A novel electrochemiluminescence immunosensor via polymerization-assisted amplification

Article abstract of DOI:10.1039/c0cc02741c

A novel electrochemiluminescent immunosensor with polymerization-assisted signal amplification has been developed and enhanced sensitivity was achieved by increasing tertiary amine loading on polymers. The Royal Society of Chemistry.

Full text of DOI:10.1039/c0cc02741c

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
A novel electrochemiluminescence immunosensor via
polymerization-assisted amplificationw
Yafeng Wu, Hongyan Shi, Liang Yuan and Songqin Liu*
Received 23rd July 2010, Accepted 1st September 2010
DOI: 10.1039/c0cc02741c
A novel electrochemiluminescent immunosensor with poly-
merization-assisted signal amplification has been developed
and enhanced sensitivity was achieved by increasing tertiary
amine loading on polymers.
glycoprotein most often associated with colorectal cancer
and is used in early detection of relapse in individuals already
treated for colorectal cancer.8
The fabrication of an electrochemiluminescent immuno-
sensor based on a polymerization-assisted signal amplification
strategy is shown in Scheme 1. A solution of N-hydroxy-
succinimidyl bromoisobutyrate (initiator) conjugated-CEA
secondary antibodies (denoted Ab2*) was first prepared and
adjusted to 1 mM in phosphate buffer solution (see ESI). The
Ab2* was introduced to a gold electrode surface through two
immunoreactions. The first, monoclonal antibody to CEA
(Ab1) were introduced to the Au electrode surface through
an acylamide-bond between the carboxylic group of
poly(o-aminobenzoic acid) previously electropolymerized on
the surface of the Au electrode (see ESI) and the amino group
of Ab1 in the presence of N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride (EDC) and N-hydroxy-
succinimide (NHS). Then, a CEA (denoted Ag) containing
solution was spotted onto the Ab1 modified electrode for
30 min to form Ag–Ab1 immunocomplexes on the surface
(Ag/Ab1/Au). The second, Ab2* was spotted on the Ag/Ab1/Au
surface for 30 min to immobilize the polymerization reaction
center of Ab2* on the electrode surface (Ab2*/Ag/Ab1/Au).
After each immunoreaction step, the electrode was immersed
into a bovine serum albumin (BSA) solution that contained
0.05% Tween-20 in order to eliminate non-specific adsorption
and block non-specific binding sites.
Electrochemiluminescence (ECL) has attracted considerable
attention due to its low cost, wide range of analytes, low
background signal, and high sensitivity.1 A number of ECL
2+
analytical methods based on ruthenium complex (Ru(bpy)3
)
with tertiary amine (TA) or diketone containing compounds
as coreactants2 have been developed for environmental assays
such as food and water testing, biological warfare agent
detection, and clinical diagnostics.3 Great efforts have been
made toward enhancement of detection sensitivity of the
Ru(bpy)32+/TA ECL system. Among these, using polymer
materials or nanoparticles as carriers for the immobilization of
Ru(bpy)32+ to enhance the ECL signal is especially promising.4
For example, by using polystyrene microspheres or beads as
carriers for the immobilization of Ru(bpy)32+, an ultrasensitive
detection methodology for DNA hybridization and C-reactive
protein immunoassay have been developed.5 With sandwich-
type immunoassay and encapsulation of hydrophobic
2+
Ru(bpy)3
compound within polymer microspheres6 or
liposomes,7 enhancement of the ECL signal was also achieved.
The enhanced signal could be used for detection of trace
amounts of various proteins contained in the influenza virus.
