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Advances in
Nanosensors for
Biological and
Environmental
Analysis
This page intentionally left blank
 Advances in
Nanosensors for
Biological and
Environmental
Analysis
EDITED BY

AKASH DEEP, MSC, MPHIL, PHD

Nanotechnology Lab
CSIR-Central Scientific Instrument Organisation (CSIR-CSIO)
Chandigarh, India
Academy of Scientific and Innovative Research (AcSIR-CSIO)
Chandigarh, India

SANDEEP KUMAR, PHD

Department of Bio and Nano Technology
Guru Jambheshwar University of Science and Technology
Hisar, India
Department of Civil Engineering
University of Nebraska Lincoln
Lincoln, United States

]
ADVANCES IN NANOSENSORS FOR BIOLOGICAL AND ISBN: 978-0-12-817456-2
ENVIRONMENTAL ANALYSIS
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List of Contributors

Valerio F. Annese Girish Chandra Mohanta

Electronics and Nanoscale Engineering CSIR-Central Scientific Instrument Organisation
School of Engineering (CSIR-CSIO)
University of Glasgow Chandigarh, India
Glasgow, United Kingdom Academy of Scientific and Innovative Research
(AcSIR-CSIO)
Gaurav Bhanjana Chandigarh, India
Department of Chemistry and Center of Advanced
Studies in Chemistry Ganga Ram Chaudhary
Punjab University Department of Chemistry and Center of Advanced
Chandigarh, India Studies in Chemistry
Panjab University
Neha Bhardwaj Chandigarh, India
Nanotechnology Lab
CSIR-Central Scientific Instrument Organisation Moondeep Chauhan
(CSIR-CSIO) Department of Environment Studies
Chandigarh, India Punjab University
Academy of Scientific and Innovative Research Chandigarh, India
(AcSIR-CSIO) Department of Chemistry and Center of Advanced
Chandigarh, India Studies in Chemistry
Panjab University
Sanjeev Kumar Bhardwaj Chandigarh, India
Nanotechnology Lab
Department of Bio and Nano Technology
CSIR-Central Scientific Instrument Organisation
Guru Jambheshwar University of Science and
(CSIR-CSIO)
Technology
Chandigarh, India
Hisar, India
Academy of Scientific and Innovative Research
(AcSIR-CSIO) David R.S. Cumming
Chandigarh, India Electronics and Nanoscale Engineering
School of Engineering
Deepanshu Bhatt University of Glasgow
Nanotechnology Lab Glasgow, United Kingdom
CSIR-Central Scientific Instrument Organisation
(CSIR-CSIO) Akash Deep
Chandigarh, India Nanotechnology Lab
Academy of Scientific and Innovative Research CSIR-Central Scientific Instrument Organisation
(AcSIR-CSIO) (CSIR-CSIO)
Chandigarh, India Chandigarh, India
Academy of Scientific and Innovative Research
(AcSIR-CSIO)
Chandigarh, India

v
vi LIST OF CONTRIBUTORS

Neeraj Dilbaghi Sandeep Kumar

Department of Bio and Nano Technology Department of Bio and Nano Technology
Guru Jambheshwar University of Science and Guru Jambheshwar University of Science and
Technology Technology
Hisar, India Hisar, India
Department of Civil Engineering
Ashraf Aly Hassan University of Nebraska Lincoln
Department of Civil & Environmental Engineering Lincoln, NE, United States
United Arab Emirates University
Al Ain, United Arab Emirates Poonma Malik
CSIR-Central Scientific Instrument Organisation
Sven Ingebrandt (CSIR-CSIO), Colony
IWE1eInstitut für Werkstoffe der Elektrotechnik1 Chandigarh, India
RWTH Aachen University
Germany Ruchi Mutreja
Chemical Biology Lab
Manpreet Kaur Biotechnology Department
Graduate Student IIT Roorkee
Indian Institute of Science Education and Research Roorkee, India
Mohali (IISER Mohali)
Mohali, India Suresh Neethirajan
BioNanoLab
Rajnish Kaur University of Guelph
Department of Physics Canada
Panjab University
Chandigarh, India Monika Nehra
Ubiquitous Analytical Techniques Department of Electronics and Communication
CSIR-Central Scientific Instruments Organisation Engineering
(CSIR-CSIO) Guru Jambheshwar University of Science and
Chandigarh, India Technology
Hisar, India
Madhu Khatri Department of Bio and Nano Technology
Faculty Guru Jambheshwar University of Science and
University Institute of Engineering and Technology Technology
(UIET) Hisar, India
Punjab University (PU)
Chandigarh, India Satish Kumar Pandey
CSIR-Central Scientific Instrument Organisation
Parveen Kumar (CSIR-CSIO)
CSIR-Central Scientific Instrument Organisation Chandigarh, India
(CSIR-CSIO)
Chandigarh, India Samadhan B. Patil
Electronics and Nanoscale Engineering
Rajeev Kumar School of Engineering
Department of Environment Studies University of Glasgow
Panjab University Glasgow, United Kingdom
Chandigarh, India
 LIST OF CONTRIBUTORS vii

Sandeep Kumar Sharma S.K. Tripathy

Department of Applied Science Department of Physics
Desh Bhagat University Panjab University
Mandi Gobindgarh, India Chandigarh, India

Sharvan Sehrawat Satish K. Tuteja

Faculty IWE1eInstitut für Werkstoffe der Elektrotechnik1
Indian Institute of Science Education and Research RWTH Aachen University
Mohali (IISER Mohali) Germany
Mohali, India
This page intentionally left blank
Preface
An ever-increasing demand of routine tests of various
biological and environmental parameters is driving the
development of portable and user-friendly diagnostic
tools. Many of such tools are nowadays available in
different forms including papers, electrodes, chips, kits,
etc. A growing significance of portable detection tools
has placed them as useful alternatives to conventional
analytical techniques like spectroscopy and biochemical
tests. In order to attain sufficient competitiveness, the
modern-day portable detection tools and diagnostic
devices are being integrated with nanotechnology. The
application of nanomaterials and nanosurfaces has
enabled the researchers to design highly sensitive nano-
sensors that can match the performance of laboratory-
based instruments. Owing to the large surface areas
and various other material properties, the nanosensors
offer many significant advantages, such as high sensi-
tivity, robust attachment of analyte specific receptors,
label-free sensing, signal reproducibility, etc. Recent
decades have witnessed the emergence of carbon nano-
tubes, graphene, metal nanoparticles, quantum dots,
and transition metal dichalcogenides as transducer sur-
faces of choice in the development of nanosensors for
both environmentally and clinically important parame-
ters. This book provides a comprehensive overview on
the most important types of nanosensor platforms
explored and developed in the recent years for efficient
detection of environmental/clinical analytes. It covers
the information in such a way that the readers could
grasp knowledge on important aspects of nanosensor
development, e.g., material synthesis, their functionali-
zation with different receptors, and techniques to record
signals. A commercial perspective of the nanosensor
market is also discussed.
Chapter 1 of the book elaborates the applications of
carbon-based nanomaterials in the development of
various nanosensors. Carbon nanomaterials have
become immensely popular among the researchers
worldwide for designing sensitive sensors on account of
their different useful properties. Carbon nanomaterials
can be synthesized in different forms such as nanosheets,
nanotubes, fullerene, and quantum dots. Consequently,
these nanomaterials have opened up numerous oppor-
tunities in order to seek the realization of nanosensors
for diverse environmental and clinical parameters. As
Chapter 2 discusses, 2-dimensional transition metal
dichalcogenide (2D TMD) nanomaterials have been
touted as promising alternatives to carbon nano-
materials in recent years. Molybdenum- and tungsten-
based disulfides are two important examples in this
category. Atomically thin 2D TMDs possessing direct
band gap property provide the development of plat-
forms for the sensing of pH, gases, heavy metals, pro-
teins, etc. Along with carbon nanomaterials, 2D TMDs
are suitable to be integrated as lab-on-chip type of
devices.
The utility of conducting polymers in the develop-
ment of sensing systems is widely recognized. Chapter 3
elaborates information on the synthesis and sensing
applications of various conducting polymers. In addi-
tion to this, the characteristics and applications of con-
ducting polymer composites with other nanomaterials
have also been outlined. Metaleorganic frameworks
(MOFs) are a class of coordination polymers which are
attracting significante attention of researchers world-
wide. Chapter 3 also focuses on this trending topic of
developing MOF-based sensors. Some extraordinary
material characteristics of MOFs (e.g., their high surface
area, tunable porosity, formation in different forms) are
the factors inspiring scientists and researchers to explore
them in the sensing of gases, proteins, explosives, anions,
cations, antigens, etc. Chapter 4 is dedicated to discuss
different biorecognition molecules and methods of their
isolation and production. Biomolecules are an impor-
tant component of many sensors wherein they help to
attain the desired selectivity during analysis. Nucleic
acids, enzymes, aptamers, and antibodies are the main
categories of biomolecules used in the development of
selective sensors. Nowadays, specific biomolecules are
available in the market to target the selective recognition
of analytes such as antigens, pesticides, heavy metals, etc.
Chapter 5 provides information about the techniques
to modify the nanosurfaces with biomolecules. The
contents of this chapter would help the readers to
ix
x PREFACE
understand different covalent and physical
attachmentebased chemistries in order to immobilize
biomolecules over surfaces of nanomaterials. A robust
immobilization of biomolecules is immensely critical to
define the stability, accuracy, and shelf life of nano-
biosensors. The utilization of nanomaterials in the
fabrication of easy-to-use paper and lateral flow sensors
is described in Chapter 6. These simple sensing devices
find important application in soil, air and, water quality
monitoring due to their low cost and convenient
handling. The advantages and existing limitations/chal-
lenges of nanosensors have been discussed in Chapter 7.
Finally, commercial aspects and an overview of currently
available sensors for various analytes are mentioned in
Chapter 8.
The nanosensor market is growing at an exponential
pace. Important features like fast response, portable
instrumentation, and cost-effectiveness are major factors
behind the escalating demand of these point-of-care
devices. They not only match the detection limits of
laboratory-based instruments but also serve the purpose
utilizing much smaller sample volumes. Nanosensors
are being accepted as preferred tools by clinical labora-
tories, regulatory agencies, and common users. Their
integration with or into smartphones and wearable de-
vices is one of trending technologies of the 21st century.
This book provides information on the most useful
nanosurfaces that are being employed for the develop-
ment of nanosensor. Critical aspects, such as the type of
nanomaterials, their conjugation with biomolecules,
methods of signal collections, applications, challenges,
advantages and limitations, and commercial aspects
have been covered. The book is written for readers from
different related backgrounds including nanotech-
nology, biochemistry, environmental science, chemistry,
material science, and biomedical engineering.
Contents
1 Carbon-Based Nanomaterials for the
Development of Sensitive Nanosensor
Platforms, 1
Monika Nehra, Neeraj Dilbaghi,
Ashraf Aly Hassan and Sandeep Kumar
2 Advances in the Synthesis and
Development of Two-Dimensional
Transition-Metal Dichalcogenide-Based
Nanosensor Platforms, 27
Sanjeev Kumar Bhardwaj, Neha Bhardwaj,
Deepanshu Bhatt, Poonma Malik and
Akash Deep
3 Conducting Polymers and
Metal-Organic Frameworks as
Advanced Materials for Development
of Nanosensors, 43
Moondeep Chauhan, Sanjeev Kumar Bhardwaj,
Gaurav Bhanjana, Rajeev Kumar,
Neeraj Dilbaghi, Sandeep Kumar and
Ganga Ram Chaudhary
4 Synthesis and Production of Different
Biomolecules for Application in the
Sensing of Environmental Pollutants, 63
Manpreet Kaur, Madhu Khatri and
Sharvan Sehrawat
5 Bioconjugation of Different
Nanosurfaces With Biorecognition
Molecules for the Development
of Selective Nanosensor Platforms, 79
Satish K. Tuteja, Ruchi Mutreja,
Suresh Neethirajan and
Sven Ingebrandt
6 Development of Disposable Sensor
Strips for Point-of-Care Testing of
Environmental Pollutants, 95
Satish Kumar Pandey, Girish Chandra Mohanta
and Parveen Kumar
7 Advantages and Limitations
of Environmental Nanosensors, 119
Rajnish Kaur, Sandeep Kumar Sharma and
S.K. Tripathy
8 Commercial Aspects of Biosensors
for Diagnostics and Environmental
Monitoring, 133
Samadhan B. Patil, Valerio F. Annese
and David R.S. Cumming
Index, 143
xi
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CHAPTER 1
Carbon-Based Nanomaterials for the
Development of Sensitive Nanosensor
Platforms
MONIKA NEHRA • NEERAJ DILBAGHI • ASHRAF ALY HASSAN •
SANDEEP KUMAR
INTRODUCTION
Carbon is one of the most commonly found ele-
ments in nature and its understanding has reached
new levels, from macroscopic to nanoscale, with
continuous advancement in nanotechnology.
The nanostructures of carbon in their different
forms have been applied in diverse fields such as
field emission displays, nanoelectronics, energy
conversion and storage, biological and chemical
sensors, and theranostics. In the 21st century,
owing to their extraordinary properties in terms
of structural perfection, carbon nanomaterials
have in fact led general science to many advanced
avenues. The structural properties of carbon mate-
rials have inspired the synthesis of novel nanoma-
terials with similar symmetries and structures, e.g.,
fullerenes, nanotubes, nanodots, and graphene.
Carbon atoms have the ability to form robust
mutual covalent bonds in different hybridization
states such as sp, sp2, and sp3. Carbon atoms also
interact with nonmetallic elements leading to
the formation of a wide range of structures from
small molecules to long chains. The major classi-
fication of carbon materials (such as carbon, dia-
mond, and graphite) is based on the way of
interconnection between carbon atoms, e.g., tetra-
hedral sp3 atom configuration in case of diamond
and hexagonal sp2 carbon atom configuration in
case of graphene monolayers. However, mixed
states also exist and lay the basis for nanocrystal-
line diamond, diamond-like carbon, and
Advances in Nanosensors for Biological and Environmental Analysis. https://doi.org/10.1016/B978-0-12-817456-2.00001-2
Copyright © 2019 Elsevier Inc. All rights reserved.
amorphous carbon. The carbon nanoallotropes
belonging to same group or same arrangement
of carbon atoms have common properties; how-
ever, there are significant differences because of
their different sizes and shapes.
A summary of carbon nanomaterials with
different morphologies but unique chemical
properties is shown in Fig. 1.1 (Yan et al., 2016).
They can be classified according to their structural
dimensionality as, e.g., (1) 0D nanostructures
(fullerenes, carbon dots, nanodiamonds, etc.),
(2) 1D nanostructures (carbon nanotubes
[CNTs]/carbon nanofibers [CNFs], etc.), and (3)
2D nanostructures (graphene, graphene nanorib-
bons, etc.). Diamond, a metastable state of car-
bon, consists of a 3D cubic lattice with 3.57 Å
lattice constant along a CeC bond length of
1.54 Å (Sque et al., 2006). In contrast, graphite
possesses a 2D layered structure having a CeC
bond length of 1.42 Å (Baughman et al., 1987).
In graphene, the layers are single atom thick and
interact through van der Waals forces having
3.35 Å interlayer spacing. Graphite corresponds
to the most thermodynamically stable form of car-
bon at room temperature. Graphene is commonly
referred as a 2D building block of sp2 hybridized
carbonaceous nanomaterials; it can be rolled
and/or distorted in order to form CNTs and fuller-
enes. The first successfully synthesized carbon
nanomaterial was C60 (also known as buckmin-
sterfullerene), through laser ablation of graphite
1
2 Advances in Nanosensors for Biological and Environmental Analysis
FIG. 1.1 Broad family of carbon nanomaterials. (Adapted from Yan, Q.L., Gozin, M., Zhao, F.Q.,
Cohen, A., Pang, S.P., 2016. Nanoscale 8, 4799e4851. Reprinted with permission from RSC.)
under helium flow (Kroto et al., 1985). However,
some reports also exist on the development of
even-numbered carbonaceous clusters before the
development of C60, but these clusters were un-
suitable for characterization because of their large
size distributions (Rohlfing et al., 1984). Fuller-
enes are viewed as the 0D form of graphitic carbon
and also referred as irregular sheets of graphene
that are curled in the form of a sphere via penta-
gons incorporation in the structure. Furthermore,
CNTs were isolated as an offshoot during the syn-
thesis of fullerene. The elongation of fullerene in
one dimension assumes the structure of CNTs
with high aspect ratios (e.g., from 102 to 107).
Different synthesis methods have been devel-
oped for the production of both single-walled
CNTs (SWCNTs) and multiwalled CNTs
(MWCNTs). These include arc discharge (Arora
and Sharma, 2014), chemical vapor deposition
(CVD) (Kumar et al., 2017a), and high-pressure
carbon monoxide method (Liu et al., 2011a).
The initial development of graphene was done
by its growth on insulating substrates by Geim
and coworkers (Novoselov et al., 2004). In the
current perspective, graphene can be referred as
the mother of all graphitic carbon. Graphene has
further been investigated as monolayer to a few-
layered nanomaterial depending on the thickness
requirement in a particular application (Liu et al.,
2018).
Carbon nanomaterials can possibly cover the
characteristics of different substances on the earth,
such as hardest to softest materials, insulators to
semiconductors and further to superconductors,
and fully light-absorbing to completely trans-
parent materials. The superiority of carbon nano-
materials is basically due to their hardness,
radiation characteristics, optical properties, elec-
tric conductivity, chemical resistance, heat resis-
tance, electric insulation, and surface/interface
properties in comparison to many other materials.
CARBON-BASED NANOMATERIALS FOR
NANOSENSOR DEVELOPMENT
Nanosensors are becoming a crucial part of mod-
ern lifestyle, particularly in healthcare due to the
demand of point-of-care devices, personalized
medicine, and cheaper and reliable diagnostic
tools. Carbon nanomaterials have motivated re-
searchers to implement them as ideal transduc-
tion materials mainly because of their geometry,
fast electron transfer kinetics, wide potential win-
dow, low residual current, fluorescent properties,
and readily renewable surfaces (Jariwala et al.,
2013). Voluminous research efforts have been
dedicated to employ carbon nanomaterials in
the development of highly sensitive and selective
nanosensors.
Fabrication of Sensing Platforms
The device architecture of electrochemical nano-
sensors can be broadly classified into two main
categories: (1) the conventional three-electrode
 CHAPTER 1 Carbon-Based Nanomaterials for Nanosensor Development 3

