Enzyme Based Biosensors Recent Advances and Applications in Healthcare 1st Edition Sanjukta Patra Debasree Kundu Manashjit Gogoi

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Sanjukta Patra
Debasree Kundu
Manashjit Gogoi Editors
Enzyme-based
Biosensors:
Recent Advances
and Applications
in Healthcare
Enzyme-based Biosensors: Recent Advances
and Applications in Healthcare
Sanjukta Patra • Debasree Kundu •
Manashjit Gogoi
Editors
Enzyme-based Biosensors:
Recent Advances
and Applications
in Healthcare
Editors
Sanjukta Patra Debasree Kundu
Department of Biosciences and Department of Biosciences and Bioengineering
Bioengineering Indian Institute of Technology Guwahati
Indian Institute of Technology Guwahati Guwahati, Assam, India
Guwahati, Assam, India
Manashjit Gogoi
Biomedical Engineering Department
North Eastern Hill University
Shillong, Meghalaya, India
ISBN 978-981-15-6981-4 ISBN 978-981-15-6982-1 (eBook)

https://doi.org/10.1007/978-981-15-6982-1
# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore
Pte Ltd. 2023
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether
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Paper in this product is recyclable.

Preface
Rapid development of science and technology in last few decades, the lifestyle of
mankind, and the environment have changed drastically. The changes in human
lifestyle and exposure to adverse environment are major causes of large number of
non-communicable diseases such as cardiovascular disease (CVD), cancer, and
diabetes. CVDs, cancer, and diabetes are the leading non-communicable diseases
and responsible for deaths of large number of people globally. Whereas poor
hygiene, lack of resources like quality drinking water, food, and medicines to
increasing number of populations cause large numbers of infectious diseases.
Biosensors have been extensively used for disease detection, diagnosis, human
health monitoring, and health management. Enzymes are drawing tremendous
attention as biological recognition elements for the development of biosensors due
to their high selectivity and specificity towards their substrate. These biosensors help
in rapid diagnosis of different diseases and their management in places where access
to sophisticated instruments and facilities is limited. In short, biosensors can play a
vital role in healthcare management in rural area and resource-limited settings. This
book reviews the applications of different enzymatic biosensors in healthcare sector,
their challenges, and future scopes.
Chapter 1 focuses on the application of different enzymatic biosensors in
healthcare sector and how they are helping in managing different diseases. It also
gives an overview of existing commercial biosensors available in the market.
Chapter 2 reviews how different types of enzymes are entrapped/immobilised in
different biological matrices for maintaining their stability and reusability for poten-
tial biosensor applications.
Chapter 3 highlights different common cardiac biomarkers detecting different
cardiac ailments along with their clinical characteristics. The chapter also explains
the challenges and future perspectives of enzymatic biosensor platforms used for the
diagnosis of heart diseases.
Chapter 4 discusses the different types of enzyme-based biosensors based on the
type of transducer used and their applications in detecting some of the most prevalent
cancer types. The chapter also highlights the various challenges faced in the cancer
diagnostics and the future roadmap for improving the performances of biosensors.
v
vi Preface
Chapter 5 elaborates different types of enzymatic biosensors for glucose sensing.

This chapter also highlights the challenges and future prospects in glucose sensing
biosensors.
Chapter 6 focuses on several enzymes-based biosensors for detection of pancrea-
titis and their limitations and future scopes.
Chapter 7 provides insight into the different types of prognostic and diagnostic
biomarkers associated with gut diseases and applications biosensor for detection of
these diseases.
Chapter 8 discusses the enzyme-based biosensors for detecting different meta-
bolic diseases and the challenges faced by these biosensors in working in real-world
settings. Future scope focusing on the advanced strategies to fulfil unmet clinical
needs are also discussed.
Chapter 9 presents different existing malarial biomarkers focusing on the protein-
based biomarkers and the application for developing advanced rapid and reliable
point-of-care (POC) detection techniques.
Chapter 10 elaborates on the significant advancements, scope, and challenges in
the application of enzymes on biosensor platforms in the diagnosis of highly
communicable infectious diseases like tuberculosis (TB) and neglected tropical
diseases (NTDs).
Chapter 11 elucidates about different piezoelectric biosensors used in the field of
healthcare including the applications of wearable health monitoring system, early
diagnostic biomarkers for Alzheimer’s disease, cancer, CVDs, Corona virus
detection, etc.
Chapter 12 presents an overview about recent advancement on the paper-based
low-cost biosensors used disease diagnosis and their potential in early diagnosis and
treatment applications.
Chapter 13 represents potential applications of wearable enzymatic biosensors
used in different healthcare applications along with their challenges and future
scopes.
Chapter 14 provides an overview on limitations, applicability and future market
scope of enzymatic biosensors, and how personal healthcare biosensors can be used
in different socio-economic settings.
In nutshell, this book depicts the current status and future prospects of enzymatic
biosensors for healthcare applications. This book deals with the basics of biosensors,
different applications of enzymatic biosensors as well as their commercial potentials.
The target group of the book is postgraduate, Ph.D. students, and advanced
researchers. It will be helpful for both novices as well as the experts working in
the field of biosensors.
Guwahati, Assam, India Sanjukta Patra

Guwahati, Assam, India Debashree Kundu
Shillong, Meghalaya, India Manashjit Gogoi
Acknowledgement
First of all, the editors would like to thank almighty God for providing us with the
motivation and strength to complete this endeavour of editing this book.
The editors would like to sincerely thank Dr. Bhavik Sawhney, Editor, Biomedi-
cine, and all the board members from Springer Nature for their approval and for
granting us this opportunity to edit this book. We would like to express our sincere
gratitude to Ms. Vaishnavi Venkatesh, Project Coordinator (Books), Springer Nature
for constantly following up on the editing process and helping us to complete this
assignment.
The editors express their heartfelt gratitude to all the contributors of this book for
their contributions. Their contributions are sincerely appreciated and gratefully
acknowledged.
Prof. Sanjukta Patra expresses heartfelt thankfulness to little Paridhi. It is her
shared time which has brought this book in black and white. The motivation of her
husband Rajesh has made the completion possible. The support of her parents,
parents in law, and siblings has kept her moving ahead.
Dr. Debshree expresses her sincere gratitude to her husband and family members
for their constant support and motivation.
Dr. Manashjit Gogoi expresses his thankfulness to his beloved sons Aditya,
Atanu, wife Mayuri who sacrificed a lot and kept him inspired to finish this task.
He is also thankful to his sister-in-law Hewali, and his parents, brothers, and in-laws
for their constant support, unconditional love, and blessings.
vii
Contents
1 Enzymatic Biosensors for Healthcare Applications . . . . . . . . . . . . . 1

Bethuel Daurai, Shrimanta S. Ramchiary, and Manashjit Gogoi
2 Choice of Enzyme Immobilization Matrices Used in Biosensor
for Healthcare Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Sanjeev Bhandari, Manashjit Gogoi, and Mrityunjoy Mahato
3 Enzymatic Biosensor Platforms for Diagnosis of Heart Diseases . . . 51
Jasmeen Kaur, Rohit Srivastava, and Vivek Borse
4 Enzyme-Based Biosensor Platforms for Detection of Cancer . . . . . . 79
Anna Anandita, Dakshita Snud Sharma, Nandini Singh,
Rajesh Kumar Singh, Vinay Sharma, and Dharitri Rath
5 Enzymatic Biosensor Platforms for Early Diagnosis of Diabetes . . . 109
Prabhjot Singh, Satish Kumar Pandey, and Nishima Wangoo
6 Enzymatic Biosensors for Detection of Pancreatitis . . . . . . . . . . . . . 127
Bethuel Daurai, Arup Jyoti Baruah, and Manashjit Gogoi
7 Enzymatic Biosensing Platforms for Gut Diseases . . . . . . . . . . . . . . 151
Damini Verma, Amit K. Yadav, and Pratima R. Solanki
8 Enzymatic Biosensor Platforms for Non-infectious Diseases:
Diagnosis of Metabolic Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Gurpreet Kaur, Naveen K. Singh, and Kuldeep Gupta
9 Protein-Based Biomarkers for the Diagnosis of Malaria
in Point-of-Care Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Babina Chakma, Priyamvada Jain, and Pranab Goswami
10 Enzymatic Biosensor Platforms for Infectious Disease Diagnosis:
Focus on Tuberculosis and Neglected Tropical Diseases . . . . . . . . . 237
Satakshi Hazra, Munna Singh Thakur, and Sanjukta Patra
11 Piezoelectric Biosensors in Healthcare . . . . . . . . . . . . . . . . . . . . . . . 255
Akshpreet Kaur, Parveen Kumar, Ankur Gupta, and Gaurav Sapra
ix
x Contents
12 Low-Cost Paper-Based Analytical Devices and Their

Application in Healthcare System . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Girish Chandra Mohanta and Satish Kumar Pandey
13 Nano-inspired Point-of-Care Enzyme-Based Wearable
Biosensors for Global Health Care . . . . . . . . . . . . . . . . . . . . . . . . . 293
Vinay Kumar and Kavita Arora
14 Commercialized Enzymatic Biosensors in Healthcare
Against the Conventional Methods . . . . . . . . . . . . . . . . . . . . . . . . . 323
Akshath Uchangi Satyaprasad
Editors and Contributors
About the Editors
Sanjukta Patra works at the Department of Biosciences and Bioengineering,

Indian Institute of Technology Guwahati, Assam, India. Holding a Ph.D. in Bio-
technology from the Central Food Technological Research Institute, Mysuru, her
areas of expertise include biosensors, enzyme and microbial technology, environ-
mental biotechnology, and metagenomics. To date, she has published 51 peer-
reviewed international research articles, authored 17 book chapters, and has three
patents to her credit.
Debasree Kundu is a Post-Doctoral Fellow at the Department of Biosciences and

Bioengineering, Indian Institute of Technology Guwahati, Assam, India. Holding a
Ph.D. in Microbiology, with a focus on microbial and enzymatic degradation of
xenobiotics, she is currently investigating pesticide-induced adaptive response in
microbes using proteomic approaches. To date, apart from having granted a patent to
her credit, she has authored 21 research articles and ten book chapters.
Manashjit Gogoi is an Assistant Professor at the Department of Biomedical Engi-

neering, North-Eastern Hill University, Shillong, Meghalaya, India. Holding a Ph.D.
in Biomedical Engineering from the Indian Institute of Technology—Bombay,
Mumbai, India, his research chiefly focuses on biosensors, point-of-care devices,
nanomedicine for drug delivery, and tissue engineering. To date, he has edited one
book, published 20 research articles, and 20 book chapters.
Contributors
Uchangi Satyaprasad Akshath Nitte University Centre for Science Education and
Research-NUCSER, Nitte University, Mangalore, Karnataka, India
Anna Anandita Department of Chemical Engineering, Indian Institute of Technol-
ogy, Jammu, Jammu and Kashmir, India
xi
xii Editors and Contributors
Kavita Arora Advanced Instrumentation Research Facility and School of Compu-

tational & Integrative Sciences, Jawaharlal Nehru University, New Delhi, India
Arup Jyoti Baruah Department General Surgery, North Eastern Indira Gandhi
Regional Institute of Health & Medical Sciences, Shillong, Meghalaya, India
Sanjeev Bhandari Physics Division, Department of Basic Sciences and Social
Sciences, School of Technology, North-Eastern Hill University, Shillong,
Meghalaya, India
Vivek Borse NanoBioSens Laboratory, Centre for Nanotechnology, Indian Insti-
tute of Technology Guwahati, Guwahati, Assam, India
Babina Chakma Global Consulting Admin, Syneos Health, Bengaluru, Karnataka,
India
Bethuel Daurai Department of Biomedical Engineering, North-Eastern Hill Uni-
versity, Shillong, Meghalaya, India
Manashjit Gogoi Department of Biomedical Engineering, North-Eastern Hill Uni-
versity, Shillong, Meghalaya, India
Pranab Goswami Biosensors and Biofuel Cell Lab, Department of Bioscience and
Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India
Ankur Gupta Department of Cardiology, PGIMER, Chandigarh, India
Kuldeep Gupta Department of Radiology and Radiological Sciences, School of
Medicine, Johns Hopkins University, Baltimore, MD, USA
Satakshi Hazra Department of Biosciences and Bioengineering, Indian Institute of
Technology Guwahati, Guwahati, Assam, India
Priyamvada Jain Medical Electronics Business Unit, TATA Elxsi, Bangalore,
Karnataka, India
Akshpreet Kaur UIET, Panjab University, Chandigarh, India
Gurpreet Kaur Department of Life Sciences, University of Hyderabad,
Hyderabad, India
Jasmeen Kaur NanoBios Laboratory, Department of Biosciences and Bioengi-
neering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India
Parveen Kumar E-waste Laboratory, CSIO – CSIR, Chandigarh, India
Vinay Kumar Department of Physiology and Cell Biology, Department of Internal
Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA
Mrityunjoy Mahato Physics Division, Department of Basic Sciences and Social
Sciences, School of Technology, North-Eastern Hill University, Shillong,
Meghalaya, India
Editors and Contributors xiii
Girish Chandra Mohanta Nanotechnology Lab (H-1), CSIR-Central Scientific

Instruments Organizations (CSIR-CSIO), Chandigarh, India
Satish Pandey Central Scientific Instrument Organization (CSIO), Chandigarh,
India
Satish Kumar Pandey Nanotechnology Lab (H-1), CSIR-Central Scientific
Instruments Organizations (CSIR-CSIO), Chandigarh, India
Sanjukta Patra Department of Biosciences and Bioengineering, Indian Institute of
Technology Guwahati, Guwahati, Assam, India
Shrimanta S. Ramchiary Department of Biomedical Engineering, North-Eastern
Hill University, Shillong, Meghalaya, India
Dharitri Rath Department of Chemical Engineering, Indian Institute of Technol-
ogy, Jammu, Jammu and Kashmir, India
Gaurav Sapra UIET, Panjab University, Chandigarh, India
Dakshita Snud Sharma Department of Chemical Engineering, Indian Institute of
Technology, Jammu, Jammu and Kashmir, India
Vinay Sharma Department of Biosciences and Bioengineering, Indian Institute of
Nandini Singh Department of Bioengineering and Biotechnology, Birla Institute of
Technology, Mesra, India
Naveen K. Singh Department of Electrical and Computer Engineering, University
of California, San Diego, CA, USA
Prabhjot Singh Centre for Nanoscience and Nanotechnology, Panjab University,
Chandigarh, India
Senior Secondary Residential School for Meritorious Students, Ludhiana, Punjab,
India
Rajesh Kumar Singh Department of Chemical Engineering, Indian Institute of
Pratima Solanki Special Center for Nanoscience, Jawaharlal Nehru University,
New Delhi, India
Rohit Srivastava NanoBios Laboratory, Department of Biosciences and Bioengi-
neering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India
Munna Singh Thakur Center for Nanomaterials and MEMS, Nitte Meenakshi
Institute of Technology, Bengaluru, Karnataka, India
Damini Verma Special Center for Nanoscience, Jawaharlal Nehru University,
New Delhi, India
xiv Editors and Contributors
Nishima Wangoo Department of Applied Sciences, UIET, Panjab University,

Chandigarh, India
Amit K. Yadav Special Center for Nanoscience, Jawaharlal Nehru University,
New Delhi, India
Enzymatic Biosensors for Healthcare
Applications 1
Bethuel Daurai, Shrimanta S. Ramchiary, and Manashjit Gogoi
1.1 Introduction
By developing the oxygen electrode in 1956, Leland C. Clark, Jr. established himself
as the originator of the biosensor idea and ushered in a new era in the sensor
technology (Clark Jr 1956; Clark Jr and Lyons 1962). Glucose was trapped by a
dialysis membrane in the Clark oxygen electrode, and the proportionality between
the concentration of glucose and the decreasing oxygen concentration was
established. A lot of different enzymes were used in this study since it was so
successful at catalyzing many different variants utilizing the same basic design.
Since this groundbreaking study, the biosensor industry has expanded dramatically,
and enzyme biosensors in particular have become one of the most widely used in the
biomedical industry (Turner et al. 1987).
A biosensor device is small or compact and contains a biological or biologically
derived sensing element that is integrated or connected to a transducer. Biosensors
are of many types and characteristics which can be categorized based on the
biological elements and the transducers used (Mohanty and Kougianos 2006).
Typically, a biosensor can be electrochemical, optical, mechanical, piezoelectric,
calorimetric, etc. based on the use of type of transducer Another component of a
biosensor may be signal processing unit. An electrochemical transducer produces
out in the form of voltage, current, or resistance (Zhang et al. 2000). To interpret
these signals corresponding to the concentration of the analyte, a signal processing
unit is required to correctly process and display the value of concentration of the
analyte. Low-power consuming signal processing unit is specifically required when
it comes to implantable biosensor and point-of-care (POC) devices (Haider and
Islam 2010).
B. Daurai · S. S. Ramchiary · M. Gogoi (✉)

Department of Biomedical Engineering, North-Eastern Hill University, Shillong, Meghalaya, India
# The Author(s), under exclusive license to Springer Nature Singapore Pte 1

