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Materials Horizons: From Nature to Nanomaterials

Shrikrishna Nandkishor Joshi

Pranjal Chandra Editors

Advanced
Micro- and Nano-
manufacturing
Technologies
Applications in Biochemical and
Biomedical Engineering
Materials Horizons: From Nature
to Nanomaterials

Series Editor
Vijay Kumar Thakur, School of Aerospace, Transport and Manufacturing,
Cranfield University, Cranfield, UK
Materials are an indispensable part of human civilization since the inception of life
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Keeping in mind the immense perspectives of various classes of materials, this
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materials research at cutting-edge interface of materials science with physics,
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This series covers a galaxy of materials ranging from natural materials to
nanomaterials. Some of the topics include but not limited to: biological materials,
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inorganic materials, inorganic-organic hybrids, metals, membranes, magnetic
materials, manufacturing of materials, nanomaterials, organic materials and
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More information about this series at http://www.springer.com/series/16122

Shrikrishna Nandkishor Joshi · Pranjal Chandra
Editors

Advanced Micro-
and Nano-manufacturing
Technologies
Applications in Biochemical and Biomedical
Engineering
Editors
Shrikrishna Nandkishor Joshi Pranjal Chandra
Department of Mechanical Engineering School of Biochemical Engineering
Indian Institute of Technology Guwahati Indian Institute of Technology (BHU)
Guwahati, Assam, India Varanasi
Varanasi, Uttar Pradesh, India

ISSN 2524-5384 ISSN 2524-5392 (electronic)

Materials Horizons: From Nature to Nanomaterials
ISBN 978-981-16-3644-8 ISBN 978-981-16-3645-5 (eBook)
https://doi.org/10.1007/978-981-16-3645-5

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Preface

Micro- and nano-manufacturing technologies deal with miniaturized fabrication

techniques that create features, products, systems, and devices having dimensions in
the range of nanometers to millimeters. These devices include functional segments,
immobile assemblies, or both. In today’s date, micro–nano-fabrication and manufac-
turing have become an integral part of applications in biochemical and biomedical
disciplines. These applications can be majorly categorized into four domains: appli-
cation in molecular biology and biochemical science, application focusing on cellular
systems, prototyping of medical devices, and development of biosensors.
Micro- and nano-manufacturing structures provide a considerably improved func-
tion for conventional devices. These fabrication techniques always offer great poten-
tials. Such enhanced and improved potentials are deliberated when these techniques
are exploited appropriately to address the right types of problems. Considering
the massive advancement of micro–nano-manufacturing technologies, this book
attempts to provide an illustrative knowledge regarding these techniques between
its two covers. It discusses vividly the principles of various advanced manufacturing
technologies applied to develop different biomedical devices. Techniques including
advanced microchannel fabrication, advanced finishing processes, and rapid manu-
facturing for configuring biomedical devices have been accounted for. The novel
areas of interest considering the mediators like nanomaterials, piezoelectric nano-
generators, localized surface plasmon resonance for sensor development, and fabri-
cation have also been discussed thoroughly. This book also collectively detailed
the varying advantages presented by these techniques and how these advantages
have been explored to date. We believe the chapters presented in the book cover
various new dimensions of micro- and nano-manufacturing technologies and their
most recent applications in biochemical and biomedical engineering. This book not
only will be helpful for the students, scholars, and faculty members, but will also
help the newcomers in the field and the industry experts.

Guwahati, India Shrikrishna Nandkishor Joshi

Varanasi, India Pranjal Chandra

v
Contents

1 Nanomaterials Based Biosensing: Methods and Principle

of Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Nirmal Kumar Katiyar, Gaurav Goel, and Saurav Goel
2 Pathways to Translate the Biomedical Prototypes . . . . . . . . . . . . . . . . 29
Tamanna Bhuyan, Surjendu Maity, Devi Rupa Saha,
Nayan Mani Das, and Dipankar Bandyopadhyay
3 Accuracy of Biosensors as Rapid Diagnostic and Biochemical
Monitoring Tools for Non-communicable Diseases
Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Norhafizah Muhammad, Lim Tiong Hoo,
Afiqah Nabihah Ahmad, Azureen Mohamad,
and Syazana Abdullah Lim
4 Rapid Manufacturing of Biomedical Devices: Process
Alternatives, Selection and Planning . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Sanchit Jhunjhunwala and Sajan Kapil
5 Advanced Finishing Processes for Biomedical Applications . . . . . . . . 105
Talwinder Singh Bedi, Ravi Kant, and Hema Gurung
6 Advanced Microchannel Fabrication Technologies
for Biomedical Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Upasana Sarma, Pranjal Chandra, and Shrikrishna N. Joshi
7 Droplet Microfluidics—A Tool for Biosensing
and Bioengineering Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
U. Banerjee, R. Iqbal, S. Hazra, N. Satpathi, and A. K. Sen
8 Advances in Microfluidic Techniques for Detection
and Isolation of Circulating Tumor Cells . . . . . . . . . . . . . . . . . . . . . . . . 173
K. Mirkale, R. Gaikwad, B. Majhy, G. Narendran, and A. K. Sen

vii
viii Contents

9 Localized Surface Plasmon Resonance Sensors for Biomarker

Detection with On-Chip Microfluidic Devices in Point-of-Care
Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
S. Z. Hoque, L. Somasundaram, R. A. Samy, A. Dawane,
and A. K. Sen
10 Development of Piezoelectric Nanogenerator Based
on Micro/Nanofabrication Techniques and Its Application
on Medical Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Aparna Zagabathuni and Subramani Kanagaraj
11 Optical Biosensors Towards Point of Care Testing of Various
Biochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Vinoth Edal Joseph and Archana Ramadoss
12 Blood Coagulation System and Carbon-Based
Nanoengineering for Biomedical Application . . . . . . . . . . . . . . . . . . . . . 279
Abhishek R. Panigrahi, Pooja Yadav, Samir K. Beura,
and Sunil K. Singh
13 Opportunities and Challenges in Medical Robotic Device
Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Neha Khatri, Mukesh Kumar, and Ranjan Jha
14 Flexible Organic Field-Effect Transistors for Biomimetic
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Vivek Raghuwanshi and Shree Prakash Tiwari
15 Methods for Surface Superfinishing of Prosthesis . . . . . . . . . . . . . . . . . 335
Atul Singh Rajput, Sajan Kapil, and Manas Das
16 Principles of Advanced Manufacturing Technologies
for Biomedical Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
G. L. Samuel, Lingxue Kong, Y. Arcot, and Pavan Pandit
About the Editors

Prof. Shrikrishna Nandkishor Joshi is a Professor of

Mechanical Engineering at the Indian Institute of Tech-
nology (IIT) Guwahati, India. He was a Visiting Faculty
at the Asian Institute of Technology, Thailand in 2015.
His research interests include micro, nano, and preci-
sion manufacturing processes with a focus on applica-
tions of lasers in manufacturing, thin-wall machining,
single point diamond turning, CAD/CAM, and manu-
facturing automation. Prof. Joshi published more than
seventy research articles in journals of international
repute and conferences. He has published two books on
Laser-based Manufacturing and authored more than ten
book chapters. He has developed a Massive Open Online
Course on “Automation in Manufacturing” and a web
course on “Mechatronics and Manufacturing Automa-
tion” as part of the Government of India’s flagship
project National Programme on Technology Enhanced
Learning (NPTEL).

Prof. Pranjal Chandra is an Assistant Professor at

the School of Biochemical Engineering, Indian Insti-
tute of Technology (BHU), Varanasi, India. He earned
his Ph.D. from Pusan National University, South
Korea and did post-doctoral training at Technion-Israel
Institute of Technology, Israel. His research focus
is highly interdisciplinary, spanning a wide range in
biotechnology, nanobiosensors, material engineering,
and nanomedicine. He has designed several commer-
cially viable biosensing prototypes that can be oper-
ated for onsite analysis for biomedical diagnostics. He
is a guest editor and an editorial board member of
various international journals. He is author of over 100
ix
x About the Editors

high impact publications including research/reviews

papers and invited book chapter. He has published
11 books on various aspects of biosensors/medical
diagnostics. Dr. Chandra is the recipient of many
prestigious awards, coveted honors, and fellowships
such as: DST Ramanujan fellowship (Government
of India); Early Career Research Award (ECRA)
(DST, Government of India); BK-21 and NRF fellow-
ship, South Korea; Technion Post-Doctoral Fellowship,
Israel; Nano Molecular Society India Young Scien-
tist Award; Biotech Research Society India (BRSI)
Young Scientist Award; Young Engineers Award 2018;
Highly Cited Corresponding authors in The Royal
Society of Chemistry (RSC), Cambridge, London; Top
10% cited article in the General Chemistry Section
RSC Journal, Cambridge, London, Gandhian Young
Technology Innovation Award (GYTI) 2020, etc. Dr.
Chandra is also listed among the world’s top 2% scientist
in a report by Stanford University, USA.
Chapter 1
Nanomaterials Based Biosensing:
Methods and Principle of Detection

Nirmal Kumar Katiyar , Gaurav Goel , and Saurav Goel

Abbreviations

CNT Carbon nanotube

D Dimensional
DNA Deoxyribonucleic acid
FAD Flavinadenine dinucleotide
FET Field Effect Transistor
NPs Nanoparticles
RI Refractive index
SPCE Screen Printed Carbon Electrode
SERS Surface-enhanced Raman scattering
SPR Surface plasmon resonance
SWCNTs Single Wall Carbon Nanotubes

N. K. Katiyar · G. Goel (B) · S. Goel

School of Engineering, London South Bank University, 103 Borough Road, London 1 0AA, UK
e-mail: [email protected]
N. K. Katiyar
e-mail: [email protected]
S. Goel
e-mail: [email protected]
S. Goel
EPSRC Centre for Doctoral Training in Ultra-Precision Engineering, University of Cambridge
and Cranfield University, Bedfordshire, UK
School of Aerospace, Transport and Manufacturing, Cranfield University,
Bedfordshire MK43 0AL, UK
Department of Mechanical Engineering, Shiv Nadar University, Gautam Budh Nagar,
Chennai 201314, India

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 1
S. N. Joshi and P. Chandra (eds.), Advanced Micro- and Nano-manufacturing
Technologies, Materials Horizons: From Nature to Nanomaterials,
https://doi.org/10.1007/978-981-16-3645-5_1
2 N. K. Katiyar et al.

1 Introduction

Biosensing is an emerging research area that is being governed by the need to

develop easy, simple, and inexpensive biosensors with instant and precise detec-
tion. Nanotechnology has brought tremendous capability to miniaturize biosensors
and to make them more affordable. On the other hand, conventional methods of
measurement are limited by the complexity of the process and requirement of skilled
manpower and time to measure a particular change or property. A simple question
one may ask, how does a sensor make life easy for humans? We can understand
this through the utilization of biosensors in our daily life, such as a diabetic person
can measure their glucose level instantly anywhere by herself/himself, pregnancy
diagnostic kits can now be used conveniently without any prior experience and are
easily affordable.
One of the simplest biosensors is the body temperature measurement sensor, which
most of the people uses in their life. There are many non-bio sensors in our daily life
such as a clock or a watch to record time, a mobile phone used to receive the data and
voice communication including the pedometer inside it, as well as a speedometer
measuring the speed of the bike. Sensors measuring a single parameter may have
different working principles but they all do the same thing, i.e. measure the magnitude
of certain property such as a thermometer based on Hg (Mercury) measures thermal
expansion, a non-contact thermometer based on infra-red radiations popularly used
recently for fast monitoring of the body temperature caused by COVID-19, and a
digital thermometer based on thermistor (thermocouple) principle. All three devices
have the same goal to measure temperature, as shown in Fig. 1.
Similarly, in the food and medical sectors, many biosensors are based on lever-
aging different working principles such as estimation of glucose in the blood sample,

Fig. 1 Temperature measurement sensor with three different working principles

1 Nanomaterials Based Biosensing: Methods and Principle … 3

cancer cell detection, bacteria detection, DNA identification, virus detection, detec-
tion of pesticides/chemicals/antibiotics, and biological toxin level detection in food
and beverages. Biosensors are becoming an integral part of our life to raise awareness
and consciousness at the early stages of a process. Biosensors either detect the target
analytes or provides exact measurements. Different target analytes require different
biosensors, and it can be associated with different working principles. Therefore,
emerging nanoscale materials have been employed in combination with newer elec-
tronic circuits to develop easy to handle nanoplatforms for sensing various types of
analytes. Nanoscale integrated biosensors can work as multiplexed biosensors, or
one sensor can detect more than one analyte. However, the prime goal behind the
development of biosensors is to improve the lower limit of detection.
Many biosensors are being used in medical and food sectors, having different work
principles such as acoustic wave biosensors, electrochemical biosensors, fluorescent
paper-based sensors, electrogenerated chemiluminescence, colourimetric detection,
Microbial fuel cell-based biosensor, etc. There are multiple biosensors, which are
part of daily use. They can be classified into various categories such as diverse
principle-based, different sectors based, and different materials based, such as paper-
based biosensors, electronic biosensors, hydrogel-based sensors, etc. This chapter
is limited in scope to discuss various essential principles being used in the devel-
opment of biosensors utilizing nanomaterials and categorization based on nano-
materials dimensions for example zero, one, and two-dimensional nanomaterials
(nanoparticles, nanofibers, and nanosheets).

