Sandwich-Type Immunosensors and Immunoassays Exploiting Nanostructure Labels: A Review

Analytica Chimica Acta 758 (2013) 118
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Analytica Chimica Acta

journal homepage: www.elsevier.com/locate/aca
Review
Sandwich-type immunosensors and immunoassays exploiting

nanostructure labels: A review
Xiaomei Pei, Bing Zhang, Juan Tang, Bingqian Liu, Wenqiang Lai, Dianping Tang
Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for
Food Safety, Department of Chemistry, Fuzhou University, Fuzhou 350108, PR China
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
Sandwich-type
immunosensors
and
immunoassays
exploiting
nanostructure labels.
Nanolabel-based
electrochemical
immunosensing and immunoassay.
Nanolabel-based
optical
immunosensors and immunoassays.
Nanolabel-based
mass-sensitive
immunosensing.
Nanolabel-based
multianalyte
immunoassays.
a r t i c l e
i n f o
Article history:
Received 11 September 2012
Received in revised form 25 October 2012
Accepted 30 October 2012
Available online 9 November 2012
Keywords:
Immunosensor
Immunoassay
Sandwich assay mode
Nanoparticle label
a b s t r a c t
Methods based on sandwich-type immunosensors and immunoassays have been developed for detection
of multivalent antigens/analytes with more than one eptiope due to the use of two matched antibodies. High-afnity antibodies and appropriate labels are usually employed for the amplication of
detectable signal. Recent research has looked to develop innovative and powerful novel nanoparticle
labels, controlling and tailoring their properties in a very predictable manner to meet the requirements of specic applications. This articles reviews recent advances, exploiting nanoparticle labels,
in the sandwich-type immunosensors and immunoassays. Routine approaches involve noble metal
nanoparticles, carbon nanomaterials, semiconductor nanoparticles, metal oxide nanostructures, and
hybrid nanostructures. The enormous signal enhancement associated with the use of nanoparticle labels
and with the formation of nanoparticle-antibody-antigen assemblies provides the basis for sensitive
detection of disease-related proteins or biomolecules. Techniques commonly rely on the use of biofunctionalized nanoparticles, inorganic-biological hybrid nanoparticles, and signal tag-doped nanoparticles.
Rather than being exhaustive, this review focuses on selected examples to illustrate novel concepts and
promising applications. Approaches described include the biofunctionalized nanoparticles, inorganicbiological hybrid nanoparticles, and signal tage-doped nanoparticles. Further, promising application in
electrochemical, mass-sensitive, optical and multianalyte detection are discussed in detail.
2012 Elsevier B.V. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Immunosensor and immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Immunosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
3
3
Corresponding author at: Key Laboratory of Analysis and Detection for Food Safety (Ministry of Education), Department of Chemistry, Fuzhou University, Fuzhou 350108,
PR China. Tel.: +86 591 2286 6125; fax: +86 591 2286 6135.
E-mail address: dianping.tang@fzu.edu.cn (D. Tang).
0003-2670/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aca.2012.10.060
X. Pei et al. / Analytica Chimica Acta 758 (2013) 118
3.
4.
2.2.
Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nanolabels-based sandwich-type immunosensor and immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Electrochemical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Mass-sensitive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.
Optical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1.
Chemiluminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2.
Electrochemiluminescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.3.
Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.4.
Surface plasmon resonance (SPR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.
Multianalyte immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Xiaomei Pei obtained her PhD degree at the Department of Chemistry, Fuzhou University in 2011.
Now she is working as a teacher at the Department of Chemistry, Jiangnan University, China. Her
main research interests focus on supramolecular
assemblies, intermolecular interaction analysis and
electrochemical immunosensors.
Bing Zhang is currently a PhD candidate at the

Department of Chemistry, Fuzhou University, China.
She obtained her Bachelor degree at the Department
of Chemistry, Liaocheng University in 2010. Her main
research interests focus on nano- and electrochemical
Juan Tang is currently a PhD candidate at the Department of Chemistry, Fuzhou University, China. She
obtained her Bachelor degree at the Department of
Chemistry, Shangrao Normal University in 2009. Her
main research interests focus on chemical sensors and
biosensors.
1. Introduction
Sensitive and specic determination of biomolecules and proteins is essential in clinical diagnosis, environmental evaluation
and food analysis [1,2]. Typically, the assay is performed by
using certain afnity ligands comprising aptamers and antibodies
that specically interact with the biomolecules and thus mediate a target-responsive signal transduction cascade [3]. Nowadays,
4
4
4
7
8
8
10
12
12
13
16
16
16
Bingqian Liu is currently a PhD candidate at the

She obtained her Bachelor degree at the Department
of Chemistry, Shandong University in 2010. Her main
research interests focus on pharmaceutical analysis
using nano- and electrochemical immunosensors and
immunoassays.
Wenqiang Lai is currently a PhD candidate at the

He obtained her Bachelor degree at the Department
of Chemistry, Fuzhou University in 2011. His main
research interests focus on nano- and electrochemical
Dianping Tang is currently a Full Professor at the

He obtained his PhD in Analytical Chemistry at Southwest University of China (2008). His research focuses
on analytical and bioanalytical chemistry, nanoand electrochemical biosensors, clinical immunoassay, and synthesis and application of multifunctional
nanomaterials. He coordinates projects related to
clinical, food and environmental immunoassays.
particular attention has been paid to using immunoassays or

immunosensors due to their advantages of high sensitivity and
specicity [4,5]. And more sophisticated analytical devices for
immunoassay, such as surface plasmon resonance, quartz crystal microbalance, optical detection methods including uorescence
and chemiluminescence, and electrochemical method, have been
studied on the basis of various signal generation principles from
complex interactions between antibodies and antigens [6,7]. The
their bioactivity and interact with their counterparts, and based

on the detection of those nanoparticles, the amount or concentration of analytes can be determined [16,17]. The enormous signal
enhancement associated with the use of nanomaterial amplifying
labels and with the formation of nanoparticleantibodyantigen
assemblies provides the basis for ultrasensitive immunosensings
and immunoassays [18]. When one antibody on the nanoparticle
reacts with the corresponding antigen, other biomolecules labeled
on the nanoparticle will be carried over, and thus participate in the
reaction. Therefore, the high immobilized amount with the antibodies can increase the possibility of antigenantibody reaction,
while that of the enzymes can enhance the measurable sensitivity
for amplication of detectable signal.
Due to the overwhelming amount of literature available,
it is our choice to underline the most recent trends in
this eld (Fig. 2). Rather than being exhaustive, this review
focuses on selected examples to illustrate novel concepts and
promising applications using nanoparticle-based labels in the
sandwich-type immunosensors and immunoassays. We divide
nanolabels-based sandwich-type immunosensors and immunoassays into electrochemical, mass-sensitive and optical, according to
their signal-transduction methods.
Fig. 1. Sandwich-type immunosensings and immunoassays exploiting nanostructure labels.
assay modes mainly contain sandwich-type assay and competitivetype assay. In a competitive format, unlabeled analyte (usually
the antigen) in the test sample is measured by its ability to compete with the labeled antigen in the immunoassay. Typically, the
detectable signal decreases with the increase of analyte concentration, which requests a high background signal toward zero analyte
[8]. In contrast, noncompetitive immunoassay formats (usually the
sandwich-type formats) give the highest level of sensitivity and
specicity because of the use of a couple of match antibodies [9].
The measurement of the labeled analyte (usually the antibody)
is directly proportional to the amount of antigen present in the
sample, thus resulting that the detectable signal increases with
the increasing target analyte [10]. Therefore, sandwich-type assay
is one of the most popular schemes in the immunosensings and
immunoassays. Although the antigenantibody reaction can cause
the change of detectable signal to some extent, the change is comparatively little. High-afnity antibodies and appropriate labels are
usually employed for the amplication of detectable signal [11].
Typical methods involve the use of an indicator system (e.g.
enzyme label) that results in the amplication of the measured
product [12]. Since there is, for sterical reasons, usually a 1:1 ratio
of enzyme and signal antibody used in the traditional enzyme
immunoassays, the detectable signal is always limited [13]. The
rapidly emerging research eld of nanoparticle labels, and the processes used to generate, manipulate and deploy nanomaterials,
provides excitingly new possibilities for advanced development of
new analytical tools and instrumentation. One major advantage in
using nanoparticle labels lies in the possibility to control and tailor their properties to meet the needs of specic applications, such
as high surface-to-volume ratio and unique conductivity, in comparison with bulk materials [14]. For example, nanomaterials can
provide unique chemical and physical properties (in comparison
with bulk materials) enabling new and advanced functions such as
good biocompatibility, high surface-to-volume ratio, and unique
optical properties.
Various nanoparticle labels including noble metal nanoparticles,
carbon nanomaterials, semiconductor nanoparticles, metal oxide
nanostructures, and hybrid nanostructures, have been developed
in the sandwich-type immunosensors and immunoassays (Fig. 1)
[15]. Antibodies (antigens) labeled with nanoparticles can retain
2. Immunosensor and immunoassay