FIG. 1.2 (A and B) A schematic representation of functionalized carbon nanomaterialebased

electrochemical biosensors. (Adapted from Yang, Y., Yang, X., Yang, Y., Yuan, Q., 2018a.
Carbon 129, 380e395. Reprinted with permission from Elsevier.)

setup and (2) the chemiresistive/field-effect tran-
sistor (FET) setup. The three-electrode setup is
commonly used for potentiometric, ampero-
metric, and impedimetric nanosensors that
comprise working, counter, and reference elec-
trodes for detection of different analytes
(Fig. 1.2) (Yang et al., 2018a). Carbon nanomate-
rials have been popularly used to modify the sur-
face of glassy carbon electrode to develop them as
a working electrode. As such, the electrochemical
sensing techniques are particularly helpful in un-
derstanding the behavior of analytes and their
associated electrochemical reaction mechanisms.
The analyte species that can undergo redox transi-
tions on glassy carbon electrodes can be detected
via different categories of electrochemical tech-
niques, e.g., amperometry and potentiometry.
The transfer of ions takes place through a conduc-
tive electrolyte medium. Carbon nanomateriale
based electrodes offer several benefits in terms of
wide potential window, good electrocatalytic ac-
tivities, and chemical inertness during redox reac-
tions. In electrochemical biosensors, carbon
nanomaterials also serve as a platform for biomol-
ecule immobilization, thereby improving electro-
chemical transduction (Hu et al., 2016).
Chemiresistive/FET sensors have attracted
considerable attention because of their enormous
benefits in terms of their fast response time, seam-
less integration with electronic manufacturing
processes, potential for miniaturization, and
parallel sensing (Bandodkar et al., 2016; Barbaro
et al., 2012). Carbon nanostructures, such as
CNTs, have been reported as excellent electrode
material for gas sensors in order to detect the gas
molecules, with high sensitivity, low device cost,
and fast response time even at room temperature
(Dube et al., 2015). In a similar manner, the
detection of gas molecules through graphene is
based on the change in their electric conductivity
as a result of the formation of surface adsorbates.
These surface adsorbates can work as either do-
nors or acceptors depending on their chemical na-
ture, preferential adsorption sites, and the
surrounding environment (Varghese et al., 2015).
The different formats of FET sensors may be
listed as ion-sensitive FET, unmodified comple-
mentary metal-oxide semiconductor, extended-
gate FET, floating-gate FET, and dual-gate FET
(Ramnani et al., 2016). Carbon nanomaterials
have been found very useful to function as the
functional channel in chemiresistor/FET nanosen-
sor configuration; for example, both the tubular
geometry of CNTs and the planar geometry of gra-
phene ensure maximum exposure of surface
atoms for the binding of target analyte molecules
to the electrode material. The Debye length (lD) is
a measure of field penetration into bulk materials
and also causes significant modulation in the elec-
tronic properties of electrode materials upon
exposure to the analytes. The lD is comparable
to the dimensions of the carbon nanostructures,
4 Advances in Nanosensors for Biological and Environmental Analysis
which ensures label-free sensing of analytes with
relatively low limits of detection and high sensi-
tivities. Carbon nanomaterials in an FET configu-
ration can detect multiple analytes concurrently
(Cullen et al., 1990). The fabrication of
graphene-FET devices requires the bulk produc-
tion of graphene films via different synthesis tech-
niques such as the CVD method, which offers
control over crystallinity, grain size, and number
of layers over the desired substrate (Srivastava
et al., 2010). CNTs can be synthesized as cylindri-
cal tubes by rolling up single and/or multiple gra-
phene sheets. The nature of CNTs, either metallic
or semiconducting, depends on their chirality
(armchair, zigzag, or chiral) and the diameter of
the tubes (Dresselhaus et al., 2004). For instance,
armchair SWCNTs are metallic in nature and,
therefore, these cannot be used for fabrication of
FET nanosensors.
Surface Functionalization of
Nanocarbon Electrodes
The surface chemistry of electrode materials has
significant importance with respect to their inter-
action with analytes present in complex solu-
tions/matrices. The surface functionalization of
carbon nanomaterials has been reported through
covalent/noncovalent interactions and/or decora-
tion with some inorganic nanomaterials (Liu
et al., 2015; Karimi et al., 2015; Balasubramanian
and Burghard, 2005). Carbon nanomaterials with
multiple functional groups at their surface/edges
can satisfy the specific requirements of different
kinds of sensors with specific intermolecular inter-
actions. For instance, graphene plays a significant
role in electrochemical sensing application
because of its high carrier mobility, exceptional
electrochemical properties (electron transfer
rates), optical properties, and structural character-
istics. Furthermore, its properties can be
controlled/modulated through adoption of suit-
able preparation methods and or functionaliza-
tion to generate specific target-sensing properties
(Kybert et al., 2014). Likewise, the presence of
functional groups such as carboxyl and amine
makes CNTs compatible in their conjugation
with biomolecules as well as other materials
such as metallic nanoparticles (Kong et al., 2001;
Gao et al., 2012). For covalent functionalization,
carboxylic (eCOOH) groups can be introduced
on the edge planes and sidewalls of the carbon
nanomaterials through oxidation. Some forms of
carbon such as reduced graphene oxide have
readily available eCOOH groups. As a matter of
concern, the covalent functionalization can deteri-
orate the sp2 structure of the honeycomb lattice of
carbon while also inducing some defects on their
surface (such as disruption in p-electronic
network), ultimately resulting in deflation of elec-
tronic properties of the materials. Noncovalent
functionalization can be a solution to this limita-
tion, which does not influence the intrinsic struc-
ture of carbon nanomaterials and also maintains
their electronic and mechanical properties
(Georgakilas et al., 2012).
The functionalization of carbon nanomaterials
helps in improving their dispersibility, biocom-
patibility, and sensing properties. In literature,
carbon nanomaterials have been used in two ma-
jor ways to modify the biosensing electrodes: (1)
modification of the bulk material, for example,
mixing the electrode material with carbon paste
and (2) modification of the electrode surface, for
example, preparation of films on premade elec-
trode through CVD or any other method. The
incorporation of carbon paste in electrode mate-
rials (e.g., other nanomaterials) enhances the
overall electrocatalytic properties and supports
improved signal transduction (Fig. 1.3) (Huang
et al., 2006). Furthermore, the application of car-
bon paste in immobilizing the enzymes can offer
protection of these protein molecules from the
external environment, while also rendering better
stability and durability in comparison to the sys-
tem having enzyme immobilized over the bare
electrode surface (Akyilmaz et al., 2017).
APPLICATIONS OF NANOCARBON
ELECTRODES FOR SENSING OF
DIFFERENT ANALYTES
In the recent years, the nanosensor platforms have
gained significant research interest for sensitive
detection of a wide variety of analytes including
both chemical (such as dopamine, ascorbic acid,
uric acid, and norepinephrine) and biological
 CHAPTER 1 Carbon-Based Nanomaterials for Nanosensor Development 5

FIG. 1.3 A schematic representation of the different strategies used to design functionalized
carbon nanotube electrodes. SWCNTs, single-walled carbon nanotubes. (Adapted from Huang,
X.J., Im, H.S., Yarimaga, O., Kim, J.H., Jang, D.Y., Lee, D.H., Kim, H.S., Choi, Y.K., 2006. Journal of
Electroanalytical Chemistry 594, 27e34. Reprinted with permission from Elsevier.)

(such as airborne bacteria, folic and pantothenic
acid, protein, and mycotoxins). The use of carbon
nanomaterials as the functional electrode surface
of nanosensor platform offers several advantages
in terms of good electrocatalytic activity, enhanced
interfacial adsorption properties, fast electron
transfer kinetics, and high biocompatibility in
comparison to traditional materials. There are
different strategies for incorporation of these
nanomaterials into electrochemical sensors, e.g.,
drop casting (Kaniyoor et al., 2009), direct growth
on a substrate (Wang et al., 2009), polymer-based
coatings (Barsan et al., 2015), use of binders such
as Nafion or dihexadecyl hydrogen phosphate
(Liao et al., 2015), and screen printing (Chen
et al., 2016). In comparison to drop casting or
dip coating, the direct growth of the carbon nano-
materials over electrode surface provides more ho-
mogeneous coating as well as supports the batch
fabrication of nanosensors (Gooding, 2005).
Furthermore, polymer-based coatings can aid in
the physical and chemical properties of the carbon
nanomaterials as well as their dispersion for depo-
sition. Additional incorporation of metallic nano-
particles in polymer matrix is sometimes desired
to maintain the requisite level of electrode conduc-
tivity (Chun et al., 2010).
Detection of Heavy Metal Ions
The quantification/detection of heavy metal ions
(including Hg2þ, Cd2þ, Cu2þ, Pb2þ, As3þ, etc.) is
a major concern among chemists, environmental-
ists, and biologists due to their toxic nature as well
as prolonged persistence in the biosphere. The
heavy metal ion contamination has mainly been
caused by the fast industrial development and
related activities such as electroplating, battery
manufacturing, mining, and smelting. The metal-
lurgical industries release various harmful toxins
into our environment, which include nonbiode-
gradable chemicals and heavy metals. The exis-
tence of these contaminants, especially heavy
metal ions in water bodies, has direct influence
over the health of living systems (Bhanjana
et al., 2015, 2017). The conventional methods
for the analysis of heavy metal contamination
are based on different techniques such as atomic
absorption spectroscopy (AAS) (Luo et al.,
2015), atomic emission spectroscopy (Zhang
et al., 2014), inductively coupled plasma mass
spectrometry (ICP-MS) (Li et al., 2015), and
X-ray fluorescence spectrometry (Sitko et al.,
2015). These techniques are very costly and not
appropriate for on-site analysis. Moreover, these
approaches can only quantify the total amount
of heavy metals and it is not possible to analyze
the bioavailable concentrations that are accessible
to the living bodies. Electrochemical detection us-
ing nanosensors is among the simple, accurate,
and sensitive methods for the detection of metal
contamination in food and environment. The
electrochemical sensing techniques offer several
6 Advances in Nanosensors for Biological and Environmental Analysis
benefits in terms of low cost, portability, high
sensitivity, short analytical time, and easy adapt-
ability for in situ detections (Bhanjana et al.,
2016). The working electrode of the three-
electrode electrochemical system can be modified
with nanomaterials for improving the sensitivity
of the nanosensors (Cheng et al., 2018). Such
nanosensors are then operated by recording
changes in their potential, current, electrochemi-
cal impedance, and electroluminescence upon
the recognition of an analyte (Simpson et al.,
2018).
Among nanomaterials, carbon nanomaterials
are most interesting materials as adsorbents/
preconcentrator agents or transducer materials
used in the development of nanosensors. The car-
bon nanomaterials can respond to both organic
and inorganic analytes. The functionalization of
carbon nanomaterials with biological recognition
elements (e.g., enzymes, antibodies, DNA, or mi-
croorganisms) enables highly specific and sensi-
tive sensing of metal ions (Wanekaya et al.,
2008). In particular, DNA-based nanosensors
have gained much research interest in the recent
years for the detection of heavy metal ions owing
to their stability in biological pH environments
(Primo et al., 2015). Wen et al. (2018) have re-
ported the application of DNA-modified gra-
phene oxide/Prussian blue nanoparticles for
arsenite detection. The interaction of graphene ox-
ide with 50 -thiolate-labeled (GT)21-ssDNA facili-
tated the generation of Prussian blue
nanoparticles on gold electrode surface. The
(GT)21-ssDNAecontaining arsenite recognition
sequence offered excellent specificity for arsenite
detection (detection limit down to 0.058 ppb)
in real water samples. DNAzyme-functionalized
carbon nanostructureebased biosensors have
also been developed for some other metal ions
including Pb2þ, Hg2þ, and Cu2þ (Zhou et al.,
2016).
The utility of carbon nanomaterials has also
been demonstrated in the development of sensi-
tive chemosensors. For example, Lu et al. (2018)
proposed a novel 3D honeycomb structure of
N-doped carbon nanosheet framework decorated
with bismuth nanoparticles (Bi-NCNF) for selec-
tive and sensitive electrochemical sensing of
Pb2þ and Cd2þ (Fig. 1.4). The proposed system
offered a detection limit of 0.04 and 0.02 mg L1
for Pb2þ and Cd2þ, respectively.
In many reported cases, the performance of car-
bon nanomaterialebased nanosensors for detect-
ing heavy metal ions has been successfully
validated with standard techniques such as AAS
and ICP-MS. A summary of the recently developed
glassy carbonebased nanosensors (electrode sur-
face modified with different carbon nanostruc-
tures) for heavy metal detection is provided in
Table 1.1, which is intended to acquaint the
readers about the main design and performance
parameters of these related technologies. The
coupling of nanosensitive platform with carbon
nanomaterials has resulted in high sensitivity,
fast response, multianalyte detection, and low
detection limits. In spite of numerous benefits,
there are a few challenges associated in this area:
(1) accurate detection of metal ions in biological
samples (involving blood, saliva, urine, etc.), (2)
issue of false-positive signal interference and
chemical fouling, (3) detection of heavy metal
ions in their complex form, and (4) continuous
monitoring of water resources for detection of
metal ions (Gumpu et al., 2015). As per the
commercialization perspective, the research ef-
forts should be directed toward reusability, mass
production, and system integration.
Detection of Food Additives and
Pesticide Residues
The innovation in food industry is increasing at an
immense speed in terms of development and
application of pesticides, food additives, and ma-
terials for food protection/processing/coating/
packaging. Pesticides are excessively used in agri-
cultural activities to enhance the production yield
by controlling pests, insects, weeds, etc. Food ad-
ditives are introduced to deliver functional attri-
butes in order to improve food life/safety.
Undesirably high concentrations of food additives
and accidental contamination of veterinary drug
residues and pesticides in foodstuff and water re-
sources have become issues of major health
concern. The pesticide residues are very toxic sub-
stances and can lead to several health issues (such
as cholinergic dysfunction) in both humans and
 CHAPTER 1 Carbon-Based Nanomaterials for Nanosensor Development 7

