CHINESE JOURNAL OF ANALYTICAL CHEMISTRY

Volume 40, Issue 1, January 2012

Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2012, 40(1), 310. REVIEW

Chemiluminescent Immunoassay and Its Applications

WANG Chen, WU Jie, ZONG Chen, XU Jie, JU Huang-Xian*
State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210093, China

Abstract: Chemiluminescent immunoassay (CLIA) has been applied in different fields, including environmental monitoring,
clinical diagnosis, food safety and pharmaceutical analysis, as a promising approach for selective, sensitive, rapid and simple analysis.
It is often necessary to detect a large number of complicated or low-abundance samples. However, traditional methods need great
consumption of time, reagents and labor, which limit their clinical applications. As a result, rapid, high-throughput, sensitive and
low-cost detection methods have become the development trend of CLIA. In this review, we summarize the recent advances in CLIA
and its applications in the last five years, and point out the future development of CLIA.

Key Words: Chemiluminescent immunoassay, Immunosensing, Rapid detection, Multiplexed detection, Review

1 Introduction concentrations of multiple pesticides and herbicides are

In recent years, chemiluminescent immunoassay (CLIA)
has gained increasing attention[14] in different fields,
including life science, clinical diagnosis, environmental
monitoring, food safety and pharmaceutical analysis[5] because
of its high sensitivity, good specificity, wide range of
applications, simple equipment and wide linear range.
Immunoreaction is the recognition between antigen and
antibody. Because of the low diffusion rate of these large
molecules and the site block problem, the immunoreaction
rate controlled by mass transport and reaction kinetics is
generally low. Traditional immunoassay always needs a long
incubation time, which in turn causes the whole analysis to
take several hours for completion, so the throughput and
application range are largely limited. Researchers have
designed different methods to shorten the analysis time by
improving mass transport and reaction kinetics. The rapid
immunoassay has expanded the applications of CLIA.
It is often necessary to detect multiple components of
complicated systems in practical use. For example, owing to
the limited specificity and sensitivity of biomarkers for
clinical diagnosis, the measurement of several biomarkers is
usually applied for enhanced diagnostic purposes[6,7]. The
simultaneously monitored to value the environmental
pollution status[8,9]. Traditionally, immunoassay is performed
as discrete tests, i.e., one analyte per assay run, and several
runs are needed to detect all the components in a complex
system. Great consumption of time, reagents and labor limits
the application of the traditional strategy. Since a
multianalyte immunosensing system has the advantages of
high throughput, short time, low consumption and low cost, it
has been very popular during the recent years. Based on
microchips[1015] in combination with automated flow-
injection systemcharge coupled device (CCD), and the
character of immobilization carriers such as temperature-
resolved strategy[16], many multianalyte immunosensing
systems have been constructed.
It is also necessary to detect samples of low abundance, so
developing highly sensitive CLIA has become a new trend.
The main approach to improving the analysis sensitivity is to
reduce the noise and enhance the signal. In order to reduce the
noise, small proteins are always used to block the active site
on the biosensing interface. Signal amplification techniques
focus on constructing functionalized interface and designing
novel trace tags. Along with the fast development of
nanotechnology, nanomaterials such as Au nanoparticles