2.1. Immunosensor
Immunosensors are afnity ligand-based biosensing solid-state
devices that couple immunochemical reactions to appropriate
transducers. Generally, an immunosensor consists of a sensing element and a transducer. The sensing element is formed by means of
immobilization of antigens or antibodies, and the binding event is
transformed into a measurable signal by the transducer [19]. The
merits of immunosensors are obviously related to the selectivity
and afnity of the antibodyanalyte binding reaction. Depending
on the transducer technology employed, immunosensors can be
divided into three principal classes: optical, piezoelectric and electrochemical. Furthermore, on the basis of the immunoassay format
used, they can be either direct (where the immunochemical reaction is directly determined by measuring the physical changes
induced by the formation of the complex) or indirect (where a sensitively detectable label is combined with the antibody or antigen of
interest) [20]. The distinction has an entirely different meaning as
the terms direct and indirect in the immunoassay eld, which both
are tracer-related. They are distinguished by whether antibody
binding is directly detected, e.g. after an enzyme-labeled antibody
is bound to an immobilized coating conjugate or an enzyme tracer
to an immobilized antibody, or whether the detection only takes
place after a secondary binding reaction, e.g. if an enzyme-labeled
secondary antibody is used to label a rs antibody bound to an
immobilized coating conjugate [2123].
For the indirect assay modes, it signicantly facilitates the
problem of signal generation. The tracer either labels the occupied binding sites of the antibodies or the free ones. As for the
sandwich-type immunosensors, the primary antibodies are usually immobilized on a solid-state support, and the sandwiched
immunocomplex is formed between the immobilized primary
antibodies and signal antibodies (usually enzyme-labeled antibodies or nanoparticle-labeled antibodies). The detectable signal
mainly derives from the labeled signal tags. In spite of many
advances in this eld, there is still a paucity of novel approaches
for improving the simplicity, selectivity, and sensitivity of clinical
immunoassays, in order to respond to the demands and needs of
modern medical diagnostics and biomedical research applications.
In this regard, the protein-mediated assembly of nanoparticles
Fig. 2. Number of published articles relative to the immunosensor and immunoassay during the period from 2002 to 2011.
is a potential tool for the fabrication of new sandwich-type

immunosensors. This approach combines tunable nanoparticle
features (size, surface functionality, and core properties) with the
unique physical and chemical properties of proteins and peptides.
2.2. Immunoassay
Immunoassays are analytical techniques based on the avidity and specicity of the antigenantibody reaction, and it has
been considered as one of the most widely used biomedical
diagnostic methods. The term immunoassay is used for tests
based on the immunoreactions, while the term immunosensor is specically employed to describe whole instruments, i.e.
immunoreaction-based biosensors. Usually, the technique consists of heterogeneous and homogeneous immunoassays. In the
heterogeneous immunoassays, the antibody or antigen is immobilized on a solid substrate (e.g. microplate), while homogeneous
immunoassays take place in the solution phase [24]. Compared
with homogenous immunoassays, the heterogeneous immunoassays are easily designed and constructed. However, they require
a step of separating antigen or antibody from the samples and
need to immobilize the antibody or antigen on the solid surface. In contrast, the homogeneous immunoassays usually involve
in the immobilization of the biomolecules on the nano-/microbeads, and take place in the solution, thus allowing the integration
of multiple liquid handling processes [25,26]. Especially combining with microuidic device, the homogeneous immunoassay
can be used for the detection of complex samples, such as urine
or blood, without the large sample consumption and sample
pretreatment, resulting in a relatively inexpensive and easy performance. Microuidic lab-on-a-chip technology has the advantages
of portability, integration, and automation. The combination of two
technologies leads to a pathway of point-of-care diagnostics using
the unprocessed serum samples.
3. Nanolabels-based sandwich-type immunosensor and
immunoassay
3.1. Electrochemical
Electrochemistry studies chemical reactions which take place
in a solution at the interface of an electron conductor (a metal
or a semiconductor) and an ionic conductor (the electrolyte), and
which involve electron transfer between the electrode and the electrolyte or species in solution. Just as the electrochemical detection
methods possess high sensitivity, low cost, low power requirement,
and high compatibility with advanced micromachining technologies, they have extensively applied in the immunosensings and
immunoassays [27]. Although the antigenantibody reaction can

cause the change in the electrochemical signal, the change is
relatively little. For the successful development of an electrochemical immunosensor or immunoassay, signal amplication and
noise reduction are very crucial [28]. High-afnity antibodies and
appropriate labels are usually employed for the amplication of
electrochemical signal. Enzyme-labeled antibodies are often used
as detection antibodies that result in amplication of the measurement signal [29]. However, the association constant of small
analyteantibody complexes may be as high as 1010 1012 M1 , and
a further increase is almost impossible owing to the limited 1:1
ratio between the detection antibody and the enzyme label. Hence,
continuing worldwide effort has been made in developing novel
nanoscaled labels capable of providing a highly detectable sensitivity. Recently, various immunosensors and immunoassays have
been devised and developed for the amplication of the electrochemical signal. Routine approaches consist of ligand-conjugated
enzyme labels and metal-containing nanolabels [3033]. Based
on the published papers, the labels mainly contain bioactive
enzymes (i.e. horseradish peroxidase and alkaline phosphatase),
electroactive materials (e.g. thionine, ferrocene derivatives, and
methylene blue), nanostructures, quantum dots (QDs), and metal
ions [3438]. In contrast, the emergence of nanotechnology opens
a new horizon for the use of nanomaterial labels for signal amplication [39,40]. The power and scope of such nanomaterials can
be greatly enhanced by coupling them with immunoreactions
and electrical processes (i.e. nanobioelectronics). Metal and/or
semiconductor nanostructures, e.g. gold, carbon tubes, silver, and
quantum dots, have been directly used as electroactive labels to
amplify the signal in the electrochemical detection of proteins
[4144]. Based on the present reports, the assay is carried out based
on a competitive-type/sandwich-type immunoassay format. For
the competitive-type immunoassay format, the detectable signals
decrease with the increment of analyte concentrations, and exhibit
a signal-off tendency. In this case, a strong background signal is
necessary. In contrast, the sandwich-type immunoassays display a
signal-enhancement (i.e. signal-on) mode with the target analyte
concentration increased. Therefore, for the multivalent antigens
with more than one epitope, the sandwich-type immunoassay is
preferable, especially for the low-concentration analyte. As indicated from Fig. 3, however, the number of using pure enzymes
or nanoparticles as labels was less than that of enzyme labels
coupling with nanolabels in the recent 10 years. Based on the
signal-generation principles, the sandwich-type electrochemical
immunosensors and immunoassays are mainly classied as amperometric, potentiometric, impedimetric and capacitometric.
Amperometric immunosensors and immunoassays are one of
most popular approaches during the electrochemical process. The
method is usually designed to measure current generated by the
Fig. 3. The percentage of published articles relative to the different labels in