FIG. 1.4 A schematic layout for fabrication of 3D N-doped carbon nanosheet framework
decorated with bismuth nanoparticles (Bi-NCNF) followed by their application for electrochemical
detection of Pb2þ and Cd2þ. EG, ethylene glycol; GCE, glassy carbon electrode; NPs, nanoparticles;
PVP, polyvinylpyrrolidone. (Adapted from Lu, Z., Dai, W., Lin, X., Liu, B., Zhang, J., Ye, J., Ye, J.,
2018. Electrochimica Acta 266, 94e102. Reprinted with permission from Elsevier.)

animals. The analysis of various types of contam-
inations (i.e., toxic food additives, veterinary drug
residues, and pesticides) that can affect our food
samples is usually carried out using methods
such as high-performance liquid chromatography
(HPLC) (Wahed et al., 2016), HPLC-mass spec-
trometry (Hoffmann et al., 2017), gas
chromatography-mass spectrometry (Jiménez-
Salcedo and Tena, 2017), and capillary electro-
phoresis (Omar et al., 2017). The development
of nanosensors has been reported based on elec-
trochemical biosensing of contaminants in food-
stuff and water. The electrochemical biosensors
offer fast operation and high selectivity, sensi-
tivity, and reproducibility (Rotariu et al., 2016;
Herzog et al., 2008).
The incorporation of carbon nanostructures en-
hances the loading of bioreceptors on the elec-
trode surface, apart from providing high
stability. Carbon nanoparticles also serve as a
relay for transfer of electrons between biomole-
cules and the electrode. Elyasi et al. (2013) re-
ported a Pt/CNT nanocompositeemodified
ionic liquid carbon electrode for specific determi-
nation of Sudan I (a coloring agent) with an excel-
lent limit of detection (0.003 mM). The
immobilization of an enzyme (from Inga edulis)
onto a carbon paste electrode containing
MWCNTs and Nafion has been reported to pro-
vide a highly selective and sensitive detection of
tert-butylhydroquinone (TBHQ, an antioxidant),
with quantification and detection limits of 1.25
and 0.41 mg L1, respectively (de Oliveira et al.,
2014). This sensing method can offer determina-
tion of TBHQ in commercial salad dressing sam-
ples with an acceptable level of accuracy (e.g.,
the relative error limiting to 5.4%). Likewise, a
gold-modified carbon paste electrode has been
demonstrated for electrochemical sensing of syn-
thetic dyes (e.g., Sunset Yellow and tartrazine) in
 8
TABLE 1.1
List of Different Carbon Nanomaterials Used in Glassy Carbon ElectrodeeBased Nanosensors of Heavy Metal Ions.
Metal Ion Details of the Electrode Interface Limit of

Advances in Nanosensors for Biological and Environmental Analysis

Detected Detection Mechanism Material/s Detection Sensitivity References
1 3
Cadmium Anodic stripping Cysteine functionalized SWCNTs 0.3 mg L (9.7  2.5)  10 Gutierrez et al.
voltammetry mA mg L1 (2017)
Differential pulse Reduced graphene oxide-chitosan/ 0.01 mg L1 d Guo et al. (2016)
anodic stripping ply-L-lysine nanocomposites
voltammetry
Square wave anodic Phenylsulfonic groupegrafted 0.08 mM d Chen et al. (2018)
stripping voltammetry MWCNTs with dye molecules
Square wave Calixarene-functionalized reduced 2  1011 M d Göde et al. (2017)
voltammetry graphene oxide
Cyclic voltammetry Ruthenium(II)-textured graphene 2.8 nM 3.43 mA mM1 Gumpu et al. (2017)
oxide nanocomposite
Arsenic Differential pulse Gold-coated, boron-doped diamond 0.005 mg L1 9.7  2.5 mA mg L1 Song and Swain
anodic stripping thin film (2007)
voltammetry
Anodic stripping Gold nanoparticleemodified carbon 0.9 mg L1 0.0176 nA mg L1 Carrera et al. (2017)
voltammetry fiber ultramicroelectrodes
Cyclic voltammetry Clay-modified carbon paste 5e40 mg L1 d Tiwari and Lee
(2017)
Flow injection analysis Gold nanoparticleedecorated 11.4 mg L1 218.1 nA mg L1 Nellaiappan et al.
carbon nanofiber-chitosane (2018)
modified carbon
Cyclic voltammetry DNA-modified graphene oxide/ 0.058 mg L1 d Wen et al. (2018)
Prussian blue nanoparticles
Fluorescence Carbon quantum dots 0.086 mg L1 d Pooja et al. (2017)
spectrophotometry
Square wave anodic Amine-functionalized graphene 0.162 mg L1 130.631 mA Yang et al. (2017)
stripping voltammetry oxideedecorated gold nanoparticles mg L1 cm2
Cyclic voltammetry Ruthenium(II)-textured graphene 2.3 nM 2.11 mA mM1 Gumpu et al. (2017)
oxide nanocomposite
 Mercury Anodic stripping CNT/asymmetric 0.36 nM d Selvan and
voltammetry N4 tetradentate Schiff base ligand Narayanan (2018)
N,N0 -bis(pyrrole-2-ylmethylene)-2-
aminobenzylamineecoated graphite
Cyclic voltammetry Polypyrrole/carbon nanofiber 0.05 mg L1 d Oularbi et al. (2017)
nanocomposite

CHAPTER 1 Carbon-Based Nanomaterials for Nanosensor Development

Cyclic voltammetry Ruthenium(II)-textured graphene 1.6 nM d Gumpu et al. (2017)
oxide nanocomposite
Lead Square wave anodic Nitrogen-doped and thiol groupe 0.3 mg L1 d Li et al. (2016)
stripping voltammetry grafted MWCNTs
Anodic stripping CNT/asymmetric 1.1 nM d Selvan and
voltammetry N4 tetradentate Schiff base Narayanan (2018)
ligand N,N0 -bis(pyrrole-2-
ylmethylene)-2-aminobenzylaminee
coated graphite
Square wave Carbon paste electrode 3.8 pM 20683 A L mol1 Alizadeh et al. (2017)
voltammetry impregnated with ion-imprinted
polymer and MWCNTs
Square wave Calixarene functionalized reduced 2  1011 M d Göde et al. (2017)
voltammetry graphene oxide
Cyclic voltammetry Ruthenium(II)-textured graphene 1.41 nM d Gumpu et al. (2017)
oxide nanocomposite
Differential pulse Reduced graphene oxide-chitosan/ 0.02 mg L1 d Guo et al. (2016)
anodic stripping ply-L-lysine nanocomposites
voltammetry
Copper Square wave anodic Graphene quantum dots/gold 0.05 nM 3.69 mgA/nM Ting et al. (2015)
stripping voltammetry nanoparticles
Differential pulse Biochar-modified carbon paste 4.0  107 M d Oliveira et al. (2015)
anodic stripping
voltammetry
Chromium Cyclic voltammetry Manganese oxide nanoflakes/ 0.3 mM 18.7 nA mM Salimi et al. (2015)
MWCNTs/chitosan nanocomposites
Linear sweep Gold nanoparticleedecorated 5.4 mg L1 1.1 nA mg L1 Tu et al. (2018)
voltammetry screen-printed carbon electrode

CNT, carbon nanotube; MWCNTs, multiwalled CNTs; SWCNTs, single-walled CNTs.

9
10 Advances in Nanosensors for Biological and Environmental Analysis

commercially available soft drinks (Ghoreishi
et al., 2012).
The detection of pesticide residues (organo-
phosphate, organochlorine, carbamates, pyre-
thrum, etc.) is generally carried out using gas or
liquid chromatography. This technique is highly
sensitive and selective, but requires expensive
instrumentation and skilled personnel. Electro-
chemical biosensors are regarded as the potential
choice for pesticide detection with desired selec-
tivity, sensibility, and reproducibility. For
example, a screen-printed electrode has been
modified with carbon nanoparticles. After subse-
quent immobilization with butyrylcholinesterase,
the electrode could detect the presence of para-
oxon in spiked wastewater samples. This biosensor
electrode was stable up to 78 days at room temper-
ature under dry conditions. The inhibition of the
enzyme activity was directly correlated with the
concentration of paraoxon (up to 30 mg L1).
Many other label-free nanosensor platforms have
also been designed for the detection of pesticides.
For instance, Li et al. (2018a) have reported the
application of acetylcholinesterase (AChE)-immo-
bilized fluorescence carbon dots for dual-mode
(fluorometric as well as colorimetric) detection
of organophosphate pesticides (Fig. 1.5). This
sensor functioned on the principle of generation
of fluorometric and colorimetric responses as a
result of the formation of a yellow reaction prod-
uct (5-thio-2-nitrobenzoic acid [TNBA]) after the
decomposition of 5,5-dithiobis(2-nitrobenzoic
acid), which was triggered by the reaction of
AChE with acetylthiocholine. TNBA functioned
as a powerful absorber for quenching the fluores-
cence of carbon dots. Hence, this method could
be used for the detection of pesticides, as the inhi-
bition in enzyme activity of AChE led to the resto-
ration of fluorescence signal alongside a reduction
in the absorbance intensity.
Research efforts have also been directed toward
the development of nonenzymatic nanosensors
for pesticides. Hsu et al. (2017) reported the
peroxidase-like activity of Ag nanoparticlee
decorated oxidized MWCNTs, which could be
exploited in the fluorometric assay of dimethoate.
This method offered good selectivity for
sensing dimethoate in a linear range of
0.01e0.35 mg mL1 from lake water and fruit
samples. Facure et al. (2017) reported a novel
enzyme-free impedimetric electronic tongue
(e-tongue) comprising graphene hybrid nano-
composites for sensing the trace levels of organo-
phosphate pesticide mixture (i.e., malathion and
cadusafos). This e-tongue system detected the
presence of organophosphate at nanomolar con-
centrations (as low as 0.1 nM) in real samples.
The use of reduced graphene oxide, containing

FIG. 1.5 Dual signalebased (fluorescence and colorimetry) detection of organophosphate

pesticides using acetylcholinesterase (AChE)-immobilized fluorescence carbon dots (CDs).
DTNB, 5,50 -dithiobis-(2-nitrobenzoic acid); OPs, organophosphates; TNBA, 5-thio-2-nitrobenzoic
acid. (Adapted from Li, H., Yan, X., Lu, G., Su, X., 2018a. Sensors and Actuators B: Chemical 260,
563e570. Reprinted with permission from Elsevier.)
 CHAPTER 1 Carbon-Based Nanomaterials for Nanosensor Development
the residual oxygenecontaining functional
groups, offered the realization of a sensing plat-
form with high conductivity. The functional
groups behaved as active sites for the sensing of
pesticide residues and increased the sensitivity of
the system. Table 1.2 lists some important carbon
nanomaterialebased electrochemical nanosen-
sors used for the detection of food additives and
pesticide residues.
Detection of Bacterial Pathogens or
Viruses
Bacterial pathogens and viruses are potential
threats to human health. Therefore the develop-
ment of tools for simple and fast detection or
diagnosis of bacterial pathogens is crucial to con-
trol their outbreak and to ensure appropriate ther-
apeutic treatments. The existing methods of
detection (including polymerase chain reaction
and enzyme-linked immunosorbent assay) are
sensitive enough and selective, but they involve
complex steps of sample preparation and take
long assay times (Berg et al., 2015; Nguyen
et al., 2017). Several evidences have been pro-
jected in the literature about the potential useful-
ness of biosensors for the analysis of bacterial
pathogens. Carbon nanomaterialemodified
glassy carbon (Dekanski et al., 2001) and pyro-
lytic graphite electrode (Banks and Compton,
2005) have been usefully exploited in this context.
In one of the important reports, Gheith et al.
(2006) suggested that lateral currents in highly
conductive SWCNT multilayers can cause the
stimulation of neural cells. Therefore CNTs have
a future in biomedical devices in which electrically
responsive cells, such as the muscle cells, endo-
crine cells, can be examined at even single-cell
level. CNTs can be used in systematic biosensors
in order to monitor the immune response in
case of immunodeficient patients (Fadel et al.,
2008). These studies led to the development of
cancer antibodyefunctionalized SWCNTs for the
thermal ablation of tumor cells (Kostarelos
et al., 2009). The selective nature of the treatment
makes SWCNTs promising for biosensing applica-
tions as antibody-functionalized SWCNTs track
only cancer cells upon irradiating them with
infrared energy. The high sensitivity and selectivity
11
of carbon-based electrochemical biosensors facili-
tate the sensing of the pathogens even in the com-
plex sample matrices (Kumar et al., 2015;
Mokhtarzadeh et al., 2017). Bhaisare et al.
(2016) reported the fluorescent detection of path-
ogenic bacteria, i.e., Staphylococcus aureus and
Escherichia coli (E. coli), in urine sample through
their strong adhesion over amine-functionalized
magnetic nanoparticles decorated with carbon
dots. Carbon nanomaterials can also be function-
alized to support the immobilization of various
selective ligands, single-stranded DNA, and even
other types of nanoparticles. For instance, CNTs
immobilized with an antimicrobial peptide
(clavanin A) have been reported to offer effective
and sensitive detection of Klebsiella pneumoniae,
Enterococcus faecalis, E. coli, and Bacillus subtilis in
concentration of 102e106 colony-forming unit
(CFU) mL1 (Fig. 1.6) (Andrade et al., 2015).
The antibody-conjugated SWCNTs have also
been proposed for highly specific and sensitive
electrochemical immunosensing of S. aureus
with a low detection limit of 13 CFU mL1
(Bhardwaj et al., 2017). The biosensors were
able to quantify S. aureus in spiked milk samples
even in the presence of other bacteria, e.g., E. coli
B, Staphylococcus epidermidis, and B. subtilis.
The recognition and quantification of cyano-
bacterial bloom are of great importance, as it
may cause many structural and functional distur-
bances to the liver because of the inhibition of
protein phosphatase (type 1 and 2A) (Zamyadi
et al., 2016). Besides complex and expensive chro-
matography and protein phosphatase inhibition
assays (Catanante et al., 2015), the electrochemi-
cal immunosensors have emerged as viable op-
tions due to their high sensitivity, simplicity, low
cost, and easy miniaturization (Zhang et al.,
2010b).
Carbon nanomaterials have also been inte-
grated in FET designebased electrochemical
immunosensors for real-time identification of
pathogens (Yamada et al., 2016). Thiha et al.
(2018) developed a lab-on-chip device for label-
free chemiresistive biosensing of Salmonella typhi-
murium using carbon nanowires functionalized
with aptamers. The device offered highly sensitive
and specific detection of Salmonella, with a
 12
TABLE 1.2
List of Different Carbon NanomaterialeModified Glassy Carbon ElectrodeeBased Voltammetric Nanosensors of Food
Additives and Pesticides.