Ltd. 2023
S. Patra et al. (eds.), Enzyme-based Biosensors: Recent Advances and Applications
in Healthcare, https://doi.org/10.1007/978-981-15-6982-1_1
2 B. Daurai et al.
Enzymes are biological catalysts that react with molecules known as substrates
and convert them into products. Binding of enzymes to their substrate is necessary
for their activation for catalysis. They are very specific to the substrate and also to the
chemical reaction. This specific nature of enzymes to bind to their respective
substrate makes them useful in biosensing. In 1894, Emil Fischer proposed the
lock and key model explaining the geometric complimentary of enzymes and
substrates. This early model failed to explain the stabilization of enzyme during
the transition stage. In 1958, Daniel Koshland described the change in the active
sites due to the interaction of enzymes and substrates. Substrates don’t bind to the
active site of the enzyme, but the amino acids of the active site are molded to the
precise position to perform catalysis. In enzymatic biosensors, enzymes are used as a
biological component to detect analytes. In an enzymatic biosensor, the enzyme is
immobilized to the transducer surface to detect the analyte. Here the analyte can also
be a substrate of the enzyme. Immobilization techniques are directly related with the
performance of the biosensor and can change the accuracy, sensitivity, selectivity,
and even the stability of the biosensor.
1.2 Types of Enzymatic Biosensors
The basic components of all biosensors are the biological sensing material and the
transducer with the option of a signal conditioner system. In the following section,
biosensors are categorized based on the use of different types of transducers and
discussed.
1.2.1 Amperometric Biosensors
1.2.1.1 First-Generation Amperometric Biosensors

In the first generation, the enzyme capable of converting a substrate into an
electroactive quantifiable byproduct is immobilized on the transducer surface
(Dzyadevych et al. 2008; Rocchitta et al. 2016). The first-generation biosensor is
based on oxidases and dehydrogenases. The rise and fall of oxygen concentration
can affect sensor response and reduce sensor linearity in this generation biosensors
(Wang 1999). In the first generation, the concentration of the measured analyte is
exactly proportional to the NADH concentration in the case of dehydrogenase
enzymes. Implantable biosensors have a challenge since NADH is necessary for
the generation of a signal in the matrix (Lee and Tsai 2009).
First-generation biosensors are sensitive and have a very quick reaction time
(Rocchitta et al. 2012). To produce a repeatable surface and sensor response, first-
generation biosensors frequently need electrode treatment, and matrix effects linked
to interference often need correction (Dzyadevych et al. 2008). Also, after repeated
use, particularly in unadulterated or complicated biological matrices, the transducer
surface of amperometric biosensors is damaged, which affects the biosensor
response (Prodromidis and Karayannis 2002).
1 Enzymatic Biosensors for Healthcare Applications 3
1.2.1.2 Second-Generation Amperometric Biosensors

In the second generation, oxidizing substances are used as mediators between the
enzyme and the transducer which carries electrons (Bartlett et al. 1991). By remov-
ing oxygen dependency and the effects of interfering molecules, this method enables
operation at low potentials. Ferricyanide and ferrocene are the two most popular and
well-known mediators (Chaubey and Malhotra 2002). Electrons are transferred to
the electrode surface from the enzyme’s redox reaction using the mediators.
Mediators are added to the sample or immobilized on the electrode (Habermüller
et al. 2000). Ideal mediators should be inert and nonreactive in normal circumstances
and do not participate in the electron transfer (Scheller et al. 1991). The redox
potential of the mediators is also an important parameter (Chaubey and Malhotra
2002). With more redox potential, the mediators will be more reactive than the other
chemicals in the sample. Immobilized mediators often have stability issues and
because of this, they are less frequently used compared to the first generation.
1.2.1.3 Third-Generation Amperometric Biosensors

Bioelectrocatalysis, which involves a direct exchange of electrons between an
electrode and an enzyme, is the basis for third-generation biosensors (Bhattarai
and Hameed 2020). Three components make up a third-generation biosensor: an
electrode (with surface to receive electrons), a reducing or oxidizing agent
(connecting element) to assure signal transmission, and an enzyme as the
biorecognition element (Prodromidis and Karayannis 2002). Performance is
enhanced by connecting the redox reaction site of the electrode surface using a
reducing or oxidizing agent. Third-generation biosensors are under research and
development stage and hence they still have limited use for analysis by the end users
in the real world (Mahdizadeh et al. 2022). Third-generation biosensors, however,
have incredibly quick reaction times and are mostly unaffected by oxygen/cofactor
(mediator) concentrations.
1.2.2 Optical Enzymatic Biosensors
Optical biosensors use enzymes as the biological recognition element. Based on the
transduction mechanism, enzymatic optical biosensor can be categorized (Choi
2004) as absorptiometry, luminescence, chemiluminescence types, etc. Absorption
by reflection or transmission of light through a material is an absorptiometry type.
Luminescence type of transducer makes use of the luminescence property of
materials (Blum et al. 1989). With separate wavelength of excitation, a separate
longer emission could be achieved. Chemiluminescence makes use of the reaction of
the reagent and analyte to give an optically readable output. There are also other
optical enzymatic biosensors like the interferometry, surface plasmon resonance
(SPR), fiber optic, acoustic photometry, etc.
Enzyme immobilization in optical biosensors is done by either chemical or
physical method. Both approaches aim to maintain the enzymes on the solid
substrate and prevent solution washing. Adsorption on solid surfaces or polymeric
4 B. Daurai et al.
gels, entrapment in inorganic or organic polymeric gels, and confinement within

semipermeable membranes are all examples of physically immobilizing an enzyme.
Enzymes may be chemically immobilized by either intermolecular cross-linking of
the biomolecules or covalently attaching the enzymes to functionalized solid
materials. While chemical immobilization may provide more stable and long-lasting
biosensors, the enzymatic performance is often not as excellent as that of physically
immobilized enzymes.
1.2.2.1 Absorbance Optical Biosensors

The Beer-Lambert law explains this kind of transducer type where the concentration
of an analyte is measured by measuring the absorbance or reflectance of light
through the analyte. Some of the biosensors used in this approach include photomet-
ric features, meaning they can measure changes in analyte or product absorbance at
certain wavelengths (Choi 2004). The typical absorbance transducers use a single
fiber or fiber bundle to provide light to the analyte-sensitive reagent phase and then
return the transmitted or reflected light through the same fiber to a measuring device
or detector. The biosensor can record these variations in transmission intensity
because changes in the chemical environment may alter how the biorecognition
element is absorbed. It becomes challenging to accurately measure transmitted light
in nontransparent measuring environments, and in these circumstances, the intensity
of the reflected light may be utilized to gauge the color of the recognition element,
analyte, or product.
1.2.2.2 Chemiluminescence
The chemiluminescence feature of an optical biosensor causes the emission of light
as a result of the interaction between the reagent material and analyte (Choi 2004).
Enzyme-linked reactions can be used in enzyme-based chemiluminescent assays,
often combining an oxidase and a peroxidase. H2O2 is produced as a result of
processes that are catalyzed by oxidases. This peroxide may then be used to oxidize
a chemiluminescent reagent, primarily luminol, producing light that can be detected
by a light sensor. This method has been used in the determination of glucose,
cholesterol, and lactate with the appropriate oxidase and dehydrogenase enzyme
for the analytes (Kuswandi et al. 2001).
1.2.3 Piezoelectric Enzymatic Biosensor
In this type of biosensor, the transduction is done by a piezoelectric quartz crystal.

The total applicability and sensitivity of this biosensing system are determined by
the enzyme (biorecognition element), the transducer, and the parameters of the assay
environment (Bunde et al. 1998). Quartz crystal microbalance (QCM) has shown
many applications in biosensing. These biosensors are different from electrochemi-
cal and optical ones, because they can quantify the attached material directly
(Pohanka 2021). They are, therefore, making them easy for construction. The change
in frequency of the quartz crystal bounded with a specific material can be correlated
to the mass of that material. The frequency may increase or decrease with the
increase or decrease in the mass of the material. Its application in healthcare has
been recently under research. Because it is label-free, the cost of a biosensor can be
reduced.
1.3 Significance of Enzymes as Biomarkers
Enzymes are biological catalysts that have extensively been utilized in industries, for
example, food and beverage industry, brewery production, and cleaning agents, to
regulate as well as speed reactions to rapidly and accurately produce different
necessary products (Godfrey and Reichelt 1982; Polaina and MacCabe 2007).
Additionally, enzymes are frequently used in laboratories, such as stereospecific
bioconversion, recycling trash into useful goods or eco-friendly replacements, etc.
(Bershtein and Tawfik 2008). There are many metabolic illnesses that are caused by
abnormalities in the enzyme metabolic system. Researchers from all around the
world have focused on the increasingly clinical uses of enzymes including acid
phosphatase, alkaline phosphate, alanine transaminase, aspartate transaminase, cre-
atine kinase, lipase, alpha-amylase, protease, lactate dehydrogenase, etc. (Patel
1995). Enzymes have already been used for the detection of diseases related to the
heart, kidneys, and liver, autoimmune disease, cancer, mental illness like
schizophrenia, etc.
“Diagnostic enzymes” is the term used for the enzymes of the human body that
are medically used for the diagnosis and prognosis of diseases (Singh et al. 2019).
Enzymes are used in diagnostics because of the high selectivity of the enzyme as a
substrate. Enzyme activities are very specific to the action of the reaction, for
example, in the hydrolysis of sugar by amylase which can help in the determination
of glucose level in detection of diseases like diabetes. Depending on the degree of
the condition, a sick state often results in tissue destruction. When this occurs,
disease-specific enzymes with increased enzyme activity are released into the blood-
stream. These increased levels of enzymes in the body fluids like blood, urine, and
saliva can be determined and can be correlated with a specific disease. In the
following section, application of enzymes as biomarkers for diagnosis of some
major diseases is discussed.
1.3.1 Enzymes as Biomarker for Cardiovascular Diseases
Cardiovascular diseases are often linked to atherosclerosis, where plaque made of fat
is accumulated in the arteries. Narrowing down of the blood vessels leads to
hypertension, heart block, heart attack, peripheral artery disorders, etc. Some of
the contributing factors for these diseases are obesity, stress, inactivity, food habit,
hereditary, and even poor mental health like depression.
Creatine kinase (CK; E.C. 2.7.3.2) catalyzes the production of adenine triphos-
phate from adenine diphosphate and creatinine phosphate. Three isoforms of CK are
6 B. Daurai et al.
CK-MM, CK-MB, and CK-BB. CK-MB are the most precise and specific
biomarkers for the detection of myocardial infarction (Panteghini 1988; Apple
1989). A normal level of CK in an adult human male is between 0.038 and
0.174 U/mL, whereas in adult human female, the normal level of CK is between
0.026 and 0.14 U/mL. Myocardial infarction, muscular dystrophy, and inflammatory
responses cause CK levels to go as high as 2 U/mL in the blood serum, which aids in
the early prognosis of illness states (Vainzof et al. 1985).
Glycogen phosphorylase (GP) (EC 2.4.1.1), an important part of carbohydrate
metabolism, is played by the glycolytic enzyme GP, which mobilizes glycogen. GP
is found in three separate physiologically distinct isoforms, GPMM, GPLL, and
GPBB (Krause et al. 1996). GPBB acts as an identifier or marker for the detection of
acute myocardial infarction, acute coronary syndrome, and even pregnancy and
preterm preeclampsia (Stejskal et al. 2007; Singh et al. 2018).
Gelatinases catalyze the breakdown of gelatin to create polypeptides, peptides,
and amino acids. The two isoforms, gelatinase A (EC 3.4.24.24) and gelatinase B
(EC 3.4.24.35), both have been used as biomarkers for the prediction and early
detection of heart disease like heart blockages and acute myocardial infarction
(Gopcevic et al. 2017). Other than heart disease, gelatinase has also been used as a
marker for detecting acute kidney damage and myocardial fibrosis when combined
with lipocalin (Gyöngyösi et al. 2017; Bulluck et al. 2018).
A digestive enzyme called salivary amylase (EC 3.2.1.1) is also reported as
biomarker for heart failure and psychological stress. When detected in urine, amy-
lase has also been found to be a helpful biomarker for renal disease (Strahler et al.
2010; Suska et al. 2012; Żyłka et al. 2016).
1.3.2 Enzymes as Biomarker for Liver Diseases
Several enzymes are released into the blood circulation when it is under stress or
injured because of hepatitis virus, fatty liver, alcohol abuse, and other certain drugs,
and measuring the amount of these enzyme catalysts helps in detection of the specific
diseases (Adams et al. 2005; Calvaruso and Craxì 2009). A few major enzymes that
aid in the identification of liver conditions and diseases are discussed in the follow-
ing section.
Aminotransferases (transaminases) convert amino acids to oxoacids by transfer-
ring amino groups. The two clinically significant enzymes included in the
aminotransferases group are alanine aminotransferase (ALT) and aspartate amino-
transferase (AST) (Huang et al. 2006). The ALT concentration range in an adult
person is between 5 and 35 U/L, and anything over this range denotes liver, heart,
and muscle damage or illness. Detection or determination is done using chromatog-
raphy, calorimetry, or spectrophotometry techniques, but recently researchers have
come up with affordable paper-based analytical devices (PADs) and POC biosensors
(Bacon 2002; Huang et al. 2006; Kang et al. 2011). The normal level of aspartate
aminotransferase (AST) (EC 2.6.1.1) in healthy human adults range from 5 to 40 U/
L (Kaplan 2002; Prati et al. 2002; Huang et al. 2006). However, with significant
injury, the level of AST may increase to as high as 20 times over the normal level.
Many other organs, tissues, and cells like the muscle fiber, blood, kidney, and
pancreas also contain AST. It can be used to track cardiac, hepatic parenchymal,
and muscular disorders in both people and animals by combining the results with
other enzymes. When an imaging method like ultrasonography or ultrasound
elastography is not available, the AST-to-platelet ratio index may be a helpful
measure to check for liver fibrosis which is a common condition in hepatitis B
(Lesmana et al. 2011).
Alkaline phosphate (ALP) (EC 3.1.3.1) is a biocatalyst in the hydrolysis of
phosphate ester bonds in an alkaline environment (Corathers 2006; Sharma et al.
2014). ALP is a helpful indicator of liver illness, especially cholestatic conditions
when there is a blockage in the bile duct, which are seen in obstructive jaundice
(Frey et al. 1990; Fargo et al. 2017).
Gamma-glutamyl transferase (GGT) (EC 2.3.2.2), which aids in the transporta-
tion of proteins and amino acids and also in breaking them down is present in all
human cells (Kunutsor 2016). Mostly found in the liver, kidney, and pancreas, GGT
in serum is produced in the liver. GGT is considered a biomarker for hepatobiliary
disease. GGT is moderately elevated because of toxic or infectious hepatitis (two to
five times) (Huber et al. 2004). GGT rises 5–30 times the normal in serum when the
intrahepatic biliary is obstructed. Other diseases and conditions like pancreatitis,
rheumatoid arthritis, alcohol abuse, muscular dystrophy, and pulmonary disease
show increase in serum GGT. GGT-to-platelet ratio index also determines liver
fibrosis in the absence of ultrasound elastography. In few researches, GGT is also
used as a biomarker for nephrotoxicity (Hu et al. 2017).
1.3.3 Enzymes as Biomarker for Cancer
Cancer is characterized as a condition in which a group of cells develops improperly,

leading to their unchecked expansion and spread. If this growth is allowed to
continue, it might be fatal. Additionally, 90% of cancer-related fatalities result
from a process known as metastasis. Several enzymes increase in concentration in
various body fluids when this condition occurs. The following are some of the
enzymes that can indicate cancer.
There are five types of acid phosphatases (ACP) (E.C. 3.1.3.2), namely, erythro-
cytic, macrophage, lysosomal, and osteoclastic. They are present in humans and
based on the origin and sequence length, they are different from one another. They
also differ based on the amount of resistance to tartrate and fluoride (Nakanishi et al.
1998). ACP levels in prostate gland are 100 times higher than in any other part of the
body. Since prostate cancer cells produce prostatic acid phosphatase in high levels, it
is utilized to track the progression of the disease (Taira et al. 2007).
Cathepsin D (CD) is a catalyst used for breaking down proteins into polypeptide.
Prepro-cathepsin-D is produced from it in the rough endoplasmic reticulum and is
present in almost all tissues, cells, and organs. Breast cancer’s prognosis is
8 B. Daurai et al.
influenced by CD, which is indicated with the overexpression of epithelial breast

cancer cells (Sun et al. 2016).
Cysteine cathepsins (CCs) are proteases that are produced during tumorigenic
phenomena like angiogenesis, apoptosis, and invasion. The increase in the concen-
tration of this enzyme indicates several types of cancers. There are 11 isoforms of
CCs (B, C, F, L, K, V, S, X/Z, H, W, and O) having different effects in the human
body (Joyce and Hanahan 2004; Turk et al. 2004). Some of the cancers where CCs
rise are in breast cancer, ovarian cancer, uterus cancer, cervical cancer, lung cancer,
and brain tumor. CCs are also used as biomarkers for inflammatory myopathies,
periodontitis, and rheumatoid arthritis (Hemalatha et al. 2013).
Cyclooxygenase-2 (COX-2) is a catalyst for the conversion of arachidonic acid to
prostaglandin H2. According to some research, COX-2 functions as a biomarker in
the development of malignancies, such as bladder, stomach, and breast cancers
(Hwang et al. 1998; Shirahama and Sakakura 2001; Van Rees et al. 2002).
Dehydrogenases are a family of enzymes that include two dehydrogenases that
are utilized for cancer prognosis: sorbitol dehydrogenase (SDH) and lactate dehy-
drogenase (El-Kabbani et al. 2004). The reaction of polyhydric alcohols D-sorbitol
and D-fructose is catalyzed by sorbitol dehydrogenase utilizing NAD+/NADH as a
mediator. It is commonly found in seminal vesicles, livers, and kidneys (Holmes
1978). An increase in SDH level in serum indicates prostate cancer and precancerous
colorectal neoplasms (Szabó et al. 2010; Uzozie et al. 2014). Some other researches
also suggest that increase in SDH also indicates liver damage and parenchymal
hepatic disease. Lactate dehydrogenase (LDH) is a catalyst in the formation of
lactate from pyruvate and vice versa during glycolysis and glyconeogenesis. It is
used as a biomarker for various cancers like breast cancer, oral cancer, prostate
cancer, pancreatic cancer, colorectal cancer, etc. (Singh et al. 2019). It has been
extensively used in researches for early detection of various cancers and for the
prognosis of acute leukemia and sickle cell disease (Kato et al. 2013; Walaa Fikry
2017).
Tartrate-resistant acid phosphatase (TRAP) hydrolyzes phosphate esters to
release oxygen. TRAP is found in high level in bone osteoclasts and lesser in
dendritic cells and activated macrophages (Hayman 2008). The two isoforms of
TRAP are TRAP5a and TRAP5b (Mira-Pascual et al. 2020). High level of TRAP5a
in serum indicates breast cancer and systemic lupus erythematosus (Chen et al. 2015,
2017). TRAP5b increases in serum in case of multiple myeloma and bone metastasis
originating from other cancers (Koizumi and Ogata 2002; Mose et al. 2003; Terpos
et al. 2003). In cancer with high incidences of bone metastases, TRAP5b can be used
for diagnosis and prognosis of breast cancer, lung cancer, myeloma, and prostate
cancer.
Thymidine kinase (TK) helps in the catalysis of thymine from thymidine. TK has
two isoforms: TK1 and TK2. TK1 is a biomarker for malignancy for biomarkers in
colon cancer, uterine cancer, prostate cancer, breast cancer, stomach cancer, chronic
lymphocytic leukemia, and acute lymphoblastic leukemia (Singh et al. 2019). Recent
research showed the use of TK1 in early detection of neoplasia (He et al. 2010).
In prostate cancer, the most common biomarkers are prostate-specific antigen,