Received 2 July 2011; accepted 10 September 2011

* Corresponding author. Email: hxju@nju.edu.cn
This work was supported by the National Basic Research Program of China (No. 2010CB732400), the National Natural Science Foundation of China (Nos. 20875044,
20821063, 21075055), the Natural Science Foundation of Jiangsu Province, China (No. BK2008014).
Copyright 2012, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.
DOI: 10.1016/S1872-2040(11)60518-5
 WANG Chen et al. / Chinese Journal of Analytical Chemistry, 2012, 40(1): 310
(AuNPs), carbon nanotubes, SiO2 nanoparticles, quantum dots
(QDs) and their nano-composite structures, have been used to
construct highly sensitive immunoassay biosensors. The wide
range of applications of nanoparticles in labeling proteins has
achieved breakthrough advancement[17,18]. Some nanoparticles
can not only directly catalyze chemiluminescent (CL)
reactions as enzyme mimics[1922], but also load a large number
of enzymes to achieve signal amplification[2329].
Nanotechnology has been widely used in CLIA to accelerate
the rapid development of CLIA.
This review introduces the basic theory of CL, summarizes
the recent progress of CLIAs whose assay time, throughput
and sensitivity have been significantly improved, and briefly
presents the prospects of the future development of CLIA.
2 Chemiluminescent immunoassay
CLIA is a method to determine the concentration of
samples according to the intensity of the luminescence that the
chemical reaction emits. CLIA combines the CL systems and
the immunoreactions. Some reagents have been used as CL
labels, and upon the introduction of the CL substrates, the
system produces chemiluminescence, so the samples can be
detected quantitatively. The most popular CL substrates are
luminol, isoluminol and their derivatives, acridinium ester
derivative, peroxidase and alkaline phosphatase (ALP).
Using enzymes to label proteins is still the mainstream in
CLIA. The most commonly used labeling enzymes are
horseradish peroxidase (HRP) and ALP. The development of
enzymes and substrates with higher activity, better stability and
more suitable kinetic curve is the hotspot in CLIA research.
The labeling technology is one of the key technologies in
modern immunoassay. The new label immunoassay
technology has been widely applied in environmental
monitoring[30,31], clinical diagnosis[32,33], food safety[34,35],
pharmaceutical analysis[36,37] and bacteria identification[38,39],
which shows great application prospects. Because
nanoparticles have favorable biological compatibility and
signal amplification effect, nanotechnology has been
extensively used in bio-labeling, for example, AuNPs can be
used to label antibodies, and enhance the CL of luminol-H2O2
system. QDs are semiconductor nanoparticles that can accept
excited lights to produce fluorescence, which have more stable
quality, better reproducibility, longer activity and less
consumption than normal labeling enzymes of CLIA. Huang[40]
used CL as energy donor to excite QDs, and realized the CL
energy transfer for sensitive detection.
CLIA has the advantages of high sensitivity of CL and
specificity of immunoreaction. It has wide application and
great prospect in clinical diagnosis, food safety and
environmental monitoring[4143].
3 Recent development of CLIA
3.1 Rapid chemiluminescent immunoassay
Traditional immunoassay usually needs several hours to
complete a run, which causes adsorption of immunoreagents
in detection channel and cross-talk between analytes. As a
result, long incubation time limits the detection throughput of
samples and the application value of CLIA. To avoid these
drawbacks and accelerate immunoassay, external forces are
employed to perform effective mixing, and higher incubation
temperatures are used to enhance immunoreaction kinetics.
Methods such as electroosmosis[44], magnetic stirring[45],
electrokinetic flow[46] and ultrasonic effects[47] can increase
mass transport so as to perform rapid CLIA. In 2007,
Morozov et al[48] designed a single microfluidic device, which
could electrophoretically capture charged analytes on an
antibody array from flow. The collected analytes were then
detected with magnetic beads. The suspension of magnetic
beads that specifically bind the analytes flowed through the
cell while a magnet attracted the beads to the surface, causing
the slide over the array surface. This electrophoretically
assisted zeptomolar microfluidic immunoassay with
magnetic-beads detection could be finished in 3 min.
The approaches driven by external forces generally need
complex or special equipment. Yang et al[49] proposed
flow-injection chemiluminescent immunoassay (FICLIA) by
combining carboxylic resin beads as immobilization support
and a micro-bubble accelerated immunoassay process (Fig.1).
This method was simple, rapid and flexible, and can
accomplish the whole assay including the pre-incubation,
detection and regeneration steps within 16 min.
Passive mixing strategies use channel geometry to
accelerate the mixing by lamination or rotation technique
without any external force[50]. When the fluid travels along a
curved trajectory, the centrifugal effect produces the
transverse flows, which enhanced the mixing.
Fig.1 Micro-bubble-accelerated immunoreaction for fast flow-
injection immunoassay[49]
Copyright 2008, Elsevier
 WANG Chen et al. / Chinese Journal of Analytical Chemistry, 2012, 40(1): 310

