Document type: Review/Account

Award Account
Account/Review for Materials Innovation

Recent Progress on the Sensing of Pathogenic Bacteria

Using Advanced Nanostructures

Gopalan Sai-Anand,*1,2,³ Arumugam Sivanesan,2,3,³ Mercy R Benzigar,2 Gurwinder Singh,1,2

Anantha-Iyengar Gopalan,4 Arun Vijay Baskar,1,2 Hamid Ilbeygi,2 Kavitha Ramadass,1,2
Venkata Kambala,5 and Ajayan Vinu*1,2

1
Global Innovative Center for Advanced Nanomaterials, Faculty of Built Environment and Engineering,
The University of Newcastle, Callaghan 2308, New South Wales, Australia
2
Future Industries Institute, Division of Information Technology, Engineering and Environment,
University of South Australia, Mawson Lakes, South Australia 5095, Australia
3
Metrohm Australia, 56 Buffalo Road, Gladesville, NSW 2111, Australia
4
Research Institute of Advanced Energy Technology, Kyungpook National University, Daegu 41566, Korea
5
Hudson Marketing Pty Ltd, Level 2/131 Macquarie St, Sydney NSW 2000, Australia
E-mail: Ajayan.Vinu@newcastle.edu.au

Received: September 14, 2018; Accepted: October 3, 2018; Web Released: October 27, 2018

Ajayan Vinu
Prof. Ajayan Vinu is a Professor of Nanomaterials and Director at Global Innovative Centre for Advanced
Nanomaterials (GICAN), The University of Newcastle. He was working as a full professor and Australian
Research Council Future Fellow at the University of Queensland and University of South Australia. Prior
coming to Australia, he had been working as a senior researcher and research group leader at the National
Institute for Materials Science (NIMS), Tsukuba, Japan since 2006 after he successfully completed two years
of the ICYS fellowship at the same institute and a few years of research at the Technical University of
Kaiserslautern (TUK), Germany. During these 15 years of research, Prof. Vinu has made a tremendous
contribution in the field of nanoporous materials and their application in sensing, energy storage, fuel cells,
adsorption and separation, and catalysis. His contribution in the field of nanoporous materials is also clearly
reflected by his international ranking by Science Watch as one of the top 15 researchers in the field and has
led to 350 original research papers in high impact factor journals with ca. 16,600 citations and an H-index
of 67.

Gopalan Sai-Anand
Dr. Sai-Anand Gopalan is currently working as a Research Associate in Global Innovative Center for
Advanced Nanomaterials, Faculty of Engineering and Built Environment, The University of Newcastle,
Callaghan Campus, New South Wales. His recent research focus is on the design and development of novel
functionalized porous materials and their widespread applications in sustainable energy technologies such
as fuel cells and supercapacitors with a special emphasis on organic optoelectronic materials and devices to
address global energy demands and environmental challenges.

Abstract tious to human beings owing to their appearance in food, water,

Ailment related to pathogenic bacteria and toxins remains a and other biological samples. Over the past several years,
significant threat to the human body. Specifically, pathogenic advanced nanomaterials-based sensing has been considered
bacteria are the main source of epidemic diseases and are infec- as an efficient and unique platform for the rapid, selective,

216 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280 © 2019 The Chemical Society of Japan
ultrasensitive, qualitative, and quantitative detection of single
or multiple pathogenic bacteria. Towards this end, various
emerging nanomaterials have been purposefully designed and
developed to integrate them onto sensor systems for the recog-
nition of pathogenic bacteria. The present review describes a
wide range of analytical techniques such as surface-enhanced
Raman scattering, electrochemistry (electrochemical and elec-
tronic), a field-effect transistor, fluorescence, calorimetry and
surface-plasmon resonance etc. which incorporate nano-
biosensor technology to develop a pathogenic bacterium based
sensor. This review also highlights the progress, trends and
strategy utilized toward the identification of harmful bacteria
by focusing on the pertinent literature available on the various
advanced nanomaterials (such as semiconducting, magnetic,
noble metal and carbon-based nanomaterials) incorporating
nano-bio sensor platforms.
Keywords: Pathogenic microorganism j Biosensor j
Nanomaterial
1. Introduction
Biocontamination and communicable ailments (generally
referred as infections or transmittable disease) are an existing
global threat leading to millions of deaths every year.1­3
Infectious diseases are generally caused by several different
classes of pathogenic microorganisms such as viruses, bacteria,
protozoa, fungi, and parasites. Among the pathogenic micro-
organisms, bacterial infections alone account for one-third of
global mortality.4 Diarrhoeal diseases caused by a variety of
bacterial organism are the second leading cause of death in
children under 5 years. Typically, each year diarrhoea kills
around ³520,000 children. Tuberculosis (TB), is another
bacterial disease found in almost every country in the world,
caused by Mycobacterium tuberculosis (M. tb), a pathogenic
bacterium in the family Mycobacteriaceae. TB causes ill-
health for nearly 10 million people each year and is recog-
nised as one of the top ten causes of death worldwide. Some of
the other high mortality causing bacteria are Streptococcus
pneumoniae, Vibrio cholera, Staphylococcus aureus, Helico-
bacter pylori, Salmonella typhi and Escherichia Coli (E. coli).
The bacterial diseases such as anthrax, relapsing fever, bru-
cellosis, clostridal, plague tularaemia etc. originate from the
respective causative microorganisms and are distributed world-
wide. Studies on the lung bacterial microbiome have unfolded
the relationship between lung microbiota and acetate respi-
ratory diseases (ARD).5 Importantly, ARD is a cause of mor-
tality, leading to the death of 4 million people every year
globally. The distribution of ARD depends on factors such as
bacterial infections, environmental conditions and infection
prevention measures.6
There are two important aspects which need to be considered
inn controlling pathogenic bacterial infection. Firstly, bacte-
rial pathogen, such as Enterrohemorrhagic Escherichia Coli
(EHEC) can produce toxins. Secondly, a small number of the
pathogenic bacteria are sufficient to initiate infection in the
body and can cause potential damage to the host system.
Development of detection strategies for different kinds of
pathogens is an important aspect of health and safety. Different
Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
analytical methods such as polymerase chain reaction (PCR),
colony count, enzyme linked immunosorbent assay (ELISA),
biosensors, electrophoresis etc. have been employed for the
detection of these pathogens. The most popular methods for
detection include culture and colony counting7 and PCR.8
Conventional methods such as surface recognition, nucleic
acid detection and enzyme mediated are quite expensive as they
require a high level sophistication and complex sample prepa-
ration even though they are quite sensitive. Therefore, new
methods are required to improve their performance. Under
these circumstances there is a growing demand for the design
and development of ultrasensitive, rapid, and selective patho-
gen detection methods capable of detecting very a few or a
single pathogenic bacterial species in target samples (water,
food or biological tissues). The recent advancement in the
biosensor technology promises access to highly selective,
reliable, and rapid pathogenic detection.
Since the invention of the first biosensor (glucose biosensor)
by L.C. Clark in 1956, sensing based on biotechnology has
emerged as an alternative to expensive chromatographic meth-
ods for the analysis of a variety of analytes. This is mainly due
to easy operation as it involves only the conversion of a
biological response into a detectable signal. A typical biosensor
comprises two parts, namely a bio-receptor and a physico-
chemical transducer, or combined into a single sensor called
a bio-transducer, a recognition-transduction component. Both
the bio-receptor or bio-recognition component and transducer
cooperate and convert a biochemical signal into to an electrical,
electrochemical, optical, piezoelectric, pyroelectric, thermal,
gravimetric or other detection signal.9 Biosensors have been
considered prominent analytical devices incorporating a bio-
logical material (e.g., tissue, microorganism, organelle, cell
receptor, enzyme, etc.) or a biologically derived material (e.g.,
recombinant antibodies, engineered proteins, aptamers, etc.) or
a bio-mimic system (e.g., combinatorial ligands, and imprinted
polymers).10 The bio-receptor usually contains an antibody or
aptamers or enzymes/proteins capable of selectively binding
the target molecule. The physicochemical transducer, which is
in direct contact with the bio-receptor, produces a physico-
chemical change when there is a biochemical interaction
between the bio-receptor and biomolecule. The continuous
investigation of various aspects of biosensors has led to several
innovations which have been applied to bacterial and viral
detection and monitoring. With advancements in nanotechnol-
ogy and nanoscience, advanced nanomaterial-based biosensors
have shown great potential in improving both sensitivity and
selectivity.
A variety of advanced nanomaterials such as metallic nano-
particles (MNP), carbon based nanomaterials, magnetic nano-
particles (MP) and quantum dots (QDs) are continuously being
utilized in biosensor design and other applications due to their
unique physical, chemical, catalytic, sensing, mechanical,
magnetic, and optical properties.11­24 These properties of the
nanomaterials are beneficial to enhance the sensitivity and spe-
cificity of the fabricated biosensors (Scheme 1). Most impor-
tantly, these technological advancements provide scope for
tuning the properties of nanomaterials by changing the size,
shape, morphology, surface charge, surface area and composi-
tion, modifying various adsorption capacities with appropriate
© 2019 The Chemical Society of Japan | 217