the sandwich-type electrochemical immunosensors and immunoassays during the
period from 2002 to 2011.
electrochemical reaction [45,46]. Most of the present amperometric methods are a subclass of voltammetric analyses in which the
electrode is held at constant potentials for various lengths of time.
Since most analytes (e.g. antigens or antibodies) cannot intristically act as the redox partners in an electrochemical reaction,
however, an electroactive label is needed for the electrochemical reaction of the analyte at the sensing electrode [4749]. Most
commercially available immunoassays were based on the catalysis of the enzyme-labeled secondary antibodies (e.g. HRP and ALP)
toward appropriate substrates to form electroactive products [50].
However, the sensitivity is still limited, which restricts the wide
usefulness in the early diagnosis of disease. Recently, great attention has been focused on signal amplication using bionanoparticle
labels or multienzyme labels, and employing DNA as an amplied
signal reporter in the sandwich-type immunoassays [5156]. In the
DNA-based immunoassays, the signal was usually amplied using
polymerase chain reaction (PCR) after target recognition of capture antibody, whereas it was pre-amplied by using nanoparticles
with a high ratio of DNA to capture antibody in the bio-barcode
immunoassay [5759]. Thus, their application is restricted due to
the complex detection procedures or conjugation processes.
Nanotechnology is multidisciplinary and interdisciplinary and
covers diverse elds including chemistry, physics, material science,
engineering, biology, and even medicine [60]. It provides excitingly new possibilities for advanced development of new analytical
methods and instruments for bioanalytical and biotechnological
applications. Currently, a vast library of nanostructures has been
synthesized and documented, with a wide variety of properties
and application. Hauch and co-authors reviewed nanotechnology diagnostics for infectious diseases prevalent in developing
countries [61]. Liu and Lin summarized recent advances in nanomaterial labels in electrochemical immunosensors and immunoassays
[15]. These nanolabels mainly consisted of nanogold, nanosilver,
nanosilica, semiconductor nanoparticles, carbon nanotubes and
so on. Gold nanoparticles (AuNPs), as a class of nanomaterial,
have many unique properties and have been widely used for
analytical and biomedical purposes. In the early 1970s, Hayatts
group utilized gold colloids as electron-dense probes in immunocytochemistry [62]. With surface modication, AuNPs can bind
with the biomolecules including peptides, enzymes, antibodies
and DNA. Gonzalez-Garcia [63] and Dequaire [64] employed gold
nanoparticles as an electrochemical label for voltammetric monitoring of protein interaction. Experimental results revealed that
gold nanoparticles with high volume-to-surface ratio and strong
surface free energy could enhance the sensitivity of the amperometric immunoassays. Recently, this method was further extended
for amperometric immunosensing of alpha-fetoprotein (AFP) on
thionine/nanogold multilayer lm-functionalized immunosensor
using biofunctional double-codied gold nanoparticles as the label
of HRP-conjugated anti-AFP secondary antibodies [65]. The detection limit (LOD) of using the bionanolabels could be 10-fold lower
than that obtained using conventional HRP-labeled anti-AFP antibodies. In this case, the doped gold nanoparticles might sever
as an intervening spacer matrix to extend the immobilized
biomolecules away from the substrate matrix in the mobile phase,
resulting in binding sites more accessible to antigens. Meanwhile,
the double-codied gold nanolabels contain many HRPanti-AFP
molecules on each nanoparticle surface due to the high surface-tovolume ratio of nanogold particles, and thus enhance the catalytic
reduction of H2 O2 .
Recent research indicated that gold nanoparticles with various
shapes displayed different electrochemical characteristics when
they were used as the label probes. Tangs group designed a new
amperometric immunosensor for detection of AFP using irregularshaped gold nanoparticle-labeled HRPanti-AFP conjugates as
trace labels on carbon nanoparticle-functionalized immunosensing
interface [66]. Compared with same-size spherical gold nanoparticles, the assay of using irregular-shaped gold nanoparticles could
exhibit higher current responses. Maybe, the irregular-shaped gold
nanoparticles could display stronger zigzag effect than that of
spherical nanoparticles, and improve the electron communication
between the nanoparticles. In addition, Choi and co-workers also
found that the effect of chemotherapeutic agents at low concentration could be successfully detected based on surface-enhanced
Raman spectroscopy (SERS) technique and cyclic voltammetry
due to the increased sensitivity provided by gold nanoowers
[67]. Mohantys group reported that Pt nanoowers have superior
catalytic activity for the Suzuki-Miyaura and the Heck coupling
reaction (Note: Suzuki-Miyaura and the Heck coupling reaction
are two important noble-metal-catalyzed processes for forming CC
bonds to produce medicines, agrochemicals and fragrances.) over
spherical counterparts [68]. Jena and Raj demonstrated that gold
nanoowers exhibited pronounced SERS and electrocatalytic activity [69]. The ower-like nanoparticles possessed unique surface
structure and high surface-to-volume ratio, which might improve
the electrochemical behavior of immunosensor [70]. Meanwhile,
nanoowers in the zig-zag orientation could show spin-polarized
metallic edge currents.
Recently, various types of the doped nanostructures, such
as magnetic particles-, semiconductor quantum dots and metal
ions-doped nanostructures, have preliminarily applied in bionanotechnology and biomedicine. For the conventional sandwich-type
enzyme immunoassays, the detection signal usually derives
from the labeled enzyme conjugated with secondary antibodies. Signicantly, if more enzyme molecules are labeled to the
detection antibodies, the sensitivity might be enhanced. Bioactive
enzyme-doped nanoparticles possess several merits: high bioelectrocatalytic intensity, good biocompatibility, and good potential for
surface modication with various biomolecules. Using this method,
there are many enzyme molecules inside and outside of the synthesized bionanolabels. The carried enzyme molecules will be
entered and participated in the catalytic reaction, suspecting, when
one antibody among them reacts with the corresponding antigen. Tang and co-workers developed two types of amperometric
immunosensor for ultrasensitive determination of cancer biomarkers using thionine-doped magnetic gold nanoparticles as labels
[7], and HRP-encapsulated nanogold hollow microspheres as labels
[10]. This high efciency makes them especially suitable for ultrasensitive bioanalysis, and negates the need for additional reagents
or signal amplication steps. Just as the advantages of hybrid
nanostructures, various hybrid nanomaterials have been used as

the labels for the development of sandwich-type amperometric
immunosensors and immunoassays, e.g. nanogold-functionalized
magnetic beads [13], poly(o-phenylenediamine)-carried nanogold
particles [51], nanogoldpolyanilinenanogold microspheres [71]
and so on. Among these methods, the primary antibodies were
usually immobilized on the modied electrodes using various techniques. In this case, it is difcult to realize the continuous testing.
Nanoparticles-based assays hold great promise in realizing highly
sensitive and selective detection at attomolar protein concentrations without requiring complex amplication methods including
polymerase chain reaction (PCR).
Usually, the nanoparticles-based assay consisted of two kinds
of particles: (i) magnetic microparticles coated with monoclonal antibodies as immunosensing probe and (ii) multifunctional
nanoparticles (i.e. nanogold particles) decorated with polyclonal
antibodies or unique barcode DNA sequences as recognition
elements. Magnetic beads with good biocompatibility and rapid
separation from the substrate solution have been extensively
applied in the elds of DNA hybridization detection, immunoassays, protein and enzyme immobilization, cell separation, and drug
delivery. The functionalized probes could pull antibodies bound to
magnetic nanoparticles from one laminar ow path to another by
applying a local magnetic eld gradient and selectively remove
them from owing biological uids without any washing step.
Mirkin and his colleagues reported a nanoparticle-based approach
for the detection of free prostate-specic antigen (PSA) at low
attomolar concentration by adding an antibody-labeled magnetic
microparticle, DNA barcodes, and conjugating a second antibody to
the DNA-conjugated gold nanoparticle [72]. Lius group reported a
nanoparticles-based assay for the highly sensitive PSA detection
at concentration as low as 500 attomolar on a single disposable
chip using light scattering method [73]. The nanoparticles-based
assay allows for the detection of low concentration levels with signal amplication, and reduces sample pretreatment requirement
owing to the presence of magnetic particles. Recently, the Tang
group devised several nanoparticle-based sandwich amperometric
immunoassays for detection of biomarkers on the functionalized
magnetic immunosensing probes based on the different nanolabels,
e.g. enzyme-coated nanometer-sized enzyme-doped silica beads
[74] and nanogold/graphene nanosheets [26].
However, recent experiments found that the bioactivity of
the labeled enzymes is usually weakened when enzymes are
labeled onto the signal antibody or nanostructures. To tackle this
issue, some newly amplied strategies without the participation
of enzymes were devised. Nanostructures, especially redox-active
nanostructures, are usually used as catalysts for electrochemical
reactions or organic/inorganic synthesis. Various nanocatalysts,
such as Fe3 O4 /MnO2 hybrid nanocrystals, ZnOSiO2 nanocomposites, Pd nanoparticles and gold nanoparticles, have been reported
for catalytic organic synthesis and electrocatalytic reaction [7578].
Among these nanocatalysts, gold nanoparticles (AuNPs) have been
found to play an important role in the catalytic processes including
low-temperature CO oxidation, reductive catalysis of chlorinated
or nitrogenated hydrocarbons, and organic synthesis. Signicantly,
AuNPs can catalyze the reduction of p-nitrophenol (NP) to paminophenol (AP) in the presence of NaBH4 [79]. Meanwhile,
the produced AP molecules can be oxidized to p-quinone imine
(QI) with the help of electron mediators, which can be reduced
again to AP via the NaBH4 . The catalytic recycling of self-produced
reactants resulted in the amplication of electrochemical signal. Tang and his colleagues reported an enzyme-free sandwich
electrochemical immunoassay for ultrahighly sensitive detection
of AFP using carbon nanotube-enriched gold nanoparticles as
nanolabels/nanocatalysts [80]. Later, this methodology was further
improved by coupling gold nanoowers [81] and multifunctional
iridium oxide nanospheres [82] with catalytic recycling of selfproduced reactants. Moreover, the enzyme-free electrochemical
immunosensors and immunoassays could also be fabricated by
using quantum dots [83] or metal ions-doped nanostructures [84]
as the labels.
Hybridization chain reaction (HCR) can also play the transduction role via an amplication approach [8587]. During this
process, the single-stranded DNA (ssDNA) molecule is a versatile
construction material that can be programmed to self-assemble
into complex structures driven by the free energy of base pair
formation without enzyme [88,89]. Typically, two stable species
of DNA hairpins coexist in the solution until an initiator strand
is introduced. The initiator triggers a cascade of hybridization
events to yield nicked double helices analogous to alternating copolymers. Zhang et al. reported the proof-of-concept of
a novel and powerful immuno-HCR assay strategy for determination of larger target analytes, e.g. proteins, by coupling the
amplication capability of the HCR with the sensitive electrochemical signal of ferrocene molecules conjugated to hairpin
probes (Fig. 4) [90]. The assay protocol mainly involves the
formation of the sandwiched immunocomplex between the immobilized capture antibodies on the magnetic beads (Ab1 -MBs)
and the secondary antibodies on the gold nanoparticles conjugated with initiator strands (Ab2 -S1-AuNPs), the HCR reaction
of DNA initiator strands on the Ab2 -S1-AuNPs between H1* and
H2* hairpin DNA molecules, and electrochemical measurement
of magnetic immuno-HCR complexes with a sequential injection mode. Based on the HCR principle, the same group utilized
hemin/G-quadruplex-based DNAzyme concatamers as electrocatalysts and biolabels to construct a sandwich-type electrochemical
immunosensor for sensitive detection of IgG1 [91]. Highlight of this
work is to adequately utilize the signal dual-amplication based on
the labeled ferrocene and the formed DNAzyme.
Potentiometric immunosensors are based on the surface charge
or potential change upon immunoreaction on the interface of the
detection device. Either antibodies or antigens in aqueous solution have a net electrical charge polarity, which is correlated to
the isoelectric points of the species and the ionic composition of
the solution [92]. If antibody complex combines with antigen, the
electrical charge of the resulting complex will be different from
that of antibody alone. This change can be measured potentiometrically against the reference electrode immersed in the same solution
[9395]. Impedimetric immunosensors, based on impedance measurements of the electrical equivalent circuit of the oscillator,
can characterize the electrical properties of immunoassay systems
non-destructively without the need for reagents and a separation step [96]. Capacitive immunosensors are based on altering
electrical conductivity at a constant voltage, caused by immunoreaction that specically generates or consumes ions [97]. These
three methods are usually adopted in the label-free electrochemical immunosensors and immunoassays. However, there are still
a few reports focusing on the nanolabels for the signal amplication of potentiometric or impedance immunosensor. Thurer
and co-workers developed a potentiometric immunoassay of proteins using CdSe quantum dot (QD) labels, and <10 fmol detection
limits [98]. The potentiometric immunoassay was performed via
CdSe quantum dot labels on a secondary antibody according to a
sandwich immunoassay protocol in a microtiter plate. The CdSe
QDs were found to be easily dissolved/oxidized in a matter of
minutes with hydrogen peroxide, allowing to maintaining the
pH at a near-neutral value. This is achieved with Cd2+ -selective
micropipet electrodes that were optimized to exhibit attractive
detection limits in conned sample volumes. Zhang et al. developed a sandwich-type impedance immunosensor for detection of
human IgG using nanogold-labeled secondary antibodies for signal amplication on a primary antibody-modied electrode with
Fig. 4. The immuno-HCR assay method.