Advances in Nanosensors for Biological and Environmental Analysis

Details of Modification of
Electrode by Carbon Food Samples Limit of
Nanomaterials Analyzed Target Analytes Linear Detection Range Detection References
(A) NANOSENSORS FOR FOOD ADDITIVES
Pt/CNT nanocomposites with Chilli sauce, chilli Sudan I 0.008e600 mM 0.003 mM Elyasi et al.
1-methyl-3-butylimidazolium powder, tomato (2013)
bromide as binder sauce, strawberry
sauce
Nanocomposite comprising Chilli powder, ketchup Sudan I 0.05e2 mM 0.03 mM Mo et al.
hydrophobic ionic liquid sample (2010)
([P6,6,6,14][NTf2]), MWCNTs,
and cationic gemini
surfactants
MWCNTs-ionic liquids gel Soft drink samples Sudan I 0.005e15 ppm 0.001 ppm Chailapakul
Sudan II 0.005e20 ppm 0.001 ppm et al. (2008)
Sudan III 0.05e20 ppm 0.005 ppm
Sudan IV 0.10e25 ppm 0.025 ppm
Immobilized peroxidase Commercial salad tert- 1.65e9.82 mg L1 0.41 mg L1 de Oliveira
enzymes (Inga edulis Mart.) in dressing samples Butylhydroquinone et al. (2014)
carbon paste containing
MWCNTs and mineral oil, and
Nafion
Gold nanoparticleemodified Commercially Sunset Yellow 1.0  107 to 3.0  108 M Ghoreishi
carbon paste available soft drinks Tartazine 2.0  106 M 2.0  109 M et al. (2012)
5.0  108 to
1.6  106 M
MWCNTs Commercially Ponceau 4R 25 mg L1 to 15 mg L1 Zhang et al.
available soft drinks Allura Red 1.5 mg L1 25 mg L1 (2010a)
50 mg L1 to
0.6 mg L1
Boron-doped diamond Commercial food Butylated 0.60e10 mM for 0.14 and Medeiros
products hydroxyanisole both 0.25 mM et al. (2010)
and butylated
hydroxytoluene
 (B) NANOSENSORS FOR PESTICIDES
Molecular imprinting polymer Wastewater samples Atrazine 1.0  1012 M to 1.5  1013 M Yola and Atar
and platinum nanoparticles/ 1.0  1010 M (2017)
carbon nitrite nanotubee
based nanocomposite
MWCNT-functionalized Standard solution Dopamine 1e70 mM 0.15 mM Mercante

CHAPTER 1 Carbon-Based Nanomaterials for Nanosensor Development

polyamide 6/poly(allylamine et al. (2015)
hydrochloride)
Butyrylcholinesterase- Wastewater samples Paraoxon Up to 30 mg L1 5 mg L1 Arduini et al.
immobilized carbon black (2015)
nanoparticles
AChE-immobilized reduced Standard solution Malathion 7.98 to 2.0  103 pg mL1 4.14 pg mL1 Zhao et al.
graphene oxide-Au Carbaryl 4.3 to 1.0  103 pg mL1 1.15 pg mL1 (2015)
nanoparticles-b-cyclodextrin
and Prussian blue-chitosan
nanocomposite
AChE controlled fluorescence Standard solution Paraoxon 0e0.5 mg mL1 0.4 ng mL1 Li et al.
carbon dots (2018a)
Ag nanoparticleemodified Lake water and fruit Dimethoate 0.01e0.35 mg mL1 0.003 mg mL1 Hsu et al.
oxidized MWCNTs samples (2017)
Screen-printed carbon Real cucumber and Carbofuran 1e250 mM 0.22 mM Jirasirichote
electrode modified with rice samples et al. (2017)
graphene oxide and Au
nanoparticles
Praseodymium molybdatee Water and vegetable/ Methyl parathion 0.002e1.55 mM and 1.8 nM Karthik et al.
decorated reduced fruit samples 1.55e114 mM (2018)
graphene oxide

AChE, acetylcholinesterase; CNT, carbon nanotube; MWCNTs, multiwalled CNTs.

13
14 Advances in Nanosensors for Biological and Environmental Analysis

FIG. 1.6 Schematic fabrication of a carbon nanotube (CNT)-based biosensor for the detection of
pathogenic bacteria: (1) formation of a cysteine self-assembled monolayer over bare gold surface,
(2) activation of electrode surface for coupling of CNTs, (3) binding of antimicrobial peptide
clavanin A (ClavA) on CNT surface, and (4) interaction of electrode surface with analyte bacteria.
(Adapted from Andrade, C.A., Nascimento, J.M., Oliveira, I.S., de Oliveira, C.V., de Melo, C.P.,
Franco, O.L., Oliveira, M.D., 2015. Colloids and Surface B: Biointerfaces 135, 833-839. Reprinted
with permission from Elsevier.)

FIG. 1.7 Fabrication of a microfluidic chip, with the application of graphene oxide
(GO) nanosheetewrapped multiwalled carbon nanotubes (MWCNTs). This chip was used for the
detection of Salmonella typhimurium bacterial cells. EDC-NHS, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide-N-hydroxysuccinimide; StAb, Salmonella typhimurium antibody. (Adapted from Singh,
C., Ali, M.A., Reddy, V., Singh, D., Kim, C.G., Sumana, G., Malhotra, B.D., 2018. Sensors and
Actuators B: Chemical. 255, 2495e2503. Reprinted with permission from Elsevier.)

detection limit of 10 CFU mL1 with reduced
sample volume requirement (5 mL). Singh et al.
(2018) reported a novel microfluidic chip devel-
oped on a graphene oxide wrapped carboxyl
MWCNT platform for the detection of S. typhimu-
rium, with a detection limit of 0.376 CFU mL1
(Fig. 1.7). This biosensor offered negligible inter-
ference even in the copresence of E. coli O157:
H7. Such microfluidic chipebased biosensing
platforms offer important feature in terms of sam-
ple volume miniaturization, reduced processing
time, and reduced use of expensive chemicals.
These microfluidic immunosensors can also be
applied for the detection or quantification of
other pathogens by immobilizing the electrode
surface with suitable bioreceptors. Future research
efforts are needed to further elaborate intricate
sensor designs and fabrication processes of carbon
nanomaterialebased FET devices and related bio-
sensors. Table 1.3 lists some important examples
of carbon nanomaterialebased biosensors for
pathogens or viruses.
TABLE 1.3
List of Different Carbon NanomaterialeBased Bionanosensors for Detection of Bacterial Pathogens or Viruses.
Details of
Modification of Bacterial Type of
Electrode by Carbon Pathogens Biosensor Limit of
Nanomaterials or Viruses Signal Bioreceptor Used Linear Range Detection References

CHAPTER 1 Carbon-Based Nanomaterials for Nanosensor Development

(A) BACTERIAL PATHOGENS
Quantum dots (green- Vibrio Fluorescence DNA sequence 50e106 25 CFU mL1 and Duan et al.
and red-emitting)- parahaemolyticus CFU mL1 35 CFU mL1 (2015)
amorphous carbon and Salmonella
nanoparticles typhimurium
SWCNTs Staphylococcus Electrochemical Antibody d 13 CFU mL1 Bhardwaj
aureus et al. (2017)
Gold tungsten wires S. aureus and Electro- Streptavidin and 102e105 102 CFU mL1 Yamada
coated with PEI and Escherichia coli chemical bio-tinylated CFU mL1 et al. (2016)
SWCNTs K-12 immune- antibodies
sensors in FET
design
Amine-functionalized S. Fluorescence d d 3  102 and Bhaisare
magnetic iron oxide aureus and E. coli 3.5  102 et al. (2016)
nanoparticles CFU mL1
decorated with carbon
dots
Polypyrrole/Au E. coli O157:H7 Electrochemical Antibody 3  101 to 30 CFU mL1 Güner et al.
nanoparticles/ 3  107 (2017)
MWCNTs/chitosan CFU mL1
nanocomposite
CNTs Klebsiella Electrochemical Antimicrobial 102e106 102 CFU mL1 Andrade
pneumoniae, peptide clavanin A CFU mL1 et al. (2015)
Enterococcus (ClavA)
faecalis, E. coli, and
Bacillus subtilis
Carbon nanowires S. typhimurium Chemiresistive Salmonella-specific 10 CFU mL1 d Thiha et al.
aptamer probes (2018)
Graphene-wrapped E. coli O157:H7 Electrochemical Monoclonal 10e108 3.8 CFU mL1 Pandey
copper(II)-assisted antibodies CFU mL1 et al. (2017)

15
cysteine hierarchical
structure
Continued
 16
TABLE 1.3
List of Different Carbon NanomaterialeBased Bionanosensors for Detection of Bacterial Pathogens or Viruses.dcont'd
Details of

Advances in Nanosensors for Biological and Environmental Analysis

Modification of Bacterial Type of
Electrode by Carbon Pathogens Biosensor Limit of
Nanomaterials or Viruses Signal Bioreceptor Used Linear Range Detection References
Carbon dote S. aureus Fluorescence S. aureus antibody 1  102 to 1  104 30 CFU mL1 Yang et al.
encapsulated CFU mL1 (2018b)
organosilica
nanocapsules
(B) VIRUSES
Graphene oxide HIV Electrochemical 38-Base synthetic 1.0  1012 to 1.1  1013 M Hu et al.
sequence (ssDNA) 1.0  106 M (2012)
Graphene quantum HBV Electrochemical Probe DNA 10 e500 nM 1 nM Xiang et al.
dots (2018)
MWCNTs/polypyrrole AIV Electrochemical DNA aptamer 5.0  1012 M 4.3  1013 M Liu et al.
nanowires/gold to 1.0  109 M (2011b)
nanoparticles
Graphene oxide DNA (H1V1) and Fluorescence ss DNA 0.1 pM to 10 nM 0.1 pM Bi et al.
protein (thrombin) (2012)
MWCNTs Influenza virus Electrochemical DNA sequence d 0.5 nM Tam et al.
(type A) (2009)
Fullerene- MTB Electrochemical DNA sequence 1015 M to 3.3  1016 M Chen et al.
functionalized 109 M (2018)
polyaniline-doped
tufted CNTs
Porous reduced HPV Electrochemical RNA (Sc5-c3) 0.2e2 ng mL1 0.1 ng mL1 Chekin et al.
graphene oxide/ Aptamers (2018)
molybdenum disulfide

AIV, avian influenza virus, CNTs, carbon nanotubes; FET, field-effect transistor; HBV, hepatitis B virus; HIV, human immunodeficiency virus; HPV, human papilloma virus;
MTB, Mycobacterium tuberculosis; MWCNTs, multiwalled CNTs; PEI, polyethylenimine; ssDNA, single-stranded DNA; SWCNTs, singe-walled CNTs.
 CHAPTER 1 Carbon-Based Nanomaterials for Nanosensor Development 17

TABLE 1.4
Carbon NanomaterialeBased Important Gas Sensors.
Gas Molecules Detection Limit/
Electrode Material Detected Sensitivity References
CNT-TFR NO2 125 ppt Kumar et al.
(2017b)
CNT-mR NO2 165 ppt Kumar et al.
(2017b)
Wood-based activated carbon NH3 100e500 ppm Travlou et al.
(2015)
3D wool-ball-like ZnO/ SO2 70 ppm Septiani et al.
MWCNTs (2018)
Platinum-decorated MWCNTs C7H8 1 ppm Kwon et al. (2016)
Polyaniline-functionalized NH3 2e10 ppm Abdulla et al.
MWCNTs (2015)
Metal oxideedecorated Formaldehyde and NH3 5 ppm Zhang et al. (2017)
graphene
Reduced graphene oxidee NO2 0.25 ppm Yun et al. (2015)
decorated yarn
Carbon adhesive tape NO2 and N2 w5 ppm Lee et al. (2018)

CNT-mR, SWCNT aligned network; CNT-TFR, SWCNT random network; MWCNTs, multiwalled carbon nanotubes; SWCNT,
single-walled carbon nanotube.

Detection of Gas Molecules (Table 1.4). The mechanism of gas detection is

The presence of hazardous gases in our surround-
ings can have serious or deadly effects on human
health and vegetation. Various types of toxic gases
in varying amounts/concentrations are generated
from natural or artificial activities such as burning
of fossil fuels, automobile exhausts, cleaning
agents (Wang et al., 2018; Mirzaei et al., 2016).
In comparison to traditional analytical methods
of gas sensing (such as gas chromatography), the
electric and electrochemical transduction offer
several benefits in terms of minimal power con-
sumption, high sensitivity, and possibility of
miniaturization. The electrochemical gas sensors
offer high sensitivity, especially in highly humid
environments. Over the past decade, several nano-
materials have been reported in electrochemical
gas sensors to detect gas molecules, e.g., carbon
nanomaterials, polymers, and metallic nanopar-
ticles (Goldoni et al., 2018). Among these mate-
rials, carbon nanomaterials have a huge
potential due to their superior electric properties
controlled by change in electric conductivity of
the electrode surface through charge transfer dur-
ing gas interaction. Kumar et al. (2017b) reported
the fabrication of SWCNT-based gas sensors by
either SWCNT random network (CNT-TFR) or
SWCNT aligned network (CNT-mR). The NO2
sensing response of CNT-mR was higher than
that of CNT-TFR due to the nature of the surface
network, mainly contributing in adsorption
capacity of the sensor.
Beyond outdoor gas pollution, the indoor air
pollutants (e.g., formaldehyde and ammonia)
are also very serious pollutants that are released
from decorative and building materials. Zhang
et al. (2017) fabricated a metal oxideedecorated
graphene oxide sensor array, provided with back
propagation neural network, for sensing indoor
air pollutants. The use of graphene can overcome
the limitation of existing electrochemical sensors
that usually operate at high temperature. The
novel physical and mechanical properties and
18 Advances in Nanosensors for Biological and Environmental Analysis

unprecedented structure of graphene make it a platform with a high sensitivity of w5 ppm.

very promising electrode material for potential
applications in gas sensing. Furthermore, neural
networks can be integrated with graphene sensors
in order to enable the detection of a target gas
from a mixture of several gases. A sensor proposed
by Zhang et al. (2017) offered detection of toxic
gaseous mixture in a concentration range of
5e500 ppm within a small period of 200 s. This
type of sensor is quite useful for real-time moni-
toring of hazardous gases. FET-based gas sensors
using CNTs have been reported due to their excel-
lent sensitivity in comparison to other existing de-
vices. For instance, Nguyet et al. (2017) designed
an n-p-n heterojunction of CNTs and SnO2 nano-
wires for the detection of NO2 gas. The sensor was
able to detect the concentration of NO2 gas down
to 20 ppb. Nonetheless, proper understanding of
the sensing mechanism of FET devices still de-
mands further investigation (Dube et al., 2015).
New trends in gas sensors are beginning to emerge
through the development of CNT sensor arrays.
Lee et al. (2018) have fabricated a carbon adhesive
tape (CAT) to detect NO2 gas molecules (Fig. 1.8).
The CAT offered a disposable, rapid (detection
time of <3 min), and robust NO2 gasesensing
Many examples have been cited in the literature
wherein carbon-based nanocomposites have
been documented to allow the development of
highly sensitive gas sensors for H2, NO2, CO,
etc. A summary of carbon nanomaterialebased
different gas sensors is provided in Table 1.4.
Detection of Organic Molecules
The sensing of toxic organic molecules (such as
pharmaceutical and biological compounds, ex-
plosives, toxins or antibiotics, and personal care
products) has significant importance from the
point of view of environmental monitoring. The
detection of biological agents has become a crit-
ical part of modern life. The unique properties
of carbon nanomaterials have been exploited for
developing nanosensors for specific and sensitive
quantitative analysis of organic molecules.
Endocrine-disrupting chemicals are the organic
molecules that mimic the natural hormones of the
endocrine system and can adversely affect human
health, such as skin sensitization, obesity, repro-
ductive failure, diabetes, immune system failure.
These chemicals can lead to the disruption of
cell function even at low doses (in nanomolar