Herein, a novel Ru(bpy)32+/TA ECL system through the
immobilization of the reductant 2-(diisopropylamino)ethylamine
(DPEA) coupling with the polymerization-assisted signal
amplification strategy was developed for immunosensing for
the first time. An ECL signal was generated whenever a
The electrode of Ab2*/Ag/Ab1/Au was then immersed into
an AGET ATRP reaction solution which contained glycidyl
methacrylate as the monomer, CuCl2 and 2,20-bipyridyl as the
reaction catalyst. Ascorbic acid was added to consume oxygen
and reduce Cu(II) complexes back to the lower oxidative state
of Cu(I) complexes, which triggered the polymerization reaction
2+
potential was applied to the electrode in the Ru(bpy)3
solution. Activators generated electron transfer for atom
transfer radical polymerization (AGET ATRP) was employed
for local accumulation of the glycidyl methacrylate monomer.
Growth of polymer materials provided numerous epoxy
groups for DPEA coupling, which in turn enhanced the
ECL signals of the Ru(bpy)32+/TA system. Carcinoembryonic
antigen (CEA) was used as a model protein, which is a
School of Chemistry and Chemical Engineering,
Southeast University Jiangning, Nanjing, 211189,
People’s Republic of China. E-mail: Liusq@seu.edu.cn;
Fax: +86-25-52090613; Tel: +86-25-52090616
w Electronic supplementary information (ESI) available: Detailed
experimental description, the preparation of secondary CEA anti-
bodies (Ab2) labeled with N-hydroxysuccinimidyl bromoisobutyrate
(initiator) (Ab2*), the reaction mechanism of AGET ATRP, the
optimization of AGET ATRP time, the surface reflectance FT-IR
spectrum of PGMA/Ab2/Ag/Ab1/Au, the optimization of the incubation
time of DPEA and polymers, the optimization of pH of the solution,
the inter-assay of the immunosensor. See DOI: 10.1039/c0cc02741c
Scheme 1 The fabrication of the immunosensor.
Chem. Commun., 2010, 46, 7763–7765 7763
c
This journal is The Royal Society of Chemistry 2010

View Article Online
for DPEA/PGMA/Ab2/Ag/Ab1/Au located at 1.11 V was
clearly observed (Fig. 2A). To ensure the signal did not
originate from nonspecific absorption of DPEA, two control
experiments were conducted. One of the control experiments
had the concentration of CEA at 0 ng mLꢀ1 (Fig. 2B), the
other used Ab2, without coupling of initiators, immobilized on
Au electrode in a similar fashion (Ab2/Ag/Ab1/Au, Fig. 2C).
Much lower ECL responses for both electrodes were obtained,
which suggested that the enhancement of ECL response in
Fig. 2A can be attributed to the presence of DPEA on the
electrode surface and that the noise signal can be neglected. To
verify the polymerization-assisted signal amplification, DPEA
was directly coupled with Ab2 to complete the sandwiched
immunoreactions, and form electrode DPEA/Ab2/Ag/Ab1/Au
(Fig. 2D). The ECL signal was more than 10-fold lower than
that of electrode DPEA/PGMA/Ab2/Ag/Ab1/Au with
polymers as the DPEA carriers. Therefore, this result confirmed
polymerization-assisted signal amplification.
Fig. 1 (A) The SEM images of Ab2*/Ag/Ab1/Au after 2 h poly-
merization. (B) X-ray photoelectron spectroscopy analysis. C 1s
core-level spectra of the Ab2*/Ag/Ab1/Au surface subjected to AGET
ATRP of GMA for 2 h.
(see ESI for reaction mechanism). After 2 h of polymerization,
several polymer islands with an average diameter of
approximately 16 nm were observed from scanning electron
microscope (SEM) images (Fig. 1A). These polymer islands
were characterized by C 1s core-level spectra of X-ray photo-
electron spectroscopy (XPS) analysis (Fig. 1B). The peak area
ratio for [C–H] : [C–O] : [OQC–O] is 3.3 : 3.2 : 1.0, which is in
good agreement with the corresponding theoretical ratio of
3 : 3 : 1 for poly-GMA (PGMA), confirming the success of
AGET ATRP reaction.9 Growth of the polymer materials
was also ascertained by the surface reflectance FT-IR
spectrum (see ESI).10 All these results suggested the presence
of PGMA layer on the Au electrode surface.