Scheme 1. Scheme showing the detection of pathogens
employing advanced nanomaterials by various analytical
techniques.
functionalization using surface chemistry by reducing sensing
time and multiplexing capability. Integration of these function-
alities in nano-biosensor devices has significantly enhanced the
sensitivity and versatility of the sensors.25,26 In a typical nano-
biosensor device, the transducer is made of a nanosized mate-
rial, i.e., nanotransducer which is then coupled to a biorecog-
nition moiety such as an antibody or aptamer. The general
mechanism of optical, electrochemical and magnetic transduc-
tion modes based on nanomaterials has been clearly detailed in
a review published in 2010.27
Modern research in biosensor technology highlights a
constant increase in the implementation of nanomaterials
either into the transducer or receptor part or both to enhance
multidetection capability and sensitivity. Since the nanomate-
rials have a very high surface to volume ratio, they can
accommodate many target-specific recognition elements and
subsequently improve the sensitivity of the biosensor device.
Precisely, in comparison with traditional biosensors, nano-
materials-based biosensor/bio-devices exhibit numerous
advantages such as fast response time, high sensitivity, reduced
size and enhanced versatility. Exploiting the advantages of the
nanomaterials, researchers have designed interaction between
biological elements and nanomaterial-based transducers and
utilized various kinds of nanomaterials for pathogen detection
and related disease diagnostics.
Considering the importance of pathogen detection in many
sectors such as the food industry, water management, envi-
ronmental quality control and biodefense and knowing the
varieties of pathogens, a number of detection strategies have
been reviewed over the past 5 years focusing individually on
multi-drug resistant pathogens,28 food borne pathogens,29 water
borne pathogens30 and whole cell bacteria.31 In conjunction
with the advancement of nanomaterials synthesis and utiliza-
tion for various applications, the field of bacterial biodetection
also flourishes with the variety of bio-sensing strategies. The
advanced technology of utilizing nanomaterials toward sens-
218 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
ing, pathogen detection and other medicinal applications (such
as cancer cell imaging, DNA marking etc.) thus receives a great
deal of attention and has produces a large quantity of pub-
lished literature.32­35 This can be witnessed by several review
articles that present the developments of biosensors based on
the nanomaterials for various pathogens including food borne
bacterial pathogens and water borne pathogens.27,36,37 Recently,
a surge of research activity can also be witnessed through
the literature on pathogen biosensors employing advanced
nanomaterials.
The main goal of this review is to provide a comprehensive
outline of the literature from the year 2013 to 2018 (a five-year
period) for ongoing and upcoming researchers in this area as
well as new researchers who want to explore this active
research area of utilizing nanomaterials for the fabrication of
efficient biosensors toward pathogen detection. This review has
several focused subsections that highlight the utility of indi-
vidual nanomaterials (such as gold nanoparticles, quantum dots
etc.) towards the construction of biosensors for bacterial
pathogens. It also discusses a broad range of analytical tech-
niques such as surface-enhanced Raman scattering (SERS),
electrochemistry, a field-effect transistor (FET), fluorescence,
calorimetry and surface-plasmon resonance (SPR) which are
being utilized in nano-biosensor technology to develop novel
pathogenic bacteria sensors (Scheme 1).
2. Semiconductor Nanocrystals
Taking advantage of the developments in semiconductor
technology and materials chemistry of surface conjugation with
pathogen detecting biomarkers, various semiconducting mate-
rials such as metal sulphides, metal oxides, etc. and organic
semiconductors are used for pathogen detections. Nanostruc-
tured semiconductors are known to exhibit a host of nonlinear
optical properties and quantum confinement effects which
consequently result in exceptional properties, like the lumines-
cence in quantum dots (QDs). With their salient properties,
semiconducting nanomaterials are utilized as the biolabel/
biomarker for bacteria detection through employing various
analytical techniques such as fluorescence, 3D microchan-
nels,38 photo-corrosion,39 sulfuration,40 matrix-assisted desorp-
tion/ionization mass spectroscopy,41 colony counting,42 spec-
trophotometry,42 flow cytometry,42 light emitting diode induced
fluorescence detection43 etc. Among the various pathogen
detection techniques used with semiconducting nanomaterials,
the fluorescence enabled spectroscopic technique dominates
because it provides low detection limits and is insensitive to
magnetic noise. However, there can be two difficulties pertain-
ing to the stability of the luminescence from pristine semi-
conducting nanocrystals, namely, the existence of surface states
arising from non-stoichiometry and unsaturated bonds and very
reactive nature owing to their high surface-area-to-volume
ratio. Therefore, controlling the surface properties of the semi-
conducting nanocrystals is highly important for achieving high
luminescence with adequate stability to be used for pathogen
detection. In the forthcoming sections, the use of individual
semiconducting materials toward pathogen detection will be
discussed.
2.1 Quantum Dots (QDs). QDs represent the II­VI, III­V,
and IV­VI group semiconductor materials, their binary, alloyed
© 2019 The Chemical Society of Japan
 Figure 1. Formation of Fe@Au functionalized nanoparticles conjugated with anti-Salmonella and the determination of Salmonella
typhimurium.51 (Adapted with permission from Ref. 51, Copyright 2014 Elsevier)
and core-shell systems with a confined size on the nanoscale
(2­20 nm) in three dimensions. QDs are photostable, fluores-
cent semiconductor nanoparticles and their emission wave-
lengths are tunable with particle size and composition.44,45 The
physicochemical properties of QDs, specifically focusing on
the cellular toxicity of cadmium-containing semiconductor
QDs are detailed.46 A review on the importance of function-
alization of QDs towards bio related applications is available.47
It has been identified that toxicity is dependent on the QDs
surface properties (including a shell, ligand, and modifica-
tions), aspect ratio and exposure time. The main advantages of
QDs in photoluminescence (PL) applications include size-
tunable PL (full-width-at-half-maximum, 25­35 nm), high
quantum yield (>20%) and long PL decay times (>10 ns).48
As compared to conventional fluorescent dyes, QDs have
superior properties like high photo-stability, dual colour
detection, good selectivity, and biocompatibility. Thus, QDs
can enhance the existing fluorescent detection schemes or
mechanisms. Besides, QDs can be functionalized with a broad
range of pathogen recognition elements.49 Notably, one of the
unique advantages of QDs over conventional fluorophores is
that QDs can be utilized for multiplexed detection or simu-
ltaneous detection of two or more bacteria. Due to these
advantages, the use of bioconjugated semiconductor QDs in
bacteria detection has been emerging over the last few years.
The problems associated with QDs, such as surface traps and
defects, could be overcome by growing epitaxial layers of
inorganic material over the QD core material to obtain a core/
shell QD structure, which can provide a substantial improve-
ment in the PL efficiency. Cadmium selenide (CdSe) cores
covered with a layer of zinc sulfide (ZnS) is considered the best
available QD fluorophore owing to exceptional performance. In
these nanostructures, the ZnS layer passivates the core surface,
protects it from oxidation, prevents leaching of the CdSe into
the surrounding medium and in turn produces a remarkable
enhancement in the PL yield. The development of core-shell
QD structures has become vital for pathogen detection.
2.1.1 Cadmium Sulfide (CdS): CdS is an essential II­VI
group semiconductor with a wide bandgap of 2.42 eV. Its
Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
biological applications such as labeling cellular structures,
tracking the path and fate of an individual cell and as contrast
agents for imaging purposes have been discussed in many
reviews.44,50 However, the specific use of CdS for pathogen
detection has not been reviewed yet.
Sulfate reducing bacteria (SRB) are a group of microorgan-
isms widely distributed in anoxic environments and are a
source of serious industrial and environmental concerns owing
to their toxicity and corrosiveness. Freitas et al. reported the
use of functionalized iron/gold core/shell nanoparticles
(Fe@Au) with different self-assembled monolayers (SAM)
and CdS nanocrystal (NC) for the amplified electrochemical
immuno-sensing on a screen printed carbon electrode of
Salmonella typhimurium in food products (milk).51 The elec-
trochemical immunoassay was developed by the immobiliza-
tion of anti-Salmonella antibodies and CdS nanocrystals and is
represented in Figure 1. From this method, bacteria detection in
a milk sample can be made in very low concentration with an
analysis time of less than 1 hour.
Qi et al. developed a novel technique based on the microbial
biosynthesis of CdS nanoparticles (NPs) and used it as a power-
ful fluorescent label for SRB detection.52 Since the CdS NPs
are synthesized both intracellularly and extracellularly, they are
expected to be present and distributed near the bacterial walls.
The detection rate of SRB, Desulforibrio caledoiens is found to
be in the range of 1*102 to 1*107 cfu/mL. With this proposed
method, the authors claimed that they were able to shorten the
detection time to 2 days over the widely used most probable
number (MPN) technique which takes about 15 days to com-
plete the process. CdS NP with suitable surface modification
has also been used for the detection of pathogens. Abdelhamid
et.al. designed a biosensing platform with novel chitosan
capped QDs (CdS@CTS) for the detection of pathogenic
bacteria (Pseudomonas aeruginosa and Staphylococcus aure-
us)53 utilizing their affinity interactions between the chitosan
molecules and bacterial membranes (CdS@CTS-bacteria).
Further, non-covalent interactions in CdS@CTS-bacteria were
probed through thermodynamic studies (negative and posi-
tive enthalpy), and matrix-assisted laser desorption/ion mass
© 2019 The Chemical Society of Japan | 219
spectrometry (MALDI-MS). Transmission electron microscopy
(TEM) images confirmed the affinity and interactions of
CdS@CTS with bacteria, P. aeruginosa (Figure 2(A)) and
S. aureus (Figure 2(B)). Two pathogenic bacteria, S. aureus
and P. aeruginosa, exhibited a wide linear dynamic range and a
low detection limit of 150 and 200 cfu/mL, respectively in a
short period (³1 min).
Figure 2. TEM images of (A) P. aeruginosa and (B)
S. aureus after incubation with (a) control, (b) CdS@MPA
and (c) CdS@CTS.53 (Reprinted with permission from
Ref. 53, Copyright 2013 The Royal Society of Chemistry)
Figure 3. Pictorial representation of (a) nanobioprobe fabrication and (b) bacterial detection scheme using fluorescent-bio targeting
bifunctional cells as bioprobes.57 (Reprinted with permission from Ref. 57, Copyright 2014 American Chemical Society.)
220 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
2.1.2 Cadmium Selenide (CdSe): CdSe has a narrow band
gap of 1.74 eV and finds a broad range of potential applications
in the fabrication of nanoscale electronic, photonic, electro-
mechanical and biomedical devices.54­56 Xiong et al. reported
the biosynthesis of CdSe QDs intracellularly57 and employed
them as nanoprobes for the detection of viruses and bacteria.
The bacteria detection involves a space-time-coupling strategy
for converting the bacteria (Staphylococcus aureus) into fluo-
rescent cells (cellular beacons). The pictorial representation of
nanobioprobe fabrication and the bacterial detection scheme is
depicted in Figure 3. Typically, a low detection limit of 8.94
ng/ml was reported. The proposed scheme has many advan-
tages like fluorescent probes being monodispersed with uni-
form size, high luminance with outstanding photostability.
Also, the probes are highly accurate, reliable, and repeatable.
There are only a few reports of the metal-accumulating bacte-
ria, Lysinibacillus fusiformis. By the suitable selection of the
antibody conjugation, this kind of new bioprobe can be extend-
ed towards the sensitive detection of various other bacterial
pathogens including pseudorabies virus, baculovirus, Salmo-
nella typhimurium bacteria, and SKBR-3 cells. CdSe based
core-shell nanomaterials were also effectively utilized for the
detection of pathogens and this will be separately presented in a
forthcoming section.
2.1.3 Cadmium Telluride (CdTe): CdTe QDs are widely
used for effectively capturing and detecting pathogenic bacte-
ria. For selective capturing of Serratia marcescens, CdTe QDs
with an average size of 4­5 nm, conjugated with Concanavalin
(CdTe QDs-Con A) were used.58 The PL intensity of CdTe
QDs­Con A showed a linear graph in the range from 10 to
90 f/ml. Similarly, there was a linear relationship between PL
intensity and logarithm of the cell population of S.Marcescens
in the range from 1 © 10 to 1 © 106 (colony forming units)
cfu/mL. Shanehsaz et al. reported the detection of Helicobacter
© 2019 The Chemical Society of Japan
pylori using CdTe QDs.59 The detection is based on fluores-
cence resonance energy transfer (FRET) where it is used in
combination with a polymerase chain reaction (PCR) system.
Similarly, Morarka et al. fabricated a 3D microchannel with
CdTe QDs with diameter 3.5 nm of uniform size distribu-
tion within the polydimethylsiloxane (PDMS) elastomer for
the rapid detection of E. coli using fluorescence technique.38
Fluorescence image of E. Coli concentration ranging from 102
to 108 cells/l was determined. Nitrocellulose based dot assay
and FRET assay are used for the detection of pathogenic bacte-
ria Staphylococcus aureus using CdTe QDs as a biolabel.60 The
detection limits of dot assay and FRET assay were 3 ng/mL and
0.5 ng/mL, respectively.
CdTe QDs with a typical size range of 2­3 nm were synthe-
sized by Beibei Wang et al. for the detection of Salmonella
Enteritidis in egg samples.61 The low detection limit that was Figure 4. Pictorial representation of the GaAs/AlGaAs
achieved was 1*102 cfu/mL. This was achieved by a simple biochip functionalized with a Bio-PEG/HDT/Neutravidin/
bio-conjugation of anti Salmonella Enteritidis with QDs. It was antibody architecture.39 (Adapted with permission from
found that the detection time can be as low as 2 hours in egg Ref. 39, Copyright 2016)
which is effective in real time analysis of food samples. On the
other hand, a sensing device based on the CdTe was able to
simultaneously detect different bacterium such as Salmonella
Enteritidis, Staphylococcus aureus, and Escherichia coli.62
This was possible by tuning the emission wavelengths of these
QDs located at 504 nm, 557 nm, and 604 nm. For the effective
use of this developed technology, real food samples (such as
soda, mineral water, apple juice, milk, tomato sauce, chicken
sauce etc.) were used for bacteria detection.
Functionalized CdTe QDs have also been utilized for
pathogen detection. Kurt et al. reported the use of carboxylic
acid functionalized CdTe QD for bacteria detection.63 Aptamer-
functionalized CdTe QDs and upconverting nanoparticles were
used for specific detection of two food pathogens, namely,
Salmonella typhimurium and Staphylococcus aureus. The Figure 5. Scheme depicting the stages involved in DNA
minimum detectable concentrations of 16, and 28 cfu/mL were bielectrode fabrication.74 (Adapted with permission from
found for Staphylococcus aureus and Salmonella typhimurium, Ref. 74, Copyright 2014 Elsevier.)
respectively. Similarly, Agarwal et al. demonstrated the multi-
plexed detection of waterborne pathogens in a circular micro-
fluidics, Escherichia Coli and Salmonella typhimurium using corrosion is dependent on the concentration of the electric
CdTe QDs.64 In that report, green and red fluorescent nano- charge of the surface. A detection limit of 103 cfu/mL is
materials were synthesized by changing the size of the CdTe achieved through this technique. In a similar way, Aziziyan
nanomaterials. The detection limits of Escherichia Coli and et al. used a GaAs/AlGaAs QDs biochip functionalized with
Salmonella typhimurium are found to be as low as 103 and a Bio-PEG/HDT/Neutravidin/antibody architecture for the
107 cfu/mL. detection of Legionella pneumophilia (Figure 4).39 Bacteria
2.1.4 Gallium Arsenide (GaAs): GaAs is a compound detection is found to be in the order of 104 bacteria/ml when
semiconductor which has a high electron mobility and a direct the bacteria is suspended in phosphate buffer saline (PBS)
band gap structure which result in improved PL signal in the solution. The biosensing of bacteria using photo-corrosion can
NIR range.65 The intrinsic luminescence of GaAs either alone control the etch depth resolution in the order of nanometres.
or in a heterostructure with other compounds such as aluminum 2.1.5 Zinc Oxide (ZnO): ZnO is a II­VI type inorganic
gallium arsenide (AlGaAs) is sensitive to the physical and semiconductor exhibiting direct wide band gap (3.3 eV), a high
chemical states of its surface and is exploited for PL based excitonic binding energy (60 meV), high refractive index, ther-
optical biosensing application. GaAs or GaAs/AlGaAs hetero- mal conductivity and many other outstanding physico-chemical
structures can be combined with a molecular recognition ele- and fascinating photo-physical properties.67­73 ZnO and nano-
ment and form promising candidates toward designing optical sized ZnO are also identified as antibacterial agents. These
biosensors for pathogen detection. Nazemi et al. reported the unique characteristics enable ZnO to be used in a broad range
synthesis of GaAs/AlGaAs heterostructures and demonstrated of applications including pathogen detection.
that their photo corrosion effect can be effectively used for the Low cost hydrothermal technique was used for the synthesis
detection of bacteria.66 By using PL technique, the detection of of flower-like ZnO nanostructures (ZnF) which were then
E. Coli was possible. It was reported that the rate of photo- immobilized with DNA molecules for the effective detection of

Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280 © 2019 The Chemical Society of Japan | 221
N. Meningitides.74 Figure 5 shows the stages involved in the
preparation of DNA bioelectrode fabrication. The nanostruc-
tures and the higher loading of DNA were found to be respon-
sible for the highly selective detection of this bacteria. The
detection limit was found to be 5 ng uL¹1. The fabricated
bioelectrode exhibited a high sensitivity of about 168.64 ng and
a wide range of 5 to 240 ng. Figure 6 presents the SEM images
of (a­c) ZNF/Pt/Si and (d) DNA/ZNF/Pt/Si bioelectrodes at
high and low magnifications before and after immobilization.
Gopal et al. also reported the synthesis of ZnO NPs for the
detection of Staphylococcus aureus from the surface of ants of
Figure 6. SEM images acquired at low and high magnifi-
cations before and after immobilization. (a­c) ZNF/Pt/Si
and (d) DNA/ZNF/Pt/Si bioelectrodes.74 (Reprinted with
permission from Ref. 74, Copyright 2012 Elsevier)
Figure 7. Synthetic route for the preparation of ZnO@BSA-PEP-MPA.76 (Reprinted with permission from Ref. 76, Copyright 2015
Elsevier)
222 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
a particular species.75 ZnO NPs assisted MALDI-MS is used
as a sensitive technique for bacteria detection. This report
demonstrated that ants could carry pathogenic bacteria such as
Staphylococcus aureus where a detection limit of 103 cfu/mL
was found.
Chen et al. designed peptide-based ZnO QDs and developed
a fluorescent nano-probe called ZnO@BSA-PEP-MPA (BSA:
stabilizing agent, MPA: near infra-red dye) with low cytotox-
icity for the detection of in vivo bacteria diagnosis through
in vivo early bacteria imaging.76 The structure and stages
involved in the preparation procedure are shown in Figure 7.
The developed strategy could achieve a detection level of
1.6 © 104 and potentially find prospects in anti-multidrug resis-
tance applications. Pan et al. reported the synthesis of ZnO,
titanium dioxide (TiO2) and silicon dioxide (SiO2) NPs for the
detection of four pathogenic bacteria (Staphylococcus epider-
midis, Enterococcus faecalis, and Escherichia coli).42 Three
different methods are used for the quantification of bacteria
such as colony counting to determine colony forming unit
(CFUs), spectrophotometer method of optical density and flow
cytometry (FCM). Among the three different methods, CFU
counting is found to be time-consuming and less accurate. The
spectrophotometry technique is also found to be the most
unreliable while FCM is considered to be an appropriate tech-
nique owing to its rapid, sensitive and simultaneous detection
of pathogen.
Like bacteria detection in a food sample, detection of the
same in a water sample is also challenging. Gedda et al. report-
ed the synthesis of ZnO NPs for bacteria detection in water.41
Dispersive liquid­liquid microextraction was used to detect
bacteria using ZnO NPs modified with polymethyl methacry-
late (ZnO@PMMA). The chemical structure and the pictorial
representation of ZnO@PMMA coupled with MALDI-MS for
© 2019 The Chemical Society of Japan