prostate cancer gene 3, and enzymes like human kallikrein 3 and human glandular
kallikrein2 (Sarkar et al. 2022). With relevant type of biorecognition element and
transduction process, many combinations of biosensors can be developed.
1.3.4 Enzymes as Biomarkers for Other Diseases
Creatinine, blood urea nitrogen, cystatin C, and uric acid are some of the common
biomarkers of kidney diseases. All of them are not enzymes but take part in an
enzymatic reaction making them feasible for an enzymatic biosensor. Salivary
enzymes have also been used to determine neurological disorders and stress levels
(Nater et al. 2006). There are many diseases where enzymes can be used as
biomarkers. Pancreatic alpha-amylase and lipase are enzymes that catalyze the
hydrolysis of starch and triglycerides, respectively. Alpha-amylase increases three
to five times in the initial days and returns back to normal, but lipase stays elevated
for a longer time. According to the American Board of Internal Medicine, the normal
range of serum alpha-amylase is 25–125 U/L and serum lipase is 10–140 U/L.
Glucose-6-phosphatase has also been used as a biomarker for hypoglycemia
(Froissart et al. 2011). The use of acid phosphatase as a biomarker in serum was
reported by Devi et al. (2017).
1.4 Enzymatic Biosensors for Various Diseases
The most researched biosensors right now are enzymatic ones, and these research
articles demonstrate several innovative developments as have been discussed below:
1.4.1 Biosensors for Cardiovascular Diseases
An aptamer-based label-free capacitive biosensor was developed using non-faradaic

impedance spectroscopy for detection of cardiac C-reactive protein (CRP) based on
charge distribution. The biosensor had low binding affinity toward RNA aptamer,
whereas it could be operated without any reagent (Qureshi et al. 2010a, b). In another
study, a POC biosensor that is used to monitor cardiovascular illness by detecting
neutrophil gelatinase-associated lipocalin (NGAL) was developed using electro-
chemical impedance spectroscopy (EIS) measurement technique (Gonzalez and La
Belle 2012). Nanowire integrated microfluidic biosensors were developed for detec-
tion of cardiovascular biomarkers (related to heart failure stage diagnosis) such as
myoglobin (Myo), creatine kinase MB (CK-MB), cardiac troponin I (cTnI), and
b-type natriuretic peptide (BNP). Though this biosensor had good specificity, it was
not cost-effective (Lee et al. 2012). A highly sensitive, stable, and controllable
biosensor for the detection of cholesterol was developed using a layer by layer of
carbon nanotube/gold nanoparticle-centered bienzyme (Cai et al. 2013). Similarly,
10 B. Daurai et al.
cholesterol enzymatic biosensor based on nanohybrid composites (CSPPy-

gC3N4H+) has been developed which was affordable, biocompatible, as well as
environmental friendly (Shrestha et al. 2017). Another study was done on fluores-
cence resonance energy transfer (FRET) biosensors for visual detection of guanosine
monophosphate (cGMP), cyclic adenosine (cAMP), and Ca2+ in cardiac cells
(Thunemann et al. 2014). More advanced and highly sensitive biosensors were
investigated for detection of cardiac troponin I (Fathil et al. 2017; Radha
Shanmugam et al. 2017). Recently, a promising potential for an electrochemical
biosensing platform based on graphene quantum dots (GQDs) for the early identifi-
cation of acute myocardial infarction (AMI) was investigated and LOD was found to
be 0.01 ng/mL (Tabish et al. 2022). Biosensors for cardiovascular disease detection
are very common and commercially available. Enzyme as a biomarker or enzyme as
a biorecognition element is shown in most of the biosensor but synthetic
biorecognition element for biosensing is also mentioned. Table 1.1 lists a compre-
hensive list of biosensors, both enzymatic and nonenzymatic type.
1.4.2 Biosensors for Liver Diseases
A multiplexed assay using giant magnetoresistive (GMR) biosensor for early detec-
tion of cirrhosis was developed by using cirrhotic protein biomarkers intercellular
adhesion molecule-1 and mac-2 (Ng et al. 2020). In another study, a surface-
enhanced Raman spectroscopy (SERS)-based biosensor was developed for detection
of liver diseases serologically by integrating convolutional neural network (CNN)
classifier with gold-silver (Au-Ag) nanocomplex-decorated zinc oxide (ZnO)
nanopillars on paper (Cheng et al. 2021). Similarly, an enzymatic biosensor for
detection of hepatocellular carcinoma (HCC) using serum biomarker alpha-
fetoprotein (AFP) had been developed for early diagnosis of liver disease (Attia
et al. 2022).
1.4.3 Biosensors for Diabetes
A glucose enzymatic biosensor for detection of diabetes was developed using

derivatives of oxidase and dehydrogenase of glucose, which rely on flavin adenine
dinucleotide (Ferri et al. 2011). In another study, several surface modifications to
nanowires of Pt were done for improving its catalytic activity toward immobilized
glucose oxidase cross-linked on the surface of the electrode using glutaraldehyde.
Three generations of these biosensors were studied utilizing cyclic voltammetry. The
response current, coefficient of determination (R21st gen = 0.834, R22nd gen = 0.955,
R23rd gen = 0.921), and glucose sensitivity improved with each successive newer
generation (Shi et al. 2010). A glucose biosensor using potentiometric transducer
made of zinc oxide (ZnO) nanowire electrode was developed for measuring glucose
concentration for detection of diabetes (Usman Ali et al. 2010). Similarly, glass
electrode glucose enzymatic biosensors have been developed for different analytes
1
Table 1.1 Recent advancements in cardiac biosensors, including technological approaches used in their operation, healthcare applications, benefits, and
drawbacks
Biosensor Technical strategy Mechanism Applications Limitations Advantages References
Aptamer-based Label-free detection Detection based on Cardiovascular Low binding Reagent less Qureshi et al.
capacitive based on charge distribution of disease affinity processing (2010a, b)
biosensor non-faradaic CRP
impedance
spectroscopy
Neutrophil Measurement-based Immobilizing Cardiovascular Detects up to the only Point-of-care Gonzalez
gelatinase- on electrochemical monoclonal disease certain level of diluted biosensor and La Belle
associated impedance antibodies (against concentration (2012)
lipocalin spectroscopy (EIS) NGAL) onto gold
(NGAL) disk electrodes
detection
biosensor
Single site- Microfluidic Detection of the Cardiovascular Low-cost efficiency Good specificity with Lee et al.
specific channels integrated b-type natriuretic disease ultrahigh sensitivity (2012)
Enzymatic Biosensors for Healthcare Applications
polyaniline with nanowire-based peptide (BNP), (diagnosis of

(PANI) biosensors myoglobin (Myo), heart failure
nanowire creatine kinase MB stages)
biosensor (CK-MB), and
cardiac troponin I
(cTnI)
Bienzyme Based on Layer-by-layer Cardiovascular The high cost of High sensitivity, Cai et al.
biosensor functionalized amassed and carbon disease assembling stability, and (2013)
carbon nanotubes nanotubes/gold (detection of controllability
(CNTs) nanoparticles cholesterol)
centered
FRET Fluorescent-tagged Förster resonance Cardiovascular Better sensitivity Visualizing cGMP, Thunemann
biosensors genetically encoded energy transfer based system requires an optimal cAMP, and Ca2+ in et al. (2014)
(tagged combination of cells
biosensor)
11
(continued)
Table 1.1 (continued)
12
Biosensor Technical strategy Mechanism Applications Limitations Advantages References

fluorescence
nanomaterials
Substrate-gate Covalent binding Cardiac troponin I Cardiovascular Limit of detection Improved the sensitive Fathil et al.
coupled immobilization of (label free detection) disease detection (2017)
FET-based MAb-cTnI via
biosensor
Flexible zinc Detection based on Multiplexed and Cardiovascular Limit of detection Early diagnosis Radha
oxide EIS and Mott- simultaneous disease Shanmugam
nanostructured Schottky analysis detection of two et al. (2017)
biosensor isoform of troponins
Nanohybrid Doping of ChOx Cardiovascular Poor long-term Cost-effective, Shrestha
composite-based engineered immobilization on disease stability biocompatible, et al. (2017)
cholesterol g-C3N4H+ CSPPy-gC3N4H+ (cholesterol eco-friendly
biosensors nanosheets with nanohybrid detection in
cylindrical composite human serum)
spongyshaped
polypyrrole
GQDs-based EIS Identification of Acute Low recovery, low Strong interactions Tabish et al.
electrochemical troponin and myocardial efficiency, the usage of between cTnI and (2022)
biosensor myoglobin infarction reagents, several steps GQDs, minimal
(AMI) in handling, and the biomarker use, and
detection length of the inquiry electrode reusability
cost of antibody
manufacturing, poor
performance and
stability under stress,
and very high
temperatures
B. Daurai et al.
with solutions (chitosan, polyethylenimine, polystyrene sulfonate, etc.). The surface

of the electrode was modified using different films of Au, Ag, Co, etc. for determin-
ing the catalytic activity of glucose oxidase using cyclic voltammetry (Qiu et al.
2012; Wang et al. 2012; Khun et al. 2012). Another study on voltammetry was done
where a working chip electrode was developed for direct electron transfer to the
electrode using a flavin adenine dinucleotide-glucose dehydrogenase enzyme
immobilized on its surface by crosslinking. The enzyme’s active site was engineered
to be less maltose-specific after it was isolated from Burkholderia cepacia
(Yamashita et al. 2013). Similarly, an Au electrode was coated with a thin cyste-
amine film covering a double layer of immobilized glucose oxidase submerged in a
chitosan (CS) solution. The glucose concentration was determined using cyclic
voltammetry (Zhang et al. 2014). In another study, carbon nanotubes (multiwalled
CNTs) coated with tiny tantalum (Ta) plates were used as electrodes. This electrode
was again coated with a Nafion film immobilized glucose oxidase. Cyclic
voltammetry transducer was then used to measure concentration of glucose (Cui
et al. 2013).
Amperometric glucose biosensors were developed using multiwalled CNTs by
co-immobilizing adenine dinucleotide flavin and glucose dehydrogenase (made
using the enzyme from Aspergillus oryzae) (Monošík et al. 2012). Another ampero-
metric glucose biosensor was developed using electrochemical polymerization of N-
phenylglycine film. It was made using the covalent immobilization of glucose
oxidase on an N-phenylglycine surface that resulted from an interaction between
the film and amino-carboxyl groups present in the glucose oxidase (Homma et al.
2014). In another study, an amperometric biosensor was made using carbon paste
electrodes coated with silicon grease paste. This silicon grease paste was modified
with glucose oxidase and maghemite (Fe2O3, γ-Fe2O3) nanoparticles. It was inserted
into the hollow of the glass electrode, after which the current was measured using an
amperometric transducer (Baratella et al. 2013).
In a study comparing chronoamperometry and cyclic voltammetry, a gold elec-
trode coated with Au-chitosan nanocomposites was electrodeposited via
chronoamperometry. A thin film of nickel (Ni) was deposited on the surface of the
electrode, and glucose oxidase immobilized on it. Chronoamperometry and cyclic
voltammetry were used for the assessment of glucose oxidase catalytic activity
(Zhao et al. 2013; Mathew and Sandhyarani 2013). In a similar study, a Pt electrode
was electrooxidized with a polyvinylferrocenium perchlorate layer to determine
glucose using cyclic voltammetry and amperometry. At the same time as Pt
nanoparticles were electrodeposited on the modified electrode with electro-
polymerization of glucose oxidase and poly(o-phenylenediamine) on the electrode
(Turkmen et al. 2014).
Optical enzymatic biosensors were developed for determination of glucose con-
centration. Based on chemiluminescence, fluorescence, and phosphorescence
techniques, the emission produced electrochemically was converted into an electri-
cal signal for detection of glucose. In a study, an optical biosensor using a poly
(luminol-aniline) nanowire composite was developed to measure glucose concentra-
tion (Li et al. 2010). In another study, optical enzymatic biosensor was developed
14 B. Daurai et al.
using modified electrode surface coated in a layer of CS and glucose oxidase inside a
produced glass carbon electrode covered with Au nanoparticles and CNTs for
detection of glucose (Haghighi et al. 2011).
Similarly, in a study, a Nafion film-wrapped electrode was taken in a glass carbon
electrode modified with CNTs. Glucose oxidase and palladium (Pd) nanoparticles
were placed on the surface of the modified electrode. The electrochemiluminescence
technique was employed to ascertain the glucose content in them (Haghighi and
Bozorgzadeh 2011). Another biosensor containing a solution of glucose oxidase, Au
nanoparticles, CNTs, and Nafion film applied to CNTs was developed to measure
glucose. It was prepared on graphite supports (5 × 5 × 30 mm) disseminated in a
solution of ethanol-Nafion (Zargoosh et al. 2012).
A glucose biosensor based on microarray plates doped with sol film using the
fluorescence intensity was developed with rubidium (Rb) fluorophores. The glucose
levels were determined as glucose oxidase might be found in the silica sol-gel
(Chang et al. 2010). In another study, a glucose biosensor was developed using a
hydrogel carrying a sol-gel matrix with glucose oxidase substrate that was
immobilized to the egg membrane with coordination polymers made of crystalline
iridium (Ir). The glucose levels were estimated using the phosphorescence technique
(Ho et al. 2014).
Zhang et al. developed a piezoelectric biosensor for real-time monitoring blood
glucose level for the prophylaxis and treatment of diabetes using a flexible self-
powered implantable skin-like glucometer. It is based on the piezo-enzymatic-
reaction coupling effect of GOx@ZnO nanowire arrays. This device converts the
mechanical energy to piezoelectric impulse for determination of blood glucose
concentration (Zhang et al. 2018). In another study of piezoelectric biosensor, a
quartz crystal microbalance (QCM) biosensor for detection of hemoglobin A1c
(HbA1c) was developed. The variations in resonance frequency of QCM allow for
the quantitative measurement of HbA1c. The HbA1c LOD with respect to hemoglo-
bin is 0.147%. This biosensor aids in the creation of beneficial HbA1c sensing
devices (Park and Lee 2018) (Fig. 1.1).
The features of the abovementioned enzymatic glucose biosensors are
summarized in Table 1.2. Enzymatic glucose sensors are typically electrochemical
and optical transducers. Electrochemical transducers were employed more com-
monly in the past due to their great sensitivity, durability, simplicity of instrumenta-
tion, cheap cost, remarkable compatibility, and possibility for downsizing. Because
the immobilization of the enzyme to the electrode in electrochemical transducers
runs the risk of losing enzymatic function, optical transducers are growing in
popularity (Mathew and Sandhyarani 2013).
1.4.4 Biosensors for Kidney Diseases
Premanode and Toumazou (2007) developed a new biosensor based on immobilized

creatininase, creatinine, and urease employing ion-sensitive field effect transistors
(ISFETs) for real-time monitoring in peritoneal dialysis. With a weak inversion at
Fig. 1.1 The custom-made fluidic modules for QCM biosensor (Park and Lee 2018)
pH 6–8 and 37 °C, the circuitry demonstrated a linear relationship between urea and
creatinine at the ranges of 0–200 and 0–20 mM, respectively (PD) (Fig. 1.2).
Oleg Palygin et al. (2015) used two different sensor designs for ex vivo and
in vivo protocols for creation of enzymatic microelectrode biosensors to measure
endogenous ATP or H2O2 in the kidney. These protocols involved the detection of
analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode and for
reduction of H2O2 on a mediator-coated Au electrode, respectively. After calibration
to known analyte concentrations, real-time amperometry is utilized to identify
changes in the final concentration (Fig. 1.3).
Pallavi Dasgupta et al. (2020) developed a practical point-of-care (POC) device
using electrochemical biosensor that uses screen-printed electrodes for estimation of
highly selective creatinine over the range of 0.2–4 mg/dL in a sample volume of
300 L without pre-processing. Creatinine is converted via a mono-enzymatic path-
way to 1-methylhydantoin, whose concentration is measured by forming a complex
with cobalt. This complex is validated via optical spectroscopy (Fig. 1.4).
As it was already discussed, several innovative biosensors have been developed
that may be used with a variety of physiological fluids that are frequently easy to get,
can be miniaturized, and are particularly tempting for the POC market for patients
with chronic kidney disease (CKD). For measuring the signs of renal disease, there
are already portable and handheld POC equipment on the market. Examples of
products that use enzymatic processes to detect creatinine electrochemically include
StatSensor (Nova Biomedical, Waltham, MA, USA) and i-STAT (crea-cartridge,
16 B. Daurai et al.
Table 1.2 Enzymatic biosensors’ overview

