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Chandra Mouli Pandey,
Bansi Dhar Malhotra
Biosensors
Fundamentals and Applications
2nd Edition
Authors
Chandra Mouli Pandey
Delhi Technological University
Department of Chemistry
Main Bawana Road
Shahbad Daulatpur -110042
Delhi
India
Prof. Bansi Dhar Malhotra

Delhi Technological University
Department of Biotechnology
Main Bawana Road
DELHI-110042
Shahbad Daulatpur -110042
Delhi
India
ISBN 978-3-11-063780-9
e-ISBN (E-BOOK) 978-3-11-064108-0
e-ISBN (EPUB) 978-3-11-063824-0
Library of Congress Control Number: 2018966600
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Preface
Biosensors have emerged recently as a vibrant technique for qualitative and quan-
titative determination of various analytes for environmental, clinical, agricultural,
food, and defence applications. Many biosensors have shown excellent character-
istics for synthetic samples and pristine laboratory specimens. However, these in-
teresting devices are not yet sufficiently robust for real-world application. The
existing limitations are related directly to the operational and long-term stability
of the biological receptor and physical transducer. Some of the other limitations
could be attributed to poor reproducibility between sensors and selectivity in
complex matrices. For practical applications, the most important obstacles are en-
countered once the sensor is used outside laboratory conditions and is applied for
in situ monitoring of real samples. The areas of development that are expected to
have an impact on biosensor technology are immobilisation techniques, nano-
technology, miniaturisation, and multi-sensor array determinations. In this book,
the basic concepts of biosensors are presented pertaining to fabrication and their
wide range of applications in different fields.
In Chapter 1, the fundamentals of biosensors are discussed. Although many
new types of transducers are being developed continuously for use in biosensors,
optical (i.e., luminescence, absorption, and surface plasmon resonance), electro-
chemical and mass (i.e., surface acoustic wave, and microbalance) transduction
methods are discussed in detail because these are traditional methods. The core
part of biosensing lies in molecular recognition. In Chapter 2, efforts are made to
describe different biological recognition elements that can be used for the develop-
ment of biosensors and their interactions for the detection of analytes. In the devel-
opment of a whole range of biosensors, the immobilisation of bioreceptors has a
key role because it ensures the high reactivity, orientation, accessibility and stabil-
ity of the surface-confined probe and avoids non-specific binding. Many methods,
such as absorption, electrochemical entrapment, biotin–avidin coupling, and cova-
lent binding, can be used for biomolecule immobilisation, and selection of these
methods is reliant on the choice of the matrix.
In Chapters 3 and 4, the applications of various conducting polymers and
nanomaterials are summarised. Biosensors based on highly sensitive and precise
nanomaterials have opened up the possibility of creating novel technologies for
rapid, early-stage detection and the diagnosis of diseases. Different characteris-
tic properties of nanomaterials have paved the way for the fabrication of a huge
range of biosensors with improved analytical capacities. Chapter 5 describes the
broad range of application of biosensors for the detection of various analytes.
Special efforts have been made on the application of biosensors for the detection
of cancer biomarkers.
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VI Preface
In Chapter 6, we explore the potential of biosensors for the design and develop-
ment of ‘smart’ sensing technologies using novel sensing strategies, such as ‘lab-
on-a-chip’, paper-based sensors, and wearable sensors.
We hope that this book will cater to the urgent requirement of students and senior
researchers venturing into this field of interdisciplinary research that has enormous
practical applications. We thank Professor Yogesh Singh (Vice Chancellor, Delhi Tech-
nological University, Delhi, India) for his interest in this work.
Contents
Preface V
1 Fundamentals of Biosensors 1
1.1 Introduction 1
1.2 Developments in Biosensors 2
1.2.1 Electrochemical Biosensors 3
1.2.1.1 Amperometric Sensors 5
1.2.1.2 Potentiometric Sensor 7
1.2.1.3 Conductometric Sensors 7
1.2.1.4 Electrochemical Impedance Spectroscopy 8
1.2.2 Optical-based Biosensor 11
1.2.2.1 Surface Plasmon Resonance 11
1.2.2.2 Chemiluminescence 13
1.2.2.3 Fibre Optic Biosensor 13
1.2.3 Piezoelectric Sensors 14
1.2.4 Calorimetric-based Biosensor 15
1.3 Conclusions 16
References 16
2 Biorecognition Elements in a Biosensor 19

2.1 Introduction 19
2.2 Immobilisation Methods 19
2.2.1 Adsorption 20
2.2.2 Covalent Bonding 20
2.2.3 Crosslinking 21
2.2.4 Entrapment 21
2.3 Principles of Biorecognition 21
2.3.1 Natural Biorecognition Elements 22
2.3.1.1 Enzymes 22
2.3.1.2 Antibodies 25
2.3.2 Semi-synthetic Biorecognition Element 27
2.3.2.1 Nucleic Acids 27
2.3.2.2 Aptamers 29
2.3.3 Synthetic Recognition Elements 31
2.3.3.1 Imprinted Polymers 31
2.4 Conclusions 33
References 33
3 Nanomaterial-based Biosensors 37
3.1 Introduction 37
VIII Contents
3.2 Metal Nanoparticle-based Biosensors 38

3.3 Nanostructured Metal Oxide-based Biosensors 42
3.4 Carbon Nanotube-based Biosensors 46
3.5 Graphene-based Biosensors 49
3.6 Quantum Dot-based Biosensors 51
3.7 Conclusions 56
References 57
4 Conducting Polymer-based Biosensors 63

4.1 Introduction 63
4.2 Application of Polyaniline in Biosensors 65
4.3 Conducting Polypyrrole-based Biosensors 74
4.4 Polythiophenes-based Biosensors 78
4.5 Conclusions 81
References 81
5 Applications of Biosensors 85
5.1 Introduction 85
5.2 Biosensors for Food/Water Safety 86
5.2.1 Biosensors for Detection of Foodborne/Waterborne
Pathogens 87
5.2.1.1 Biosensors for Escherichia Coli Detection 87
5.2.1.2 Biosensors for Salmonella Detection 90
5.2.2 Biosensors for Mycotoxin Detection 94
5.2.2.1 Biosensors for Aflatoxin Detection 94
5.2.2.2 Biosensors for Ochratoxin Detection 96
5.3 Biosensors for the Defence Industries 99
5.4 Biosensors for Clinical Diagnostics 100
5.4.1 Biosensors for Glucose Detection 101
5.4.2 Biosensors for Cholesterol Detection 103
5.4.3 Biosensors for Cancer Detection 106
5.5 Biosensors for Environmental Monitoring 109
5.6 Conclusions 110
References 111
6 Challenges and Prospects 119

6.1 Introduction 119
6.2 Challenges in Biosensing 119
6.2.1 Preparation of Samples 119
6.2.2 Separation of the Sample 120
6.2.3 Immobilisation of Biomolecules on Suitable Matrices 121
6.2.4 System Miniaturisation 121
Contents IX
6.3 Future Prospects 121