FIG. 1.8 (A) Schematic layout for the fabrication of a bare carbon adhesive tape (CAT) chip and
analysis of its electric conductivity through a current analyzer. (B) Application of CAT chips for the
detection of NO2 and N2 through their controlled exposure. (Adapted from Lee, S.W., Lee, W., Lee,
D., Choi, Y., Kim, W., Park, J., Lee, J.H., Lee, G., Yoon, D.S., 2018. Sensors and Actuators B:
Chemical 266, 485e492. Reprinted with permission from Elsevier.)
 CHAPTER 1 Carbon-Based Nanomaterials for Nanosensor Development 19

level) (Annamalai and Namasivayam, 2015;
Braun et al., 2014). Therefore biosensors should
be efficient enough to accurately determine even
trace levels of these chemicals/toxins. Yan et al.
(2018) reported a self-powered sensor for specific
detection of bisphenol A (BPA) using molecular
imprinted polymer-modified graphitic carbon
nitride photoanode. This photofuel cell configura-
tion offered electrocatalytic sensing of BPA, with
accessible range from 5 to 200 mM and with a
detection limit of 1.3 mM. Jiang et al. (2013) re-
ported a DNA-functionalized SWCNT/Nafion
composite for the detection of BPA, with a detec-
tion limit of 5 nM. The sensor offered high selec-
tivity without any fouling and/or activity loss in
the presence of other interferences such as acetate,
chlorides, metal ions, phosphate, and phenolic
compounds.
Nitroaromatic and nitramine-type compounds
are released into the environment mainly from
dye manufacturing, pesticides, plasticizers, and
military activities. These hazardous chemicals
contaminate soil and water with longer persis-
tence. A novel biosensor based on reduced gra-
phene oxide/CNT nanocomposite has been
reported for rapid and highly sensitive sensing
of 2,4,6-trinitrotoluene (TNT) (Castro et al.,
2018). Briefly, boron-doped diamond electrode
was modified with a mixture of MWCNT/reduced
graphene oxide. The synergistic effect between
MWCNTs and reduced graphene oxide offered
detection of TNT concentrations within
0.5e1100 mM with a detection limit of
0.019 mM. Pan et al. (2016) explored the active
sites of carbon nanomaterials for highly selective
and/or anti-interference detection of nitroaro-
matic compounds, such as nitrobenzene, dinitro-
benzene, and trinitrobenzene, with a low
detection limit of 0.88e1.8 mg L1. Likewise, car-
bon nanodots have been proposed for the detec-
tion of different nitroaromatic compounds as
illustrated in Fig. 1.9 (Ren et al., 2018).
Beyond industrial pollutants, water contamina-
tion due to antibiotics (tetracycline, nitrofurans,
etc.) is one of the major concerns. This type of wa-
ter pollution is caused by household wastewater
and commercial livestock farming. In particular,
nitrofuran drugs are commonly used in the treat-
ment of urinary tract diseases of humans and an-
imals. However, several reports are available on
mutagenic, toxic, and carcinogenic effects of these
drugs and their metabolites. Based on useful prop-
erties of CNTs in terms of good chemical stability,
high surface area, and electric conductivity, Chiu
et al. (2018) developed a screen-printed carbon
electrode sensor after modifying it with a

FIG. 1.9 Selectivity of carbon nanodots in sensing 2,4,6-trinitrophenol (TNP); nitrobenzene (NB);
2,4,6-trinitrotoluene (TNT); 3-nitrophenol (3-NP); toluene; 3-nitrotoluene (3-NT); 2,4-dinitrotoluene
(DNT); 2-nitrophenol (2-NP); and 4-nitrophenol (4-NP). (A) Ultravioletevisible absorption spectra of
carbon nanodots dispersed in analytes containing ethanol-water solution, (B) relative
fluorescence emission intensity of carbon nanodots when tested for different nitroaromatics, and
(C) fluorescence spectra of the probe during the selective analysis of TNP. (Adapted from Ren, G.,
Yu, L., Zhu, B., Tang, M., Chai, F., Wang, C., Su, Z., 2018. RSC Advances. 8, 16095e16102.
Reprinted with permission from RSC.)
20 Advances in Nanosensors for Biological and Environmental Analysis

nanocomposite of MWCNTs with conducting pol- based on the nanocomposite of mesoporous

y(melamine). The sensing electrode offered excel-
lent electrochemical detection of nitrofurans in
lake water and milk samples. Zhou et al. (2012)
reported an aptamer-MWCNT-modified glassy
carbon paste electrode for detecting tetracycline.
The carboxyl groupemodified MWCNTs were
incubated with amine-modified antitetracycline
aptamer (76-base sequence). The sensor showed
good sensitivity and selectivity for tetracycline,
with a detection limit of 5 nM upon testing spiked
milk samples. An electrochemical aptasensor
carbon-gold nanoparticles and CNFs offered an
ultrasensitive detection of kanamycin and strepto-
mycin, with detection limits of 87.3 and 45.0 pM,
respectively (Li et al., 2018b). Besides the good
conductivity of CNFs, the mesoporous carbon-
gold nanoparticles helped in improving the
efficiency of electron transfer because of their mes-
oporous structure as well as excellent electron
transfer kinetics. Some literature on the sensing
of toxic organic molecules using carbonaceous
nanomaterials is summarized in Table 1.5.

TABLE 1.5
Summary of Some Recent Carbon NanomaterialeBased Electrochemical Nanosensors for
Small Organic Molecules.
Details of Electrode
Fabrication Analyte Linear Range Limit of Detection References
(A) ENDOCRINE-DISRUPTING CHEMICALS
Molecular imprinted Bisphenol A 5e200 mM 1.3 mM Yan et al.
polymer-modified (2018)
graphitic carbon
nitride
Magnetic molecularly Bisphenol A 0.07e10 mM 8.8 nM Messaoud
imprinted polymer- et al. (2018)
modified Au
nanoparticles/carbon
black nanoparticles
Fe3O4/MWCNT Parabens (butyl, 0.5e150 Between 0.03 and Pastor-Belda
composite ethyl, isopropyl, ng mL1 2.0 ng mL1 et al. (2018)
isobutyl, methyl,
pentyl, phenyl,
propyl, and
benzylparaben)
Silver nanoparticles/ Hydroquinone 2.5e260 mM 0.16 mM Goulart et al.
MWCNTs Catechol 20e260 mM 0.2 mM (2018)
Bisphenol A 5.0e152 mM 2.4 mM
Phenol 2.4e152 mM 3.0 mM
(B) NITROAROMATIC COMPOUNDS
MWCNT/reduced 2,4,6- 0.5e1100 mM 0.019 mM Castro et al.
graphene oxide Trinitrotoluene (2018)
Hydroxyl-rich carbon Nitrobenzene 0.2e12.3 mg L1 1.3 mg L1 Pan et al.
submicrospheres Dinitrobenzene 0.01e16.8 mg L1 0.88 mg L1 (2016)
Trinitrobenzene 0.2e1.0 mg L1 1.8 mg L1
(continued)
 CHAPTER 1 Carbon-Based Nanomaterials for Nanosensor Development 21

TABLE 1.5
Summary of Some Recent Carbon NanomaterialeBased Electrochemical Nanosensors for
Small Organic Molecules.dcont'd
Details of Electrode
Fabrication Analyte Linear Range Limit of Detection References
Nitrogen- and sulfur- 2,4,6- 0.0008e5.1 ppm 0.1 ppb Zhang et al.
co-doped graphene Trinitrotoluene (2018)
nanoribbons
Carbon nanodots 2,4,6-Trinitrophenol d 0.127 mM Ren et al.
(2018)
(C) ANTIBIOTICS
CNFs/mesoporous Kanamycin and 0.1e1000 nM 87.3 and 45.0 pM Li et al. (2018b)
carbon-gold streptomycin
nanoparticles
MWCNTs and Nitrofurantoin 0.05e2.0 mM 0.012 mM Chiu et al.
conducting Nitrofurazone 0.05e5.0 mM 0.014 mM (2018)
poly(melamine) Furaltadone 0.05e2.0 mM 0.007 mM
nanocomposite Furazolidone 0.05e2.0 mM 0.006 mM
Aptamer-MWCNTs Tetracycline 10 nM to 100 mM 5 nM Zhou et al.
(2012)
Fluorescent carbon Oxytetracycline d 0.06 mM Qiao et al.
dots (2018)

CNFs, carbon nanofibers; MWCNTs, multiwalled carbon nanotubes.