2 What Makes a Sensor Good?

The quality of a biosensor depends on some of its salient features such as selectivity,
stability, reproducibility, sensitivity, and linearity. Besides these properties, the sensor
must be economical. Modern technology is integrated with nanoscience/technology,
which makes biosensors ultrasensitive, and they can detect even a single biomolecule.
Fast detection: The quick response of a sensor has its importance, especially in point-
of-care diagnostic. Also, during a life-threatening incident, the rapid response of the
sensor is essential such as heartbeat monitoring, in-vivo monitoring, etc.
Selective detection: Selective detection of analytes by a biosensor is equally crucial
because most of the biological samples (e.g. Blood) and food (e.g. Beverage), etc.
are the pool of biochemicals. A biosensor needs to detect only the particular analytes,
for which it is tuned, and it must be neutral to other interfering analytes. It is known
as the selectivity of the biosensor.
Reproducibility: Reproducibility is the ability of a biosensor to produce similar or
identical results every time the measurement is repeated, or we can say there is a
negligible change in the result during multiple measurements.
4 N. K. Katiyar et al.

Stability: The stability of a biosensor is an essential factor and indicates the consis-
tency in results after a biosensor is deployed for a long time (storage/transportation).
Better stability guarantees consistency, reproducible results, and precision regardless
of secondary factors such as environmental conditions.
Accuracy of biosensor: The human body is prone to respond to slight chemical
change. Therefore, a sensor must be accurate to quantify the measurement.
Minimum sample requirement: The sampling requirement for sensing is an impor-
tant parameter and the smallest quantity requirement is the best especially in forensic
science, where destructive testing and availability of samples could be very limited.

3 Methods for Biosensors Formulation

A biosensor is a device having three parts, as shown in Fig. 2, the first part being
the biological recognition system, which is highly specific towards the analytes and
provides quick response on interaction with analytes. The second part is a transducer,
which takes the response from the recognition system, and converts it into an electric
signal, which is calibrated with the standard value of the response. The third part
deals with the amplification of the weak signals and displays on the controlling unit,
although it is not necessary for all biosensors.
Recently developed biosensors are based on various types of nanomaterials and
possess advantage due to their high surface energy to volume ratio, and delivers
response on minor changes over the surface such as metallic nanoparticle change their
surface plasmon response on binding with other molecules, nanowires change their
frequency of vibration on binding with another biomolecule, fluorescence efficiency
variation in nanosheets on the landing of analytes, etc., as shown in Fig. 3.

Fig. 2 Classification of biosensor parts and working principles

1 Nanomaterials Based Biosensing: Methods and Principle … 5

Fig. 3 Schematic of functionalization with receptor. a nanoparticles 0D, b nanowire 1D,

c nanosheet 2D

4 Working Principle

The following sections discuss the various working principles for the different biosen-
sors based on various nanomaterials (i.e. 0D, 1D, and, 2D). These working principles
may be used individually or in combination to achieve optimum results.

4.1 Nanoparticles (0D Materials)

The materials with all three dimensions less than 100 nm are considered nanopar-
ticles [1, 2]. They have unique and sensitive properties suitable for developing the
biosensors such as high surface energy to bind with other molecules, surface plasmon
resonance, etc. The nanoparticles-based biosensor is further divided into two cate-
gories: Label-based biosensor and Label-free biosensor. In the former technique,
nanoparticles functionalized with the specific receptor (enzyme, fluorescence, anti-
body, etc.) directly bind to the analytes. On the other hand, in the Label-free technique,
the nanoparticles directly interact with the analytes to give the response.

4.1.1 Surface Plasmon Resonance-Based

The surface plasmon resonance (SPR) is a versatile character of the metallic nanopar-
ticles. This phenomenon is recognized by the collective oscillation of the valance
electrons when incident electromagnetic radiation oscillation matches the surface
electrons, and it appears as an absorption band, the schematic of oscillation shown
in Fig. 4 [3]. The position of the absorption band is very sensitive to the dielectric
constant of the surrounding medium, nanoparticle shape, size, and ligand interactions
(increase or decrease surface electron density). The shifting of the band towards a
lower wavelength (high energy) is referred to as a blue shift and shifting of the band
towards a higher wavelength (lower energy) is referred to as redshift. The shift of
6 N. K. Katiyar et al.

Fig. 4 Surface plasmon resonance on nanoparticles

the SPR band by more than 2 nm in the visible range (~500 nm wavelength) can
be recorded easily. On the change of SPR band position (by analytes-nanoparticles
coupling), the reaction jar reflects the colour change, which can be identified through
the naked eyes, as shown in Fig. 5 [4]. A slight change of refractive index (RI) around
the nanoparticle’s surroundings can be detected as a signal, and this phenomenon is
being utilized to develop many different types of biosensors. Similarly, a change in
the size of the nanoparticle also alters the surface plasmon band and can be used for
the development of the sensor. The spherical gold nanoparticles suspension having an
average diameter of 10 nm appears as red colour (SPR band at 520 nm). On the other
hand, silver nanoparticles suspension having the same size appears as yellow colour
(SPR band at 395 nm) [5, 6]. Therefore, the core-shell nanoparticles (Au nanopar-
ticles with Ag surface) optical properties can be tuned to the cyan-green region by
altering the Ag layer thickness. Also, these nanoparticles are surface modified using
engineered antibody-conjugated, which are prone to bind to harmful biotoxin staphy-
lococcal enterotoxin A (SEA) [5, 7]. It is a small protein mostly found in staphylo-
coccal food poisoning [8]. Therefore, a solution assay containing these nanoparticles
functionalized with engineered conjugate antibody works as a biosensor; the assay
changes their colour on binding with biotoxin SEA, which may be observed through
naked eyes as shown in Fig. 5.

4.1.2 Surface-Enhanced Raman Scattering Based

The Surface-Enhanced Raman Scattering (SERS) is a phenomenon that enhances

signal intensity by many orders of magnitude (~106 –1014 ) and overcomes the inherent
weakness of a Raman signal. It is highly sensitive and selective due to unique finger-
print spectra. Therefore, even just a single molecule [9] can be detected using this
technique.
1 Nanomaterials Based Biosensing: Methods and Principle … 7

Fig. 5 a Schematic of synthesis core-shell nanoparticles (Au@AgNPs), their functionalization

with conjugated-antibody, and binding with SEA, b Ag@AuNPs change in colour before and after
binding with SEA, c Au@AgNPs, change in colour before and after binding (printed with the
permission) [7]

SERS can be utilized for developing different biosensors such as in-vivo glucose
detection [10, 11] nucleic acid and proteins [12], DNA detection [13], and rapid
detection of bacteria [14, 15]. The detection methodology with label-based and label-
free techniques utilizing nanoparticles is shown in Fig. 6.

4.1.3 Magnetic Nanoparticles Based

Magnetic nanoparticles-based sensing is also being actively used for biosensing since
the last decade. They are alternatives to fluorescent biosensors. Magnetic nanopar-
ticles show super magnetic behaviour at nanoscale dimensions due to a reduced
8 N. K. Katiyar et al.

Fig. 6 Schematics of label-based and label-free surface-enhanced Raman scattering phenomena

for designing biosensors

number of domains, and magnetization can flip direction instantly. However, in the
case of bulk, the external magnetic field is required to demagnetize and flip the direc-
tion, as shown in Fig. 7 [16]. For nanoparticles, there is no thumb rule to determine
magnetic behaviour (diamagnetic, paramagnetic, ferromagnetic) [17]. In the past few
years, different types of magnetic nanoparticles are used for biosensing such as Co,
Mn, Fe, Ni, Fe2 O3 , Fe3 O4 , FePt, FePt-Ag, CdS, etc. [18]. The magnetic behaviour
of nanoparticles is now being integrated with transducers’ materials, which impart
analytical merits such as low limit detection, high signal to noise ratio, enhanced
sensitivity, etc. [19].
The separation of analytes from a large sample or other words concentration
enhancement of analytes utilizing functionalized magnetic nanoparticles is a compe-
tent technique nowadays. In this technique, a nanoparticle functionalized with the
receptor directly binds with the targeted analytes in a sample. Furthermore, analytes,
along with magnetic nanoparticles, separates from the solution using an external
magnetic field. In this way, centrifugation can be avoided from sample preparation,
which brings other impurities along with targeted analytes [17].
1 Nanomaterials Based Biosensing: Methods and Principle … 9

Fig. 7 Schematics of the magnetic materials hysteresis loop of bulk magnetic vs nanoparticles
(Ms: magnetic saturation, Mr: remnant magnetization, Hc: coercivity)

4.2 Nanowire (1D Materials)

A nanowire is a thin wire with a diameter below 100 nm. Nanowires made from
various materials such as Ag, Au, Pt, carbon nanotubes, etc. have been developed.
The frequency of vibration, stress response of piezoelectric nanowire, and an array
of nanowires endows with photonic properties such as wire arrangement helps to
trap the light in a broad range of wavelengths, and these properties of nanowires are
utilized in biosensor development.

4.2.1 Nanomechanical-Based Sensors

The deformation and vibrations in a nanowire are sensitive properties, and the
utilization of these unique properties is helpful in the development of biosensors.
The nanowires functionalized with the receptors are purposely made to bind with
biomolecules of interest. Whenever the analytes or biomolecules bind with the
receptor on nanowires, it induces the stress and nanowire bend shown by a schematic
in Fig. 8. The deflection of the nanowire can be measured through optical (laser deflec-
tion, optical interferometric, etc.) or electrical techniques (piezoelectric, etc.). Simi-
larly, whenever biomolecules land onto a nanowire, which reduces their vibration
frequency due to mass increase, which can be measured through optical techniques
[20, 21].
10 N. K. Katiyar et al.

Fig. 8 Nanomechanical biosensor principle. a static; bending of nanowire, b dynamic; changing

the vibration of the nanowire

4.2.2 Opto-Thermo-Mechanical Based

The working principle of Opto-thermo-mechanical based biosensor is slightly

different from the thermomechanical based biosensor. In this method, the array of
the nanowire is vertically planted over the silicon–silicon dioxide membrane. In this
way, the high surface area achieved makes it highly sensitive to respond to even a few
analyte molecules. These nanowires are capable of trapping light of broadband due
to nanostructures. Also, nanowires are functionalized with the single-stranded probe
DNA. When analyte molecules come in contact with a target single-stranded DNA,
the relevant molecules bind together, changing the mass detected by the device. Also,
the vibration frequency of nanowire changes due to the addition of mass. 90% of the
laser light exposed over the surface of the array nanowire gets trapped or absorbed
due to nanostructure array, which activates the efficient opto-thermomechanical exci-
tation of the resonator. An optical signal of laser light (variation in frequency) is used
to read for interpretation of the results. In this way, there is no requirement for wires
to connect with the biorecognition platform [22].