(from Ref. [90] with permission.)
high resistance [99]. On the formation of the sandwiched immunocomplex, the resistance decreased with the increment of analyte
concentration.
Conductometric immunosensors, which consist of a planar glass
support with interdigitated gold electrode pairs on one surface
in a planar conguration, have been introduced by Watson et al.
[100]. The principle of the detection is based on the changes
of the electrical resistance between two parallel electrodes by
many biochemical reactions in solution. Conductometric enzyme
immunosensors can detect products of enzymatic reactions due
to increasing conductivity of the enzyme membrane. When
enzyme-labeled antibodies were immobilized on the electrode and
conjugated with antigens in sample solution, the antigenantibody
complex coating on the surface of the electrode inhibits the biocatalytic efciency of the immobilized enzyme, the conductivity of the
supporting electrolyte was changed. The antigenantibody reaction can change the enzyme activity and hence the immunosensor
signal due to the hindrance of access of a substrate or the electron
transduction between the electrode and the enzyme active site.
Liu et al. developed a new conductometric immunoassay for hepatitis B surface antigen (HBsAg) based bioelectrocatalytic reaction
on a microcomb-type electrode by using double-codied nanogold
particles as labels [101]. The double-codied nanogold particles
were prepared by using nanogold-labeled anti-HBs antibodies
conjugated with horseradish peroxidase (HRP). The formation of
the immunocomplex changed the direct electrical communication
between the carried HRP and the electrode, and thus local conductivity variations could be assayed based on the bioelectrocatalytic
reaction of the carried HRP in 0.01 M PBS (pH 7.0) containing 60 M
H2 O2 , 0.08 M KI and 0.1 M NaCl. Recently, Tang et al. designed a
simple and sensitive conductometric immunosensor for detection
of AFP using carbon nanoparticles (CNPs) as labels [102]. With a
sandwich-type immunoassay format, the CNP-labeled HRPantiAFP conjugates on the transducer were increased with the increase
of AFP in the sample, and the conductivity of the immunosensor
was decreased in the H2 O2 KI system with a LOD of 0.05 ng mL1

AFP.
3.2. Mass-sensitive
The mass-based immunosensors usually involve in the quartz
crystal microbalance (QCM) technique. The QCM immunosensors
measure the resonant frequency (f) using the standard oscillator
technique, and the frequency change (fx ) is usually explained
by Sauerbrey equation, which states that the decrease in f is
linearly proportional to the increase in surface mass (m) loading of QCM [103]. This Sauerbrey equation, however, holds only
for the case of rigid coated material. The qualitative aspects of
the resulting data and the magnitude of the observed effect are
shown to be independent of lm thickness for values as low as
When an overlayer is thick, the relationship between the
115 A.
fx and m is no longer linear and corrections are necessary. In
QCM immunosensing and immunoassays, the detection sensitivity can be improved by increasing the coverage of biomolecules
and by decreasing the steric-hindrance effect on the sensor surface [104106]. Thus, antibody immobilization method and its
stabilization are very important. These methods of efcient antibody immobilization on QCM immunosensor are classiable into
three main categories: (i) immobilization of the antibody on the
crystal precoated with a suitable material; (ii) immobilization via
entrapment in polymer membranes; and (iii) immobilization via
glutaraldehyde cross-linking [107]. These features are particularly
important in the QCM immunosensors because high sensitivity can
only be achieved using active, thin and rigid layers.
In the QCM immunosensings and immunoassays, the assay is
usually based on a label-free assay format. Despite some advantages, the use of QCM for determination of trace biological target
is still encumbered by its relatively low intrinsic sensitivity [108].
Great efforts have been made to further improve the sensitivity of the QCM-based immunosensors. At present, there are
two strategies for enhancement of the sensitivity in the masssensitive immunosensors. The rst method is the immobilization
of biomolecules mentioned above. Usually, the technique mainly
consists of adsorption, covalent conjugation, self-assembly, and
encapsulation. Another important strategy is the mass amplication technique, which comprise the deposition of an amplied
mass on the probe surface via a chemical route activated by the
adsorbed analytical targets. Typical approaches mainly contain
the use of a catalytic label that induces insoluble precipitates
to deposit on the probe or the use of a massy label that have
mass much larger than the analyte. Alfonta et al. constructed a
sandwich-type EQCM immunosensor for detection of the cholera
toxin employing horseradish peroxidase and GM1-functionalized
liposomes as the catalytic recognition labels [109]. However, the
method is limited due to involving in the bioactivity of the enzymes.
To tackle this issue, Chu and her colleague devised a dendritic
amplication procedure for sandwich-type QCM immunosensing by using nanogold particles-labeled secondary antibodies as
the amplication probes [110]. The assay does not require any
commercial label reagents, and can be implemented with easy
and low cost. Just as the use of nanoparticle labels, the sensitivity was largely improved. Recently, Xia et al. reported a new
QCM immunoassay protocol based a double amplication mode
by using gold nanoparticles-labeled anti-microcystin-LR antibodies
on the primary antibodies-functionalized dendritic surface [111].
The dendritic surface increased the surface coverage of the probe,
and enhanced the immobilization amount of primary antibodies.
In the presence of target analyte, gold nanoparticles-labeled secondary antibodies could be conjugated onto the probe surface, thus
resulting in the large frequency shift. The mode was further developed for detection of inuenza by Miller group [112].
In these methods, the primary antibodies are immobilized
on the surface of gold substrate. The emerging research eld of
nanoparticle-enabled bio-barcode technology provides excitingly
new possibility for advanced development of new analytical tools
and instrumentation for bioanalytical applications. Magnetic particles are attractive because they have good biocompatibility and
can be separated very readily from reaction mixtures in an external
magnetic eld. Shen et al. described a new method for detection of Escherichia coli O157:H7 by using a QCM immunosensor
based on beacon immunomagnetic nanoparticles and catalytic
growth of colloidal gold (Fig. 5) [113]. The designed E. coli
O157-immunosensing probes play crucial roles in the detection
system and have three functions: separation, conjugation, and
mass enhancement (magnetic nanoparticles). The frequency shift
of QCM immunosensor is amplied for three times, and the QCM
immunosensor has a high sensitivity, with a detection limit of
23 CFU mL1 in PBS and 53 CFU mL1 in milk. Moreover, the total
analysis time is approximately 4 h. This method of detection using
a QCM immunosensor can be easily developed and used.
In addition, the QCM technique is often used as a characterization method in the nanoparticle-labeled sandwich-type
immunoassays. The reaction between the antigens and the antibodies can obviously cause the frequency change of QCM probe.
Liu et al. reported a sandwich-type immunoassay method by using
nanogold-patterned mesoporous CoFe2 O4 nanocomposites as the
labels of secondary antibodies for the amplication of detectable
signal [114]. The fabrication process of the sandwiched immunocomplex was characterized by using the QCM method. Meanwhile,
the results were in accordance with those obtained by the electrochemical method. Most recently, Akter et al. also used the QCM
technique to demonstrate carbon nanotube-attached horseradish
peroxidase for the precipitation process of 4-chloro-1-naphtol
during the formation of the sandwich-type immunoassays [115].
Selective capture of an analyte by a functionalized crystal surface increases the effective surface mass, and the ensuing decrease
in resonance frequency allows measurement of the binding