6.3.1 Paper-based Biosensors 122
6.3.2 Wearable Sensors 123
6.3.3 Biosensors for Detection of Cancer Biomarkers 123
6.4 Conclusions 124
References 125
Abbreviations 129
Index 133
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1 Fundamentals of Biosensors
1.1 Introduction
The role of biological and biochemical processes is paramount in clinical diagnostics,

medical applications, bioreactors, food quality control, agriculture, control of indus-
trial waste water, mining, and the military defense industry [1, 2]. However, the conver-
sion of biological data to measurable electrical signals is currently a tedious and time-
consuming process [3]. In this context, biosensors have been explored widely because
they can be used to convert a biochemical process into a measurable signal [4, 5]. The
basic difference between the biosensor and physical/chemical sensor is that its recog-
nition element is biological [6]. With advances in device technology, the use of biosen-
sors has increased and they can be used to detect what many others traditional
sensing systems cannot. Nowadays, many biosensors are being produced industrially
and utilised to develop large scale multi-valued sensing systems [7]. Much research is
being conducted in the field of biosensors, with an estimated 60% annual growth rate,
with the major contribution coming from the healthcare industry [8].
The history of biosensors began through the development of an enzyme elec-
trode by Clark [9]. Thereafter, researchers from various fields (physics, chemistry,
and material science) have come together to develop more sophisticated, reliable,
and mature biosensing devices [10]. Biosensors can be used in the fields of medi-
cine, agriculture, biotechnology, defence as well as the war against bioterrorism
[11]. Depending on the area of application, various definitions and terminologies
are being used to define a biosensor. The most common cited definitions are those
by Higson (a chemical sensing device in which a biologically derived recognition
entity is coupled to a transducer, to allow the quantitative development of some
complex biochemical parameter) and Frazerare [an analytical device incorporat-
ing a deliberate and intimate combination of a specific biological element (that
creates a recognition event) and a physical element (that transduces the recogni-
tion event)] [9]. In general, a biosensor is an analytical device that incorporates a
biological sensing element integrated with a physico-chemical transducer that
measures the sensitivity and specificity of a biochemical reaction to deliver com-
plex bioanalytical measurements with a simple, easy-to-use format. A biosensor
consists of two main components: a bio-element and a sensor element (Figure 1.1).
For the fabrication of a biosensor for non-specialist markets, the following condi-
tions are required [3, 5]:
– The desired analyte should be specific and stable under a normal storage
condition.
– The sensor should be accurate, precise and show high sensitivity in a repro-
ducible way, and linearity must be obtained with different concentrations.
https://doi.org/10.1515/9783110641080-001
2 1 Fundamentals of Biosensors
Biocomponent Biosensors Transducers
Antibody
Optical Mass based EC
Enzyme
SPR Piezoelectric Amperometric
Cell
Fiber optic Potentiometric
DNA
Conductometric
Phase
Figure 1.1: Scheme showing the classification of biosensors (DNA: deoxyribonucleic acid;
SPR: surface plasmon resonance; and EC: electrochemical).
– Physical parameters such as pH, temperature should be optimised, which will

lead to sample analysis with minimal pre-treatment.
– The biosensor should be small and biocompatible so that it can be used for in-
vasive monitoring in clinical diagnostics.
– The fabricated biosensor should be portable, cost-effective, small, and capable
of being used by semi-skilled operators.
– The biosensor should provide real-time analysis so that it can be employed for
rapid measurements of analytes from human samples.
Biosensors are composed mainly of two elements: bioreceptors and transducers.

– Bioreceptors are biological recognition elements that consist of an immobilised
biocomponent that can detect the specific target analyte (e.g., enzyme sub-
strate, complementary DNA, antigen).
– The second and the most important part of the biosensor is the transducer,
which converts a biochemical signal into an electrical signal, resulting from the
interaction of the analyte with the bioreceptor. The intensity of signal general
as a result of the biochemical reaction is directly or inversely proportional to
the analyte concentration.
1.2 Developments in Biosensors
For the development of biosensors, the selection of suitable transducers, immobilisa-

tion methods, and bioreceptors are crucial [6]. There is enough scope relating to the
innovation in the fabrication of a biosensor for application of clinical diagnostics.
Further, this multi-disciplinary field of science and technology is predicted to result
in miniaturised, cheaper, and faster biosensors that not only provide accurate infor-
mation but also feedback to the real world for necessary actions [12]. Further, on the
basis of the transducing elements, biosensors can be classified into four types: elec-
trochemical (EC), optical, piezoelectric, and thermal sensors (Figure 1.2).
Transducers used in sensors

EC
Amperometric
Potentiometric
Conductometric
Capacitive
ISFET/MOSFET
Transducer
Optical
Interferometric
Potential Response Refractometric
Fluorimetric
Luminometric
SPR
Amplifier Physical
Surface acoustic wave
Signal Magnetic
Caulometric
Qscillating quartz crystal
Viscometric
Microelectronics
Figure 1.2: Schematic of a biosensor (ISFET: ion-selective field-effective transistor and MOSFET:
metal oxide semiconductor field-effect transistor).
1.2.1 Electrochemical Biosensors
EC biosensors have been explored widely in the fabrication of biosensors because

they allow analyses of biomolecules with high specificity, sensitivity, and selectivity,
have low response time and are cost-effective [13]. An EC biosensor can be used for
clinical analyses, in online control processes for industry or environment, or even in
vivo studies. According to a recommendation from the International Union of Pure
and Applied Chemistry in 1999, ‘An electrochemical biosensor is a self-contained in-
tegrated device, that is capable of providing specific quantitative or semi-quantitative
analytical information using a biological recognition element (biochemical receptor)
which is kept in direct spatial contact with an electrochemical transduction element’
[14]. ‘EC sensing’ usually requires a working electrode (WE), reference electrode (RE),
and a counter (or auxiliary) electrode (CE). In the EC biosensor, the reactions are de-
tected only in the proximity to the electrode surface. Thus, the electrode has a signifi-
cant role in the overall performance of EC biosensors. Further, the detection ability of
the biosensor is based on the properties of the electrode (electrode material, its sur-
face modification or its dimensions) [15].
The transduction element in the EC system is the WE, where the biochemical
reaction takes place. The CE (conductive and steady) acts as a connection to the
electrolytic solution for applying current to the WE. For the RE, silver/silver chlo-
ride has been commonly used, which is kept at a distance from the reaction site to
maintain a desirable and stable potential (Figure 1.3). During the redox reaction of
the molecule at the electrode surface, electrons are transferred from the analyte to
the WE, or from the electrode to the analyte.
CE Potentiostat
RE WE
Figure 1.3: Assembly of different electrodes

in an EC biosensor.
The direction of flow of electrons depends on the properties of the analyte,

which can be controlled by applying the electric potential to the WE [16]. An
oxidation reaction is said to occur if the WE is driven to a positive potential,
where the flow of current depends on the concentration of the analyte (elec-
troactive species) diffusing to the surface of the WE. On the other hand, if
the WE is driven to a negative potential, then a reduction reaction occurs.
The third electrode (i.e., CE or the auxiliary electrode) is often used to mea-
sure the current flow because it functions as a cathode whenever the WE is
operating as an anode and vice versa. The surface area of the CE is often
larger than that of the WE, and the half-reaction at the CE should be rapid
so that it may not limit the process at the WE. The auxiliary electrode is ad-
justed so that it can balance the reaction occurring at the WE, and this con-
figuration allows the potential of the WE to be measured against a known
RE. The commonly used auxiliary electrodes are fabricated from electrochem-
ically inert materials such as gold, platinum or carbon.
EC biosensors measure the current produced as a result of the oxidation and

reduction reactions. The three electrodes are connected to a potentiostat, which
controls the potential of the WE and measures the resulting current. In an EC reac-
tion, a potential is applied to the WE and the resulting current is measured versus
time. In a solution, the equilibrium concentrations of the reduced and oxidised
forms of a redox couple are linked to the potential (E) via the Nernst equation,
Equation 1.1:
RT Coxi
E = E0 + ln (1:1)
nF Cred
where E0 is equilibrium, F is the Faraday constant, T is absolute temperature, and

Coxi and Cred are concentrations of the oxidation and reduction centres, respectively.
When the potential is applied to the WE, the redox couples present at the electrode
adjust their concentration ratios according to the Nernst equation. The resulting
electrical signal is related to the recognition process by the target analyte and is
proportional to the analyte concentration. An EC biosensor has many advantages
such as speed, simplicity, low cost, high sensitivity, and relatively simple instru-
mentation [17, 18]. The EC sensor, where the electrode is used as the transduction
element, represents an important subclass of sensors and is based on the nature of
the EC changes detected during a biorecognition. EC biosensors fall into one of four
categories: amperometric, potentiometric, impedance and conductometric [19].
1.2.1.1 Amperometric Sensors