4.2.3 Field-Effect Based (Semiconductor) Biosensing

A material having its conductivity between conductor and insulator is known as a

semiconductor, which is defined in terms of a bandgap. Therefore, a nanowire having
a bandgap and its ability to bind different analytes over the surface makes it more
popular to design a wide range of biosensors. Silicon is a popular semiconductor
used in most electronics applications. Cui et al. [23], reported for the first time to use
the silicon nanowire building block to develop different sensors, which can detect
protein, viruses, nucleic acid, etc. Silicon nanowire acts as a resistor when the number
1 Nanomaterials Based Biosensing: Methods and Principle … 11

Fig. 9 Semiconductor-based nanowire biosensor configured with receptor over nanowire surface

of charge molecules binds over nanowire, and it leads to depletion or accumulation

charge carrier, as shown in Fig. 9. As a result, the conductivity of the nanowire can be
harnessed as a signal, and it is ultrasensitive up to a single virus/molecule detection
[24, 25]. The bio-molecules induce charges in an aqueous solution (proteins, nucleic
acid) that can bind easily with appropriate receptor functionalized over the surface
of the nanowire [26]. The different materials nanowire used for various bio analyte
detection such as silicon nanowire for gastric cancer detection from exhaled vola-
tolome [27], troponin detection [28], Follicle-Stimulating Hormone detection [29],
etc.
Similarly, the carbon nanotubes are very sensitive to response even at single-
molecule interaction. There are carbon nanotubes (SWCNTs) and multi-wall carbon
nanotubes (MWCNTs) being used for biosensor formulation. The SWCNTs having
a high electrical transduction property and utilized paper-based chemiresistor sensor
for the detection of human immunoglobulin G (HIgG) up to the picomolar level [30].

4.3 Nanosheet Based (2D)

A two-dimensional thinnest material, having a thickness of less than a few nanometers

is referred to as a nanosheet or in short 2D nanostructure materials. These materials
12 N. K. Katiyar et al.

are having unique and excellent mechanical, thermal, optical, and electrical properties
that have attracted researchers to harness their properties for many applications, one
of them is to develop biosensors.

4.3.1 Fluorescence-Based Biosensing

Fluorescent molecules absorb light and in turn excites the electrons of the molecules,
causing them to jump to a higher energy level. Upon returning from an excited state
to a ground state, the energy is released in the form of radiation or heat. The light-
emitted in this process is referred to as fluorescence, and molecules are known as
fluorophore. However, when the fluorophore interacts with the quencher (another
molecule), the resultant complex may become non-fluorescent. A German scien-
tist Theodor Förster proposed and explained the mechanism of two light-sensitive
(donor–acceptor) molecules interaction, which may emit light or can absorb [31]. It
is known as Föster (Fluorescence) resonance energy transfer (FRET). The electroni-
cally excited state of a donor may transfer energy to an acceptor via the non-radiative
dipole–dipole interaction. Energy transfer efficiency is inversely proportional to the
sixth power of the distance between donor and acceptor making FRET extremely
sensitive to small gaps [31].
The two-dimensional nanomaterials are extensively in use for biosensing because
2D nanosheets can act as a platform, where biomolecules can easily land or adsorb
and alter the fluorescence efficiency (donor–acceptor). Now a day, many 2D materials
are in practice to harness fluorescence property for use in biosensing, including the
use of Vanadium disulfide (VS2 ) nanosheet for detection of cytochrome c.

4.3.2 Field-Effect Based (Semiconductor)

The field-effect transistor technology is a milestone in developing tiny and simplest

biosensors. There are many-layered structure materials such as molybdenum disul-
fide (MoS2 ), Graphite, Vanadium disulfide (VS2 ), boron nitride (BN), tungsten disul-
fide (WS2 ) that are easy to exfoliate in different solvents to prepare nanosheets.
Additionally, graphene-based FET’s are highly sensitive towards the detection of
the analyte. Graphene has an extraordinary ability of the electron-transport prop-
erty [32]. Therefore, graphene channels show conductivity change in response to
the minor adsorption of analyte molecules on the sensor surface. Chen et al. [33]
described field-effect transistor-based detection of Ebola shown in Fig. 10. The
reduced graphene oxide bridge between the source and the drain electrode, and
layer protected with Al2 O3 to protect underline rGO. The gold nanoparticles conju-
gated with antibody immobilized over the reduced graphene oxide layer, are highly
prone to bind with Ebola virus glycoproteins. Therefore, the exposure of EGP (Ebola
glycoprotein), makes the dynamic response of FET measured and the drain-voltage
current to decrease with time as more protein binds with the platform (see Fig.10b).
The detection limit of such a biosensor is 1 ng/ml EGP.
1 Nanomaterials Based Biosensing: Methods and Principle … 13

Fig. 10 a Schematic of reduced graphene oxide-based Field-Effect Transistor (FET) biosensor.

Ebola antibody conjugated with gold nanoparticles and Al2 O3 coating for passivation, b dynamic
response of FET sensor on the exposure of Ebola glycoprotein (EGP) (reprinted with permission)
[33]

4.3.3 Optical Biosensors

Graphene-based materials are mostly studied due to their extraordinary optical prop-
erty such as surface plasmon and absorption polarization, which endowed the sensor
to detect even single cells [34]. The distinction of graphene biosensors based on their
electrical and optical properties are summarized in Table 1.

Surface Plasmon Based Biosensing

The best property of optical sensors is that they respond ultra-fast and gives result
in real-time. The electronic properties of graphene are excellent because the Fermi
surface of graphene lies in the overlapped/intersection of the empty conduction band

Table 1 Difference in electrical and optical principle-based graphene biosensor

Property Graphene electrical biosensor Graphene optical biosensor References
Principle Receptor functionalized Broad band absorption of [35–37]
graphene changes conductivity graphene and their sensitivity
on binding with analytes. with any change near to
Monitor the drain-source graphene surface or refractive
conductivity in Field effect index
transition mode
Advantage Fast response time Unlabelled sample
High surface area Accurate detection
Lower detection limit High spatial resolution
Disadvantage Only measure current Light absorption of monolayer
Sample damage graphene too low. Aggregation
of graphene affects the optical
properties
14 N. K. Katiyar et al.

and the filled valence band and also in the middle of the p band, where electrons are
the valence electrons and have the mobility of about 1/300 the speed of light known
as Dirac Fermion [38, 39]. These fermions show a linear energy-momentum relation-
ship near the Dirac point, and this endowed graphene with optical resonance prop-
erty at any frequency in the ultra-violet, infra-red region. Therefore, many different
biosensors have been developed, in which the fibre optic sensor is the simplest. The
fibre optic integrated with graphene makes use of optical fibre used for transmission
of light and to receive the output. The change in intensity, wavelength, frequency,
etc. after interaction with the graphene platform received through the optical fibre is
analyzed [40, 41]. There are many graphene SPR based biosensors used for a variety
of purposes, such as specific protein detection [42], gas detection [43], and different
chemical- biological species [44].

Polarization Absorption Enhanced

Another interaction of light with graphene depends on the number of layers and
prismatic total internal reflection (TIR) structure. In the graphene, the prismatic TIR
structure shows the characteristic polarization absorption and broadband absorp-
tion enhancement [45]. When the refractive index of the medium increases than
the graphene, it starts to show a variation in the optical power of two polarization
states [34, 46]. Also, by utilization of change in polarization states, many sensitive
biosensors have been designed [39].

4.3.4 Gravimetric-Based Biosensors

The natural frequency of a piezo crystal or film is inversely proportional to its thick-
ness, and also, we can make it oscillatory due to piezoelectricity by applying elec-
tricity using a simple circuit. Also, whenever any mass is bound to the piezoelectric
film, its frequency gets reduced. This method can be utilized in different modes
such as resonating crystal, in which well-known quartz crystal microbalance works,
surface acoustic wave mode, etc. [47]. This type of sensor sensitivity depends on
the film thickness. Therefore, many piezoelectric polymer films have been devel-
oped, which can be drawn in very thin films to develop highly sensitive biosensors.
The Polyvinylidene Fluoride (PVDF) polymer is one such well known piezoelectric
polymer [47].

4.4 3D Nanomaterials

Bulk materials with nanophase domains or equiaxed clear grains can be called 3D
nanomaterials [48]. Suppose a material has been rolled to form inside nanostructuring
due to deformation; it becomes a nanomaterial. However, if we extend 2D rapheme in
1 Nanomaterials Based Biosensing: Methods and Principle … 15

the third dimension by stacking without any raphemelization between two rapheme
sheets, it becomes bulk graphite. The properties of these stacked sheets are like the
graphite, while it is having 2D nano rapheme sheets by its virtue. Therefore, the
author’s points of view are that a 3D nanomaterial will fall in the category of 0D,
1D, and 2D. It is just consolidation of nanoparticles/rods/sheets.

4.5 Electrochemical Sensor

The electrochemical sensors work as electrochemical transduction, where electro-

chemical reactions (oxidation–reduction) results in the transfer of electrons, which
can be detected through amperometry or voltammetry. It can detect enzymes,
cells, tissues, gas, ligand, etc. In principle, it is a conventional electrochemical cell
containing three separate electrodes: the working electrode (WE), the counter elec-
trode (CE), and the reference electrode (RE), and these three-electrodes can be printed
on paper, polymer, etc. as shown in Fig. 11.
Nowadays, screen-printed carbon electrode (SPCE) biosensors are so famous
due to easy, point-of-care, small fluid volume (analyte) [49]. The working electrode
is functionalized with a biorecognition system (enzyme, etc.) utilizing nanoscale
materials, and the analytes directly interact with the working electrode. As a result
of this, the electrochemical cell becomes active and produces a signal.
Zelada-Guillén et al. [50] utilized carbon nanotubes as an ion-to-electron trans-
ducer, and covalently bonded aptamers for the detection of Staphylococcus aureus.
The measurement of electromotive force (EMF) or potential, generated on the
antigen–antibody interaction is known as a potentiometric biosensor. On the other
hand, the measurement of the ionic strength of the solution, which affects the conduc-
tivity, is known as a conductometry biosensor [51]. Similarly, amperometric trans-
ducers measure the direct current from redox reaction under a constant potential
applied to WE. The activity of the recognition element varies before and after the
interaction with a target molecule. The product must be electroactive and undergoes a
redox process [52]. However, if the biosensor measures the current-potential relation-
ship, and the reference of potential at zero current applied is known as a voltammetric

Fig. 11 Screen printed electrode-based biosensor

16 N. K. Katiyar et al.

biosensor. Many analytes can be detected with their characteristic potentials. It has
been divided into many such as cyclic voltammetry (CV), linear sweep voltammetry
(LSV), and differential pulse voltammetry (DPV), etc. Currently, the most popular
nanoscale materials being utilized for enhancing the sensitivity of biosensors are
Au nanoparticles (NPs), Pt NPs, alloy nanoparticles, carbon nanotubes, graphene,
MoS2 nanosheet, VS2 nanosheet. The utilization of different nanoscale materials
and various working principles used for analytes detection have been summarized in
Table 2.

4.6 Electrochemical Glucose Biosensor: How Nanoscale

Materials Appear to Play a Role?