Liu et al[51] developed a three-dimensional helical glass antibody immobilized PMs. After the vessels immersed in a
tubes for rapid mixing of immunoreagents (Fig.2). water bath for incubation, the mixtures were introduced into
Immunoreation kinetics is another crucial factor to accelerate four separation channels to perform online separation and
immunoassay, and infrared radiation can be used for fast washing. After the introduction of the substrate, the mixture
heating and temperature control[52,53]. The immunoreaction was delivered to record CL signals. The supporting-resolution
could be finished within 90 s because of the dual acceleration strategy used glass tubes and PMs as supports for antibodies
by the improved mass transport and enhanced reaction immobilization and automatically separated different analytes
kinetics. by a simple wash and magnetic capture step. This novel
automated support-resolution strategy and flow-through CL
3.2 Multiplexed detection of chemiluminescent multiplex immunoassay system indicated a promising
immunoassay practicability in automated rapid screen and clinical diagnosis.
Owing to their large surface areas and easy handling as
Multianalyte immunosensing system possesses the suspension and capture by magnet[5760], the magnetic particles
advantages of high throughput, short time, little consumption
and low cost[54], which is the goal of immunoassay, and is also
the hotspot of recent research. Multiple immunoassay
detection has two models: simultaneous detection and
sequential detection. Simultaneous detection usually combines
zone-resolved strategy and CCD equipment to detect all the
components in a single assay run. Sequential detection
provides a simple way to detect samples one by one in a
period. In recent years, with the development of multichannel
electrochemical workstation, multiarray piezoelectric sensor
and parallel photodiode array, electrochemical and optical
sensor arrays have been widely designed in multiplexed
detection. Multiplexed detection has experienced rapid
development since the main problems of sequential flow
injection analysis were resolved.
Ju et al[12] proposed a flow-through multianalyte CL Fig.2 Dually accelerated immunoreaction by infrared heating and
immunosensing system with substrate zone-resolved passive mixing[51]
technique, which could detect carcinoma antigen 125 (CA 125) Copyright 2009 American Chemical Society

and CEA in a single run. The capture antibodies were

immobilized on an UltraBind aldehyde-active membrane to
act as an immunoreactor. Then the mixture of CA 125, CEA,
and their corresponding tracers, HRP-labeled anti-CA 125 and
ALP-labeled anti-CEA was introduced for online incubation.
The substrates for HRP and ALP were delivered to perform
substrate zone-resolved immunoassay. The research group
also brought up the concept of channel-resolved approach[13],
and developed an automate multianalyte CLIA. This system
used moveable optical shutter to detect the signals of three
parallel detection channels, which could detect three tumor
markers sequentially. Later on, they integrated channel and
substrate zone-resolved strategies, and proposed
two-dimensional resolution for CL multiplex immunoassay[14].
They designed two channels to detect four analytes
simultaneously (Fig.3). To achieve the goal of detecting more
analytes at the same time, this group further designed the
sampling[55] and supporting-resolution strategies[56]. In the
sampling-resolution strategy, researchers immobilized the
capture antibodies on the paramagnetic spheres (PMs), then
Fig.3 Channel and substrate zone two-dimensional resolution for
they dispended the samples into four stirring vessels multiplex immunoassay of tumor markers[14]
containing tracer antibodies and corresponding capture Copyright 2007 American Chemical Society
 WANG Chen et al. / Chinese Journal of Analytical Chemistry, 2012, 40(1): 310