Amperometric sensors (AS) measure the current change resulting from the oxidation
and reduction of an electroactive species while a constant potential is being applied.
The change in the current is related to the concentration of the species in solution. In
an AS, all the three types of the electrode (WE, RE, and CE) are employed [20]. The
WE used in AS applications are gold, indium-tin-oxide, carbon, and platinum,
whereas silver/silver chloride is used as the RE, which has a fixed potential that con-
trols the potential of the WE. Along with the RE, the CE can also be used to measure
current flow. During oxidation and reduction of the molecule at the inert metal elec-
trode, electrons are transferred from the analyte to the WE, and the flow of the elec-
trons is governed by the properties of the analyte. An oxidation reaction occurs when
the WE is driven to a positive potential, whereas a negative potential results in the
reduction reaction [21]. Different analytes [enzyme, nucleic acid(s) (NA), antibodies]
can be integrated with the amperometric transductor for clinical diagnostics [22].
However, the major limitation in using this transduction is that electroactive interfer-
ence in a sample matrix can result in the generation of false current reading. To over-
come this limitation, various methods, such as dilution of the sample, changing the
medium of the analyte, coating the electrode with various conducting polymers, or
the addition of a suitable mediator, have been adopted [23].
1.2.1.1.1 Cyclic Voltammetry

In cyclic voltammetry (CV), the WE potential is swept at a specific sweep rate (in
volts/second), and the resulting current is recorded versus time. Usually, the sweep
is reversed at a specific switching potential. Hence, it is named ‘CV’ [3]. The sweep
rate is constant and the initial and switching potentials are known, so the time can
be easily converted to potential, and current versus applied potential can be re-
corded (Figure 1.4). CV is applied to gain information on the EC reactions of electro-
active species having a known redox potential. The current is monitored at the WE
(because the CE conducts electricity from the signal source to the WE) during a po-
tential scan against a constant RE potential. The electrolytic solution is used to pro-
vide ions to the electrodes during a redox process [24].
700.0μ
EPC
600.0μ
500.0μ
400.0μ
IPC
300.0μ
Current (A)
200.0μ
100.0μ
0.0
IPA
–100.0μ
–200.0μ
–300.0μ
EPA
–400.0μ
–0.7 –0.6 –0.5 –0.4 –0.3 –0.2 –0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
Potential (V, vs Ag/AgCl)
Figure 1.4: Cyclic voltammogram of a bare gold electrode in phosphate buffer saline (100 mM,
pH 7.4, 0.9% sodium chloride) solution containing 5 mM [Fe(CN)6]3−/4− at a scan rate of
30 mV/s (EPA: potential of the anodic peak current; EPC: potential of the cathodic peak current;
IPA: anodic peak current; and IPC: cathodic peak current).
Figure 1.2 describes a model CV of a bare gold electrode where the resulting
current versus applied potential curve is predicted for an ideal, reversible system.
The peak current in this voltammogram is given by the Randles–Sevcik equation
(Equation 1.2):
Ip = 2.69 × 105 An3=2 D1=2 u1=2 C (1:2)

where Ip is peak current (A), n is electron stoichiometry (eq/mol), A is the electrode

area (cm2), D is diffusion coefficient (cm2/s), C is concentration (mol/cm3), and υ is
the scan rate (V/s). The potential at the mid-point of the two peaks in the voltammo-
gram can be represented by Equation 1.3:
ðEp anodic + Ep cathodicÞ RT 1=2

= Eo + lnðDR1=2 DR jDo1=2 D1=2
o Þ (1:3)
2 nF
where E o is the redox potential and DO and DR are the diffusion coefficients for the
oxidised and reduced halves of that redox couple, respectively. Finally, the separa-
tion between the two peaks of the voltammogram is given by Equation 1.4:
RT 59
Δ Ep = Ep anodic − Ep cathodic = 2.3 = mVðat 298 KÞ (1:4)
nF n
CV is a versatile diagnostic tool used to assess the reversibility of the EC reaction

and its diffusion-controlled character. Other than sensing applications, it can be
used to characterise the processes that take place at the sensing electrode. Hence,
by conducting CV of an electrode, one could easily deduce the concentration, diffu-
sion coefficient, the number of electrons per molecule of analyte oxidised or re-
duced, and the redox potential for the analyte.
1.2.1.2 Potentiometric Sensor

Potentiometric transduction was first reported in the year 1969 where the enzyme-
based sensor was used for detection of urea [25]. It relies on the utilisation of an ion-
selective electrode (ISE) and ion- sensitive field-effect transistor (ISFET) for obtaining
the analytical information. In these sensors, the biological recognition element con-
verts the recognition process into a potential signal to provide an analytical signal.
The potentiometric biosensor consists of two electrodes: an indicator electrode
(which is used to develop a variable potential from the recognition process) and an
RE (usually silver/ silver chloride, which provides a constant half-cell potential) [26].
In an EC cell during the recognition process, the transduction of the potentiometric
biosensor depends on the accumulation of a potential difference between the indica-
tor electrode and the RE. High impedance voltammetry is used to measure the poten-
tial difference or the electromotive force between the two electrodes. The WE consists
of a permselective ion-conductive membrane which is sometimes called an ISE [12].
The measurement of the potential response of a potentiometric device is governed by
the Nernst equation, in which the logarithm of the concentration of the substance
being measured is proportional to the potential difference.
1.2.1.3 Conductometric Sensors

A conductometric sensor measures the change in the electrical conductivity of the
cell solution using two electrodes that are separated by a certain distance or by a
medium [27]. The conductometric sensor has been used widely in enzyme sensors
in which the enzymatic reaction results in a change of ionic strength and the con-
ductivity of a solution between two electrodes. An alternating current (AC) source
is used to across the electrode for conductivity measurement and, thus, the
change in the ionic composition provides a conductance which is measured using
an ohmmeter. Recently, micro-/nanoelectronic devices (field-effect transistor)
based on conductometric transduction have been used for the detection of NA hy-
bridisation, and immunosensors [3]. The advantages of the conductometric device
are that no RE is required, it is inexpensive, and there is possibility of miniaturisa-
tion or direct electrical response. In spite of these interesting features, this trans-
duction method is less sensitive compared with the other EC methods, and is
strongly dependent on the response to buffer capacity [28]. However, conducto-
metric-based transduction has received a great deal of attention and the sensitiv-
ity problem has been overcome. Subsequently, some research has been directed
towards the improvement of performance and sensitivity.
1.2.1.4 Electrochemical Impedance Spectroscopy

Impedance can be defined as the ability of the circuit to resist the flow of electri-
cal current. In EC impedance, the current of the cell is measured by an AC poten-
tial to an EC cell. The impedance is a measure of the impeded flow of ions
through solutions, interfaces, and coatings [29]. When a sinusoidal potential is
applied, the response to this potential results in the generation of an AC signal
which can be considered to be a sum of sinusoidal functions. The impedance can
be calculated by setting the input potential and measuring the induced current.
Impedance methods are powerful because they are capable of sampling electron
transfer at high frequency and mass transfer at low frequency [30]. Impedance
measurements are made at an open circuit potential in which the current flows
in an electrified interface resulting from an EC reaction [31]. The electrons are
transferred across the electrified interface as illustrated in Figure 1.5A and this
process always contains a non-Faradaic component, which is expressed as
Equation 1.5:
½0 + ne − ! ½R (1:5)
where n is the number of electrons transferred, O is the oxidant and R is its

reduced product. The charge transfer has Faradaic and non-Faradaic components.
The Faradaic component arises from the electron transfer via a reaction across the
interface that overcomes an appropriate activation barrier, namely the polarisa-
tion resistance (Rp) along with the uncompensated solution resistance (Rs) [32]
The non-Faradaic current results from charging the double-layer capacitor (Cdl).
When the charge transfer occurs at the interface, the mass transport of the
(A) IHP OHP (B)