The glucose level detection in the human body is a very important task, which changes
abruptly in a diabetic person, and needs careful monitoring. The electrochemical
amperometric biosensors are so common in daily use. The working principle of
this type of biosensor is based on a simple enzyme-catalysed reaction of glucose as
follows:
Glucose oxidase (Enzyme)
Glucose + O2 −−−−−−−−−−−−−→ Gluconic acid + H2 O2 (1)

reduction
O2 + 4H+ + 4e− −−−−→ 2H2 O (2)

oxidation
H2 O2 −−−−→ O2 + 2H+ + 2e− (3)

Therefore, we can detect the consumption of oxygen or the generation of H2 O2

electrochemically. The H2 O2 oxidizes and produces electrons, and these electrons
(anodic current) can be monitored electronically. These were classed as the first
generation of glucose biosensors. In this process, the high potential required to apply
at which other drugs may present in the blood sample becomes electroactive at such
potential resulting in inaccuracy. Therefore, the influence of coexisting electroactive
compounds reduced by a coating over the electrode surface with polymer layer
(poly-(phenylenediamine), polyphenol, Nafion, cellulose acetate, etc.), and tuning
the operational electrode potential of targeted analytes oxidation range preciously.
However, the whole process depends on oxygen. Consequently, slight tension in
oxygen and stoichiometry alter the sensor response [63]. Therefore, to counter this
problem in a glucose biosensor, a mediator (non-physiological electron acceptor)
is inserted between glucose oxidase enzyme and electrode, which carry forward
electron from the reaction center to the electrode surface.
These are classed as the second-generation glucose biosensors and are based on
the following equations as illustrated further in Fig. 12.
1 Nanomaterials Based Biosensing: Methods and Principle … 17

Table 2 Some biosensors researching nanoscale materials, method and analytes

Materials Method Analytes Lower Dimension References
or principle to be measured limit of of
detection nanomaterials
Ag@Au core Surface plasmon Biotoxin 0.2 nM 0D [7]
shell resonance staphylococcal
nanoparticles enterotoxin A
(SEA)
FeCo Giant Endoglin 83 fM 0D [53]
nanoparticles Magnetoresistive detection from
immunosensor urine
Carboxyl Superconducting breast cancer 106 cells 0D [54]
functionalized quantum cells with
iron oxide interference uncertainty
nanoparticles 20%
Manganese-doped Hall Sensor Rare cells: Individual 0D [55]
ferrite (MnFe2 O4 ) MDA-MB-468 cell
cancer cells
(whole blood)
Carbon nanotube Chemiresistor Proteins 6.6 pM 1D [30]
detection
Polyaniline Electrochemical DNA detection 150 pM 1D [56]
nanofibres (Cyclic
(PANI-NF) voltammetry)
functionalized
with gold NPs
Nickel oxide Field effect Alcohol 100 ppm 1D [57]
(NiO) nanofibres detection
Gold NanoWire Optical and Bacteria in 103 cell/ml 1D [58]
arrays electrocheical kidney
infection
Platinum Electrochemical electrocatalytic 5 × 10–6 1D [59]
Nanowires (Amperometer) reduction of M
hydrogen
peroxide
(H2 O2 )
Graphene Field Effect Ebola virus 1 ng/ml 2D [33]
Transistor detection
Graphene Field Effect Acetylcholine 2.3 M 2D [60]
Transistor
Layered titanate Electrochemical Catalytic 0.6 M 2D [61]
sheets based reduction of
H2 O2
Flower-shaped Electrochemicacal Glucose 1.37 M 2D [62]
copper oxide thin (amperometric)
sheet
18 N. K. Katiyar et al.

Fig. 12 Sequence of reaction occur in second-generation glucose biosensor [63]

Glucose + Glucose oxidase(oxi) → Gluconic acid + Glucose oxidase (red) (4)

Glucose oxidase (red) + 2M(oxi) → Glucoseoxidase (oxi) + 2M(red) + 2H+

(5)

2M(red) → 2M(oxi) + 2e− (6)

Many mediators are being used, such as ferricyanide, conducting organic salts,
quinone compounds, transition-metal complexes, phenothiazine and phenoxazine
compounds, etc. which are insoluble, non-toxic, and chemically stable. The mediator
is fast enough to react with enzymes and transfer electrons, but still, oxygen remains
a problem due to self-diffusing. It limits the accuracy of low glucose concentration.
Further improvements in the biosensor have been made by placing wiring as
nanomaterials to fast electron transfer from the centre of the redox enzyme to the
electrode. It is fast communication between the redox enzyme and the electrode.
Patolsky et al. [64, 65] have constituted the glucose oxidase enzyme over the carbon
nanotubes, which vertically connects to the electrode, and acts as a wire to fast
transfer charge, as shown in Fig. 13.
It is known as the third generation of glucose biosensors based on amperometry.
Modern technology is revolutionizing very fastly. These biosensors can now be screen
printed with multilayers (electrode, enzyme, mediator, binder, etc.) strips for one-time
use.

5 Biological Recognition Element

The biological recognition elements are different types of receptors or bio-probes,

which are highly specific towards their stimuli. The bio probes are immobilized over
the surface of nanoscale materials, which can form covalent bonds with analytes
1 Nanomaterials Based Biosensing: Methods and Principle … 19

Fig. 13 Carbon nanotube (CNT) as a connector for electrical communication in glucose biosensor;
(FAD: flavinadenine dinucleotide) [64]

or adsorbed and bind with Van der-Waal forces, etc., such as antibodies, enzymes,
and cells. The most straightforward process is the physical adsorption of bio-probes.
However, the salts, pH, and other contaminants appear as a nuisance, which creates
noise in the output signals and losses in the precision quantification or detection.
Contrarily, the chemical immobilization of bio-probes has attractive features, such
as strong covalent bonding with nanoscale materials and is highly specific towards
the target analytes; which increases precision and the probability of detection.
Each biosensor needs a unique combination of biorecognition element and trans-
ducer, which depends on the target analyte. A schematic map is shown in Fig. 14 for
selecting a suitable biorecognition element.

Fig. 14 Map of selecting biorecognition element for developing biosensor (reprinted with
permission) [66]
20 N. K. Katiyar et al.

Antibodies
Antibodies are different types of proteins that are largely used as biorecognition
elements. They are more specific and selective towards analytes that are being used
for developing various types of biosensors. The antibodies may be categorized into
two kinds monoclonal and polyclonal. Monoclonal antibodies can recognize a single
epitope of a target molecule, whereas polyclonal antibodies can recognize different
epitopes of the same target. The antibodies are more susceptible to temperature and
required to store at low temperatures. Otherwise, it gets denatured; as a result, it can
lose the binding ability.
Deoxyribonucleic acid (DNA)
In modern biosensors, DNA is being used as a probe in the biorecognition platform. It
is a short DNA sequence, immobilized over the transducer surface, that can interact
with the specific target DNA sequence, or it can bind to a complementary DNA
sequence. In the case of metal ions detection, the phosphate backbone of DNA
molecules have a negative charge and easily binds with the positively charged metal
ions [67].
Cells
The living cells are being used as biorecognition (bio-receptor), which is an econom-
ical alternative to enzymes and antibodies. Designing a biosensor recognition plat-
form becomes easier by using cells in comparison to the other bio-receptors. The
different types of cells with distinct functional strategies are in use for developing
biosensors, as shown in Fig. 15. The cells are immobilized over the transducer in
such a way; the viability of the cell does not affect, such as direct immobilization on
the electrode, hydrogel entrapment, and biofilm formation, etc. [68].
Enzymes
The enzymes are biocatalytic biomolecules, which can enhance the biological reac-
tion and also liberate the measurable product. During the biocatalytic reaction, the
intermediate product to the final product formation process is easily monitored
through the transducer, electrochemical transducers (amperemeter, voltammeter,
etc.).

6 Challenges

There are still many challenges to developing a special type of biosensors, such as
particular virus-detection biosensors. The virus is a tiny biological entity capable of
destroying human body cells. Its detection at an early stage of infection has not been
very much possible as yet, primarily because the affected person does not realize
the asymptotic symptoms. However, early diagnosis of virus infection/biochemical
disbalance/bacterial infection, etc. can be detected through in-vivo biosensor, and
1 Nanomaterials Based Biosensing: Methods and Principle … 21

Fig. 15 Schematic showing different cell types obtained (left), and functional strategies of cell-
based biosensors (right) (reprinted with permission) [68]

it will be a milestone in the clinical sector. However, the materials currently being
utilized in making biosensors are foreign materials for the human body and their
acceptance into the human body is still a challenge. Therefore, the utilization of
materials in biosensing needs to be compatible with the human body, or the whole
biosensor must be biological. Also, many biosensors are complicated and required
to be stored at low temperatures. Therefore, the new technology needs to develop
sensors that can be stored and operated at room temperature.

Research Data statement

Data sharing is not applicable to this article as no new data were created or analyzed
in this study.

Acknowledgements Authors are very much thankful to the Research support provided by
the UKRI via Grants No.: (EP/L016567/1, EP/S013652/1, EP/S036180/1, EP/T001100/1 and
EP/T024607/1), Royal Academy of Engineering via Grants No. (IAPP18-19\295, TSP1332 and
EXPP2021\1\277), EU Cost Actions (CA15102, CA18125, CA18224 and CA16235) and Newton
Fellowship award from the Royal Society (NIF\R1\191571). SG is particularly thankful to European
Regional Development Funds (ERDF) sponsored A2i project at LSBU that has catalyzed several
industrial partnerships. The work used Isambard Bristol, UK supercomputing service accessed by
Resource Allocation Panel (RAP) grant as well as ARCHER resources (Project e648).
22 N. K. Katiyar et al.

Questions and Answers to Check Your Reading

Objective Types

(1) Surface plasmon resonance belongs to which type of biosensor?

(a) Transducer (b) Recognition system

(c) Signal Amplification (d) none

Ans: Transducer

(2) What is an aptamer in a biosensor?

(a) Transducer (b) Recognition system

(c) Signal Amplification (d) none

Ans: Recognition system

(3) Please fill in the blank. An analyte can be detected through ___________working principles.

(a) one (b) two

(c) three (d) many

Ans: Many

(4) A nanoscale material is defined to have its _______ ?

(a) one dimension <100 nm (b) two dimensions <100 nm

(c) three dimensions <100 nm (d) All dimensions <100 nm

Ans: one dimension <100 nm

Subjective Types
(5) What is a biosensor?
A biosensor is an analytical device, which is successfully used for the qualita-
tive and quantitative estimation of several biologically important substances.

(6) What are the various types of biosensors?

Biosensors can be classified by their transduction mechanisms such as Electro-
chemical, Piezoelectrical, Thermometric, Optical, and Physical; one can also
1 Nanomaterials Based Biosensing: Methods and Principle … 23

classify them by the mode of biorecognition, e.g., enzyme-based, antibody-

based, nucleic acid-based, aptamer-based and whole cell-based. Also, the
transducer-based classification can be further sub-divided. For example,
electrochemical biosensors can be amperometric, potentiometric, conducto-
metric, etc. In addition, biosensors can be defined by both the recognition
mode and transducer, e.g., enzyme-based electrochemical biosensors or, more
specifically, enzyme-based potentiometric biosensors.

(7) Describe the main components of biosensors and explain their function.
The biosensor consists of a biorecognition element, transducer, amplification,
and display.
The biorecognition system detects the analytes; the transducer converts action
of recognition system into the signal; the amplification enhances the signal
strength to read easily; display shows the result.

(8) Define precision and selectivity for a sensor.

Precision: The ability to have high reproducibility upon the repetition of
measurements of the same sample. The sensor has good reproducibility of
its response (low standard deviation).
Selectivity: The sensor performance towards targeted analytes. A
sample has many chemical/biomolecules, but sensor detects particular
analytes/biomolecules, for what it is tuned.

(9) What are nanomechanical biosensors (cantilever biosensors)? How have these
been used for biosensor development? How can the biorecognition event be
transduced?
These are microfabricated finger-like transducers that can bend/oscillate
(depending on the material) as a result of a physical/electrical stimulation. The
bending/oscillation of these devices is proportional to their mass. Cantilever-
based biosensors have been used for hybridization assay, for the screening of
the effect of drugs on the growth of bacteria, for affinity (antibodies) based
assay by immobilizing on the surface of the cantilever DNA probes, drugs
or antibodies. Transduction can be optical or piezoelectrical. In optical, the
reflection of a laser beam is used to evaluate the bending of the cantilever. In
the case of piezoelectrical changes in the oscillation frequency of cantilever
(made with piezoelectric material) are used to detect changes in the mass onto
them.