event.
3.3. Optical
Optical immunosensings and immunoassays combine
antibodyantigen interaction with optical measurements are
one of the most popular protocols for bioanalysis due to the
advantages of applying visible radiation, nondestructive operation mode and the rapid signal generation and reading [116].
It has been developed using different techniques including
chemiluminescent, electrochemiluminescence, uorescence and
surface-plasmon resonance (SPR). Creating such nanoparticle
labels remains a challenge. Notably, the harsh environmental
conditions into which these nanoparticles are placed often causes
inactivation of the sensitive biological targets. To deal with these
challenges various surface modications and immobilization
procedures such as physisorption, afnity interaction, covalent
conjugation, and entrapment in sol-gel matrices, have been
explored and developed (Fig. 6). Further, these have now been
applied to the conjugation of secondary antibodies.
3.3.1. Chemiluminescence
Chemiluminescence (CL) methods have become very popular in routine clinical analysis as well in clinical and biomedical
researches ascribed to the advantages as no radioactive wastes,
the relatively simple instrumentation required, the very low detection limit and wide dynamic [117119]. CL immunoassay, combing
the chemiluminescent systems and the immunoreactions, is a
method to determine the concentrations of samples according
to the intensity of the luminescence that the chemical reaction emits [120]. Nowadays, sandwich-type CL immunosensors
using enzyme as label for signal amplication is still the mainstream. In the system, the CL reagents in the base solution react
with hydrogen peroxide released from the enzymatic reactions
to produce a CL light signal. Therefore, the high sensitivity of
the CL enzyme immunosensor is determined by the amount of
the enzyme labeled on the secondary antibody. Bionanotechnology, the emerging research eld of manipulating matter at the
molecular or atomic level, has provided excitingly new possibilities for advanced development of CL enzyme immunosensor
[121]. Among these nanomaterials, gold nanoparticles are one of
the most widely used labels because of their several advantages,
such as rapid and simple chemical synthesis, easy preparation in a
wide range of sizes, good capability. Thus, they can be used as an
excellent bio-labeling for CL immunoassay. Zhang and his workers proposed a novel and sensitive CL immunoassay by employing
a new CL enhancer, bromophenol blue, for the determination of
alpha-fetoprotein based on magnetic beads and gold nanoparticles modied with HRP-labeled anti-AFP antibodies [122]. The
detection limit is 1 order of magnitude lower than that obtained
without using gold nanoparticles and much lower than that typically achieved by enzyme-linked immunosorbent assay (ELISA).
Ambrosi group reported a novel double-codied gold nanolabel
(DC-AuNP) for detection of human IgG based on gold nanoparticle modied with anti-human IgG peroxidase-conjugated antibody
[123]. Yangs group proposed a similar enhanced CL immunoassay
by using double-codied gold nanoparticle as labels for detection
of -fetoprotein (AFP) (Fig. 7) [124]. However, a new potential signal enhancer, 4-(4 -iodo)phenylphenol, was introduced for further
signal amplication. The proposed immunoassay presented high
sensitive and provided a linear response range of AFP from 0.008
to 0.3 ng mL1 with an extremely low detection limit of 5 pg mL1 ,
much lower than those achieved by the classical enzyme-linked
immunosorbent assay. Recently, irregular gold nanoparticles were
synthesized and applied in the CL immunoassay of IgG by Wang
Fig. 5. Schematic illustration of QCM immunosensor based on beacon immunomagentic nanoparticles and catalytic growth of gold nanoparticle labels.
and his workers [125]. It is found that the catalytic efciency on

luminol CL of irregular gold nanoparticles is 100-fold greater than
that of spherical gold nanoparticles. Gold nanoparticles can not
only load a large number of enzymes or proteins to achieve signal amplication, but also can directly catalyze CL reactions as
enzyme mimics [126]. Fan and his works reported a new oxidative
gold metal dissolution-based CL immunoassay by using a magnetic bead-based CL metal immunoassay with a colloidal gold label
[127]. A large number of Au3+ from each gold label are released
after oxidative gold metal dissolution and then quantitatively
determined by a simple and sensitive Au3+ -catalyzed luminol CL
immunoassay. However, colloidal gold used as the label for CL analysis has big drawbacks that the dissolution of colloidal gold requires
extremely severe conditions (e.g. highly concentrated HNO3 HCl
or HBrBr2 ). Further, Lis group found silver particles were more
suitable for CL analysis than gold particles. Thus, they proposed
a CL metal immunoassay for detection of human IgG based on
silver deposition on colloidal gold labels [128]. The human IgG
was indirectly determined by a sensitive combined CL reaction of

Ag+ K2 S2 O8 Mn2+ H3 PO4 luminol.
Nanomaterials such as carbon or silica materials with high
surface-to-volume ratio and good compatibility can also be used
as an efcient bio-label for CL enzyme immunoassay. Zhang and
his cooperators investigated a novel CL immunoassay method
based on multiple enzyme layers assembled multiwall carbon
nanotubes (MWCNTs) as signal amplication labels employing
luminolH2 O2 HRPbromophenol blue enhanced CL system [129].
In the study, horseradish peroxidase was assembled onto MWCNTs templates layer-by-layer through electrostatic interactions.
A linear range from 0.02 to 2.0 ng mL1 was obtained with the
detection limit of 8.0 pg mL1 which was 2 orders of magnitude
lower than standard ELISA method. Functionalized mesoporous silica nanoparticles (MSN) have gained great interest due to their
unique properties, including good monodispersity, large surface
area, high pore volume, controlled pore structure, and high thermal and mechanical stability [130]. Chens group reported an
Fig. 6. Antibody-functionalized nanoparticles with differently conjugated approaches.
10
Fig. 7. Schematic protocol of the developed sandwich-type CL enzyme-linked immunoassay by using magnetic beads.
ultra-sensitive CL immunosensor of carcinoembryonic antigen

using HRP-functionalized mesoporous silica nanoparticles as labels
[131]. Because the large surface area of MSN carriers increased
the amount of HRP bound per sandwiched immunoreaction, the
conjugated provided a much higher signal and increased sensitivity. The analysis showed a linear response within the range of
0.140 ng mL1 .
3.3.2. Electrochemiluminescence
Electrochemiluminescence (ECL) technique involves the generation of species at electrode surfaces that then undergo
electron-transfer reactions to form excited stated, and light is
produced when the excited molecule decays to the ground state
[132]. It combines the electrochemical and luminescent techniques.
Compared with the conventional CL, the ECL technique not only
holds the advantages of sensitivity and wide dynamic range, but
also exhibits several advantages of the electrochemical method
including the simplicity, stability, facility. Since the rst detailed
ECL investigations described by Hercules and Bard et al. in 1960
[133135], ECL analysis has received considerable attention and
been applied in many elds, such as environmental pollutant
determination, pharmaceutical analysis, and immunoassay [136].
ECL-based immunoassays have attracted intensive and extensive
research interests due to their important applications in clinical
diagnosis with the promising advantages, such as simplicity, high
sensitivity, reproducibility, rapidity and low background [137,138].
For immunoassay, most of the biological targets are not ECL-active,
so ECL tags are required to label the biomolecules with ECL reagent,
such as Ru complex, luminol and its derivatives, quantum dots and
dendrimer-encapsulated palladium nanoparticles (Fig. 8) [139].
After ECL from Tris(2,2 -bipyridyl)ruthenium(II) ([Ru(bpy)3 2+ ])