Diffusion layer
Rs
Cd
Rp Zw
Electrode
IHP Inner Helmholtz plane

OHP Outer Helmholtz plane
Cation
Solvent
Adsorbent
Electron
Rs
Cdl
WE CE
Rp Zw
Figure 1.5: A) The electrified interface in which the electrode is negatively charged; counter cations
are aligned along the electrified surface and B) the electrode–solution interface can be modeled by
an equivalent circuit (Randles circuit).
reactant and product has a major role in determining the rate of electron transfer,
which depends on the consumption of the oxidants and the production of the re-
ductant near the electrode surface. The mass transport of the reactants and the
products provides another class of impedance, Warburg impedance (Zw), which
can be explored electrochemically and can be shown in the form of a peak current
in a voltammogram [33]. The electrochemical impedance circuit (EIC) shows that
each circuit component corresponds to each interfacial part (Figure 1.5). The
EIC first proposed by Randles displays the high-frequency components (Rs) and
low-frequency components (e.g., Zw). The left-to-right arrangement of the EIC is
necessary because the impedance data are typically displayed in this manner.
Moreover, the activation barrier at a particular potential can be represented by the
Rp but, at the standard (or formal) electrode potential, the barrier becomes
the charge-transfer resistance (Rct). A Faradic impedance spectrum (Nyquist plot)
includes a semicircle region observed at a higher frequency corresponding to
an electron-transfer limited process on the Z’ axes, and is followed by a
linear straight line at 45° to the real axes at lower frequencies, revealing a diffu-
sion-limited electron-transfer process, as shown in Figure 1.6 [34].
The complex impedance plot can be represented as a sum of the real (Z’) and
imaginary (Z’’) components that mainly originate from the resistance and
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fmax ]
[Z w
–lm [Z] y0
]
[Z
x/2 x0 x0 45º Re [Z]
0 R∞ R0
x
x w
Rs r Rct
Figure 1.6: Nyquist plot with a depressed arc where the polarisation is due to a combination of ki-
netic and diffusion processes.
capacitance of the cell and, for the parallel circuit, it can be calculated using
Equations 1.6 and 1.7:
Rp ωCd R2p
Z = Z ′+ jZ ′′ = Rs + + (1:6)
1 + ω2 C2 R2p 1 + ω2 C2 R2p
1
Cd = (1:7)
2πfmax Rct
In electrochemical impedance spectroscopy (EIS) the Zw represents the bulk

properties of the electrolyte solution and can be estimated from the Nyquist
plot to describe the electrical response at the electrode. It can be expressed
as an intercept of the straight line having a slope of 45° using Equations 1.8
and 1.9:
Rp λ
Zw ðωÞ = Wint ½i − j (1:8)
ð2ωÞ1=2
kf kb
Wint = Rs Rp − R2p iCd where, i = 1=2
+ 1=2
(1:9)
Do DR
where, λ is defined as the Warburg coefficient, kf and kb are forward and backward
electron transfer rate constants, respectively, and Do and Dr are diffusion coeffi-
cients of the oxidant and reductant, respectively.
EIS has been used widely by many research teams to detect cancer/ tumour
cells, viruses, bacteria and pathogens. The EIS biosensor could become a powerful
tool for clinical diagnostics in the near future, and can be used for real-time moni-
toring because it can provide label-free detection.
1.2.2 Optical-based Biosensor
During the last decade, much research has been conducted in the field of optical
biosensors, especially in the area of food safety, security, life science, environmen-
tal monitoring and medicine [35]. The high specification, sensitivity, small size, and
cost-effectiveness of optical biosensors may provide an alternative tool to other con-
ventional analytical methods. The first optical chemical sensor was developed for
measurement of the concentration of carbon dioxide and oxygen, and was based on
analyses of the changes in the absorption spectrum. After that, a large variety of
optical sensors, such as ellipsometry, interferometry, spectroscopy (luminescence,
phosphorescence, fluorescence, Raman), optical waveguide structures, and surface
plasmon resonance (SPR) have been used in biosensor applications [36–38]. Among
them, the most commonly used optical biosensors are based on SPR or fluorescence
integrated with the optical fibres [39]. These sensors rely on a change in the refrac-
tive index, absorbance and fluorescence properties of analyte molecules or a
chemo-optical transducing medium. In the last decade, there has been enormous
growth in the research and technological development of optical biosensors be-
cause they provide direct, real-time and label-free detection of many chemical and
biological substances. Despite this technological development, the commercialisa-
tion of this sensor for field application has been slow. The major problem with this
transduction is related to the interaction of the biological molecule with the trans-
ducer surface, and the integration of optical biosensors into a miniaturised device.
Second, the cost is presently a major hurdle for realistic mass production of
biosensors.
1.2.2.1 Surface Plasmon Resonance

SPR biosensors utilise surface plasmon waves (SPW, which are electromagnetic) that
detect changes when the target analyte interacts with a biorecognition element on
the sensor surface [40]. During the interaction of the target analyte with the immobi-
lised biomolecule on the sensor surface, there is a change in the refractive index at
the sensor surface. The excitation of SPW by an optical wave results in the resonant
transfer of energy into the SPW, and SPR manifests itself by resonant absorption of
the energy of the optical wave. This change produces a variation in the propagation
constant of the SPW, which is detected using a spectrophotometer [40, 41]. The high
signal from the electromagnetic field in the dielectric, the propagation constant of
the SPW, and consequently the SPR condition, are very sensitive to variations in the
optical properties of the dielectric adjacent to the metal layer supporting the SPW
(transducing medium) [42]. Therefore, by monitoring the interaction between the
SPW and the optical wave, the change in the optical parameters of the transducing
medium can be measured. The building blocks of an SPR instrument are shown in
Figure 1.7. The two most common detection approaches used in SPR sensors are
Flow channel
Sensor chip
with gold film
Polarised Reflected
light Prism light
Optical
Light detection
source unit
Sensorgram
Resonance
Intensity
signal
Angle Time
Figure 1.7: Basic components of an instrument for SPR biosensing. A glass slide with a thin gold
coating is mounted on a prism. Light passes through the prism and slide, reflects off the gold, and
passes back through the prism to a detector. Reproduced with permission from M.A. Cooper, Na-
ture Reviews Drug Discovery, 2002, 1, 515. ©2002, Nature Publishing Group [45].
(a) the measurement of the intensity of the optical wave near the resonance and
(b) measurement of the resonant momentum of the optical wave, including angular
and wavelength interrogation of SPR [43, 44].
Various biorecognition elements, such as proteins, antibodies, NA, and en-
zymes, have been integrated with SPR biosensors. The important feature of a
SPR biosensor is that it can provide label-free sensing without radioactive and
fluorescence tagging, which makes it highly attractive for real-time monitoring
[46]. Also, SPR-based transduction can be used for interaction without exhibit-
ing any unique properties of fluorescence or characteristic absorption and scat-
tering bands. However, the major drawback with this transduction is its
specificity due to non-specific interaction with the biorecognition element. Sec-
ond, SPR is not suitable for studying small analytes because they yield inade-
quate responses, and the SPR measures the mass of the material binding to the
sensor surface. SPR biosensors have been used to detect the binding of an ana-
lyte as small as ≈2 kDa, but direct detection is not possible because these mole-
cules generate insufficient changes in a bound mass [6]. With recent
improvements in the signal-to-noise ratio, it has been possible to measure the
binding of such small analytes. To date, SPR has been used widely in fundamen-
tal biological studies, health-science research, drug discovery, clinical diagnos-
tics as well as environmental and agriculture monitoring.
1.2.2.2 Chemiluminescence
Chemiluminescence is the energy of a chemical reaction which produces an emis-
sion of light, usually described as luminescence. During a chemical reaction, when
the atom or molecule relaxes from its excited state to its ground state, luminescence
is produced as a side product of the reaction [47]. In chemiluminescence biosensor,
light is generated as a result of the binding event between the analyte and the im-
mobilised biomolecule which is detected using a photomultiplier tube. This trans-
ducer-based platform has been widely used to identify specific biochemical
reactions and this property. The chemiluminescence-based biosensor has been
widely used for immunosensing and NA hybridisation studies as it provides en-
hanced sensitivity along with simple instrumentation, fast dynamic response prop-
erties, and a wide calibration range [48]. In spite of the interesting features,
chemiluminescence transduction has a few drawbacks, such as a low quantitative
accuracy due to short lifetime and its application in real time monitoring.
1.2.2.3 Fibre Optic Biosensor