CONCLUSIONS AND FUTURE several benefits in terms of reduction of sample

PERSPECTIVES
This chapter has discussed the role of diverse
nanosensitive platforms based on carbon nano-
materials for environmental analyses and food
safety applications. Carbon nanomaterials offer
several interesting properties important for
sensing different pollutants such as heavy metal
ions, food additives, pesticides, biological patho-
gens and viruses, toxic gaseous molecules, and
small organic molecules. Graphene and CNTs
offer highly selective and sensitive detection of
numerous environmental analytes without any
strict requirement of labeling or amplification
steps. Beyond excellent electric properties of car-
bon nanomaterials, there are several other param-
eters that make them ideally suitable for rapid,
multianalyte, and field-deployable environmental
monitoring, e.g., low cost manufacturing, less po-
wer consumption, compact size, and robustness.
The miniaturization of sensing platforms can offer
volume and bioreceptor as well as high-
throughput analysis with enhanced sensitivity.
In this context, latest research trends can be
observed in the development of lab-on-a-chip tech-
nology that offers accurate and compact sensing
in point-of-care assessment.
However, there are several factors that can
limit the performance of carbon nanomaterials
for analyte detection, such as (1) selecting CNTs
of the right nature (either metallic or semicon-
ducting), diameter, and length; (2) optimizing
graphene-based sensors, as the sensitivity of gra-
phene depends on the number of layers, edge
structure, impurities, etc.; and (3) controlling
the level of agglomeration during device devel-
opment. With the ever-happening advancement
in materials technology, it has now become
possible to fabricate biosensors that can offer
consistent as well as reproducible sensing
performances.
22 Advances in Nanosensors for Biological and Environmental Analysis
ACKNOWLEDGMENTS
Monika Nehra thanks the University Grant Com-
mission, India for providing financial assistance
in the form of JRF (award No. 3608 dated
February 29, 2016). Sandeep Kumar thanks the
Department of Science and Technology (Govern-
ment of India), University of Nebraska (Lincoln),
Daugherty Water for Food Institute (DWFI), and
Indo-US Science and Technology Forum (IUSSTF)
for financial support through Water Advanced
Research and Innovation (WARI) research grant
vide letter No. IUSSTF/WARI/2018/F-029-2018
dated January 03, 2018, along with DST-PURSE
sanctioned to Guru Jambheshwar University of
Science and Technology (GJUS&T), Hisar under
PURSE program No. SR/PURSE Phase 2/40(G).
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CHAPTER 2
Advances in the Synthesis and
Development of Two-Dimensional
Transition-Metal Dichalcogenide-
Based Nanosensor Platforms
SANJEEV KUMAR BHARDWAJ • NEHA BHARDWAJ • DEEPANSHU BHATT •
POONMA MALIK • AKASH DEEP
INTRODUCTION
The development of advanced nanosensors, to a
large extent, is based on the application of nano-
materials as transducer and biomolecule loading
surface. In this context, two-dimensional (2D)
layered nanomaterials have taken the center stage
during the past decade. Graphene, of course, has
been the material of choice to develop a large
number of modern nanosensors. The atomically
thin 2D materials have characteristic intralayer co-
valent and interlayer noncovalent interactions,
which induce interesting properties in them
(Akinwande et al., 2014). The 2D nature of gra-
phene is responsible for its large surface area
and strong intra-/intermolecular interactions.
The successful application of graphene in diverse
fields motivated the scientific community to
explore other 2D forms of different nanomateri-
als. As a result, many new members of the 2D
nanomaterial class have been discovered with po-
tential utility in areas such as field-effect transis-
tors (FETs) (Singh et al., 2017), ultracapacitors
(El-Kady et al., 2016), solar cells (Hsieh et al.,
2015), and biosensors (Abraham et al., 2015;
Tuteja et al., 2016). Other than graphene,
transition-metal dichalcogenides (TMDs) consti-
tute the new category of 2D layered materials
(Chhowalla et al., 2015). As such, the 2D TMDs
are a class of materials with around 40 members,
Advances in Nanosensors for Biological and Environmental Analysis. https://doi.org/10.1016/B978-0-12-817456-2.00002-4
Copyright © 2019 Elsevier Inc. All rights reserved.
which has a generalized molecular formula of
MX2 (M ¼ transition metal [e.g., groups IVeVII:
Mo, Ti, Hf, V, Nb, Ta, and W], X ¼ accompanying
chalcogen [e.g., S, Se, and Te]) (Chhowalla et al.,
2013, 2015). The 2D TMD nanosheets have
drawn increasing interest across vast disciplines
of science owing to their important properties
(e.g., high surface area, phase transformation,
polymorphism, and photoluminescence [PL])
and applicability in diverse fields (e.g., as insula-
tors [e.g., HfS2], semiconductors [e.g., MoS2 and
WS2], metallics [e.g., NbS2 and VSe2], and semi-
metallics [e.g., WTe2 and TiSe2]) (Zhu et al.,
2017; Hu et al., 2017). In their bulk forms,
TMDs exist as layered materials with three atomic
layers of a transition metal sandwiched between
two chalcogenide layers interconnected with cova-
lent intralayer and van der Waals interlayer forces
(Kolobov and Tominaga, 2016). The atomically
thin and active 2D individual layers can be iso-
lated with the help of different techniques. In
contrast to their bulk counterparts, 2D TMD
sheets display new material properties as a result
of localization of charge carriers along the x- and
y-axes, as the interaction forces along the z-axis
do not exist anymore (Sorkin et al., 2014). In
particular, the increased bandgap of 2D TMDs
makes them a better alternative for electronic and
electrochemical applications (Xiao et al., 2012).
27
28 Advances in Nanosensors for Biological and Environmental Analysis
Simultaneously, some of the 2D TMDs also
display both fluorescent emission and fluores-
cence quenching abilities, which have been
exploited to use these novel materials in the fabri-
cation of sensors (Hu et al., 2017). Upon reduc-
tion to a monolayer thickness, the TMDs (such
as MoS2) can experience a vital change in their
bandgap property (e.g., from indirect to direct)
(Mak et al., 2010). Such thickness-dependent
modulations in bandgap properties of 2D TMDs
have been exploited for the fabrication of next-
generation electronic and optoelectronic devices.
The subsequent parts of this chapter discuss
various TMDs such as MoS2, WS2, MoSe2, and
WSe2, with respect to their synthesis, structure,
properties, and utilization in sensing applications.
STRUCTURE AND PROPERTIES OF
TWO-DIMENSIONAL TRANSITION-
METAL DICHALCOGENIDES
In most cases, 2D TMD nanostructures are basi-
cally layered sheets with a layer thickness of
6e7 Å (Presolski and Pumera, 2016). Structurally,
they contain a layer of transition-metal atom
sandwiched between two layers of chalcogen
atoms. They are characterized with strong in-
plane covalent bonding and weak van der Waals
interlayer interactions (Shen et al., 2017). The
number of layers have a strong influence on the
properties of 2D TMDs. For example, MoS2
FIG. 2.1 Typical structures of layered transition-metal dichalcogenides (Choi et al., 2017).
(Reprinted with permission from Elsevier.)
undergoes an indirect to direct bandgap transition
as it is converted from bulk to a monolayer form.
For instance, an indirect bandgap of 1.3 eV of bulk
MoS2 changes to a direct bandgap of 1.8e1.9 eV
in a monolayer form (Mak et al., 2010). Further-
more, the TMDs exhibit various polymorphs,
often showing 1T (trigonal), 2H (hexagonal),
and 3R (rhombohedral) crystal phases (Fig. 2.1)
(Enyashin et al., 2011; Late et al., 2015). 1T and
2H are the two prominent phases when TMDs
are exfoliated into a single layer. It is possible to
achieve phase transformation in TMDs by
applying certain strategies; for example, the 3R
phase of MoS2 can be transformed into the 2H
phase by heating. Likewise, the 2H form can be
transformed into 1T through intercalation of suit-
able alkaline metals (Li, K, or Na) during the exfo-
liation process.
The number of layers in the TMD materials has a
big influence on their properties. Single-layered
TMDs possess large surface area, which aids in
higher physical adsorption of biomolecules onto
their surface (important for nanosensing applica-
tions). Single-layered TMDs may also show a fluo-
rescence quenching ability; that is, they can
consume the emission of other fluorophores when
brought in proximity (10 nm). This behavior of
TMDs has been exploited to use them in the devel-
opment of optical biosensors (Hu et al., 2017).
Some of the semiconducting 2D TMDs have a use-
ful bandgap that makes them to show PL as well.
 CHAPTER 2 2D TMD-Based Nanosensor Platforms
Molybdenum-Based Two-Dimensional
Dichalcogenides
Important molybdenum-based dichalcogenides
can be named as MoS2, MoSe2, and MoTe2.
Among these, MoS2 is the most widely investi-
gated material owing to its excellent semiconduct-
ing properties with naturally widespread
distribution (in bulk form) (Jiang, 2015; Lv
et al., 2015). The structure of MoS2 (SeMoeS)
is characterized by one atomic layer of Mo that
is sandwiched between two atomic layers of S.
The thickness of a single MoS2 layer is around
6e7 Å, with each Mo atom hexagonally packed
and each sulfur atom coordinated to three Mo
atoms (Huang et al., 2015b). The 2D MoS2 can
be categorized broadly into four different poly-
types: 1T, 1H (single-layered MoS2), 2H, and 3R
(few-layered MoS2) (Fig. 2.2) (Voiry et al.,
2015). This classification is based on the lattice ar-
rangements of Mo and S atoms (Galvan et al.,
2016). 2H MoS2 is the most stable and abundant
polytype, whereas 1T MoS2 and 3R MoS2 are
metastable in nature (Song et al., 2015). Both
2H MoS2 and 3R MoS2 polytypes exhibit trigonal
prismatic coordination around Mo atoms with a
difference of two (in the former) and three
S-Mo-S units (in the latter) per elemental cell
(Sebenik et al., 2000). In general, 2H MoS2 exhibits
n-type semiconducting behavior and possesses
values of indirect bandgap and electron mobility
FIG. 2.2 Schematic illustration of (A) 2H-type
structure of MoS2 and (B) 1T-type structure of MoS2
(Huang et al., 2013). (Reprinted with permission from
RSC.)
29
as 1.29 eV and 100 cm2 V1 s1, respectively, at
room temperature. This type is also known for
its diamagnetic properties due to the splitting of
4d orbitals and two 4d electrons occupying
4dz2. In contrast, 1T MoS2, with an octahedral
coordination geometry, possesses a metallic
behavior (zero bandgap) and paramagnetic prop-
erties (Gao et al., 2013). As some specific poly-
types of 2D MoS2 (e.g., 1T MoS2) could allow
facile ion intercalation within them, it becomes
easier to obtain them in an exfoliated form
(Song et al., 2015). Moreover, direct intercalation
of certain species (e.g., lithium ions, metallocenes,
metal clusters, and polymers) within 2H MoS2 has
been proposed to obtain 1T MoS2 (Pandey et al.,
2016). It may be noteworthy that the intercalation
strategy can also be applied to modulate the semi-
conducting properties of 2D MoS2. For instance,
the intercalation of polypyrrole polymers within
n-type MoS2 leads to its conversion into p-type
MoS2 (Bissessur and Liu, 2006).
Apart from MoS2, MoSe2 is another emerging
member of the 2D TMD family. MoSe2 is a narrow
bandgap semiconductor having a lamellar crystal
structure with a basic crystal unit built of SeeMoe
Se sandwich layers. A low bandgap (1.1e1.5 eV)
and substantial electron mobility (z50 cm2 V1
s1) of MoSe2 has encouraged the researchers to
employ this material in the fabrication of sensitive
photodetectors, switchable transistors device, and
humidity detectors (Zappa, 2017).
Tungsten-Based Two-Dimensional
Dichalcogenides
Tungsten-based dichalcogenides such as WS2 and
WSe2 have been explored as layered dichalcoge-
nides. The thin sheets of these materials exhibit
high in-plane carrier mobility and electrostatic
modulation of conductance. They possess a
trigonal prismatic structure and are indirect
bandgap semiconductors (bandgap of 1e1.3
eV). The metal-metal bond length of WS2 (3.15 Å)
is quite similar to that of MoS2 (3.16 Å), whereas
that of WSe2 is relatively larger (3.28 Å). In its
bulk form, WSe2 consists slabs of trilayers, with
ionic-covalent metal-chalcogenide bonds within
each trilayer. The lattice structure of WSe2 consists
of W atoms confined in a trigonal prismatic
30 Advances in Nanosensors for Biological and Environmental Analysis
coordination sphere neighbored to Se atoms. In
contrast to MoS2, WSe2 supports p-type doping
(Eftekhari, 2017). Atomically flat 2D WSe2 has
emerged as a vital nanomaterial for applications
in electronics, optoelectronics, nonlinear optics,
FETs, photodetectors, etc. The layered WS2 (S-W-
S, trigonal prismatic coordination) is an indirect
band semiconductor and has been reported with
great promise for the development of FET, cata-
lyst, lithium ion batteries, etc. According to optical
measurements, a single-layer WS2 material ex-
hibits a direct bandgap of about 2.0 eV. Owing
to low water solubility, WS2 has not been reported
much for biological application.
SYNTHESIS OF TWO-DIMENSIONAL
TRANSITION-METAL
DICHALCOGENIDES
The synthesis of 2D TMDs has been reported via
both top-down and bottom-up approaches as dis-
cussed in the following sections.
Exfoliation
Exfoliation methods are most frequently used to
synthesize single- or few-layer 2D TMDs. Mechani-
cal exfoliation is a more conventional method to
obtain atomically thin 2D TMDs. This procedure
involves rubbing of a fresh surface of a layered crys-
tal against another solid surface (such as Si/SiO2
substrate), which subsequently releases the desired
nanosized flakes. Single to quindecuple layers (15
layers) of MoS2, WSe2, and TaS2 have been pre-
pared by mechanical exfoliation. This method
can yield pristine, clean, and high-quality end
products (Li et al., 2013). However, it is difficult
to control the size and thickness of the final nano-
sheets and the technique is not suitable to be
scaled up at commercial level (Zeng et al., 2012).
Exfoliation with the aid of liquid agents is
another widely applied method for synthesizing
2D TMDs. It can be processed with solvent-
assisted and stabilizer/surfactant-assisted reac-
tions. The solvent-assisted exfoliation commonly
involves the use of ultrasonic energy or shear
forces to overcome the interlayer bonding of the
TMDs. For instance, MoS2, WS2, TaSe2, NbSe2,
and NiTe2 have been converted into ultrathin
nanosheets by processing the exfoliation reactions
in solvents such as N-methyl-pyrrolidone, isopro-
pyl alcohol, and even water (Coleman et al.,
2011). The use of organic solvents offers better
product yield than with water. In order to obtain
functionalized and better water-dispersible prod-
ucts, stabilizer- or surfactant-assisted exfoliation
has been suggested. A variety of ionic surfactants
(such as sodium cholate, sodium dodecyl sulfate
[SDS]) (Smith et al., 2011a), polymers (such as
poly(vinylpyrrolidone)) (Lei et al., 2016), and
biomolecules (such as bovine serum albumin)
(Jia et al., 2017) have been successfully employed
for exfoliation of TMDs.
Chemical exfoliation has been proposed for
the efficient, large-scale, and size-controlled syn-
thesis of single-/few-layered 2D TMDs, such as
MoS2 nanosheets (Fig. 2.3) (Yuwen et al., 2016).
This is basically an intercalation-exfoliation assis-
ted synthesis method that involves the insertion
of a suitable intercalating species (such as lithium
ion) into the interlayer cavity of the TMDs. The
intercalation results in the expansion of the inter-
layer distance that weakens the van der Waals
forces. A subsequent step of ultrasonication exfo-
liates the layers. The commonly chosen intercalat-
ing agents are able to produce gaseous molecules
upon ultrasonication treatment, which imparts a
peeling force to cleave layers and provides a suffi-
ciently high yield of dispersed nanosheets (Brent
et al., 2017). Apart from the popularly used
lithium species, some other alkali metals, e.g., so-
dium and potassium, have also been explored for
the purpose (Shen et al., 2017).
Stable dispersions of MoS2 and WS2 have been
obtained with lithium intercalation and exfolia-
tion technique. Li intercalation provides high
yield, controlled synthesis, and water-dispersible
products but the procedure is time consuming
and may leave residual lithium ions in the end
product (Ambrosi et al., 2015). The application
of surface tensionereducing surfactants, e.g., so-
dium cholate, has also been suggested as another
chemical exfoliation method for the synthesis of
single-/few-layered MoS2 (Smith et al., 2011b).
Nonetheless, the surfactant impurities in the final
product tend to disturb its electronic properties
and biocompatibility.
 CHAPTER 2 2D TMD-Based Nanosensor Platforms
FIG. 2.3 Ultrasonication-assisted lithium intercalation method for the synthesis of two-
dimensional transition-metal dichalcogenide nanosheets (Yuwen et al., 2016). (Reprinted with
permission from RSC.)
Electrochemical Methods
Electrochemical methods have been demon-
strated for the synthesis of large lateral sized
(50 mm) MoS2 nanosheets. Species such as OH,
O2, and SO4 2 can be electrochemically interca-
lated while exfoliating bulk MoS2 crystals in
Na2SO4 solution (Liu et al., 2014). Gases such
as O2 and SO2 are released at the anode, which
expand the interlayers. Electrochemical synthesis
is useful to obtain the exfoliated MoS2 flakes in
much larger lateral sizes than other methods
(5e50 mm) (Shen et al., 2017). As an experi-
mental example of this process, a bulk MoS2 crys-
tal and a platinum wire can be taken as the
working and counter electrodes, respectively. The
electrolysis reaction can be performed in the pres-
ence of 0.5 M Na2SO4 as an electrolyte (Liu et al.,
2014). An electrolytic method was reported for
the synthesis of few-layered 2D MoS2 nanosheets,
which involved the electrodissolution of a molyb-
denum metal sheet in the presence of Naþ and S2
ions (Kukkar et al., 2016). The process allowed the
formation of Naþ-intercalated MoS2, which could
then be exfoliated via ultrasonication to yield
good-quality few-layered MoS2 nanosheets.
Bottom-Up Methods
The bottom-up synthesis method is related to the
fabrication of multifunctional nanomaterials by
the self-assembly of atoms or molecules (Biswas
et al., 2012). It is a simple and less expensive tech-
nique to yield various 2D TMDs. Moreover, such
methods are also useful to grow TMD layers and
nanosheets directly onto different types of sub-
strates. Chemical vapor deposition (CVD) and
wet chemical synthesis routes are widely used
for the bottom-up production of 2D TMDs
(Dong and Kuljanishvili, 2017).
31
Chemical vapor deposition
The CVD technique generates high-quality single-
layer uniform thin films of 2D TMDs. This
method provides reproducible growth of thin
films on solid substrates with specific patterns
and uniformity (Zhan et al., 2012). In its simplest
form, the technique involves the co-evaporation
of metal oxide and chalcogen precursors over a
suitable substrate. The vapor phase reaction leads
to the formation of stable 2D TMD sheets. In
another “two-step CVD method,” a metal film
(of, e.g., Mo, W, Ti) is first deposited onto a suit-
able substrate (usually Si/SiO2), which is then
made to react with chalcogen (S, Se, Te) vapor
in an inert atmosphere (300e700 C) (Choi
et al., 2017). Several types of inorganic precursors
(such as oxides [MoO3] (Lee et al., 2012), chlo-
rides [MoCl5] (Yu et al., 2013), tert-butyl molyb-
denum (Mo(S-t-Bu)4) (Cheon et al., 1997), and
hexacarbonyl molybdenum (Mo(CO)6) (Pol
et al., 2008)) have been used for the CVD growth
of 2D MoS2 films on a variety of substrates
including silicon, silicon dioxide (Man et al.,
2016), gold (Li et al., 2016), sapphire (Yu et al.,
2013), and even graphene (for MoS2-graphene
composites) (McCreary et al., 2014).
The CVD of a single-layered 2D MoS2 nano-
sheet over mica has been found to result in the
generation of PL emission properties due to
intrinsic compressive strain originating from the
lattice mismatch between MoS2 and the substrate
(Ji et al., 2013). Such MoS2 nanosheets are useful
for their application in the fabrication of thin film
optical nanosensors. A variant of CVD, known as
metal-organic CVD, is also used for uniform and
large-scale growth of 2D TMDs. The technique
employs metal-organic or organic compound as
the source material and is useful to obtain
32 Advances in Nanosensors for Biological and Environmental Analysis
controllable morphology and composition of the
desired nanosheets (Choi et al., 2017). Similar to
CVD, atomic layer deposition, which is a gas-
phase chemical process, is also used for the
layer-by-layer deposition of atomically thin films
of 2D MoS2, WS2, and TiS2 (Choi et al., 2017).
Wet chemical method
Hydrothermal and solvothermal methods are the
two major types of wet chemical procedures used
for the synthesis of 2D TMDs. The hydrothermal
method is one of the simplest methods and has
been practiced to generate 2D TMD nanosheets
of varying sizes and thicknesses. This method is
also useful to create nanocomposites containing
2D MoS2 in combination with other nanomateri-
als such as graphene (Tuteja et al., 2016), gold
nanoparticles (AuNPs) (Huang et al., 2014b),
and palladium nanoparticles (Li and Du, 2017).
An example of hydrothermal synthesis of 2D
MoS2 nanosheets is described in Fig. 2.4.
In solvothermal methods, organic solvents are
preferred instead of aqueous reagents and the
treatment is carried out at high temperatures.
Such conditions enable effective nucleation and
growth process to drive the nanomaterial synthe-
sis. The type of organic ligand used in the reaction
is a key factor to define morphology, size, and
FIG. 2.4 Schematic of a hydrothermal method for the preparation of MoS2 nanosheets (Guo
et al., 2015). (Reprinted with permission from RSC.)
dispersibility of the synthesized 2D TMDs (Shen
et al., 2017).
BIOSENSING APPLICATIONS OF TWO-
DIMENSIONAL TRANSITION-METAL
DICHALCOGENIDES: MATERIAL
PREPROCESSING
The successful biosensing applications of 2D
MoS2 necessitate their conjugation with biomole-
cules, e.g., antibodies, enzymes, and nucleic acids
(DNA and RNA) (Justino et al., 2015). Therefore it
becomes important to render the materials with
proper aqueous dispersibility. In most cases, after
their synthesis the 2D TMDs are hydrophobic in
nature and tend to aggregate (restack) in solution
due to interlayer cohesion forces (van der Waals
and hydrophobic interactions) (Gupta et al.,
2015). Therefore significant research has been
directed to modify the surface properties of 2D
TMDs in order to make them suitable for bio-
conjugation. The absorption of surfactant mole-
cules onto the hydrophobic planes of bulk
TMDs has been reported to disrupt the interlayer
van der Waals forces. This strategy can prevent
layer restacking and help in maintaining TMDs
in nanosheet form. Similarly, the use of ionic sur-
factants also helps stabilize the aqueous
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heirlooms, and these were pucka (good old Indian word!) and not
those of other people.
If he could only hold on to the end, and put in his time fairly and
squarely, he might yet see Aurea at Wynyard—though at present his
prospects were blank; all he had to his name was his weekly wages,
and these wages, figuratively, bore him into the presence of Miss
Parrett. What an old bully she was! how she brow-beat and hectored
her unfortunate sister, and what a jabbering impostor! talking
incessantly of all she did, and was going to do, but leaving
everything in the way of work to Miss Susan and her niece—whilst
she trotted round spying and scolding.
As Wynyard reclined against the bank smoking, absorbed in his
reflections—and Joss was equally engrossed in an adjacent ditch—a
far-away sound broke faintly on their ears. In a few seconds this had
resolved itself into the regular “thud, thud, thud,” of a galloping horse,
and here he came into sight—a chestnut in a lather, with streaming
reins, and exultant tail, carrying an empty side saddle.
Wynyard instantly recognised Aurea’s weedy thoroughbred, and,
flinging away his cigar, ran forward, but the animal, bound for his
stable, was not thus to be captured and detained; with a snort of
defiance, he made a violent swerve, and tore on, hotly pursued by
Joss.
 CHAPTER XXIV
ON YAMPTON HILL