was rst reported in 1972 [140] in acetonitrile using tetrabutylammonium tetrauoroborate as the electrolyte, Ru(bpy)3 2+ was
quickly developed as an important ECL emitter with outstanding
applications due to its superior properties including high sensitivity
and stability under moderate conditions in aqueous solution [141].
Nowadays, ECL immunoassay based on the system of Ru(bpy)3 2+ as
label and tripropylamine (TPA) or diketone as coreactant are most
widely used. Wus group developed a novel ECL immunosensor
based on the Ru(bpy)3 2+ -TPA ECL system through immobilization of the reductant 2-(diisopropylamino)ethylamine (DPEA) with
polymerization-assisted signal amplication for determination of
carcinoembryonic antigen [142]. Growth of the polymer materials
on the electrode surface provided numerous epoxy groups on the
side chain of poly-glycidyl methacrylate, which allowed the accumulation of ECL coreactant DPEA on polymers acrylamide bonds.
The coupled DPEA on polymers sensitized the ECL of Ru(bpy)3 2+ in
solution. The proposed immunosensor is extremely sensitive with
a detection limit of 0.5 pg mL1 .
In comparison to the solution-phase Ru(bpy)3 2+ ECL system,
solid-state Ru(bpy)3 2+ ECL can reduce the consumption of expensive ECL reagent, enhance the ECL signal, simplify experimental
design and create a regenerable sensor [143]. Great efforts have
been made toward immobilization Ru(bpy)3 2+ on a solid electrode surface. One of the efcient methods is using Ru(bpy)3 2+ as
electroactive ECL labels immobilized onto the electrode through
a series of recognition reactions. Typically, Ru(bpy)3 2+ can acted
as electroactive ECL labels in the sandwich-type immunoassay.
However, it is difcult for Ru(bpy)3 2+ directly label the antibody
because there is no functional group on the Ru(bpy)3 2+ molecule
11
Fig. 8. Schematic representation of preparations of tracing tag, and ECL annihilation strategy by electrocatalytic reduction toward dissolved O2 at PdNPs@PMM5/SWNH
nanohybrids.
that can covalently connect with biological substances. Nanoparticles provide new possibilities for application of Ru(bpy)3 2+ as
label in ECL immunoassay because of their special physical and
chemical properties. Especially, silica nanoparticles are considered as a good matrix because of their biocompatibility, chemical
stability and their surfaces are easy functionalized and modied.
Sardesais group reported a ECL immunosensor for detection of
protein cancer biomarkers using Ru(bpy)3 2+ doped silica nanoparticle as signal amplication [144], which could be prepared by
the Stber method [145147]. The prepared Ru-silica nanoparticle can be used as the signal amplication to label proteins in ECL
immunoassays due to the relatively easy modied silica surface.
For example, Ru-silica nanoparticle has been respectively modied with gold and nanoporous gold nanoparticles by Yuan and
Zhangs groups and applied as the labels for determination of cancer
markers [148,149]. Thus, stable and sensitive ECL biosensors based
on Ru-silica nanoparticles can be successfully prepared. Moreover,
Ru(bpy)3 2+ can be encapsulated in liposome as the label in ECL
immunoassay. Bard and Wangs group has, respectively, proposed
a novel ECL immunoassay with Ru(bpy)3 2+ -encapsulated liposome
as the label [150,151]. Great signal amplication was achieved since
liposome could encapsulate large amount of reporter molecules
and avoid the loss of biological activity. A cation-exchange polymer, such as Naon, can effectively immobilize Ru(bpy)3 2+ on the
surface of nanostructure materials. Maos group reported a new
ECL immunosensor based on Ru(bpy)3 2+ doped-TiO2 (Naon functionalized TiO2 ) nanoparticles labeling for ultrasensitive detection
of human chorionic gonadotrophin (HCG) [152]. The proposed
immunosensor can perform the ultrasensitive detection of HCG
with a low detection limit of 0.007 mIU mL1 .
Luminol as one of the most efcient ECL reagents is the best
well known. In the ECL system, luminol-H2 O2 ECL system has
aroused some concern due to its low oxidation potential, inexpensive reagent consumption and the high emission yields [153].
However, H2 O2 as coreactant suffered from difculty in labeling
and unstable in the detection solution. Fortunately, it is worth
to be mentioned that H2 O2 is the product of some substrates
with the catalysis of corresponding enzymes [154]. For example,
glucose oxidase (GOD) can catalyze the oxidation of glucose to
gluconolactone in the presence of oxygen, producing H2 O2 simultaneously [155]. Moreover, glucose is stable in the detection solution.
Thus, addition of glucose instead of H2 O2 could improve stability
and prolong the lifetime of the immunosensor.
The nanostructures can promote the evolution of highperformance ECL immunosensors. They can be used as carriers to
load a large amount of ECL label and thus afford substantial ECL
signal amplication [156]. Generally, luminol could not be directly
used as a label (except conjugated with gold nanoparticles) in the
ECL immunosensor of luminol due to the fact that the functionalization of luminol has lower CL efciencies than the parent compounds
[157]. Thus, there are growing considerable interest in employing
nanoparticles as carriers for the immobilization of GOD for signal
amplication. Xu et al. reported a cathodic ECL of luminol based on
gold nanorods multilabeled with glucose oxidase and secondary
antibody [158]. The gold nanorods were not only used as carriers
of secondary antibody and GOD but also catalyzed the ECL reaction
of luminol, which further amplied the ECL signal for luminol in the
presence of glucose and oxygen. A linear relationship between ECL
signals and the PSA was obtained in the range from 10 pg mL1 to
8 ng mL1 . Yuans group developed an ECL immunoassay of luminol
based on synergetic catalysis effect of enzyme and Pd nanoparticles for signal amplication [159]. Greatly enhanced ECL signal
was achieved by using bioconjugates featuring GOD labels and
secondary antibodies linked to functional carbon nanotubes-Pd
nanoparticles, which exhibit attractive catalysis activity when the
modied electrode was detected in the working buffer containing
proper amounts of glucose. Moreover, their group also proposed
an ECL immunosensor based on glucose oxidase supported on Au
nanoparticles decorated multi-walled carbon nanotubes as labels
[160].
Quantum dots (QDs), as a new kind of ECL luminophores, have
been extensively studied due to their numerous advantageous
features including high quantum yield, low photobeaching, high
photochemical stability, size-tunable emission and broad excitation spectra for multicolor imaging, and feasibility for surface
modication [161]. Bards group found that Si could generate
light emission during potential cycling or pulsing (ECL) [162].
Great attentions have been paid to applying QDs as ECL labels for
12
bioassays. Particularly, the modied QDs have been successfully

used as ECL labels in immunoassays.
Most of the ECL immunoassays of QDs are based on the quenching, inhibition, or enhancement of the ECL intensities via the well
studied coreactant ECL systems which took S2 O8 2 , H2 O2 , SO3 2
as the coreactants [163]. The coreactant is a species that, upon
oxidation or reduction, produces an intermediate that can react
with an ECL luminophore to produce excited states. CdTe QDs as
the luminophor and K2 S2 O8 as the coreactant is the most widely
used system in sandwich enhanced ECL immunoassays. Lis group
developed an ECL immunoassay at a nanoporous gold leaf electrode and using CdTe quantum dots as labels [164]. Zhangs group
reported a novel ECL immunosensor for sensitive detection of
human chorionic gonadotrophin antigen (HCG-Ag) using CdTe QDs
functionalized nanoporous PtRu alloys as labels for signal amplication [165]. Due to signal amplication from the high loading
of CdTe QDs, 4.67-fold enhancements in ECL signal for HCG-Ag
detection was achieved compared to the unamplied methods.
Qians group proposed a versatile immunosensor using CdTe quantum dots coated silica nanosphere as a label for ultrasensitive
detection of a biomarker [166]. Due to signal amplication from
the high loading of CdTe QDs, 6.6-fold enhancements in ECL for
IgG detection were achieved compared to the method without
using silica nanosphere as the carrier. Wangs group reported a
Near-Infrared ECL immunosensor by using CdTe/CdS QDs tagged
silica nanospheres as signal amplication for sensitive detection
of biomarker [167]. All of the reported works basing on K2 S2 O8 as
the coreactant provided an effective way with good stability and
sensitivity for protein detection.
QDs ECL quenching principles were also employed in sandwich
ECL immunoassay for protein analysis. Tians group reported an ECL
immunoassay method for ultrasensitive detection of prostate protein antigen, by remarkably efcient energy-transfer induced ECL
quenching from the CdS QDs sensitized TiO2 nanotube array to the
activated CdTe QDs functionalized multi-wall carbon nanotubed
composite [168]. The ECL intensity decrement was logarithmically
related to the concentration of the PSA in the range of 1.0 fg mL1 to
10 pg mL1 with a detection limit of 1 fg mL1 . Guos group investigated an ECL immunosensor for determination of alpha fetoprotein
(AFP) based on the label CdSe/ZnS QDs effectively scavenging the
ECL of graphene-CdS QDs-alginate composite [169]. The quenched
ECL intensity depended linearly on the logarithm for AFP concentration in the range from 0.05 to 500 fg mL1 with a detection limit
of 20 ag mL1 . Songs group reported an ECL immunoassay based on
CdS nanocrystals functionalized TiO2 nanotube arrays [170]. The
ultrasensitive rabbit IgG detection is achieved on the composites
via an efcient ECL quenching process by CdTe QDs introduced
by sandwiched immunoreaction, and shows a detection limit of
1.0 fg mL1 .
3.3.3. Fluorescence
Fluorescence is by far the method most often applied and is the
dominant analytical approach in a large variety of schemes. Fluorescence immunoassay, as one of the most common approaches
in the eld of optical biosensors, combines the high sensitivity
of uorescence detection with the high selectivity of immunoassay. Nanoparticle-based signal amplication of sandwich-type
immunosensors has attracted wide interest due to its unique optical, electronic, and biocompatible properties. -NaYF4:Yb,Er has
been recognized as one of the most efcient luminescent materials, which proves to be an ideal choice for biolabeling in various
biotechniques [171]. Lius group reported a immunoassay based
on the -NaYF4:Yb,Er nanophosphors [172]. High sensitivity is
achieved for the proposed immunoassay and as low as 0.1 ng mL1
goat anti-human immunoglobulin G can be detected.
Semiconductor nanocrystals (quantum dots, QDs) have