The ECL intensity was associated with the accessibility and
steric hindrance of epoxy groups on the electrode. At 2 h of
polymerization, the polymer film (PGMA/Ab2/Ag/Ab1/Au)
thickness detected by ellipsometry reached a maximum of
about 14.3 nm. At that time the accessibility of epoxy groups
on the electrode was the largest and the steric hindrance was
the smallest (see ESI). The ECL intensity also depended on the
incubation time of the polymer materials modified electrode in
DPEA containing solutions and the pH value of the detection
solution (see ESI). Under optimal conditions, the ECL
intensity of DPEA/PGMA/Ab2/Ag/Ab1/Au depended on
the loading amount of DPEA on the polymers, which was
related to the amount of Ab2* attached on the electrode
surface, thus permitting the quantification of antigen trapped
by Ab1/Au.
Growth of the polymer materials on the electrode surface
provided numerous epoxy groups on the side chain of PGMA,
which allowed the accumulation of ECL coreactant DPEA on
polymers through acylamide bonds. The coupled DPEA on
2+
polymers sensitized the electrochemiluminescence of Ru(bpy)3
in solution (see ESI for reaction mechanism). Fig. 2 presents
the ECL response of the different electrodes. The ECL peak
Fig. 3 shows the ECL curves at different CEA concentrations
in incubation solutions. The ECL intensity was proportional
to the logarithm of CEA concentrations over 7 orders of
magnitude from 0.001 to 1000 ng mLꢀ1. The linear regression
equation is I = 4162 + 1495 log(cCEA/ng mLꢀ1) with a
detection limit of 0.5 pg mLꢀ1 at a signal-to-noise ratio of
3 (R2 = 0.994), where I is ECL intensity, c is the CEA
concentration in the incubation solution. This suggested that
Fig. 2 ECL response of electrodes. (A) DPEA/PGMA/Ab2/Ag/Ab1/
Au, (B) Ab1/Au was incubated with CEA antigen at 0 ng mLꢀ1 for
30 min, (C) Ab1/Au was incubated with Ab2 without initiator for
30 min, (D) Ab1/Au was incubated with Ab2 directly coupled with
DPEA for 30 min. The concentration of CEA is 0.01 ng mLꢀ1
,
Fig. 3 The ECL intensity versus the logarithm of the concentration of
CEA. The detection solution is 1 mM Ru(bpy)32+ in 0.1 M phosphate
solution (pH 7.5) at a scan rate of 50 mV sꢀ1, AGET ATRP time is 2 h,
the incubation time of DPEA and polymers was 5 h, the incubation
time of immunoreaction is 30 min.
2+
detection solution is 1 mM Ru(bpy)3 in 0.1 M phosphate solution
(pH 7.5) at a scan rate of 50 mV sꢀ1, the emission window was placed
in front of the photomultiplier tube biased at 600 V. AGET ATRP
time is 2 h, the incubation time of DPEA and polymers was 5 h.
c
7764 Chem. Commun., 2010, 46, 7763–7765
This journal is The Royal Society of Chemistry 2010

View Article Online
electrochemiluminescence). This result showed that our
method was comparable and acceptable for CEA detection.
Electrochemiluminescence gave similar results when compared
to the proposed method, that is, the developed immunoassay
may provide a promising alternative tool for determining CEA
in human serum in the clinical laboratory.
In summary, we have successfully developed a highly
sensitive and selective electrochemiluminescent immunosensor
for detection of cancer biomarkers via a polymerization-
assisted signal amplification strategy. The surface in situ
polymerization provided numerous epoxy groups on the side
chain of PGMA for local accumulation of ECL coreactant
DPEA. The attached DPEA sensitized the solution phase ECL
2+
behaviors of Ru(bpy)3 and allowed accurate quantification
Fig. 4 Assay results of clinical sera using the proposed and reference
of CEA with a wide range of 7 orders of magnitude and a low
detection limit of 0.5 pg mLꢀ1
electrochemiluminescent methods.