The optical fibre or the optrodes have found considerable interest in the fabrication
of ultrasensitive biosensor. It consists of a light source, a biorecognition element
and, an optical fibre which is used to transmit light and acts as the substrate, and a
detector (e.g., spectrophotometer) [49]. The optical fibre-based biosensor comprises
of a flexible optical fibre consisting of small wires that are made up of glass/plastic
having different geometrical morphology. These fibres transmit light signals over
long distances with minimum lost value. Due to their remarkably strong, flexible
and durable structures, it can be conveniently used in harsh and hazardous condi-
tions. These optical fibres also permit the transmission of multiple signals synchro-
nously and by this means it can obtain various capabilities for sensing of the
analyte. The high quality and low cost of the optical fibres make them a suitable
transducer for sensing applications. In principle during the interaction of the ana-
lyte with the biorecognition elements at the surface of the fibre, a biochemical reac-
tion takes place which results in a change of the optical properties which
is correlated to the analyte concentration [50]. Due to their high sensitivity, fast re-
sponse, high selectivity, and low detection limits, these optoelectronic are consis-
tently being used for miniaturised high-performance sensor and small fibre optical
sensors. Compared to the EC biosensing approach, optical fibres-based biosensor
provides higher sensitivity, safety, freedom from electromagnetic interference and
can be used for real-time monitoring. This transduction has been used for remote
sensing and single molecule detection as it is reagent-less and flexible [3]. In spite,
of these interesting, features the poor stability of biorecognition element and sensi-
tivity to ambient light are certain disadvantages associated with optical fibre-based
transduction.
1.2.3 Piezoelectric Sensors
Piezoelectric sensors are sensitive mass-to-frequency transducers that are of