(MP) have been extensively used for development of uidic

immunoassay methods. Lin et al[61] immobilized antibodies on
PMs and coated tubes, respectively, to detect AFP, and
compared the analytical merits. The advantages of MP became
conspicuously apparent. Moreover, using MP as solid phases,
this method detected 59 human samples and obtained a good
correlation with the results of a commercial
electrochemiluminescent immunoassay kit.
In recent years, owing to the development of poly-N-
isopropylacrylamide (PNIP), thermo-sensitive PNIP can be
separated when the temperature is lower than the lower critical
solution temperature (LCST). Kang et al[16] proposed a CLIA
method based on temperature- and substrate-resolved
technologies to detect four proteins (Fig.4). They used two
different homogeneous carriers, which are thermo-sensitive
PNIP and magnetic beads (MB), and two different CL Fig.4 Homogeneous temperature- and substrate-resolved
substrates for ALP and HRP. One pair of capture antibodies technologies for the CL detection of four proteins[16]
Copyright 2009 the Royal Society of Chemistry
was immobilized on the PNIP and another one was bonded to
the MB. The mixture of ALP- and HRP-labeled antibodies
was introduced to form four sandwich immunocomplexes, and
then the substrates were delivered and the CL signals were
recorded sequentially. However, this method involves
complex manipulation, which brings difficulties to the
automatic operation, and the assay needs long detection time.
Liu et al[62] developed a microplate CL enzyme
immunoassy (CLEIA) for the detection of staphylococcal
enterotoxin B (SEB). With the help of a file wheel, the
analysis speed was rapid, approximately 2 min per 96-sample
plate (1 s per sample). This high-throughput detection is ideal
for clinical practice. However, this sequential detection
Fig.5 New 96 4 well microtiter plate for multiplexed
method may cause the time accumulation effect, which is a
chemiluminescent imaging immunoassay[64]
long interval between the detection of the first sample and the
Copyright 2007 American Chemical Society
last one.
The zone-resolved strategy is a method to immobilize
The application of lab-on-a-chip method to CLIA helps to
different immunoreagents on different places of the
develop multianalyte systems. The integration of the chip and
immunoreactor, so the immunoreactions can take place in
CLIA system enhances the detection capacity, and achieves
different places, and then with the help of the array detector,
multiplexed detection, which is available for medical
the multiplexed detection can be achieved. The application of
diagnosis and drug screening. Sun et al[10] designed a
CCD makes the CLIA more convenient, and also accelerates
three-dimensional chip, which could detect 96 samples by a
the development of the CL imaging detection technique. The
CCD camera. Wolter et al[11] overcame the drawbacks of CCD
integration of CLIA and CCD image sensor can satisfy the
static detection, such as time consumption and difficulties in
requirement to simultaneously detect a large amount of
automation, and developed a new flow-through CL microarray
samples in a short time. Urbanowska et al[63] used a 96-well
system. They designed a 6 7 microarray and immobilized
plate as the solid phase to immobilize antibodies and detected
the capture antibodies on poly(ethylene glycol)-modified glass
six proteins by CL imaging detection. Magliulo et al[64]
substrates. Cell recognition was carried out by binding
designed a new polystyrene 96-well microtiter plate format, in
species-specific biotinylated antibodies. CL detection was
which each main well contains four sub-wells in the bottom.
accomplished by a streptavidin-HRP catalyzed reaction of
The capture antibodies were immobilized on the microtiter,
luminol and H2O2. The sampling, washing and separation
and the samples and the peroxidase-labeled antibodies were
process all used the low-through technique with the help of a
added to form the sandwich immunocomplexes. After the
hand operated distribution valve and a changeable flow cell.
introduction of CL substrate, the signal was recorded by a
The CL signal was recorded by a CCD camera. The whole
CCD image sensor, and 96 4 of the samples were detected
assay was finished in 13 min (Fig.6)
simultaneously (Fig.5).
 WANG Chen et al. / Chinese Journal of Analytical Chemistry, 2012, 40(1): 310
Fig.6 A flow-through chemiluminescence microarray readout
system for the detection of three bacteria[11]
Copyright 2008 American Chemical Society
3.3 Highly sensitive chemiluminescent immunoassay