attracted a great deal of attention due to some unique properties such as size-controlled uorescence, high uorescence
quantum yields, and stability against photobleaching. QDs were
functionalized with antibodies and used as uorescent probes for
antigens [173]. Cuis group developed a versatile immunoassay
using CdTe QDs as electrochemical and uorescent labels for
sensitive protein detection [174]. Tu and his workers developed
a CdSe/ZnS QDs-based optical immunosensor for human serum
albumin detection (Fig. 9) [175]. The detection limit for the
CdSe/ZnS QD-based immunosensor developed in this study was
approximately 3.2 105 mg mL1 . Kermans group developed a
QDs-based immunosensor for the detection of prostate-specic
antigen (PSA) using uorescence microscopy [176]. Fluorescence
imaging of the substrate surface illuminated the QDs, and provided
a very sensitive tool for the detection of PSA in undiluted human
serum samples with a detection limit of 0.25 ng mL1 . Lis group
proposed a portable uorescence biosensor based on quantum
dots and a lateral ow test trip (LFTS) for detection nitrated ceruloplasmin [177]. A lateral ow test trip, also called a dry-reagent strip
biosensor, has been becoming a powerful tool for protein analysis
and has attracted increasing attention. Due to the advantages
derived from QDs and LFTS, a rapid, sensitive, selective, and onestep strategy has been developed. And the portable uorescence
biosensor displays rapid responses for nitrated ceruloplasmin with
the concentration as low as 1.0 ng mL1 .
Nanoparticles possessing the ability of efcient uorescence
quenching have been hold for great promised for biosensors. Especially, gold nanoparticles have a very strong ability for uorescent
quenching because they can monitor receptor or ligand binding
and release events through changes in the uorescence intensity or lifetime [178]. Cui and his co-workers have developed a
uoroimmunoassay based on the uorescence quenching of uorescein isothiocyanate caused by gold nanoparticles coated with
anti--fetoprotein monoclonal antibody, where the sandwich-type
immunocomplex was separated by a magnetic eld using magnetic
nanoparticles [179]. Yus group reported a similar immunoassay based on the uorescence quenching of uorescein by gold
nanoparticles coated with antibody dissociated by the mixed solution of sodium hydroxide and trisodium citrate [180]. Compared
with the method provided by Cui and co-workers [174], the method
does not need the complicated preparation of the magnetic beads
and magnetic nanoparticles coated with antibody.
3.3.4. Surface plasmon resonance (SPR)
SPR is one kind of physical optics phenomena produced by optical coupling of thin metal lms [181]. Since the rst application
of SPR technology for a biosensor in 1983 [182], SPR biosensors
have attracted increasing attention and have been widely used in
various elds including medical diagnostics, environmental monitoring, food safety, and security [183]. SPR technique combining
with immunosensors have recently attracted a lot of attention especially for direct detection of biomolecular interactions because of
their high sensitivity and real-time monitoring [184,185]. However,
the use of the SPR method is hindered and the system displayed
limited sensitivities when the change of the refractive index as a
result of binding process is often small, which might be caused by
the binding of small molecular weight materials [186,187]. With
the development of life science, there is a growing requirement for
higher sensitive detection technologies in analysis and the detection of biological samples with low concentration and molecular
weight has attached more and more importance [188]. Thus, it is
urgent to improve the sensitivity of the SPR immunosensors for
extending the range of application of the tool.
Gold nanoparticle has been known to provoke an outstanding
shift in the angle of plasmon resonance, and it combined sandwich
13
Fig. 9. Schematics and real picture of a QD-based optical biosensing system, including diode laser, lens, long-pass lter, and photodiode.
assay are the techniques most commonly applied for enhancing the
sensitivity of SPR immunosensor. Lius group reported an enhanced
SPR immunosensor based on the sandwich assay and colloidal-Auenhanced assay for determining the concentration of human C4
[189]. In the colloidal-Au-enhanced sandwich assay, the human
C4 had good response in the concentration range 0.055 g mL1
and the lowest concentration is 40-fold lower than that obtained
by the direct assay. Chois group developed a ultra-sensitive surface plasmon resonance based immunosensor for prostate-specic
antigen (PSA) using gold nanoparticle-antibody complex [187].
For the detection of PSA, the use of the proposed Au nanoparticle complex enabled 103 -fold signal enhancement in parallel
with femto-level detection. Uludags group reported a point-of-care
immunosensor for the detection of the cancer biomarkers (total
prostate-specic antigen, tPSA) using SPR sensors with gold signal
amplication [190]. Gold nanocomposites are also excellent agents
functioned as an amplier to increase the sensitivity of the SPR

immunosensor. Liangs group reported a novel SPR sensor based on
a sandwich immunoassay to detect AFP by employing Fe3 O4 @AuAFP secondary antibody conjugates as the amplication reagent. A
signicant increase in sensitivity was therefore afforded through
the used of Fe3 O4 @Au-secondary antibody conjugates as an amplier. The experimental results demonstrate that the designed SPR
immunosensor possessed a good sensitivity and a high selectivity
for AFP detection [183].
3.4. Multianalyte immunoassays
Multianalyte immunoassay, in which two or more analytes
are measured simultaneously in a single assay, is requested
in clinical laboratories [191,192]. Compared with the traditional single-analyte immunoassay, multiplexed immunoassay
14
Fig. 10. Schematic diagrams of immunosensors array with 4 12 cells and CL imaging immunoassay procedure.
with the advantages of shortened analysis time, simplied signals,

decreased sampling volume, improved test efciency, and reduced
cost as compared to parallel single-analyte assays is a promising
analytical method in sample analysis [92,192,193]. For the successful development of multiplexed immunoassays, the rst major
concern is how to discriminate each signal for one special analyte from the multiple antigenantibody reactions. Nowadays, the
present methods are mainly based on the multilabel and spatially
resolved assay protocol [194198]. Parallel ultra-microelectrode
arrays with small inter-electrode distances behave similarly to conventional macroscopic electrodes on the typical time-scales used
for electrochemical immunosensors [199]. Jus group reported two
sandwich-type multiplexed immunoassays for simultaneous electrochemical detection of multiple biomarkers on an immunosensor
array based on various nanostructure labels, e.g. carbon nanotubeenriched gold nanoparticles as the labels [200], or gold nanoparticle
labels [187]. Zongs group reported a chemiluminescence image
immunoassay of multiple tumor markers fro cancer screening by
using gold nanoparticle-based bioconjugated with a high molar
ratio of HRP as detection antibodies (Fig. 10) [201]. Arrays of
individually addressable electrodes can be used to determine different analytes or to provide spatially resolved measurements.
Although modication of the individual electrodes with different
biological recognition elements would enable the construction of
miniaturized immunosensor arrays, the directed immobilization of
functional proteins on individual microscopic regions is still a challenge. Hence, multilabel methods based on enzymes, metal ions,
redox tags, and quantum dots, have been used for development of
multiplexed immunosensings and immunoassays [201205].
An alternate strategy for multiplexed immunoassay can be performed by use of a single-enzyme label [206], which offers an
advantage in simplicity as compared to multiple labels but requires
sufcient separation to prevent signal interference (cross-talk)
between neighboring electrodes. Wilson has described enough
spatial separation of electrodes which can perform individual

immunoassay of multiple proteins without amperometric crosstalk [207]. Jus group immobilized an electron-transfer mediator
on the electrode to avoid cross-talk when a single-enzyme label
for multiplexed immunoassay of proteins was used [208]. Various signal amplication technologies using nanomaterials have
been developed for ultrasensitive detection of proteins. One of
the most popular strategies is enzyme-functionalized nanoparticles used as tracers to enhance the sensitivity of detection by
loading a large amount of enzymes toward an individual sandwich immunological reaction event. Du reported a multiplexed
electrochemical immunoassay of phosphorylated proteins based
on enzyme-functionalized gold nanorod labels and electric elddriven acceleration [194]. Jeon developed a new gravimetric
immunoassay for sensitive detection of multiple protein biomarkers using multifunctional hybrid nanoparticles (Fe3 O4 /TiO2 /SiO2 )
as the labels on silicon microcantilever arrays [209]. However,
these methods usually employed conductive nanomaterials for the
detection with no serious consideration of enzyme stability, which
is critical and important for the delity of ELISA signals. To tackle
this issue, Kim reported a multiplexed immunoassay based on
the approach of nanoscale enzyme reactors (NERs), in which the
enzyme adsorption into mesoporous silica with the bottleneck pore
structure was followed by the chemical crosslinking of adsorbed
enzyme molecules [210]. The NER approach could stabilize the
enzyme activity and maintain high enzyme loading by preventing
the enzyme leaching via a ship-in-a-bottle effect.
Magneto-controlled molecular electronics and bioelectronics
have become new tools for simultaneous monitoring of multiple biomolecules in food, environmental and clinical samples
[72,211,212]. Magnetic sorting protein assay systems with varying throughputs have been built and employed for multiple
immunoassays [213,214]. Batch-type magnetic separators have
been fabricated on a single chip for trapping and directed sequential
elution of magnetic particles in owing uids [215217]. Tang

reported a new ow-through multiplexed immunoassay protocol for simultaneous electrochemical determination of CEA and
AFP in biological uids using biofunctionalized magnetic graphene
nanosheets as immunosensing probe and multifunctional nanogold
hollow microspheres as distinguishable signal tags [218]. The assay
was based on the catalytic reduction of H2 O2 at the various peak
potentials in the presence of the corresponding mediators.
Quantum dots (QDs) have shown great potential in the multiplexed immunoassays because of their unique advantages:
nanoscale size similar to proteins, broad excitation spectra for
multicolor imaging, robust, narrowband emission, and versatility in surface modication [174,219221]. Compared with organic
uoropores, QD provide tunable, symmetrical and narrow emission bands with high photostability. Cooper and co-worker
reported upon the application of quantum dot barcodes prepared by layer-by-layer biological self-assembly of quantum
dot-biotin and quantum dot-streptavidin on magnetic beads for
qualitative multiplexed immunoassays [222]. More signicantly,
different-sized QDs can be excited with a single wavelength
excitation light. Therefore, QD are considered one of the most
promising materials in the multiplexed immunoassays. Zhang
developed a new cross-talk-free duplex uoroimmunoassay for
cancer-related biomarkers based on dual-color quantum dots
as detection elements [223]. Zeng and colleague constructed
a multiple homogeneous immunoassay based on a quantum
dots-gold nanorod FRET nanoplatform by using green QDs and
red QDs as distinguishable signal tags [224]. More signicantly,
multicolor quantum dots could be also used as the labels for
multiplexed uorescence polarization immunoassay [225]. In the
multiplexed electrochemical immunoassays, the coding bioassay
relies on the use of different inorganic-colloid nanocrystal tracers,
whose metal components yield well-resolved, sensitive stripping
voltammetric signals. Viswanathans group devised an electrochemical immunosensor for multiplexed detection of food-borne
pathogens using three metal sulde (CdS, PbS, and CuS) nanocrystals as distinguishable tags on a carbon nanotube-modied screen
15
printed electrode [226]. Kong et al. developed an electrochemical

