The tissue with a temperature of 40–50 °C is considered sub-lethal damage

and at a temperature less than 40 °C is considered normal tissue. However, necrosis
depends on tissue type, temperature level, time duration, and heat energy accumu-
lated. In this analysis damage integral Eq. (12)derived using the Arrhenius equation
[21] is used to evaluate the ablation volume and results are compared with isothermal
temperature contour of 50 °C. The thermal damage integral of tissue is calculated
using Eq.(12).
{t [ −Ea ]
Ω(t) = Ae RT (12)
0
where Ω(t) is the tissue damage integral, E is the activation energy for irreversible
tissue damage, A is the tissue frequency factor, T is the temperature, and R is the
universal gas constant. For liver tissue E = 2.577 × 105 Jmol−1 , A = 7.39 × 1039
s−1 .
The fraction of tissue damage is expressed by Eq. (13),
θd = 1 − e−Ω(t) (13)
where θd is the fraction of tissue damage, its value lies between (0, 1). The value
of tissue damage Ω(t) =1 and 4.6 corresponds to 63.2 and 99% probability of cell
death respectively.
2.2 Boundary Conditions and Analysis
The boundary conditions employed on computational domains for this analysis are
as follows:
The initial voltage in the computational domain is 0 V and an impedance of
Z = 60Ω is maintained in the analysis. Grounded (V = 0) boundary condition is
employed on the one of the outer surfaces of tissue, and analysis is carried out by
changing electrical potential from 20–40 V which is given to the electrode. Stainless
Steel (SS) trocar of the RF applicator is subjected to insulated boundary condition,
n. J = 0.
For thermal analysis, the initial temperature employed in the computational
domain is the same as body temperature, T = Tb . The outer boundaries of tissue
which remains unaffected also subjected to temperature boundary condition of
T = Tb . The electric energy applied generates heat in the tissue to necroses the
tumor cells.
For the fluid domain at the inlet, the temperature boundary condition T = Tb is
employed with the pressure of P = 3333.33(Pa). The inlet blood velocity consid-
ered in the analysis is u(x) = u(y) = 0; u(z) = 35, 70, and 90 mm/s. At the
liver–blood interface, blood velocity is u = v = z = 0. At the outlet of blood vessel,

P = 3333.33(Pa) is employed to carry out an investigation.
The prepared models are imported into COMSOL Multiphysics FEM software
to perform coupled analysis of thermoelectric, fluid field problem. In this study,
electric currents AC/DC module, biological heat transfer module, and laminar fluid
flow module are used to simulate RF ablation of hepatic tumors. Multiphysics has
been used to couple modules considered for analysis.
Physics-dependent meshing has been done on domains using tetrahedral elements
of a minimum size 1 mm and a convergence test has been performed to find an
optimum number of elements. The relative error obtained for the mesh with 189,385
elements and higher density mesh is <0.5%. Therefore, the analysis is carried out
at this mesh size and solved for 545,255 degrees of freedom. The implicit time-
dependent analysis is performed for 8 min with time step 0.01 to 5 s using Backward
Differential Formula (BDF) of second order.
2.3 Validation
Validation of the model has been done with the experimental work of B. Chinn et al.
(2000) [5]. Lesion volume by occlusion of the portal vein is 8.6 ± 3.8cm 3 . Results
obtained by placing simple hepatic artery at 21 and 26 mm from tumor centre produce
9.13 and 9.57 cm3 respectively using 50 °C isothermal contours. Analysis shows
≤ 10% relative error with experiment results. The results also compared with the
study performed by Tungjitkusulmon et al. (2003) [7]. Ablation volume obtained
in his work with no blood vessel, 10 mm diameter blood located 5 mm and 1 mm
away from the probe is 19.2, 18.6, and 17.2 cm3 respectively. However, in the present
work results are 21.8, 17.5, and 17.1 cm3 respectively with a maximum difference
of 13.54%.
3 Results and Discussion
The HCC cases diagnosed increased 115% from the year 2002 to 2012, in many
cases using open surgery to eliminate cancer tumors is difficult due to the critical
location of the tumor. Hence, percutaneous techniques like RF ablation evolved as a
safe and efficient technique to destroy hepatic tumors without causing much damage
to normal tissue. The necrosis volume in RF ablation primarily depends on applied
voltage, duration of treatment, tumor physical properties, and relative location blood
vessel with respect to the tumor.
The effect of voltage on temperature distribution and an ablation volume is evalu-
ated by varying input voltage from 20–40 V at 500 kHz. Figures 2 and 3 show results
obtained by locating a 10 mm diameter blood vessel at 26 mm from the tumor center
with a blood flow velocity of 70 mm/s. Temperature-dependent property models are
given in Eqs. (9), (10) and (11) are used in the analysis. The temperature increases
with an increase in source voltage, temperature rises to the maximum value within
70 s for all voltages and remains almost constant for the remaining duration as shown
in Fig. 2.
Maximum and minimum temperatures observed in the analysis are 119 °C at
40 V and 52 °C at 20 V respectively. Figure 3 shows a change in ablation volume
with time for different input voltage conditions. Maximum and minimum ablation
volumes obtained in the study are 12.3 cm3 at 40 V and 1.44 cm3 at 20 V respectively
when calculated using volume integral of 50 °C isothermal contours. Figure 4 shows
ablation volume obtained using tissue damage integral, 99% probability of cell death,
50 °C isothermal contours when a blood vessel of 10 mm diameter located at 21 and
26 mm.
Ablation volume decreases with a decrease in relative distance between tumor and
blood vessel. The ablation volume corresponds to 50 °C isothermal contours and 99%
cell death damage integral is 12.3, 13 cm3 , and 13.8, 14.5 cm3 when a blood vessel
is located at 21 and 26 mm respectively from tumor center. Results obtained using
tissue damage integral and isothermal contours differ by 6% and ablation volume
Fig. 2 Effect of volume on

temperature distribution over
time
Fig. 3 Ablation volume 14

observed with time at 12
various voltages
Ablation Volume (cm 3 )
10
20 V
8
25 V
6
30 V
4 35 V
2 40 V
0
0 100 200 300 400 500
Time (sec)
Fig. 4 Effect of blood 14

vessel location and cell death
models on ablation volume 12
Ablation Volume (cm3)

at 40 V
10
8 50⁰C @ 21 mm
50⁰C @ 26 mm
6
θd ≥ 0.99 @ 26 mm
4
θd ≥ 0.99 @ 21 mm
2
0
0 100 200 300 400 500
Time (sec)
variation with blood vessel location is 12.19% as shown in Fig. 4. Maximum ablation
volume for each condition is attained before 5 min, however; analysis is carried out
for 8 min to make sure that the heat energy required for complete ablation is supplied.
Figure 5 shows the effect of time on the ablation volume accumulated. Temperature
above 100 °C causes tissue charring, carbonization, and increases impedance which
further restricts the accumulation of necrosis volume. Hence source voltage is tuned
to 35 V to keep the temperature below 100 °C.
Blood perfusion is another critical parameter which significantly affects lesion
volume. Figure 6 shows temperature distribution and ablation volume with time
considering different perfusion rates for tumor. The analysis is carried out at 35 V,
locating 10 mm diameter blood vessel 26 mm away from the tumor center. The perfu-
sion rate in normal tissue is significantly different from the tumor; hence perfusion
Fig. 5 Ablation
accumulated over time in RF
ablation at 40 V
Fig. 6 Effect of tumor 19

109
perfusion variation on
ablation volume and 17 Ablation Volume 107
Ablation Volume (cm3 )
Temperature (degC)
temperature at 35 V Temperature 105
15
103
13 101
99
11
97
9 95
0.005 0.007 0.009 0.011 0.013 0.015
Blood Perfusion (1/s)
rate has been varied from normal tissue perfusion, 6.4 × 10−3 to tumor perfusion
0.1168 s −1 .
A maximum temperature of 108.5 °C is observed when tumor and normal tissue
perfusion is considered to be the same and a minimum temperature of 97 °C is
observed when actual perfusion rates given in Table 1 are considered. Ablation
volume obtained is 17.8 and 9.13 cm3 when tumor perfusion is 6.4 × 10−3 and
0.1168 s −1 respectively. Maximum temperature and ablation volume decrease with an
increase in perfusion rate as shown in Fig. 6. Therefore, any uncertainty in considering
perfusion rate causes significant error in the estimation of temperature distribution
and necrosis volume.
The thermal conductivity of the tumor is another critical parameter which can
influence ablation volume. Therefore, the effect of thermal conductivity on temper-
ature distribution and an ablation volume is evaluated at 35 V by locating the 10 mm
diameter blood vessel at 26 mm from the tumor center. Results shown in Fig. 7
indicate the variation of ablation volume and temperature with thermal conductivity.
Temperature reduces with increase in thermal conductivity whereas ablation volume
increases as shown in Fig. 7. The error in the estimation of ablation volume is <2%
by considering the same thermal conductivity for tumor and tissue. The electrical
conductivity of tumor is another influencing characteristic of RF ablation proce-
dure. Results in Fig. 6 show the effect of electrical conductivity on temperature
distribution and ablation volume. Both temperature and ablation volume increases
with an increase in electrical conductivity as shown in Fig. 8. Considering the same
electrical conductivity for both tumor and tissue produces 126 °C temperature and
necrosis volume of 16.8 cm3 , otherwise, temperature and volume are 97 °C and
9.13 cm3 respectively. Therefore, uncertainty in the electrical conductivity of the
tumor overestimates ablation volume.
Analysis has been carried out to evaluate the effect on ablation volume by locating
a blood vessel of diameters 6, 10, and 13 mm at 21 and 26 mm from the tumor centre.
Ablation volume is evaluated for 30, 70, and 90 mm/s as shown in Table 2. Changing
velocity of blood flow from 30 to 90 mm/s reduced ablation volume by 3, 1.7, 3.8,
Fig. 7 Effect of variation in 9.2 103

thermal conductivity on
9.1 102
temperature and ablation
Ablation Volume (cm 3 )

volume with time
Temperature (degC)
101
9
100
8.9
99
8.8
Ablation Volume 98
8.7 Temperature 97
8.6 96
0.46 0.48 0.5 0.52 0.54 0.56
Thermal Conductivity (W/m.K)
Fig. 8 Effect of variation in 17

electrical conductivity on 127
16 Ablation Volume
temperature and ablation
Ablation Volume (cm3 )
volume with time 15 Temperature 122
Temperature (degC)
14 117
13
112
12
107
11
10 102
9 97
0.1 0.15 0.2 0.25 0.3 0.35
Electrical Conductivity (W/m.K)
1.75, 2.59, and 1.89% when 6, 10 and 13 mm diameter blood vessel located at 21
and 26 mm distance from tumor respectively.
The results shown in Table 2 state that the increase in diameter of the vessel when
located at 21 mm from the tumor center showed a significant decrease in ablation
volume. But the increase in diameter of the blood vessel has no considerable change
Table 2 Results obtained for the ablation volume using tissue damage integral at 99% cell death
after 8 min duration considering temperature dependent property models at 35 V
Blood vessel diameter (mm) 6 6 10 10 13 13
Location of blood vessel (mm) 21 26 21 26 21 26
Flow velocity (mm/s) Ablation volume (cm3 )
30 7.14 7.55 7.23 7.54 7.13 7.54
70 7.36 7.42 7.12 7.43 7.07 7.40
90 7.28 7.53 7.24 7.35 6.87 7.48
in ablation volume. Increase in velocity of blood flow reduces ablation volume as

shown in Table 2. Variation of density and specific heat of tumor has no major effect
on temperature distribution and ablation volume. Therefore, input voltage, perfusion
rate, and electrical conductivity are the most important parameters along with cell
death models.
4 Conclusions
RF ablation is an effective and safe method of treating surgically inoperable tumors

in critical locations. Predicting lesion volume accurately prior to surgery allows for
lower recurrence rates and more effective treatment planning. This study looked
at the factors that influence lesion volume. Temperature and lesion volume increase
significantly as voltage increases, but temperatures above 35 V exceed 100 °C, which
is undesirable due to increased impedance and carbonization. The most influential
parameter is blood perfusion; considering the same perfusion for tumor and tissue
doubles the ablation volume, causing a prediction error. When thermal conductivity
is changed, it causes a 2% change in temperature and ablation volume. Increasing the
electrical conductivity of the tumor results in a 1.5-fold increase in ablation volume.
When compared to blood vessels at 26 mm, large blood vessels located 21 mm from
the tumor center cause localized cooling and reduce ablation volume by 12.19%. As
a result, this study may be useful to medical practitioners in effectively planning RF
ablation treatment.
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Lab-On-Chip Electrochemical Biosensor
for Rheumatoid Arthritis
Rahul Kumar Ram, Nirmita Dutta, Jai Shukla, and Gorachand Dutta
Abstract With the current prevalence rate of 0.5–1%, rheumatoid arthritis (RA)
is the most common type of autoimmune arthritis. The chances of occurrence of
this chronic multifactorial disease are more in females than males, significantly
increasing with age (Crowson et al. in Arthritis Rheum 63:633–639, 2011;Eriksson
et al. in Arthritis Care Res (Hoboken) 65:870–878, 2013; Smolen JS, Aletaha D,
Barton A, et al. (2018) Rheumatoid arthritis. Nature Reviews Disease Primers 2018
4:1 4:1–23. 10.1038/nrdp.2018.1;Vollenhoven in BMC Med 7, 2009;). Rheumatoid
arthritis primarily affects the lining of the synovial joints and can cause progressive
disability, premature death, and socioeconomic burdens. Currently, there is no cure
for RA. Hence the treatment strategy aims to speed up diagnosis and rapidly achieve
a low disease activity state (LDAS) (Guo Q, Wang Y, Xu D, et al. (2018) Rheuma-
toid arthritis: pathological mechanisms and modern pharmacologic therapies. Bone
Research 2018 6:1 6:1–14. 10.1038/s41413-018–0016-9). Presently, even though
different techniques like ELISA (enzyme-linked immunosorbent assay), radioim-
munoassay, fluorescence-based analysis, surface-enhanced Raman scattering, and
chemiluminescence-based analysis are prevalent as reliable diagnostic tools for
rheumatoid arthritis biomarker detection, they have their own shortcomings. The
immediate need for rapid, accurate, cheap, and early diagnosis have urged scien-
tists to develop new advanced technologies, of which biosensors are one of the
most reliable platforms. This review discusses the clinical as well as pathophys-
iological features of rheumatoid arthritis along with the application of different
electrochemical nanobiosensors for its rapid and early diagnosis.
Keywords Rheumatoid Arthritis · Electrochemical nanobiosensors · Biomarker ·

Diagnosis · Point of care
R. K. Ram · N. Dutta · J. Shukla · G. Dutta (B)

NanoBiosensors and Biodevices Lab, School of Medical Sciences and Technology, Indian
Institute of Technology, Kharagpur, West Bengal, India
158 R. K. Ram et al.
1 Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder that

causes cartilage, bones, and joints to deteriorate over time [1–3]. It affects three
times as many women as it does men [4–7]. Inadequate treatment leads to the accu-
mulation of irreparable joint damage, putting patients at risk for the rest of their
lives [3]. They have discomfort and deformity in their joints, and in severe cases
of sickness, they may require assistance from family members or nurses to move
around and complete daily duties. RA is a multifactorial disease [8] that advances
rapidly in the first few days after the onset [9]. Lack of markers, signs, or symptoms
to specifically diagnose RA causes a considerable delay in treatment administra-
tion, resulting in permanent joint degeneration within a few days after beginning.
Furthermore, the presently utilized diagnostic processes have limits with regard to
point-of-care applications and use in remote places, where patients have limited
access to advanced healthcare facilities. To overcome the limits of traditional detec-
tion methods, a variety of physical diagnostic instruments have emerged in recent
years, the majority of which are based on biosensors because of the advantages they
present, such as high sensitivity, high specificity, cost-effectiveness, and ease of use.
Biosensors are devices with both organic and inorganic components that analyze and
offer qualitative and quantitative information about the type of analyte present in the
sample. The necessity of biosensors is in high demand in the field of diagnostics,
and they will be incredibly useful in the early diagnosis of rheumatoid arthritis and
other chronic inflammatory disorders.
This review presents a broad insight into the pathophysiology of rheumatoid
arthritis and significant diagnostic biomarkers associated with it. Different electro-
chemical nanobiosensors were reviewed, including enzymatic, non-enzymatic, and
label-free nanobiosensors for rheumatoid arthritis diagnosis using several surface
modification strategies. Besides, the conventional diagnostic techniques for rheuma-
toid arthritis biomarker detection and their drawbacks are also discussed. Further-
more, the advancement of recent technologies in the field of biosensors that have
simplified the instrumentation system, sped up the diagnosis time, made it more
specific and sensitive, have also been discussed briefly.
2 Rheumatoid Arthritis Pathophysiology
“Arthr”- means joints, “-it is” refers to inflammation, and “rheumatoid” derives from
rheumatism, which refers to musculoskeletal illness in general. Rheumatoid arthritis
is a chronic systemic inflammatory autoimmune condition that primarily affects the
joints, although it can also affect the skin, lungs, and heart.
The etiology of rheumatoid arthritis is not well known. But it is believed that the
interaction between some susceptible genes of the immune proteins like HLA-DR1
and HLA-DR4 with some environmental factors like smoke from cigarettes or a
Lab-On-Chip Electrochemical Biosensor for Rheumatoid Arthritis 159
pathogen like a bacterium that resides inside the gut can trigger the development
of the disease [10]. These environmental influences can cause alterations of a few
proteins, namely vimentin or Type II collagen, and also IgG antibodies.
One of the processes involved in the modifications of these self-antigens is citrul-
lination. Citrullination is a process in which the amino acid arginine in a protein
changes to citrulline. These modified proteins are thought to be a threat to the body
by the immune cells and therefore they gather these self-antigens up and take those
to the lymph nodes. There, the self-antigens are presented to the CD4+ T-helper
cells to activate them. Because of the T-helper cells, the neighboring B-cells which
present these self-antigens are activated too and begin to multiply and differen-
tiate into plasma cells that produce the required autoantibodies. T-helper cells along
with the antibodies enter the circulation to reach the joints. Cytokines, for example,
interferon-γ (IFN-γ) and interleukin-17 (IL-17), are released by T-cells that recruit
macrophages in the joint area. Inflammatory cytokines, such as tumor necrosis factor-
alpha (TNF-α), IL-6, and IL-1, are also produced from the macrophages which,
combined with T-cell cytokines, encourage synovial cells to multiply. This rise in
the number of immune cells along with synovial cells results in the formation of a
pannus, which is a bulging and thick synovial membrane consisting of granulation
or scar tissue composed of fibroblasts, inflammatory cells, and myofibroblasts. The
pannus is capable of dissolving bone as well as destroying cartilage, along with other
soft tissues over time. Activated synovial cells also secrete proteases that can disin-
tegrate proteins present in the articular cartilage. This causes the bones to present
underneath to be stripped off of their protective cartilage and rub against one another
[2, 11].
Additionally, the level of receptor activator of nuclear factor kappa-B ligand
(RANKL), on the surface of T-cells, is increased by inflammatory cytokines. RANKL
is required to bind to RANK, a protein found on the surface of osteoclasts, and this
association is needed to break down bone. High amounts of T-cells are responsible
for carrying out this attachment between RANKL and RANK, and therefore affect
more degradation of bone.
Furthermore, during the course of the disease, immune complexes are formed
when antibodies like anti-cyclic citrullinated peptide antibodies or rheumatoid factor
(RF) bind to their targets, and they fill the space inside the synovial fluid. On doing
so, they turn on the complement system, which promotes inflammation in the joints
and causes injury [2, 11].
Eventually, chronic inflammation results in angiogenesis, allowing the entry of
additional inflammatory cells. Several joints get inflamed and damaged when the
disease starts to advance. However, the inflammatory cytokines don’t merely linger
in the confined joint region. Instead, they infiltrate the bloodstream and cause extra-
articular disorders by reaching many organ systems. They accelerate the degra-
dation of protein in the skeletal muscle and produce collections of round-shaped
lymphocytes and macrophages with a core area of necrosis, in various parts of the
skin and other internal organs. Inflammation of blood vessels may result in various
kinds of vasculitis, creating a risk for coronary artery heart disease resulting from
atherogenesis [2, 11].
Fig. 1 Overview of Rheumatoid Arthritis pathophysiology
In reaction to inflammatory cytokines, the liver produces considerable amounts