Figure 8. Illustration of ZnO@PMMA-DLLME towards
extraction of pathogenic bacteria.41 (Adapted with permis-
sion from Ref. 41, Copyright 2014 The Royal Society of
Chemistry)
the extraction of bacteria is shown Figure 8. A minimum fluo-
rescence detection level of 9.7 © 103 and 1.7 © 104 cfu/mL for
S. aureus and P. aeruginosa was reported. A high surface area
of ZnO@PMMA NPs was found to be the primary reason for
the high sensitivity of MALDI-MS spectra.
2.1.6 Core-Shell Quantum Dots: It has been well demon-
strated through several research studies that a fraction of sur-
face atoms in QD nanocrystals are passivated by the stabilizing
organic ligands. Hence, these passivated core QD nanocrystals
exhibit surface trap/defect states which can act as fast non-
radiative channels for photogenerated charge carriers and
decrease the fluorescence quantum efficiency. One of the
strategies to alleviate the problems in QD nanocrystals is to
form a shell layer of a second semiconductor, resulting in core/
shell QDs. Core/shell QD-based nanomaterials with both core
and shell made of semiconducting materials are commonly
synthesized and used as biomarkers for the sensitive detection
of various bacteria. Various semiconducting nanomaterials
such as cadmium sulfide (CdS), CdS0.5Se0.5, CdSe/ZnS@SiO2,
cadmium telluride (CdTe), CdTe/Cd(OH)2, GaAs/AlGaAs,
PbS, ZnO, ZnS, ZnS/CdSe, ZnS:Mn were utilized for bacteria
detection by exploiting the unique physicochemical character-
istics of the nanomaterials through different kinds of analytical
methods/techniques.
2.1.6.1 Zinc Sulfide (ZnS)/Cadmium Selenide (CdSe);
ZnS/CdSe is the most commonly used core/shell semiconduct-
ing nanomaterial for the detection of bacteria using fluores-
cence techniques. Arshad et al. reported the use of CdSe/ZnS
QDs for the detection of Vibriyo harveyi in solution and in
animal cells.77 Techniques such as fluorescence microscopy,
elastase assay, polyacrylamide gel electrophoresis (PAGE), and
comet assay are used to evaluate the interactions of QDs with
Vibriyo harveyi. The appearance of bright yellow fluorescence
when different concentrations of QDs were applied confirms
the perfect attachment of the QDs to the bacteria. It was also
demonstrated that the toxicity level of CdSe/ZnS QDs is
genetically and cytotoxically safe for labelling the bacteria
allowing live imaging and tracking of the microorganisms.
Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
In another report, the stability of the sensors were signifi-
cantly enhanced by using oligonucleotide microarray combined
with CdSe/ZnS QDs as fluorescent labels.78 The bacteria was
identified with PerkinElmer Gx microarray scanner displaying
sensitivity of 10 CFU/mL. Fluorescence detection of E. Coli
0157: H7 bacteria was obtained by using CdSe/ZnS QDs with
carboxylic functional groups.79 The 72-mer aptamer is used as
the probe and sensing element where detection limit of 102
cfu/ml could be achieved. Drugs are susceptible to the infec-
tion by bacteria and other contaminations. Therefore, it is
essential to detect bacteria in drug samples. Wu et al. used
ZnS/CdSe QDs conjugated with the rabbit anti goat antibody
for the detection of E. Coli in drugs.80 The detection process
took less than four hours to complete which is acceptable for
ensuring the rapid detection of bacteria. Detection of Salmo-
nella bacteria from chicken breast samples using CdSe/ZnS
QD was reported by Kim et al.81 A microfluidic nanosensor
based on CdSe/ZnS QD immobilized with anti-salmonella
polyclonal antibodies was used for the detection and showed
the detection limit of 103 cfu/mL of borate buffer and food
extract. In another report, the same group employed streptavi-
din functionalized CdSe/ZnS core-shell QDs for the detection
of Salmonella.82 The detection limits were found to be 1.4*103
cfu/mL in bovine serum albumin (PBS­BSA) and 4*103 in
food extracts. Light emitting diode induced fluorescence detec-
tion (LIF) can also be used for bacteria detection. Wang et al.
reported the synthesis of CdSe/ZnS QDs for the detection of
Salmonella typhirium.43 The detection was based on a micro-
fluidic chip and a LIF microsystem. The detection limit was
found to be 37 cfu/mL. The simultaneous detection of E. Coli
and Salmonella in ground beef was also successfully achieved
by Wang et al. using ZnS/CdSe QDs.83 The detection limit was
found to be 10 cfu/g after 24-hour enrichment. It used a bead
free technique for the detection and detection time was less
than 3 hours without enrichment.
Qi et al. reported the synthesis of ZnO for the determination
of SRB. From ZnO, firstly an intermediate such as ZnO/ZnS
is formed during sulfuration and then later ZnS arrays are
formed. The proposed method takes only 4 days versus 15 days
using the conventional most probable number (MPN) tech-
nique. ZnS nanoparticles were also utilized for the bacteria
detection towards SRB detection.40 Using this method, the
population of SRB can be monitored selectively and can be
employed without any biological markers or sophisticated
instruments. Importantly, the time taken for detection is only
3 days and is much less than MPN method.
2.2 Inorganic Metal Oxides. Bahadoran et al. reported the
use of SiO2-TiO2 hybrid for the detection of the Salmonella
bacteria in human blood.84 Here, the SiO2-TiO2 nanomaterial
was used for making a double ring add-drop filter micro ring
resonator which in turn was used to detect bacteria. The scatter-
ing matrix method was utilized for the detection of the output
signal by inductive calculation. The achieved sensitivity is
95500 nm/refractive index unit (RIU), and the detection limit
is 1 © 10¹8 RIU.
3. Magnetic Nanoparticle Based Bacteria Detection
Crystal violet or gentian violet (CV; 4,4¤,4¤¤-dimethylamino-
triphenylmethane) is a triarylmethane based deep purple dye
© 2019 The Chemical Society of Japan | 223
particularly used in Gram’s method for classifying bacteria.85 multiplex PCR methodology for the sensitive and concurrent
Budin et.al. modified CV functionalized with trans-cyclooctene recognition of the three food pathogens, viz. Salmonella
(CV-TCO) for the recognition of Gram-positive bacteria and enterica, Listeria monocytogenes, and Escherichia coli. Silica
evaluated three samples Staphylococcus aureus (S. aureus), magnetic nanoparticles have been utilized as carrier platform
Escherichia coli (E.coli) and the combination of two species.86 for immobilization along with various electrochemical report-
Interestingly, they observed that CV-TCO could act as a host ers (anti-Fluorescein­HRP, Streptavidin­HRP and anti-Digox-
anchor to immobilize tetrazine (Tz)-modified magnetic nano- igenin­HRP) specific to each pathogen. This study was aimed
particles. Gram stain, being the most standard technique for at detecting low concentrations of Salmonella enterica, Listeria
differentiating bacteria was utilized for the sensitive detection of monocytogenes and Escherichia coli (Figure 10). It was report-
Gram-positive pathogens using micro nuclear magnetic reso- ed that this method could successfully detect bacteria in less
nance (μNMR). It was observed that orthogonal CV is useful in than 50 min with LOD ranging from 12­46 pg/¯L.89 The
distinguishing biological samples. In addition, the implemented authors envisage that this versatile sensing strategy could find
standard Gram strain method along with labeling of magneto- applications in microfluidic-based devices.
fluorescent nanoparticles modified with tetrazine (MFNP-Tz) Xia et.al. investigated bio-accessibility of seven types of
for strain bacteria can enhance the detection and character- soil with various amounts of As, Cd, and Pb. They showed that
ization of bacteria both by μNMR and optical imaging.86 The presence of Pb in the soil was the main control parameter for
proposed strategy can be further extended to attach small
molecule ligands for the specific recognition of other bacterial
samples.
Chan et.al. reported biofunctional polystyrene magnetic
beads (particles) for ultra-sensitive detection of E. Coli
O157:H7 through conjugating magnetic bead concentration
on a nanoporous alumina membrane-based electrochemical
immunosensor. The beads were modified by specific antibody
to enhance sensitivity. Figure 9 presents the functionalization
process for nanoporous alumina membrane. By utilizing an
external magnetic field, E. Coli O157:H7 cells could be easily
localized in a confined area on the nanoporous membrane to
form a sandwich architecture of antibody-modified magnetic
beads. Impedance measurements were used to evaluate the effi-
cacy of the conjugated and non-conjugated E. Coli O157:H7
by applying a magnetic field. By using this conjugation-
concentration strategy, the authors could attain the detection
limit of 10 CFU/mL (Figure 9).87
Among the different strategies available for simultaneous
detection (such as time-resolved fluorescence (TRF), biosen- Figure 9. Pictorial representation for biofunctional mag-
sors, multiplex quantitative polymerase chain reaction (PCR), netic bead concentration of E. Coli O157:H7 in the bio-
etc.) electrochemical genosensors have been most actively chip. (a) Polystyrene beads were conjugated with E. Coli
employed for identifying pathogens, offering a futuristic pros- O157:H7; (b) Concentrated in a confined region under the
pect for simultaneous detection, owing to their high sensitivity, influence of an magnetic field and (c) Removal of magnetic
specificity and cost effectiveness.88 Brandao et al. combined field.87 (Adapted with permission from Ref. 87, Copyright
electrochemical magneto-sensing together with triple-tagging 2013 Elsevier)

Figure 10. Electrochemical approach of triple-tagging magneto genosensing on silica magnetic particles by varying the quantity of
electrochemical reporter (from 1 to 30 ¯g/assay).89 (Reprinted with permission from Ref. 89, Copyright 2015 Elsevier)