(10) What is an enzyme? What is the function of an enzyme, when used as a

biorecognition element?
The enzymes are protein and act as a biological catalyst. In the biorecognition
system, it is attached to the transducer and catalyzes analyte conversion faster
and efficiently. Correlate the analyte to a measurable event (loss in a substrate
or generation of a product).
24 N. K. Katiyar et al.

(11) What are the two main problems associated with the use of the Oxygen
electrode (Clark electrode) as a transducer in the glucose electrochemical
biosensor?
This is dependent on the starting concentration of oxygen in the starting solu-
tion. Use high potential: sensitive to interference from other molecules. The
electrochemical reaction consumes oxygen (this can produce false positive).
(12) What are the main advantages and disadvantages of the transducer’s func-
tionalization via the adsorption process instead of covalent bonding?
Physical adsorption is a simple and easy process. However, pH variation and
other analytes in the sample create interference in the signal, which appears
as noise and reduces the probability of accurate detection.

Some further unsolved problems:

(1) Explain a label-based and a label-free biosensor.
(2) What is meant by spectral interrogation schemes in LSP sensors?
(3) Describe Glucose biosensors. First, second and third generation of ampero-
metric sensors.
(4) Achieving selectivity and sensitivity in SPR biosensors. Immobilization
strategies in SPR. Improving signal for small analytes.
(5) Describe applications of carbon nanotubes and nanowires for biosensing.

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Chapter 2
Pathways to Translate the Biomedical
Prototypes

Tamanna Bhuyan, Surjendu Maity, Devi Rupa Saha, Nayan Mani Das,
and Dipankar Bandyopadhyay

1 Introduction

Recent decades have witnessed remarkable technological advancements in healthcare

devices that have been responsible for early and precise diagnosis, more successful
treatments, and helping patients to lead healthier lives [1, 2]. However, because
new technology primarily drives healthcare expenditures and product risks against
benefits, a careful evaluation of primary critical paths, including product initiation,
formulation, design, final validation, and product launch, is increasingly necessary
for clinical practice [3–5]. The target audience for these activities is scientists, clin-
icians, and technologists to translate novel medical technologies from academia to
the market [6, 7]. The global medical device industry is significantly technology-
driven and progressing rapidly. However, in India, it is still in the nascent stage with
restricted levels of availability of infrastructure, penetration, lack of trained human
resources, adoption and out-of-pocket expenditures [8–10].

T. Bhuyan · S. Maity · D. R. Saha · N. M. Das · D. Bandyopadhyay (B)

Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam 781039,
India
e-mail: [email protected]
S. Maity
e-mail: [email protected]
D. R. Saha
e-mail: [email protected]
D. Bandyopadhyay
Department of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati,
Assam 781039, India
N. M. Das
Department of Physics, Sipajhar College, Darrang, Assam 784145, India

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2022 29
S. N. Joshi and P. Chandra (eds.), Advanced Micro- and Nano-manufacturing
Technologies, Materials Horizons: From Nature to Nanomaterials,
https://doi.org/10.1007/978-981-16-3645-5_2
30 T. Bhuyan et al.

Currently, the Indian healthcare industry is significantly import-dependent,

comprising only 1.7% of the world market, and valued at approximately USD 10
billion. Over the past few years, the Indian government has raised public expenditure
on health to 2.5% of the Gross Domestic Product that has substantially registered
a compound growth rate of 25%. As per the market evaluations, the Indian health-
care industry comprises over 800 manufacturers, of which 65% of companies having
a turnover of USD 1.5 million. Interestingly, 2% of the company’s turnover was
measured to be more than USD 73 million, which has been expected to reach close
to USD 25 billion by 2025 [11–14]. This presents an exciting opportunity to under-
stand the nature of this industry and to play a considerable role in providing quality
universal healthcare to the nation.
The path for transforming an idea into a user-centered medical product is essen-
tially based on multiple factors, including basic research, invention, patent applica-
tion, additive manufacturing, clinical testing, market trends, capitalization, regulatory
requirements and commercialization [15–22]. The roadmap for prototype translation
toward product commercialization comprises of six consecutive stages: (i) Initia-
tion and risk analysis, (ii) formulation and feasibility, (iii) design and development,
(iv) verification and validation, (v) product launch, and (vi) post-launch assessment
[23–25].
The six stages of lifecycle to develop and commercialize user-centric medical
device has been schematically represented in Fig. 1. Employing such systematized
approaches ensures the development of effective and well-designed medical devices
that are in tune with medical practitioners, patients, and lay caregivers. Earlier,
companies adopted the licensing intellectual property when the prototype was still
in the research phase. However, in recent years, the assessment of preclinical data
has been made mandatory by the venture capital groups to minimize their risks
before investing [15]. The funding agencies for laboratory research largely support
hypothesis-driven studies and experiences high commercial risk. On the contrary,
major granting agencies undergo relatively lower commercial risk while translating
prototypes from the clinical-stage toward marketing [26–28].
For these reasons, planning ahead for translation is the finest way to obviate
costly downfalls that can endanger long-term profits [29, 30]. Currently, numerous
companies are familiar with the effects of prototype translation, while they are also
curious to understand the intricacies of such transformations toward medical device
industries for developing clinically effective devices [31, 32].
In view of the commercialization complexities that lead to many innovations being
“lost in translation”, this chapter unpacks an overview of the methodical translational
strategies to drive promising biomedical prototypes from laboratories to market.
The chapter introduces the current technological trends of several commercialized
biomedical sensors outlining their basic working principle, component-based clas-
sification and their impact on the healthcare industry. This chapter allows a better
understanding of the strategic planning required to design medical devices for early-
stage valuation and implementation. The chapter also discusses the rigorous testing
needed for passing clinical trials, premarket approval process, post-market surveil-
lance procedures and other essential regulatory regimes of the Indian medical device
2 Pathways to Translate the Biomedical Prototypes 31

Fig. 1 The six stages of lifecycle to commercialize the user-centric medical device

industry. Through this discussion, we also highlight the gaps and challenges that exist
between universities, industry partners and medical practitioners, and articulate the
role of government to drive medical devices growth by combating the difficulties
with prototype translation.

2 Diagnostic Biosensors: An Overview

Since the discovery of the first glucose biosensor in 1962, biosensors have progressed
tremendously, leading to their vital use in almost every discipline in today’s world [33,
34]. In the past two decades, several integrated biosensors have led to the formation of
sophisticated medical devices, which has increased the early detection of infections,
decreasing the patient mortality rate and encouraging the researchers to imbibe more
advanced technologies in the domain of biosensors [35–37].
(i) Technological Breakthroughs
Over the past few decades, the discovery of biosensors has gained tremendous atten-
tion in the field of healthcare devices. An overview of the advancements in biosensors
technology till date has been summarized in Table 1 [38–45].
32 T. Bhuyan et al.

Table 1 A summary of a few reported biosensors

Serial No. Year Events
1 1962 First electrode-based biosensor for glucose detection present in biological
samples
2 1970 Discovery of ISFET (Ion sensitive field effect transistor) based biosensor
3 1983 First SPR (surface plasmon resonance) based immunosensor
4 1984 Amperometric biosensor mediated with ferrocene for detection of glucose
5 1992 First hand-held-based blood biosensor
6 2008 Aptamer-antibody-based chip immunoassay to detect C-reactive protein
present in spiked serum
7 2013 Detection of (HER2) human epidermal growth factor receptor-2 found in
patients with breast cancer using aptamer-based electrochemical
nanobiosensor
8 2014 Silicon-based optical biosensor for cancer therapy
9 2015 Detection of CTCs (circulating tumor cells) in case of breast, melanoma and
prostate cancers using cluster chips
10 2016 Detection of blood glucose non-invasively using low powered lasers-based
wearable biosensors
11 2017 Detection of glucose using ZnO (zinc oxide) nanorods on FTO (fluorine
doped tin oxide) based biosensor
12 2018 Detection of prostate cancer biomarkers using gold nanorods-based
electrochemiluminesence (ECL) aptasensors
13 2019 Functional tattoos to detect concentration and pH of albumin and glucose
present in human serum
14 2020 Detection of CTCs (circulating tumor cells) present in patients suffering from
neck and head cancer using cellulose nanocrystals magnetized by iron oxide
NPs (nanoparticles) based biosensors

(ii) Components of Medical Biosensors

A diagnostic biosensor typically comprises an analyte, bioreceptor, transducer, elec-
tronic system, and a display, as presented in Fig. 2. Realizing the appropriate role
of every biorecognition element during the preliminary developmental stages is
necessary to promote novel biosensor technology’s clinical success. Choosing the
biorecognition elements that are sensitive to the bio-analyte concentrations facilitates
the detection of diseases at an early stage [46, 47]. The performance of the biosensing
elements depends on factors like transport and storage conditions. Therefore, in the
initial design phase, it is vital to analyze the target bio-analyte and its corresponding
biosensor utilization because an appropriate bioreceptor selection will contribute to
the development of efficient biosensors [48, 49]. An early emphasis on the clinical
trials during the biosensor development stage has the potential to ameliorate the
accessibility of patient-centric medical devices in low-resource regions.
2 Pathways to Translate the Biomedical Prototypes 33

Fig. 2 Components of a diagnostic biosensor

The success of innovative biosensor development depends on an overall biosensor

performance and requires a crucial understanding of selecting an optimal biorecog-
nition element in the preliminary design stage. There are numerous biorecognition
elements that range from naturally occurring to synthetic constructs [47, 50]. The pros
and cons of selecting natural, pseudo-natural and synthetic biorecognition elements
have been outlined in Fig. 3.
34 T. Bhuyan et al.

Fig. 3 Selecting various biosensing elements

(iii) Biotransducer-Based Classification

Biotransducers are components in the biosensing system that determines the stim-
ulus discharged from the analyte-bioreceptor interactions and generates an electrical
signal following electrochemical, mass, or temperature changes [51, 52]. Transducer-
based biosensors are categorized based on their physicochemical transduction and
are mainly of four types: electrochemical, optical, mass-based, and calorimetric, as
shown in Fig. 4.
(iv) Biosensors and Healthcare
Over the past 60 years, the contributions of the biosensors have provided a greater
outlook to develop highly multi-functional, and low-cost point of care testing devices
to measure the medication records taken by patients. The capability of such devices
to provide rapid real-time testing have not only allowed the patients to evade labor-
intensive medical laboratory analyses but also have assisted the clinicians in exam-
ining minute details of the patient’s health [53]. In the upcoming years, further
advancements in detecting methods will lead to the design of highly sensitive sensors
for disease-related sensing of biomolecules. Conclusively, it could be realized that
design and development of real-time biosensors, particularly microfluidic integrated
biosensors, photonics-based biosensors, and biochip integrated biosensors, would
improve the efficacy of translating healthcare innovations, thus facilitating rapid
estimation of sample and accurate testing [54–57].
 2 Pathways to Translate the Biomedical Prototypes

Fig. 4 Classification of transducer-based biosensors

35
36 T. Bhuyan et al.