of CRP or ESR proteins, which are inflammatory markers, and a lot more hepcidin,
a protein that causes anemia. In the meantime, the fibroblasts that are present in the
lung interstitium get activated and multiply, producing a fibrotic tissue that hinders
alveolar gas exchange. At later stages, inflammation of pleural cavities leading to
pleural effusion can occur, which can further obstruct lung expansion (Fig. 1).
3 Diagnostic Biomarkers
The novel notion of “window of opportunity” demonstrates that early detection of RA

is critical for preventing erosion and stopping the progression of radiologic abnor-
malities. In this context, the identification of biomarkers with diagnostic potential in
the initial stages of the disease remains a hot topic [12, 13].
Rheumatoid factor (RF) and antibodies against cyclic citrullinated proteins (anti-
CCP) are currently used in the ACR/EULAR 2010 criteria for RA diagnosis. Other
diagnostic biomarkers that can aid in the early diagnosis of RA have also been
discovered.
A reaction that is mediated by peptidyl arginine deiminase causes the citrullination
of vimentin, which results in the generation of anti-vimentin antibodies. To increase
the test’s quality and to test the notion that additional alterations could affect vimentin
antigenicity, a mutation where arginine residues were replaced with glycine, was
performed, resulting in the creation of mutant citrullinated vimentin antibodies (anti-
MCV) [14].
A 2010 meta-analysis indicated that there is no difference between anti-CCP and
anti-MCV for the diagnosis of RA, based on 14 trials. Anti-MCV may thus be a line
2 test, performed in patients suspected of this autoimmune disease but negative for
anti-CCP and RF [15].
Antibodies to carbamylated proteins (anti-CarP) were discovered in the blood
of RA patients in 2011. Anti-CCP (generation II), RF (IgM), and anti-CarP were
investigated in a study of 2086 patients with early RA. The anti-CarP test had a
sensitivity of 44% and a specificity of 89%, compared to 54% sensitivity and 96%
specificity for anti-CCP and RF who had 59 and 91%, respectively [16].
The 14-3-3 eta protein is a member of the 14-3-3 protein family. It is present
intracellularly but becomes externalized during the inflammatory process, where it
becomes citrullinated [17].
The specificity and sensitivity of 14-3-3 eta protein for rheumatoid arthritis were
found to be 93 and 77%, respectively, in a trial of 619 individuals [18]. The presence
of protein 14-3-3 eta, as well as RF and anti-CCP, enhances the diagnosis rate from
72% (RF + anti-CCP) to 78% (RF + anti-CCP + 14-3-3eta) in the preliminary
stages of the disease.
C-reactive protein (CRP) is a sensitive marker of systemic inflammation that
is elevated in people with RA. Therefore it could be another potential marker for
increased RA risk [19]. It has been proposed to mediate part of the complement
activation in RA. In a study, serial measurement in male and female blood donors
showed an increase in CRP levels particularly within 2 years of RA diagnosis [19–21]
(Fig. 2).
Fig. 2 Diagnostic biomarkers for Rheumatoid Arthritis

4 Conventional Diagnostic Techniques for Rheumatoid

Arthritis Biomarker Detection and the Challenges
Associated
4.1 Imaging
The standard method for determining the number of anatomic alterations in RA

patients is plain radiography. At a later stage of the RA process, radiographic symp-
toms of RA such as joint space narrowing, erosions, and subluxation appear. Early
signs of RA include synovitis, which is a good predictor of bone degradation [11]. In
early-stage RA, soft-tissue edema and modest juxta-articular osteoporosis may be the
first radiological findings of hand joints [22]. These abnormalities are characteristic
of synovitis, although they cannot be seen in all patients on conventional radiographs,
and they are not precise enough to be used in routine synovitis assessments [23]. Plain
radiography is insensitive for detecting bone deterioration, which is a feature for the
diagnosis of RA, due to the delayed onset of radiographic changes [24]. In the initial
stages of RA, sonography is a reliable test that detects more erosions and in a greater
population of patients than radiography [25]. The development of magnetic resonance
imaging (MRI) imaging has increased the diagnostic capability for diagnosing RA
sooner and distinguishing it from non-RA disorders. In one study, traditional radio-
graphy had a sensitivity of just 13% in detecting bone erosion, whereas MRI and
ultrasonography had a sensitivity of 98 and 63%, respectively, in detecting bone
erosion [26]. Furthermore, when it comes to detecting synovitis of the hands and
wrists in RA, MRI is more sensitive than clinical examination. Sonography and MRI
are more sensitive and appear promising, but they can only be performed in a few
centers [12, 27].
4.2 ELISA-Based Detection
Antibodies against CCP (ACPA) had been detected using first-generation ELISAs,
which had a specificity of about 85 percent for RA and a sensitivity of 65–70%.
Synthetic peptides with a ring configuration due to the formation of disulfide
bridge within molecules are used as antigens in second-generation ELISAs, with
the citrulline epitope in a prominent position. The specificity of these CCPs has
improved to 96–98% without affecting sensitivity [28].
Fibrinogen and fibrin are also considered as citrullinated antigens. Filaggrin and
citrullinated fibrin exhibit a close cross-reactivity, according to studies using citrul-
linated peptide derivatives of the two proteins [29]. The identification of antibodies
against citrullinated fibrinogen in patients with rheumatoid arthritis has been demon-
strated to have high diagnostic specificity and sensitivity in several studies [30]. The
sensitivity of ELISA for RA was around 75%, with a specificity of 98 %. For some
time, an ELISA based on mutant citrullinated vimentin (MCV) has been commer-
cially available for the detection of rheumatoid arthritis, with diagnostic sensitivity
and specificity comparable to that for ACPA.
The challenges faced with ELISA-based methods are longer incubation time, a
larger sample volume, and more intermediate sample preparation processes.
4.3 Chemiluminescence and Fluorescence-Based Detection
Sandwich detection techniques based on chemiluminescence have also been devel-

oped for the detection of CRP and RF. Optical biosensors based on fluorescence
and chemiluminescence technologies have demonstrated their ability to detect
biomarkers associated with RA with decreasing LODs, long dynamic ranges, and
high specificity. They are becoming a significant technology that allows for the
provision of a reliable, highly sensitive, reusable, and fully automated solution for
the diagnosis of RA [31, 32].
Despite their advantages in the field of diagnosis, they have certainly added disad-
vantages too. These include high expenses, longer assay duration, less compatibility,
and limitation to a certain application [33].
4.4 Radioimmunoassay-Based Detection (RIA)
CRP is a long-established acute phase reactant, whose serum levels can elevate
following most types of tissue injury, infection, or inflammation. The serum CRP
levels can be persistently elevated in chronic active inflammatory diseases such as
RA [20]. Radioimmunoassay determines the concentration of an antigen in a sample
based on the competitive binding between radiolabeled antigen and unlabeled antigen
for its specific high-affinity antibody. Solid-phase RIAs had been developed for the
quantification of CRP in body fluids such as human serum and cerebrospinal fluid.
The findings showed that CRP exhibited the properties of an acute phase protein in
infection or inflammation, and that it could be used to monitor the clinical course of
the disease [34, 35].
There are numerous challenges while using RIA. The first is the unavoidable radia-
tion hazard that comes along with it. This is why laboratories require a special license
to handle radioactive materials used in this assay. Also, special storage arrange-
ments for radioactive material are mandatory. Highly skilled and trained persons are
required to run the assay, and proper radioactive waste disposal arrangements are
necessary.
4.5 Surface-Enhanced Raman Scattering-Based Assay

(SERS)
The use of SERS-based approaches in clinical diagnosis is gaining popularity. SERS-

based immunoassays have already been used to quantify a number of biomarkers.
A sandwich immunocomplex platform is a more frequently used detection approach
using a surface-enhanced Raman scattering-based assay. Magnetic beads have
recently been used to build a fast SERS-based test method. To aid in the early diag-
nosis of RA, a SERS-based immunoassay was built for the early diagnosis of RA in
human serum. The assay was based on the formation of a sandwich-type immuno-
complex involving CCP-conjugated magnetic beads, ACPA from the serum sample
and anti-human IgG (or anti-ACPA)-conjugated SERS nanotags [36, 37].
This detection technique has the limitation of the requirement of a highly reliable
system, and it is also an expensive one.
5 Importance of Early Diagnosis of Rheumatoid Arthritis
Early diagnosis of rheumatoid arthritis can have an impact on the disease’s progres-
sion. If discovered during the first 2 years of disease development, it can help
avoid articular damages and bone and joint degradation. When disease-modifying
antirheumatic medicines (DMARDs) are prescribed early after a diagnosis, it is
possible to achieve remission. The mechanism of action of DMARDs is unique,
interfering with crucial pathways in the inflammatory cascade [38]. Early diagnosis
of RA is critical for the beginning of treatment, otherwise, the disease may advance
to severe forms, necessitating more vigorous therapy with potentially dangerous side
effects [12].
6 Biosensor as a Device for “Rapid and Early Diagnosis”
According to IUPAC, the definition of biosensors encompasses “integrated receptor-

transducer devices, which are able to provide selective quantitative or semi-
quantitative analytical information using a biological recognition element” [39].
Biosensors are basically a type of an analytical cum sensing tool that combines
organic i.e., the biological component, inorganic, i.e., the transducer, and the signal
processing components to detect a specific analyte or molecule. A biosensor gives an
amplified output and processed information about both the presence and the quantity
of the analyte in the sample. It does so by integrating a recognition element which
can be an antibody or aptamer, into a transducer which in turn generates the signal
in the sensor [40, 41].
Fig. 3 Working principle of a biosensor
Recent advances in the fields of nanotechnology and microfluidics are opening up

new avenues for the development of multidimensional biosensors, which is the next
upcoming step to the world of biosensors [42, 43]. Some of these physicochemical-
based biosensors have already been commercialized as rapid diagnostic tools;
however, some are yet to be made available on the market as they are still in the stage of
development. This is because some are based on the principle of CRISPR-based tech-
nology, isothermal amplification, microfluidics, microarray, and many others, which
are quite modern technologies and would require further research but are predicted
to be revolutionary technologies that would make the detection of analytes so much
easier [44]. Various nanomaterials like silica-based nanoparticles, metallic nanopar-
ticles, nanotubes, TiO2 , graphene, and ZnO show promising results in increasing
sensitivity and selectivity even when there is an extremely low limit of detection
(LOD), which suggests the better scope of enhancing biosensor efficiency in the
future [45–47] (Fig. 3).
6.1 Working of a Biosensor
Biosensors are made up of three parts:
6.1.1 An Element for Recognition
It recognizes a specific analyte or multiple analytes present in the sample.

6.1.2 A Transducer
It is responsible for generating the electric signal. The receptor unit sends data to a
transducer unit, which converts the data into an analytical form.
6.1.3 A Signal Processor
It analyzes the sample’s chemical information. This information could have been
derived from the system’s physical properties or from any type of chemical reaction
of a species present in our system [48].
Apart from this, the working electrode, a part of the transducing system, also
plays a key role in the biosensor. The working electrode may be made up of diverse
types of materials, from expensive metal electrodes, namely silver, gold, mercury,
and platinum, to less expensive materials like carbon paste, screen-printed electrodes,
and glassy carbon. The bioreceptor molecules can also be of several types like anti-
bodies, enzymes, dyes, metal ions, and nucleic acids. These are made to attach to the
electrodes for producing an efficient signal and for better detection of our desired
biomarkers or analytes. The working electrode is applied with a potential with respect
to the reference electrode (e.g., Ag/AgCl, saturated calomel, etc.), while the elec-
trical circuit is completed by the counter electrode (e.g., platinum wire, carbon, etc.).
When a negative potential is applied, the working electrode releases electrons into
the solution, and the electroactive molecules get reduced. On the application of a
positive potential, the electroactive molecules get oxidized on the working electrode
[49].
Further analysis of the reaction is done through various electrochemical tech-
niques like cyclic voltammetry, differential pulse voltammetry, chronoamperometry,
square wave voltammetry, electrochemical impedance spectroscopy, etc.
6.2 Types of Biosensors
Based on the distinct types of systems for generating a signal, biosensors can be
classified as:
6.2.1 Electrochemical Biosensors
An electrochemical sensor is a device that reads a sample’s chemical information

and converts it into an analytical signal. They involve the integration of a receptor
and a transducer component in a single device that can be easily miniaturized and are
mostly robust, sensitive, and can produce output signals rapidly. They have become
a favorable platform for field analysis or point-of-care diagnosis [50].
6.2.2 Optical Biosensors
These are compact analytical devices that have an optical transducer integrated with
a biorecognition element [51]. Its working principle is based on the fact that the
intensity of adsorbed or emitted light can also change based on the physical quantity
of the analyte that is provided. Molecular interaction between the analyte particles
and substrate receptors will change the refractive index of the medium. The amount
of analyte that is present in the sample can influence the amount of change in the RI,
thus allowing us to measure the target analyte quantitatively. These biosensors are
also used to determine association and dissociation kinetic interactions along with
the affinity of the receptor [52].
6.2.3 Piezoelectric Biosensors
These are the biosensors that base their analysis on affinity-based interaction
recording. The sensors in these devices are the piezoelectric platform or piezoelectric
crystal, whose oscillations change when the desired analyte binds on its surface [53,
54]. The reduction in the oscillatory frequency is directly proportional to the amount
of analyte in contact with the crystal, making it an efficient device for the detection
of an analyte without the requirement of a label.
6.2.4 Thermometric Biosensors
These biosensors work on the principle of the amount of heat generation that arises in
a medium due to a biochemical reaction. This generated heat change in the medium
is then converted into an electric signal allowing quantitative measurement of the
analyte [55].
7 Electrochemical Biosensors
Electrochemical sensors have recently grown to have enormous potential to be the

best-suited platform for biomedical applications. Modifications with various nano-
materials have increased the efficiency of biosensors in identifying a wide range of
biomolecules with high specificity and sensitivity. The biosensor consists of a trans-
ducer that helps in converting biological events into electrical signals. Amperometric
and potentiometric parameters are two of the most commonly used parameters in
electrochemical sensing. Potentiometric analysis converts the analytical data gath-
ered after biorecognition processes into potential, whereas amperometric analysis
is involved in monitoring the constant potential current generated from the reduc-
tion or oxidation of an electroactive species [49, 56]. As a result, they are widely
used in diagnosing diseases to detect appropriate marker proteins, DNA sequences,
or antibodies. Various electrode modifications have been made so that the surface
compatibility of biological species is improved. Electrochemical sensors are made
to be extremely sensitive and can be easily miniaturized and have a short analytical
time. It is also necessary to have a simple instrumentation system in them [57].
An electrochemical sensor is a device that reads a sample’s chemical information
and converts it into an analytical signal. The sample information could have come
from the system’s physical properties or from reactions taking place in a species
that is present in the system. The receptor unit sends data to the transducer unit,
where it gets converted into an analytical form. In electrochemical biosensing for
target analytes, a three-electrode system is typically used. Electrochemical sensing
has been extensively studied in order to identify markers of various diseases such as
cancer, hepatitis, acquired immunodeficiency syndrome, cardiac disease, and urinary
infections [58–63]. Electrochemical biosensors that have been miniaturized and are
implantable have now been regarded to be of immense importance for the detection of
specific metabolites like triglycerides, cholesterol, blood glucose, and various other
protein biomarkers without patient intervention or altering their physiological state
like resting or sleeping or exercising [64–67].
7.1 Measurement Methods in Electrochemical Biosensors
7.1.1 Amperometry
This is a type of electrochemical technique that continuously measures the current