Construction of sensor Transducer LOD Reference
Glucose oxidase immobilized on Amperometry 2.0 μmol/L Baratella
a carbon paste electrode covered et al. (2013)
with surface-active maghemite
nanoparticles
Sol-gel-encased glucose oxidase Fluorescence 60.0 μmol/ Chang et al.
was put onto a microarray with a L (2010)
Rb-fluorophore layer
Fe phthalocyanine-modified Cyclic voltammetry 30.0 μmol/ Cui et al.
CNTs covered with titanium L (2013)
dioxide layer and glucose
oxidase immobilized therein
A glassy carbon electrode Electrochemiluminescence 50.0 nmol/ Haghighi and
covered with multi-walled CNTs L Bozorgzadeh
and glucose oxidase immobilized (2011)
on Pd nanoparticles
Glucose oxidase is immobilized Electrochemiluminescence 0.5 μmol/L Haghighi
on a chitosan layer on multi- et al. (2011)
walled CNTs that are covered in
gold nanoparticles on the surface
of glassy carbon electrodes
Egg membrane covered with Phosphorescence 10.0 μmol/ Ho et al.
glucose oxidase immobilized in L (2014)
hydrogel and crystalline Ir
substrate
Covalently immobilized glucose Amperometry 30.0 μmol/ Homma et al.
oxidase on a polymerized N- L (2014)
phenylglycine layer on the
electrode surface
Glucose oxidase immobilized on Potentiometry – Khun et al.
an Au-glass electrode using a (2012)
chitosan-magnetic coating
Gelatin, silicon oxide, polyvinyl Cyclic voltammetry – Shi et al.
alcohol, Prussian blue, and a self- (2010)
assembled monolayer of
cysteamine were used to
immobilize glucose oxidase onto
a Pt nanowire electrode
Immobilized glucose oxidase on Electrochemiluminescence 30.0 nmol/ Li et al.
composite poly(luminolaniline) L (2010)
nanowire-coated graphite
electrode
Chitosan-Au nanostructures on Cyclic voltammetry, 100.0 nmol/ Mathew and
an Au electrode are covered in a chronoamperometry L Sandhyarani
nickel hydroxide coating with (2013)
glucose oxidase immobilized on
it
Co-immobilized flavin adenine Amperometry 4.0 μmol/L Monošík
dinucleotide and glucose et al. (2012)
(continued)
Table 1.2 (continued)

Construction of sensor Transducer LOD Reference
dehydrogenase on a multi-walled
carbon nanotube electrode in a
chitosan layer
Glucose oxidase mounted on a Cyclic voltammetry, 0.3 μmol/L Qiu et al.
glassy carbon electrode with an differential pulse (2012)
Au thin film coating voltammetry,
amperometry
Pt nanoparticles covered with Pt Cyclic voltammetry, 20.0 μmol/ Turkmen
electrode modified by amperometry L et al. (2014)
polyvinylferrocenium
concurrently immobilized
glucose oxidase with poly(o-
phenylenediamine)
Immobilized glucose oxidase on Potentiometry – Usman Ali
ZnO-Ag nanowire et al. (2010)
Glucose oxidase immobilized on Cyclic voltammetry 0.2 μmol/L Wang et al.
a glassy carbon electrode covered (2012)
with magnetic-silica-Au core-
shell nanoparticles
For direct electron transfer, flavin Voltammetry – Yamashita
adenine dinucleotide-glucose et al. (2013)
dehydrogenase is immobilized
on the surface of a functioning
chip electrode
Carbon nanotube film on Chemiluminescence, 1.0 μmol/L Zargoosh
graphite supports with glucose fluorescence et al. (2012)
oxidase immobilized on Au
nanoparticles
Immobilized glucose oxidase on Cyclic voltammetry 50 and Zhang et al.
a thin cysteamine layer 300 μmol/L (2014)
overlaying an Au electrode
Glucose oxidase is immobilized Differential pulse 10.0 μmol/ Zhao et al.
on a silicon oxide electrode using voltammetry L (2013)
phytic acid nanoparticles
Coupling effect of GOx@ZnO Piezoelectric – Zhang et al.
nanowire arrays (2018)
Mass changes of QCM caused by Quartz crystal 0.147% Park and Lee
size enlargement of conjugated microbalance (QCM) HbA1c (2018)
Au nanoparticles with thiol-
terminated SAMs on QCM
sensor surface due to gold
staining by hydrogen peroxide
(H2O2) generated from
enzymatic HbA1c assay
18 B. Daurai et al.
Fig. 1.2 Block diagram of the sensor system (Premanode and Toumazou 2007)
Fig. 1.3 Diagram of the biosensor’s protocol system (Palygin et al. 2015)
Abbott Point of Care Inc., Princeton, NJ, USA). The StatSensor is a simple device
with significant potential that can quantify creatinine from a finger-prick sample, in
contrast to the i-STAT device which has a volume requirement of 100 L and makes
finger-prick sampling difficult. Despite these encouraging developments, POC sens-
ing technology development remains difficult, necessitating substantial further
research efforts. Notably, interaction with a number of the substances present in
bodily fluids, including ascorbic acid, glucose, fructose, and ketone bodies, may
have an impact on the measurement’s accuracy. Even when dealing with a complex
Fig. 1.4 Serum creatinine electrochemical biosensor schematic (Dasgupta et al. 2020)
matrix like whole blood, conflicting reactions and signals may be reduced by using
antibody-based sensors rather than enzymatic ones.
1.4.5 Biosensors for Neurological Diseases
Biosensors for neurological disease (ND) serve two purposes: they may be utilized
to find new biomarkers and to be used as diagnostic instruments. These sensors
include a wide variety of technologies, from experimental proofs of concept to
commercially available kits. These sensors have detection efficiencies that are on
par with or beyond those of the traditional test. Multistep immunoassays are a
common part of the processes employed in clinical labs nowadays to diagnose
ND. Due to the immunoassay’s reliance on antibody-antigen (Ab-Ag) interaction,
these tests are easily adapted for use in biosensor, including Roche Diagnostics’
portable therapeutic drug monitor and Biacore® SPR (surface plasmon resonance).
With the use of microarrays from firms like Affymetrix®, Agilent (gene-based), and
Ciphergen, extensive biomarker discovery is now taking place (protein based).
There are also studies on detection of neurological diseases but gene- and
DNA-based biosensors have more commercial presence. Table 1.3 depicts a quick
list of biosensor firms and platforms available commercially. Due to their prevalence
in the diagnostic society, description of these biosensors is focused in this chapter.
20 B. Daurai et al.
Table 1.3 Platforms and technologies for biosensors

Company Platform probe/technology Target analyte type
Affymetrix® GeneChip® DNA-based microarray Genetic
Agilent DNA-based microarray, uses 60-mer probes Genetic
Applied DNA-based microarray and protein Genetic, proteomic
Biosystems biomarker discovery
Biobarcode Nonenzymatic amplification Genetic, proteomic,
immunogenic, small
molecules, complex targets
Cepheid DNA-based microarray Genetic
Ciphergen ProteinChip: protein, peptide, and Ab Proteomic, immunogenic
microarrays
Luminex xMAP technology: cDNA or peptide/ Genetic, proteomic,
protein/Ab-coated beads immunogenic
Plexigen GeneCube™, geneCard™: genetic-, Genetic, proteomic,
proteomic-, and immunogenic-based immunogenic
Roche TDM technology: Ab-based handheld Drug analytes, potentially any
Diagnostics device Ag-Ab pair
Third Wave Invader® technology: nucleic acid Genetic
Technologies quantitation
The binding contact between Ag and Ab is detected by immunosensors. On the

other hand, immobilized immunogenic peptides are employed to identify specific
Abs in biological fluids. Immunoglobulins or immobilized Abs are used for
detecting the presence of particular Ags. These sensors make use of the ease of
different physical transducers while minimizing, and in some instances like
eliminating, time-consuming washing and separating processes. After exposure to
an acid (such as HCl or H2SO4), a base (such as tetraethylamine), a denaturant (such
as urea), or a high salt concentration, many immunosensors may be recovered. The
probes may be cost-effectively reused after being rejuvenated.
Surface plasmon resonance (SPR) is used by a broad variety of immunosensors to
identify and quantify neurological biomarkers. The interaction between particular
anti-ADDL Abs and amyloid-β-derived diffusible ligands (ADDLs) has been
quantified using an optical biosensor based on SPR for the detection of Alzheimer’s
disease (AD) (Haes et al. 2005). To assess the pathogenic threshold for HD, normal
and enlarged polyglutamine tracts have been characterized using Biacore® SPR
devices (Bennett et al. 2002). Although the HD trait marker is easily recognized,
other markers are required to better understand the age of illness start and
progression.
SPR is often used to find cancer biomarkers. There are several instances of
prostate-specific antigen (PSA) detection, a marker for prostate cancer, at nano- to
femtomolar levels (Yu et al. 2004; Huang et al. 2005). Additional instances of PSA
detection using microcantilever and amperometric technology are available (Sarkar
et al. 2002; Lee et al. 2005; Wee et al. 2005). The piezoelectric immunosensor array
for acute leukemia is another cancer diagnostic concept using clinical

immunophenotyping (Wang et al. 2004).
Chips made from proteins or peptides have been utilized to find biomarkers. The
Protein Chip platform is provided by Ciphergen Biosystems, Inc. for the identifica-
tion and evaluation of biomarkers. Using this platform, a wide variety of biomarkers
have been established for the diagnosis of Alzheimer’s disease (AD) (Carrette et al.
2003; Haes et al. 2005). Using surface-enhanced laser desorption/ionization time-of-
flight (SELDI-TOF) mass spectrometry, Ciphergen protein arrays have discovered a
unique panel of early-stage AD biomarkers from CSF samples. Multiplexed analyte
detection and quantification employing protein (e.g., Ab) and nucleic acid (e.g.,
cDNA) complexed probes are both possible with Luminex bead-based array devices.
Seven hundred and fifty-six cervical cancer patient sera were studied utilizing
Luminex systems. Human papillomaviruses (HPVs) come in more than 100 varieties,
many of which are responsible for proliferative illnesses like cervical cancer. In this
investigation, in situ affinity-purified recombinant HPV proteins against 27 Abs
were simultaneously detected (Waterboer et al. 2005). According to the Luminex
xMAP® technology requirements, the assay may test up to 100 distinct Ags concur-
rently in each reaction well. The collaboration between Luminex Corporation and
Zeus Scientific for providing test kits for autoimmune disease was announced in
May 2005 by Luminex Corporation. High-throughput gene expression profiling is
offered by SuperArray Bioscience Corporation using Plexigen’s geneCube™ tech-
nology, which makes use of stacked detection arrays. The arrays may screen for a
broad range of targets in bodily fluids and can be functionalized using nucleic acids,
proteins, or antibodies. In a few of days, the system can examine tens of thousands of
samples.
Based on the BMI-1 oncogene pathway, the study team discovered an 11-gene
signature for the chance of developing cancer. The expression of these cancer-related
genes was examined using an Affymetrix® microarray (Glinsky et al. 2005). The
11-gene signature was linked to a higher chance of survival by the authors when it
was paired with a statistical analysis of patient therapeutic outcome data. Addition-
ally, they were successful in doing so for 11 distinct cancer types. To find disease
signatures, the same approach is being used to study AD and other NDs.
1.5 Challenges and Future Scope
Since their introduction into the medical industry, biosensors have seen significant
advancement. However, technology constantly leaves behind certain options that
require additional study and development. Recognizing tiny molecules, improving
sensitivity and selectivity in detecting various analytes, cross-linking, and
immobilizing bioreceptors on the substrate are the key problems. Multiple
biomarkers must be detected simultaneously using a single device in order to
diagnose PCs (Shergujri et al. 2019). Microfluidics and biomarkers’ pattern software
can be combined in the near future to create promising devices for use in this field
(Tothill 2009). Biosensor sensitivity amplification has been a significant problem.
22 B. Daurai et al.
There are various methods for increasing sensitivity. To increase the sensitivity of
these devices, nanorods with high aspect ratios are chosen over spherical
nanoparticles. Observing protein and protein interactions is more difficult than
observing nucleic acid interactions since new amplification tools are not yet accessi-
ble. This situation is further enhanced by the complex interaction of the proteins and
their increased nonspecific binding to solid supports. Consequently, it is anticipated
that the potential for protein-nanoparticle hybrids would be completely promoted by
nanoparticles that include mixed monolayers (Wang 2005).
Most sensors require calibration prior to each usage as well as incubation in
buffer solutions. Some sensors have a maximum concentration at which they can
detect an analyte. This component is also influenced by the biosensor’s stability.
Because bioreceptors are sensitive to variations in temperature, pressure, and pH
value, long-term preservation and use of bioreceptors is a significant difficulty. Any
modifications to the regular surroundings could cause the senses to become less
sensitive. As the environmental circumstances change over time, biosensors with a
higher stability range are considerably more favored. The stability of the biosensors
in a changing environment is currently a significant challenge. Biosensors have been
needed to detect numerous analytes for specificity in some cases. Multiple analytes
may be required for the validation of some specific malignant cells. Therefore, it is
necessary to build a dual-sensing capability in order to increase the biosensor’s
specificity (Liu et al. 2017).
Due to their immense potential in both research and commercial applications,
biosensors are quickly becoming a necessity in today’s society. Reduction in cost
and detectability of a variety of analytes are the advantages that can be achieved by
combination of microfluidic technology and 3D printing. According to the World
Health Organization (WHO), biosensor devices should be affordable, sensitive,
specific, quick, dependable, equipment-free, and easily distributable (Ding et al.
2015).
Enzymatic biosensors can be specialized for detection and diagnosis of diseases
for treatment and lifesaving procedures (Sarkar et al. 2022). Despite the benefits of
utilizing enzymes, several drawbacks, such as the enzyme’s quick loss of activity as
a result of interactions with the electrode surface, result in a biosensor’s lifetime of
only 2–4 weeks. However, this can be avoided if the enzyme is properly stabilized
by selecting an appropriate matrix and a better method to immobilize the enzyme.
1.6 Conclusion
As enzymes are very specific to their respective substrates, use of an enzyme to

detect a substrate or vice versa is very applicable in healthcare. Enzymes have been
extensively used in amperometric, optical, and even piezoelectric biosensors.
Biosensors based on enzymes have several uses in the fields of food, medicine,
and environmental monitoring. Electrochemical and optical types of transductions
are one of the most common types. In optical type, physical and chemical process of
immobilization of enzyme is used. In electrochemical type, the enzymes themselves

are used in the reaction to determine the action of the analyte on the enzyme.
In healthcare, they can be used for estimation of various analytes. Enzymatic
biosensors as an amperometric biosensor have been extensively used for detection of
glucose using glucose oxidase as the biorecognition element. Commercially avail-
able biosensors are also mostly based on enzymatic biosensor because of their
sensitivity, specificity, and necessary substrate combination of selectivity.
The present trend in biosensor is to reduce the cost which is mainly due to the
high cost of biological identification element. This will make biosensors available
for point-of-care application. The development of tiny, self-contained devices for a
variety of chemical or biological analyses, which play an increasingly significant
role in our contemporary society, is centered on enzyme-based biosensors
employing optical or electrochemical transducers. They will undoubtedly become
more significant in the coming years.
Acknowledgments We sincerely acknowledge the generous funding received from the Depart-
ment of Biotechnology, India (No. BT/PR24652/NER/95/795/2017; Dated: 06/03/2019), sanction
to Dr. Manashjit Gogoi for carrying out this piece of work.
References
Adams LA, Angulo P, Lindor KD (2005) Nonalcoholic fatty liver disease. CMAJ 172:899–905
Apple FS (1989) Diagnostic use of CK-MM and CK-MB isoforms for detecting myocardial
infarction. Clin Lab Med 9:643–654
Attia MS, Mohamed AA, El-Saady MM et al (2022) A new method for early diagnosis of liver
cancer using a biosensor embedded in an alginate polymer thin film. J Mater Chem C 10:6464–
6472. https://doi.org/10.1039/D2TC00209D
Bacon BR (2002) Treatment of patients with hepatitis C and normal serum aminotransferase levels.
Hepatology 36:s179–s184
Baratella D, Magro M, Sinigaglia G et al (2013) A glucose biosensor based on surface active
maghemite nanoparticles. Biosens Bioelectron 45:13–18. https://doi.org/10.1016/J.BIOS.2013.
01.043
Bartlett PN, Bradford VQ, Whitaker RG (1991) Enzyme electrode studies of glucose oxidase
modified with a redox mediator. Talanta 38:57–63
Bennett MJ, Huey-Tubman KE, Herr AB et al (2002) A linear lattice model for polyglutamine in
CAG-expansion diseases. Proc Natl Acad Sci U S A 99:11634–11639. https://doi.org/10.1073/
PNAS.182393899/SUPPL_FILE/3938SUPPTEXT.PDF
Bershtein S, Tawfik DS (2008) Advances in laboratory evolution of enzymes. Curr Opin Chem Biol
12:151–158
Bhattarai P, Hameed S (2020) Basics of biosensors and nanobiosensors. In: Wu A, Khan WS (eds)
Nanobiosensors: from design to applications. Wiley, pp 1–22
Blum LJ, Gautier SM, Coulet PR (1989) Design of luminescence photobiosensors. J Biolumin
Chemilumin 4:543–550
Bulluck H, Maiti R, Chakraborty B et al (2018) Neutrophil gelatinase-associated lipocalin prior to
cardiac surgery predicts acute kidney injury and mortality. Heart 104:313–317
Bunde RL, Jarvi EJ, Rosentreter JJ (1998) Piezoelectric quartz crystal biosensors. Talanta 46:1223–
1236. https://doi.org/10.1016/S0039-9140(97)00392-5
24 B. Daurai et al.
Cai X, Gao X, Wang L et al (2013) A layer-by-layer assembled and carbon nanotubes/gold