224 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280 © 2019 The Chemical Society of Japan
 Figure 11. Combinatorial strategy using Apt-MB and AuNCs@Van for the quantification of SA.92 (Adapted with permission from
Ref. 92, Copyright 2016 American Chemical Society.)
bio-accessibility of Pb. Further studies for a simulated human
digestive system revealed no impact of As or Cd on Pb bio-
accessibility.90 It was proved that proximity ligation assay
(PLA) could be utilized using magnetic particle based substrate-
free sensing, without the aid of thermocyclic or optical
equipment.91
Cheng et.al. devised a strategy to combine inexpensive and
widely available aptamer and antibiotic-based dual recognition
units with magnetic enrichment for the sensitive detection of
Staphylococcus aureus (SA) in the presence of other bacteria
with higher contents. To capture SA, magnetic beads modified
aptamer (Apt-MB) were utilized. A single step process has
been applied for vancomycin-stabilized fluorescent gold nano-
cluster (AuNCs@Van) synthesis. Apt-MB and AuNCs@Van
were further used for the quantification of SA in milk and
human serum samples with a high recovery ratio. It has been
shown that around 70 cfu/mL of SA in the mixture can be
detected via this technique (Figure 11). The as-developed
approach can be used as a dual recognition technique for the
specific and sensitive detection of SA in diagnosing infectious
diseases.92
3.1 Iron Oxide Nanomaterials. Kwon et.al. devised a
facile method for detection of Salmonella bacteria in milk
using magnetophoretic chromatography. To accomplish this,
gold-coated Fe3O4 magnetic nanoparticles (Au/MNCs) were
suitably surface functionalized with amine-reactive N-hydroxy-
succinimide (NHS) linkers to immobilize the antibody. After
10 min of magnetophoretic chromatography, 100 cfu/mL of
Salmonella bacteria could be detected with the naked eye.
The proposed method sensitivity is limited to 100 cfu/mL,
which has been validated using absorption spectroscopy. It was
believed that the reported sensitivity could be further enhanced
by improving impregnation of MNCs with fluorescent tags.
This technique can also be applied to rapid on-site detection
as it can be assembled with a permanent magnet and precision
pipette for food safety monitoring.93
In another study, Jin et.al. described a similar magnetic
separation technique via anchoring antibody onto Fe3O4 mag-
Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
netic nanoparticles (MNPs) and TiO2 nanocrystals (TNs) as
optical nanoprobes to capture Salmonella bacteria in a buffer
solution or milk through optical sensing (UV-vis spectroscopic
detection) (Figure 12). The UV-visible spectrum of the ob-
tained complex revealed the high sensitivity of the assay
toward low concentration of Salmonella bacteria. The detection
scheme is illustrated in Figure 12. The proposed measurement
process was found to be much quicker, cost efficient and less
hazardous over conventional methods due to the absence of a
cell culturing process with a detection limit of over 100 cfu/mL
for Salmonella bacteria in milk.94
Kim et.al. also proposed a detection platform for the rapid
and selective capture of pathogenic Salmonella, based on a
theoretically optimized model. They have reported the prepa-
ration of water-soluble superparamagnetic Fe3O4 magnetic
clusters coated with hydrophobic ligand (oleic acid) to provide
conjugation sites for antibody immobilization followed by
evaporation-induced self-assembly (EISA) strategy to modify
the magnetic properties. Two different antibodies: H- and O-
antigens were immobilized on the cluster surface to enhance
the selective binding. Typically, the results suggested that O-
antibody-coated polysorbate 80-coated magnetic nanoclusters
(PCMNCs) exhibited the highest capture efficiency (³99%)
for Salmonella,95 while the capture efficiency for H-antibody
modified magnetic nanoparticles was relatively lower (³57%).
Shukla et.al. demonstrated a method for rapid detection of
Cronobacter Sakazakii (C.sakazakii) in both culture and infant
formula. They have coated immunoglobulin G (lg G) onto
carboxyl Fe2O3 magnetic nanoparticles. The proposed assay
exhibited a detection sensitivity of 3.3 © 103 CFU/mL of
C.sakazakii in pure culture within 2 h 30 min, while an indirect
non-competitive enzyme-linked immunosorbent assay exhib-
ited a detecting ability of 6.2 © 105 cfu/mL of C.sakazakii in
pure culture after 17 h and 103 cfu/mL of C.sakazakii was
observed in infant formula. However, the developed method
suffered from the limitation of multiplexing efficiency.96
Tian et.al. utilized two kinds of magnetic particles, micron-
sized and nano-sized particles as capture and detection parti-
© 2019 The Chemical Society of Japan | 225
 Figure 12. The schematic representation using optical nanoprobe (left) and (right) TEM images acquired for Salmonella bacterium
after binding to (a) bare MNPs, (b, c) antibody-immobilized MNPs and antibody-immobilized TiO2 and (d) A magnified portion of
the square region from (c).94 (Reprinted with permission from Ref. 94, Copyright 2012 The Royal Society of Chemistry.)
cles, respectively through a novel and cost-efficient Blu-ray
optomagnetic technique. The proposed method was able to
detect Salmonella bacteria as low as 8 © 104 CFU/mL within
3 h of process time. The observed sensitivity limit was 20 times
lower by using the as-developed technique compared to volu-
metric magnetic stray field detection device immunoassay. The
newly developed method exhibited high compatibility to dif-
ferent types of pathogens. Moreover, no visible cross-reaction
has been observed with other bacteria.97
Xu et.al. proposed a detection technique using
immunosensor-based on a screen printed interdigitated micro-
electrode (SP-IDME) coupled with 130 nm iron oxide (Fe3O4)
magnetic beads (MBs) and glucose oxidase (GOx) antibody
labeling for the capture of foodborne and iatrogenic pathogens
(Escherichia coli O157:H7 and Salmonella Typhimurium). It
was reported that as developed approach was capable of detect-
ing the E. Coli O157:H7 and Salmonella Typhimurium with
LOD of 2.05 © 103 CFU g¹1 and 1.04 © 103 CFU g¹1, respec-
tively.98 Yang et.al. utilized dual recognition agent to increase
the specificity of the Staphylococcus aureus (S. aureus), by
applying vancomycin, as a strong antibacterial agent function-
alized with magnetic beads and alkaline phosphate (ALP)-
tagged rabbit immunoglobulin G(ALP-lg G). It has been ob-
served that the S. aureus detection was 12­1.2 ©106 CFU mL¹1
with LOD as low as 3.3 CFU mL¹1, during 75 min of process
time. Interestingly, the presence of other bacteria did not have
any effect on S. aureus detection. However, utilizing vanco-
mycin in the process produces a drug-resistant pathogen that
may require further sterilization.99
S. Typhimurium and S. aureus are considered to be the root
cause of many food borne diseases. In order to address and
provide a feasible solution, Zhang et al. developed a Raman-
based biosensing platform for the concurrent detection of
pathogens, S. typhimurium and S. aureus. For this purpose,
gold nanoparticles (GNPs) have been suitably incorporated
with Raman molecules (mercaptobenzoic acid (MBA) and
Dithiobis(2-nitrobenzoic acid)) (DNTB)). GNPs and aptamer
have been applied as the signal probe for S. typhimurium and
S. aureus, correspondingly. The SERS intensity of MBA,
226 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
DNTB were identified at 1582 cm¹1 and 1333 cm¹1 that corre-
sponds to the S. typhimurium and S. aureus, respectively.
Under optimal conditions, this simple approach presented the
LOD of 35 CFU mL¹1 for S.aureus and 15 CFU mL¹1 for
S.typhimurium that is high when compared to the existing
methods.100 This method is straightforward and rapid, results
in high sensitivity and specificity, and can be used for the
detection of practical samples.
Wang et.al. introduced a technique for developing porta-
ble and sensitive pathogen detection using a personal glucose
meter (PGM). Sequential steps were followed for the preparat-
ion of glucoamylase-quaternized magnetic nanoparticles (GA-
QMNPS) through the chemical modification of Fe3O4 with
SiO2 (Fe3O4@ SiO2). Fe3O4@SiO2 is further surface modified
with N-trimethoxysilypropyl-N, N, N-trimethylammonium
chloride to obtain QMNPS. Implemented glucoamylase-
quaternized magnetic nanoparticles (Fe3O4) (GA-QMNPS)
interacted with the pathogenic bacteria and released GA as a
result. The presence of free GA, after magnetic separation,
could catalyze the hydrolysis of amylose. The proposed
approach was very sensitive to low concentration down to
20 cell mL¹1 suitable for clinical application. However, there is
no result on detection ability of their approach towards various
microbes and pathogenic bacteria.101
A gold-coated nanoparticle-labelled electrochemical bio-
sensor has been developed by Wang and Alocilja for E. Coli
O157:H7 detection in broth samples. Magnetic gold nano-
particles (AuNPs) were conjugated with polyclonal antibodies
to segregate E. Coli O157:H7 from broth samples. The amount
of E. Coli bacteria was quantified by measuring the content of
AuNPs coated on a screen-printed carbon electrode via an elec-
trochemical method (Differential pulse voltammetry, DPV).
The dynamic linear range of 10­106 CFU mL¹1 has been
reported within 1 h of detection and extraction.102
Wang et.al. developed a low-cost magnetic assisted SERS
biosensor for single-cell detection of S. aureus based on apta-
mer recognition. The as-developed biosensor contained two
main parts including a SERS substrate (Ag-coated magnetic
NPs) and SERS tag (AuNP-DTNB@Ag-DTNB core-shell plas-
© 2019 The Chemical Society of Japan
 Figure 13. Colorimetric immunoassay for rapid detection of S. pullorum based on blue-SiNPs and MNPs.104 (Adapted with
permission from Ref. 104, Copyright 2015 American Chemical Society.)
monic NPs or DTNB-labelled inside-and-outside plasmonic
NPs, DioPNPs). DioPNPs exhibited 10 fold higher Raman
intensity compared to AuNR-DTNB and observed good linear
range from 10 to 105 cells/mL for S.aureus detection.103
A colorimetric immunoassay antibody-coated magnetic
nanoparticle (IgG-MNPs) has been developed by Sun et al.
for quantitative Samonella pullorum (S. pollorum) detection.
Blue-SiNPs (Tetraethyl orthosilicate as Si source) surface-
modified with anti-Salmonella pullorum (S. pollorum) has been
utilized as detection probes. Magnetic nanoparticles (MNPs)
has been applied to anchor polyclonal antibodies against
(S. pollorum) considered as a capture probe (Figure 13). The
application range for the as-prepared assay was 4.4 © 102
CFU mL¹1 to 4.4 © 107 CFU mL¹1 with LOD of 4.4 © 101
CFUmL¹1 for S. pollorum. Interestingly, in this assay, no
enzyme catalysis was required and exhibited a high selectivity
toward S. pollorum among other pathogenic bacterium.104
Rahman et.al. studied the impact of various nanoparticles on
multiplex polymerase chain reaction (PCR) method for recog-
nition and strain typing of Salmonella enterica serotype typhi
(S. Typhi) (targeting Variable Number of Tandem Repeats
[VNTR]) by employing different nanomaterials such as citrate
capped gold nanoparticles, rhamnolipid protected gold and
silver and magnetic iron oxide nanoparticles. It was observed
that appropriate incorporation of nanoparticles significantly
reduced non-specificity of PCR. The results demonstrated the
enhancement of target DNA template. It is worth mention, the
improvement relied heavily on the nature of applied metal
nanoparticles. However, there is no clear discussion on the exact
mechanism of diagnosis using different metal nanoparticles.105
Ozlap et.al. utilized a quartz crystal microbalance sensor
(QCM) for the recognition of Salmonella enterica serovar
Typhimurium cells in milk samples. The results demonstrated
that the integration of aptamer-magnetic and QCM-sensor tech-
nique provided a quick and sensitive detection of Salmonella in
food contaminants with a detection range from 100 to 4 © 104
CFU/mL cell and LOD of 100 cells in less than 10 min. Further
Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
Figure 14. Rapid capture of selected pathogen through
gold-coated iron oxide nanoparticle treatment.107 (Adapted
with permission from Ref. 107. Copyright 2015 Elsevier.)
treatment of QCM surface with NaOH solution could regen-
erate the aptamer-sensor allowing each crystal to be reused.106
Niemirowicz et.al. reported the effect of gold-coated iron
oxide nanoparticles and their derivatives on restricting various
pathogens growth and assisting in capturing them (Figure 14).
The results revealed a substantial reduction of E. Coli,
S. aureus; P. aeruginosa and sandida albicans growth after
exposing them to MNPs for 24 h. However, the phenomena
depend on MNPs concentrations, pathogens strains, surface
chemistry and the surrounding environment.107
Antibody/enzyme-conjugated MNPs have been utilized for
S. typhimurium detection by Mun et al. using 100 nm-antibody
and horseradish peroxidase-conjugated magnetic MNPs (Ab-
HRP-MNPs). The response range of this technique was 104
CFU/mL with LOD of 10 CFU/mL. An 80 nm iron oxide core
and 10 nm silica and APTES EDC/NHS treated MNPs also
have been used.108
Mezger et al. demonstrated a rapid and sensitive molecular
detection approach for detection of bacteria causing urinary
tract infection using padlock probe, and on-bead isothermal
rolling cycle amplification (RCA) integrated with on-disk auto-
mated aptomagnetic nanoparticles. Interestingly, the change
of temperature and laborious DNA preparation were not
required. Optomagnetic read out has been utilized for ampli-
© 2019 The Chemical Society of Japan | 227
fication product detection and demonstrated highly sensitive
and multiplexing capability. Implementing RCA monomers
enhanced the sensitivity by 10 fold, along 3 fold improvement
for magnetic incubation. Therefore, the proposed approach
exhibited 10 times higher sensitivity than the sample prepara-
tion amplification and detection process was more rapid (4 h
including preparation time) compared to 7 days for other
methods.109
Liu et al. prepared a magnetic nanoparticle-labeled immu-
nochromatographic test strip (MNP/ICTS) for the food-borne
pathogen, vibrio parahaemolyticus (v. parahaemolyticus). The
specific antibody has been immobilized onto the superpara-
magnetic nanoparticles. The observed sensitivity was 1.58 ©
102 CFU/mL for v. parahaemolyticus in about 4.5 h.100 Lin and
Hamme developed an assay for quantitative and qualitative
detection of Salmonella DT104 by utilizing antibody affinity
binding; popcorn-shaped gold nanoparticle (GNPoPs) labeling,
surface enhanced Raman spectroscopy (SERS), and inductively
coupled plasma mass spectrometry (ICP-MS). The LOD of
100 CFU/mL has been noticed within 40 min (Figure 4).110
Although the developed assay is very sensitive in detecting
Salmonella DT104, a higher concentration of bacteria causes
less accurate detection due to bacteria binding to antibody-
conjugated GNPoPs.
Lee et al. incorporated a 3D-printed helical microchannel
and magnetic nanoparticle clusters to detect E. Coli bacteria
in milk samples. The process includes dispersing free MNCs
and MNCs-EC components into buffer mixture, followed by
injecting the resultant solution into a helical microchannel
device. The LOD was reported around 10 CFU/mL in buffer
solution and 100 CFU/mL in milk sample.111 A colorimetric
technique has been developed using Pt-coated magnetic nano-
particles and magnetophoretic chromatography for pathogenic
bacteria detection. The Pt/MNPs were coated by monoclonal
E. Coli O157:H7 antibodies to anchor E. Coli bacteria in
milk. After separating Pt/MNPs-EC from free Pt/MNPs via a
magnetic field, tetramethylbenzidine (TMB) solution has been
added to the separated part. Catalytic oxidation of TMB by Pt
led to colour changes of the solution visible to the naked eye.
The sensitivity observed was 10 CFU/mL in 30 min.112
Hardiansyah et al. developed a multifunctional hybrid core-
shell nanostructure composed of iron platinum (FePt), silica
(SiO2) decorated by gold nanoparticles (AuNPs) (Au-FePt@
SiO2-N NPs) for label-free surface-enhanced Raman scattering
(SERS) sensing of biomolecules (adenine) and gram positive
pathogenic bacteria (S. aureus). Remarkable intensity has been
observed in SERS spectra for anchored adenine and S. aureus
on Au-FePt@SiO2-N NPs. The proposed SERS platform
displayed the potential application for simultaneous magnetic
separation and bio-sensing.113 Horikawa et al. demonstrated
bacteria detection in stagnant liquids using magnetoelastic
(ME) bisentinels. ME bisentinels are made of pristine ME
resonator grafted with an engineered phage-based molecular
recognition to bind with pathogenic bacteria of interest. Mag-
netic properties of biosentinels enable them to move through
liquid via external magnetic force. After forming a chemical
bond between bisentinels and pathogen bacteria, the presence
of pathogen was identified by noticing the changes in the
biosentinel resonant frequency.114
228 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
4. Noble Metal Based Nanostructures
for Bacteria Detection
Noble metal nanoparticles such as platinum, gold, and silver
are fascinating materials with unique electrical, optical, mag-
netic, surface, and chemical properties depending on their size
and morphology. Due to their unique physico-chemical prop-
erties at the nanoscale, noble metal nanoparticles have been
continuously explored in the evolution of new kind of sensors.
Furthermore, nanoparticle-based biosensors are particularly
attractive because the synthetic process of nanoparticles is
straightforward and does not require advanced fabrication
methodologies. They also offer very high surface areas due to
their minuscule size and are typically used as suspensions in
solutions, where they effectively interact with the analytes.
Because of their large surface area, excellent biocompatibility
and ease of functionalization, various noble metal nanomate-
rials, especially gold/silver nanoparticles, have been used as
excellent carriers for loading different bio-recognition moieties
such as enzymes, antibodies, aptamers, organic ligands, and
antimicrobial agents which can dramatically amplify and trans-
duce the signals in a biosensor device. In the forthcoming
sections, we will focus on the noble metal nanomaterial such as
gold silver, platinum, and bimetallic nanomaterials, used for
the quantification of pathogenic bacteria using various ana-
lytical techniques.
4.1 Gold Nanostructures for Pathogen Detection.
Fleischmann et al. discovered the phenomenon of surface-
enhanced Raman spectroscopy (SERS) which is considered to
be among the most popular ultrasensitive analytical techniques
due to its high detection capability and sensitivity.115 Noble
metal colloidal particles and nanostructured surfaces have
become the most commonly used platform for SERS. The
SERS phenomenon can be generally explained by a combina-
tion of an electromagnetic and a chemical enhancement and
out of which electromagnetic enhancement predominantly con-
tributes to SERS. Electromagnetic enhancement exploits the
Raman scattering from molecules in close neighbourhood to a
nanostructured surface due to the coupling effect of surface
plasmon (gold) via the oscillating electric field of the incident/
scattered radiation. The possibility of observing Raman signals
with enhancements in the order (106­1014) is normally weak
and the ability to obtain molecular recognition of target bacte-
ria at very low concentrations allow SERS to be unique for
ultrasensitive pathogen sensing. High selectivity, sensitivity
and informative spectral characteristics derived from Raman
spectroscopy render SERS-based methods to be a feasible
alternative for bacterial detection than traditionally used optical
sensing methods. In this part of the review, we will discuss
the SERS-based detection of pathogenic bacteria using the
advanced plasmonic nanomaterials including gold and silver.
The plasmonic nanomaterials include both solution based
nanoparticles and nanomaterials prepared in the form of solid
platform. On reviewing this sub-topic, our observation is that
the majority of the plasmonic nanomaterials use SERS tech-
nique to identify pathogenic bacteria.
Papadopoulou et al. reported the SERS-based label-free
sensitive detection of unmodified single and double stranded
DNA (ssDNA and dsDNA) down to nanomolar concentration
© 2019 The Chemical Society of Japan
by adsorbing Au and Ag colloids. Herein, magnesium sulfate
(MgSO4) was used as an aggregating agent mainly because of
the low-affinity of sulfate ions for metal (Au and Ag) colloids
and the tendency to aggregate even at a higher concentration
without displacing the adsorbed DNAs. Five DNA sequences
that correspond to different strains of E. coli bacterium were
used.116 The authors were able to differentiate SERS spectra of
five sequences corresponding to various E.coli strains without
using advanced multivariate data analysis techniques. On the
other hand, Zhang et al. demonstrated the rapid concentration
and SERS-based detection and identification of bacteria cells
at a concentration range of 105 CFU/mL using magnetic­
plasmonic iron oxide-Au core-shell (Au-MNPs) nanoparticles
via applying external point magnetic field.117 The authors
coated a known quantity (10 uL of 105 CFU/mL) of prepared
Au-MNPs on the silica substrate to enable sensitive SERS-
active substrate. The authors could effectively differentiate
three different Gram-negative bacterial strains (Acinetobacter
calcoaceticus, E. coli K12, and Pseudomonas aeruginosa)
through principal component analysis (PCA). With beneficial
plasmonic and magnetic properties and the existence of an
external magnetic field, Au-MNPs could be condensed to form
a highly compact dot (60 times more concentrated) in less than
5 min. The proposed Au-MNP sensor platform offers many
advantages over the existing sample preparation method
including low-cost, easy operation and rapid detection and
concentration of bacterial samples. The authors also envisage
the potential applications in various field applications such as
environmental monitoring, food safety etc.
Lin et al. also prepared highly sensitive SERS filter-like
substrate using AuNPs encapsulated mesoporous silica
(AuNPs@MS) through a convenient one-pot method with a
high specific surface area and it utilized towards SERS appli-
cation.118 This simple surfactant free SERS substrate was
prepared by dispersing Au NPs into the mesoporous silica
matrix and subsequent thermal treatment at 600 °C for 6 h to
remove the organic protecting agents. S.aureus can be effec-
tively localized on the prepared AuNPs@MS substrates within
a few seconds. The resultant SERS spectra exhibited character-
istics peaks at 1158 and 1520 cm¹1 attributing to the cell wall
of S.aureus. SERS intensity increased significantly (over 900
fold) for AuNPs@MS with Au wt% = 16 when compared with
that of the pure mesoporous silica without nanoparticle loading.
Keeping in the mind the low pathogenic concentration
(<100 CFU/mL) in the cases like food poisoning and other
infectious diseases, Drake et al. employed two different nano-
particles labels, iron-oxide nanoparticles for magnetic trap-
ping and 4-mercaptobenzoic acid (MBA) coated SERS active
AuNPs (MBA-AuNPs), for the quantification of pathogenic
Staphylococcus aureus (S. aureus).119 MBA-AuNPs exhibited
signature peaks at 1077 and 1584 cm¹1 assigned to the aromatic
ring vibrations, respectively.119 These kinds of nanoparticles
were conjugated with antibodies that can accurately bind to
protein A on the S. aureus which led to an ultrasensitive SERS
detection of 1 S. aureus cell/mL within 10 min. This work
encouraged other researchers to work on the mixed pathogenic
multiplexed system to enable simultaneous detection of several
bacterial species. On the other hand, Fan et al. designed and
developed the multifunctional popcorn-shaped iron magnetic
Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
core-gold plasmonic nanoparticle shell for the label-free SERS-
based detection, magnetic separation, imaging and selective
photothermal destruction of MDR Salmonella DT104, a multi-
drug-resistant bacteria (MDRB), by utilizing an excitation
source of 670 nm light.120 They have modified the surface of
the prepared core-shell nanoparticle with an antibody which is
highly specific for the detection of MDR Salmonella DT104.
The experimental results demonstrate promising results with a
limit of detection as low as 102 CFU/mL for MDR Salmonella
DT104 using the hybrid nanostructures.
With an aim of enhancing the Raman signal, Chae et al.
reported the SERS detection of infectious bacteria, E. Coli
(gram negative) and listeria monocytogenes (gram positive)
through the electrodeposition of AuNPs onto indium tin oxide
substrate (ITO)121 and verified the successful formation through
appropriate spectroscopic and microscopic techniques. The
antibody against E. Coli was immobilized on AuNPs to bind
the bacteria on the SERS substrate selectively through anti-
body-antigen interaction. Like any other SERS based nano-
biosensors, the observed shift in Raman peak after E. Coli
binding was used for the detection of bacteria. Concentration
range from 101 to 105 were detected with R2 = 0.99.
Xu et al. developed unique quasi-3D (Q3D) plasmonic
nanostructure arrays of plasmonic nanoholes via electron beam
lithography (EBL) and used as a sensitive and reproducible
SERS substrate to rapidly differentiate seven different strains
(50, 3355, 605, 551, 846, 12310 and 3256) of the marine
pathogen Vibrio parahaemolyticus.122 The fabricated Q3D was
patterned on the ITO/glass (³140 nm) substrate through EBL
followed by metallization. The main advantage of this tech-
nique is that herein in-situ acquisition of SERS spectra and
barcoding were utilized towards the direct identification of
bacterial sample, Vibrio parahaemolyticus. The authors envis-
aged the potential usage of this technique in broad sectors such
as in environmental monitoring, homeland security etc. Chen
et al. also demonstrated a hybridization chain reaction-SERS
coupled nano-biosensing platform via target DNA (tDNA)
triggered self-assembly of Au nanoparticles (Au NPs) probes
on DNA nanowires for the signal amplification of Bacillus
thuringiensis DNA sequence and sensitive detection ranging
from 50 pM to 500 pM with good linearity (S/N = 3).123 The
proposed SERS sensing platform exhibited a high sensitivity
and specificity for the detection of tDNA and many advantages
such as simple operation, and room-temperature implementa-
tion without the assistance of an enzyme over the standard
enzyme reaction and RCA method.
Lin et al. reported a highly sensitive SERS filter-like sub-
strate prepared via a one-step method by embedding AuNPs
within mesoporous silica (AuNPs@MS). The AuNPs@MS acts
as a simultaneous matrix for the rapid filtration of the bacte-
ria in a few seconds and subsequent detection by SERS. This
bifunctional substrate was able to generate 900 fold greater
SERS signal by concentrating the Staphylococcus aureus
bacteria (Figure 15) than the typical SERS method.124
Lin et al. reported an integrated SERS nanoprobe and a
microfluidic dielectrophoretic (DEP) device for the faster
multiplex detection of multiple pathogens, Salmonella enterica
serotype Choleraesuis and Neisseria lactamica. These probes
were purposefully prepared through the immobilizing antibody
© 2019 The Chemical Society of Japan | 229
 Figure 15. One-step method for the preparation of AuNPs@MS to serve as SERS filter like substrate.124 (Adapted with permission
from Ref. 124. Copyright 2014 Elsevier.)