generated from the oxidation or reduction in an electroactive species during a
biochemical reaction [68, 69]. Clark oxygen electrodes may serve as the founda-
tion for the most basic amperometric biosensors, in which the oxygen concentration
available determines the amount of current that is produced. At a given potential,
this is measured when the oxygen gets reduced at a platinum working electrode in
comparison to an Ag/AgCl reference electrode. The current peak calculated over a
linear potential range is proportional to the bulk concentration of the electroactive
species, which is the analyte [39, 69, 70]. Since all protein analytes are not able
to undergo redox reactions in electrochemical reactions, these devices rely heavily
on mediated electrochemistry at the working electrode where the analyte undergoes
electrochemical reaction [68, 69].
7.1.2 Potentiometry
This technique measures the charge potential accumulation at the working electrode
in an electrochemical cell compared to the reference electrode when there is no
or little current flowing between them [39, 69, 70]. Potentiometry, in other words,
gives information about the electrochemical reaction and the ion activity in it [71].
The Nernst equation governs the relationship between potential and concentration
in potentiometric measurements.
7.1.3 Conductometry
This technique assesses an analyte’s (e.g., electrolyte solutions) or medium’s (e.g.,

nanowires) ability to conduct electrical current between reference nodes or elec-
trodes. Most conductometric devices are strongly associated with enzymes, where
an enzymatic reaction between two electrodes changes the conductivity and the ionic
strength of a solution. When the concentration of a charged species present in the
solution changes, in a reaction involved with enzymes, that change can be measured
using conductometric devices [72]. The variable ions in clinical samples, as well as
the need to measure the change in small conductivity in a medium with high ionic
strength, limits the application of conductometric tools [39, 73].
7.1.4 Voltammetry
Voltammetry is the study of currently produced by a system between two electrodes

while applying controlled potential changes to the working electrode as a function
of time. This is typically done with a potentiostat, which is capable of applying
varying potentials to the working electrode with respect to a reference electrode
while measuring the current that flows as a result of the electrode reaction. It is
possible to use reducing and/or oxidizing potentials depending on the procedure.
The current is referred to as a cathodic current when it undergoes a reduction. When
there is an oxidation, the current is referred to as an anodic current. Voltammetry is
a subset of ammeter technology [74].
7.1.5 Impedimetry [Electrochemical Impedance Spectroscopy (EIS)]
EIS can investigate particular processes that may affect the conductive or resistive
properties in an electrochemical system. Hence, EIS is a helpful method in the exam-
ination of biosensor transduction substances, like polymer degradation studies. The
current response (I) is measured by applying a small potential (U) that varies sinu-
soidally [75]. In other words, imaginary as well as real impedance components like
resistance or reactance can be evaluated by EIS after their integration [73, 76, 77].
7.1.6 Field-Effect Transistor (FET)
The field-effect transistor is a form of transistor that controls the conductivity of a

channel between two electrodes (source and drain) in a semiconducting material by
using an electric field (at the gate). When the voltage from the drain to the source
is lower than the voltage between the gate and the source, in linear mode, FET
switches between conductive and non-conductive states similar to a variable resistor.
However, FET acts as a source providing constant current and is frequently used to
amplify the voltage, in saturation mode. The amount of constant current in this mode
is determined by the voltage between the gate and the source. FET devices can work
in low signals and also have high-impedance applications. This could be the reason
they will be preferred more in the future when the field of electrochemical biosensors
expands [69, 73].
Due to qualities such as easy instrumentation, requirement of low sample volume,
easy miniaturization, portability, efficient analysis, high selectivity and sensitivity,
electrochemical detection is anticipated to be used more in the future than other
quantitative detection methods.
8 The Function of Nanostructures in a Sensor
Nanostructures involve nanoparticles, nanotubes, nanowires, nanopores, self-

adhesive monolayers, and nanocomposites that share dimensions with biological
molecules such as DNA and proteins [74]. By combining nanostructures and
biomolecules, an interface composed of nanostructural and biological material with
favorable characteristics and efficient performance is created [78, 79].
8.1 Biocompatibility of Nanoparticles
Nanomaterials have distinctive chemical, physical, and electronic properties by virtue

of their small size, which distinguishes them from larger-scale materials. Nanopar-
ticles tend to have a large surface area, as well as high surface energy, because of
which they can adsorb biomolecules on their surface strongly. On the surface of
the biosensor, this property goes a long way in biomolecule stabilization. When
biomolecules are adsorbed onto the surface of nanomaterials, the biological potency
of the biomolecules is retained, this is because of the biocompatibility property
of nanoparticles [80]. Nanoparticles possess charge in them and hence they can
electrically adsorb oppositely charged biomolecules [74].
8.2 Nanoparticles as Catalysts for Reactions
Compared to bulk materials, because of the high surface energy of nanoparticles, they
tend to be more active. Nanoparticles, especially the ones that are metal in nature, are
used as catalysts in most reactions since they have powerful catalytic effects. This is
because they are efficient in surface activity [74, 81, 82].
8.3 Nanoparticles for Facilitating Better Electron Transfer
A critical step when constructing a biosensor would be when redox proteins, for
example, enzymes, attach to the surface of the electrode. Redox proteins tend to
have a protein shell on their active sites that don’t conduct electricity. This makes it
difficult for any type of electron exchange to take place from the active site of the
protein to the surface of the electrode and vice versa. However, when nanoparticles,
particularly metal nanoparticles come into the picture, the scenario gets altered,
because these provide an effective way to transfer electrons between the electrode
and the protein [74, 81, 82].
8.4 Labeling of Biomolecules With Nanoparticles
The use of nanoparticles to label biomolecules is becoming increasingly essential

for the construction of electrochemical biosensors. Biomolecules that have been
labeled with nanoparticles can retain their biological activity. The analyte binds
with its receptor, and in doing so the analyte concentration can be determined with
the help of nanoparticles electrochemically detecting the binding levels [74]. When
biomolecules labeled with nanoparticles dissolve, the concentration of ions that get
dissolved is estimated with the help of voltammetry stripping. This estimation allows
us to calculate metal effects, that in turn facilitates the measurement of analytes in
trace amounts [84].
8.5 Nanoparticles as Direct Reactants
Nanoparticles that have a high surface energy tend to be more active than bulk
materials. They also tend to execute unique chemical properties [74]. This advantage
can be exploited in several modern electrochemical devices.
9 Overview of Electrochemical Nanobiosensor

for Rheumatoid Arthritis Detection
For the detection of biomarkers associated with rheumatoid arthritis, the application
of electroanalytical-based immunodetection methods has played a significant role in
the past few years. Among the discerning antigens for diagnosing rheumatoid arthritis
like citrullinated fibrinogen, citrullinated α-enolase, citrullinated vimentin and CCPs
have been the most commonly selected antigens for the capture and detection of
ACPAs.
In the work shown by Selvam et al. [33], the use of a nanomatrix probe, namely,
the polyaniline (PANI), nanowire-gold nanoparticle (AuNP), nanomatrix enhanced
the capacity of loading of ACPA on the nanomatrix which further improved the
sensitivity of the ACPA immunosensor. Also, an enhanced electrocatalytic perfor-
mance was observed, which was attributed to the molybdenum disulfide-PANI matrix
(MoS2 /PANI) modification on the screen-printed electrode (SPE). Additionally, the
PANI base matrix made it easier to immobilize CCP on the electrode surface by
forming stable amide bonds between CCP and PANI. Bovine serum albumin (BSA)
was further used as a free-site blocking agent. ACPA detection was carried out
in phosphate buffered saline and 10% human serum with an LOD of 0.16 and
0.22 IU/mL, respectively, and a logarithmic dynamic range of 0.25–1500.0 IU/mL
using the BSA/CCP/PANI/MoS2 /SPE-based immunosensor. The acquired experi-
mental results revealed increased LODs for a broad range of analytical dynamic
ranges. Because CCP was immobilized, the ACPA immunosensor could discrim-
inate ACPA from other human serum proteins and demonstrated high selectivity.
With low storage stability, the ACPA immunosensor could be stored for three weeks
at 4 °C. It was established that the total time required for ACPA detection, including
CCP immobilization and the final addition of PANI-Au-ACPA, was less than 2 hours.
The constructed sensor has many advantages as compared to other reports.
According to Pruijn et al. [85], ACPAs are the most specific serological indicators
for diagnosing RA, and their presence in patient serum supports clinicians in making
initial treatment decisions. According to research conducted recently, the ACPA
response in RA patients is polyclonal and heterogeneous [86]. An artificial chimeric
peptide synthesized using filaggrin and fibrin was adsorbed to the surface and used
as a biorecognition element on an electrochemical transducer composed of multi-
walled carbon nanotubes and polystyrene (MWCNT-PS) in the research presented by
de Gracia Villa et al. [87]. According to the amperometric response, the transducer
construction process demonstrated good repeatability and reproducibility from an
electrochemical standpoint. The resulting immunosensor was first tested in rabbit
sera that had been injected with the synthetic peptide before being used in human
sera.
In a work conducted by Ma et al. [88], an electrochemical immunosensor without
the use of any label was prepared for the capture of ACPA, where they used nitrogen-
doped graphene modified with AuNPs, as an electrode substrate material and CCP
as the biological recognition material. The negatively charged gold nanoparticles
(AuNPs) electrostatically interacted with the positively charged amine groups of
CCP to immobilize CCP on the electrode surface. CCP was used as a probe to
capture ACPA on the surface through affinity binding. The combination of antigen
and antibody created an insulating barrier that prevented the transfer of electrons.
As a result, the square wave voltammetry response of the sensor decreased with the
increase in ACPA concentration. The proposed ACPA immunosensor has a wider
linear range of 0.125–2000 pg mL−1 and a lower LOD of 0.0125 pg mL−1 owing to the
strong conductivity of nitrogen-doped graphene and the favorable biocompatibility
of AuNPs. It can also be employed in human serum samples with satisfying test
Table 1 Comparison of electrochemical nanobiosensor for the detection of ACPA with other
reported works
Detection assay Linear range Detection References
limit
Surface-enhanced Raman scattering 0–100 pgmL−1 13 pgmL−1 [35]
Electrogenerated chemiluminescence 1–15,000 pgmL−1 0.2 pgmL−1 [86]
immunosensor
Electrochemical immunosensor 0.125–2000 pgmL−1 0.0125 [85]
(BSA/anti-CCP/AuNPs/N-G/GCE pgmL−1
Electrochemical square wave voltammetry 0.25–1500.0 IU/mL 0.16 IU/mL [32]
results making it a worldwide applicable platform in the field of biological analysis

(Table 1).
CRP is a well-known hallmark of inflammation. A conductive hybrid nanomate-
rial made of AuNPs and ionic liquid functionalized MoS2 (IL-MoS2 ) with a large
specific surface area was developed and used to adsorb capture antibodies against
CRP in a study conducted by Ma et al. [88]. Subsequently, using π- π stacking,
1,5-diaminonaphthalene (DN) was attached onto GO, where iridium nanoparticles
(IrNPs) were loaded next. This hybrid has been described to mimic catalase and
peroxidase activity, and therefore, was used as a tag to label secondary antibodies
against CRP. The combination of a large surface area provided by AuNPs/IL-MoS2
and enhanced electrocatalytic properties of IrNPs/GO-DN toward H2 O2 reduction
caused an extremely sensitive detection. The linear range of this immunosensor
was 0.01 to 100 ng mL−1 , with an LOD of 3.3 pg mL−1 . Low cost, great sensi-
tivity, low interference, and low LOD were all features of this sensor. This CRP
immunosensor can be used to analyze real serum samples and produce excellent
findings, demonstrating that the immunosensor has biomedical detection capacity.
In a study conducted by Tang et al. [91], one-pot biomimetic mineralization was
used to create a novel nanoflower composed of BSA-anti-CRP antibodies-copper
phosphate hybrid (BSA-Ab2 -Cu3 (PO4 )2 ) as a signal amplifier for an electrochem-
ical non-enzymatic immunoassay for the detection of CRP. Inorganic phosphate
ions were first used for electrochemical transduction in this study, in the form of
the nanoflower composite BSA-Ab2 -Cu3 (PO4 )2 , as mentioned by the authors. The
composite had a 3D hierarchical porous nanoflower structure with a large specific
surface area that could immobilize more antibodies and BSA for non-specific site
blocking, significantly raising the sensitivity of the developed biosensor. Cu3 (PO4 )2
hybrid nanoflowers can provide a large amount of PO4 3− for molybdate reactions,
resulting in molybdophosphate precipitates and the generation of redox currents for
much more robust electrochemical signal readout without the requirement of the
enzyme. The fabricated immunosensor performed well in terms of detection, with
a linear range of 5 pg/mL−1 ng/mL and an LOD of 1.26 pg/mL. Furthermore, this
technique has shown great feasibility for clinical sample analysis (Table 2).
Table 2 Comparison of electrochemical nanobiosensor for the detection of CRP with other reported
works
Detection assay Linear range Detection limit References
Label-free immunoassay 0.2–80 ngmL−1 0.04 ngmL−1 [89]
Fluorescent aptasensor 0.05–100 ng mL−1 0.01 ngmL−1 [90]
Molecularly imprinted 0.0625–1 mg L−1 0.0625 mg L−1 [91]
Surface plasmon resonance-based 1.2–80 ng mL−1 1.2 ngmL−1 [92]
immunoassay
Enzyme-free electrochemical 5 pg/mL–1 ng/mL 1.26 pgmL−1 [88]
immunoassay
Electrochemical immunosensor 0.01–100 ng mL−1 3.3 pgmL−1 [87]
10 Real Sample Detection
Sample obtained from the patient, for instance, blood and urine have several surface
fouling components and this component tends to foul the surface of the detector and
show interference. Due to surface fouling noises are introduced in the signal given
by the sensor and results into reduces sensitivity. To overcome this microfluidic and
mems-based devices are incorporated for preprocessing of the sample.
Blood plasma separation: Blood tends to clot outside the human body resulting
in surface blocking of the analytical surface of the sensor. To overcome this blood and
plasma are separated using microfluidic or mems devices. This can be demonstrated
by the application of capillary forces in a microfluidic channel. (https://link.springer.
com/article/10.1007/s10404-017-1907-6). Furthermore with a single junction design
by Tripathi et al. (https://www.nature.com/articles/srep26749) efficient blood plasma
separation can be obtained by varying the flow rate at the bifurcation exploiting
Zweifach-Fung bifurcation law at elevated dimensions. Another approach to separate
blood from the plasma could be using the di-electrophoresis. Dielectrophoresis force
is experienced by a polarized body when it is subjugated to the electric field. Using
these techniques mems devices can be designed. One of the examples is C-MEMS
devices developed by Pramanick et al. (https://iopscience.iop.org/article/10.1149/
MA2018-01/13/1030). In this device, a microchannel was made between a carbon
and ITO electrode. These two electrodes were in capacitive coupling and on the
application of voltage varying at 1 MHz resulted in different velocities of blood and
plasma. Similarly adding a hydrophobic patch in the microchannel results in the
formation of a self-build filter that results in the separation of plasma from blood
(https://www.mdpi.com/2306-5354/8/7/94).
Surface Fouling: Surface fouling affects the sensor surface greatly that causes
the sensor to reduce its effectiveness and results in decreased sensitivity. To sensur
11 Conclusion
Early detection of rheumatoid arthritis can have an impact on the disease’s progres-
sion. Early diagnosis is critical for achieving recovery, where medications can have
a much more significant effect on reducing disease severity. Also, more aggressive
therapy could be avoided, hence lowering the risk of therapy-induced side effects
[12].
Biosensors are considered to be immensely potential diagnostic and decision-
making tools that could aid in improving health monitoring at both the individual and
community levels. The incorporation of nanostructures in the fabrication of electro-
chemical sensors has resulted in the construction of a highly efficient platform that
makes use of both nanotechnology and electrochemical-based techniques. Metal
nanostructures increase the loading capacity of the bioreceptors in electrochem-
ical nanobiosensors, enhancing the sensor’s sensitivity. The electrocatalytic effect of
metal nanoparticles, on the other hand, aids in quicker electron transfer. Rapid transfer
of electrons minimizes response time and increases the sensor’s productivity. As a
result, detections at the attomolar levels are feasible. Nanotechnology and microflu-
idics tools and advancements can contribute to the creation of numerous biosensors
that will provide us with the advantage of rapid diagnosis with extremely low LOD
and more precision at the point-of-care, letting us take a multidimensional approach
in solving various health-related issues [42, 43].
12 Futuristic Strategy for the Detection of Rheumatoid