interest for potential applications in analytical chemistry. A piezoelectric de-
vice is sensitive to changes in the mass, density, or viscosity of samples in
contact with its active surface. The piezoelectric effect is observed when the
pressure applied to a dielectric material deforms its crystal lattice. The piezo-
electric transduction provides mechanical and electrical forces to a biological
medium usually in the form of progressive or standing acoustic waves, which
may be of different types [51]. Understanding of the properties is crucial for
the selection of the perfect acoustic wave for a given analyte. The mechani-
cal force causes the separation of the cationic and anionic centres and hence
changes the dipole moment of a molecule. There are two types of piezoelec-
tric sensors: i) bulk wave devices and ii) surface acoustic wave (SAW) devi-
ces [52]. The SAW can be compared with bulk wave devices that are capable
of fundamental oscillations at higher frequencies. Current research indicates
that SAW devices are capable of lower detection limits [53]. Therefore, to de-
velop viable, practical piezoelectric biosensors, it is important to consider a
complete sensor-development process from day one of the sensor-design pro-
cess. Extensive research conducted over the decades in the area of piezoelec-
tric biosensors has led to the development of theoretical and experimental
knowledge. However, efforts are underway for the commercialisation of this
transduction-based biosensor. Recent research on piezoelectric sensors has
shown their potential use in medical diagnostic tools and laboratory experi-
ments [51]. However, the main advantage of using this type of transduction
is that it provides a multi-domain sensing mechanism and has good tempera-
ture stability in comparison with EC and optical biosensors [18, 54]. For de-
velopment of the complete device, it is essential to devise specific design
concepts that relate to the nature of a piezoelectric transducer-biological
film-enclosure interface. In a piezoelectric sensing mechanism, the mechani-
cal character of the piezoelectric material, the enclosure of a biosensor as
well as sample handling systems may interfere with the sensing mechanical
motion [55]. This may lead to a decrease in the sensor performance. The
quartz crystal microbalance (QCM) is a good example of a piezoelectric bio-
sensor in which measurement of the mass of a thin-surface coating is based
on the ‘piezoelectric effect’ (Figure 1.8) [56]. The QCM biosensors are compat-
ible with the protocol of biopolymer polymerisation that provides patterned
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From the start Darcy sat in his place and watched each actor in the
little scene before him as if he were somehow outside of it all,
detached from the whole thing, as if the outcome were of little
moment to him, only the persons.
Darcy had not asked for a lawyer. In fact, he had refused one. He
would not ask anybody to help him, nor tell anything that would give
a clue to where he had been or what he had been doing. He had
told them he would plead his own cause when the time came.
So the evidence went on. Witnesses were sworn in and testified to
the most unpleasant details in a well constructed tale of horror. Tyke
was clever, but Bill was sharp, and what the two of them could not
think out the canny Cottar did. They had left no question unprepared
for, no weak places in their line of evidence. They even had an old
flashlight of Darcy’s they had found where he had left it last at one
of their meeting places, and most carefully had they preserved it
without handling that the finger prints might be observed. Obligingly
Darcy put out his fingers for the impression, that smile of half
amusement on his lips. So well he understood the revenge that was
working all this elaborate network of lies to catch him.
Yet as the evidence went on he began to realize how cleverly it had
been done, and how only a miracle of some kind could save him. He
sat gravely watching it all, listening. Now and then jotting down a
note for his own reply when his time came, but for the most part,
gravely listening, and the day went on and blacker grew the
evidence against him. The excitement in the court-room was great.
There were not wanting gruesome details and Darcy’s face grew
stern and his soul sick within him. To think that Joyce should,
through him, be mixed up in a loathsome mess like this! He would
rather have died a thousand deaths than to have had her name
connected in such wise.
The spectators were strained to the highest point. Nan, heavily
veiled and weeping, was most affected. When it came her turn to
testify she told of the beautiful relation between herself and Joyce,
but said that Joyce was very secretive and went out a good deal
evenings, staying late. Once during Bill’s blunt testimony she
screamed and fainted and had to be taken out, but insisted on
coming back again. And hourly the look of suffering grew on Darcy’s
face, as if the ordeal were actual physical pain. But once, there was
a little relaxing of the strain, when old Noah Casey took the stand,
and was asked to swear that he would tell the truth, the whole truth
and nothing but the truth.
He climbed into his place and laid a trembling, knotted hand upon
the book, but when they asked him to swear he smiled about upon
them and shook his head.
“My mother taught me not to swear,” he said serenely, while the four
who were sponsoring him frowned and cursed beneath their breath.
He stood there looking about on the throng, his quick bright eyes
travelling from one face to another, half suspicious of them all, half
frightened like a wild thing of the woods. And when the people
laughed he laughed with them at himself. The difficulty about the
oath over, he told his story, eagerly, somewhat like a child, in short
hurried sentences, his bright eyes still hurrying over the audience,
his long nervous fingers fingering the brim of his old felt hat. “I was
going acrost the medder—” he began, “ahint of the graveyard—”
and Gene’s lawyer helped him out with questions. “You saw a bundle
on the ground like a human body—” the bright eyes focused on the
lawyer an instant.
“No, it was broken glass. Leastways that’s what I thought I saw.
They tell me—” The lawyer hurried into another question, and the
Judge interrupted:
“Suppose you look around, Noah, and tell me if you can see the man
you saw that night digging in the graveyard?”
The bright eyes focused on the Judge, and then turned quickly
toward Tyke. The lawyer hastened with his assistance.
“Was it this man, Noah?” he pointed to the prisoner.
Noah Casey turned around toward the box where Darcy sat and saw
Darcy for the first time:
“What! Him?” he asked, pointing with a long finger at Darcy who
regarded him with a grin of friendliness.
“Why, no, that’s Darcy Sherwood. I know him. I’ve knowed him since
he was a little tad. Oh, no, it wa’n’t Darcy. He’s a good boy. He
wouldn’t do such a thing. The man I saw had red ha—”
But Gene’s lawyer raised his voice:
“Your Honor, I am disappointed in this witness. Mentally he does not
seem to be quite all that I supposed—”
“Undoubtedly—” said the Judge under his breath, and Noah was
hustled off the scene.
But the afternoon came on and somehow the false witnesses were
making a pretty good case of it against Darcy. The Judge’s eyebrows
were drawn in a heavy frown and his breath came quick and deep.
Those who knew him well knew that he was troubled, and it was
just then that Dan’s note was handed up.
No one but Darcy noticed the twinkle that came in the Judge’s eyes,
and he wondered and tried to puzzle it out. The Judge was his friend
he knew, and wanted to see him cleared, but surely all hope was
gone. The evidence was all on one side. Why prolong the agony? It
almost seemed as if the Judge were trying to keep the case going,
trying to make time. He asked the most trivial questions and tripped
up the lawyer again and again, holding a witness far beyond
necessity.
All at once the Judge drew a long breath and a light came in his eye.
He sat back as if he were done, and ordered that the prisoner be
allowed to speak for himself.
The leather door at the back of the court-room had swung
noiselessly but that moment, and little Lib had entered, straight and
beaming, and behind her walked a lady, and Dan Peterson. Darcy
gave one glance and then arose, and there was a new light in his
face. It was almost as if he had come to a triumphant moment,
instead of being about to plead for his life in the face of indubitable
evidence against him. Those who were watching noticed with a
shock that he actually had a kind of smile on his face, and a look of
something—could it be peace? What utter nonsense! Perhaps he
was going out of his mind. Any one might, having to listen to such a
list of his own horrible crimes!
But Darcy was speaking in his quiet tone:
“It almost seems a pity to add anything after such well established
evidence as you all have heard. If I didn’t know I wasn’t guilty I
would almost think I was after listening to what has been said. So I
won’t try to argue in my own favor. I see Miss Joyce Radway herself
has just come in, and I’m going to ask if she may come up here and
tell you whether I ever abducted her, or murdered her, or buried
her.”
Then indeed there was a great stir in the court-room. People
stretched their necks to see, and rose up in their seats, but the
Judge commanded silence. Under cover of the confusion Tyke
attempted an escape, but was stopped by order of the watchful
Judge.
Joyce came to the front of the room, proudly escorted by Lib who
held her hand to the very witness stand and then stood by with glad
eyes to watch her.
Joyce turned and faced the excited throng, then looking toward her
old friend, Judge Peterson, she spoke in clear, ringing tones that
everybody could hear:
“Your Honor, I haven’t seen Mr. Sherwood but once since I left home
a year ago to go to Silverton and teach. Mr. Sherwood does not
know I saw him then. He was making a speech in a religious service
in the city where I happened to be one evening, and it was a good
speech too. I wish you could have heard it. I tried to get up to speak
to him but the crowd was so great that he was gone before I got to
the platform.”
She turned her face toward the court-room a little more, looking
down at the seats where the witnesses sat, and noticing with
startled eyes the man of the loud voice who had addressed her as
“girlie” on that memorable morning one year ago.
“I don’t know what you have been trying to do to my old friend, Mr.
Sherwood, or where you got such utter lies. I went away from
Meadow Brook because I wanted to teach and I knew my relatives
were opposed to my doing it. I did not realize that I could be
misunderstood or make trouble for anybody by doing so, but my
going certainly had nothing whatever to do with Darcy Sherwood.
We have seldom seen each other since we were school children
together, and he has always been most kind and gentlemanly to me
whenever I have met him.
“I happened to see an old copy of the Meadow Brook News this
morning and read to my horror what you were saying about him and
me. It made me sick that my old friends and my relatives could allow
such an awful charge to be made on such a man as Darcy
Sherwood. I had to get somebody to take my place in school while I
came here, and I was afraid I wouldn’t get here in time before you
did something dreadful you could never be forgiven for. But I’m glad
I came, and I’m—ashamed of you all.”
If any one had been looking at Darcy then they would have seen a
wonderful look in his eyes, but everybody’s attention was centred on
Joyce. There had not been such a sensation in Meadow Brook in
years as the dead coming to life just in time to save a tragedy.
The Judge stood up and addressed Darcy. His voice was trembling.
He was very unjudgelike in his manner.
“My boy, you are free from the charge and the court dismisses the
case.” He was smiling and there was something like a mist in his
eyes.
Darcy inclined his head slightly.
“Your Honor, I thank you. I am glad to be exonerated from a crime
that I did not commit; but I want to ask your permission now to
confess to one that I did. I want to take the penalty, whatever it is,
and be cleared in the eyes of the law forever.”
The court-room grew suddenly hushed. People who had risen and
begun to adjust wraps and pick up their gloves sat down again. All
ears were strained to hear every word.
“You have my permission,” said the Judge looking instantly grave
and anxious. “Is the attorney here to take down the confession? Mr.
Robinson—”
There was a little stir in the room while the attorney came forward,
and then Darcy went on:
“For several months prior to the time last spring when I left town I
had been in the bootlegging business.”
“Oh!—Ah!” were whispered here and there with nods of previous
conviction from people who had been half disappointed to have the
trial turn out so well.
“I gave it up because I had come to feel that it was wrong. I confess
it now because I want to pay the penalty of what I have done.
Judge, I will be glad if you will put this through as soon as possible.
I am ready to take what is coming to me.”
The Judge bowed gravely. There was no denying that he looked
relieved.
“This will have to go through the regular routine of course,” he said,
“but it can be run through quickly. Mr. Robinson, you get the items,
number of cases sold and so on; prepare the indictment, and we’ll
try to get it through tomorrow.”
The Judge straightened up and looked about him, his eyes resting
on the four witnesses, holding their hats ready for a speedy
departure, and at that moment the district attorney jumped up
briskly.
“Your Honor,” he said. “I ask that these four witnesses be held for
perjury.”
A murmur of satisfaction went rippling over the court-room as
people rose to go out.
People were rushing around Joyce as she came out, still escorted by
little Lib, proud as a small peacock. Nan was the first to envelop her
in a smothering embrace, weeping copiously upon her neck with
loud show of affection. Many old friends lingered, waiting just to
watch her dear face alight with the relief and triumph of the
moment. The minister and his wife were close behind Joyce and
eagerly asked her to come home with them.
“No,” put in Nan decidedly, “She’s coming to her own home of
course. Everything in your room is just as you left it, darling—”
Joyce couldn’t help smiling at the affectionate appellative.
“For pity’s sake get rid of that awful child, and come on,” whispered
Nan loudly. “Don’t let her hang on you like that. Let’s get out of this
terrible crowd! How curious people are! Come on home!”
Poor little Lib dropped Joyce’s arm as if she had been shot, but Joyce
quickly caught the little cold hand and drew it back close within her
arm, her own warm fingers keeping the little hand clasped tight.
“I want her here, Nan. She is my dear little friend,” said Joyce
pleasantly.
“For mercy’s sake! You always had such queer friends, Joyce,” she
laughed disagreeably. “Well, never mind, bring her along, only come
on!”
But Dan Peterson’s hand was on Joyce’s shoulder.
“No, Mrs. Massey, Joyce is coming with us. Father wants to see her
right away at home,” and off he carried Joyce and Lib in his own big
shiny car, while Nan tried to hide her chagrin by taking to herself
reflected glory, and trying to make a little social hay while the sun
shone.
Joyce went home with the Petersons and was presently sitting in
Judge Peterson’s library, learning about her inheritance, and being
prepared for the reading of the will which was to come after supper
as soon as the Masseys could be summoned to the hearing.
But all the time her mind was on the listen, and she was hoping that
Darcy would come. Surely, surely he would come and speak to her,
just thank her or something. He had been busy with the attorney
when she left the court-room, and had flashed her just one