Figure 16. (a) Preparation sequence of NAEBs. (b) TEM images, and (c) absorption spectra of AuNPs and various Raman-reporter-
labelled NAEBs.125 (Reprinted with permission from Ref. 125. Copyright 2014 Wiley-VCH.)

onto nanoaggregate-embedded bead (NAEBs) surfaces and suit-
ably coated with silica and AuNPs. (Figure 16). Each NAEB
provides highly enhanced Raman signals due to the existence
of well-defined plasmonic hot spots at the interface between
AuNPs enabling efficient single bacterium detection.125
Lin and Hamme developed a SERS active nanohybrid
system by coupling antibody conjugated Raman reporter
labeled gold nanoparticles to single-walled carbon nanotubes
(SWCNT). The nanohybrid system was used to selectively
detect and eradicate multiple drug resistant Salmonella
(MDRS) typhimurium DT104 bacteria. 670 nm light source
was used for the photothermal killing of bacteria by creating
irreparable damage to more than 99% Salmonella DT104 at the
concentration of 105 CFU/mL.126 The same group, by follow-
ing a similar strategy, reported the SERS-based detection and
killing of E. coli using an antibody coupled gold nanopopcorns
attached to SWCNTs.127
Capture and detection technique has been gaining momen-
tum recently owing to their selective sensing capability. Najafi
and co-worker established a combined nano-immunomagnetic
separation capture and SERS detection method for E. coli
O157 in a liquid sample (apple juice). Antibody-functionalized
iron oxide-gold bimetallic nanoparticles were employed for the
separation, concentration, and detection of E. coli with a detec-
tion limit of 102 CFU/mL.128 On the other hand, Szymborski
and co-worker prepared a new class of SERS substrate by
sputter coating 90 nm gold on top of an electrospun polymer
mat. The substrate showed a good SERS enhancement factor in

230 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280 © 2019 The Chemical Society of Japan

Figure 17. Preparation procedure of QSY 21 derivatized
IO­Au NOVs as nanotags toward SERS measurements and
their attachment to E. coli bacterial cells.60 (Adapted with
permission from Ref. 60. Copyright 2015 The Royal
Society of Chemistry.)
the order of 106. The bacteria from the contaminated sample is
adsorbed on gold coated polymer mat by filtering the solution
through the SERS mat using a filter funnel, and subsequently,
SERS measurement was carried out. This new class of SERS
substrate was able to selectively detect E. Coli and S. aureus in
contaminated water and blood samples.129
Cao and co-workers also successfully demonstrated the pre-
paration of large-area SERS active substrates using porous
polymer monolithic layers. A porous polymer layer was
initially formed on the glass surface through a simple photo-
initiated polymerization process using glycidyl methacrylate
and ethylene dimethacrylate as the starting material. Followed
by, the thiol groups were introduced onto the surface of the
porous polymer using cysteine molecules, which were then
exploited to uniformly adsorb the preformed gold nanoparti-
cles which eventually resulted in a sensitive and reproducible
large area SERS substrate. The resultant strong SERS signal of
E. Coli demonstrated the sensitivity of prepared large area
SERS substrate.130
In 2014, Madiyar et al. reported a synergistic approach for
the concentration, detection and kinetic monitoring of E. coli
using an integrated system which is the combination of nano-
structured dielectrophoresis (DEP), microfluidics and nanotag-
labelled SERS. First, a microfluidic chip with nanoelectrode
array was developed using vertically aligned carbon nanofibers
(VACNFs). SERS nanotags were prepared by functionalizing
the iron oxide-gold (IO-Au) core-shell nanoovals (NOVs) of
³50 nm size with QSY21 Raman reporter and an antibody
specific towards E. coli DHα5 cells (Figure 17). Then, SERS
tags and the E. coli DHα5 cells containing solution was mixed
together in the microfluidic chip while applying an AC voltage
between VACNFs and the transparent ITO top electrode. Here,
the ITO surface acts as a cover slip for the microfluidic chip
and as an electrode material to complete a DEP device. The
applied potential concentrates the bacterial cells on top of the
VACNFs which are in turn bound by the SERS nanotags due
to selective immunochemistry reaction. The SERS signal was
recorded on a confocal Raman microscope and a portable
Raman probe while DEP capturing and was confirmed with
Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
fluorescence microscopy measurements. The SERS measure-
ments were efficient enough for the detection of a single bacte-
rium. A limit of detection of 210 CFU mL¹1 using a portable
Raman system was obtained within a rapid DEP capture time
of ³50 s.60
A Graphene-AuNPs nanohybrid system was successfully
developed by Mevold et al. and subsequently demonstrated the
SERS-based sensitive detection of bacteria. In their approach,
the authors first prepared poly (diallyldimethylammonium
chloride) (PDDA) functionalized graphene nanosheets. Citrate
protected AuNPs were adsorbed onto the surface of graphene-
PDDA sheets facilitated by electrostatic interaction. The char-
acterization results showed that the particle size of AuNPs was
in the range of 15­20 nm immobilized on the graphene-PDDA
sheets, while the zeta potential value of AuNPs/graphene-
PDDA ratio was found to be 7.7­38.4 mV. Finally, AuNPs/
graphene-PDDA nanohybrid was successfully used for the
sensitive detection of S. aureus.102
4.2 Silver Nanostructures for Pathogen Detection.
Nanostructures based on silver (Ag) have been given special
emphasis and find extensive applications in pharmaceutical,
food, health, energy sectors etc. owing to their intriguing prop-
erties as compared to other noble metals. This attracted a para-
mount importance in the field of sensing. Silver nanoparticles
(Ag NPs) prepared through numerous methods including
chemical, physical and biological procedures have been suc-
cessfully utilized for human pathogens. Liu et al. used silver
nanoparticles grown on porous anodic alumina templates as a
highly reproducible SERS platform and successfully demon-
strated the label-free and rapid detection of Staphylococcus
Aureus, Klebsiella Pneumoniae, and Mycobacterium Smegma-
tis.131 Multivariate data analysis techniques such as PCA, linear
discriminant analysis (LDA), clustering analysis, and SVM
were used to differentiate the various bacterial species. Negri
et al. developed novel Ag nanorod uniform arrays by oblique
angle vapour deposition (OAD) and used as a robust platform
for the rapid SERS detection of various pathogenic bacterial
species such as E. coli, E. coli O157: H7, E. coli DH 5¡,
S. aureus, S. epidermidis, and S. typhimurium 1925-1, a poul-
try isolate.132 This multivariate statistical method was em-
ployed to differentiate the various bacterial species. Sundaram
et al. demonstrated the SERS detection of S. Typhimurium,
E. coli, S. aureus and L. innocua from chicken wash using
biopolymer encapsulated AgNPS deposited on a mica sheet as
a SERS platform.133 Principal component analysis (PCA)
model was utilized to classify the foodborne bacteria types.
Xiao et al. reported an interesting single step self-referencing
method for the SERS-based detection of pathogenic bacteria
using silver nanocubes as Raman enhancer.134 In the reported
self-referencing scheme, SERS signal after target binding was
referenced against blank Raman tag with no washing/sepa-
ration required to a sensitivity of 102 CFU/mL. Cheng et al.
demonstrated a novel and simple dielectrophoretic approach to
assembling microparticles to form nanogaps which are then
used to trap the 20 nm silver nanoparticles rapidly.135 This
approach was successfully utilized in the bead-based SERS
for the rapid identification of bacteria from diluted blood.135
Cho et al. demonstrated a simple, fast and sensitive tech-
nique (10 CFU/mL) for the detection of E. Coli O157:H7 strain
© 2019 The Chemical Society of Japan | 231
 Figure 18. Plausible mechanism showing Ag-SP and Ag-SP-S contact with hydrophilic and hydrophobic microbes.137 (Reprinted
with permission from Ref. 137. Copyright 2014 American Chemical Society.)
using membrane filtration and silver intensification steps along
with SERS. Prior to SERS measurement, the target bacteria
were captured and separated by two different antibody bear-
ing magnetic nanoparticles and Raman reported labeled gold
nanoparticles.136
Ho et al. reported a tricomponent nanohybrid system com-
posed of exfoliated nanoscale silica platelets (SP) stabilized
by silver nanoparticles (Ag) and subsequently encapsulated
by non-ionic surfactant poly (oxyethylene) alkyl ether. This
hydrophobic nanohybrid system was then used to selectively
detect hydrophobic bacteria such as E. coli and Mycobacterium
smegmatis via SERS technique (Figure 18).137
Betancourt et al. reported a layer-by-layer polyelectrolyte
encapsulation of Mycoplasma pneumoniae cells within Ag NPs
in a matrix of poly (allylamine hydrochloride) and poly
(styrene sulfonate) and subsequently detected the bacteria by
SERS technique. Multivariate distribution of the Raman spec-
tra distinguished the 3 strains of M. pneumoniae with high spe-
cificity.138 Sivanesan and co-worker developed a silver­gold
bimetallic SERS platform by electro-deposition method and
subsequently coated with the antibiotic vancomycin to selec-
tively capture bacterial species from a blood sample. This kind
of novel antibody functionalized bimetallic SERS substrate
successfully differentiated four different pathogenic bacteria,
namely E. coli, S. enterica, S. epidermidis, and B. megaterium
from blood samples.139 Xiaomeng and co-workers developed a
sensitive SERS substrate by growing silver nanorod arrays on
glass substrates. The substrates were fabricated by the oblique
angle deposition technique using an electron beam evapora-
tion method. The as-prepared SERS substrate was used to
detect one of the major biomarkers of P. aeruginosa, pyocyanin
at the very low concentration of 5 ppb or 2.38 © 10¹8 mol/L
in both aqueous solutions and spiked clinical sputum samples.
Most importantly, this technique would allow the dynamic
monitoring of pyocyanin excretion during the growth of
P. aeruginosa.140
Ag NPs were also effectively utilized for the detection of
bacteria from drinking water. Zhou and co-workers demon-
strated the SERS-based detection of living bacteria in drinking
water by synthesizing AgNPs on the cell wall of bacteria.
232 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
Initially, silver ions were adsorbed onto the cell wall of bacteria
followed by reduction with hydroxylamine hydrochloride to
form AgNPs on the cell wall surface. The SERS signal of
bacteria varied with respect to the zeta potential of the bacterial
cell wall. When the cell surface became more negative, the
SERS signal increased which is attributed to the adsorption of
more silver ions on the cell surface. With this approach, the
Raman signal of bacteria was enhanced 30-fold over that of
mixed colloid-bacterial suspension. The assay time and reac-
tants volume needed to quantify bacteria in a real environment
was found to be 10 min and 1 mL, respectively. Besides, a
droplet of 3 ¯L sample is more than sufficient for every SERS
measurement. Furthermore, the authors used this approach
combined with hierarchy cluster analysis to differentiate 3
strains of E. coli and 1 strain of S. epidermidis.130
In a similar fashion, Chen et al. reported the formation of
AgNPs on the surface of bacterial cell wall using the com-
bination of Triton X-100, cell membrane disruption reagent,
and sodium borohydride. In a typical experiment, the bacterial
cells were mixed with 0.1% Triton X-100 for 5 min and then
incubated with sodium borohydride for about 5 minutes before
the addition of silver nitrate solution. The pre-treatment of
bacterial cells with Triton X-100 resulted in the disruption of
the cell wall and released the intracellular matrix allowing the
successful discrimination of common disease-causing bacteria
including E. coli, P. aeruginosa, Methicillin-resistant S. aur-
eus, L. monocytogenes and L. innocua. By using L. innocua as
a model sample, the limit of detection of the assay was found to
be 103 CFU/mL. The proposed analytical method proves to be
a rapid tool for selective and label-free identification of
pathogenic bacterium.141
Mycoplasma pneumoniae is a cell wall-less bacterial patho-
gen of the human respiratory tract accounting to more than 20%
of all community-acquired pneumonia (CAP). A silver nanorod
array based SERS platform was developed for the detection of
M. pneumoniae with high specificity and sensitivity in simu-
lated and clinical throat swab samples. The methodology was
able to differentiate the two major genotypes of M. pneumo-
niae. The lowest possible detection limit of 5.3 « 1 cells/¯L
was also achieved.142
© 2019 The Chemical Society of Japan
 Figure 19. Pictorial representation of step by step sensor chip development.144 (Reprinted with permission from Ref. 144.
Copyright 2014 The Royal Society of Chemistry.)
Kumar and co-workers reported a method to fabricate highly
sensitive Ag nanorod (AgNRs) array based SERS substrates
atop a poly(dimethylsiloxane) (PDMS) substrate for the sensi-
tive detection of P. aeruginosa. To do this, a suspension was
coated onto the AgNRs and grown on a PDMS substrate. This
AgNR-PDMS buckled system potentially increase the number
of active spots thereby providing improved entrapment of
P. aeruginosa. It is to be noted that this buckled AgNR-PDMS
array system showed 11-fold enhancement in the Raman signal
as compared to that of AgNR-PDMS film. This remarkable
enhancement in SERS can be assigned to the formation of
dense active hotspots surrounding the AgNRs and improved
bacterial interaction with the metal surface. These modified
AgNR-PDMS array substrates can potentially be used in
chemical and biological sensing applications.143
In 2015, Srivastava et al. reported a highly sensitive SERS
nanobiosensor chip for the specific and quantitative detection
of E. Coli using T-4 bacteriophages as the target capturing
agent (Figure 19). The authors employed glancing angle depo-
sition (GLAD) to prepare plasmon enhanced and SERS active
nanosculptured thin films (nSTFs) of silver on a silicon (Si)
wafer. Then, T-4 bacteriophages specific towards E. Coli were
covalently immobilized on the chip surface. It was revealed
that the fabricated sensor was found to be accurate detection
of E. coli with ultra-small concentrations of bacteria down to
the level of a single bacterium. Further, the sensor was very
accurate and selective towards E. Coli in the presence of other
bacterial species such as Chromobacterium violaceum, Para-
coccus denitrificans and Pseudomonas aeruginosa.144 This
was possible because of utilization of GLAD and enhanced
plasmonic nSTFs of silver coated on Si towards the realization
of Raman bands.
In 2015, Wang et al. developed a new multifunctional SERS
chip capable of capturing, discriminating, and deactivating the
pathogenic bacteria. The AgNPs decorated SERS chip was
grown on a silicon wafer by etching the wafer in hydrofluoric
acid followed by AgNPs deposition. It was found that Si-H on
Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
the surface of silicon wafer facilitates the reduction of silver
ions and subsequently leads to the formation of AgNPs. The
SERS substrate was then functionalized with 4-mercaptophen-
ylboronic acid (4-MPBA), where the boronic acid group acts
as a bacteria binding moiety by reversibly binding to the wall
of bacteria. Further, the benzene ring of the MPBA serves as
a marker band whose SERS intensity increases upon bacte-
ria binding. The primary advantages of the fabricated SERS
chip include outstanding reproducibility (standard deviation
<11.0%), bacterial-capture efficiency (60%) at low concentra-
tions (500­2000 CFU/mL), a low detection capability (1.0 ©
102 cells/mL), and high antibacterial activity (97%).145
Wu et al. developed a combined SERS and chemometric
analysis method for the differentiation and classification of
bacteria. A sensitive SERS substrate was prepared by fabricat-
ing silver nanorods (AgNR) on glass substrates by oblique
angle deposition (OAD) in a custom-designed electron beam
evaporation (e-beam) system. The AgNR substrates were
coated with vancomycin (VAN) and subsequently used for
the SERS analysis of 27 different isolates from 12 species. The
VAN AgNR substrates produced reproducible SERS spectra for
the bacteria. By taking advantage of variation in the surface
protein of each bacteria, the differentiation of bacterial species
was demonstrated by using chemometric analyses of the ob-
tained SERS spectra. Further, the authors performed an analyti-
cal step within the species cluster to be able to differentiate
serotypes and strains. The proposed method was also able to
discriminate gram-positive and gram-negative bacteria with
high sensitivity and specificity and accuracy for the prediction
of actual test samples.146
Zhou et al. reported a label-free in-situ method for the
identification of live and dead bacteria by SERS. These new
nanostructures were prepared by coating AgNPs on the surface
of bacteria (Bacteria@AgNPs) and their utility for rapid count-
ing of live and dead bacteria by SERS. Interestingly, the
AgNPs bind only to the live bacteria and not to the dead bacte-
ria. Thus, the authors were able to selectively detect the gram-
© 2019 The Chemical Society of Japan | 233
negative bacteria in a suspension of live and dead bacteria.147
Duan et al. reported a SERS-based aptasensor for quantitative
detection of pathogenic bacteria.148 In this approach, Au@Ag
core/shell nanoparticles (NPs) functionalized with target bac-
teria specific aptamer 1 (apt 1) were employed for both the
SERS substrate and also the capturing agent. A second type of
aptamer (apt 2), functionalized with X-Rhodamine (ROX) as
Raman reporter molecule, which is also specific towards the
target bacteria and simultaneously acts as a recognition element
was also used. When both the Au@Ag-apt 1 and apt 2-ROX
encounter the target S. typhimurium, all three form a complex
structure where the bacteria is sandwiched between both the
aptamers. Thus, the proximity of Au@Ag NPs and ROX results
in an improved SERS signal which is directly proportional to
the concentration of bacterial species present in the mixture.
The concentration of S. typhimurium with a limit of detection
of 15 cfu/mL can be achieved with this technique. The meth-
odology was also extended to detect S. typhimurium species in
food samples.
4.3 Carbon Nanomaterials for Bacteria Detection. 4.3.1
Carbon QDs: Nanostructured biosensors based on carbon
based nanomaterials (such as carbon nanotubes, graphene, ful-
lerenes) have been constantly utilized toward bacterial detec-
tion due to their remarkable properties including aspect ratio
and surface modification. Table 1 summarizes the details of the
carbon nanomaterials based detection of bacteria with their
linear concentration range and limit of detection. A novel
hybrid detection strategy, utilizing carbon dots magnetized with
iron oxide (Fe3O4) nanoparticles and functionalized using
nitrogen rich chitosan was developed to detect S. aureus and