Arthritis
The first electrochemical dual biosensor was recently developed for the simulta-
neous determination of RF and ACPA based on sandwich-type bioassays for the
target compounds captured between Fc (IgG) or biotin-CCP attached to carboxylated
MBs or neutravidin-functionalized MBs, respectively, and HRP-labeled detector
antibodies [96]. Since there have been no other such demonstrations of electrochem-
ical biosensors based on a multiplex system for the detection of rheumatoid arthritis,
therefore there is a scope for building a novel one. The biosensor can use the benefit
of the simultaneous diagnosis of multiple biomarkers to increase the specificity and
sensitivity and therefore increase the diagnostic rate to a higher level. Also, the use of
nanoparticles will open many advantageous possibilities that will make the biosensor
more efficient.
While fabricating these sensor platforms, the real emphasis is given to the rapid
and cost-effective point-of-care testing (POCT) of rheumatoid arthritis markers with
real samples. Therefore, there has been a high demand to upgrade such systems
for their implementation in bench-to-bedside applications. Multiple strategies which
have been evolved in the area of biosensor development research can be incorporated
to enhance and modify the parameters of these biosensors by either changing the
electrochemical properties, the surface properties or the biorecognition element of

the sensor [97].
Dutta and Lillehoj developed a novel label-free and wash-free electrochemical
biosensor for the one-step detection of Pf HRP2 biomarker in a whole blood sample
without any pretreatment, using a signal amplification step based on electrochemical-
chemical (EC) redox cycling [98]. This innovative sensing platform has not only
simplified the protocol for detection but also decreased the detection time to 5 mins. A
similar strategy can be applied while fabricating the nanobiosensors for the detection
of rheumatoid arthritis biomarkers and thus can be implemented in rapid and user-
friendly point-of-care testing.
Acknowledgements Authors gratefully acknowledge the Start-Up Research Grant (SRG) funded
by the Science & Engineering Research Board (SERB) (SRG/2020/000712), Department of Science
and Technology (DST) (Government of India, Ministry of Science and Technology), (Technology
Development and Transfer, TDP/BDTD/12/2021/General), Indo-German Science & Technology
Centre (IGSTC) (IGSTC/Call 2019/NOMIS/22/2020-21/164), and Institute Scheme for Innova-
tive Research and Development (ISIRD) (IIT/SRIC/ISIRD/2019–2020/17), Indian Institute of
Technology Kharagpur (IIT Kharagpur), India for the financial support.
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35471-8
Transdermal Injection with Microneedle
Devices in Healthcare Sector: Materials,
Challenging Fabrication Methodologies,
and its Limitations
A. Gowthami, B. S. Sreeja, and S. Radha
Abstract In recent years, the management of many health disorders takes place at
home either by community nurses or by patients independently. However, the medi-
cation management inside domestic healthcare situations may be difficult, mainly
while therapy is administered via injection. The large percentage of transcutaneous
injuries during needle handling has become a hazard in healthcare settings. The
proper incineration of needle waste disposal after treatment is a major concern in
the medical field. The cost-effective, biocompatible, portable, microfluidic devices
are a promising technology for monitoring and diagnosing health conditions. One
of the microfluidic device systems is a Microneedle (MN), which is an alternative
method of an oral and conventional hypodermic needle for biomedical applications.
The development of microminiaturized needles with scale dimensions in the order
of 1 mm or less with a biocompatible material is a challenging aspect of today’s
scenario. Microneedle-based devices are customized for a wide range of applica-
tions, including disease detection, drug delivery mechanisms, and metabolic pathway
monitoring. The different types of microneedles are developed based on the appli-
cations and their fabrication methodologies are selected based on the material and
geometrical structure. Numerous fabrication processes of these microneedle devices
from small-scale to large-scale production, with regulatory approval for commer-
cialization, is a challenging perspective. This chapter mainly focuses on the various
types of microneedles and the selection of materials for the microneedle type, the
benefits of microneedle technology in various health sectors, along with a critical
assessment of its possible impact on healthcare being investigated and discussed. It
also elaborates on the different challenging microfabrication technologies and their
limitations for various types of microneedle devices.
Keywords Transdermal delivery · Microneedles · Fabrication · Applications ·

Types
A. Gowthami (B) · B. S. Sreeja · S. Radha

Materials and MEMS Laboratory, Department of Electronics and Communication Engineering,
Sri Sivasubramaniya Nadar College of Engineering, Chennai, Tamilnadu 603110, India
184 A. Gowthami et al.
1 Introduction
An effective pharmaceutical depends not only on its active component but also on the
delivery mechanism to the body. Therefore, it is important to consider an appropriate
delivery method based on the drug characteristics. In general, the most convenient
and simple way of drug delivery is through oral administration, but it can be difficult
to use it with a complex pharmaceutical drug. This challenge can be overcome by
the use of injections and it is a rapid start of pharmaceutical action. However, the
injection with a hypodermic needle requires specialization, and patient compliance is
limited. The future best drug delivery strategy should therefore be as straightforward
as oral administration and have a high solubility and great health benefits as a like
injection.
The transdermal administration of a drug has the benefit of continuous pharma-
ceutical release to the exact site of action without any alteration in drug concentra-
tion. Yet, the skin outermost layer called stratum corneum acts as barrier and makes
drug delivery challenging [1]. Microneedles (MNs) serve as a delivery system for
transdermal medication; they are simple to use on oneself and have a high level of
medication bioavailability. Additionally, it is a non-invasive and painless approach
that aids in the quick passage of drug through the stratum corneum. This tech-
nology has unique features such as safety, patient compliance, self-administration,
increased permeability, and better performance [2]. Although it has numerous bene-
fits, it also has some drawbacks. The sensitive skin may subject to irritation or allergy.
In some cases, the tips of microneedles may break and causes issues inside the skin.
These limitations are solved by using excellent microneedle material selection. The
microneedles are produced in such a way that it bypasses the stratum corneum and
deliver the entire dose of loaded drug directly into the epidermis layer [3]. More-
over, the medication dosage, delivery rate, and effectiveness of drug passage can
be controlled by geometry and drug composition. Studies have been done so far on
microneedles designed to deliver drugs and cosmetics that were made with a variety
of fabrication techniques [4].
2 Patient Monitoring: Limitations and Challenges

of Therapeutic Monitoring in the Health Sector
In most countries, chronic diseases are associated with high healthcare costs and
lower societal productivity. Modern technology tools should be exploited to their
full potential in an effort to minimize healthcare costs, improve patient moni-
toring through continuous assessment of symptoms and indicators of disease, and
ensure compliance with self-management initiatives and chronic disease prevention.
Because of the inability to notice difficulties linked with the drug therapy, inadequate
monitoring might result in the emergence of Adverse Drug Events [5].
Transdermal Injection with Microneedle Devices in Healthcare … 185
In general, oral delivery is still the most preferred method for the administra-
tion of active pharmaceutical ingredients because of the advantages such as self,
pain free administration, more safe, and better patient compliance. Even though oral
administration has many benefits, it has some drawbacks, such as the possibility of
first-pass effect, the drug concentration may be reduced [6]. In addition, drug absorp-
tion may be limited by intestinal permeability and solubility, which may naturally
limit the bioavailability of medications. These problems repeatedly appear during
the delivery of biopharmaceuticals. As a result, intravenous (IV) injection is one
of the most promising drug delivery systems, as it can accomplish precise dosing
with the balance of high bioavailability. This system can cause some discomfort,
and sometimes skin-pricking leads to blood drawing. One of the major problems is
the sharp biomedical waste generated after usage of injection. The transdermal route
has been investigated as another potential route for improving peptide medication
delivery in order to potentially overcome some of these drawbacks [7].
Transdermal drug administration has gained popularity during the past ten years as
a result of its advantages over traditional oral dosing forms. By 2025, the market for
transdermal medication delivery is anticipated to increase and reach around $95.57
billion [8]. The different approaches of drug administration are depicted in Fig. 1.
Transdermal Drug Delivery (TDD) is a painless method of injecting the drug into the
stratum corneum layer and the drug reaches into the dermis layer without any skin
damage. TDD is superior to other traditional drug delivery methods in several ways.
By offering a non-invasive alternative to parenteral routes, it can get around problems
like needle fear. Numerous placement choices on the skin for transdermal absorption
are possible due to the skin’s enormous surface area and ease of access. It keeps medi-
cation concentrations consistent despite repeated dosage administration, and plasma
level instability brought on by oral dosing and injections, and it makes it simple
to administer drugs with short half-lives. TDD bypasses pre-systemic metabolism,
resulting in increased bioavailability [9]. In general, transdermal systems are econom-
ical and affordable, and the market available patches are used for the medication for
up to 7 days. These patches are cost-effective when compared to other therapies.
With little risk of systemic toxicity, the transdermal route allows the use of powerful
drugs [10].
Furthermore, the World Economic Forum has named this distribution platform as
one of the “Top 10 Emerging Technologies of 2020.” The World Economic Forum’s
support for the quick commercialization of MN products presently through regula-
tory evaluation and development strengthens that position. To guarantee that MN
technology may have a favorable influence on patients and doctors across the whole
medical profession, it is crucial that regulatory oversight for this growing technology
is thorough and that all aspects of commercialization are thoroughly handled [11].
Fig. 1 Different approaches of drug administration
3 Evolution of Microneedle
Direct drug administration through the skin is referred to as transdermal delivery. The
most popular method involves using hypodermic needles, which can cause pain to
patients, needle aversion, and even the potential for infectious disease transmission
[12]. These conventional injections can be substituted by microneedle technology,
which is described as the non-invasive delivery of drugs through the skin surface. This
technology has received interest from various research organizations and businesses.
Microneedle delivery system is an appealing substitute for drug delivery technique
to overcome the drawbacks of current traditional approaches [13]. The microneedle
technology eliminates the needle phobia and more attracted the attention of the
healthcare community. This technology also ensures the proper safe needle disposal
is an added factor.
Microneedle patches are considered as minimally invasive devices that pierces
the stratum corneum layer of the skin without pain and drawing blood. Microneedle
devices are made to pierce the epidermis and deliver a medicine directly to the micro-
circulation beneath. These typically feature a variety of 50–900 µm long micron-
sized projections. Needle length increment leads to more pain incredibly. Normally
the needles have at least a certain length to overcome any deformation in the epidermis
layer [14]. As compared to traditional needles, it attracts the industry with benefits
such as non-hazardous, patient comforts, less painful, and cost-effective. Various
designs of microneedles are progressed under research for efficient drug delivery
Fig. 2 Important developments in microneedle devices. Reproduced with permission from [16–31]
to avoid breakage of the needle. Many researchers reported that the sharpness and
strength of the needle were decided by the proper material composition [15].
It is still challenging and difficult to exhibit the research idea at industry level.
Some important problems and difficulties should be swiftly taken into account in
order to move this novel technology from the lab level to practical products in
the pertinent markets. Few of the innovative and significant advancements in MN
research have been outlined in Fig. 2 [16–31].
4 Microneedle Classification
Based on fabrication, microneedles are generally divided into two categories, In-
plane and Out-of-plane microneedles. In-plane MNs, needles are placed parallel
to the substrate surface, and for out-of-plane MNs, needles are protruded out of the
substrate surface. Out-of-plane MNs are ideal for two-dimensional geometry creation
through wafer-level processing, but In-plane MNs are very difficult to produce with
two-dimensional geometry [32].
Based on design, microneedles are classified as Solid, Hollow, Coated, Dissolving,
and Hydrogel forming MN. The different types of microneedle representation are
displayed in Fig. 3.
Solid Microneedles
Solid MNs are normally work in the principle of Poke with patch approach, it punc-
tures a hole in the skin of the stratum corneum. They provide passage for the drugs to
enter into the epidermis layer. The drug penetration or drug delivery rate depends on
the depth of the opened pores. The micropores created on the skin that remain open for
a week may cause infections. It is a major drawback for these types of microneedles
Fig. 3 Representation of Microneedle types a—Solid MNs; b—Hollow MNs; c—Coated MNs;
d—Dissolving MNs; e—Hydrogel forming MNs [33]. Reproduced with permission from
Sharma et al., Materials Science and Engineering C, (2019). Copyright © 2019 Elsevier
[34]. The materials used for developing solid MNs are typically silicon, metals, and
polymers. Silicon is a fragile material and may break in the skin from an extended-
release patch. The fabrication process for silicon-based solid MNs is very costly.
The first silicon-based solid microneedles were manufactured using microfabrica-
tion technology. Some other fabrication methodologies are wet etching technology
using mixtures of chemicals called HNA (Hydrofluoric acid, Nitric acid, Acetic acid)
and reactive ion etching [36]. Some advantages of metal-based solid MNs are cost-
effective and they have good mechanical strength. The frequently used materials
for the fabrication of metal-based solid MNs such as stainless steel [35], titanium,
and nickel. Polymer MNs are fabricated based on a mold-based method and usually
made up of biocompatible polymers such as polyvinyl pyrrolidone, poly-lactic acid,
poly-glycolic acid, and their copolymers. The fabrication method of polymer-based
solid MNs is very inexpensive and easy for mass production.
Coated Microneedles
Based on the "coat and poke" strategy, these MNs are fabricated and the drug solution
is applied to the microneedles before they are punctured into the skin. The coated
drug will be dissolved and deposited into the skin surface by entering into the dermis
layer. The amount of drug penetration into the skin mainly depends on the needle size
and thickness of the drug-coated layer. But the important constraint is only a limited
amount of drug can be coated on the body of the microneedle. Recent innovations in
coating methods are developed for efficient Coated MNs in the field of drug delivery,
biopharmaceuticals, and disease diagnosis [37]. It follows a single-step process of the
fabrication either by dipping or micromolding techniques, with the use of materials
such as stainless steel, titanium, and polymer.
Dissolving Microneedles
Dissolving microneedles are created using biocompatible (or) bio-degradable
polymer (e.g., sugar, polyvinyl-pyrrolic (PVP), polyvinyl alcohol (PVA), poly-lactic
acid (PLA), and poly-glycolic acid (PGA). They are fabricated based on the “poke
and release” principle. The microneedle patch is loaded with the drug and it is inserted
into the skin. The drug enters into the dermis layer and the dissolution of the drug
takes place and it is released after a period of time. The dissolution of the drug
depends on the polymer composition and the fabrication of the molding process.
The preferred fabrication process for the dissolving microneedles such as micro-
molding fabrication, which is a one-step process always convenient for patients [15].
It involves a polymer solution poured into mold structures and allowing the mold to
dry under ambient conditions. A less expensive fabrication process with better patient
compliance is the major benefit of dissolving MNs. The stability can be improved
by processing the polymer under high temperature and extreme pH. The hazard of
polymers deposition into the skin can be avoided by the proper selection of biocom-
patible polymers for drug delivery. Water-soluble polymers are preferably used for
eliminating biohazardous sharp waste in the skin layers [38]. Some literature works
with the solid microneedle developed as coated or dissolving patches are displayed
in Fig. 4.
Hollow Microneedles
Hollow MNs are designed with hollow holes or lumens in the middle of the needles
[39]. These empty spaces are filled with a drug solution. Some designs of hollow
MNS have holes at the tip of the needles to deliver the drug into the inner layers of
the skin using methods like diffusion, pressure, electrical assistance, or mechanical
vibration. These hollow microneedles can bypass the stratum corneum and directly
enters into the epidermis and deeper dermis layer. The principle called “Poke and
flow” is suitable for the hollow microneedle to deliver the drug into the skin [3, 40].
Based on internal bores or lumens, the hollow microneedles can be classified
as side opened single lumen, tip opened single lumen, side opened double lumen
and tip opened double lumen. Preferably, side opened double-lumen-based hollow
microneedles are more suitable for drug transport. Hollow MNs occupy larger doses
Fig. 4 Solid microneedles composed of stainless steel (i and ii) and titanium (iii and iv) b Coated
microneedles composed of stainless steel (i and ii), silicon (iii and iv) and titanium (v) c Dissolving
microneedles composed of CMC (i), HPMC (ii) and PLGA (iii) d Hydrogel microneedles composed
of HA (i and ii), PVA (iii) and alginate (iv) [1]. Reproduced with permission from Jung, J.H., Jin,
S.G. J. Pharm. Investig. 51, 503–517 (2021). Copyright © 2021 Springer
of the drug as compared to solid MNs, as it carries more amount of drug in the space
inside the needle. The benefits of the hollow microneedle are that the diffusion of
a drug is deeper into the skin, closer to blood vessels. Generally, it is preferred for
high molecular weight substances like peptide or proteins, DNA or RNA molecules,
and vaccines [41]. Continuous flow rate to be maintained with the adjustment of
microneedle bore [42]. The major restrictions with the hollow type needles are flow
resistance and clogging of the solution in the needle tip. Change in the hollow bore
will increase the flow rate but it leads to a decrease in the strength and sharpness of
the needle. To increase the needle strength, at times a metal coat is applied at the
needle tip. The ongoing research studies with these limitations led to the innovative
design of hollow microneedles. Side-opened sharp-tip hollow microneedles [43] are
Fig. 5 Hollow microneedles made of silicon and polymers [44]. Reproduced with permission from
Yeu-ChunKim et al., Advanced Drug Delivery Reviews, (2012). Copyright © 2012 Elsevier
repeatedly investigated as it leads to less prone of clogging and tranquil to insert.