nanoparticles-based bienzyme biosensor for cholesterol detection. Sens Actuators B Chem
181:575–583. https://doi.org/10.1016/J.SNB.2013.02.050
Calvaruso V, Craxì A (2009) Implication of normal liver enzymes in liver disease. J Viral Hepat 16:
529–536
Carrette O, Demalte I, Scherl A et al (2003) A panel of cerebrospinal fluid potential biomarkers for
the diagnosis of Alzheimer’s disease. Proteomics 3:1486–1494. https://doi.org/10.1002/PMIC.
200300470
Chang G, Tatsu Y, Goto T et al (2010) Glucose concentration determination based on silica sol–gel
encapsulated glucose oxidase optical biosensor arrays. Talanta 83:61–65. https://doi.org/10.
1016/J.TALANTA.2010.08.039
Chaubey A, Malhotra B (2002) Mediated biosensors. Biosens Bioelectron 17:441–456
Chen Y-G, Janckila A, Chao T-Y et al (2015) Association of tartrate-resistant acid phosphatase-
expressed macrophages and metastatic breast cancer progression. Medicine 94:e2165
Chen JH, Wu YY, Huang TC et al (2017) Tartrate-resistant acid phosphatase 5a activity as a novel
biomarker for flare of systemic lupus erythematosus. Acad J Biotechnol 5. https://doi.org/10.
15413/ajb.2017.0101
Cheng N, Chen D, Lou B et al (2021) A biosensing method for the direct serological detection of
liver diseases by integrating a SERS-based sensor and a CNN classifier. Biosens Bioelectron
186:113246. https://doi.org/10.1016/J.BIOS.2021.113246
Choi MMF (2004) Progress in enzyme-based biosensors using optical transducers. Microchim Acta
148:107–132
Clark LC Jr (1956) Monitor and control of blood and tissue oxygen tensions. Trans Am Soc Artif
Intern Organs 2:41–48
Clark LC Jr, Lyons C (1962) Electrode systems for continuous monitoring in cardiovascular
surgery. Ann N Y Acad Sci 102:29–45
Corathers SD (2006) The alkaline phosphatase level: nuances of a familiar test. Pediatr Rev 27:382
Cui HF, Zhang K, Zhang YF et al (2013) Immobilization of glucose oxidase into a nanoporous TiO2
film layered on metallophthalocyanine modified vertically-aligned carbon nanotubes for effi-
cient direct electron transfer. Biosens Bioelectron 46:113–118. https://doi.org/10.1016/J.BIOS.
2013.02.029
Dasgupta P, Kumar V, Krishnaswamy PR, Bhat N (2020) Serum creatinine electrochemical
biosensor on printed electrodes using monoenzymatic pathway to 1-methylhydantoin detection.
ACS Omega 5:22459–22464. https://doi.org/10.1021/ACSOMEGA.0C02997/SUPPL_FILE/
AO0C02997_LIVESLIDES.MP4
Devi CS, Nadiger HA, Krishna TS, Naidu JN (2017) Serum acid phosphatase level-is it a marker for
diagnosis of malaria. Int J Res Med Sci 5:4400–4402
Ding X, Srinivasan B, Tung S (2015) Development and applications of portable biosensors. SLAS
Technol 20:365–389. https://doi.org/10.1177/2211068215581349
Dzyadevych SV, Arkhypova VN, Soldatkin AP et al (2008) Amperometric enzyme biosensors:
past, present and future. IRBM 29:171–180
El-Kabbani O, Darmanin C, Chung R-T (2004) Sorbitol dehydrogenase: structure, function and
ligand design. Curr Med Chem 11:465–476
Fargo MV, Grogan SP, Saguil A (2017) Evaluation of jaundice in adults. Am Fam Physician 95:
164–168
Fathil MFM, Md Arshad MK, Ruslinda AR et al (2017) Substrate-gate coupling in ZnO-FET
biosensor for cardiac troponin I detection. Sens Actuators B Chem 242:1142–1154. https://doi.
org/10.1016/j.snb.2016.09.131
Ferri S, Kojima K, Sode K (2011) Review of glucose oxidases and glucose dehydrogenases: a bird’s
eye view of glucose sensing enzymes. J Diabetes Sci Technol 5:1068–1076. https://doi.org/10.
1177/193229681100500507
Frey CF, Suzuki M, Isaji S (1990) Treatment of chronic pancreatitis complicated by obstruction of
the common bile duct or duodenum. World J Surg 14:59–69
Froissart R, Piraud M, Boudjemline AM et al (2011) Glucose-6-phosphatase deficiency. Orphanet J

Rare Dis 6:1–12
Glinsky GV, Berezovska O, Glinskii AB (2005) Microarray analysis identifies a death-from-cancer
signature predicting therapy failure in patients with multiple types of cancer. J Clin Invest 115:
1503–1521. https://doi.org/10.1172/JCI23412
Godfrey T, Reichelt J (1982) Industrial enzymology: the application of enzymes in industry. U.-
S. Department of Energy
Gonzalez SI, La Belle JT (2012) The development of an at-risk biosensor for cardiovascular
disease. Biosens J 1:1–5
Gopcevic K, Rovcanin B, Kekic D et al (2017) Gelatinases A and B and antioxidant enzyme
activity in the early phase of acute myocardial infarction. Folia Biol (Praha) 63:20
Gyöngyösi M, Winkler J, Ramos I et al (2017) Myocardial fibrosis: biomedical research from bench
to bedside. Eur J Heart Fail 19:177–191
Habermüller K, Mosbach M, Schuhmann W (2000) Electron-transfer mechanisms in amperometric
biosensors. Fresenius J Anal Chem 366:560–568
Haes AJ, Chang L, Klein WL, van Duyne RP (2005) Detection of a biomarker for Alzheimer’s
disease from synthetic and clinical samples using a nanoscale optical biosensor. J Am Chem Soc
127:2264–2271. https://doi.org/10.1021/JA044087Q/ASSET/IMAGES/MEDIUM/
JA044087QN00001.GIF
Haghighi B, Bozorgzadeh S (2011) Enhanced electrochemiluminescence from luminol at multi-
walled carbon nanotubes decorated with palladium nanoparticles: a novel route for the fabrica-
tion of an oxygen sensor and a glucose biosensor. Anal Chim Acta 697:90–97. https://doi.org/
10.1016/J.ACA.2011.04.032
Haghighi B, Bozorgzadeh S, Gorton L (2011) Fabrication of a novel electrochemiluminescence
glucose biosensor using Au nanoparticles decorated multiwalled carbon nanotubes. Sens
Actuators B Chem 155:577–583. https://doi.org/10.1016/J.SNB.2011.01.010
Haider MR, Islam SK (2010) Ultra-low-power sensor signal processing unit for implantable
biosensor applications. In: International conference on electrical & computer engineering
(ICECE 2010), pp 302–305
Hayman AR (2008) Tartrate-resistant acid phosphatase (TRAP) and the osteoclast/immune cell
dichotomy. Autoimmunity 41:218–223
He E, Xu XH, Guan H et al (2010) Thymidine kinase 1 is a potential marker for prognosis and
monitoring the response to treatment of patients with breast, lung, and esophageal cancer and
non-Hodgkin’s lymphoma. Nucleosides Nucleotides Nucleic Acids 29:352–358
Hemalatha T, UmaMaheswari T, Krithiga G et al (2013) Enzymes in clinical medicine: an
overview. Indian J Exp Biol 51(10):777–788
Ho ML, Wang JC, Wang TY et al (2014) The construction of glucose biosensor based on crystalline
iridium(III)-containing coordination polymers with fiber-optic detection. Sens Actuators B
Chem 190:479–485. https://doi.org/10.1016/J.SNB.2013.08.100
Holmes RS (1978) Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase,
aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues. Comp
Biochem Physiol B 61:339–346
Homma T, Ichimura T, Kondo M et al (2014) Covalent immobilization of glucose oxidase on the
film prepared by electrochemical polymerization of N-phenylglycine for amperometric glucose
sensing. Eur Polym J 51:130–135. https://doi.org/10.1016/J.EURPOLYMJ.2013.12.005
Hu Y-C, Liu H, Liu X-Y et al (2017) Value of gamma-glutamyltranspeptidase-to-platelet ratio in
diagnosis of hepatic fibrosis in patients with chronic hepatitis B. World J Gastroenterol 23:7425
Huang L, Reekmans G, Saerens D et al (2005) Prostate-specific antigen immunosensing based on
mixed self-assembled monolayers, camel antibodies and colloidal gold enhanced sandwich
assays. Biosens Bioelectron 21:483–490. https://doi.org/10.1016/J.BIOS.2004.11.016
Huang X-J, Choi Y-K, Im H-S et al (2006) Aspartate aminotransferase (AST/GOT) and alanine
aminotransferase (ALT/GPT) detection techniques. Sensors 6:756–782
26 B. Daurai et al.
Huber R, Hockenjos B, Blum HE (2004) DDB treatment of patients with chronic hepatitis.
Hepatology 39:1732–1733
Hwang D, Byrne J, Scollard D, Levine E (1998) Expression of cyclooxygenase-1 and
cyclooxygenase-2 in human breast cancer. J Natl Cancer Inst 90:455–460. https://doi.org/10.
1093/jnci/90.6.455
Joyce JA, Hanahan D (2004) Multiple roles for cysteine cathepsins in cancer. Cell Cycle 3:1516–
1519
Kang HS, Um SH, Seo YS et al (2011) Healthy range for serum ALT and the clinical significance of
“unhealthy” normal ALT levels in the Korean population. J Gastroenterol Hepatol 26:292–299
Kaplan MM (2002) Alanine aminotransferase levels: what’s normal? Ann Intern Med 137:49–51
Kato GJ, Nouraie SM, Gladwin MT (2013) Lactate dehydrogenase and hemolysis in sickle cell
disease. Blood 122:1091–1092
Khun K, Ibupoto ZH, Lu J et al (2012) Potentiometric glucose sensor based on the glucose oxidase
immobilized iron ferrite magnetic particle/chitosan composite modified gold coated glass
electrode. Sens Actuators B Chem 173:698–703. https://doi.org/10.1016/J.SNB.2012.07.074
Koizumi M, Ogata E (2002) Bone metabolic markers as gauges of metastasis to bone: a review. Ann
Nucl Med 16:161–168
Krause E-G, Rabitzsch G, Noll F et al (1996) Glycogen phosphorylase isoenzyme BB in diagnosis
of myocardial ischaemic injury and infarction. Mol Cell Biochem 160–161:289–295
Kunutsor SK (2016) Gamma-glutamyltransferase—friend or foe within? Liver Int 36:1723–1734
Kuswandi B, Andres R, Narayanaswamy R (2001) Optical fibre biosensors based on immobilised
enzymes. Analyst 126:1469–1491
Lee C-A, Tsai Y-C (2009) Preparation of multiwalled carbon nanotube-chitosan-alcohol dehydro-
genase nanobiocomposite for amperometric detection of ethanol. Sens Actuators B Chem 138:
518–523
Lee JH, Hwang KS, Park J et al (2005) Immunoassay of prostate-specific antigen (PSA) using
resonant frequency shift of piezoelectric nanomechanical microcantilever. Biosens Bioelectron
20:2157–2162. https://doi.org/10.1016/J.BIOS.2004.09.024
Lee I, Luo X, Huang J et al (2012) Detection of cardiac biomarkers using single polyaniline
nanowire-based conductometric biosensors. Biosensors 2:205–220. https://doi.org/10.3390/
BIOS2020205
Lesmana CRA, Salim S, Hasan I et al (2011) Diagnostic accuracy of transient elastography
(FibroScan) versus the aspartate transaminase to platelet ratio index in assessing liver fibrosis
in chronic hepatitis B: the role in primary care setting. J Clin Pathol 64:916–920
Li G, Lian J, Zheng X, Cao J (2010) Electrogenerated chemiluminescence biosensor for glucose
based on poly(luminol–aniline) nanowires composite modified electrode. Biosens Bioelectron
26:643–648. https://doi.org/10.1016/J.BIOS.2010.07.003
Liu T, Li N, Dong JX et al (2017) A colorimetric and fluorometric dual-signal sensor for arginine
detection by inhibiting the growth of gold nanoparticles/carbon quantum dots composite.
Biosens Bioelectron 87:772–778. https://doi.org/10.1016/J.BIOS.2016.08.098
Mahdizadeh B, Nouri A, Baharinikoo L, Lotfalipour B (2022) Enzymatic glucose biosensors: a
review on recent progress in materials and fabrication techniques. Anal Bioanal Chem Res 9:1–
19
Mathew M, Sandhyarani N (2013) A highly sensitive electrochemical glucose sensor structuring
with nickel hydroxide and enzyme glucose oxidase. Electrochim Acta 108:274–280. https://doi.
org/10.1016/J.ELECTACTA.2013.07.010
Mira-Pascual L, Patlaka C, Desai S et al (2020) A novel sandwich ELISA for tartrate-resistant acid
phosphatase 5a and 5b protein reveals that both isoforms are secreted by differentiating
osteoclasts and correlate to the type I collagen degradation marker CTX-I in vivo and in vitro.
Calcif Tissue Int 106:194–207
Mohanty SP, Kougianos E (2006) Biosensors: a tutorial review. IEEE Potentials 25:35–40
Monošík R, Streďanský M, Lušpai K et al (2012) Amperometric glucose biosensor utilizing
FAD-dependent glucose dehydrogenase immobilized on nanocomposite electrode. Enzym
Microb Technol 50:227–232. https://doi.org/10.1016/J.ENZMICTEC.2012.01.004
Mose S, Menzel C, Kurth AA et al (2003) Tartrate-resistant acid phosphatase 5b as serum marker of

bone metabolism in cancer patients. Anticancer Res 23:2783–2788
Nakanishi M, Yoh K, Uchida K et al (1998) Improved method for measuring tartrate-resistant acid
phosphatase activity in serum. Clin Chem 44:221–225
Nater UM, la Marca R, Florin L et al (2006) Stress-induced changes in human salivary alpha-
amylase activity—associations with adrenergic activity. Psychoneuroendocrinology 31:49–58
Ng E, Le AK, Nguyen MH, Wang SX (2020) Early multiplexed detection of cirrhosis using giant
magnetoresistive biosensors with protein Biomarkers. ACS Sens 5:3049–3057. https://doi.org/
10.1021/acssensors.0c00232
Palygin O, Levchenko V, Evans LC et al (2015) Use of enzymatic biosensors to quantify endoge-
nous ATP or H2O2 in the kidney. J Vis Exp 2015:e53059. https://doi.org/10.3791/53059
Panteghini M (1988) Serum isoforms of creatine kinase isoenzymes. Clin Biochem 21:211–218
Park HJ, Lee SS (2018) A quartz crystal microbalance-based biosensor for enzymatic detection of
hemoglobin A1c in whole blood. Sens Actuators B Chem 258:836–840. https://doi.org/10.
1016/j.snb.2017.11.170
Patel PD (1995) Enzymes as diagnostic tools. In: Enzymes in food processing. Springer, pp
292–314
Pohanka M (2021) Quartz crystal microbalance (QCM) sensing materials in biosensors develop-
ment. Int J Electrochem Sci 16:2
Polaina J, MacCabe AP (2007) Industrial enzymes. Springer
Prati D, Taioli E, Zanella A et al (2002) Updated definitions of healthy ranges for serum alanine
aminotransferase levels. Ann Intern Med 137:1–10
Premanode B, Toumazou C (2007) A novel, low power biosensor for real time monitoring of
creatinine and urea in peritoneal dialysis. Sens Actuators B Chem 120:732–735. https://doi.org/
10.1016/J.SNB.2006.03.051
Prodromidis MI, Karayannis MI (2002) Enzyme based amperometric biosensors for food analysis.
Electroanalysis 14:241–261
Qiu C, Wang X, Liu X et al (2012) Direct electrochemistry of glucose oxidase immobilized on
nanostructured gold thin films and its application to bioelectrochemical glucose sensor.
Electrochim Acta 67:140–146. https://doi.org/10.1016/J.ELECTACTA.2012.02.011
Qureshi A, Gurbuz Y, Kallempudi S, Niazi JH (2010a) Label-free RNA aptamer-based capacitive
biosensor for the detection of C-reactive protein. Phys Chem Chem Phys 12:9176–9182. https://
doi.org/10.1039/C004133E
Qureshi A, Gurbuz Y, Niazi JH (2010b) Label-free detection of cardiac biomarker using aptamer
based capacitive biosensor. Proc Eng 5:828–830. https://doi.org/10.1016/J.PROENG.2010.
09.236
Radha Shanmugam N, Muthukumar S, Chaudhry S et al (2017) Ultrasensitive nanostructure sensor
arrays on flexible substrates for multiplexed and simultaneous electrochemical detection of a
panel of cardiac biomarkers. Biosens Bioelectron 89:764–772. https://doi.org/10.1016/J.BIOS.
2016.10.046
Rocchitta G, Secchi O, Alvau MD et al (2012) Development and characterization of an implantable
biosensor for telemetric monitoring of ethanol in the brain of freely moving rats. Anal Chem 84:
7072–7079
Rocchitta G, Spanu A, Babudieri S et al (2016) Enzyme biosensors for biomedical applications:
strategies for safeguarding analytical performances in biological fluids. Sensors 16:780
Sarkar P, Pal PS, Ghosh D et al (2002) Amperometric biosensors for detection of the prostate cancer
marker (PSA). Int J Pharm 238:1–9. https://doi.org/10.1016/S0378-5173(02)00015-7
Sarkar S, Gogoi M, Mahato M et al (2022) Biosensors for detection of prostate cancer: a review.
Biomed Microdevices 24:1–12
Scheller FW, Schubert F, Neumann B et al (1991) Second generation biosensors. Biosens
Bioelectron 6:245–253
Sharma U, Pal D, Prasad R (2014) Alkaline phosphatase: an overview. Indian J Clin Biochem 29:
269–278
28 B. Daurai et al.
Shergujri MA, Jaman R, Baruah AJ et al (2019) Paper-based sensors for biomedical