3 Planning for Device

Transforming a laboratory-based prototype into a marketable device requires some

vital and systematic planning. It is noted that the healthcare devices are directly
involved with monitoring and saving human lives as well as improving the lifestyle.
Therefore, the focus has been shifted toward meeting the industrial standard with
high accuracy, and low interference that is possible by managing strategic planning
ahead of time [58]. The process of strategic planning for prototype translation is
described below.
(i) Translate Ideas to Invention
It is crucial to select the idea around the laboratory-based prototype. For that, one
must survey the problems associated with the society or community. For example,
the survey on the detection of diseases or any time-taking biomarker analysis through
blood or urine may give you the clues for the new ideas, which might be transformed
into future inventions [59–61].
(ii) Risk Analysis of Product
Once the idea or target is fixed, the risk analysis of the product becomes a pivotal
factor in giving the idea a green signal. A detailed market survey should be done
to analyze the potential of the device. For example, the idea must be unique, cost-
effective, less time-consuming, and highly accurate. The product must have a sellable
market, or the market should be created by spreading the awareness of the device.
Errors related to medical devices create a genuine risk to patients that can be reduced
through understanding and evaluation of device-related events [62–64].
(iii) Prototyping
The prototype should be protected by filing for Intellectual Property (IP) and patent
the idea after finalizing the idea and completing the feasibility of the product in the
market. This step would provide the developer with legal freedom from the competitor
to develop the product. In India, the patent filing trends in the medical device domain
has roughly doubled with 1104 filings in 2014, as against 640 filings in 2005. This
specifies the progressive and consistent IP awareness and strategies of the foreign
manufacturers to capitalize on the flourishing Indian medical device industry [63].
(iv) Laboratory Research to Prototype Development
The planning should be conducted thoroughly after analyzing the risk and
marketability of the product. This step would provide information regarding the
clear pathway to develop the laboratory-based product through proper research work.
Besides, it reveals all the parameters associated with the development of the device
and where the optimizations are required. The involvement of all kinds of accessories
such as instruments, chemicals, reagents is discussed in detail. Once such searches
have been performed and anticipate to protect and follow the idea, the project and
preclinical research have to be separated from fundamental research projects keeping
it confidential until submission of patent protection.
2 Pathways to Translate the Biomedical Prototypes 37

(v) Formulating Budgets and Time Chart

Formulating budgets is a very crucial entity for the development of any product.
Allocation of money should be planned diligently to maintain the seamlessness of the
development. For example, all the reagents, chemicals, electronics, and instruments
should be bought with the allocated money. A time chart should be formed, and
the weekly targets must be fixed for the entire time frame. The headroom approach
holds promise for rational planning of business decisions, financial sustainability
with commercial negotiations based on timing along the development cycle for a
new product [65].

4 Initial Implementation Stages

Once the developer is wholly convinced with the product, which has the market to sell
and is financially viable, only then they go forward with the initial implementation
stage. This stage consists of writing a project for funding to set up protocols for
product-related developments. The linear stage-gate model provides a comprehensive
elucidation of the activities related to the development of medical devices. Some of
the parameters are listed below.
(i) Funds
At this stage, writing the project report is the most critical work to do because it
would attract funding for development. The detailed description of the device, patent
or IP document, the working principle, and market survey might help the report to
be more approachable to the funding agencies. Besides, the strong partnerships may
be formed by signing memorandums with other companies for the developments, if
needed.
(ii) Potential Revenue
A detailed report must be prepared to project on the potential revenue by selling the
products. This may provide ideas toward the sustainability of the company and the
product. Additionally, it articulates the potential market available for the device. An
estimation of the potential commercial viability of medical devices can be carried
out by following the Headroom approach combined with return on investment [66].
(iii) Regulatory Compliances
A proper investigation must be conducted for the regulatory compliance on the
medical product or healthcare device, which are being developed. A medical device
or product should pass all the rules set by the governing authority. Failing to do so
may compromise the future of the device and deny the license to market the inven-
tion. Thus, a detailed study on regulatory compliance is required to ensure safety for
the patients. Additionally, it may provide the required approval from the regulatory
authority and conduct the audit to go smoothly [67].
38 T. Bhuyan et al.

(iv) Quality Management System (QMS)

QMS is the foundation of any company, which provides an excellent base to develop
everything on it. ISO 13,485 is the internationally recognized standard and adopted by
most of the medical device companies such as Johnson & Johnson, Novartis AG, and
Abbott Laboratories. The QMS would help to standardize the procedure, forms, docu-
ments and templates that would cover all activities throughout the product develop-
ment cycle, including product design, manufacturing, risk management, addressing
the complaint, clinical data storage, loading and supply, among others [68].

5 Design and Development

(i) Traceability of Device Design

A strategic approach of designing is followed while doing innovation, which at large,

diverges, way beyond the concept of the product design in the context of strategy and
management. Systematic management of skills, works power, workman-handling,
documentation, organization, investments reliability, etc., is the part of this strategy
while looking at the cutting edge approaches and developments in the strategic design
deals in the case of medical devices.
(a) Strategic Design and Development of Product: Inspiration, ideation and
implementation are the main three spaces of the new business plan that can be
developed thinking a strategic plan. Looking at the parameters, the strategic
design possesses complex learning and thinking to solve a mainland problem
with a correct rational approach. Thus the strategic design becomes a strategic
practice, and the practitioners or the researchers literally deals in designing a
practice. An organization now aim to perform the strategic plan keeping a view
on the problem assigned to achieve “what” it is and “how” it is, which uplifts
design or rather a practice, up to a strategic path than mere thinking of a purely
tactical approach, hence the emergence of strategic design [69, 70]. Thus a
strategic design in the case of a medical device does accumulate the feasibility,
desirability, viability, design practices, principles and tools at extending while
implicating the complete design [71–73].
The Gartner’s Hype Circle: Following the strategic plan for the innovation
through research, the scientist found the best way to deal with the strategy and
that is determined by what is called the Gartner’s hype circle [74, 75]. The study
says that the hype circle seems to be very well managing while following the
higher-level concepts of device program such as IT methodologies and manage-
ment disciplines. In general, the Hype circles produce innumerable numbers
of plan circles to track the concerned innovation maturity and future potential.
Hype circles manage the decision to implement the typical progression of inno-
vation by maintaining the trends and ideas, strategies, standards, management
concepts, competencies and capabilities, as shown in Fig. 5.
2 Pathways to Translate the Biomedical Prototypes 39

Fig. 5 The Gartner’s hype

model

(b) Defining Design Inputs: Once the strategic design is confirmed, then comes
the design input. In general, the platform where the academic push and the
industrial pull starts operating (Fig. 5) for satisfying the goal of innovation
and marketing, respectively. The academic push is concerned with mostly
the design, prototype, manufacturing and product engineering. The lab-based
research work is now ready for device makers, and the designers or the archi-
tect starts thinking about the design. They undergo a lot of hits and trials in
designing a perfect geometrical shape and ready to get hold of the next step of
prototyping. The prototyping also needs a lot of trials, although less than the
design part and finally comes with device product manufacturing with perfect
engineering [76]. The design inputs go hand in hand with the development.
Development is the stage where the ideas cherished are transformed into defi-
nitions and then turned into geometrical shapes. Design and development all
stay with the proper market concerns, and they must be flexible to be imple-
mented according to the relevant prevailing concepts and information. In this
regard, physicians or medical practitioners can come up with really valued
ideas to be implemented or incorporated once the design and development
process is carried out.
The practitioners are dealing with the real public, and within the area of exper-
tise, they are exposed to vivid ideas and shortcomings. The physicians repre-
sent the customer, and engineers implement the design engineering according
to their needs, and hence the practitioners are media of data generation and
the engineers innovate and develop based on real facts and requirements. Thus
collecting knowledge of years of experience from the medical practitioners
can deeply impact the device design and manufacturing inputs. We can now
generalize that the device input and performance characteristics are used as the
basis for device design, as represented in Fig. 6.
 40

Fig. 6 The mind-map of the medical device concept

T. Bhuyan et al.
2 Pathways to Translate the Biomedical Prototypes 41

We may define the design inputs as the supersets of the following subsets,
which are:
• Ensure requirements are appropriate by addressing user needs and intended
use in terms that are measurable.
• One needs to address incomplete, ambiguous, or conflicting requirements.
• The document, review and approve input requirements.
• Lastly, Human factors.
(c) Specifying Design Outputs: The design outputs are the fruits that are gained
after the successful implementation of strategic design, design input and devel-
opment. These are all at a time come out with the assemblies and subassemblies
of thoughts, components, parts and pieces. A successful device output is a care-
fully executed mission of assembled ideas and implementing them step by step
with proper documentation. A similar approach has been demonstrated as a
case study of translational research, as shown in Fig. 7. These documents are
clearly abbreviated and further explaining the new user with proper guidance
and exact work streaming to redesign the same with repetition. Such a docu-
mented idea leads to the basis of the device master record (DMR). Thus the
DMR stands alone as the complete recipe for the new device. Further, the docu-
mented literature about the device output is described very well in the FDA
21 CFR Part 820.30(d) and ISO 13485:2016 7.3.4 state about Design Outputs
[77].

Fig. 7 The case study for translational research

Other documents randomly have
different content
 — Sinä et vastaa siihen mitä kysyin. Sinä tahallasi vältät sitä. Sinä
tiedät sen jo. Ehkäpä se roisto jo on kihloissa!

— Mutta rakas Jenny, piika kuulee, muista ettei se enää ole

Maali… En minä mitään tiedä. Hautajaisissa vain oli muuan tyttö,
suuren talon tytär, kansanopiston käynyt, joka tavattoman selvästi
osoitti ihailunsa Toivoon ja kuiskailtiin, että se taitaa pyrkiä Lainan
paikalle. Se leikki koko illan lasten kanssa…

— Se hävytön…! purskahtaa Jenny täti puhumaan. — Se hävytön!

Mutta sehän on julkeata! Ruveta jo Lainan hautajaisissa… Mutta
minä sanon sen, että minun täytyy matkustaa sinne varjelemaan
lapsia siltä ilkiöltä… Jaa, minähän en voi, kun minun täytyy ottaa
tuntini taas, mutta Hilja saa lähteä. Mitä hänellä täällä on tekemistä.
Sopii hyvin, että hän pakenee pois ihmisten hampaista.

— Voi, rakas Jenny, kuinka vähän sinä tunnet elämää. Jos Toivo
tahtoo mennä naimisiin, niin mitä luulet meidän voivan tehdä
hänelle! Mitä sinä voit Signelle ja tohtorille…

— Sehän on ihan eri asia. Kaikki sinä sekoitat. Minä olisin voinut
erottaa heidät, jos olisin tahtonut. Minä en tahtonut, sillä tohtori on
kelpo mies ja konsulinna antoi suostumuksensa.

— Mutta, rakas Jenny, älä nyt suutu noin. Piika kuulee.

— Jaa, se on kanssa kaunis juttu, että Maali on poissa. Kyllä täällä

on ollut kaunis elämä keittiössä sillaikaa.

— Mutta kyllä Maalikin viime aikoina oli käynyt sietämättömäksi.

Hän oli hermostunut aivan yksinkertaisesti. Nykyaikana on
palvelijoillakin hermot.
 — Älä ensinkään puolusta Hiljaa. Sellaista elämää ei millään voi
puolustaa… Voi, Herra Jumala sentään…

— Älä nyt joka toisena sananasi sano "Herra Jumala".

— Mutta minä avuksihuudan Häntä! Tämä ei ole mitään

turhaanlausumista. Jollet sinä anna minun puhua rauhassa, niin
minä lähden… Kuinka sinä olet noin kauhean äreä!

— Etkö sitten luule, että minulla on ollut kestettävää…

— No niin, minä tiedän… Kerro nyt Lainasta. Kun en minä

ensinkään tahdo ymmärtää, että se on totta… Eikö Laina sitten
ennen kuolemaansa pyytänyt ehtoollista, tai osoittanut edes joitakin
merkkejä, että ikävöi sovitusta Jumalan kanssa?

— Ei. Sehän se juuri on surkeaa. Ensimmäisen päivän hän itki

lasten takia, itki niin, että sitä oli kauhea katsella. Mutta sitten hän
tyyntyi ja kovettui. Hän ei voinut ymmärtää Jumalan tarkoitusta eikä
nöyrtynyt ottamaan vastaan kohtaloaan sitä ymmärtämättä. Kyllä se
oli surkea kuolema. Niin nuori kun hän oli ja niin kovettuneena
nukkui pois. Toivo puhui ja Toivo veisasi kauniilla äänellään. Ei
mikään auttanut. Hän vain pyysi häntä menemään pois. Toivo oli
onneton. Hän pelkäsi todella, että Laina joutui pahaan paikkaan,
helvettiin. Lainan kasvoihin jäi ruumiinakin sellainen outo kovuus.
Kuolleiden kasvoilla voi olla niin kaunis rauha, niinkuin kaikkien
meidän kuolleiden on ollut. Mutta näissä oli jotakin aivan toista. Se
lääkäri, joka tuli kaupungista ja ehti perille vasta Lainan kuoltua,
seisoi pitkän aikaa ääneti ja katseli kuollutta. Se oli vanha mies ja
nähnyt monen kuolevan. Minä kysyin häneltä, oliko hän ennen
nähnyt sellaista ilmettä kuolleilla. "Joskus", hän sanoi. "Se on
kysymys, joka on jäänyt vaille vastausta." Ehkä se oli niin.
 — Mutta sehän on kauheata! Hän oli siis ihan kääntynyt pois
Jumalasta?