Predominantly used materials for the fabrication of hollow MNs are silicon, metals,
polymers, glass, and ceramic. These microneedles are fabricated using processes
like deep X-ray photolithography, laser micromachining, deep reactive ion etching,
metal electroplating, two-photon polymerization, and a unified molding technique.
The different types of the fabricated hollow microneedle are shown in Fig. 5.
Hydrogel forming Microneedles
This type of microneedle is made up of super-swelling polymers and these materials
have a better capacity for water absorption. When the MN is injected into the skin,
these polymers absorb the water and get swelled due to the presence of body fluid.
This result in the development of conduits and these allow the discharge of drugs into
the microcirculation from the reservoir. After injection, needles quickly absorb inter-
stitial fluid from the tissue, causing the medication to diffuse from the patch through
enlarged MNs. Specific polymeric components, such as poly methyl vinyl ether-
co-maleic acid (PMVE/MA), carboxymethylcellulose (CMC), and amylopectin, are
combined in aqueous solutions to create these types of microneedles [45].
5 Existing Methods: For Microneedle Fabrication
In the past few decades, the field of MN manufacturing technologies has experienced
a steady flow of research and invention, with both conventional and cutting-edge
methods being researched. The selection of an appropriate manufacturing method
for microneedles depends on drug type and dose, targets for use, optimized geom-
etry and material. Most of the existing fabrication methodologies are very costly,
comprise tedious procedures, and are challenging to implement for batch produc-
tion. Most of the future research focuses on the cost-effective, repeatable fabrication
process to enhance the usage of microneedle usage in the market. In the case of the
financial side, micromolding or solvent casting methodology is more preferred [46].
However, MEMS-based metal or silicon microneedles are manufactured to obtain
better precision and to ensure the repeatability of needle production. The various
MN fabrication techniques are summarized in Table 1 [47–65].
Table. 1 Available microneedle manufacturing techniques

References Fabrication method Material used for fabrication Type of MN Geometry
[47] 3D microlens mask Glass Solid Pyramidal
Lithography
[48–50] Deep Ion reactive Silicon Solid, Conical,
etching, wet or dry Hollow Bevel
etching
[51, 52] Laser ablation, etching, Polyhydroxyalkanoate, PMMA Solid, Conical
and micromolding Hollow
[53, 54] Sacrificial PLA mold, Metal Hollow Conical,
Micromolding and Pyramidal
Selective
Electrodeposition
[55, 56] Photolithography PEGDA Solid, Cylindrical,
Hollow, conic,
Dissolving Pyramid
[57–59] 3D Printing PEGDA, PVP, PEGDMA, PLA Hollow, Square
(microstereolithography, Solid, Pyramidal,
Two-photon Coated Conical
polymerization)
[60, 61] Soft Lithography Polycarbonate MN master Solid, Square
mold, thermoplastic Hollow Pyramid
polyurethane (TPU), PDMS
[62, 63] Atomized spraying Polyvinyl alcohol, Dissolvable, Pyramidal
process polyvinylpyrrolidone, Hydrogel
carboxymethylcellulose,
hydroxypropylmethylcellulose,
sodium alginate
[64, 65] Pulling pipette Glass Hollow, Conical
Solid
6 Benefits of Microneedle Technology in Various Health

Sectors
In recent years, the use of needle-free transdermal administration methods moved

closer to reality. Particularly, microneedle technologies came into reality and gained
an attention of the healthcare community because it eliminates the issue of needle
phobia. In addition, the usage of many biocompatible material made needles ensures
the safety issues typically connected with needle disposal. Due to the small size of
the needles, physical harm to the skin is low and the pores close within a few hours
of the initial treatment. However, numerous studies have shown that as compared to
conventional injection systems, the risk of infection from the use of microneedles is
significantly lower. The introduction of needles made up of biocompatible polymer
can further lessen the risk of infection from germs that may have unintentionally
been pulled into the channel during patch administration [14]. Currently, the usage
of microneedles is being expanded into new domains, such as disease diagnostics,
immunobiological administration, and cosmetic applications as described in Fig. 6.
Immunobiological administration
Due to the COVID-19 pandemic, there has been a huge surge in research into
microneedles and their wide benefits for the vaccine delivery and medications.
Microneedles as a method of transdermal administration were created in 1976, but
this idea was only recently applied to vaccination. Becton, Dickinson, and Company
created SoluviaTM as the first intradermal vaccine. The vaccination antigen can
be delivered into the dermis layer of the skin using the microneedle patch, which
consists of a 30-gauge metallic microneedle implanted 1.5 mm into the skin. This
Fig. 6 Various microneedle fabrication methodologies and their applications

method was used to provide the influenza vaccine [66]. Cassie Caudill et al fabri-
cated the microneedle patch using 3D printing and the transdermal vaccination
provides many benefits over conventional vaccination such as less skin damage,
self-implementation, minimally invasive, the potential to reduce cold chain depen-
dency and there is no need of professional administration as like hypodermic needle
[58].
Transdermal vaccination using biodegradable microneedles is a rapidly emerging
area of research and application [67]. Dissolving microneedle patches made up of
polymer material is very effective for vaccine delivery as they dissolve quickly in
the skin and will not leave any biomedical waste [68].
Disease Treatment and Diagnosis
The most popular use for microneedles is still the transdermal drug delivery for
disease therapy. In case of cancer disease, the effect of chemotherapy or surgery can
cause serious side effects and in some anticancer therapy, only lesser amount of drug
reaches the tumor site. The development of MNs offers a practical and minimally
invasive method for administering drugs topically to treat surface malignancies. In
general, MNs can lessen the effects of systemic toxicity as well as enhance drug
distribution in the diseased position. Through MNs, the antigens enter the body and
set off a generalized response that stimulates the immune cells, killing cancer cells
[55]. Lan et al reported a method to cure cancer by developing a dissolving MN
patch for the transdermal delivery of cisplatin to kill cancer cells. The cancer cells
are killed by the dissolving patch loaded with pH responsive lipid nanoparticles by
injecting directly into the tumor site. The author ensures the safety after admin-
istration and this method exhibits minimal side effects and less systemic toxicity
[69]. In another study, researchers developed polymer-based dissolving microneedle
patches for the treatment of breast cancer. The PVA and PVP microneedles are loaded
with the combination of doxorubicin and docetaxel in aid for the photothermal and
chemotherapy and increase the efficiency of cancer treatment [70].
For diabetes patient, the complex and multiple daily injections will reduce the
patient compliance and increases the risk of hypo glycemia. The use of hypo-
dermic needles in conventional ways has been discovered to cause tissue trauma
and extremely discomfort. Nowadays, microneedle patches are used promisingly
for diabetes management, which is an added benefit for patients with uncontrolled
glucose levels or those at high risk of hypo glycemia. MN patches will release the
medication when the blood glucose level raises and acts as an emerging drug delivery
system that is being used to treat diabetic patients successfully [5]. Yu et al. devel-
oped insulin loaded biodegradable dissolving microneedle patch for the management
of diabetes. The MN patch is made up of alginate and hyaluronate, these are non-
cytotoxic and also improve the mechanical functionality. The system exhibits a more
sustained release of insulin and avoids steep fall in glucose levels [71]. Chang et al
developed a swellable microneedle patch for the diabetes management and insulin
delivery. Additionally, the technology is capable of extracting ISF instantly to detect
both cholesterol and glucose levels and this value is observed to be the same as that
measured using a traditional glucometer [72].
Microneedle patches were also applied in the treatment of other diseases. For illus-
tration, Alzheimer disease is treated with tip-loaded dissolving microneedles encap-
sulated with a Donepezil hydrochloride (DPH) drug, resulting in efficient treatment
when compared to oral administration. Problems such as neuropathic disorder are
treated with analgesic microneedle patches as an alternative to clinical treatments.
It was reported that the MN patches transdermally distribute anti-calcitonin gene-
related peptide (A-CGRP) to reduce localized neuropathic pain by blocking CGRP
receptors [73].
Cosmetic applications
Since 2005, when microneedles were first employed in the cosmetics business,
a number of new products have been created, greatly increasing the usage of
these tiny needles to treat skin disorders like wrinkles, scars, seborrheic keratosis,
depigmentation, reducing fat, and other uses [74, 75].
The skin pigmentation is called seborrheic keratosis or lentigo and is usually
observed in elder adults of age >50 and these can be cured by injection of retinoic
acid into the skin. Hibrobe et al. created an all-Trans retinoic acid (ATRA) loaded
microneedle patch (ATRA-MNs) and the patch was applied on the wound site for
once in a week and continuously monitored for the next 4 weeks. This study has
demonstrated that microneedles are used as a safe and effective treatment for senile
lentigo and seborrheic keratosis [76].
A microneedle can deliver active cosmetic molecules straight into the skin by
forming microchannels that do not enter the nerve with better effectiveness and safety.
An innovative cosmetic patch loaded with retinyl retinoate and ascorbic acid is fabri-
cated for anti-wrinkle purposes. The best example of a microneedle-based equipment
is “Dermaroller,” and other approved microneedle devices for cosmetic procedures
are “Derma Pen” [77]. These microneedles assisted equipments are utilized in home
environments, and they are easily sterilizable for repeated usage. This area is still in
a wide range of research with innovations for the delivery of cosmeceutical agents
[78].
7 Microneedles: Challenges Involved in Their Development
Although various uses for microneedles have been proposed, only a small number
of items have actually hit the market. When creating microneedles for the transport
of both small and large molecules, safety and efficacy must be taken into account.
Metallic microneedles may cause irritation, erythema, discoloration, or other negative
side effects because metal traces are left behind beneath the skin. The above issues
could arise if the microneedle is used repeatedly at the same location. The variation
in skin thickness or use of microneedles at different sites may differ in bioavail-
ability, which should be taken into consideration when developing the microneedles
[4, 79, 80].
Currently, research is focused on developing new technologies for the safe admin-
istration of existing molecules, thereby reducing development time and increasing
success rates. For this reason, many in the pharmaceutical industry strive for the
successful development of microneedles as transdermal drug delivery systems [79].
The fabrication of various microneedle types encounters a variety of difficul-
ties. The use of solid microneedles made up of metal may irritate the skin or cause
metallic particles to remain in the skin. Additionally, after usage, they could leave
behind biohazardous sharp waste, thus careful destruction is required. Dissolving
microneedles dissolve completely into the skin and do not leave any waste. The
major challenges in the development will be effective needle penetration, drug inter-
action into the skin, and loading the drug abundantly at the tip. Researchers are also
becoming interested in the usage of hollow microneedles because they have the ability
to administer a wider variety of molecules than other types of devices. However, this
particular sort of microneedle lacks sufficient strength, so the researcher needs to
emphasize overcoming this issue.
8 Conclusion and Future Perspectives
MNs provide numerous chances for the intradermal delivery of a variety of medi-
cations, including biopharmaceuticals. MN approach is a painless, efficient, secure
form of drug delivery when compared to other intravenous methods. Moreover, this
method can be useful for medications with low oral bioavailability. MNs come in
a variety of types and can be created from a vast range of materials. They can be
customized to treat particular diseases because of this feature. Since the academic and
patent literature has accumulated mounting evidence showing that microneedles of
many different designs can be successfully used to deliver drugs, vaccines, and active
substances, the industrial effort to develop microneedle devices is likely to inten-
sify. It is expected that newer applications of microneedle technology will become
more popular. As the number of novel pharmaceuticals with biological origins keeps
increasing, microneedles will significantly increase the transdermal delivery market
value and become more significant during the succeeding years.
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An Economical and Efficient Method
for the Fabrication of Spiral Micromixer
Ekta Tripathi, Pallab Sarmah, Promod Kumar Patowari, and Sukumar Pati
Abstract Micromixers have been widely used due to its vast applications in biolog-
ical, chemical, electronics, and mechanical fields. Micromachining through common
fabrication methods is a challenging issue, therefore it is emerging as the latest
research area. This work presents the rapid and cost-effective method of micromixer
fabrication. It includes two steps of fabrication. Initially, a mold of spiral micromixer
is fabricated on an aluminum workpiece using wire electrical discharge machining
(Wire-EDM). In the Wire-EDM process, molybdenum wire of diameter 180 µm is
used as a tool electrode. Pulse-on-time (Ton ) of 15 µs, pulse-off-time (Toff ) of 6 µs,
peak current (IP ) of 4 A and table feed (S) of 55.6 m/s are set as the cutting parameters.
After machining the mold, in the second step final micromixer is fabricated using
soft lithography technique with poly-di-methyl-siloxane (PDMS). It is confirmed by
visualizing the flow inside the fabricated micromixer that a spiral micromixer may
be used for mixing for various applications.
Keywords Spiral · Micromixer · WIRE EDM · PDMS · Soft lithography
1 Introduction
Micromixing has grown rapidly during the past few decades due to the developments
in the MEMS and microfluidics fields. Microfluidic chips have gained popularity due
to the growing demand for automatic and rapid agricultural, biomedical, and environ-
mental screening. These chips demand micromixing to carry out lab-on-chip biomed-
ical tests [1]. Active micromixers use an external source, such as an electric field,
magnetic field, acoustic field, etc., to initiate the mixing of two fluids, whereas passive
micromixer does not require external means for mixing. It utilizes pressure energy to
E. Tripathi (B) · P. Sarmah · P. K. Patowari · S. Pati

Department of Mechanical Engineering, National Institute of Technology Silchar, Silchar, Assam,
India
204 E. Tripathi et al.
mix the fluids in the microchannel [2–4]. Fabrication of passive micromixers is easy
because they are less sophisticated and require only the introduction of grooves or
modification of the curvature, which can enhance mixing [5–9]. To increase mixing
efficiency, chaotic advection or secondary flows are generated in various innova-
tive designs of micromixers such as sinusoidal, square wave, zigzag, spiral, split,
and recombine micromixers [10–14]. Regardless of the Reynolds number, spiral
micromixers offer superior mixing than serpentine and straight channels [15].
The ever increasing demand for these microchannel devices has shifted the
focus of the researchers towards developing less time-consuming, accurate, and
precise microchannels. Microfluidic devices are generally fabricated on glass, metals,
polymers, or silicon-based materials as per the need for biomedical, electronic,
and mechanical-related applications. However, it is a difficult task to fabricate
these micromixers because of the required precision. Many fabrication techniques
have been developed to fabricate the channel, such as laser ablation, 3D printing,
photolithography, and etching [16–19].
Wangikar et al. fabricated microchannel molds having obstacles using photo-
chemical machining on copper and brass. First, control parameters such as etchant
concentration, etching temperature, and time are optimized to get high material
removal rate, low surface roughness, and low edge deviation. The mold is fabri-
cated with the optimized value of the control parameters [19]. Arockiam et al. [20]
fabricated serpentine micromixers having a width of 700 µm on the glass slide. The
microchannel design was cut from the double-sided polymer-based adhesive tape
which was sandwiched between two glass slides. Tarlet et al. [21] used laser stere-
olithography for mold fabrication having a width of 1.1 mm and soft lithography
for channel fabrication. Mondal et al. [22] also fabricated serpentine microchannel
using wire electric discharge machining (Wire-EDM) and soft lithography technique
and suggested that WEDM could be useful for fabricating the channel with accurate
dimensions.
It is established from the literature study that various attempts have been made to
fabricate the micromixer using the non-traditional machining process. However, no
significant work has yet been presented for the fabrication of a micromixer with a
spiral shape. It motivates the authors to fabricate the spiral micromixer using CNC
Wire-EDM mold. Wire-EDM is a non-traditional machining process that provides
higher accuracy with a faster production rate [22]. It is a commonly used machine
for cutting, so it can be easily accessed in any manufacturing laboratory to cut mold.
Thus, in this work, an attempt has been made to fabricate the PDMS micromixer by
soft lithography technique using a mold machined by Wire-EDM. A computational
study on a spiral micromixer is first presented at Re = 100, and then the detailed
methodology of fabrication of the spiral micromixer using the Wire-EDM process
and soft lithography technique is suggested. Wire-EDM is used to fabricate the mold,
whereas the soft lithography process is used to fabricate the PDMS micromixer.
An Economical and Efficient Method for the Fabrication of Spiral … 205
2 Design and Computational Study
The schematic design for the spiral micromixer is presented in Fig. 1. Two fluids
(water and water-dye) enter into the main channel through Y joining. The width
and depth of the channel are kept at 0.6 mm each. Fluids flow through a straight
section of 3 mm and then through spiral sections of different radii of curvatures.
The minimum and maximum radii of curvature of the spiral sections are 1 mm and
4.1 mm, respectively.
To observe the efficiency of the desired micromixer at higher Reynolds numbers,
a computational study is performed in ANSYS-fluent software at Re = 100. N-S
and mass transfer equations are solved by considering various boundary conditions
for obtaining the velocity and concentration field continuity. Laminar and species
transport model is selected. At the mixer wall, outlet, and inlets, no-slip boundary
conditions, atmospheric pressure, and uniform velocities are applied respectively.
The mass fractions of water in inlet 1 is 1, and in inlet 2 is 0. The SIMPLE algorithm
is utilized for the pressure and velocity coupling for the simulation. The relative
convergence criteria are selected as 10–5 . The mixing index (M) is calculated as
follows [11–13],
[ ( )2
|
| 1 ∑ N
CJ − C
M =1− | (1)
N J =1 C
where N is the number of sampling points, C J and C are normalized concentration

and expected normalized concentration, respectively.
After performing the mesh independent test, 2,733,430 elements are selected
to perform numeric simulations. Mixing characteristics are analyzed in the spiral
micromixer through computational study. Figure 2 shows the concentration contours
of water in the micromixer. The blue color denotes water (concentration = 1), whereas
Fig. 1 Schematic diagram of spiral micromixer

Fig. 2 Representation of concentration contour of water in spiral micromixer
red denotes water dye (concentration = 0). Green color (concentration = 0.5) repre-
sents the complete mixing. When fluid flows through any curve trajectory, it experi-
ences the centrifugal force, leading to the generation of transverse dean flows. The
dimensionless Dean number (De = x 0.5 Re, where x is the ratio of the hydraulic radius
of the channel and radius of curvature; Re is Reynolds number) is used as a measure-
ment parameter for these flows. Due to these centrifugal forces, induced secondary
forces generate a vortex in the fluid flow, promoting the mixing. Hence, from Fig. 2,
86.7% mixing of fluids at the outlet is observed at Re = 100. Microchannels providing
more than 85% mixing can be used for practical applications [7].
3 Experimental Procedure
After confirming the mixing effectiveness of the micromixer, it is fabricated using

Wire-EDM and soft lithography techniques. The fabrication of the micromixer is
done in the order of: (i) mold fabrication using Wire-EDM and (ii) micromixer
fabrication using soft lithography technique.
3.1 Mold Fabrication Using Wire-EDM
Wire-EDM is used to create the mold for the proposed micromixer. To create the
profile, the design is first created with the required dimensions in the RR CAM
plug-in software of CNC unit of the Wire-EDM machine. It is then converted into
a machine file (.rrw) for profile cutting. Aluminum (Al) is chosen as the material
for the workpiece, while a molybdenum wire of a diameter of 180 µm is used as a
Table 1 Set of process parameters

Parameter Pulse-on-time Pulse-off-time Peak current Table feed (S) Wire diameter
(Ton ) (Toff ) (IP )
Value 15 µs 6 µs 4A 55.6 µm/s 180 µm
tool electrode. Based on the different pilot experiments, the process parameters are
chosen to cut the spiral profile as tabulated in Table 1. The photographic image of
the mold for spiral micromixer cut by Wire-EDM machine is shown in Fig. 3. The
dimensions of the fabricated mold are observed using an optical microscope. The
average width of the spiral channel after machining is 603.32 µm, whereas the other
measured dimensions are shown in Table 2. The obtained dimensions show less than
a 3% average deviation from the required dimensions (Fig. 4).
Fig. 3 Photographic image

of spiral mold fabricated in
Wire-EDM
Table 2 Comparison of
Desired dimensions Obtained dimensions % deviation
required and obtained
(µm) (µm)
dimensions with percentage
deviation L1 5500 5378.45 2.2
L2 3000 2890.604 3.6
R1 3400 3508.419 3.1
R2 1100 1104.77 0.4
R3 1700 1646.78 3.1
R4 4100 4153.994 1.3
L3 2000 1906.687 4.6
Fig. 4 Microscopic image of mold fabricated in Wire-EDM
3.2 Spiral Micromixer Fabrication Through Soft