Table 1. Summary of carbon nanomaterials based detection of bacteria with their linear concentration range and limit of detection.

Target Nanomaterials Detection Linear range LOD

Reference
pathogen utilized strategy (CFU/mL) (CFU/mL)
E. coli O157:H7 Holey reduced graphene oxide FM, SEM and AFM ® 803 18

(hRGO)
Salmonella Paratyphi A SWCNT-DNAzyme assembly FM 103­107 103 167

E. coli K-12 SWCNT ­ immobilised EC 102­105 102 168

specific antibodies
Heat killed O157-H7 Chitosan-MWNT-SiO2/ EC 4.12 © 102­ 250 169

THI nanocomposite 4.12 © 105

Neisseria gonorrhoeae PANI-nFe3O4-CNT DPV 1 © 10¹19 M­ 1 © 10¹19 M 170

nanocomposite 1 © 10¹6 M
S. aureus ZrO2 and graphene DPV 1 © 10¹13­ 3.23 © 10¹14 M 159

nanocomposite 1 © 10¹6 M
Heat killed E.coli Au-SiO2-CHI-SH/Fc/C60 CV 3.2 © 101­ 15 171

O157:H7 nanocomposite 3.2 © 106

E. coli O157:H7 and SWCNT-aptamer assembly Electromotive force 0.2 © 10¹6 0.2 172

S.aureus
V. parahaemolyticus,
Soot CNP ­ dye loaded Confocal laser scanning
S. aureus and 102­106 25­50 173
aptamers microscopy
S. Typhimurium
Uropathogenic E. Coli GCE/f-MWCNT-Chit@Th CV 102­109 50 174

V. parahaemolyticus and Quantum dot-CNP-aptamer FM 50­106 25, 35 175

S. typhimurium
Aeromonas MWCNT­Chi­Bi-PbNP- DPV 0.3 © 10¹12 M­ 1 © 10¹14 M 176

aptamer 0.01 © 10¹12 M

E. coli mAb-AuNP@f3-SWCNT SERS 102­106 100 127

B. anthracis ssDNa/AuNp/Gr/GCE DPV 10 ­10¹7 M

¹12
2 © 10¹13 M 177

Enterobacter sakazakii AuNP/ERGO/SPCE-HRPab CV 103­109 119 178

S. aureus Graphene-modified Piezoelectric frequency 4.1 © 101­ 41 162

IDE-SPQC-aptamer shift 4.1 © 105

Listeria monocytogenes QD­GO nanoplatform FM 102 to 106 fg/¯L 100 fg/¯L 163

Shewanella oneidensis Ab/AuNPs/BSA/GO Anodic stripping 70 © 107 to 12 164

voltammetry 7 © 107
E. coli Gr-Pt hybrid NP-aptamer DPV 1.5 © 10¹7 and 1.12 © 10¹9 M 165

2.25 © 10¹6 M
M. tuberculosis rGO-AuNP-aptamer CV 1.0 © 10¹15 and ® 179

1.0 © 10¹9 M
FM ­ fluorescence microscopy, SEM ­ scanning electron microscopy, AFM ­ atomic force microscopy, CV ­ cyclic voltammetry,
DPV ­ differential pulse voltammetry, EC ­ electric current.