— Jollei Jumalalla nyt sittenkin olisi armoa sellaisillekin…

— Ei ole! Sehän on niin selvästi sanottu.

— Kun minusta sittenkin tuntuu, että on…

Sisarukset itkevät. Kello käydä raksuttaa. Ulkoa katsoo

punaraitaisten uudinten takaa kylmä, säteilevän kirkas päivä.

— Oliko hän paljon muuttunut?

— Hän oli käynyt ihan samanlaiseksi kuin hän oli ennen tyttönä.
Kaunis hän oli arkussaan. Me puetimme hänet hänen
morsiusvaatteisiinsa. Ne olivat olleet lakanan sisällä siitä asti kun hän
ne hääiltana riisui. Ne olivat kuin uudet. Kaunis hän oli, paitsi että
kasvoissa oli se sellainen sovittamaton kysymys. Pitäjäläisiä kävi
joukoittain häntä katsomassa. Toivo kustansi kalliin arkun,
kalleimman minkä voi saada. Helat ja levyt olivat puhdasta hopeaa.
Siitä puhuttiin pitkin pitäjää.

— Voi Herra Jumala sentään, etten minä koskaan saa nähdä

Lainaa! vaikeroi Jenny täti ja vuolaina vuotavat sisarusten kyyneleet.
— Mutta kuka saattoi aavistaa, että Hilja tuottaisi meille tällaisen
häpeän! alkaa Jenny täti äkkiä. — Kuka sitä olisi uskonut, kun
viimeksi istuimme täällä yhdessä. Että sinun, hänen äitinsä, pitää
istua rautatievaunussa kuuntelemassa, kuinka vieraat ihmiset
puhuvat hänestä mitä häpeällisimpiä asioita. Mitä he oikeastaan
sanoivat?
 — Johan minä kerroin, en minä jaksa niitä toistaa. He tulivat
sisään päivälliseltä Riihimäellä — kaksi keski-ikäistä herraa, ja
rupesivat puhumaan Hiljasta ja hänen suhteestaan Mataristoon.

Jenny täti kiehui kiukkua.

— Minä olisin sinun sijassasi noussut, mennyt heidän luokseen,

sanonut kuka olen ja antanut heidän kuulla kunniansa. Sinä kuuntelit
kiltisti, sinä!

— Onneksi! Siitähän olisi tullut täydellinen skandaali, jos minä

vielä olisin ruvennut sotkemaan.

— Mistä he sitten tietävät, ettei Hilja ole kihloissa?

— Eihän hän kanna sormusta.

— Onko koko maailma sitten ehtinyt huomata sen!

— Tiedäthän, kuinka huhut leviävät.

— Mutta mitä Hiljan kihlaus kuuluu kehenkään! Mitä oikeutta

kenelläkään on sekaantua meidän asioihimme?

— Sinä puhut kuin suuri lapsi! Mitä oikeutta? Sama oikeus kuin
poimia marjoja toisen metsästä tai herneitä toisen hernemaasta, jos
se vain on tarpeeksi kaukana omistajan asunnosta.

Sisarukset vaikenivat. Rouva Haapanen hypisteli jotakin Jenny

tädin tuomisista, mutta ajatteli toisia asioita.

— Mitä sinä kehoitit minua lähtemään ulkomaille! tuli Jennyltä

taasen kuin putouksena. — Jollen olisi lähtenyt, olisin kyllä vartioinut
Hiljaa. Ja jahka minä saan sen Matariston käsiini, niin kyllä hän
kuulee. Täällä hän esiintyi kuin viaton lammas ja vei sitten koko
perheen kunnian. En kehtaa enää näyttää silmiäni kadulla. Lydiakin
kohtelee minua suorastaan halveksien. Vaikka hänellä itsellään on
paljon syytä. Hän oli Helsingissä, hän olisi saanut pitää Hiljaa
silmällä.

— Minun täytyy vielä käydä Bredellä. Se ei ole hauskaa se.

— Ei ole, myönsi Jenny täti. — Kai minunkin pitäisi mennä sinne,

mutta koko kylpymatkani menee hukkaan, jos nyt saan
mielenliikutuksia. Liian aikaiseen me ylpeilimme Armaan hienoista
sukulaisista.

— Kyllä Armaskin olisi saanut odottaa kunnes oli tavannut Hiljan.

— Kuka heistä oikeastaan rikkoi välin?

— En minä tiedä, vaikka Hiljan äiti olenkin. Hilja ei sano mitään.

Hänhän on niin muuttunut.

— En ymmärrä Hiljaa. Hän ottaa asian aivan tyynesti. Häntä ei

edes hävetä, vaikka hän tietää, mitä hänestä puhutaan.

— Häntä ei hävetä, kun ei hän kadu. Siinä se on! Hän sanoo, että
jollei Armas välittänyt hänestä sen enempää, niin oli parasta, että
meni näin.

— Sen hän voi sanoa! Ei mikään mies kärsisi sellaista.

— Armas olisi saanut odottaa kunnes olisi tavannut Hiljan, sanon

sen vielä, toisti äiti.

— Sinä siis luulet, että Armas purki?

 — Mistä minä tiedän. Ja onhan se jokseenkin yhdentekevää.

— Taas sinä kiukustut! Ehkä minä lähden nyt. Voinhan syödä

aamiaista vaikka…

— Että sinä nyt taas viitsit. Meillä on sieniomelettia. En tosin tiedä

miten tämä uusi "neiti" osaa sitä valmistaa, mutta…

Tuli taas äänettömyyden hetki, jonka aikana kumpikin sisaruksista

yritti käydä käsiksi joihinkin tavaroihin.

— Saisin minä sen Matariston käsiini! uhkasi Jenny täti äkkiä.

— Hilja vain kehuu häntä, sanoi äiti. — He ovat yhä

kirjevaihdossa.

— Mitä sinä sanot!

Jenny täti kimmahti pystyyn.

— Eilenkin häneltä tuli kirje ja Hilja kiirehti itse asemalle viemään

siihen vastausta.

— Ja sinä sallit sen tapahtuvan! huusi Jenny täti, silmät suurina

päässä.

— Mitä minä sitten sille voin.

— Etkö sinä olekaan Hiljan äiti? Sinulla on oikeus ottaa haltuusi

hänen kirjeensä, avata ne tai lähettää ne avaamattomina takaisin.

— Mitä sinä puhut! Ei nykyajan tyttöjen kirjeitä avata.

— Mitä se mokoma sitten kirjoittaa?

 — Mistä minä tiedän. Hilja menee omaan huoneeseensa
lukemaan.

— Jollei sinussa ole äitiä saamaan selville mitä se roisto — en sano

paremmin — vielä uskaltaa kirjoittaa, niin kyllä minä otan selvää.
Missä Hilja säilyttää kirjeet?

— Laatikossaan. Olen minä ne joskus nähnyt pöydälläkin.

— Etkä ole lukenut!

— Mutta, rakas Jenny, näethän itse, miten Hilja on vanhentunut.

Kyllä hän on kärsinyt tarpeekseen. Mitä minä nyt rupeaisin
urkkimaan hänen kirjeitään. Eihän mitään enää voi muuttaa. Huhut
ovat kuin irti päästetyt linnut. Ota nyt metsästä lintu kiinni!

— Aikooko Hilja sitten nyt mennä kihloihin tämän Matariston

kanssa?

— Hän sanoo, ettei.

— Mitä he sitten aikovat? Mitä tämä kaikki on ollut?

— Hilja sanoo sitä ystävyydeksi.

— Ystävyydeksi!

Jenny täti lausui sanan ihmeissään, ikäänkuin olisi kuullut sen ensi
kerran eläessään.

— Niin, niin, niinkuin hän ja Elsa ovat ystävät! Tai sinä ja minä.

— Onko hän hullu! huusi Jenny täti. — Sinä ja minä olemme

sisarukset ja Elsa on tyttö — mutta nuori tyttö ja nuori herra, jotka
eivät ole edes sukua! Tiedätkö, sinä olet sinäkin syypää Hiljan
kevytmieliseen katsantokantaan. Minä sanoin sinulle aina: pidä kiinni
ovet eteiseen, koska sen ylioppilaan pitää kulkea sitä tietä. Äläkä
anna Hiljan rajattomasti viipyä tansseissa. Sinun kasvatuksesi on
ollut leväperäistä!

— Loppujen lopuksi kaikki on minun syyni! sanoi Aline rouva ja

nousi suuttuneena.

— Minä en sanonut niin! Jos tahdot ymmärtää sen niin, niin tee se
vain, mutta minä en sitä sanonut!

Jenny nousi hänkin ja meni Hiljan kamariin. Hänen täytyi istuutua

tuolille itkemään. Yhtä paljon kuin hän viime keväänä oli surrut sitä,
että Hilja antoi rakkautensa Armaalle, yhtä paljon sattui häneen nyt
Armaan kadottaminen. Huone puhui selvään, että Armas oli
pyyhittävä pois Haapasten suvusta. Kaikki kauniit taulut, kaikki
hienot koristeet, yksin lamppukin olivat poissa. Huone oli karu ja
autio. Itkettyään aikansa Jenny täti pyyhki silmänsä ja huusi
ruokasaliin:

— Missä Armaan lahjat ovat?

Hän sai toistaa kysymyksen pariin kertaan ennenkuin Aline vastasi.

Hän ei ollut vielä unohtanut äskeistä pientä kahakkaa.

— Kopassa vinnillä, vastasi hän vihdoin.

Jenny täti meni kirjoituspöydän luo. Siellä oli Lainan kuva

rippikouluajoilta ja sen yläreunaan pistettynä vielä tuore
sypressinoksa. Se oli tietysti hautajaisseppeleestä. Jenny tädin
kyyneleet alkoivat taasen valua. Ja kaikki Armaan kuvat poissa! Ja
kaunis venetsialainen maljakko poissa! Äkkiä hänen silmänsä sattui
kirjepinkkaan, jonka päällimmäisen kirjeen ympärillä oli halpa,
harmaa kotelo. Käsiala oli selvä ja tavallinen. Hän ei hetkeäkään
häikäillyt. Hänen kyyneleensä kuivuivat, jännityksen ja uteliaisuuden
ilossa kävi hän käsiksi kirjeeseen ja näki sen olevan juuri sen, jota
hän halusi nähdä. Alla oli "ystäväsi Matti" — se roisto uskalsi vielä
nimittää itseään Hiljan ystäväksi! Kaiken sen jälkeen mitä oli
tapahtunut! Sen jälkeen, että hän oli ryöstänyt kunnian kokonaiselta
perheeltä ja Hiljalta koko hänen tulevaisuutensa!

Täti kävi lukemaan, ensin yksinään, mutta jaksamatta kantaa

kirjeen omituista salaisuutta, huutaen sisarensa jakamaan sitä. Ja
Aline rouva, joka ei itse puolestaan olisi avannut kirjettä, oli valmis
kuuntelemaan, kun toinen luki sen hänelle. Hänellekin oli suhde niin
epäselvä ja kummallinen, että pelkkä uteliaisuus veti häntä
pääsemään sen perille.

Rakas Hilja!