Lithography Technique
After fabricating the mold, it is the time to fabricate the micromixer by casting it
with PDMS in the following steps. PDMS mixture is initially prepared in a beaker
by combining Silicone Elastomer base and curing agent in a mass proportion of
1:10. Weight of these two materials is measured in precision analytical balance.
Manual stirring of the mixture is done for about 10–15 min for proper mixing. While
mixing, air bubbles get trapped inside the mixture. It is necessary to remove these
bubbles. For this purpose, the mixture is held in a vacuum desiccator for almost
an hour. The mold is kept inside the small square glass container where the mixed
solution is poured. After that, it is kept in an open atmosphere for 24−30 h to let the
mixture solidify. The PDMS microchannel is then removed from the master mold.
A continuous microchannel with fine edges and accurate dimensions is obtained
as shown in Fig. 5a. Then, inlet and outlet holes are punched through the PDMS
microchannel using biopsy punch. However, it is open from one side. A closed
microchannel is essential to observe the fluid flow inside the mixer. So, one more
sheet of PDMS is needed to be prepared to cover it. Therefore, a fine sheet of PDMS
solution is prepared and left in an open atmosphere for 8–10 h. The prepared one-
sided open PDMS microchannel is glued to it when it is in semi-solidified condition.
It is again put in an open atmosphere for 20–24 h for drying. After bonding, the final
spiral PDMS micromixer is ready for use and which is shown in Fig. 5b. Thus, the
spiral micromixer is successfully fabricated using mold fabricated by wire-EDM.
Fig. 5 a Open-sided spiral micromixer, b Final spiral PDMS micromixer
3.3 Flow Visualization in the Fabricated Spiral Micromixer
To check the functionality of the fabricated PDMS micromixer, fluid flow is visual-
ized in it, as shown in Fig. 6. A twin syringe pump is used for delivering the fluids
inside the channel. To conduct the experiment, water is used as an inlet fluid. As it
is a colorless fluid, blue and red colors are added to it for better visualization. Water
with red and blue colors is injected from the syringe into the micromixer. As seen
from the inset of Fig. 6, red and blue color fluids are observed in the inlets whereas,
in the outlet, dark violet color is seen. The dark violet color at the outlet depicts the
proper mixing of fluids which is also depicted through computational analysis as
observed in Fig. 2.
Fig. 6 Experimental arrangement for flow visualization

4 Conclusion
In this work, the spiral micromixer of PDMS is successfully fabricated using the soft
lithography method with the mold machined by Wire-EDM. The mold is machined
on an aluminum workpiece using a molybdenum wire of 180 µm at the parametric
conditions as Ton of 15 µs, Toff of 6 µs, IP of 4 A, and S of 55.6 m/s. The observed
machining time is 4.2 min. The average deviation between obtained and desirable
dimensions is less than 3%. The precision of the mold determines the accuracy
of the micromixer. So, through Wire-EDM, it is possible to manufacture mold with
accurate dimensions. Finally, in the fabricated PDMS micromixer, flow is visualized,
confirming the micromixer’s functionality in various applications.
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Damping Estimation and Analysis
for High Performance Inertial MEMS
for Early Detection of Neurological
Disorders During Pregnancy
Sonali Biswas, Anup Kumar Gogoi, and Moushumi Biswas
Abstract High performance inertial MEMS require appropriate damping estimation

and control. In any MEMS device, the ratio of the surface area to volume is large
for which we need to do dynamic performance analysis. Inertial MEMS sensors
find its suitability in varied applications and bio-motion sensors are no exception.
One of such low frequency applications is the early detection of neurological disor-
ders, targeted specially for pregnant women suffering from tremors, epilepsy and
seizures. It becomes very important to capture the most feeble tremor (0 g) to a
sudden jerk (±6 g) where the person usually falls. During Pregnancy, most of the
drugs for curing neuro-disorders are not suitable to be consumed. Therefore, high
performance micro-sensors are required for early detection of tremors (2−12 Hz)
and seizure (0.5−29 Hz). An attempt has been made to design a micro sensor with
adequate damping estimation by comparing it with the existing models. The proposed
MEMS sensor simulated for the target application has an air gap of 23.5 μ which
produces around 0.7 damping ratio. Finally, time dependent analysis is done by
comparing both with intended damping and very low damping value. We obtain
smooth characteristics for the intended damping and overriding oscillations appear
for very low damping. Hence, the proposed inertial MEMS sensor has a dynamic
range of ±6 g, frequency range of 0−25 Hz and damping ratio of 0.7. Therefore, the
proposed microsensor makes it suitable for early detection of neurological disorders
especially during pregnancy addressing maternal health care.
Keywords Inertial MEMS · Dynamic · Damping · Neurological · Tremor ·

Seizures
S. Biswas (B)
Jorhat Engineering College, Assam Science and Technology University, Jorhat, India
A. K. Gogoi
Indian Institute of Technology Guwahati, Amingaon, Guwahati, India
M. Biswas
Silchar Medical College and Hospital, Silchar, India
214 S. Biswas et al.
1 Introduction
Inertial MEMS require adequate damping control for high performance applica-
tions [1]. The performance enhancement of inertial sensors is of utmost importance
for diagnosing neurological disorders especially during pregnancy. Pregnancy is the
most important and beautiful phase in the life of every woman. This crucial period
can however be critical if some of the neurological disorders of the mother, which
need urgent attention remain undetected. Therefore, early diagnosis of some of the
deadliest neurological disorders becomes very essential to preserve the health of
both the mother and the baby which can be fatal during this period. The focus of this
research is early detection of some neurological disorders like epilepsy, parkinsons,
etc. where tremor of very low frequency occurs. Tremor is basically roughly sinu-
soidal in nature. It may be mentioned that tremor occurs in frequency from 0.5 to
12 Hz and its causes may be many [2]. During pregnancy, the tremor action can occur
at almost 0−6 g signifying jerks. Moreover, during this period some women have
convulsions for the first time. There may be many factors which can trigger seizure,
out of which hormonal changes, water retention and stress are the most common.
The combination of any neurological disorder and pregnancy is very risky and fatal.
There are certain drugs available for neuro-degenerative disorders for balancing the
hormone levels but such doses are rarely preferred both by patients and medical prac-
titioners to avoid risks. This is mainly because the physical health does not support
all types of medications. Also, the EEG procedure requires a longer time with a
large number of electrodes on the skull which adds to one’s discomfort. Thus, early
diagnosis requires continuous monitoring. Such wearable sensors must be small and
light weight and can measure low frequency range and must have high performance
w.r.t sensitivity and resolution [3].
There may be several aspects of design modification to cater to a particular appli-
cation, out of which adequate damping control is very important for high performance
applications. In fact, damping is the most critical aspect one has to investigate for
high performance device design [4, 5]. It is very important for micro-sensors to
have a fine control of gap dimensions. Many Researchers have been working on
several aspects of Squeeze film damping. Blech has studied adiabatic processes [4,
5]. Starr was the first to suggest damping control in solid-state inertial sensors [6].
The squeeze film detail was given by Bao [1]. Renolds equation is mainly used for the
analysis [4, 7]. Different Researchers have proposed various Mathematical models
for determining damping [7–9]. Investigations are focussed mainly onthe assump-
tion of correct damping coefficient and damping ratio, for high performance MEMS
applications.
Therefore, estimation of correct damping ratio becomes very important. Hence
an attempt has been made to design an inertial micro sensor of low frequency range
from 0.1 to 25 Hz and ±6 g by estimating its damping for appropriate dynamic
behaviour.
Damping Estimation and Analysis for High Performance Inertial … 215
2 Theoretical Background
Acceleration can be determined by Inertial MEMS sensor in terms of displacement.

Thus, the equation of motion can be written in terms of Eq. 1 [11]. Here m is the
mass, z is the displacement, ζ is the damping ratio, ωn is the natural frequency. It
is the damping ratio and natural frequency which determines the dynamic behavior
[11].
z̈ + 2ς ωn ż + ωn2 z = −a (1)
/
k
ωn = (2)
m
c
ς= (3)
cc
Here C and Cc are the damping coefficient and the critical damping coefficient
respectively.
We have the transfer function that can be written as
ωn2
H (s) =
s 2 + 2ς ωn s + ωn2 (4)
In this system underdamped means ζ < 1 and Fig. 1 shows the settling time and
overshoot.
The time history of the step response of a second-order system found from the
inverse Laplace transform of H(s)/s is given as [1]
Fig. 1 Underdamped
second-order response with
settling time and overshoot
[11]
1
z(t) = 1 − √ e−ςωn t Sin(ωd t + β)
1 − ς2 (5)
where ωd and β are the damped frequency of oscillation and phase lag respectively.
By taking the derivative of Eq. (5) and equating to zero we obtain Mp, the
Maximum Overshoot [11].
− √πζ
Mp = e 1−ζ 2
(6)
Taking logarithm on both sides of Eq. (6) we obtain the damping ratio [11]
|ln M p|
ζ = (7)
π2 + |ln(M p)|2
The peak time is given by [11]

π
tp = (8)
ωd
2.1 Damping Ratio Estimation for High Performance MEMS

Device
The damping ratio is one of the most important parameters of inertial sensors. The
device geometry, pressure, density and fluid viscosity decide the damping. The
damping ratio should limit its value such that the device should not collapse nor
the system becomes too slow and sluggish. Appropriate damping gives a very good
frequency response.
The basic mechanism of air damping for micromechanical devices mainly are of
two types viz. (1) squeeze film (2) slide film.
2.2 Squeeze Film Damping
When the thin film of air which is trapped in the gap interacts with the proof mass,
then the squeeze damping occurs as shown in Fig. 2 [10].
The prediction of damping coefficient is based on some theoretical models for
a given geometry and other related parameters. Different models provide damping
coefficients of different levels of precision [4, 7, 9].
Fig. 2 Squeeze film damping phenomenon
3 Damping Analysis of the Proposed Structure
3.1 Estimation of the Damping Ratio
In any micro inertial sensor, the necessary damping is provided by the gas in the
gap between mass and encapsulation. Mostly the intended value of the damping
ratio which gives stability is 0.6−0.7. Here, the gap is designed to obtain the value
of damping ratio of 0.7. For different models proposed in literature, the degree of
accuracy varies. The geometry chosen was of area 3200 × 3200 μm, with a height
of 250 μm.
3.2 Time Domain Analysis of the Microstructure
The Time Domain Analysis was done for sinusoidal input for different types of
damping. The total response characteristic is the summation of both steady state and
transient response.
4 Results and Discussion
The damping estimation and analysis are carried out by simulating the proposed
quad geometry having a proof mass height of 250 μm using software COMSOL. The
specifications and boundary conditions when given as input and simulated squeeze
film damping results in a pressure distribution which gives an air gap dimension of
18.6 μm. Then with this gap height, the damping coefficient is obtained using the
models proposed by Blech [4], Veijola [7] and Andrew [9] and also obtained by
Table 1 Damping ratio for

Height of the air gap(μm) From simulations
different air gap heights
18.6 ≥1
23.5 0.69
30 0.31
simulation. It was observed that for the air gap height of 18.6 μm, the damping ratio
obtained was greater than 1 which was not desirable. This is because we know for
an almost flat and stable response characteristic damping ratio should be around 0.7.
We then tried to simulate by increasing the thickness of gap height in steps as shown
in Table 1 and finally obtained the desired damping ratio near to 0.7 for a gap height
dimension of 23.5 μm. We, therefore, propose the air gap height design dimension of
23.5 μm as it gives the desired damping ratio. Thus, we can conclude that damping
is controlled by the dimension of the air gap height chosen in the design and we need
to design the air gap height for obtaining the necessary damping.
Also, as we know that Squeeze film damping effect is the interaction of the silicon
proofmass and the air gap, so an attempt was made to obtain the air gap height by
doubling the height of the proofmass (500 μm). In this case, the simulated air gap
obtained was 16.6 μm for which we got a damping ratio of 0.7. From which we can
conclude that by varying the height of the proofmass the air gap can be controlled to
achieve the desired damping ratio.
The simulation results of displacements versus time and total force versus time
are obtained for different air gap heights.
The simulation when done for the air gap height of 18.6 μm, we obtain the
displacement time graph as shown in Fig. 3 and the total force versus time is shown
in Fig. 4. From the response characteristic, we observe that for the chosen dimension
of air gap height, the damping ratio obtained was greater than 1. This shows an
overdamped behaviour where the response very slowly tries to reach the equilibrium.
So, we can conclude that we need to lower the damping ratio in our case and for
this, we need to make necessary design modifications.
Next, we increase the height of the air gap to 23.5 μm and simulate it to obtain the
displacement time graph as shown in Fig. 5 and the total force versus time is shown
in Fig. 6. In this case, we obtain a damping ratio of almost 0.7 which is desirable as
it offers a higher response time, fast settling time and good stability. The response
clearly shows that there is initially some overshoot and then slowly it settles down.
Therefore, we conclude that the air gap height in the proposed sensor should be
designed as 23.5 μm in order to have a control over the damping and maintain the
damping ratio as 0.7.
On further increasing the air gap height to 30 μm we obtain the displacement time
graph as shown in Fig. 7 and the total force versus time characteristic is plotted in
Fig. 8. The response characteristic shows that there is a higher amplitude and after
two overshoots it settles down. Therefore, the damping ratio of 0.31 is not suitable
for the design as the amplitude is very high and will not produce a flat response.
Fig. 3 The characteristic graph for displacement obtained with air gap height of 18.6 μm
Fig. 4 Dynamic characteristics with air gap dimension of 18.6 μm

Fig. 5 The characteristic graph for displacement obtained with air gap height of 23.5 μm
Fig. 6 Dynamic characteristics with air gap dimension of 23.5 μm
Finally, the dynamic characteristic is obtained for both low damping value and the
estimated damping value as shown in Fig. 9 and Fig. 10. It is observed that a smooth
response is obtained with the estimated damping as shown in Fig. 10, as compared
to the one obtained with the very low damping as shown in Fig. 9.
Fig. 7 The characteristic graph for displacement obtained with air gap height of 30 μm
Fig. 8 Dynamic characteristics with air gap dimension of 30 μm

Fig. 9 Dynamic characteristic for very low damping value
Fig. 10 Dynamic characteristic for estimated damping value

5 Conclusion
A detailed analysis of the damping aspects for the proposed MEMS inertial sensor
for a low frequency range of 0−25 Hz has been carried out. It has been shown that for
a thin film height of 23.5μ, the desired damping ratio of around 0.7 is achieved. The
time domain analysis obtained through simulation reveals very smooth characteristics
for sinusoidal input with the intended damping value whereas it shows overriding
oscillations for very low damping. Thus, high performance design of MEMS sensors
can be achieved by appropriate damping control.
From simulations, we also conclude that the air gap height can be varied by
changing the height of the proof mass. Thus, by varying the height of the proofmass,
the air gap can be controlled to achieve the desired damping.
Therefore, the proposed design with appropriate damping can be suitably used
for sensing low g acceleration value (0 to ± 6 g), diagnosing feeble tremors to
sudden jerks in case of neurological disorders especially during pregnancy. Thus,
early diagnosis of such disorders with the help of high performing micro inertial
sensors ensures safety for both the mother and the baby.
Acknowledgements I would like to thank IIT Guwahati for providing the research facilities and the
doctors/Associate Professors from Silchar Medical College, Assam India, namely Dr. Moushumi
Biswas and Dr. Debajit Das for helping me with medical related works. I got to know more on
pathological tremor and other neurological disorders specially for Pregnant woman. Dr. Biswas is
an expert in Community medicine and has immense contribution towards society specially in rural
areas. I would like to thank her specially for arranging my visit to Guwahati Medical and Silchar
Medical for case studies.
References
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motor control. J Neurology, Brain 123 (8), 1545–1567
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tivity for Medical Diagnostic, In proc. Advances in Systems, Control and Automation, Lecture
Notes in Electrical Engineering, Singapore: Springer 442:481–490
4. Blech JJ (1983) On isothermal squeeze films, vol105. p 615
5. Lu Q, Fang W, Wang C et al (2021) Investigation of a complete squeeze-film damping model
for MEMS devices. MicrosystNanoeng 7:54. https://doi.org/10.1038/s41378-021-00279-6
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on solid-state sensor and actuator workshop. pp. 44–47
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Affinity Biosensing: Modeling
of Adsorption Kinetics and Fluctuation
Dynamics
Olga Jakšić
Abstract A vast number of processes that are crucial for biosensing in microflu-
idic MEMS devices for health applications are based on affinity bonding between
analyte biomolecules and functionalized adsorption sites. Whether it is applied for
the development of new drug discovery or new sensors for point of care devices,
the final design relies greatly on modeling these interactions. This chapter aims
to present a compendium of models used for the representation of these interac-
tions. It addresses modeling in time and frequency domain, from a deterministic and
from a stochastic point of view, with respect to monocomponent and multicompo-
nent monolayer adsorption in microfluidic MEMS devices with or without direct
flow-through and mass transfer effects. The goal is to contribute to sequential and
concurrent multiscale modeling by offering a collection of theoretical kinetic models
like pseudo first order and pseudo second order kinetic models. The text includes
the criteria for the domains of viability of the presented models, stochastic simula-
tion algorithms and comparative analysis of analytical vs numerical modeling with
artificial intelligence-assisted approach complementing these methods.
Keywords Adsorption kinetics · Fluctuation dynamics · Immunosensing ·