234 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280 © 2019 The Chemical Society of Japan
E. coli. Fluorescent spectroscopy, and MALDI-MS were used
for bacterial detection, and results show a detection limit of
3 © 102 and 3.5 © 102 for both bacteria respectively.149 Natural
biomass was used to develop carbon dots which can act as
fluorescent probes for bacterial detection. Punicagranatum,
when subjected to HTC in an autoclave, led to the formation of
carbon dots which were then used for fluorescent imaging of
bacterium Pseudomonas aeruginosa. This method is very
inexpensive as the final material is used without any chemical
or magnetic treatment and the process also signifies the
importance of natural biomass for the development of suitable
materials for bacterial detection.150 Another interesting and
recent study on bacterial detection was based on amphiphilic
carbon dots. The detection of different bacteria relies on the
affinity of these dots towards the surface of the bacterium cell.
When tested for Escherichia coli MG1655 wild type, Salmo-
nella typhimurium (strain ATCC14028), Pseudomonas aerugi-
nosa PAO1 wild type, Bacillus cereus, and PET28a-FtsA-GFP
strains, the intensity and spectral positions in fluorescence
spectra were different for all bacteria. This indicates the ability
of such carbon dots to distinguish among the various bacterial
strains.151 A combination of the microwave-synthesized carbon
dots and antibiotic vancomycin provides a pathway for fluores-
cent detection of Staphylococcus aureus showing a linear range
relation between 3.18 © 105 and 1.59 © 108 CFU mL¹1 and
detection limit of 9.40 © 104 CFU mL¹1. This probe shows
higher affinity for gram positive bacteria which is due to the
ligand receptor interactions between the vancomycin and
bacterial cell walls. Recovery experiments with orange juice
demonstrate the capability of this sensing approach in real-
world detection of Salmonella aureus.152
4.3.2 Graphene: An aptamer free sensing platform was
developed by incorporating chitosan in reduced graphene
sheets and then electrodepositing a film on tin oxide electrode
that can detect the charge transfer resistance produced by the
presence of sulfate reducing marine bacteria (SRB). The
impedimetric measurements using electrochemical impedance
spectroscopy (EIS) demonstrates the capability of the sensor
within a linear range of 1.0 © 104 CFU mL¹1 to 1.0 © 108
CFU mL¹1. This device is quite selective, and the further
enhancement is possible by immobilizing aptamers or anti-
bodies on the surface.153
A label-free SERS probe built by attaching nanopopcorn to
graphene oxide can detect methicillin-resistant Staphylococcus
aureus (MRSA) at a low concentration of 10 CFU mL¹1. An
enhancement in SERS signal was observed due to the assembly
of graphene to nanopopcorn which is attributed to the large
surface area with high aspect ratio of graphene and lighten-
ing rod effect and surface plasmon excitations produced in
the nanopopcorn. During the detection process using aptamer
modified SERS probe, it was found that the MRSA bacteria
tends to form aggregates inside the graphene oxide sheets.154
A wireless graphene silk nanosensor was synthesized and
transferred onto tooth enamel using water-soluble silk. It was
reported that the dissolution of silk is faster on muscle than
tooth enamel. This thin film nanosensor on tooth enamel, when
functionalized with antimicrobial peptides (AMP) biorecogni-
tion molecules and fitted with inductive coil becomes ex-
tremely sensitive and can even detect a single bacterium. AMP-
Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
coated graphene nanosensor was shown to have stronger
affinity/bonding for the single bacterium as compared to the
only graphene nanosensor.155
A highly sensitive thermally reduced monolayer graphene
oxide (TRMGO) FET immunosensing device built by ultra-
sonic incorporation of graphene oxide monolayer sheets on
FET functionalised gold electrodes can detect E. Coli between
concentration ranges of 10 CFU per mL to 103 CFU/mL. Scan-
ning electron microscopy (SEM) and atomic force microscopy
(AFM) data suggest the semiconducting role of TRMGO sheets
which get plugged into the electrode gaps. The device senses
the response from specific binding of E.coli cells to anti E.coli
antibodies coated/immobilised on its surface, and its sensitiv-
ity of detection was shown to be more when tested for E.coli
O157:H7 (7.3%) as compared to E.coli DH5α (1.4%) and
Dickeya dadantii 3937 (1.3%).156
An antimicrobial peptide, magainin I functionalised holey
reduced graphene oxide (hRGO) based FET device was report-
ed for gram negative bacteria with a detection limit of 803
CFU mL¹1 for E. Coli O157:H7. The presence of abundant
oxygen functionalities along with rich electronic properties
accounted for its superior bacterial detection when compared
with three other similar carbon nanomaterials, reduced GO,
pristine SWNTs and oxidized SWCNTs. Fluorescence micros-
copy, SEM, and AFM were used to visualize the bonding
between FET device and bacterium. From AFM images, it is
quite clear that bacterium is well attached to the surface of
hRGO.18
A selective fluorescent aptamer-monolayer GO combined
bacterium sensor was developed with a detection limit of 100
CFU mL¹1 and a range of 103 to 108 CFU mL¹1 for Salmonella
typhimurium. Systematic evolution of ligands by exponential
enrichment (SELEX) procedure was used to select single strand
DNA specific to S. typhimurium which was then dissolved in
phosphate buffer and GO to make a standard testing solution.
The addition of target bacteria to fluorophore 5-carboxyfluores-
cein (FAM) ­aptamer ­GO sensor solution results in fluores-
cence quenching of FAM-aptamer by GO which is detected by
the spectrophotometer.157
A polymethyl methacrylate (PMMA) fabricated graphene
device when functionalized with a linker molecule immobilizes
the E. coli antibodies on the surface can successfully iden-
tify the bacterium E. coli at a very low concentration of 10
CFU mL¹1. One end of the linker molecule (1-pyrenebutanoic
acid succinimidyl ester) forms π-π interactions with graphene,
and the other one is connected to the antibody. Conductance
measurements show a linear relationship to E. coli concen-
tration within a range of 0­105 CFU mL¹1 and as the antibody
on the FET device is specific to E. Coli, it shows no conduc-
tance response for P. aeruginosa in the same concentration
range. This FET device also responds very well to real-time
detection of glucose metabolism induced by bacteria which
highlights its potential application for real sample analysis.
E. Coli aided glucose metabolism studies showed that the
device picked up the response only when glucose and bacteria
are present together and not with glucose alone.158
4.3.2.1 Graphene-Modified Electrode; Graphene-coated
carbon electrodes, when electrodeposited with zirconium
dioxide (ZrO2), can effectively immobilize a single strand
© 2019 The Chemical Society of Japan | 235
DNA probe sequence on the surface for rapid and sensitive
detection of S. aureus DNA sequence with a detection limit
of 3.23 © 10¹14 M and offers a detection range of 1 © 10¹13 to
1 © 10¹6 M. The high surface area along with conductive
environment provided by graphene and high affinity of ZrO2
for DNA sequence together makes a perfect combination for
efficient immobilization of DNA sequences on the surface.159
A metal hybrid nanocomposite constructed using electro-
deposition of platinum nanoclusters (nPt) on Pt/Ir electrode
with a surface coated with GO can easily incorporate In1A
aptamers on its surface, and the assembly can be utilized for
successful detection of L. innocua using cyclic voltammetric
and impedimetric measurements. High surface area, conduc-
tivity and electrocatalytic properties of the biosensor make it
possible to detect bacterium at a low detection limit of 100
CFU mL¹1 with an amplified signal.160
Anti- Vibrio parahaemolyticus (VP)/N-(4-aminobutyl)-
N-ethylisoluminol (ABEI) immobilized on the surface of a
nano Fe3O4 - graphene oxide (nano@Fe3O4@GO) combination
and then coated onto a glassy carbon electrode offers a stable,
selective, highly sensitive and rapid sensing capability for the
detection of Vibrio parahaemolyticus in seawater and seafood
samples. The detection ability of this assembly was recorded
using electrochemiluminescence of ABEI. The presence of a
two-dimensional structure and high conductivity of nano Fe3O4
- GO increases the electrochemiluminophorisity of ABEI and
anti-VP, thereby enhancing the detection sensitivity of the
sensor. The sensor is capable of detecting VP within a range
of 10­108 CFU mL¹1 with a detection limit of 5 CFU mL¹1 and
5 CFU g¹1 for seawater and seafood respectively.161
Another aptamer-based framework for detection of S. aureus
is constructed by cross-linking graphene to interdigital Au
electrodes connected to a series electrode piezoelectric quartz
crystal (SPQC). The attachment of S. aureus aptamer to the
surface of graphene offers binding sites for the corresponding
antigens of the bacterium. Frequency shifts in the oscillation of
SPQC produced due to antibody-antigen reaction were found to
be linear in the range of 4.1 © 101­4.1 © 105 CFU mL¹1 with
the lowest shift detected at 41 CFU mL¹1. The bacterial detec-
tion limit for a real sample of milk (41 CFU mL¹1) is in 100%
agreement with the plate count value indicating the potential
application of such a sensor in a real world scenario.162
A fluorescence sensing platform generated by a combination
of GO-QD with surface immobilized specific ssDNA possesses
excellent ability to detect multiple genes of Listeria mono-
cytogenes. High amplification using linear-after-the-exponen-
tial (LATE) ­ polymerase chain reaction (PCR), quenching
effect of GO, and high quantum yield of QDs make it possible
to detect bacterium DNA at a low concentration of 100 fg/¯L
in 1.5 hours within a range of 102 to106 fg/¯L.163
Bovine serum albumin (BSA)-modified GO when loaded
with AuNP’s can immobilize antibodies on the surface for
highly sensitive electrochemical detection of Shewanella with-
in a range of 70 © 107 to 7 © 107 and a detection limit of
12 CFU mL¹1. This conjugate can reduce silver ion to metallic
silver which can be quantified using anode stripping voltam-
metry. To validate the specificity of the sensing platform
for S. oneidensis, stripping response of two other bacteria
B. subtilis, E. coli and S. oneidensis in a similar concentration
236 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
Figure 20. (a) rGO nanocomposite membrane (b) incorpo-
ration of the AuNP film, (c) immobilisation (d) combina-
tion, and (e) detection of target DNA.166 (Adapted with
permission from Ref. 166. Copyright 2014 The Royal
Society of Chemistry.)
of 7 © 106 CFU mL¹1 was measured. S. oneidensis gave a very
high response as compared to the other two bacteria demon-
strating the specificity of the sensor. The analysis results for
river water spiked with the bacterium provided a linear corre-
lation within a range of 2.31 © 103 to 2.43 © 107 CFU mL¹1
and can potentially be used for such practical samples.164
An electrochemical genosensor prepared by modifying
glassy carbon paste electrode (GCPE) with graphene-platinum
hybrid nanoparticle (Gr-Pt hybrid NP) and then immobilized
with ssDNA on the surface can detect guanine oxidation
signals from E. coli oligonucleotides before and after hybrid-
ization at the surface of the assembly. Analytical signals from
differential pulse voltammetric exhibited a linear range of
detection between 1.5 © 10¹7 and 2.25 © 10¹6 M and a LOD
of 1.12 © 10¹9 M.165
An electrochemical sensing platform developed for the
specific DNA insertion sequence IS6110 of M. tuberculosis
involving components such as aptamer, rGO and AuNPs can
detect the bacterium in a linear range of 1.0 © 10¹15 and
1.0 © 10¹9 M (Figure 20). The combination, when coated on
GCE, could enhance the effective surface area, conductivity,
and electron transfer capability of the electrode and was con-
firmed by the increase of peak current in cyclic voltammetry
measurements. Further, the attachment of capture probes and
then target probes led to decrease in peak current indicating the
success of the detection process. The sensitivity of the sensor
was amplified using an Au-PANI nanocomposite which dis-
plays good biocompatibility and outstanding electrochemical
activity.166
A combination of a glass microfluidic system and aptamer
functionalized GO offers a straightforward and rapid method
for simultaneous detection of Staphylococcus aureus and
Salmonella enteric with a linear detection range from 104­
106 CFU mL¹1 and 42.2­675 CFU mL¹1 for each bacterium,
respectively (Figure 21). PDMS was used to set up a top layer
with microchannels for reagent delivery and middle layer with
microwells was used for incubation and detection. The chro-
matographic paper was introduced in microwells for adsorption
of aptamer-GO assembly, and the whole assembly sits on a
glass bottom layer. A detection limit of 11 CFU mL¹1 and
linear range of 9.4­150 CFU mL¹1 was detected for L. Acid-
ophilus using fluorescence measurements.180
© 2019 The Chemical Society of Japan

Figure 21. Scheme showing PDMS/paper hybrid micro-
fluidic system using aptamer-functionalized GO bio-
sensors. (a) layout of microfluidic biochip, (b) and (c)
principle of the one-step ‘turn-on’ detection approach.180
(Adapted with permission from Ref. 180. Copyright 2013
The Royal Society of Chemistry.)
A magnetic nanohybrid biosensor (GMCS) developed by
combining GO and magnetic chitosan can detect pathogenic
bacteria Pseudomonas aeruginosa and Staphylococcus aureus
using matrix-assisted laser desorption/ionization (MALDI-MS)
and fluorescence spectroscopy. GMCS interacts with bacteria
via hydrogen bonding, hydrophobic and electrostatic interac-
tions, acid­base, π-π, and polar functional groups interactions
which make it highly sensitive for fluorescent analysis of blood
samples.181
A potentiometric aptamer ­ GO/RGO biosensor was con-
structed for single cell detection of Staphylococcus aureus.
When coated on GCE, GO and RGO can be covalently or
non-covalently functionalized with corresponding S. aureus
aptamers. The results proved that the covalently functionalized
sensors provide the best results with a detection limit of 800
CFU mL¹1 as compared to non-covalent functionalised sam-
ples which gave a poor 107 detection limit. Also, the device is
very selective towards S. aureus, and this is reflected by no
potentiometric response for E. coli, a gram negative bacteria
and L. casei, a gram positive bacteria testing.182
An impedance measuring device for detection of Salmonella
was built using a combination of reduced GO and COOH-
modified MWNTs coated on a GCE. The electrode surface was
further modified with covalently bonded amide linkage to an
anti-salmonella aptamer. The rGO-MWCNT nanocomposite
is proposed to be connected through ³-³ interactions and
provides excellent electronic properties, a high surface area
and binding sites for the accommodation of anti-salmonella
aptamer. The sensor is able to detect spiked in chicken within
a range of 75­7.5 © 105 CFU mL¹1 with a lower detection limit
of 25 CFU mL¹1.183
A sensing device constructed using a combination of rGO
and AuNP and ssDNA linked to this nanocomposite is capable
of measuring S. aureus in the range of 10­106 CFU mL¹1 and
Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
with a detection limit of 10 CFU mL¹1. This assembly is stable
due to the linkage of ssDNA with rGO using ³-³ stacking and
AuNP using Au-S bonding. EIS signals for this system were
significantly enhanced due to the high electronic and surface
properties of rGO and AuNPs, respectively. When tested for
three different bacteria, E.coli, Salmonella and S. aureus, the
probe showed excellent specificity towards S. aureus with ¦Ret
value of nearly 200 ³ as compared to only 20 ³ for other two
bacteria.123
DNA sequence of Klebsiella pneumonia carbapenemase
(KPC) can be precisely detected by an electrochemical bio-
sensor constructed using ssDNA, graphene, GCE and Au-NP
(ssDNA/Au-NP/Gr/GCE). The synergistic effect produced by
combining Gr/GCE with Au-NP led to higher conductivity,
electrochemical and enhanced surface properties which help in
detecting bacteria within a linear range of 10¹12­10¹7 M at a
detection limit of 2 © 10¹13 M using differential pulse voltam-
metry.177 E. sakazakii, a foodborne bacterium can be detected
using a screen-printed carbon electrode (SPCE) modified with
ERGO-AuNP and [BMIM]PF6-HRP-anti-E. Sakazakii on the
surface. SEM images showed the formation of 100 nm size
AuNP on the surface of ERGO while using AFM it was con-
firmed that HRP antibody had been successfully immobilized
on the AuNP/ERGO system. This sensor platform provides a
high surface area, conductivity and electrocatalytic function
which helps to detect bacterium within a range of 103 to 109
CFU mL¹1 reaching the lowest detection limit of 119 CFU mL¹1
as shown using cyclic voltammetric measurements.178
Europium loaded graphene quantum dots (Eu-GQD) of size
3 and 10 nm with the enhanced surface to volume ratio can
adequately bind Bacillus anthracis (B. anthracis) at very low
concentrations of 10 pM which are six orders of magnitude less
than the infectious dose of the bacterium at 60 ¯M. Fluores-
cence measurements showed that the smaller Eu-QD (3 nm)
with larger surface to volume ratio is 1.5 times faster (5.2 s) in
detecting B. anthracis when compared to 10 nm size dots (8 s).
Such a GQD platform has potential for the effective detection
of pathogenic Bacillus Anthrax.184
4.3.3 Carbon Nanotubes: An electrochemical impedance
immunosensing platform of AuNPs and poly(amidoamine)-
multiwalled carbon nanotubes-chitosan nanocomposite film
modified GCE (AuNPs/PAMAM-MWCNT-Chi/GCE) pro-
vides a sensitive mechanism for detecting Salmonella typhi-
murium in a linear range of 1.0 © 103 to 1.0 © 107 CFU mL¹1
with a LOD of 5.0 © 102 CFU mL¹1. The interaction of bacte-
rium with immobilized antibodies on the sensing probe causes
change in the electron transfer resistance which can be mea-
sured using electrochemical impedance spectroscopy. The real
sample testing of the sensor was done using fat-free milk, and
the recoveries of the bacteria were within a range of 94.5­
106.6% indicating the potential use of this sensor in prac-
tical utilities. Drastic increase in electron transfer resistance
(806 ohms) with S. typhimurium as compared to E.coli and
S. aureus (100 ohm) demonstrated the specificity of the sensing
platform.185
The binding of aptamer with SWCNTs in a covalent fashion
offers enhanced detection of S. aureus as compared to the non-
covalent interaction between the two. In the covalently bonded
system, a detection range of 8 © 102 to 108 CFU mL¹1 and a
© 2019 The Chemical Society of Japan | 237
Table 2. Comparison of virtual and real sample analysis for carbon based nanomaterials toward detection of pathogen.