immunoassay for simultaneous determination two tumor markers using CdS/DNA and PbS/DNA nanochains as distinguishable
tags [227]. Due to the difference in the oxidation potentials of
these metal components, they exhibited sharp and well resolved
stripping voltammetric peaks at various peak potentials. Further,
some metal ions comprising cadmium, copper and zinc usually display various voltammetric characteristics at the different
applied potentials. Favorably, these metal ions can be stripped
from the corresponding metal nanoparticles under the harsh conditions. Zhus group designed a new multianalyte electrochemical
immunoassay for detection of two human cardiopathy biomarkers using metal-ions (i.e. Cd2+ and Zn2+ ) functionalized titanium
phosphate nanospheres as distinguishable signal tags [228]. Unfortunately, the QDs-based electrochemical detection often requires
harsh detection conditions, including nanocrystals dissolution,
high potential accumulation, and deoxygenation, which is not suitable for clinical application.
An alternative approach is to utilize enzyme-catalyzed metal
ion deposition for the amplication of detectable signal. Compared
with AuNPs, silver nanoparticles (AgNPs) can be oxidized at more
negative potential with a relatively sharp peak, which is favorable
to obviating the interference of reducing species and improving
the detection precision and sensitivity [229]. The silver deposition can be carried out with the aid of enzyme, nanoparticles or
other reduction agent. Lai and co-worker reported several methods for electrochemical stripping analysis of silver nanoparticles in
the sandwich-type multiplexed immunoassays by coupling different silver deposition techniques and nanoparticles labels, e.g. silver
nanoparticles catalytically deposited by gold nanoparticles and
enzymatic reaction [230], nanogold label-induced silver deposition
[231], and silver-nanoparticle-enriched carbon nanotube labelsinduced silver deposition [232]. Just as these advantages of silver
nanoparticles, Woo et al. constructed a multiplexed immunoassay using uorescent-surface enhance Raman spectroscopic dots,
which were composed of silver nanoparticle-embedded silica
nanospheres, organic Raman tagging materials, and uorescent
Table 1
Comparison of analytical properties of various sandwich-type immunosensors and immunoassays for the detection of CEA using different nanoparticle labels.
Method
Linear range (ng mL1 )
LOD (ng mL1 )
Labelsa
Ref.
Electrochemiluminescence
Amperometric immunosensor
Electrochemical immunoassay
Fluorescence immunoassay
Fluorescence immuosensor
Electrochemical immunosensor
Chemiluminescence
Magnetic immunoassay
ELISA
Chemiluminescence
0.0180
0.01200
0.1100
5.0 106 5.0 104
0.110
11000
0.1100
0.5160
0.001100
0.00050.5
0.00150
0.00110
0.00550
0.055000
0.00010.002
0.0050.5
0.02120
0.250
3.060
0.140
0.00520
0.00011.0
0.00550
0.0052.0
0.180
0.0112
0.0033
0.00011
0.0033
2.0 106
0.01
0.5
0.04
0.08
0.001
0.00012
0.0001
0.0008
0.002
0.0024
0.00005
0.0041
0.0012
0.058
1.5
0.093
0.04
0.0017
0.00004
0.001
0.0032
0.03
0.005
Nano-ZnO + GOx
AuPt nanochain + HRP
CdS/DNA nanochains
QD labels
B-Galactosidase
Europium chelates
Without labels
Without labels
Pt hollow spheres
Nano-Au
Polyaniline nanobers
Ru-Silica-Au composite
Nano-Au
Carbon nanotubes
Gold nanoparticles
Gold nanoparticles
Hollow Pt nanoparticles
PbS nanoparticles
HRP without nanolabels
Silver-carbon nanotube
HRP-SiO2
Pt-HRP
Carbon nanotube-HRP
AuAg-GOx
GOx-SiO2
Fe3 O4 -Au
Au-SiO2 -CNT
[239]
[240]
[227]
[241]
[242]
[243]
[244]
[245]
[28]
[246]
[247]
[149]
[248]
[249]
[250]
[251]
[252]
[253]
[254]
[232]
[131]
[255]
[256]
[155]
[257]
[258]
[259]
GOx: glucose oxidase; HRP: horseradish peroxidase; CNT: carbon nanotube.
16
dyes [233]. Brady and colleague presented a strategy for the

synthesis of multiplexed spectral encoder beads based on combination of different surface enhanced Raman signatures generated
by dye-functionalized silver nanoparticle tags [234]. For detection purposes, the use of these nanoparticle assemblies as an
encoder will allow for the straightforward simultaneous identication of multiple analytes, limited in principle only by the number
of available unique molecular dye signatures. These multiplexed
encoder beads are envisioned to have applications that supplement
more ubiquitous uorescence-based bioassay schemes that require
small spectral bandwidths and large numbers of multiplexed
analytes.
In addition, nanoparticle labels can be used in the other
sandwich-type multiplexed immunoassays for the amplication
of detectable signal, e.g. colorimetric immunoassay [235,236],
immunochromatographic strips [237], and thermally addressed
immunosorbent assay [238]. As the discussion is coming to the
end, to further elucidate the advantages of nanoparticle labels, the
analytical properties of selected immunosensors and immunoassays with and without nanoparticle labels are also compared by
using carcinoembryonic antigen (CEA), as an example (Table 1).
The results strongly highlighted one sentence made by Royce W.
Murray, former Editor of the journal of Analytical Chemistry, in an
editorial series: Nanoparticles will be part of the heart and soul
of analytical chemistry and science at large, far off into the future
[260].
4. Concluding remarks
Inherent sensitivity, simplicity, speed, and cost benets continue to be strong driving forces for the development of
sandwich-type immunosensings and immunoassays. Herein we
have described a variety of nanoparticle labels for the amplication
of detectable signal in different sandwich-type immunosensors
and immunoassays. Despite historic achievements in the elds of
label-free bioassays, labeling techniques will continue to play a
leading role in this eld. Nanoparticle labels offer very elegant ways
of interfacing biomolecule recognition events with inherent signal amplication. Along with developing of labeling techniques,
biofunctionalized nanoparticles have paved the way for the development of highly sensitive diagnosis devices because of unusual
properties of nanoparticles. Especially, coupling enzyme labels
with nanoparticle labels is one of the most exciting and challenging aspects of this eld. Success will play a vital role in advancing
numerous scientic disciplines, including biomedicine, biology,
chemistry, environment science, toxicology, and materials science.
Due to different shapes and sizes probably possessing different
properties, designing and developing new strategies are necessary
to achieve a better understanding of the nanofabrication process,
and to control particle aggregation and surface interactions. To
achieve a better understanding of nanolabels-based sandwich-type
immunosensings and immunoassays, great efforts will need to
be made worldwide to design and develop new strategies that
improve the properties of nanolabels. In addition, developing uses
for a variety of nanomaterials coupled with enzyme labels and
micro-/nano-uidic devices will offer advanced miniaturized highthroughput and cost-effective multiplex assays for tagging of a wide
variety of important chemical and biological targets. Sandwichtype immunosensors and immunoassays exploiting nanoparticles
will become a common technology increasingly accepted as
labeling reagents in bioanalysis and bioimaging because of its
advantages. In spite of distinct advantages of the sandwich-type
assay approach, e.g. lower detection limit and more robust assay,
it cannot be applied for haptens because it requires as analytes
multivalent antigens with more than one epitope.
Acknowledgments
This work was nancially supported by the National Natural
Science Foundation of China (41176079, 21075019), the National
973 Basic Research Program of China (2010CB732403), the Doctoral Program of Higher Education of China (20103514120003),
the National Science Foundation of Fujian Province (2011J06003),
and the Program for Changjiang Scholars and Innovative Research
Team in University (IRT1116); as is a critical pre-publication review
undertaken by Dr. Miro Manuel, University of the Balearic Islands,
Spain.
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Sandwich-Type Immunosensors and Immunoassays Exploiting Nanostructure Labels: A Review

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Contents lists available at SciVerse ScienceDirect

Analytica Chimica Acta

Sandwich-type immunosensors and immunoassays exploiting

X. Pei et al. / Analytica Chimica Acta 758 (2013) 118

Bing Zhang is currently a PhD candidate at the

Bingqian Liu is currently a PhD candidate at the

Wenqiang Lai is currently a PhD candidate at the

Dianping Tang is currently a Full Professor at the

particular attention has been paid to using immunoassays or

X. Pei et al. / Analytica Chimica Acta 758 (2013) 118

their bioactivity and interact with their counterparts, and based

2. Immunosensor and immunoassay

X. Pei et al. / Analytica Chimica Acta 758 (2013) 118

is a potential tool for the fabrication of new sandwich-type

immunoassays [27]. Although the antigenantibody reaction can

X. Pei et al. / Analytica Chimica Acta 758 (2013) 118

Fig. 3. The percentage of published articles relative to the different labels in

X. Pei et al. / Analytica Chimica Acta 758 (2013) 118

nanostructures, various hybrid nanomaterials have been used as

X. Pei et al. / Analytica Chimica Acta 758 (2013) 118

Fig. 4. The immuno-HCR assay method.

was decreased in the H2 O2 KI system with a LOD of 0.05 ng mL1

X. Pei et al. / Analytica Chimica Acta 758 (2013) 118

in resonance frequency allows measurement of the binding

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was indirectly determined by a sensitive combined CL reaction of

Fig. 6. Antibody-functionalized nanoparticles with differently conjugated approaches.

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ultra-sensitive CL immunosensor of carcinoembryonic antigen

After ECL from Tris(2,2 -bipyridyl)ruthenium(II) ([Ru(bpy)3 2+ ])

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bioassays. Particularly, the modied QDs have been successfully

Semiconductor nanocrystals (quantum dots, QDs) have

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functioned as an amplier to increase the sensitivity of the SPR

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with the advantages of shortened analysis time, simplied signals,

spatial separation of electrodes which can perform individual

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Linear range (ng mL1 )

LOD (ng mL1 )

GOx: glucose oxidase; HRP: horseradish peroxidase; CNT: carbon nanotube.

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