MEMS · Microfluidics · Neural network fitting · Protein-Peptide Binding ·
Stochastic analysis · Surface Plasmon Resonance (SPR)
1 Introduction
Generally speaking, affinity-based biological or biochemical sensing may relate to

interactions between biomolecules from a biological material (microorganisms, cells
of multicellular organisms, their organelles, tissues, extracellular and intracellular
targets–receptors, enzymes and transporters, antibodies, nucleic acids), a biologi-
cally derived material (engineered proteins and aptamers) or a biomimetic analogue
O. Jakšić (B)
Center of Microelectronic Technologies, Institute of Chemistry, Technology and Metallurgy,
National Institute of the Republic of Serbia, University of Belgrade, Beograd, Serbia
226 O. Jakšić
(synthetic receptors, biomimetic catalysts, combinatorial ligands, imprinted poly-

mers). The MEMS devices investigating these interactions are the focus of research
directed to multitudinous applications, for instance, the development of lab-on-a-chip
devices for high throughput screening and identifying compounds of clinical interest
for therapeutics [1, 2], the development of organ on chip MEMS devices such as a
liver or kidney on a chip [3–9] or the development of lab on a disc MEMS microflu-
idic devices [10]. Microfluidic chips allow for food safety sensing, peptide analysis,
medical diagnosis, DNA purification, PCR activity, pregnancy, glucose estimation
[11] and many more. For all of these applications, the insight into the biomolecular
interactions is of prime importance.
Surface plasmon resonance (SPR), localized SPR and SPR imaging have proven
themselves as prominent techniques in optical biosensing and screening of biomolec-
ular interactions [12, 13]. Mechanisms that ensure high quality readout of SPR-based
refractometric biosensors are related to surface phenomena. The establishing, prop-
agation and extinction of surface plasmon polaritons at a conductor–dielectric inter-
face are greatly affected by every, even tiny, change on the interface between the
plasmonic material of the sensor surface and the surrounding medium with targeted
analytes. Thus the presence of analyte molecules on the sensor’s active surface is
detectable with an extremely high precision. Compared to a majority of other detec-
tion techniques, SPR-based biosensing proved superior in terms of the dynamic
range and the detection limit (even reaching single-molecule sensitivity) [14, 15].
In pharmacology, SPR-based biosensing is the technique of choice for the drug
profiling and screening, antibody characterization, proteomics, and immunogenicity,
for different kinds of pharmaceutical analysis, ADMET studies (Absorption, Distri-
bution, Metabolism and Excretion of chemicals processed by a living organism,
accompanied by Toxicology tests), to name just a few [16].
SPR-based refractometric biosensing allows for the rapid detection, with high
specificity, high-throughput analysis of biomolecular interactions, in real time, on
the label-free assays, in compact, lightweight devices. As such, it allows for quality
laboratory testing, but also for lab-on-a-chip testing in point of care (PoC) or bedside
testing and patient treatment [17, 18]. Preventive measures and therapeutic recom-
mendations in healthcare are often related to oral supplements and food. SPR-based
refractometric biosensing also allows for food and feed quality analysis, by allowing
a fast and accurate detection of the presence of antibiotics, microbes, toxins, pesti-
cides and biomolecules in food, but also of food adulteration and genetically modified
food [19]. In addition, the substrates that support the formation and propagation of
surface plasmon polariton plasmonic waves and the technique of light manipulation
by structures whose parts are significantly smaller than the light’s wavelength bring
benefits and magnify the signal in other sensing techniques such as surface enhanced
Raman scattering (SERS). The development of microfluidic MEMS devices for
healthcare applications is a multidisciplinary meeting point for scientists involved
in engineering, biotechnology, medicine, physics, chemistry, material science, etc.
Although the advancement in such a multidisciplinary field is already noticeable,
there is ample room for further improvements posed by ever growing needs.
Affinity Biosensing: Modeling of Adsorption Kinetics and Fluctuation … 227
In a chemical sense, for screenings and analyses related to human health, the
interactions between proteins and/or peptides plays a great role, for instance in phar-
macology, preventive medicine and sports medicine, but also in everyday life, for the
digestion of food and food supplements and the design of health-promoting foods
[20]. Real-time surface plasmon resonance imaging of protein adsorption–desorption
kinetics is not new, the studies of surface enzymatic reactions on peptide microar-
rays date back to 2004 [21]. However, the topic still remains a subject of vivid
research scrutiny. The current research has new foci of interest, like monitoring
and preventing unwanted adsorption of proteins on walls of microfluidic chips [22].
Once released from the parent protein, depending on the sequence of amino acids
they consist of, bioactive peptides are able to alter the physiological systems in the
human body. Among others, there are anti-obesity and satiety peptides, antihyperten-
sive, antithrombotic, antioxidant, hypocholesterolemic, antimicrobial, cytomodula-
tory, immune-modulatory peptides, opioid peptides, to name just a few, and screening
for new bioactive peptides is one of the attractive research goals [23]. Previous
modeling of enzymatic reactions on peptide microarrays has been often based on
linear, first order kinetic models. However, the domain of validity of these models
is limited [24]. This is especially so when it comes to the detection of extremely
low concentrations such as the detection by the use of nanobiosensors capable of
detecting a single molecule [25].
In a mathematical sense, for mathematical modeling, scaling down to nanoscale
dimensions and to extremely low concentrations makes a great difference. The calcu-
lation apparatus valid for moderate analyte concentrations is usually based on the
Lagergren model of adsorption. It allows for an easy estimate of all figures of merit in
time and frequency domain (sensor response, sensitivity, linearity, and selectivity),
no matter whether we use a deterministic or a stochastic approach in our analysis.
Models valid for extremely low concentrations are generally analytically unsolvable
and one must resort to extensive numerical computations instead. Apart from the
deterministic analytical or numerical approach, the stochastic approach, which takes
into account the true nature of the binding process, is needed, for modeling the fluc-
tuation kinetics in transient states and fluctuation dynamics in equilibrium. Artificial
neural network fitting also complements these methods [26].
Subsequent sections are devoted to modeling. First, in Sect. 2, a general intro-
duction to the system used for modeling biomolecular interactions, along with the
notation and surmises, is given. Then, Sect. 3 outlines modeling in time domain.
The possibility for emerging oscillations in a binding process is discussed in Sect. 4.
Afterwards, in Sect. 5, an overview of the modeling of equilibrium fluctuations
in frequency domain is given. Section 6 gives a summary of the text, along with
conclusions and comments on future prospects.
228 O. Jakšić
2 Affinity Binding and Biosensing: Preamble
When speaking of a biosensor, we refer to a self-contained integrated device capable

of providing specific quantitative or semi-quantitative analytical information using a
biological recognition element which is in direct spatial contact with a transduction
element (IUPAC definition, 1996). In a modern sense, it is a sensor that integrates
a biological element with a physicochemical transducer to produce an output signal
(e.g. electronic or optical) proportional to the amount of an analyte. This signal is
then conveyed to a processing block which is often capable of conditioning and
exhibiting other complex functionalities like sending data to cloud, etc. The specific
biochemical reaction that we focus on here is physisorption, a reversible stochastic
process of binding and unbinding between bioreceptor molecules positioned at the
sensing surface (adsorbent) and freely moving adsorbate particles (target analyte
molecules) in a surrounding fluid. Most often, the readout mechanism of such affinity
devices is based on reversible adsorption. The one that we address here is electro-
magnetic (through surface plasmon resonance, SPR), however, it can be electrical
(e.g. piezoelectric), electrochemical, mechanical, etc., as shown in Fig. 1.
Figure 1 is a schematic representation of the side view of a microfluidic channel
with monocomponent target analyte and sensing surface functionalized with linker
molecules in order to support bioreceptor adsorption sites.
Depending on the readout mechanism, the transducer part in the block diagram and
the measured signal are different. Recent advancement in refractometric transducers
is presented in a review of optical biosensing schemes in [27] and in works [28–30]
alongside other transducer techniques. The focus here is on modeling the binding
mechanisms. The target analyte concentration in a microfluidic channel or chamber
may be modeled and considered a constant, as in Fig. 1, but more realistically, it has
some spatial distribution, as shown in Fig. 2, where flow is depicted with horizontal
arrows, their lengths being proportional to the velocity of the fluid.
Fig. 1 Side view of a microfluidic channel with monocomponent target analyte and sensing surface
functionalized with linker molecules in order to support bioreceptor adsorption sites
Fig. 2 Side view of a microfluidic channel magnified and denoted in accord with two compartment
model, showing inhomogeneous spatial distribution of target analytes
In microchannels and microreactors, binding and unbinding of target analyte

molecules to and from bioreceptor molecules positioned on the surface is addition-
ally affected with mass transfer in the flow direction along separate compartments
(convection), and mass transfer between the compartments (diffusion). In modeling
of binding kinetics in the flow-through chamber, by using the two compartment
models, target analyte concentrations in the outer compartment, far from the sensing
surface and the inner compartment, adjacent to the sensing surface, are modeled
differently. However, the general stoichiometric equation is the same for all models
discussed here.
The general stoichiometric equation, (1), represents multicomponent adsorption
of r a different target analytes in a fluid, whose molecules are denoted with Ag on a
multimodal surface with r c different classes of adsorption centers with bioreceptors,
denoted with Af . After binding to a bioreceptor, target analyte molecule forms an
occupied adsorption center Aa . k a and k d are the association rate constant and the
dissociation rate constant respectively. The process takes place without dissociation
or mutual interaction of target analyte molecules.
ka,i
→
A g,i + A f, j Aa,i i = 1, ...ra j = 1, ...rc (1)
←
kd,i
Based on the interpretation of this system of stoichiometric equations one gets

different systems of differential equations, i.e. different analytical models, different
reaction rate equations and different propensity functions in chemical master
equations. Next sections address these models and some of their solutions.
230 O. Jakšić
3 Time Domain: Transients and Steady States
3.1 Monocomponent Case
Lagergren pseudo first order (PFO) equation. Written in terms of the number of
adsorbed target analyte molecules N, this reaction rate equation, (2) refers to the
adsorption on homogeneously functionalized surface, meaning there is one type of
adsorption center with bioreceptors. The rate Eq. (2) depicts a process whose transient
response, (3) for initial condition equal to zero, exhibits an exponential relaxation
towards the steady state N s , with a time constant τ [31].
dN
= ka N0 (M − N ) − kd N (2)
dt

N (t) = Ns 1 − e−t/τ = Mka N0 τ 1 − e−(ka N0 +kd )t (3)
M is the number of adsorption centers with bioreceptors when the surface is fully
populated and N 0 is the overall number of target analyte molecules in a microfluidic
chamber. Adsorption rate in this model is modeled as greater than it truly is.
Riccati pseudo second order (PSO) equation. This more correct model takes into
account the depletion of the overall number of target molecules due to adsorption,
as shown in (4). It is still the process with exponential relaxation towards the steady
state, denoted with β, (5), except that the number of transients is infinite (6).
dN
= ka (N0 − N )(M − N ) − kd N = ka (N − β)(N − γ ) (4)
dt
/
β = ka (N0 + M) + kd − (ka (N0 + M) + kd )2 − 4ka2 N0 M /2ka
(5)
β = [ka (N0 + M) + kd − k]/2ka
∞
1 − e−kt ∑
N (t) = β =β+ (β − γ )(β/γ )i e−ikt (6)
1 − (β/γ ) 1 − e−kt i=1
The drawback of this model is that it assumes a uniform concentration distribution

of the analyte target molecules in the microfluidic channel and assumes that the mass
transfer effects are negligible, which is usually not the case.
Two compartment model. Again, the reaction rate equation relates the change in
the number of bound (adsorbed) target analyte molecules on its left hand side with
the difference in adsorption and desorption rates on its right hand side. The analyte
concentration in the vicinity of the sensing surface, C s , does not equal the analyte
concentration in the rest of the microfluidic chamber, C 0 . Its change is caused by

the adsorption and the mass transport between the compartments as shown in Fig. 2
[32–36].
dN C0 + kkmdA N
= ka' k'
(M − N ) − kd N (7)
dt 1 + a (M − N )
km A
/
D2v
km = 1.467
3
[m/s] (8)
Lh
Rate constant of adsorption is now denoted with apostrophe in order to mind different
units (now m3 /s) and k m is the mass transfer coefficient, assumingly constant too
(8). D is the diffusion coefficient of the target analyte molecules, v mean convec-
tion velocity, L is the length of the sensing area and h is the height of the sensing
microchamber.
However, whether modeled as a process with pseudo first order kinetics (described
with linear first order differential equations, first proposed by Lagergren) or modeled
as a process with second order kinetics (described with nonlinear Riccati equations),
adsorption is genuinely a stochastic process.
Analytical stochastic approach. By setting and solving the Chemical Master Equa-
tion in terms of the probability generating function one gets insight into all properties
of the system, all its moments. Adsorption process with PFO kinetics has binomial
probability distribution function PDF (11), its mean value of the number of adsorbed
molecules N equals the deterministic solution N (3) and its probability generating
function PGF, F(s,t), (9) can be written in terms of the surface coverage, G, (10),
(the number of adsorbed molecules normalized with the overall number of adsorption
centers on the surface M) [37] .
∑
M
M
F(s, t) = [(1 − G) + sG] M = (1 − G)(M−N ) G N s N |s| ≤ 1 (9)
N
N =0

G(t) = ka N0 τ 1 − e−(ka N0 +kd )t (10)
M
PN (t) = (1 − G)(M−N ) G N (11)
N
For adsorption process with PSO kinetics such an analysis partial differential equation
stemming from the Chemical Master equation is generally not solvable, so one must
resort to the numerical stochastic approach.
Numerical stochastic approach. Once set and coded, the stochastic simulation
algorithm SSA [38], may serve successfully for simulation of adsorption processes
with PFO as well as ones with PSO. In SSA, the time instances of transitions due
232 O. Jakšić
to adsorption or desorption are also stochastic, not equally spaced in time. SSA
is based on the use of built-in numerical routines for generating random numbers
with exponential distribution (which is related here to the mean residential time of
the adsorbed molecules on adsorption centers), applied with different parameters
(which are here related to instantaneous sorption rates).
The algorithm has the following steps [39]
• set initial number of adsorbate particles and use the reciprocal value of the initial
rate of adsorption (1/k a N 0 M in case all adsorption centers are initially empty)
as a parameter for calculation of the first time step towards the first transition
(in case of zero initial condition, the adsorption is only possible) and update the
corresponding number of the adsorbed molecules in the next time step accordingly.
• for every next time step and every next update of the instantaneous number of
adsorbed molecules, the random number with exponential PDF is calculated twice,
for both sorption constants, once using the reciprocal value of the instantaneous
adsorption rate as a parameter, and also using the reciprocal value of the instanta-
neous desorption rate as a parameter. Whichever number is smaller, it determines
the next transition and the next transition update.
The adsorption process, as all natural processes, is ergodic, the mean value
of multiple iterations of SSA (averaging over an ensemble), is in accord with
time average, and for adsorption process with PFO, it is also in accord with the
deterministic solution (3).
The approaches presented so far can be scaled up to multicomponent adsorption.
3.2 Binary Analyte Mixture
Lagergren pseudo first order (PFO) equation. The system of differential Eq. (12),
has a characteristic polynomial (13) whose roots represent the time constants in the
transient response (14)
⎛ ⎞
d Ni ∑
2
= ka,i N0,i ⎝ M − N j ⎠ − kd,i Ni i = 1, 2 (12)
dt j=1
P2 (s) = s2 + bs + c = (s − z1 )(s − z 2 )

b = k1 + k2 = ka,1 N0,1 + kd,1 + ka,2 N0,2 + kd,2 (13)
c = k1 k2 − ka,1 N0,1 ka,2 N0,2 = k1 k2 − kl,1 kl,2
Ni = Ns,i + Nt1,i e z1 t + Nt2,i e z2 t i = 1, 2

Mk k
Ns,i = zl,i1 z2d, j j = 3 − i (14)
N = (−1)(i+1) l, j ( d,3− j i )
k k +z
ti, j z i (z 1 −z 2 )
Riccati pseudo second order (PSO) equation. The set of two nonlinear Riccati
differential Eq. (15) is conveniently solved numerically for determining the full time
response. The solution to stationary states in analytical form is calculated by
⎛ ⎞
d Ni ∑
2
= ka,i N0,i − Ni ⎝ M − N j ⎠ − kd,i Ni i = 1, 2 (15)
dt j=1
P3 (N1s ) = N1s
3
+ b2 N1s2
+ b1 N1s + b0
kd,1 kl,2 (16)
N2s = N1s k k +N k k −k k
d,2 l,1 1s ( d,1 a,2 d,2 a,1 )
kl,1 kd,2 +kl,2 kd,1

b2 = − M − N01 − kkd,1
ka,2 kd,1 −ka,1 kd,2 a,1
b1 = N01 M − kl,1 kd,2 +kl,2 kd,1 +ka,1 kd,2 M+kd,1 kd,2
ka,2 kd,1 −ka,1 kd,2
(17)
b0 = 2
N01 M ka,2 kkd,1a,1−k
kd,2
a,1 kd,2
Two compartment model. Mass transfer in microfluidic flow-through devices with

binary mixture of analyte target molecules, with concentrations C 1 and C 2 , compet-
itively fighting for same adsorption centers on the surface with the overall capacity
M is modeled with the set of Eq. (18) [40, 41] .