Plate Count Analysis Real Sample Analysis

Target Linear detection Limit of
pathogen Real Linear range LOD
range detection Ref.
sample (CFU/mL) (CFU/mL)
(CFU/mL) (CFU/mL)
Salmonella Paratyphi A 103­107 103 City water 104­108 104 167

Heat killed O157-H7 4.12 © 10 ­4.12 © 10

2 5
250 Water 3.06 © 103­3.46 © 105 ® 169

Heat killed E.coli O157:H7 3.2 © 10 ­3.2 © 10

1 6
15 Synthetic stool 3.2 © 102­2.3 © 106 ® 171

E. coli O157:H7 0.2 © 10¹6 0.2 Milk/fruit juice 4­104 4 172

S. aureus 0.2 © 10¹6 0.2 Pig skin 8 © 102­108 800 172

V. parahaemolyticus, 102­106 25­50 Milk Salmon 1 © 103­0.9 © 105 ® 173

S. aureus and 0.9 © 103­1 © 105

S. Typhimurium
V. parahaemolyticus and 50­106 25, 35 Shrimp 1.1 © 103­1.2 © 105 1.1 © 103 175

S. typhimurium Chicken 0.9 © 103­1.0 © 105 0.9 © 103

Aeromonas 0.3 © 10¹12 M­ 1 © 10¹14 M Tap water 101­106 100 176

0.01 © 10¹12 M
S. aureus 4.1 © 101­4.1 © 105 41 Milk 4.1 © 101­4.1 © 105 41 162

Shewanella oneidensis 70 © 107 to 7 © 107 12 River water 2.31 © 103 to 2.43 © 107 ® 164

LOD of 8 © 102 CFU mL¹1 was observed, while the non-
covalently functionalized sensor responded to a bacterium only
at a high concentration of 107 CFU mL¹1. The number of avail-
able binding sites on the surface is a contributing factor for
different responses of the sensor towards the bacterium.186
A sensor based on the Clavanin A immobilized CNT that
can differentiate gram positive and gram negative bacteria
was developed by Andrade et al.187 Electrochemical responses
using EIS showed the ability of the sensor to differentiate
Klebsiella pneumoniae, Enterococcus faecalis, Escherichia coli
and Bacillus subtilis within a range of 102 to 106 CFU mL¹1.
The development of SWNT fluorescent probes in combination
with M13 virus also offers a much safer, cheaper, and non-
invasive method for fluorescent optical imaging of bacterial
infections. M13-SWNT probe can distinguish between the F-
positive and F-negative strains and can be suitably tuned to
sense specific detection of F-negative strain. Staphylococcus
aureus intramuscular infections in mice were successfully
detected with this probe using FIT. Such biologically function-
alized probes without any chemical treatment/modification
could prove to a be a great potential in the field of bacterial
detection in the future.188
Salmonella Paratyphi A bacteria could also be detected
using a non-covalent assembly of SWNT, and a DNAzyme
labeled aptamer with a detection limit of 103 CFU mL¹1 and
linear range of 103 to 107 CFU mL¹1. Systematic evolution of
ligands by exponential enrichment (SELEX) procedure was
used to derive the single strand DNA aptamers form oligonu-
cleotides which were shown to have high affinity and specific-
ity against Salmonella Paratyphi A using fluorescent assays.
When applied to the real-world situation of a water sample, it
was found that the bacterial detection limit was 104 CFU mL¹1
with a linear range of 104 to 108 CFU mL¹1. This indicates the
potential use of such materials for bacterial detection in real
samples (Table 2).167 The fabrication of immobilized anti-
bodies streptavidin and biotin in the crossbar junction of
SWNT with PEI makes it possible to detect E. Coli K-12
precisely. The reaction between antibodies and the bacteria was
recorded as changes in the electric current which resulted in a
linear range of 102­105 CFU mL¹1 with a detection limit of
102 CFU mL¹1. There is a negligible change in current when
measured for S. aureus (7.25 nA) as compared to E. Coli
(65.76 nA) indicating the specificity of this sensor to E. Coli.168
A layer by layer (LBL) methodology to construct an
immunosensor for detecting heat killed Escherichia coli
O157:H7 was successfully achieved using a chitosan-
MWNT-SiO2/THI nanocomposite and GNP multilayer films.
ELISA was used to observe the covalent linkage of E. Coli
O157:H7 antibody to the surface of GNP monolayer. Current
measured by using a cyclic voltammetric method records a
linear range of 4.12 © 102­4.12 © 105 CFU mL¹1 with a detec-
tion limit of 250 CFU mL¹1. There is a significant degree of
agreement between measurements when the sensor is tested
for real samples of milk and water. The qualitative data using
milk showed 94.4% similarity, and it was also found that the
quantitative analysis of water is also in good agreement with
the plate count data.169
A combination of PANI-nFe3O4-CNT nanocomposite and
biotinylated nucleic acid sequence of 20 bases immobilized on
its surface using biotin-avidin coupling offers a very sensitive
genosensor for the precise detection of Neisseria gonorrhoeae.
The facile electrochemical synthesis of the nanocomposite
involves the fabrication of polyaniline (PANI)-iron oxide nano-
particles and MWCNT on an indium tin oxide (ITO) coated
glass tube which was then characterized using TEM, FTIR,
XRD, and SEM. Differential pulse voltammetric measurement
results indicate a detection limit of 1 © 10¹19 M and a detection
concentration range of 1 © 10¹19 to 1 © 10¹6 M (Table 2).170
Potentiometric aptasensors using SWCNT and specific aptam-
ers were also developed for successful detection of both gram
positive (S. aureus) and gram negative (S. typhi and E. coli)
bacteria in real samples. The assembly includes carboxylated

238 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280 © 2019 The Chemical Society of Japan
SWCNT deposited on a GCE and then covalently linked to
­ (CH2)5NH2 3¤ end of aptamers on the surface establishing
stable CO-NH bonding. SWCNT provides a transducing effect
which minimizes instrument noise and allows low concen-
tration of bacteria to be detected, whereas the actual sensing
function is performed by the specific aptamer and is recorded
as changes in the electromotive force for each individual
bacterium. When tested for liquid samples such as semi-
skimmed milk or apple juice and a solid sample of pig skin, it
was found that the aptasensors are more selective than sensitive
towards bacterial detection (Table 2).172
It was also demonstrated that uropathogenic Escherichia coli
(UPEC) can be detected within a wide linear range of 102­
109 CFU mL¹1 and LOD of 50 CFU mL¹1 using an immuno-
sensor constructed by immobilizing thionines dye on function-
alized MWNT-chitosan composite coated on GCE. The peak
current in the cyclic voltammetric measurement increases with
the increase in UPEC cells in the range 102­106 CFU mL¹1
with a determined current sensitivity of 7.16 © 0.04 ¯A. This
electrochemical immunosensor can also measure the current
from peroxide reduction in a simulated urine sample inoculated
with 109 cells of E. coli, thereby showing its applicability in
real sample analysis (Table 2).174
A sensitive dendritic nanotip with a LOD of 103 CFU mL¹1
was also constructed by using SWCNTs coated Si nanowires
that was designed with the help of UV lithography and etching.
The bacterial detection capability of the system is evident from
the reduction in electric current when target bacteria BCG
combine with fluorescence antibodies at the dendritic tip. Both
fluorescent and electrical measurements showed the efficient
binding of the bacteria onto the tip.189 A biosensor construct-
ed by a combination of MWCNT-chitosan-bismuth complex
with lead nanoparticles and surface attached DNA probes can
also rapidly detect Aeromonas at a very low concentration of
1 © 10¹14 M. The interaction of target bacterium DNA and
probe DNA is recorded using differential pulse voltammetry
signals. The probe is capable of detecting Aeromonas within a
range of 101­106 and a detection limit of 101 in real water
samples spiked with the bacterium.190
A rapid and selective detection of E. Coli was also possible
using a sensor based on immobilization of monoclonal E. coli
antibodies on the surface of gold nanoparticles combined with
thiol terminated SWCNT’s. This sensor is capable of detect-
ing bacterium in a range of 102­106 CFU mL¹1 with a lower
detection limit of 102 CFU mL¹1. The whole process used for
constructing the device offers a high conductivity and plenty of
binding sites for monoclonal antibodies. These immobilized
antibodies coated on nanohybrid structure detect E. coli and
form microbial clusters on the surface, and this leads to
enhancement in SERS.191 The strong electromagnetic and
chemical effects attributable to the components of the hybrid
nanostructure significantly increase SERS-active enhancements
and hence enable detection and quantification of E. coli in
water for environmental monitoring.
4.3.4 Carbon Nanoparticles: A multiplexed FRET system
from fluorescence dyes and carbon soot nanoparticles was
designed for simultaneous detection of V. parahaemolyticus,
S. aureus and S. Typhimurium with a linear range of 102­
106 CFU mL¹1 and a detection limit of 25­50 CFU mL¹1. Three
Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
different fluorescence dyes (fluorescein amidite, cyanine, and
6-carboxy-X-rhodamine) were attached to 5¤ end of three
aptamers, and the combination was then attached to soot carbon
nanoparticles through ³-³ stacking interactions. Confocal laser
scanning microscopy provided fluorescence images of the
bacterial binding to the specific aptamers and TEM images
showing the soot nanoparticles. The aptasensors gave con-
sistent results when the plate count was compared with a real
sample analysis using milk and salmon.173 Gold nanoparticles
when combined with the selective RNA sequences can further
enhance the capability of E. Coli detection by 189% as com-
pared to without nanoparticles. The whole assembly is sensitive
to electrochemical signals relying on the conductive behavior
of CNTs and high surface area of gold nanoparticles for
bacterial detection.192
4.3.5 Fullerenes: Fullerenes (C60) are stable caged mole-
cules with sp2 carbon atoms arranged in the form of hexagons
and pentagons. C60 are considered promising members of the
carbon family owing to their exceptional properties such as
stable redox properties, high thermal/mechanical stability, ease
of functionalization, conductivity etc.193 In recent years, C60,
derivatized C60, and its porous counterparts have been utilized
in energy and environmental applications.194,195 Besides, read-
ers are strongly encouraged to refer to some of the articles/
reviews based on advanced fullerene nanostructures highlight-
ing their state-of-the-art trends and developments.196­202 In
particular, their biocompatibility and unique features (such as
signal amplification) enable the specific application of C60 in
biosensing based platforms. Based on the available literature,
C60 derivatives are powerful antioxidants capable of scaveng-
ing and detoxifying free radicals (reactive oxygen/nitrogen
species). They are very good photosensitizers and thus can be
utilized in photodynamic therapy to heal cancer and eradicate
microorganisms. In one specific study, heat killed E. Coli
O157:H7 can be detected within a concentration range of 3.2 ©
101­3.2 © 106 CFU mL¹1 with a detection limit of 15 CFU mL¹1
by employing an efficient sensitive and biocompatible electro-
chemical immunosensor through the immobilization of biotin
using C60 based biocompatible platform and enzyme function-
alized Pt nanochains tracing tag. The immunosensing platform
comprises multi-components that include C60, ferrocene (Fc),
and thiol functionalized chitosan (CHI-SH) nanocomposites
(CHI-SH/Fc/C60). In addition, Au embedded SiO2 was also
utilized to assemble Au-SiO2-CHI/Fc/C60 nanocomposite.171 It
is interesting to note the concentration value reported in this
work is well below the threshold limit and the proposed sys-
tem has been successfully implemented in analyzing synthetic
stool samples. The values obtained for this immunosensor is
very well comparable to the conventional plate count method
indicating they can be used in practical utilities.171
5. Conclusion and Future Prospects
The field of pathogenic bacteria detection using advanced
nanomaterials in biosensors is continuously evolving which is
evident from significant research emphasis focussed on the
development and utilization of more efficient materials for the
fabrication of next level sensing devices which will possess a
rapid and controlled sensitivity, higher magnitudes of detection
levels, longer life, and relative ease of operational use. With
© 2019 The Chemical Society of Japan | 239
respect to this, advanced nanomaterials have gained paramount
importance in the recent past due to their unique physicochem-
ical properties such as small size, high surface to volume ratio,
tuneable semiconducting band gap, fast reaction rate, magnetic
activity, optical activity, and antibacterial surface. More impor-
tantly, these materials can be suitably functionalized with
relative ease for the detection of specific target pathogenic
bacteria. The amalgamation of nanotechnology into the field of
biosensing is based on the underlying mechanism of initial
binding/reaction of the nanoparticles with the specific bio-
logical component of the pathogenic bacteria which is then
subsequently and rapidly converted into a detectable signal.
This approach is quite user friendly and the fact that nano-
technology allows the fabrication of devices with much smaller
sizes makes it easier for handling which is an added advantage.
In this review article, we have reviewed and summarized
the recent literature reported on the detection of pathogenic
bacteria by using biosensor devices fabricated using advanced
nanomaterials such as metal/metal oxides, magnetic core-shell
nanostructures, metal selenides, metal tellurides, carbon dots,
quantum dots, magnetic nanostructures, graphene, etc. Graph-
ene based nanoparticles were the focus of the research inves-
tigations in the field of biosensing for bacterial detection for the
past few years. However, several other promising nanomate-
rials such as metal/metal oxides nanoparticles, quantum dots,
magnetic nanoparticles and other carbon-based nanomaterials
have recently attracted great attention due to their superior
characteristics. Therefore, more exploratory research needs to
be carried out for the design of next level of biosensors based
on these nanomaterials for the selective detection of pathogens.
Each class of nanomaterials discussed in this review has its
own peculiar advantages which helps in improving the qual-
itative and quantitative sensitivities for the pathogenic bacterial
detection. For example, semiconductor based NPs exhibit
unique optical properties while magnetic NPs offer the ease of
detection using NMR. On the other hand, noble metal NPs
possess high surface to volume ratio whereas carbon based NPs
show a large linear detection range etc. The unique properties
of such nanoparticle based devices provide a platform for an
easier and quicker detection of the pathogenic bacteria as
compared to the conventional microbial culture and growth
based techniques which can take up to six days for the overall
detection process to complete.176 There is no doubt that the
combination of nanotechnology and biosensing offers an attrac-
tive platform for the development of sophisticated devices,
however, there are still several associated challenges that need
to be addressed for enhancing the overall performance of these
devices. By far the most important criteria that should be con-
sidered is to lower the cost involved in the fabrication of such
devices. For example, nanomaterials derived from semicon-
ductors (CdS, CdTe and GaAs), metal/metal oxides (Au, Ag
and ZnO, SiO2), magnetic materials (Fe2O3) provide highly
promising results at the lab scale but these could prove too
expensive when applied on a commercial scale such as clinical
labs. Besides this, the interference generated due to the pres-
ence of nanoparticles could also pose an issue during the
analysis of the real samples which might be constituted of other
components along with the pathogenic bacteria. The potential
toxicity arising due to the presence of nanoparticles must be
240 | Bull. Chem. Soc. Jpn. 2019, 92, 216–244 | doi:10.1246/bcsj.20180280
minimised and or identified by employing sophisticated tech-
niques that allow only a specific and time bound nanoparticle
interaction with target pathogens without any side interac-
tions. Not only these, more research also needs to be done on
improving the reproducibility of nanomaterial based biosensing
devices. An integration of such advanced research in the field
of advanced nanomaterials based biosensing should respond to
these obstacles/challenges which will allow for the fabrication
of sensing devices and further offer more specific and environ-
mentally friendly methods with higher accuracy and precision
to detect bacterial pathogens. Although the research and devel-
opment on coupling of nanotechnology with biotechnology for
designing advanced bio-sensing devices for detecting patho-
genic bacteria is gaining momentum, much work should be
done in integrating the recent advancement in the nano-
technology with emerging bio-nanotechnological approaches.
For example, work on 1D, 2D and 3D nanosheets with different
elemental frameworks have been receiving a lot of attention
these days but the applications of these nanostructures with or
without the functionalization of biomolecules are quite limited.
Focusing on these areas would bring next generation devices
for the selective sensing and the detection of pathogenic
bacteria.
Thus, rapid pursuance of advanced nanotechnology for
pathogenic bacterial detection would certainly benefit from
future research undertaken for the fabrication of advanced
biosensing devices which could be scalable for large oper-
ations. The most important aspect also lies in the application
of such nano biosensing devices for analysis of pathogenic
bacteria in real samples for rapid translation into the commer-
cial world.
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