applications. In: Paul S (ed) Biomedical engineering and its applications in healthcare, pp
355–376. https://doi.org/10.1007/978-981-13-3705-5_15/COVER
Shi Y, Li X, Ye M et al (2010) Glucose oxidase immobilization on different modified surfaces of
platinum nanowire for application in glucose detection. Adv Nat Sci Nanosci Nanotechnol 1:
035004. https://doi.org/10.1088/2043-6254/1/3/035004
Shirahama T, Sakakura C (2001) Overexpression of cyclooxygenase-2 in squamous cell carcinoma
of the urinary bladder. Clin Cancer Res 7:558–561
Shrestha BK, Ahmad R, Shrestha S et al (2017) In situ synthesis of cylindrical spongy polypyrrole
doped protonated graphitic carbon nitride for cholesterol sensing application. Biosens
Bioelectron 94:686–693. https://doi.org/10.1016/J.BIOS.2017.03.072
Singh N, Rathore V, Mahat RK, Rastogi P (2018) Glycogen phosphorylase BB: a more sensitive
and specific marker than other cardiac markers for early diagnosis of acute myocardial infarc-
tion. Indian J Clin Biochem 33:356–360
Singh RS, Singh T, Singh AK (2019) Enzymes as diagnostic tools. In: Advances in enzyme
technology. Elsevier, pp 225–271
Stejskal D, Lacnak B, Jedelsky L et al (2007) Use of glycogen phosphorylase BB measurement with
POCT in the diagnosis of acute coronary syndromes. A comparison with the ELISA method.
Biomedical papers of the Medical Faculty of Palacky University in Olomouc, p 151
Strahler J, Mueller A, Rosenloecher F et al (2010) Salivary α-amylase stress reactivity across
different age groups. Psychophysiology 47:587–595
Sun T, Jiang D, Zhang L et al (2016) Expression profile of cathepsins indicates the potential of
cathepsins B and D as prognostic factors in breast cancer patients. Oncol Lett 11:575–583
Suska A, Alehagen U, Lundström I, Dahlström U (2012) Salivary alpha-amylase activity, a new
biomarker in heart failure? J Clin Exp Cardiol 2:005
Szabó Z, Hämäläinen J, Loikkanen I et al (2010) Sorbitol dehydrogenase expression is regulated by
androgens in the human prostate. Oncol Rep 23:1233–1239
Tabish TA, Hayat H, Abbas A, Narayan RJ (2022) Graphene quantum dots-based electrochemical
biosensing platform for early detection of acute myocardial infarction. Biosensors 12:77. https://
doi.org/10.3390/BIOS12020077
Taira AL, Merrick G, Wallner K, Dattoli M (2007) Reviving the acid phosphatase test for prostate
cancer. Oncology (Williston Park) 21:1003–1010
Terpos E, de la Fuente J, Szydlo R et al (2003) Tartrate-resistant acid phosphatase isoform 5b: a
novel serum marker for monitoring bone disease in multiple myeloma. Int J Cancer 106:455–
457
Thunemann M, Schmidt K, de Wit C et al (2014) Correlative intravital imaging of cGMP signals
and vasodilation in mice. Front Physiol 5:394. https://doi.org/10.3389/FPHYS.2014.00394/
PDF
Tothill IE (2009) Biosensors for cancer markers diagnosis. Semin Cell Dev Biol 20:55–62. https://
doi.org/10.1016/J.SEMCDB.2009.01.015
Turk V, Kos J, Turk B (2004) Cysteine cathepsins (proteases)—on the main stage of cancer? Cancer
Cell 5:409–410
Turkmen E, Bas SZ, Gulce H, Yildiz S (2014) Glucose biosensor based on immobilization of
glucose oxidase in electropolymerized poly(o-phenylenediamine) film on platinum
nanoparticles-polyvinylferrocenium modified electrode. Electrochim Acta 123:93–102.
https://doi.org/10.1016/J.ELECTACTA.2013.12.189
Turner A, Karube I, Wilson GS (1987) Biosensors: fundamentals and applications. Oxford Univer-
sity Press
Usman Ali SM, Nur O, Willander M, Danielsson B (2010) A fast and sensitive potentiometric
glucose microsensor based on glucose oxidase coated ZnO nanowires grown on a thin silver
wire. Sens Actuators B Chem 145:869–874. https://doi.org/10.1016/J.SNB.2009.12.072
Uzozie A, Nanni P, Staiano T et al (2014) Sorbitol dehydrogenase overexpression and other aspects
of dysregulated protein expression in human precancerous colorectal neoplasms: a quantitative
proteomics study. Mol Cell Proteomics 13:1198–1218
Vainzof M, Zatz M, Otto PA et al (1985) Serum CK-MB activity in progressive muscular

dystrophy: is it of nosologic value? Am J Med Genet 22:81–87
Van Rees BP, Saukkonen K, Ristimäki A et al (2002) Cyclooxygenase-2 expression during
carcinogenesis in the human stomach. J Pathol 196:171–179
Walaa Fikry ME (2017) Lactate dehydrogenase (LDH) as prognostic marker in acute leukemia
quantitative method. J Blood Disord Transfus 8:1–8
Wang J (1999) Amperometric biosensors for clinical and therapeutic drug monitoring: a review. J
Pharm Biomed Anal 19:47–53
Wang J (2005) Nanomaterial-based electrochemical biosensors. Analyst 130:421–426. https://doi.
org/10.1039/B414248A
Wang H, Zeng H, Liu Z et al (2004) Immunophenotyping of acute leukemia using an integrated
piezoelectric immunosensor array. Anal Chem 76:2203–2209. https://doi.org/10.1021/
AC035102X
Wang AJ, Li YF, Li ZH et al (2012) Amperometric glucose sensor based on enhanced catalytic
reduction of oxygen using glucose oxidase adsorbed onto core-shell Fe3O4@silica@Au mag-
netic nanoparticles. Mater Sci Eng C 32:1640–1647. https://doi.org/10.1016/J.MSEC.2012.
04.055
Waterboer T, Sehr P, Michael KM et al (2005) Multiplex human papillomavirus serology based on
in situ–purified glutathione S-transferase fusion proteins. Clin Chem 51:1845–1853. https://doi.
org/10.1373/CLINCHEM.2005.052381
Wee KW, Kang GY, Park J et al (2005) Novel electrical detection of label-free disease marker
proteins using piezoresistive self-sensing micro-cantilevers. Biosens Bioelectron 20:1932–
1938. https://doi.org/10.1016/J.BIOS.2004.09.023
Yamashita Y, Ferri S, Huynh ML et al (2013) Direct electron transfer type disposable sensor strip
for glucose sensing employing an engineered FAD glucose dehydrogenase. Enzym Microb
Technol 52:123–128. https://doi.org/10.1016/J.ENZMICTEC.2012.11.002
Yu F, Persson B, Löfås S, Knoll W (2004) Surface plasmon fluorescence immunoassay of free
prostate-specific antigen in human plasma at the femtomolar level. Anal Chem 76:6765–6770.
https://doi.org/10.1021/AC048937W/SUPPL_FILE/AC048937WSI20040720_055958.PDF
Zargoosh K, Chaichi MJ, Shamsipur M et al (2012) Highly sensitive glucose biosensor based on the
effective immobilization of glucose oxidase/carbon-nanotube and gold nanoparticle in nafion
film and peroxyoxalate chemiluminescence reaction of a new fluorophore. Talanta 93:37–43.
https://doi.org/10.1016/J.TALANTA.2011.11.029
Zhang S, Wright G, Yang Y (2000) Materials and techniques for electrochemical biosensor design
and construction. Biosens Bioelectron 15:273–282
Zhang Y, Li Y, Wu W et al (2014) Chitosan coated on the layers’ glucose oxidase immobilized on
cysteamine/Au electrode for use as glucose biosensor. Biosens Bioelectron 60:271–276. https://
doi.org/10.1016/J.BIOS.2014.04.035
Zhang W, Zhang L, Gao H et al (2018) Self-powered implantable skin-like glucometer for real-time
detection of blood glucose level in vivo. Nanomicro Lett 10:1–11. https://doi.org/10.1007/
S40820-017-0185-X/FIGURES/6
Zhao W, Ni Y, Zhu Q et al (2013) Innovative biocompatible nanospheres as biomimetic platform
for electrochemical glucose biosensor. Biosens Bioelectron 44:1–5. https://doi.org/10.1016/J.
BIOS.2012.12.036
Żyłka A, Gala-Błądzińska A, Dumnicka P et al (2016) Is urinary NGAL determination useful for
monitoring kidney function and assessment of cardiovascular disease? A 12-month observation
of patients with type 2 diabetes. Dis Markers 2016:8489543
Choice of Enzyme Immobilization Matrices
Used in Biosensor for Healthcare 2
Applications
Sanjeev Bhandari, Manashjit Gogoi, and Mrityunjoy Mahato
2.1 Introduction
Immobilization of the enzymes means confining the specific enzyme molecules to a

suitable support solid matrix, which are mostly inert polymer and inorganic materials
(Sirisha et al. 2016). Enzymes have low reusability factor and low structural
stability; thus, developing an enzyme-based sensor with high reproducibility is a
challenging task (Kawaguti et al. 2006; Jesionowski et al. 2014). These limitations
lead to confined and conditional uses of the enzyme-based sensor within narrow pH
range, low thermal stability, and the loss of catalytic activity after one cycle (Liese
and Hilterhaus 2013; Gray et al. 2013; Dicosimo et al. 2013). Studies have been
conducted to find appropriate matrices which allow to retain enzyme structure under
different sensing environments (Bornscheuer 2003; Betancor and Luckarift 2008;
Badalo et al. 2006; Chen et al. 2008). In this regard, researchers have explored
various nanostructure-/nanocomposites-based matrices for immobilization of the
enzymes (Kim et al. 2006).
Enzyme-based biosensors have been applied for sensing urea, glucose, choles-
terol, penicillin, or other disease biomarker (Bostick and Hercules 1975; Ismail and
Adeloju 2010; Raja et al. 2011). Biosensor is an analytical device used to detect
biomarkers like antibodies, enzymes, and oligonucleotides at low concentrations
(Malhotra and Chaubey 2003; Ispas et al. 2012). Biosensors also have wide
applications in detecting toxins (in food and water), environmental monitoring,
S. Bhandari · M. Mahato (✉)

Physics Division, Department of Basic Sciences and Social Sciences, School of Technology, North-
Eastern Hill University, Shillong, Meghalaya, India
e-mail: mmahato@nehu.ac.in
M. Gogoi
Department of Biomedical Engineering, School of Technology, North-Eastern Hill University,
Shillong, Meghalaya, India
# The Author(s), under exclusive license to Springer Nature Singapore Pte 31

Ltd. 2023
S. Patra et al. (eds.), Enzyme-based Biosensors: Recent Advances and Applications
in Healthcare, https://doi.org/10.1007/978-981-15-6982-1_2
32 S. Bhandari et al.
Fig. 2.1 Different types of enzyme immobilization techniques. (Reprinted with permission from
Sirisha et al. (2016), Advances in Food and Nutrition Research 79 (2016))
agriculture, etc. (Angela Leung et al. 2007; Kalyani et al. 2020; Zhang et al. 2018)
due to their high specificity, high sensitivity, rapid response, and low cost (Amine
et al. 2006).
There are several ways to immobilize enzyme on the substrate, namely, adsorp-
tion, covalent bonding, entrapment, and ionic binding (Cordeiro et al. 2011).
Adsorption method involves hydrophobic interaction, whereas covalent binding
involves the formation of covalent bonds between the matrix and side chain amino
acids (such as arginine, aspartic acid, etc.) (Hsieh et al. 2000; Fu et al. 2011).
Entrapment method involves binding of the enzyme to the polymeric network
using covalent or non-covalent bonds, in which the enzymes are bound with water
as shown in Fig. 2.1 (Foulds and Lowe 1986). The water-soluble matrices such as
polysaccharide derivatives, glass, synthetic polymers, etc. are used for immobiliza-
tion of enzymes using adsorption methods (Wu and Lia 2008; Rosa et al. 2002).
Different types of entrapment techniques such as gel entrapment, microencapsula-
tion, and fiber entrapment are used for enzyme immobilization (Dinelli 1972;
Wadiack and Carbonell 1975).
2 Choice of Enzyme Immobilization Matrices Used in Biosensor for. . . 33
2.1.1 Required Properties of Matrices for Enzyme Function
Immobilization of the enzyme is done by selecting the appropriate matrix. Hence,

the supporting matrix must have the following properties (Sirisha et al. 2016):
1. The enzyme should be operational under various conditions and the matrix
should have mechanical and thermal resistance.
2. The selected matrix should enhance the enzyme specificity (Magner 2013).
3. Pore diameter should be of the appropriate range, because if pore size is large,
then surface area will decrease and if pore size is small, the protein will be
excluded (Hartmann and Kostrov 2013; Tran and Balkus 2011).
4. The matrix after immobilization should behave inertly and not interfere in any
reaction.
5. The matrix should be stable and highly regenerable.
6. The hydrophobicity of the matrix should be minimized so that the protein is
prevented from denaturing (Zhou and Hartmann 2012; Rodrigues et al. 2013).
7. The support matrix should have antimicrobial properties.
2.2 Variety of Reported Matrices
2.2.1 Nanocellulose (CNC)/Gold Nanoparticle (AuNP)