' km,i ACi +kd,i Ni
2
M− Nj
d Ni '
(
km,i A+ka,i M− 2j=1 N j ) j=1
= ka,i − kd,i Ni , i = 1, 2 (18)
dt
Analytical stochastic approach. Knowing that (12)–(14) refer to the mean values
of the number of bound target analyte molecules in adsorption process with PFO
kinetics, we can normalize N 1 and N 2 with M and calculate expectation E (19),
variance i.e. dispersion D (20) and relative fluctuations δ (21) in terms of the surface
coverages G1 and G2 . (19)–(21) are given here for a general response where readout
signal is linearly proportional to the numbers N 1 and N 2 , like the refractive index
change where RIC = w1 N 1 + w2 N 2 , [37] .
E R I C = w1 M G 1 (1 − G 1 ) + w2 M G 2 (1 − G 2 ) (19)

D R I C = M w21 G 1 + w22 G 2 − (w1 G 1 + w2 G 2 )2 (20)
/
w21 G 1 + w22 G 2 − (w1 G 1 + w2 G 2 )2
δR I C = √ (21)
M(w1 G 1 (1 − G 1 ) + w2 G 2 (1 − G 2 ))
Numerical stochastic approach. The SSA outlined for the case of monocompo-
nent adsorption can be scaled up for estimations of the response of adsorption of
234 O. Jakšić
multicomponent mixtures [26]. These calculations, however, depend on the specific

numerical values used in simulations, and may be CPU and time consuming.
ANN assisted approach. Artificial neural network ANN fitting provides us with the
tool that harnesses advantages of both worlds, analytical and numerical. In [26]
the time response of the binary adsorption has been prioritized over numerical
solutions. After training an ANN with examples obtained from a pool of experi-
mental data or numerical simulations, we can use the generated ANN fitting func-
tion without concerns regarding calculations over a vast parameter space where
differential equations may unexpectedly become stiff.
3.3 Ternary Analyte Mixture
Lagergren pseudo first order (PFO) equation. The system of differential Eq. (22),
has a characteristic polynomial (23) whose roots represent the time constants in the
transient response given by (24)-(28)
⎛ ⎞
d Ni ∑
3
= ka,i N0,i ⎝ M − N j ⎠ − kd,i Ni i = 1, 2, 3 (22)
dt j=1
P3 (s) = s 3 + c2 s 2 +1 s + c0 = (s − z 1 )(s − z 2 )(s − z 3 )

3 3 3 3
kl, j
c2 = kj = kl, j + kd, j c0 = kd,1 kd,2 kd,3 1 + kd, j
j=1
⎛
j=1 j=1
⎞ j=1
⎜ ⎟ (23)
3 ⎜ ⎟
⎜ 3
⎟ 3
c1 = ⎜kl,i kd,y ⎟ + kd,1 kd,2 kd,3 1
⎜ ⎟ kd, j
j=1
⎝ y=1 ⎠ j=1
y /= j

Ni (t) = Mkl,i Ai + Bi e−z1 t + Ci e−z2 t + Di e−z3 t i = 1, 2, 3 (24)
−kd,(i+1)mod3 kd,(i+2)mod3
Ai = i = 1, 2, 3 (25)
z1 z2 z3

z 1 + kd,(i+1)mod3 z 1 + kd,(i+2)mod3
Bi = i = 1, 2, 3 (26)
z 1 (z 1 − z 2 )(z 1 − z 3 )

z 2 + kd,(i+1)mod3 z 2 + kd,(i+2)mod3
Ci = i = 1, 2, 3 (27)
z 2 (z 2 − z 1 )(z 2 − z 3 )

z 3 + kd,(i+1)mod3 z 3 + kd,(i+2)mod3
Di = i = 1, 2, 3 (28)
z 3 (z 3 − z 1 )(z 3 − z 2 )
Two compartment model. Mass transfer in microfluidic flow-through devices with

ternary mixture of analyte target molecules, with concentrations C 1 , C 2 and C 3 ,
competitively fighting for same adsorption centers on the surface with the overall
capacity M, is modeled with the set of Eq. (29) [42].
⎛ ⎞
d Ni km,i ACi + kd,i Ni ∑
3
'
= ka,i 3 ⎝M − N j ⎠ − kd,i Ni i = 1, 2, 3
dt '
k m,i A + ka,i M − j=1 N j j=1
(29)
Analytical stochastic approach. We know that (24)-(28) refer to the mean values
of the number of bound target analyte molecules in the adsorption process with PFO
kinetics. Thus we can normalize N 1 , N 2 and N 3 with M and calculate expectation
E (19), variance i.e. dispersion D (20) and relative fluctuations δ (21) in terms of
the surface coverages G1 , G2 .and G3 . (30)-(32) here are given for general response
where readout signal is linearly proportional to the numbers N 1 , N 2 and N 3 , like the
refractive index change where RIC = w1 N 1 + w2 N 2 + w3 N 3 [37].
∑
3
ERIC = M wi G i (1 − G i )i = 1, 2, 3 (30)
i=1

D R I C = M w12 G 1 + w22 G 2 − (w1 G 1 + w2 G 2 )2 (31)
/
3 2
3
i=1 wi G i − wi G i
2
i=1
δR I C = √ 3 (32)
M i=1 wi G i (1 − G i )
3.4 Mixture with Arbitrary Number of Analytes
Lagergren pseudo first order (PFO) equation. The general solution to Chemical
Master Equation implemented on the stochastic analysis of adsorption of a mixture
of an arbitrary number of analytes, derived in [37], provides the tool for complete
insight into the system behavior, its means and all its higher moments. If we denote
with r the number of components in a mixture of adsorbates and with N f the number
of free bioreceptors or alternatively the number of unoccupied adsorption centers
236 O. Jakšić
on the surface, then consequently the fraction of bioreceptors which is free or the
fraction of the surface would be Gf :
∑
r ∑
r
Nf = M − Ni G f = 1 − Gi (33)
i=1 i=1
The general solution to the probability generating function F implies that the
process of multicomponent adsorption of analytes with pseudo first order kinetics
has multinomial probability distribution function:
M
∑
r
Fs1 ,...sr ,t = [1 + G 1 (s1 − 1) + ... + G r (sr − 1)] M
= Gf + G i si (34)
i=1
∑ M N

r
Fs1 ,...sr ,t = Gf f (G i si ) Ni (35)
N1 , ..., Nr , N f
N1 +...+Nr +N f =M i=1
It is in accord with the conditions
F(s1 , ...sr , t = 0) = 1F(1, ...1, t) = 1 (36)
The corresponding probability distribution function is
M! N

r
PN1 ,...Nr (t) = r Gf f G iNi (37)
Nf! i=1 Ni ! i=1
Hence, the expectation, variance and all higher moments are known.
Riccati pseudo second order (PSO) equation. The solution to the matrix Riccati
equation relevant for modeling of the process of multicomponent adsorption of
analytes with pseudo second order kinetics, in an analytical form, is not derived
yet. What is easily feasible is the determination of number of adsorbed molecules
in the stationary state, for each component in the mixture. In order to calculate
the number of adsorbed molecules in the stationary state for one component in the
mixture with r components, one has to solve a polynomial with the rank r + 1. The
set of the differential equations is such that for the mixture of r components, there
are r + 1 solutions, r + 1 sets of r numbers that correspond to the stationary state
of each component.
4 Multiple Steady States and Stability
The possibility of multiple solutions when determining stationary states in the adsorp-
tion process needs to be critically considered from a physical point of view. If the
adsorption process is stable by its nature, there is only one stationary solution and all
others, although present in mathematical models, are rejected as physically impos-
sible. If multiple stationary states are physically possible, then it is necessary to
determine the conditions for transitions between them. For that, it is necessary to
apply a stochastic approach.
Many natural processes are truly stochastic and, due to inherent random mecha-
nisms, fluctuate around some stationary state. In experiments and practical applica-
tions, these fluctuations can appear reproducible and resemble chemical or biological
reactions that actually exhibit oscillatory behavior. Population dynamics in general
can be oscillatory. Since the interest in oscillatory reactions is still growing in many
areas, the results of the analysis of the stability of the adsorption process, carried out
in the paper [43], are also summarized here.
In [43], stability was tested in several ways:
• By analyzing the Jacobian in Taylor expansion of a set of deterministic equations,
• By applying a stochastic approach, analyzing random perturbations around the
stationary state,
• By applying the Langevin method with special attention to the correct modeling
of the Langevin forces and the relationship between the reaction flux and the
cross-correlation function,
• Using stochastic analysis, based on the basic chemical equation, and
• Using stochastic simulation algorithms.
The conclusion of the comparative analysis of those results is that the process
of monocomponent adsorption and the process of binary adsorption with pseudo
second order kinetics both have one stationary state which is a stable focus. Hence,
monocomponent adsorption, as well as binary adsorption, is a stable process.
A generalization of this conclusion to the stability of adsorption of a mixture of
an arbitrary number of analytes is possible. It is based on the fact that an arbitrary
mixture can be viewed as a binary mixture by extracting one analyte from it. For the
rest of the mixture, positive real numbers, which represent fictitious rate constants,
can be defined, and, in a mathematical sense, satisfy the conditions that apply to a
binary mixture.
Figure 3 shows monocomponent adsorption with zero initial conditions in time
domain, obtained in three different ways. The first one is by the use of SSA, stochastic
numerical approach, where multiple iterations are shown by green lines. Then, the
mean value of these multiple iterations is calculated (magenta line) and compared
with the deterministic solution (blue circles). In this case, they coincide with the
solution of the chemical master equation as obtained by the stochastic analytical
approach.
238 O. Jakšić
Fig. 3 Monocomponent adsorption with zero initial conditions (no adsorbed analyte at the
beginning of the process) in time domain calculated in three different ways
5 Frequency Domain: Equilibrium Fluctuations
All models developed for the time domain are not informative enough when it comes
to the fluctuation dynamics in equilibrium. In equilibrium, random processes fluctuate
around the steady state and the dynamics of those fluctuations greatly affects the
operation of the whole device. Next subsections present a collection of related results.
It will be shown that all natural processes with the exponential relaxation towards
the stationary state have the power spectral density of intrinsic fluctuations in a form
of typical Lorentzian curve, which, presented graphically, exhibits a flat region at
low frequencies, LFNM (low frequency noise magnitude), and a slope after some
distinctive knee related to the cut off frequency. Time constants and stationary states
calculated for temporal response are the key data for the determination of power
spectral densities of intrinsic fluctuations in the number of adsorbed molecules in
the frequency domain.
5.1 Monocomponent Case
One compartment model. The analytical expression for the power spectral density
of the fluctuations in the number of adsorbed molecules is [44–46]
P S D0 4kd Ns τ 2 4vdes,(N =Ns ) τ 2

P S D(ω) = = = (38)
1+ω τ2 2 1+ω τ2 2 1 + ω2 τ 2
It has the same form for adsorption processes with PFO and PSO, and for the
adsorption processes in flow through microchannels with mass transfer due to the
convection and the diffusion, but the numerical values for the time constants and
the stationary states are calculated in accord with the models developed for the time
domain, as presented previously.
Two compartment model. The laminar flow of analyte solutions in microchannels
and mass transfer caused by it, affect the fluctuation dynamics in equilibrium so that
the system needs more time to reach the equilibrium once it has been drawn from
it. The expression for the power spectral density of the fluctuations in the number of
adsorbed molecules also has the form written in (38) but the time constant is greater
than the time constant for systems with PFO [48].
1 kd ka' M
τ= + 2 (39)
ka' C0 + kd ka' C0 + kd Akm
The reasoning for calculating the low frequency noise magnitude is the same as in
(38). It is the quadruple of the product of desorption rate in equilibrium and the
squared time constant, but desorption rate in equilibrium is calculated in accordance
with the models presented previously.
5.2 Binary Analyte Mixture
The analytical expression for the power spectral density of the fluctuations in the
number of adsorbed molecules, in case of adsorption processes with PFO and PSO,
has two important time constants and three changes of slope after the LFNM flat
region, one zero and two poles [47]:

P S D0 1 + ω2 τ I2I I
P S D(ω) = (40)
1 + ω2 τ I2 1 + ω2 τ I2I
τ 2τ 2
P S D0 = 4 w12 kd,1 N1s + w22 kd,2 N2s I 2 I I (41)
τI I I
For transducer elements whose signals are proportional to the number of adsorbed
analyte molecules, a scaling factor wi is used, as before RIC = w1 N 1 + w2 N 2 .
The expression for the third time constant, τIII , is also the same for adsorption
processes with pseudo first order kinetics and processes with pseudo second order
kinetics:
/
w12 kd,1 N1s + w22 kd,2 N2s
τI I I = (42)
(w1 M22 − w2 M21 )2 kd,1 N1s + (w2 M11 − w1 M12 )2 kd,2 N2s
However, the expressions for the stationary states and the expressions for the time
constants are calculated differently.
240 O. Jakšić
Lagergren pseudo first order (PFO) equation. Stationary states in expressions

for the power spectral density (41)–(43) are calculated by using the expressions
developed for the analysis in time domain for that case, that is expressions (13), (14).
The same expressions (13), (14) are also used for calculations of time constants
τ I = z 1−1 , τ I I = z 2−1 (43)
Riccati pseudo second order (PSO) equation. For the determination of the
stationary states expressions (16), (17) are used and for the determination of the
time constants, the following expressions are valid:
/ −1
τ I,I I = 2 M11 + M22 ± (M11 − M22 )2 + 4M12 M21 (44)
Constants M ij are calculated after following expressions
M11 = k1 + ka,1 (M − 2N1s − N2s ), (45)
M22 = k2 + ka,2 (M − 2N2s − N1s ), (46)
M12 = ka,1 (N01 − N1s ), (47)
M21 = ka,2 (N02 − N2s ), (48)
6 Conclusions and Future Prospects
In this Chapter, a survey of modeling methods of adsorption kinetics and fluctua-

tion dynamics in affinity-based biosensors is presented which are applicable for the
design and application of healthcare microfluidic devices and systems. Various cases
are considered regarding the number of analytes in the fluid (one, two, three or an
arbitrary number). Different approaches to the analysis are presented (deterministic,
stochastic, artificial neural networks-assisted) in time domain and frequency domain.
New advancements with respect to enriched functionalities, novel adsorbate mate-
rials and multi-functional sensing surfaces, new integrations in digital healthcare
systems, novel design of microfluidic systems with independent but interconnected
customizable microfluidic chips and new regulations regarding personal data protec-
tions are on their way too [48–50]. New advancements in modeling can be expected in
the domain of stronger implementations of artificial intelligence and neural network
fitting for optimal experiment design, optimal methodology design, and integrations
of existing models into more complex sequential and concurrent multiscale models.
Acknowledgements This research was funded by the Ministry of Education, Science, and
Technological Development of Republic of Serbia, grant number 451-03-68/2022-14/200026.
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Lecture Notes in Electrical Engineering 989

MEMS and Microfluidics

ISSN 1876-1100 ISSN 1876-1119 (electronic)

The significance of medical MEMS is increasing exponentially from past two

Silchar, India Dr. Koushik Guha

N/MEMS Biosensors: An Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Transdermal Injection with Microneedle Devices in Healthcare

About the Editors

and Visiting Fellow to Department of Electrical and Computer Engineering, National

Valanukonda Bhavya Bhargavi Department of Electronics and Communication

Gorachand Dutta NanoBiosensors and Biodevices Lab, School of Medical

Rahul Kumar Ram NanoBiosensors and Biodevices Lab, School of Medical

Keywords Biosensors · N/MEMS · Health and well-being · BioMEMS ·

Fig. 2 A diagram showing

Fig. 3 Performance parameters in biosensor development

Reproducibility: Reproducibility is the capability of the biosensor module to

3 N/MEMS Biosensor (BioMEMS)

N/MEMS features scalability, ultra-high sensitivity, energy efficiency, and compat-

3.1 Mass Accumulation

transducer that can be used as a primary element in a typical N/MEMS biosensing

Usually, silicon/polysilicon is used to construct such a transducer. In recent years,

or into a body. Such materials can be a part of N/MEMS-enabled biosensors. These

3.3 N/MEMS Resonators for Ultra-Precise Bio/Chemical

3.4 Multiple Analyte Detection Using N/MEMS

Figure 6 shows a platform parallel processing using N/MEMS sensors. As seen,

4 Summary, Outlook, and Conclusion

Keywords Microfabrication · Biosensor · Microfludic channel · Optical detection

A microfluidic platform consists of a group of precisely defined, fabricated microflu-

Sample Sample Sample Sample

Fig. 1 Basic component of lab-on-a-chip technology

Utilizing the photolithography process, integrated circuits and microchannels can

Table 1 LOC Technologies in healthcare [2]

One of the major fields of application of lab-on-a-chip is molecular biology;

LOCs are used in a variety of chemical and molecular diagnostic procedures,

1.1.1 Optical Detection Techniques for the Detection of the Analyte

on DNA adsorption on silica beads in particular buffer conditions, are portable to

1.1.3 Molecular, qPCR, and PCR Detection Using LOC Devices

1.2 Genomic Application

Fig. 3 Microfluidic RT-PCR assay

Multiple biosensors must be integrated in conjunction with DNA microarrays for

1.4 Biochemical Applications

Fig. 4 Basic element of biosensors

1.7 Cell Research

1.9 Drug Design and Development

2 Conclusion and Future Directions

1. Mark D, Haeberle S, Roth G, von Stetten F, Zengerle R (2010) Microfluidic lab-on-a-chip

Subhash Turaka, Valanukonda Bhavya Bhargavi, Naga Nikhila Nandam,

Keywords Isolation · Red Blood Cells · Centrifugation · Microfluidics ·

S. Turaka · V. B. Bhargavi · N. N. Nandam · S. B. Syed · J. Sateesh (B)

2 Red Blood Cells and Their Functions

3 Laboratory Techniques for the Segregation of RBCs

The conventional laboratory approach for the separation of RBCs is centrifugation

Centrifugation is a methodology that utilizes centrifugal force to remove blends or

3.2 Density Gradient Centrifugation (DSC)

In hematology, difficulties such as partitioning erythrocytes into age-dependent frac-

Antibodies can cluster cells or particles in a process termed agglutination, in addi-

3.4 Phase Partitioning

The topology of microvascular networks is complicated, with many bifurcating

4 Microfluidics and Applications

To comprehend and deal with microfluidics, it is necessary to first comprehend the