gorgeous smile as she looked back at him. Had she been mistaken?
Surely there was a promise in that glance, that he would see her
again. She wondered why everything seemed to have suddenly gone
so flat. She ought to go back of course on the night train and be
ready to teach on the morrow, but her heart was not willing to go—
not yet—and Judge Peterson presently settled the matter by saying
that she would be needed the next day for the technicalities of the
settlement of the estate.
So she sent a telegram to Harrington:
“Will be back to teach Monday morning.
Cannot possibly come sooner.
(Signed) J. Radway.”
CHAPTER XXXI
The will was read at last.
Gene and Nan, glowering in the corner of Judge Peterson’s
comfortable library, learned with dismay that their part was only a
small patrimony which Mary Massey had in her own right. The rest,
house, and meadow lands, and money enough to keep her
comfortably were all Joyce’s left her by her mother from her own
father’s estate.
It had been her mother’s wish that Joyce should not know that she
had anything but herself to depend upon until she grew up. She felt
that so she would the better come up unspoiled and independent.
So she had placed the property in her sister’s hands in such a way
that unless Mary Massey died Joyce would not know that she had
anything until she came of age. Judge Peterson was the other
trustee of the property and was to use his own discretion about
telling Joyce in case of her aunt’s death before her majority. But
during the interval of Joyce’s absence from Meadow Brook Joyce had
come to her majority, so there was no longer any hindrance to her
entering into her inheritance at once.
Mary Massey had not told her son the whole thing for reasons of her
own, but she had left a letter explaining the matter to him, and
reminding him of what she had always told him, that she had very
little to leave him, but commending Joyce to his tender care, and
saying she had little fear but that Joyce would always be generous
with him.
Gene and Nan arose silently when the business of the will was
concluded. They had a look of withdrawing. A hurt, stricken look.
Joyce sprang up and went over to them, saying eagerly:
“Of course, Gene, you’ll stay in the house.”
“It’s your house,” said Gene. “Of course we’ll get right out.”
“Please don’t,” said Joyce earnestly. “At least not unless you don’t
want to stay, of course. The house was your mother’s home. All
these technicalities of law don’t change the matter a mite to me. I
know Aunt Mary expected us all to live there, and she knew I would
say so. Even though it is mine it’s just the same as yours. Besides,
I’ve another little house of my own in Silverton and I presume I shall
go back there and go on teaching. I should not be happy doing
nothing. The house may have been bought by my father’s money,
but it was made into a home by your mother’s loving care, and it’s
yours as much as it is mine. As long as you live I want you to feel
you can live in it if you want to.”
“You could sell it,” said Gene, still independently, “or rent it. I will
pay you rent,” stiffly.
Joyce laughed.
“No, indeed. You won’t pay me rent. If you try to I’ll pay you for
your mother’s love and care. How would that be? And I don’t want
to sell the house. I love it. I like to think it’s there for me to come
home to now and then. And Nan, you don’t need to feel hampered
there. You can arrange things just as you like, just as if it were your
own. Just treat me like a sister, that’s all, and share with me in what
there is.”
“I’m sure that’s very generous in you, Joyce,” said Gene, feeling a
sense of shame over the way he had always treated her. “We’ll think
about it. Aren’t you coming home with us now?”
“Why, I’ll be down tomorrow, I guess,” she said pleasantly. “I want
to get all these papers signed and everything fixed. I must be back
on my job Monday, you know.”
“I shouldn’t think you’d have to teach now,” said Nan, an envious
note in her voice. “Why don’t you just telegraph them you aren’t
coming back, and quit?”
“Why, that wouldn’t be honorable,” laughed Joyce. “And besides I
like it. I wouldn’t leave the kiddies for anything till the term is up.”
“You’re a queer girl,” said Nan speculatively. “I don’t see why you
don’t just want to have a good time.”
“Why, I’m having a beautiful time,” said Joyce, wide-eyed. “I’m doing
what I’ve always wanted to do.”
They went home, and the evening passed and still Darcy did not
come. Joyce began to wonder if he were not coming at all. If,
perhaps, she might be going to have to write him a note or call him
up or something; for she did not mean to go back to Silverton
without telling him in so many words how glad she was that he had
found her Lord.
Joyce spent Friday in going over her things in the old home and
packing up what she wanted to take back with her to Silverton, but
she went back at night to Judge Peterson’s. The minister and his
wife came in to call that evening and they had a beautiful talk, but
all the time Joyce was listening for a step that did not come, and
wondering. Was Darcy still shy? Surely he would come just once
after all that had happened, after she had come home to make
things straight and set him free.
She thought she heard the minister speak his name as they were
about to leave, talking to Dan Peterson over by the door, and Dan
said, “Yes, tomorrow,” that was all. She wanted to ask, but
something held her back, and no one mentioned Darcy. Was it his
second trial that was coming off tomorrow she wondered?
She did not sleep well that night. She kept waking and thinking of
Darcy. Was he going to have to go to prison after all for
bootlegging? It seemed so hard now that he had begun his new life.
She wished she dared ask about the law.
The next morning at the breakfast table she followed a sudden
impulse and told the Judge and his wife all about Darcy’s speech in
the meeting on the night of the rain. Dan had gone into the hall to
answer the telephone and when he came back he tiptoed in and
stood by the door quietly, as if some one were praying. His eyes
were down and his face looked strangely tender as if he were
hearing a miracle. Neither the Judge nor Dan were much on religion,
but Mrs. Peterson was a saint if there was one, and her face glowed
with joy over the story. The old Judge cleared his throat three times
before he growled out the words:
“Yes, Darcy’s all right, Darcy’s all right.” His glasses seemed to be
blurred and he had to take them off and polish them before he could
see right again. “He needn’t have confessed that at all. It was all
over and forgot. But still, I like him better for it. Great stuff in him!”
Then Joyce summoned courage.
“Will he have trouble again, with this other trial? What will be the
penalty? Will he have to go to prison?”
“Oh, no, oh, no! Nothing like that,” said the Judge hastily. “Oh, no,
he’ll just have to pay a penalty. Probably about five hundred dollars
or something like that, according to the amount of stuff handled.
Know how much it was, Dan?”
“About,” said Dan gruffly.
“Great boy, Darcy!” said the Judge emphatically. “He’ll be all right
this morning. Case comes off before noon, doesn’t it, Dan? Where’s
my note book?”
“Yes, before noon,” said Dan, and then they both went out and
Joyce, with relieved heart, went to singing and playing on the old
tinpanny, yellow-keyed square piano, singing with all her heart, the
song they sang that night in the meeting:
“Free from the law, O sinner, receive it,
Free from the law, O brother, believe it—!”
and Mrs. Peterson, up in her room making her bed and plumping up
the pillows, said to herself happily:
“Bless her dear little heart. I wish we could keep her here.”
Darcy came home with Judge Peterson at noon to lunch. He seemed
a new Darcy. His face was alight, and his smile was joyous.
He did not talk much during the meal, but what he said rippled with
humor, and kept them all in gales of laughter. The new gravity that
was upon him when he was silent sat well there, and gave him an
air of one who had found a solid foundation.
As they arose from the table he looked at Joyce and said in a tone
that every one could hear:
“Miss Joyce, it’s a gorgeous afternoon and I thought perhaps you’d
like to take a walk through some of the old paths. I have something
I’d like to tell you, and if you don’t mind being seen in company with
a law-breaker like me we might stroll down to the old woods.”
Joyce looked up with her face flooded with glory: “Oh, but you’re not
under the law now, you know,” she said brightly.
“No, not in any sense, thank the Lord,” said Darcy reverently. “Not
even under the law of the land.”
They walked down the long, smooth road together past the bridge
and round the turn, and there on the hill before them lay the woods
in all the beauty of the springtime verdure. They had been talking of
the trial, Darcy telling her all that had gone on before she arrived,
and also of the second trial where he had paid the fine and been set
free. But now, at the turn of the road, they stopped and looked. The
scene was so lovely, one could but exclaim at the beauty of the
hillside. The fields were green and dotted with violets where they
walked, and all about were spicy odors of the spring, with exquisite
perfumes in the making. Before them rose the woods, pale greens
stippled with red buds on their tips, and a background of darker
pines. And now they spoke of Aunt Mary and the day they had spent
together.
As they entered the woods frail anemones scattered their pathway,
and hepaticas, blue and white, met them in groups where maiden
hair hid, and little curled fern fronds stuck up through the black
mould. As they walked down that pine-strewn path, flower broidered
and dim, each was conscious of the last time they had passed that
way. They had gone out from that arched silence a boy and girl,
they were reëntering man and woman.
For a few moments neither spoke. Then Darcy, as if he were
treading holy ground:
“It was here you sat when I first saw you.”
Joyce flashed him a golden look.
“And you there?” she pointed. “I have always wanted to go there
and sit myself,” she said, “with my feet hanging over that rock.”
“Let’s go!” he said, and caught her hand.
He helped her down to a comfortable spot, where the mossy rock
shelved over the water, and the little brook babbled over bright
stones in a quiet, musical way.
Sitting there he told her of the deep experience of his heart. Of how
that day and the story of the blind man had lingered with him all
those years, until he came to understand that he was blind, and he
was a sinner and needed a Saviour. He told her how her eyes had
pierced his soul, like the thought of God searching him, and how he
had finally surrendered. He spoke of the peace and joy that were his
now, and of his Bible.
And then Joyce told him how she had come in out of the rain and
heard him talking, and tried to reach him and failed.
The sun dropped lower in the west and the long shadows came
within their sweet retreat. Finally, they sat in silence, just listening to
the birds, and the tinkle of the water, feeling how good it was to be
here after the years.
Suddenly Darcy began to speak again:
“Joyce, I’m going to tell you something. You may think perhaps I
oughtn’t to tell you, that I have no right to speak of such things—
that I am unworthy. But somehow I think you ought to know. After
I’ve told you I’m not going to presume upon it. I know as well as
you do that I’m unworthy. But I’ve loved you all my life, and it’s kept
me from a great deal that I might have done if I hadn’t. I never
dreamed of you as mine, not in any material sense of the word. I
always knew I wasn’t big enough and good enough for you, but I’ve
kept you like a shrine in a temple, a place to worship at. You can’t
know what it’s meant to me. I’m telling you this because it’s the only
way I can thank you for what you did for me. You saved my life, and
I want you to know that all that a man has to give a woman, that I
have given to you. There will never be any other girl for me!”
Joyce’s head was turned away. She was trying to keep the blinding
joy of her heart from leaping to her eyes.
“And you refuse to let me give anything back to you?” she asked in a
little faltering voice.
“What do you mean, Joyce?” He lifted his eyes and looked at her
anxiously.
“I mean, does it mean nothing to you that I have loved you too,
ever since the day we were here last?”
He caught her hand.
“Joyce! Do you mean that? You loved me all that time? But of course
you did not know me, did not know that I was—”
“I loved you,” said Joyce firmly. “And I love you now. I didn’t know it
was that when I came home to save you, but I guess it was there all
the time only I hadn’t told myself about it—yet—”
“Oh, Joyce, my darling!”
He gathered her close to his heart and closed his eyes in an ecstasy
of joy.
“It makes me feel so humble!” he said at last looking into her eyes.
“To think that I, a sinner, a law-breaker—”
She laid her fingers on his lips.
“‘For ye are not under the law,’” she quoted softly, “‘but under
grace.’ Have you forgotten that He puts His righteousness upon us?”
They came back from their walk in the twilight with the stars looking
down upon them and a new moon shining in a clear sky, but they
did not see it. They were walking hand in hand and talking of many
precious things.
The Petersons had been waiting dinner for them almost an hour, but
they were serenely unconscious of the fact.
They went to church the next day and sat in the old Peterson pew,
side by side, with the Judge and Mrs. Peterson and Dan, for the
delectation of all eyes, but they didn’t know that either. They were
as happy as any two people could be in this world.
Monday morning, with the first ray of light, Darcy was up and at his
car, and before the people of Meadow Brook had begun to think
about waking up he and Joyce were on their way to Silverton. Joyce
had around her shoulders her gray fox neckpiece. Nan had
ostentatiously thrown it out the window in the gray of the morning
when they stopped there for Joyce’s trunk saying: “Here, Joyce,
you’ll need this. It’s chilly. I had it put carefully away for you in
camphor all winter.”
They had the road to themselves for the first two hours and Darcy’s
racing engine flew out along the road as smoothly as perfect steel
and well oiled bearings could make it. They drove into Silverton at
ten minutes to eight, and went straight to Joyce’s little house to
leave her trunk, much to the wonder and delight of Mrs. Bryant who
hadn’t known what to make of Joyce’s absence.
Joyce was wearing on her finger a splendid diamond. Darcy had
routed a jeweller friend out of bed late Saturday night to get it, and
paid for it with a check that almost cleaned his bank account out
entirely, but he wore a look on his face of utter happiness.
They drove up to the school house five minutes before the bell rang,
and Professor Harrington stood on the steps talking to a teacher.
Joyce was still in her new spring suit and pretty, becoming little hat,
with the gray fox around her neck, and Harrington felt his resolve
slowly melting away from him. How could one be cold to a girl who
looked like that? She certainly was stunning in those clothes. He had
thought all along that clothes would make a big difference. But who
the deuce was the big, good-looking giant who brought her.
And then the giant stooped and kissed Joyce, and he frowned.
“Was that your brother?” he asked as Joyce came flying up the walk
afraid she was going to be late.
Joyce lifted a saucy face and smiled:
“No, Mr. Harrington,” she said sweetly. “That is the man I am going
to marry. Have you time to come down and meet him?”
THE END
TRANSCRIBER’S NOTES:
Obvious typographical errors have been corrected.
Inconsistencies in hyphenation have been
standardized.
Archaic or variant spelling has been retained from the
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Advanced Materials and Techniques for Biosensors and