Nanocomposite
CNC/AuNP matrix for enzyme immobilization was prepared using the following
two steps by Mahmoud et al. (2009). At first, CNCs were prepared by grounding flax
fibers with a 20-mesh screen to obtain 0.85 mm fiber size followed by mixing of flax
fibers (5 g) in 20 mL acid solution. Later, H2O2 (10 mL) was added dropwise on the
prepared mixture. The solution temperature was cooled till 45 °C for 3 h followed by
dilution with deionized water (DI) and centrifuging (10,000 rpm) to achieve a pH 6.
Firstly, the prepared CNC resin was mixed with ion-exchange resin, namely,
DOWEX MR-3 for 2 days, and removed using filtration (Whatman 541) followed
by sonication for 35 min. Secondly, gold(III) chloride trihydrate (HAuCl43H2O,
10 mM) and R-CD were added to CNC (23 mg/mL) along with sodium borohydride
(0.1 M, 20 μL) till a reddish color of AuNPs/CNCs is obtained. The solution was
centrifuged (10,000 rpm) and washed three times to remove unbound AuNPs. The
resulting AuNP/CNC colloid was incubated in an ethanol solution (0.05 M) of
thioctic acid (Thc) for 24 h. The CNC/AuNP (0.5 mL, 6 mg) was mixed with
CGTase enzyme (7–8 mg/mL) in a 0.1 M phosphate buffer (0.5 M NaCl at
pH 7.2) followed by centrifuging to get a supernatants. The enzyme loading capacity
of the nanocomposite was found to be 220 mg/g of AuNPs/CNCs. The maximum
CGTase binding activity was leveled at 50,000 U/g of AuNPs/CNCs.
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“Then that devil found out about it! For he was a devil, Mr.
Sheringham,” Mrs. Plant said, looking at Roger with wide eyes, in
which traces of horror still lingered. “I could never have imagined that
anyone could be so absolutely inhuman. Oh! It was hell!” She
shuddered involuntarily.
“He demanded money, of course,” she went on after a minute in
a calmer voice; “and I paid him every penny I could. You must
understand that I was willing to face any sacrifice rather than that my
husband should be told. The other night I had to tell him that I had no
more money left. I lied when I told you what time I went into the
library. He stopped me in the hall to tell me that he wanted to see me
there at half-past twelve. That would be when everyone else was in
bed, you see. Mr. Stanworth always preserved the greatest secrecy
about these meetings.”
“And you went at half-past twelve?” Roger prompted
sympathetically.
“Yes, taking my jewels with me. I told him that I had no more
money. He wasn’t angry. He never was. Just cold and sneering and
horrible. He said he’d take the jewels for that time, but I must bring
him the money he wanted—two hundred and fifty pounds—in three
months’ time.”
“But how could you, if you hadn’t got it?”
Mrs. Plant was silent. Then gazing unseeingly at the rose bed, as
if living over again that tragic interview, she said in a curiously
toneless voice, “He said that a pretty woman like me could always
obtain money if it was necessary. He said he would introduce me to
a man out of whom I—I could get it, if I played my cards properly. He
said if I wasn’t ready with the two hundred and fifty pounds within
three months he would tell my husband everything.”
“My God!” said Roger softly, appalled.
Mrs. Plant looked him suddenly straight in the face.
“That will show you what sort of a man Mr. Stanworth was, if you
didn’t know,” she said quietly.
“I didn’t,” Roger answered. “This explains a good deal,” he added
to himself. “And then, I suppose, Jefferson came in?”
“Major Jefferson?” Mrs. Plant repeated, in unmistakable
astonishment.
“Yes. Wasn’t that when he came in?”
Mrs. Plant stared at him in amazement.
“But Major Jefferson never came in at all!” she exclaimed. “What
ever makes you think that?”
It was Roger’s turn to be astonished.
“Do I understand you to say that Jefferson never came in at all
while you were in the library with Stanworth?” he asked.
“Good gracious, no,” Mrs. Plant replied emphatically. “I should
hope not! Why ever should he?”
“I—I don’t really know,” Roger said lamely. “I thought he did. I
must have been mistaken.” In spite of the unexpectedness of her
denial, he was convinced that Mrs. Plant was telling the truth; her
surprise was far too genuine to have been assumed. “Well, what
happened?”
“Nothing. I—I implored him not to be so hard and to be content
with what I had paid him and give me back the evidence he’d got,
but——”
“Where did he keep the evidence, by the way? In the safe?”
“Yes. He always carried the safe about with him. It was supposed
to be burglar-proof.”
“Was it open while you were there?”
“He opened it to put my jewels in before I went.”
“And did he leave it open, or did he lock it up again?”
“He locked it before I left the room.”
“I see. When would that be?”
“Oh, past one o’clock, I should say. I didn’t notice the time very
particularly. I was feeling too upset.”
“Naturally. And nothing of any importance occurred between his
—his ultimatum and your departure upstairs?”
“No. He refused to give way an inch, and at last I left off trying to
persuade him and went up to bed. That is all.”
“And nobody else came in at all? Not a sign of anybody else?”
“No; nobody.”
“Humph!” said Roger thoughtfully. This was decidedly
disappointing; yet somehow it was impossible to disbelieve Mrs.
Plant’s story. Still, Jefferson might have come in later, having heard
something of what had taken place from outside the room. At any
rate, it appeared that Mrs. Plant herself could have had no hand in
the actual murder, whatever provocation she might have received.
He decided to sound her a little farther.
“In view of what you’ve told me, Mrs. Plant,” he remarked rather
more casually, “it seems very extraordinary that Stanworth should
have committed suicide, doesn’t it? Can you account for it in any
way?”
“No, I certainly can’t. It’s inexplicable to me. But, Mr. Sheringham,
I am so thankful! No wonder I fainted when you told us after
breakfast. I suddenly felt as if I had been let out of prison. Oh, that
dreadful, terrible feeling of being in that man’s power! You can’t
imagine it; or what an overwhelming relief it was to hear of his
death.”
“Indeed I can, Mrs. Plant,” Roger said with intense sympathy. “In
fact, what surprises me is that nobody should ever have killed him
before this.”
“Do you imagine that people never thought of that?” Mrs. Plant
retorted passionately. “I did myself. Hundreds of times! But what
would have been the use? Do you know what he did—in my case, at
any rate, and so in everyone else’s, I suppose? He kept the
documentary evidence against me in a sealed envelope addressed
to my husband! He knew that if ever he met with a violent death the
safe would be opened by the police, you see; and in that case they
would take charge of the envelope, and presumably many other
similar ones, and forward them all to their destinations. Just imagine
that! Naturally nobody dared kill him; it would only make things
worse than before. He used to gloat over it to me. Besides, he had
always a loaded revolver in his hand when he opened the safe, in
my presence at any rate. I can tell you, he took no chances. Oh, Mr.
Sheringham, that man was a fiend! Whatever can have induced him
to take his own life, I can’t conceive; but believe me, I shall thank
God for it on my bended knees every night as long as I live!”
She sat biting her lip and breathing heavily in the intensity of her
feelings.
“But if you knew the evidence was kept in the safe, why weren’t
you frightened when it was being opened by the inspector?” Roger
asked curiously. “I remember glancing at you, and you certainly
didn’t seem to be in the least perturbed about it.”
“Oh, that was after I’d had his letter, you see,” Mrs. Plant
explained readily. “I was before, of course; terribly frightened. But not
afterwards, though it did seem almost too good to be true. Hullo! isn’t
that the lunch bell? We had better be going indoors, hadn’t we? I
think I have told you all you can want to know.” She rose to her feet
and turned towards the house.
Roger fell into step with her.
“Letter?” he said eagerly. “What letter?”
Mrs. Plant glanced at him in surprise. “Oh, don’t you know about
that? I thought you must do, as you seemed to know everything.
Yes, I got a letter from him saying that for certain private reasons he
had decided to take his own life, and that before doing so he wished
to inform me that I need have no fears about anything, as he had
burnt the evidence he held against me. You can imagine what a
relief it was!”
“Jumping Moses!” Roger exclaimed blankly. “That appears to
bash me somewhat sideways!”
“What did you say, Mr. Sheringham?” asked Mrs. Plant curiously.
Roger’s dazed and slightly incoherent reply is not recorded.
CHAPTER XXIV.
Mr. Sheringham is Disconcerted
Roger sat through the first part of lunch in a species of minor
trance. It was not until the necessity for consuming a large plateful of
prunes and tapioca pudding, the two things besides Jews that he
detested most in the world, began to impress itself upon his
consciousness, that the power of connected thought returned to him.
Mrs. Plant’s revelation appeared temporarily to have numbed his
brain. The one thing which remained dazzlingly clear to him was that
if Stanworth had written a letter announcing his impending suicide,
then Stanworth could not after all have been murdered; and the
whole imposing structure which he, Roger, had erected, crumbled
away into the sand upon which it had been founded. It was a
disturbing reflection for one so blithely certain of himself as Roger.
As soon as lunch was over and the discussion regarding trains
and the like at an end, he hurried Alec upstairs to his bedroom to talk
the matter over. It is true that Roger felt a certain reluctance to be
compelled thus to acknowledge that he had been busily unearthing
nothing but a mare’s nest; but, on the other hand, Alec must know
sooner or later, and at that moment the one vital necessity from
Roger’s point of view was to talk. In fact, the pent-up floods of talk in
Roger’s bosom that were striving for exit had been causing him
something very nearly approaching physical pain during the last few
minutes.
“Alexander!” he exclaimed dramatically as soon as the door was
safely shut. “Alexander, the game is up!”
“What do you mean?” Alec asked in surprise. “Have the police
got on the trail now?”
“Worse than that. Far worse! It appears that old Stanworth was
never murdered at all! He did commit suicide, after all.”
Alec sat down heavily in the nearest chair. “Good Lord!” he
exclaimed limply. “But what on earth makes you think that? I thought
you were so convinced that it was murder.”
“So I was,” Roger said, leaning against the dressing table. “That’s
what makes it all the more extraordinary, because I really am very
seldom wrong. I say it in all modesty, but the fact is indisputable. By
all the laws of average, Stanworth ought to have been murdered. It
really is most inexplicable.”
“But how do you know he wasn’t?” Alec demanded. “What’s
happened since I saw you last to make you alter your mind like this?”
“The simple fact that Mrs. Plant received a note from old
Stanworth, saying that he was going to kill himself for private
reasons of his own or something.”
“Oh!”
“I can tell you, it knocked me upside down for the minute.
Anything more unexpected I couldn’t have imagined. And the trouble
is that I don’t see how we can possibly get round it. A note like that is
a very different matter to that statement.”
“You know, I’m not sure that I’m altogether surprised that
something like this has turned up,” Alec said slowly. “I was never
quite so convinced by the murder idea as you were. After all, when
you come to look at all the facts of the case, although they certainly
seemed to be consistent with murder, were no less consistent with
suicide, weren’t they?”
“So it appears,” Roger said regretfully.
“It was simply that you’d got the notion of murder into your head
—more picturesque, I suppose—and everything had to be construed
to fit it, eh?”
“I suppose so.”
“In fact,” Alec concluded wisely, “it was an idée fixe, and
everything else was sacrificed to it. Isn’t that right?”
“Alexander, you put me to shame,” Roger murmured.
“Well, anyhow, that shows you what comes of muddling in other
people’s affairs,” Alec pointed out severely. “And it’s lucky you hit on
the truth before you made a still bigger idiot of yourself.”
“I deserve it all, I know,” Roger remarked contritely to his hair-
brush.
It was Alec’s turn to be complacent now, and he was taking full
advantage of it. As he lay back leisurely in his chair and smoked
away placidly, he presented a perfect picture of “I told you so!” Roger
contemplated him in rueful silence.
“And yet——!” he murmured tentatively, after a few moments’
silence.
Alec waved an admonitory pipe.
“Now, then!” he said warningly.
Roger exploded suddenly. “Well, say what you like, Alec,” he
burst out, “but the thing is dashed queer! You can’t get away from it.
After all, our inquiries haven’t resulted in nothing, have they? We did
establish the fact that Stanworth was a blackmailer. I forgot to tell
you that, by the way. We were perfectly right; he had been
blackmailing Mrs. Plant, the swine, and jolly badly, too. Incidentally,
she hadn’t the least idea that his death might be anything else than
suicide, and Jefferson didn’t come into the library while she was
there; so I was wrong in that particular detail. I’m satisfied she was
telling the truth, too. But as for the rest—well, I’m dashed if I know
what to think! The more I consider it, the more difficult I find it to
believe that it was suicide, after all, and that all those other facts
could have been nothing but mere coincidences. It isn’t reasonable.”
“Yes, that’s all very well,” Alec said sagely. “But when a fellow
actually goes out of his way to write a letter saying that——”
“By Jove, Alec!” Roger interrupted excitedly. “You’ve given me an
idea. Did he write it?”
“What do you mean?”
“Why, mightn’t it have been typed? I haven’t seen the thing yet,
you know, and when she mentioned a letter it never occurred to me
that it might not be a hand-written one. If it was typed, then there’s
still a chance.” He walked rapidly towards the door.
“Where are you going to now?” Alec asked in surprise.
“To see if I can get a look at this blessed letter,” Roger said,
turning the handle. “Mrs. Plant’s room is down this passage, isn’t it?”
With a quick glance up and down the passage, Roger hurried
along to Mrs. Plant’s bedroom and tapped on the door.
“Come in,” said a voice inside.
“It’s me, Mrs. Plant,” he replied softly. “Mr. Sheringham. Can I
speak to you a minute?”
There came the sound of rapid footsteps crossing the floor and
Mrs. Plant’s head appeared at the door.
“Yes?” she asked, not without a certain apprehension. “What is it,
Mr. Sheringham?”
“You remember that letter you mentioned this morning? From Mr.
Stanworth, I mean. Have you still got it, by any chance, or have you
destroyed it?”
He held his breath for her reply.
“Oh, no. Of course I destroyed it at once. Why?”
“Oh, I just wanted to test an idea. Let me see.” He thought
rapidly. “It was pushed under your door or something, I suppose?”
“Oh, no. It came by post.”
“Did it?” said Roger eagerly. “You didn’t notice the postmark, did
you?”
“As a matter of fact I did. It seemed so funny that he should have
taken the trouble to post it. It was posted from the village by the
eight-thirty post that morning.”
“The village, was it? Oh! And was it typewritten?”
“Yes.”
Roger held his breath again. “Was the signature written or
typewritten?”
Mrs. Plant considered.
“It was typewritten, as far as I remember.”
“Are you sure of that?” Roger asked eagerly.
“Ye-es, I think so. Oh, yes; I remember now. The whole thing was
typewritten, signature and all.”
“Thank you very much, Mrs. Plant,” Roger said gratefully. “That’s
all I wanted to know.”
He sped back to his own room.
“Alexander!” he exclaimed dramatically, as soon as he was
inside. “Alexander, the game is on again!”
“What’s up now?” Alec asked with a slight frown.
“That letter sounds like a fake, just the same as the confession. It
was all typewritten, even the signature, and it was posted from the
village. Can you imagine a man in his sane senses deliberately
going down to the village to post his letter, when all he had to do was
to push it under her door?”
“He might have had others to post as well,” Alec hazarded,
blowing out a great cloud of smoke. “Would Mrs. Plant’s be the only
one?”
“H’m! I never thought of that. Yes, he would. But still, it’s rather
unlikely that he should have posted hers as well, I should say. By the
way, it was that letter which accounted for her change of attitude
before lunch. She knew then that she had nothing to fear from the
opening of the safe, you see.”
“Well, how do matters stand now?”
“Exactly as they did before. I don’t see that this really affects it
either way. It’s only another instance of the murderer’s cunning. Mrs.
Plant, and possibly, as you say, one or two others, might raise
awkward questions at Stanworth’s sudden death; therefore their
apprehensions must be allayed. All that it really does as far as we
are concerned, is to confirm the idea that the murderer must have a
very intimate knowledge of Stanworth’s private affairs. Of course it
shows that the safe was opened that night, and it brings our old
friends, the ashes in the hearth, into prominence once more as being
in all probability the remains of the blackmailer’s evidence. Curious
that that first guess of mine should have turned out to be so near the
truth, isn’t it?”
“And what about Jefferson?” Alec asked quietly.
“Ah, yes, Jefferson. Well, I suppose this affair of the letter and the
fact that he did not break in on Mrs. Plant and Stanworth in the
library that night and consequently was not helped by that lady—I
suppose all this gives him credit for rather more brains than I had
been willing to concede him; but otherwise I don’t see that his
position is affected.”
“You mean, you still think he killed Stanworth?”
“If he didn’t, can you tell me who did?”
Alec shrugged his shoulders. “I’ve told you I’m convinced you’re
barking up the wrong tree. It’s no good going on repeating it.”
“Not a bit,” Roger said cheerfully.
“So what are you going to do?”
“Exactly what I was before. Have a little chat with him.”
“Rather ticklish business, isn’t it? I mean, when you’re so very
uncertain of your ground.”
“Possibly. But so was Mrs. Plant for that matter. I think I shall be
able to handle friend Jefferson all right. I shall be perfectly candid
with him, and I’m willing to wager a small sum that I shall be back
here within half an hour with his confession in my pocket.”
“Humph!” Alec observed sceptically. “Are you going to accuse
him directly of the murder?”
“My dear Alec! Nothing so crude as that. I shan’t even say in so
many words that I know a murder has been committed. I shall simply
ask him a few pointed and extremely pertinent questions. He’ll see
the drift of them all right; Master Jefferson is no fool, as we have
every reason to know. Then we shall be able to get down to things.”
“Well, for goodness’ sake do bear in mind the possibility (I won’t
put it any stronger than that) that Jefferson never did kill Stanworth
at all, and walk warily.”
“Trust me for that,” Roger replied complacently. “By the way, did I
tell you that Mrs. Plant received that letter just before going into
lunch? It caught the eight-thirty post from the village.”
“Did it?” Alec said without very much interest.
“By Jove!” Roger exclaimed suddenly. “What an idiot I am! That’s
conclusive proof that Stanworth couldn’t have posted it himself, isn’t
it? Fancy my never spotting that point before!”
“What point?”
“Why, the first post out from the village is five o’clock. That letter
must have been posted between five and eight-thirty—four hours or
more after Stanworth was dead!”
CHAPTER XXV.
The Mystery Finally Refuses to Accept
Mr. Sheringham’s Solution
Roger had no time to waste. Mrs. Plant, Alec, and himself were
all to leave by the train soon after five o’clock; the car would be
ready to take them into Elchester at half-past four. Tea was to be at
four, and the time was already close on three. He had an hour left in
which to disentangle the last remaining threads. As he stood for a
moment outside the morning-room door it seemed to Roger as if
even this narrow margin were half an hour more than he needed.
Jefferson was still at work among the piled-up papers. He
glanced up abstractedly as Roger entered the room and then smiled
slightly.
“Come to offer me a hand again?” he asked. “Devilish good of
you, but I’m afraid there’s absolutely nothing I can turn over to you
this time.”
Roger drew a chair up to the other side of the table and seated
himself deliberately.
“As a matter of fact, I hadn’t,” he said slowly. “I wanted to ask you
one or two questions, Jefferson, if you would be good enough to
answer them.”
Jefferson looked slightly surprised.
“Questions? All right, fire away. What can I tell you?”
“Well, the first thing I want to ask you,” Roger shot out, “is—
where were you at the time that Stanworth died?”
A look of blank astonishment was followed in Jefferson’s face by
an angry flush.
“And what the devil has that got to do with you?” he asked
abruptly.
“Never mind for the moment what it has to do with me,” Roger
replied, his heart beating a little faster than usual. “I want you to
answer that question.”
Jefferson rose slowly to his feet, his eyes glittering ominously.
“Do you want me to kick you out of the room?” he said in a strangely
quiet voice.
Roger leaned back in his chair and watched him unmoved.
“Do I understand that you refuse to answer?” he said evenly. “You
refuse to tell me where you were between, say, one and three
o’clock on the morning that Stanworth died?”
“Most decidedly I do. And I want to know what the hell you think
that has to do with you?”
“It may have nothing and it may have everything,” Roger said
calmly. “But I advise you to tell me, if not for your own sake at least
for the lady’s.”
If this was a chance shot, it had certainly got home. Jefferson’s
face took on a deeper tinge and his eyes widened in sheer fury. He
clenched his fists till the knuckles showed up white and menacing.
“Damn you, Sheringham, that’s about enough!” he muttered,
advancing towards the other. “I don’t know what the devil you think
you’re playing at, but——”
A sudden bluff darted into Roger’s mind. After all, what was a
man like Jefferson doing as secretary to a man like Stanworth? He
decided to risk it.
“Before you do anything rash, Jefferson,” he said quickly, “I’d like
to ask you another question. What was Stanworth blackmailing you
for?”
There are times when bluff pays. This was one of them. Jefferson
stopped short in his stride, his hands fell limply to his sides and his
jaw drooped open. It was as if he had been struck by a sudden and
unexpected bullet.
“Sit down and let’s talk things over quietly,” Roger advised, and
Jefferson resumed his seat without a word.
Roger reviewed the situation rapidly in his mind.
“You see,” he began in conversational tones, “I know quite a lot of
what’s been going on here, and in the circumstances I really have no
alternative but to find out the rest. I admit that it places me in rather
an awkward situation, but I can’t see that I can very well do anything
else. Now what I suggest, Jefferson, is that we both put our cards on
the table and talk the thing over as two men of the world. Do you
agree?”
Jefferson frowned. “You don’t appear to give me much option, do
you? Though what it has to do with you, I’m really hanged if I can
see.”
It was on Roger’s lips to retort that Jefferson would very probably
be hanged if he didn’t, but fortunately he was able to control himself.
“I should have thought that would have been obvious,” he said
smoothly. “I can hardly leave things as they are, can I? Still, we’ll
pass over that for the present. Now Stanworth was, as I know, a
blackmailer, and there can be no doubt as to that affecting the
situation in no small degree.”
“What situation?” Jefferson asked in puzzled tones.
Roger glanced at him shrewdly. “The situation,” he said firmly. “I
think we both understand to what I am referring.”
“I’m blessed if I do,” Jefferson retorted.
“Of course if you take up that attitude——!” Roger said
tentatively. “Still, perhaps it’s a little early to get down to brass tacks,”
he added, after a moment’s pause. “We’ll confine ourselves to the
other aspect for a time, shall we? Now Stanworth, I take it, had some
definite hold over you. Would you mind telling me exactly what that
was?”
“Is this necessary?” Jefferson demanded shortly. “My private
affair, you know. Why the deuce should you want to concern yourself
in it?”
“Don’t talk like that, Jefferson, please. You must see what course
you’ll force on me if you do.”
“Damned if I do! What course?”
“To put the whole thing in the hands of the police, naturally.”
Jefferson started violently. “Good God, you wouldn’t do that,
Sheringham!”
“I don’t want to do so, of course. But you really must be frank with
me. Now please tell me all about your relations with Stanworth. I
may tell you, to save you trouble, that I am already in full possession
of all the similar facts with regard to—well, the lady in the case.”
“The devil you are!” Jefferson exclaimed in undisguised
astonishment. “Oh, well, if I’ve got to tell you, I suppose I must.
Though what in the name of goodness it can have to—— However!”
He leaned back in his chair and began to fiddle abstractedly with
the papers in front of him.
“It was this way. My regiment was in India. Pal of mine and I were
both in love with the same girl. No bad blood or anything like that.
Good friends all the time. He got her. Wanted to get married at once,
but very hard up, of course. We all were. He’d got a lot of debts, too.
Damned fool went and drew a check on another man’s account.
Forged it, if you like. Absurd thing to do; bound to come out. There
was a hell of a row, but we managed to keep it confined to a few of
us. Chap came and confessed to me; asked what on earth he’d
better do. They hadn’t found out who’d done it yet, but when they did
it would be all up with him. Lose the girl and everything; she was
fond enough of him, but straight as a die herself. Couldn’t have stood
the disgrace. Well, what could I do? Couldn’t stand by and see all
this happen. Went to the colonel and told him I’d done the blessed
thing. Only thing to do.”
“By Jove, you sportsman!” Roger exclaimed involuntarily.
“Sportsman be damned! Wouldn’t have done it for him alone. I
was thinking of the girl.”
“And what happened?”
“Oh, it was hushed up as much as possible. I had to send in my
papers, of course, but the fellow didn’t prosecute. Then that hound
Stanworth got wind of the story somehow and managed to lay his
hands on the check, which had never been destroyed. Perfect
godsend to him, of course. Gave me choice of taking on this job with
him or letting the whole thing be passed on to the police. No
alternative. I had to take on the job.”
“But why on earth did he want you as his secretary? That’s what I
can’t understand.”
“Simple enough. He wanted to push his way in among the sort of
people I knew. I was a sort of social sponsor for him. Damned
unpleasant job, of course, but what could I do? Besides, when I took
the job on I didn’t know anything about him. Thought he was just a
new-rich merchant and I was his only victim in the threatening line.
Soon found out, of course; but too late to back out then. That’s all.
Satisfied?”
“Perfectly. Sorry I had to ask you, but you see how it is. Well, I’m
dashed if I can blame you. I’d have done the same thing myself. But
I’d like to have the story of it from your own lips.”
“Just told you the story.”
“No, the other one, I mean.”
“What other one?”
“Oh, don’t beat about the bush like this. You know perfectly well
what I’m driving at. I’ll put it in the original form, if you like. Where
were you during the night that Stanworth died?”
Jefferson’s angry flush returned.
“Now look here, Sheringham, that’s too much. I’ve told you things
I never dreamed I’d have to tell anyone, and I’m not going to have
you probing any farther into my business. That’s final.”
Roger rose to his feet. “I’m sorry you take it like that, Jefferson,”
he said quietly. “You leave me no alternative.”
“What are you going to do then?”
“Tell the police the whole story.”
“Are you mad, Sheringham?” Jefferson burst out angrily.
“No, but I think you are, not to trust me,” Roger retorted, hardly
less so. “You don’t think I want to tell them, do you? It’s you who are
forcing me to do so.”
“What, through not telling you what—what I was doing that
night?”
“Of course.”
There was a short pause, while the two glared at each other.
“Come back in a quarter of an hour,” Jefferson said abruptly. “I’ll
think it over. Have to consult her, of course, first.”
Roger nodded acquiescence to this proposal and hurried out of
the room. Exultantly he sought Alec.
“I told you so, Alexander,” he cried triumphantly, as soon as he
was fairly inside the room. “Jefferson’s on the point of confession!”
“He’s not!” Alec exclaimed incredulously.
“He is indeed. And there’s a lot more to it than that. I’ve bluffed
him into believing that I know a lot more than I do really, and he’s
going to tell me all sorts of other things as well. He’s let one cat out
of the bag already. I can tell you. Mrs. Plant is in it, after all!”
“Oh, rot!” Alec replied with decision. “That’s out of the question. I
know she isn’t.”
“Don’t be so absurd, Alexander,” Roger retorted somewhat
nettled. “How can you possibly know?”
“Well, anyhow, I’m sure she isn’t,” Alec replied obstinately.
“But my dear chap, friend Jefferson has just gone off to consult
her as to whether he shall tell me the whole story or not. I threatened
him with the police, you see, if he didn’t.”
“I suppose you taxed him outright with the murder, did you?”
“No, Alexander, I didn’t,” Roger answered wearily. “The word
murder was never so much as mentioned. I simply put it that I
wanted to know what he was doing on the night of Stanworth’s
death.”
“And he wouldn’t tell you?” Alec asked, somewhat surprisedly.
“He certainly would not. But he told me a lot of other things. He
was in Stanworth’s power all right. I haven’t got time to tell you the
whole story, but there’s motive enough for him to kill Stanworth
himself, even without the introduction of Mrs. Plant’s side of it. Oh,
the whole thing’s as plain as a pikestaff. I can’t understand why
you’re so sceptical about it all.”
“Perhaps I make a better detective than you do, Roger,” Alec
laughed, a trifle constrainedly.
“Perhaps,” Roger said without very much conviction. He glanced
at his watch. “Well, I’d better be getting back. I wonder if you’d
believe it if I showed you Jefferson’s confession in writing! Would
you?”
“I very much doubt it,” Alec smiled.
Jefferson was no longer alone in the morning room when Roger
returned to it. To the latter’s surprise Lady Stanworth was also there.
She was standing with her back to the window and did not look
round at his entrance. Roger shut the door carefully behind him and
looked inquiringly at Jefferson.
That gentleman did not waste time.
“We’ve talked the matter over,” he said curtly, “and decided to tell
you what you want to know.”
Roger could hardly repress an exclamation of surprise. Why
should Jefferson have imported Lady Stanworth into the matter?
Obviously she must be involved, and deeply, too. Could it be that
Jefferson had taken her into his confidence with regard to Mrs.
Plant? How much did she know, if that were the case? Presumably
everything. Roger felt that the situation was about to prove not a little
awkward.
“I’m glad,” he murmured, half apologetically.
Jefferson was carrying the thing off well. Not only did he appear
to be feeling no fear at all, but his manner was not even that of
defiance. The attitude he had adopted and which sat perfectly
naturally upon him was rather one of dignified condescension.
“But before I answer you, Sheringham,” he said stiffly, “I should
like to say, both on behalf of this lady and myself, that we consider
——”
Lady Stanworth turned to him. “Please!” she said quietly. “I don’t
think we need go into that. If Mr. Sheringham is incapable of
understanding the position into which he has forced us, there can
hardly be any need to labour the point.”
“Quite, quite,” Roger murmured still more apologetically, and
feeling unaccountably small. Lady Stanworth was perhaps the only
person in the world who consistently had that effect upon him.
“Very well,” Jefferson bowed. He turned to Roger. “You wanted to
know where I was on the night that Stanworth shot himself?”
“On the night of Stanworth’s death,” Roger corrected, with a slight
smile.
“On the night of Stanworth’s death then,” Jefferson said
impatiently. “Same thing. As I said before, I fail entirely to see how it
can concern you, but we have decided under the circumstances to
tell you. After all, the fact will be common knowledge soon enough
now. I was with my wife.”
“Your wife?” Roger echoed, scarcely able to believe his ears.
“That is what I said,” Jefferson replied coldly. “Lady Stanworth
and I were married secretly nearly six months ago.”
CHAPTER XXVI.
Mr. Grierson Tries His Hand
For some moments Roger was incapable of speech. This
disclosure was so totally unexpected, so entirely the reverse of
anything that he had ever imagined, that at first it literally took his
breath away. He could only stand and stare, as if his eyes were
about to pop out of his head, at the two entirely unmoved persons
who had sprung this overwhelming surprise upon him.
“Is that what you wished to know?” Jefferson asked courteously.
“Or would you wish my wife to confirm it?”
“Oh, no; no need at all,” Roger gasped, doing his best to pull
himself together. “I—I should like to apologise to you for the apparent
impertinence of my questions and to—to congratulate you, if you will
allow me to do so.”
“Very kind,” Jefferson muttered. Lady Stanworth, or Lady
Jefferson as she was now, bowed slightly.
“If you don’t want me any more, Harry,” she said to her husband,
“there are one or two things I have to do.”
“Certainly,” Jefferson said, opening the door for her.
She passed out without another glance at Roger.
“Look here, Jefferson,” exclaimed the latter impulsively, as soon
as the door was closed again, “I know you must be thinking me the
most appalling bounder, but you must believe that I shouldn’t have
tackled you in that way if I hadn’t got very solid and serious reasons
for doing so. As things have turned out, I can’t tell you at present
what those reasons are; but really it’s something of the greatest
possible importance.”
“Oh, that’s all right, Sheringham,” Jefferson returned with gruff
amiability. “Guessed you must have something up your sleeve. Bit
awkward, though. Ladies, and all that, y’know,” he added vaguely.
“Beastly,” Roger said sympathetically. “As a matter of fact, that’s
a development that had never occurred to me at all, you and Lady
Stanworth being married. If anything, it makes things very much
more complicated than before.”
“Bit of a mystery or something on hand, eh?” Jefferson asked
with interest.
“Very much so,” Roger replied, gazing thoughtfully out of the
window. “Connected with Stanworth, and—and his activities, you
understand,” he added.
“Ah!” Jefferson observed comprehendingly. “Then I’d better not
ask any questions. Don’t want to learn anything more about that side
of things. Seen too many poor devils going through it already.”
“No, but I tell you what,” Roger said, wheeling suddenly about. “If
you could answer a few more questions for me, I should be more
than grateful. Only as a favour, of course, and if you refuse I shall
understand perfectly. But you might be able to help me clear up a
very tricky state of affairs.”
“If it’s anything to do with helping somebody Stanworth got hold
of, I’ll answer questions all night,” Jefferson replied with vigour. “Go
ahead.”
“Thanks, very much. Well, then, in the first place, will you tell me
some details regarding your wife’s relations with Stanworth? It
doesn’t matter if you object, but I should be very glad if you could
see your way to do so.”
“But I thought you said you knew that story?”
Roger did not think it necessary to explain that the lady to whom
he had been referring was not Lady Jefferson. “Oh, I know most of it,
I think,” he said airily, “but I should like to hear it all from you, if I
could. I know that she was in Stanworth’s power, of course,” he
added, making a shot in the twilight, “but I’m not quite clear as to the
precise way.”
Jefferson shrugged his shoulders. “Oh, well, as you seem to
know so much, you’d better have the whole lot straight. Stanworth
nosed out something about her father. His brother was in love with
her, and Stanworth gave her the option of marrying him or having her
father shown up. He could have had the old earl put in the dock, I
believe. Naturally she chose the brother, who, by the way, didn’t
know anything about Stanworth’s activities, so I understand. Quite
an amiable, rather weak sort of a fellow.”
“And since then, of course, Stanworth had the whip hand over
her?”
Jefferson winced. “Yes,” he said shortly. “Even after her father
died, she wouldn’t want the family shown up.”
“I see,” said Roger thoughtfully. So Lady Stanworth had little
enough reason to love her brother-in-law. And since Jefferson fell in
love with her, her cause would naturally become his. Truly he had
motive and to spare for ridding the world of such a man. Yet,
although Jefferson and his wife might easily have concocted the
story of his whereabouts that night, Roger already felt just as
convinced of the former’s innocence as he was before of his guilt.
The man’s manner seemed somehow to preclude altogether the idea
of subterfuge. Had he really killed Stanworth, Roger was sure that he
would have said so by the time that matters had reached this length,
bluntly and simply, just as he had told the story of his own downfall.
But in spite of his convictions, Roger was not such a fool as not
to put the obvious questions that occurred to him.
“Why was your marriage secret?” he asked. “Did Stanworth know
about it?”
“No; he wouldn’t have allowed it. It would have looked like a
combination against him. He wanted us separate, for his own ends.”
“Did you hear the shot that killed him?” Roger said suddenly.
“No. About two o’clock, wasn’t it? I’d been asleep two hours.”
“You did sleep with your wife then, in spite of the necessity of
preserving secrecy?”
“Her maid knew. Used to go back to my room in the early
morning. Beastly hole-and-corner business, but no alternative.”
“And only Stanworth’s death could have freed you, so to speak?”
Roger mused. “Very opportune, wasn’t it?”
“Very,” Jefferson replied laconically. “You think I forced him
somehow to shoot himself, don’t you?”
“Well, I—I——” Roger stammered, completely taken aback.
Jefferson smiled grimly. “Knew you must have some comic idea
in your head. Just seen what you’ve been driving at. Well, you can
rest assured I didn’t. For the simple reason that nobody or no threats