For practical detection, the sensitivity and specificity of
existing detection methods are not very satisfying, so highly
sensitive and selective detection methods have shown their
importance. The development of nanotechnology has provided
great opportunity for the improvement of highly sensitive
CLIA. Nanoparticles have been widely applied in bio-labeling
to develop many signal amplification methods for highly
sensitive analysis.
Zhang et al[21] found that gold colloids with nanoparticles of
different sizes could enhance the CL of the luminol-H2O2
system, and the most intensive CL signals were obtained with
38-nm-diameter AuNPs. Wang et al[22] synthesized specially
shaped, irregular gold nanoparticles (IGNPs) and found their
catalytic efficiency on luminol CL to be 100-fold greater than
that of spherical AuNPs. They established the sandwich-type
analytic method for rapid, simple, selective and sensitive
immunoassay using the IGNPs-modified anti-IgG as probes.
Ambrosi et al[65] reported a novel double-codified nanolabel
(DC-AuNPs) based on AuNPs modified with anti-human IgG
HRP-conjugated antibody. The DC-AuNPs label showed
excellent specificity and offered enhanced performances.
Yang et al[24] introduced this DC-AuNPs into CLIA, and also
added 4-(4-iodo) phenylphenol (IPP) as a new signal
enhancer (Fig.7). The combination of the remarkable
sensitivity of the enhanced CL method and the use of AuNPs
as an anti-AFP-HRP carrier for the enzymatic signal
amplification provided a linear response range of AFP from
0.08 g L1 to 0.3 g L1 with an extremely low detection limit
of 5 ng L1.
Because AuNPs have small size, they cannot carry a large
number of enzymes. As a result, scientists have started to try
many other nanomaterials to load more enzymes. Bi et al.[27]
used multiple enzyme layers assembled multiwall carbon
nanotubes (MWCNTs) as signal amplification labels to
develop an ultrasensitive CLIA method. In this study, HRP
was assembled onto MWCNTs templates layer-by-layer
through electrostatic interactions with poly(dimethyldially-
lammonium chloride) (PDDA), and further conjugated with
AFP secondary antibodies as the enzyme label. Under
optimum conditions, a linear range from 0.02 g L1 to 2.0 g
L1 was obtained with a detection limit of 8.0 ng L1, which
was two orders of magnitude lower than those of the standard
enzyme-linked immunosorbent assay method.
As enzyme-labeled antibodies possess bigger steric
hindrance, people began to link enzymes and antibodies in
certain proportion to the surface of nanomaterials directly,
which insured that a large number of enzymes can be
modified on per nanomaterial, so people began to link
enzymes to the surface of nanomaterials directly, and
immobilize antibodies as proportion, which insured that the
nanomaterials can link a large number of enzymes and also
decrease the stereospecific blockade. Wu et al[28]
coimmobilized HRP and AFP onto the surface of SiO2
nanoparticles with the help of -glycidoxy-
propyltrimethoxysilane (GPTMS) as the linker. The improved
particle synthesis using a seed-particle growth route yielded
particles of narrow size distribution, which allowed consistent
loading of HRP and anti-AFP on each microsphere and
ensured subsequent immunosensing possessed high sensitivity
and reproducibility. Use of large silica nanoparticles as a label
could reduce the physical adsorption and load a large amount
of HRP, which enhanced the sensitivity. This strategy
provided a simple, cost-effective, specific and potent method
for the detection of AFP in practical samples. Yang et al[66]
proposed a novel CL analytical device making use of the high
loading ability of three-dimensional ordered nanoporous SiO2
film. The nanoporous SiO2 film was prepared with
self-assembly of polystyrene spheres as a template and 5-nm
SiO2 nanoparticles on a glass slide followed by a calcination
process. Its functionalization with streptavidin was achieved
by using GPTMS as a linker, and then recognized with
biotin-labeled antibody to further construct highly efficient
CLIA (Fig.8).
Fig.7 Ultrasensitive enhanced chemiluminescence enzyme
immunoassay amplified by double-codified gold
nanoparticles labels[24]
Copyright 2009 Wiley InterScience
 WANG Chen et al. / Chinese Journal of Analytical Chemistry, 2012, 40(1): 310

theories and methods have been developed rapidly.

Exploitation of enzymes and substrates with higher catalytic
activity, better stability and more suitable kinetics,
development of new labeling skills and construction of new
CLIA methods are the subject of focus for future research.

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