It was not the horse, but the horse’s rider that was of consequence.
Where was she? What had happened? Spurred by an agony of
apprehension, Wynyard ran in the direction from which the runaway
had appeared. In five minutes’ time a speck, and then a figure came
into sight, and this presently resolved itself into Miss Morven—
apparently unhurt. She, too, had been running; her habit was
splashed, she carried her hat in her hand, her beautiful hair was
becomingly loosened, and she had a brilliant colour.
As Wynyard slowed down to a walk, she called to him—
“Have you seen my horse?”
“Yes; he must be in Ottinge by this time,” was the comforting
rejoinder.
“Why didn’t you stop him?”
“It would have wasted a lot of time, and I wanted to see what had
happened to you.—I was afraid you’d had a spill.”
This was not the ever silent and respectful chauffeur to whom Miss
Morven had been hitherto accustomed; but no less a person than
Lieutenant Wynyard, late of the Red Hussars, who, in a cheery
voice, addressed her as an equal—as no doubt he was. So be it.
She instantly decided to abandon herself to the situation. Possibly he
would now confide something about himself, and how and why he
came to be in her aunts’ service. So, after a momentary hesitation,
she replied—
“Oh no, I only got off to open a gate, and Rufus broke away. I
suppose I shall have to walk home!”
To this Wynyard secretly and joyfully agreed, but merely said—
“I see you are alone.”
“Yes; father and I rode over to Shrapton-le-Steeple; he wanted to
see Mr. Harnett, a literary friend, and Mr. Harnett had so much to
show and to say that he persuaded father to stay and dine, as there
is a moon, and I came home by the short-cut. I must be three miles
from Ottinge?” and she halted and deliberately looked about her.
“Yes,” he replied; “a good three miles.”
(Oh, a very good three miles, during which he would have Aurea’s
undivided company—what a piece of luck!)
For some little time the couple proceeded in silence—a sensitive
silence. During the interval since their last meeting, they had
accomplished a vast amount of very special thinking—many
disturbing, dominating, and dangerous thoughts had entered the
young lady’s brain, and she said to herself—
“I must keep perfectly composed, and if ever he intends to speak
freely, now—now is the time! To think of us two alone on Yampton
Hills, three miles away from home!”
Somehow those three miles held a thrilling prospect. Wynyard, for
his part, was longing to utter what was in his mind; here was his one
grand opportunity; and yet for several hundred yards a strange
silence hung between them, though the man was burning to speak
and the girl was longing to listen; meanwhile moments, precious as
life itself, were ebbing fast! At last the conversation began to trickle;
the topics were the choir, the boy scouts, old Thunder’s pig, and Mrs.
Hogben’s face-ache—a spent cartridge in the path introduced sport
and shooting.
“I wonder why men are so keen on killing things?” said Aurea.
“I believe we inherit it from our ancestors, who had to kill wild
creatures or starve. I must say I like shooting.”
“Oh, do you!”—a blank pause—“the only sport I can imagine any
pleasure in, is hunting.”
“Do you hunt?”
“No; I only wish I did; but Aunt Bella thinks it so improper for a
woman to follow the hounds, and father could not escort me.”
“But parsons do hunt.”
“They did; a vicar of Ottinge actually kept hounds. Father says he
only left a dozen dusty books in the library, but a hundred dozen of
sound wine in the cellar.”
“Yes, those were the good old days!”
“I’m not so sure that they were superior to our own times. What do
you say?”
“That I hope you will always have a good time, Miss Morven.”
Miss Morven coloured and bit her lip, but resumed—
“If I only might hunt, I would be bound to have a good time.”
“Is your horse a clever jumper?”
“No; he either blunders on his head, or sits down.”
“Doesn’t sound very promising!” and they both laughed. “Anyway, it’s
a rotten, bad country,” said Wynyard, with a contemptuous wave of
his hand; “the uplands are full of rabbit holes, and as for the lowlands
—you’d want a boat! You should see Leicestershire—big fields and
sound turf.”
“Yes; but I’m afraid I can’t hunt in Leicestershire from Ottinge,” she
answered, with a smile; “and I have some hopes of sport this winter.
Mrs. Waring, who is tremendously keen, wants me to go out with
her.”
“On a pillion?”
“No; her brother has a capital horse, not up to his weight, that would
just carry me. He is so anxious that I should try it; it jumps
beautifully.”
“And what does Miss Parrett say?”
“I think Mrs. Waring may talk her over, and Mr. Woolcock promises to
look after me.”
This information roused Wynyard’s ire, his face hardened, and his
tone was dry as he said—
“Woolcock is too heavy to hunt, except pounding along the road. He
must weigh seventeen stone!”
“Very likely; but he is going to do a cure before the season opens.”
“Why not a couple of hours with the garden roller, and save the
donkey?”
Miss Morven took no notice of this impudent suggestion—merely
flicked her habit with her hunting crop, and he continued—
“Westmere is a fine old place.”
“Yes, isn’t it? The hall and galleries are real Tudor, and the park is
lovely.”
“How would you like to live there?”
“I?” and as she turned to him her air was lofty. “What a—a—an
extraordinary question!”
“Yes,” he replied, with hasty penitence; “please forgive me, it was
more than extraordinary, it was impertinent.”
“By the way (it was, after all, the girl who broke the ice), I must ask
you to excuse me for my inquisitive question the other day in the
garden.”
“You wanted to know why I hung on at Ottinge, with little or nothing
to do?” and he paused. “I think you do know, Miss Morven, in fact,
I’m sure you know. I’d be only too glad to speak out, but my hands
and tongue are tied. I’ve given a promise I’m bound to keep, and
between you and my absolute confidence, there stands at present
an enormous obstacle.”
“Oh!”
“I ought to tell you that I’m not what I seem.”
“Of course,” with a touch of impatience, “you are a gentleman by
birth; I’ve always known that.”
“Nor am I here in my own name—only my christian name; but I’ve
never done anything to disgrace it, I give you my word of honour.” As
he came to a halt and faced her, and the setting sun shone into his
truthful eyes and touched his crisp brown locks, the glow of the
evening air seemed to give added force to his personality. “I’ve
played the fool—the silly ass—and I’ve got to pay. How I wish I might
talk to you openly, and tell you all about myself!”
“I wish you might,” repeated the girl, and her voice shook; an
emotional tension had crept into the situation—her pulses beat
wildly, and her mind was in a tumult.
“You cannot imagine what it is to be in my fix,” he continued,
speaking with low, passionate intensity; “for months and months to
love some one with all my soul, and never be able to open my lips.”
“It must be trying,” she answered, now moving on, with her eyes on
the ground.
“And when I’m free, I may be too late!” he said gloomily.
“You may,” she assented; “for how could some one guess?”
“That’s it! That is what is the awful part of the whole thing; but, look
here, Miss Morven, let me state a case. Supposing you knew a
fellow in such a hole, and felt that you cared for him, and could trust
him and stick to him, as it were, blindly for a time; supposing he were
your social equal, and had a clean record, and that you knew he
worshipped your very shadow—would”—and here he looked straight
into her face—“you wait?” To this question, impetuously delivered,
there followed a silence.
“This is a sort of problem, isn’t it?” she faltered at last, “like the Hard
Cases in Vanity Fair?”
“No, by Jove, part of it is God’s truth! but I’m only talking like an idiot.
Of course no girl that ever was born could do it.”
“I’m not so sure,” she murmured, with her eyes on the ground, her
heart beating in hurried thumps.
“Miss Morven—Aurea,” he went on, now moved out of all discretion,
and casting self-control to the winds, “you are the only girl I’ve ever
cared for in all my life. I fell in love with you the first moment I ever
saw you, when you danced with Mackenzie in the Manor drawing-
room. This meeting to-day has been the one good turn luck has
done me in three years—and I seize upon it perhaps unlawfully;
perhaps it’s not just cricket, my talking to you in this way, but it’s my
only chance, so I snatch it, for I may never see you alone again—
and all is fair in love and war.”
At this moment he caught sight of a stout figure, far below, labouring
up the winding lane; it was Miss Morven’s maid, Norris. He
recognised her bright blue gown. Oh, the precious moments were
numbered, and it was now—or never!
“What do you say?” he demanded, coming to a standstill.
“But what can I say?” she rejoined, lifting her startled eyes to his. “I
don’t know anything about you. You cannot even tell me your name.”
(Naturally she did not mention that it was already known to her.) “It’s
all rather bewildering, isn’t it?”
“Yes, it’s simply crazy,” he admitted; “here am I, your aunts’
chauffeur, receiving weekly wages, living like a working man, telling
you, with the most audacious and astounding impertinence, that I’ve
been in love with you for months. You know that I’m of your own
class, but who I am, or where I come from, I refuse to disclose. No
wonder you feel dazed.” They were now within sight of the village
and of Norris. “Look here, Miss Morven,” he continued, and his voice
was a little hoarse, “I see your maid coming, and my priceless,
precious time is running out. Let me ask you one question;
supposing I were not Owen, the chauffeur, but had fair prospects,
good friends, and say a thousand a year to start with—would you—
marry me?”
Aurea knew perfectly well that she would marry him if he had only a
few hundreds a year, no prospects, and no friends; but she took
refuge in that nice, useful, and evasive word “Perhaps.”
“Perhaps”—he stooped and kissed her gloved hand—“perhaps—will
keep me going! Even if I don’t see you, I shall live on that one little
word for the next eighteen months. I don’t suppose I shall have
another opportunity of speaking to you.”
Here he was interrupted by Norris, who suddenly appeared round a
bend in the lane, puffing like a grampus, her hat on the back of her
head, and her face crimson from exertion.
“Oh, Miss Aurea!” she screamed, as she halted and held her fat
sides, “such a cruel fright as you give me—and the three men gone
by the road looking to find your corpse! and I ran this way, after your
horse come home all loose and wild. Are you hurt?”
“No; he only broke away when I was opening a gate, and I had to
walk,” replied the young lady with wonderful composure.
Norris threw a sharp glance at the chauffeur.
“And couldn’t he have helped you? What was he about?”
“I’m afraid, like yourself, I was looking for Miss Morven,” he replied.
Norris turned from him with a sniff of disparagement, and,
addressing herself to her mistress, said—
“And where’s the Rector?”
“He is dining with Mr. Harnett.”
“Tut, tut, tut! And he will stay half the night talking books, and there
are a brace of grouse for dinner—kept to the hour—and all he will
get at Harnett’s will be green vegetables, like a goat—he’s a
sexagenarian!”
At this Aurea laughed and the chauffeur smiled; he was now walking
alongside of Miss Aurea, as much at his ease as if he were a
gentleman! Norris turned on him abruptly, and said—
“Look here, young man, you’d better be getting on—it’s your supper-
time, and Mother Hogben won’t keep it hot for you.”
“It’s very good of you to think of my supper,” he replied, with a touch
of hauteur; “but I’m not in any hurry.”
He spoke to her as her superior; his was the voice and air of the
ruling and upper class, and Norris’ dislike to the insolent young
ne’er-do-well suddenly flared into active hatred. Meanwhile, he
walked with them to the very end of the lane, and opened the side
gate for Miss Morven and herself; and as he held it, he took off his
cap to Miss Aurea and said—
“Good-night—miss.”
As mistress and maid crossed the lawn, the latter burst out—
“I can’t abide that young fellow, with his fine manners and his taking
off his cap like a lord! Miss Aurea dear, I’m thinking the Rector would
not be too well pleased to see you in the lanes a-walking out like any
village girl along of your aunts’ chauffeur.”
“Norris, how dare you speak to me in such a way!” cried Aurea
passionately. And yet, why be furious? She had been “a-walking out”
precisely like any other country girl.
“Well, well, well, dearie, don’t be angry. I’m only giving you a hint for
your good, and I know you are a real lady, as proud as proud, and as
high-minded as a queen or an angel. Still and all, I’m mighty glad
that none of our talkers happened to come across you!”
 CHAPTER XXV
LADY KESTERS AT THE DRUM