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Chapter 5 elaborates different types of enzymatic biosensors for glucose sensing.

Guwahati, Assam, India Sanjukta Patra

1 Enzymatic Biosensors for Healthcare Applications . . . . . . . . . . . . . 1

12 Low-Cost Paper-Based Analytical Devices and Their

About the Editors

Sanjukta Patra works at the Department of Biosciences and Bioengineering,

Debasree Kundu is a Post-Doctoral Fellow at the Department of Biosciences and

Manashjit Gogoi is an Assistant Professor at the Department of Biomedical Engi-

Kavita Arora Advanced Instrumentation Research Facility and School of Compu-

Girish Chandra Mohanta Nanotechnology Lab (H-1), CSIR-Central Scientiﬁc

Nishima Wangoo Department of Applied Sciences, UIET, Panjab University,

B. Daurai · S. S. Ramchiary · M. Gogoi (✉)

# The Author(s), under exclusive license to Springer Nature Singapore Pte 1

1.2 Types of Enzymatic Biosensors

1.2.1 Amperometric Biosensors

1.2.1.1 First-Generation Amperometric Biosensors

1.2.1.2 Second-Generation Amperometric Biosensors

1.2.1.3 Third-Generation Amperometric Biosensors

1.2.2 Optical Enzymatic Biosensors

gels, entrapment in inorganic or organic polymeric gels, and conﬁnement within

1.2.2.1 Absorbance Optical Biosensors

1.2.3 Piezoelectric Enzymatic Biosensor

In this type of biosensor, the transduction is done by a piezoelectric quartz crystal.

1.3 Significance of Enzymes as Biomarkers

1.3.1 Enzymes as Biomarker for Cardiovascular Diseases

1.3.2 Enzymes as Biomarker for Liver Diseases

1.3.3 Enzymes as Biomarker for Cancer

Cancer is characterized as a condition in which a group of cells develops improperly,

inﬂuenced by CD, which is indicated with the overexpression of epithelial breast

In prostate cancer, the most common biomarkers are prostate-speciﬁc antigen,

1.3.4 Enzymes as Biomarkers for Other Diseases

1.4 Enzymatic Biosensors for Various Diseases

1.4.1 Biosensors for Cardiovascular Diseases

An aptamer-based label-free capacitive biosensor was developed using non-faradaic

cholesterol enzymatic biosensor based on nanohybrid composites (CSPPy-

1.4.2 Biosensors for Liver Diseases

1.4.3 Biosensors for Diabetes

A glucose enzymatic biosensor for detection of diabetes was developed using

polyaniline with nanowire-based peptide (BNP), (diagnosis of

Biosensor Technical strategy Mechanism Applications Limitations Advantages References

with solutions (chitosan, polyethylenimine, polystyrene sulfonate, etc.). The surface

1.4.4 Biosensors for Kidney Diseases

Premanode and Toumazou (2007) developed a new biosensor based on immobilized

Table 1.2 Enzymatic biosensors’ overview

Table 1.2 (continued)

1.4.5 Biosensors for Neurological Diseases