Sydämellinen kiitos kirjeestäsi. Olen niin onnellinen, että

voit kantaa kaikki niin tyynesti. Minä puolestani en voi
tarpeeksi ihmetellä johdatusta kohtalossani. Kaiken on
täytynyt tapahtua, jotta minä kypsyisin. Kaikki tapahtumat
liittyvät toistensa lomiin. Jumalan sormi piirtää selvästi tieni.
Kaikki on niin yksinkertaista ja selkeää, etten käsitä miten
minun niin kauan täytyi haparoida pimeydessä. Mutta miksi
sinun täytyi joutua uhriksi, sitä en vieläkään ymmärrä. Ehkä
siksi, että vain viattomat kelpaavat uhreiksi. Varmaan tällä
kaikella on ollut tarkoituksensa sinuunkin nähden, vaikkemme
vielä sitä tajua.
 Minä ymmärrän nyt, mitä Jumala on ihmiselle. Ei hän ole
sitä, mitä meille aikojen sivu on opetettu. Hän on ydinvoima
meissä itsessämme, se elävä vesisäije, joka kaiken elämän
lähteestä on vuodatettu jokaiseen ihmiseen. Hän on hengen
terveys ja tarmo ja vain pysymällä yhteydessä hänen
kanssaan, voimme me elää. Me sanomme rukoukseksi sitä
kaipausta, pyrkimistä ja kohoamista, jolla henkemme etsii
ikuista lähdettään. Sanoin väärin, kun sanoin, että jo
ymmärrän mitä Jumala on ihmiselle. Aavistan sitä vasta.
Tunnen jo niin suurta onnea tässä aavistuksessa.

Edessäni on ehkä pitkä sairaus. Jumala varaa minulle aikaa

syventyä uuteen elämään.

Olin viikon päivät kotona. Emme näy pääsevän

sopusointuun omaisteni kanssa. Kunnioitan ja rakastan heitä,
ymmärrän, että heidän täytyy olla sillä kannalla kuin he ovat,
mutta he eivät ymmärrä minua eivätkä ikinä voi oppia
hyväksymään kantaani. Heidän mielestään on minun
jumalanpelkoni perkeleestä. Me tuotimme toisillemme
mielipahaa joka päivä. Ehkä olisin jaksanut katsoa asioita
paremmin yläpuolelta, jos olisin ollut terve. Katsoin parhaaksi
lähteä pois. He surevat tähteni, mutta sille ei voi mitään.
Täällä parantolassa on hyvä. Täällä ei ole paljon potilaita.

Sinua huvittaa ehkä kuulla mitä tuloksia oli käynnistäni

Karoliinan luona. Hän ei ensin ollut tuntevinaan minua — tai
ehkä olinkin niin paljon muuttunut. Pian hän kysyi, koska voin
maksaa lainani. Sanoin, että nyt aion ruveta papiksi. Hän
huomautti, ettei se enää merkitse hänelle mitään, kosken
ruvennut silloin kun hän tahtoi. Olin vähän häijy, sanoin
 hymyillen, että minun täytyi odottaa siksi kunnes Jumala
tahtoi. Lopulta hän itse pyytämällä pyysi minua pitämään
lainan. Karoliina on hiukan omituinen, mutta sääli häntä käy.
Ei kukaan välitä hänestä paitsi hänen rahojensa takia. Kaikki
omaiset odottavat hänen kuolemaansa, päästäkseen kiinni
perintöön.

Olen paljon ajatellut niitä asioita, joista sairauteni aikoina

puhelimme. Minulle on käynyt yhä selvemmäksi, että naisen
ja miehen suhteen täytyy perustua ystävyyteen. Ystävyys on
paljon hienompi tunne kuin se, jota sanotaan rakkaudeksi ja
josta uusi ihmiselämä saa alkunsa. Uuden ihmiselämän ei tule
saada alkuaan huumeessa ja humalassa, vaan itsetietoisesta
ja tahtoen. Me emme saa siitä ja syntyä synnissä, vaan
pyhyydessä ja kauneudessa. Etkö usko, että jos uusi polvi
kasvaisi siinä katsantokannassa, niin siitä tulisi onnellisempi
kuin nykyinen? Ei syntyisi niin paljon ihmisiä, mutta syntyisi
onnellisempia ihmisiä.

Hyvästi nyt, rakas Hilja, sinä, jonka kautta Jumala on

tehnyt minulle niin paljon ihmeitä. Yhtä sinulta vielä pyydän:
etkö sallisi minun kirjoittaa Parisiin? Lähetä minulle osoite ja
suostumuksesi. Kaikki tulee jälleen hyväksi, kun saan selittää,
miten kipeä olin ja mitä ihmiset ymmärtämättömyydessään ja
ajattelemattomuudessaan voivat päättää sairaasta ihmisestä.
He suorastaan ajavat hänet onnettomiin ajatuksiin ja tekoihin.
En voisi sen suurempaa onnea ajatella, kuin että te taasen
löytäisitte toisenne.

Sydämelliset terveiset ja Jumala olkoon kanssamme.

Ystäväsi Matti.
 Kun sisarukset olivat lukeneet kirjeen, joutuivat he aivan ymmälle.
Jos suhde siihen asti oli ollut heille epäselvä, kävi se nyt yhä
epäselvemmäksi.

*****

Hilja oli lähtenyt viemään kirjettä asemalle.

Oli polttavan kylmä aamu pari päivää ennen joulua. Pistettyään

kirjeen postivaunun laatikkoon, astui hän hitaasti läpi
asemahuoneiden ja hidastutti hidastuttamistaan kulkuaan. Portaille
hän kokonaan pysähtyi. Sädehtivänä oli talvinen Helsinki hänen
edessään. Kun ne, jotka olivat olleet saattamassa matkustavia, olivat
kiirehtineet alas kadulle, jäi Hilja miltei yksin portaille. Pari kantajaa
puheli toisella puolen pääovea, käsillään lämmitellen korvanlehtiään,
ja vanha tupakanmyyjä, kellahtavaksi haaltuneessa turkissaan,
asettui telttatuolilleen portaiden syrjälle.

Sinertävän kirkas auringonpaiste iski kipunoita lumesta, joka narisi

jalkojen ja reenanturoiden alla. Ihmisten hengitys asettui kuuraksi
heidän turkinkauluksiinsa, hiuksiinsa, viiksiinsä. Kivimuurien seinät,
katot, kaidepuut, portaat — kaikki oli kuin kuuran päällystämää ja
kauttaaltaan liimattua täyteen välkkyvää jäähilettä. Ilman täytti
lumen narina, kulkusten kilinä, ihmisten kiire ja taivaan sinen ja
maan kimmellyksen oudon kaukainen soitto.

Tori oli täynnä kuusia. Rouvia, herroja ja lapsia kierteli puiden

ympärillä. Kuusia kannettiin pois olkapäillä ja vedettiin kelkoilla.
Sinipunaiseen puettu pelastusarmeijanainen käveli edestakaisin
apajansa ympärillä, jonka hän oli pystyttänyt asemahuoneen
edustalle. Kappaleen matkan päähän asettui rivi ajureja, nähtävästi
odottamaan uutta junaa. Ihmisiä tuli ja meni, siinä oli nuoria,
vanhoja, köyhäpukuisia, rikaspukuisia. Kauempaa torilta, ajuririvin
takaa, kohosi vihreitä heinäkuormia, joita maalaiset olivat tuoneet
kaupan. Elävää lehmääkin kaupattiin etäämpänä, kesken pakkasen.
Savu nousi talojen mustista piipuista ilmaan. Myyjät olivat
lämpimäkseen tehneet tulen kuormien ja myymätelttojen väliin.
Torin poikki astui nuori nainen, kantaen kainalossaan pientä
ruumisarkkua. Se oli kääritty paperiin, vain hopeanväriset jalat
näkyivät. Nainen astui nopeasti, vieden puuhkaa korvalta toiselle.
Hänellä oli yllä vain lyhyt takki.

Hilja katseli eteensä niinkuin ihminen kirkontornista katselee

maisemaa allaan ja hänen mielessään liikkuivat asiat, joista hän
vasta oli kirjoittanut Matille. Hän oli noussut varhain ja kirjoittanut
pitkän kirjeen.

Niin, siinä vierii elämä, josta emme tiedä mistä se tulee ja minne
se menee. Tuossa rientävät kaikki nuo ihmiset, jotka sikiävät
huumauksessa, elävät riemussa, tuskassa, vihassa, rakkaudessa,
kaipauksessa ja jotka kuolevat. Jokainen kantaa povessaan
kokonaista maailmaa, jokainen on keskipiste, jonka ympärille elämä
kehrää ihmeellistä seittiänsä. Vieraina, ehkä vihaten he rientävät
toistensa ohi. Sovussa he kerran kaikki löytävät toisensa maan
povessa. Ja kuoleman jälkeen — mitä seuraa silloin? Ikuinen lepo.
Yhdistyminen Jumalaan.

Me emme tunne tulevaa elämää. Tämän me tunnemme ja se on

ainoa, josta me tiedämme mimmoinen se on. Se on sittenkin
suruineen, kärsimyksineen, taisteluineen rikas ja täyteläinen ja
ihanaa on sitä elää.

Siitä ei ole kauan kun hän oli lapsi ja kaikki oli peitettynä ja
salaperäisenä hänen edessään. Häneen sattui, kun hunnut revittiin
pois ja hänelle näytettiin elämän paljas pohja. Hänestä tuntui, ettei
kannata elää elämää, joka on niin rumaa. Hän tunsi kuinka hän
vanheni tuossa rumuudessa niinkuin kukka näivettyy. Mutta nyt hän
on päässyt sen tilan yli, hänen päänsä on selkeä ja hänen ruumiinsa
on nuori ja työnjanoinen. Hän haluaa elää läpi elämän ilon, tuskan ja
työn. Hän haluaa sen liasta ja loasta ponnistaa kohti kirkastusta. Hän
koettaa pyrkiä omistamaan itselleen Kristuksen katseen, sen suuren,
syvän ja säälivän, jonka avulla hän näki läpi ihmisen, katsoi yli
pikkuseikkojen, näki suuret piirteet, ymmärsi ihmisen ja antoi hänelle
anteeksi.

Maailma on tänään säteilevän kirkas. Joulu tekee tuloaan!

Surupuvussaan erottautui Hilja asemahuoneen vaaleaa seinää

vastaan. Hän oli laihtunut ja ihon kalpeus loisti esiin mustasta. Silmät
olivat ikäänkuin suurentuneet ja katsoivat jonnekin kauas. Hän oli
niin ajatuksissaan, ettei huomannut ajuririvin siirtyvän
asemahuoneen eteen, asemapalvelijoiden kiirehtivän paikoilleen ja
ihmisjoukkojen taas rientävän ohitseen. Elämän yksityiskohdat olivat
lakanneet olemasta hänelle, hän näki kaikki suurina, kaukaisina
viivoina.

Pitkä, ulkomaalaiselta vaikuttava herra, päässä ruskeankirjava

matkalakki, yllä pakkaspäivään liian ohut ruskea päällystakki,
samettikaulus pystyssä, likeni kantajilleen ovea. Äkkiä hän pysähtyi
kuin naulittuna. Kantaja jäi oudoksuen odottamaan häntä. Herran
katse riippui surupukuisessa naisessa, joka ovenpielessä seisoi
auringonpaisteessa. Ja ikäänkuin nainen olisi sen tuntenut, kääntyi
hän päin.

— Armas!
 Hilja ei muistanut, että heidän välinsä oli rikki, että heidän piti olla
toisilleen kaksi vierasta ihmistä.

Kaikki, mitä oli tapahtunut siitä, kun he erosivat pyyhkiytyi pois

hänen mielestään, ja hän karkasi kihlattuaan vastaan syli avoinna,
sydän ylitsevuotavana.

Joukko, joka tungeskeli asemahuoneella ja sen edustalla, näki

kahden säihkyvän onnellisen ihmisen astuvan portaille
auringonpaisteeseen. He neuvottelivat hajamielisinä naurahdellen
matkatavaroista, päättelivät sinne tänne, jättivät vihdoin kaikki tyyni
kantajan haltuun ja astuivat käsi kädessä alas portaita. He hymyilivät
ja säteilivät. He kuuluivat toisilleen ja tunsivat olevansa kahden
tähtien alla.
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