Jane Norris, who had been Aurea’s nurse, was now her maid and
housekeeper, a most efficient individual in both capacities. Jane was
a woman of fifty, with a round, fat face, a complacent double chin, a
comfortable figure, and a quantity of ginger-coloured hair—of which
she was unreasonably vain. Jane had also a pair of prominent brown
eyes (which gave the impression of watchfulness), a sharp tongue, a
very sincere affection for her child, and an insatiable appetite for
gossip. She was left in sole charge of the Rector and Rectory when
Aurea was absent, and considered herself a person of paramount
importance in the community, not only on account of her position at
the Rectory, but also for being the happy possessor of a real fur
coat, a gold watch, and, last, but by no means least, considerable
savings. Her circle was naturally contracted and select; her
intimates, the village dressmaker, Miss Poult—who had many clients
in the neighbourhood—Mrs. Frickett, of the Drum; and Mrs. Gill, the
schoolmistress. (Mrs. Hogben, who took in washing, needless to say,
was not in her set.) Miss Norris had a flair for uncloaking scandals,
and was a veritable Captain Cook in the way of making marvellous
and unsuspected discoveries. She had always been particularly
anxious to explore the chauffeur’s past and to learn what she called
the “geography” of this young man. Hitherto the young man had
defeated her efforts, and baffled her most insidious inquiries. He did
not drink or talk or give himself away; he did not carry on with girls,
or encourage them. Oh, it was an old head on young shoulders, and
there was something about him that was not fair and square—and
she was bound to know it!
Miss Norris had been occasionally disturbed by a vague
apprehension (resembling some persistent and irritating insect) that
her mistress was interested in this good-looking stranger, but she
thrust the idea angrily aside. Miss Aurea was not like those bold,
chattering minxes who were always throwing themselves in his way!
She was really ashamed of herself, and her wicked mind. Of course,
Miss Aurea would make a grand match, and marry young Woolcock
—who was just crazy about her, as all the world knew—and she
would go with her as maid to Westmere Park. But the vision of her
young lady and the chauffeur talking to her so earnestly in the hill
lane had excited her fears, and she resolved to give Miss Aurea
something to think of, and put her from speaking to the upsetting,
impudent fellow—who got more notice and made more talk in
Ottinge than the Rector himself!
Aurea, who had been accustomed to Norris ever since the days of
socks and strapped shoes, regarded her as a friend, and even
suffered her to gossip (mildly) as she dressed her hair, for she said
to herself—
“The poor thing has no one else to talk to all day long”—Simple
Aurea!—“being set in authority over the other servants, and must
have some safety-valve.”
The night after her walk with Wynyard, Aurea slept but little; she was
thinking, and wondering, and happy. As she dressed, she was
unusually abstracted, and when Norris began her coiffure, she did
not as usual read the Psalms for the day, but sat with crossed hands
in a trance of meditation, whilst her maid brushed her soft and
lustrous locks. After twice clearing her throat with energetic
significance, Norris began—
“So Mrs. Ramsay is letting the house for six months, I hear?”
“Yes,” was the languid reply.
“To a sort of county inspector; the chauffeur fellow showed him in—
he has a finger in every one’s pie.”
“I don’t know what you mean, Norris.”
“Well, anyway, he did a lot for Mrs. Ramsay,” she answered, with
significance. “He was in and out at all hours—some think he is good-
looking—and ladies like him.”
“What ladies?”
“Well, now, Miss Aurea, you know I don’t intend any harm, but the
talk is that your aunt, Miss Susan, makes too great a pet of him.
Why, half his day he’s helping her in the garden or potting plants in
the greenhouse; and she lends him books, and talks and makes a
fuss of him, just as if he were in her own station.”
Norris’ speech was so rapid, such a cataract of words, that her
young mistress had not been able to interrupt; at last she broke in—
“How wicked of people!” endeavouring to wrench her hair away.
“Poor Aunt Susan—so good, unselfish, and kind—not even spared!
Oh, it’s too abominable! I’m ashamed of you, Norrie; how can you
listen to such things?”
“Indeed, Miss Aurea, I said just what you said, and that Miss Susan
was too old; but they say there is no fool like an old one—and some
folk will gossip. And there was Mrs. Lambert, who married a boy that
was at school with her own son. You know there’s not much to talk of
here—now the Ramsays are gone. As for the young man, as I told
you to-night, I never held a good opinion of him; he’s too secret and
too off-hand to please me. He goes out of a night for exercise, so he
says, walking the country till daybreak; but that’s just a blind. Who is
he with?—tell me that?”
Aurea remembered, with a sudden stinging pang, how she and her
father had overtaken him one evening escorting Dilly Topham. Dilly
had been crying, and she was holding his hand!
“Why, I saw him myself in the theatre at Brodfield,” resumed Norris,
“and he had a young woman with him—so he had.”
“And why not?” bravely demanded Aurea, but her lips were white.
“The two were in a box, and he sat back—but I knew him—and
afterwards they walked together to the Coach and Horses Hotel, the
best in Brodfield. She was tall and slim, and wore a long coat and
black lace scarf over her head—I call it very bold in the public street.”
“One of his friends,” explained Aurea, with a stoical indifference her
heart belied; and to cut short any further disclosures, she released
herself from her handmaiden’s clutches and knelt down to say her
prayers.
By a disagreeable and curious coincidence, Miss Morven received
that same evening ample confirmation of Norris’ arraignment!
Lady Kesters had decided to pay her brother another visit, and wrote
to announce that, as she and Martin were within fifty miles, she
would fly down to see him for a few hours.
“I’ll come to Brodfield by train and motor over. Don’t
breathe a word to the Parretts. I can put up at the Drum
and meet you there. I’ve ever so much to say and hear;
your letters are miserable, and I’ve not seen you for more
than two months. Martin is off to America in October—he
has to look after some business—and I am going with him,
as I want to see the country, but I shudder to think of the
crossing. Uncle Dick is at Carlsbad. If you come over to
the churchyard about six to-morrow, I shall be there. I’ll
hire a car for the day and get back to Brodfield for the
night, and rush to Rothes next morning with the milk; if
you will make an appointment, I can meet you, and go for
a stroll and a talk.”
A smart Napier and a motor-veiled lady were not now a startling
novelty in Ottinge—it was the highway to many places; but the 40
h.p. motor and lady who put up at the Drum was a refreshing novelty
—and a novelty invested in mystery.
The Drum jutted out obtrusively; the front faced down the road
towards the Manor, and one side was parallel to the street, and
whoever entered or left was well in evidence. Lady Kesters asked for
dinner and a sitting-room, as if such were a matter of course! The
sole sitting-room was just across the passage from the bar and
overlooked the street. It was oak-panelled, very low, the walls were
decorated with cheap prints and faded photographs of cricket
groups, there was a round table, three or four chairs, and an
overpowering atmosphere of stale beer.
“Oh, let me see—I’ll have some tea and roast chicken,” announced
the traveller.
“Chicken, ma’am?” repeated Mrs. Frickett, and her tone was
dubious. “I don’t know as I can run to that. The hens is roosting now.”
“Oh, well,”—impatiently—“bacon and eggs. I’ll go and take a turn
about the village.”
With her veil drawn over her face, Lady Kesters walked out, went
slowly up to the church, and critically inspected the Parsonage.
Then, just inside the churchyard, she discovered her brother sitting
on a tombstone. As he sprang to meet her, she exclaimed—
“Are you smiling at Grief?”
“Hullo, Sis, this is most awfully good of you! How are you? Very fit?”
“Yes. Do come out of this horribly dismal rendezvous, and let us go
down one of the lanes, and talk.”
“Aren’t you tired?”
“No, only hungry. I’ve ordered a meal at the Drum. I’m tired of sitting
in a train or motor, and glad of a walk. Well, Owen, so far so good—
six months are gone—hurrah!”
“Yes, thank goodness, but it’s been a pretty stiff job.”
“An uphill business, and terribly dull! Again I repeat, would you like to
move? You could so easily better yourself.”
“No, I stop on till the car breaks up.”
Lady Kesters raised her eyebrows.
“Well, I can only hope that blest epoch will be soon! I met Miss
Susan, you know, and the crafty old thing was fishing to find out who
you are? She has her suspicions, but I gave her no assistance. The
niece was with her—Miss Aurea——” She paused expressively, then
went on, “Owen—she’s a remarkably pretty girl.”
He nodded.
“Yes, I understand your reason for remaining in Ottinge; it is beautiful
—simplicity itself.” She looked at her brother attentively. “Are you
making love to her?”
“I—her aunts’ chauffeur?”
“Nonsense! Are you in love with her?” she persisted. “Come, tell the
truth, my dear boy. Why should you not take me into your
confidence? Are you?”
“Well—I am.”
“And she?”
“Don’t I tell you that I’m only her aunts’ chauffeur, and my tongue is
tied? All the same, Sis—it’s beastly hard lines.”
“Then, Owen, you really ought to go away; you’ll soon forget her and
Ottinge. I’ll find you another opening at once.”
“No, I won’t stir yet,” he answered doggedly.
“You are wrong, and on your head be it! I wish you could come out to
America with us; but foreign countries are barred.”
“Why are you and Martin off there?” artfully changing the subject.
“Partly business—chiefly, indeed. He has not been well, and I can’t
allow him to go alone; but, anyway, I’m looking forward to the trip.
Tell me, how are you off for money?”
“All right; I fare sumptuously on a pound a week and washing extra.”
“I suppose you live on bacon? That’s to be my dinner.”
“Bacon—eggs—fowl—steak. Mrs. Hogben is a mother to me, and a
real good sort.”
“I must say I think you look rather thin, Owen.”
“I’m glad of it; I’m as fit as a fiddle, and made sixty runs last week for
Ottinge. They little dream that I was in the Eton Eleven! Hullo! here
are some people coming. I say—what a bore!”
No less than two couples now approached arm in arm; as they
passed, they stared hard, and even halted to look back.
“What will they think, Owen?” and she laughed gaily.
“I don’t care a blow what they think!” he answered recklessly; “but all
the same you’d better return to the Drum alone.”
“Well, mind you come in this evening—I start at nine; you can
pretend my chauffeur is your pal—pretend anything!”
“Oh, I’m good enough at pretending; it’s now my second nature!
Joking apart, you ought to be going back to the inn, and getting
something to eat.”
 CHAPTER XXVI
THE OBSTACLE

At seven o’clock Wynyard went boldly to the Drum and inquired for
the lady who was stopping there.
Mrs. Frickett stared at him with a stony expression in her dull grey
eyes. She had heard of his airs and his impudence from Norris.
“Will she see you?” she asked, and her tone was aggressively
insolent.
“Oh yes,” was the ready answer; “it’s business.”
“Oh, if it’s business——” and she gave an incredulous sniff and,
flinging open the parlour door, ushered him into the presence of his
sister.
Lady Kesters had removed her cap and motor coat, and was seated
at the table in a careless attitude, leaning her head on her hand and
smoking a cigarette. The door was exactly opposite to the taproom,
and the assembled crowd enjoyed a rare and unexpected spectacle.
A woman smoking—ay, and looking as if she were well used to it
and enjoying herself—a lady, too—there was a string of pearls round
her throat, and the hand that supported her dark head was ablaze
with diamonds. Ottinge had heard and read that females were taking
to tobacco, and here was the actual demonstration before their
gloating eyes. A fine, handsome young madam, too, with a car in the
yard—ay, and a friend to visit her! They craned over to catch a
glimpse of the figure ushered in by Mrs. Frickett. The man’s back
and shoulders had a familiar look. Why, if it wasn’t Owen, Miss
Parrett’s chauffeur! The immediate result of this astounding
discovery was a deadly and expressive silence.
Since Wynyard had parted with his sister he had made up his mind
to tell her all about Aurea. He longed to share his secret with some
one, and who could be better than Leila? She would give him her
sympathy and—what was more—a helping hand; if any one could
unravel a hopeless tangle, it was she. After a little commonplace
talk, in a few abrupt sentences he commenced to state his case.
“Ah!” she exclaimed as he paused, and she dabbed the end of her
cigarette on the old oak table, “so it’s all coming out now! You show
your good sense, Owen, in confiding in me—two heads are better
than one. I’ve seen the young lady; she is distractingly pretty—and I
think I approve.”
“Think!” The words were a text upon which her brother delivered to
his astounded listener an address of such emotional eloquence, that
she sat and stared in bewildered silence.
As he spoke, he strode about the room, carried away by his adorable
subject—Aurea’s beauty, her cleverness, her unselfishness, her
simple and single-hearted disposition, her good influence in Ottinge,
her delicious voice, and her entrancing smile. Oh, it was a wonderful
relief to share with another the raptures so long bottled up in his own
breast!
In the middle of his discourse, the door, which was flung open to
admit “two lemonades,”—Owen had warned his sister against the
deadly Drum coffee,—revealed to a profoundly interested tap, young
Owen, the shover, “a-walkin’ and a-talkin’ and a-carryin’ on like old
Billy, and in such a takin’ as never was seen.”
“She’s his sweetheart, ’tis sure!” suggested one sightseer.
“Nay, more likely his missus,” argued another; “she was a-laughin’ at
him!”
As the door closed Leila threw her cigarette into the grate with a
quick, decided gesture, and, leaning both elbows on the table, said,
as she looked up at her brother—
“It’s an extraordinary entanglement, my dear boy. You are in love—
for the first and only time in your life. Of course I can believe as
much of that as I like!”
“You can!” His voice was sharp and combative.
“In love with an angel. I may tell you that she really is a fellow-
creature! You think she likes you, but for one solid year and a half
you may not impart to her who you are, or where you come from, or
even your name—I mean your surname. You are at liberty to inform
her that you are ‘Owen St. John Willoughby FitzGibbon’—a nice long
string!—but must never breathe the magic word ‘Wynyard.’”
“No, you know I can’t,” he answered irritably.
“You are her aunts’ servant now, though you will be, if you live, Sir
Owen Wynyard of Wynyard; but you may not give her the faintest
hint, as you must stick to your bargain with Uncle Dick and he to his
with you. Now, let me consider,” and she held up a finger: “if you
speak, and reveal your identity, and become engaged, you lose a
fortune.”
“Yes,” he agreed, a trifle dryly.
“If you don’t speak, you run a great chance of losing the young lady!
Mr. Woolcock is on the spot, and as willing as Barkis. Westmere is
close by—an ever-enticing temptation—and he has the goodwill of
the girl’s relations.”
“Yes, that’s a true bill; it’s wonderful how you grasp things.”
“What grounds have you for supposing the girl would wait for
eighteen months in absolute ignorance of who you were? Have you
ever spoken to her, as her equal?”
“Yes, once,” and he described their walk two days previously. “I
stated a similar case; I made the most of my time, and asked her
what she would do under such circumstances.”
“My dear Owen,”—and she looked at him with an expression of
wonder in her eyes—“I am simply staggered at your presumption!”
“Yes, so was I; but, you see, it was my only chance, and I snatched
it.”
“And what did she answer. That it was evident you were an
uncertified lunatic!”
“No; she said ‘Perhaps.’”
“‘Je m’en vais, chercher un grand peut-être,’ as some one said on
his death-bed.”
“Don’t talk French—or of death-beds, Sis.”
“No, I won’t. I see that your divinity is a clever, modern young
woman, who refuses to commit herself. Look here, Owen, I won’t
tease you any more; this situation is such that it even baffles the
activity of my clever and contriving mind! I’m afraid I can do nothing
at present; but when we return from America, I shall make a point of
cultivating General and Mrs. Morven, on account of the girl. I’ll
cultivate the girl for your sake, and ask her to stay in Mount Street.
Possibly she may open her heart to me, and tell me everything! I
have a wonderful knack of extracting similar confidences even from
my housemaids! I shall listen sympathetically, advise sagaciously,
and urge her to stick to you!”
“Yes, I know that once you take a thing in hand, Sis, it goes like an
express train; but you will be away for six months—six months is a
long time.”
“Time!”—springing to her feet—“and talking of time, I must be off.
Ring the bell, my dear boy, and order the car at once.”

Miss Morven had been dining at the Manor. She had endured a long,
leaden evening playing draughts with her Aunt Bella; she played so
carelessly that Bella had repeatedly huffed her, and eventually won
with six kings to the good! After their niece’s departure, the sisters
were for once unanimous in their opinion: they had never seen
Aurea looking so well, as that night.
“What a rose-blush complexion, what clear, glowing eyes!” said
Susan, with enthusiasm.
“Yes,” agreed Miss Parrett, who was putting away the draught-board,
“she’s got my skin, and her mother’s eyes. I’ve often been asked if I
were painted!” she announced, with serene complacency.