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LaunchPad for Fundamentals of Abnormal Psychology Ronald

A Mathematical Primer of Molecular Phylogenetics 1st

Planning, Time, and Self-Governance: Essays in Practical

The Virtues of Freedom: Selected Essays on Kant 1st

How Open Source Ate Software: Understand The Open Source

Carbon-based Smart Materials

Nano Devices and Sensors

Intelligent Materials and Structures

Fundamentals and Applications

Prof. Bansi Dhar Malhotra

Library of Congress Control Number: 2018966600

Bibliographic information published by the Deutsche Nationalbibliothek

© 2019 Walter de Gruyter GmbH, Berlin/Boston

2 Biorecognition Elements in a Biosensor 19

3.2 Metal Nanoparticle-based Biosensors 38

4 Conducting Polymer-based Biosensors 63

6 Challenges and Prospects 119

6.3 Future Prospects 121

The role of biological and biochemical processes is paramount in clinical diagnostics,

Biocomponent Biosensors Transducers

– Physical parameters such as pH, temperature should be optimised, which will

Biosensors are composed mainly of two elements: bioreceptors and transducers.

1.2 Developments in Biosensors

For the development of biosensors, the selection of suitable transducers, immobilisa-

Transducers used in sensors

1.2.1 Electrochemical Biosensors

EC biosensors have been explored widely in the fabrication of biosensors because

Figure 1.3: Assembly of different electrodes

The direction of flow of electrons depends on the properties of the analyte,

EC biosensors measure the current produced as a result of the oxidation and

where E0 is equilibrium, F is the Faraday constant, T is absolute temperature, and

1.2.1.1 Amperometric Sensors

1.2.1.1.1 Cyclic Voltammetry

Ip = 2.69 × 105 An3=2 D1=2 u1=2 C (1:2)

where Ip is peak current (A), n is electron stoichiometry (eq/mol), A is the electrode

ðEp anodic + Ep cathodicÞ RT 1=2

CV is a versatile diagnostic tool used to assess the reversibility of the EC reaction

1.2.1.2 Potentiometric Sensor

1.2.1.3 Conductometric Sensors

1.2.1.4 Electrochemical Impedance Spectroscopy

½0 + ne − ! ½R (1:5)

where n is the number of electrons transferred, O is the oxidant and R is its

(A) IHP OHP (B)

IHP Inner Helmholtz plane

In electrochemical impedance spectroscopy (EIS) the Zw represents the bulk

1.2.2 Optical-based Biosensor

1.2.2.1 Surface Plasmon Resonance

1.2.2.3 Fibre Optic Biosensor

1.2.3 Piezoelectric Sensors

Piezoelectric sensors are sensitive mass-to-frequency transducers that are of

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