.
the proposed immunosensor is extremely sensitive, especially
for detection in low abundance of biomarkers. In addition, the
proposed immunosensor has a wider linear range and much
lower detection limit compared to other amplified sensing
strategies, such as linear range of 0.05 to 6 ng mLꢀ1 and a
detection limit of 0.02 ng mLꢀ1 for the dual signal amplification
strategy using multibioconjugates of HRP–Ab2–carbon
nanospheres as labels and functional graphene sheets as
biosensor platform to increase the primary antibodies loading;11
linear range of 0.01 to 160 ng mLꢀ1 and a detection limit of
0.01 ng mLꢀ1 for an electrochemical immunosensor using
thionine-doped magnetic gold nanospheres as labels and
horseradish peroxidase as enhancer.12 The high sensitivity
might be due to the fact that a large number of DPEA
molecules could be attached onto polymers. To further
investigate the selectivity of the proposed immunosensor for
CEA detection, the immunosensor was incubated in 1 ng mLꢀ1
The project is supported by the National Natural Science
Foundation of China (Grant Nos. 20675013, 20875013),
the National Basic Research Program of China (No.
2010CB732400), and the Specialized Research Funds for the
Doctoral Program of Higher Education (200802860035).
Notes and references
1 (a) H. Zhong, X. Lei, X. Hun and S. S. Zhang, Chem. Commun.,
2009, 6958; (b) H. Dai, Y. U. Chi, X. P. Wu, Y. M. Wang,
M. D. Wei and G. N. Chen, Biosens. Bioelectron., 2010, 25,
1414; (c) Y. L. Chen, B. Qiu, Y. Y. Jiang, Z. Y. Lin, J. J. Sun,
L. Zhang and G. N. Chen, Electrochem. Commun., 2009, 11, 2093;
(d) T. T. Jia, Z. M. Cai, X. M. Chen, Z. J. Lin, X. L. Huang,
X. Chen and G. N. Chen, Biosens. Bioelectron., 2009, 25, 263;
(e) D. O. Hull, B. Bajrami, I. Jansson, J. B. Schenkman and
J. F. Rusling, Anal. Chem., 2009, 81, 716; (f) J. Zhang, H. L. Qi,
Y. Li, J. Yang, Q. Gao and C. X. Zhang, Anal. Chem., 2008, 80,
2888.
2 (a) J. P. Yuan, T. Li, X. B. Yin, L. Guo, X. Z. Jiang, W. R. Jin,
X. R. Yang and E. K. Wang, Anal. Chem., 2006, 78, 2934;
(b) W. Zhan and A. J. Bard, Anal. Chem., 2007, 79, 459;
(c) J. G. Li, Q. Y. Yan and H. X. Ju, Anal. Chem., 2006, 78, 2694.
3 M. M. Richter, Chem. Rev., 2004, 104, 3003.
4 (a) W. J. Miao, Chem. Rev., 2008, 108, 2506; (b) H. Wei and
E. K. Wang, TrAC, Trends Anal. Chem., 2008, 27, 447;
(c) M. Zhou, J. Roovers, G. P. Robertson and C. P. Grover, Anal.
Chem., 2003, 75, 6708; (d) D. Dong, D. Zheng, F. Q. Wang,
X. Q. Yang, N. Wang, Y. G. Li, L. H. Guo and J. Cheng, Anal.
Chem., 2004, 76, 499; (e) M. Zhou and J. Roovers, Macro-
molecules, 2001, 34, 244.
5 (a) W. J. Miao and A. J. Bard, Anal. Chem., 2003, 75, 5825;
(b) W. J. Miao and A. J. Bard, Anal. Chem., 2004, 76, 5379.
6 M. So, E. G. Hvastkovs, B. Bajrami, J. B. Schenkman and
J. F. Rusling, Anal. Chem., 2008, 80, 1192.
7 (a) X. M. Zhou, D. Xing, D. B. Zhu and L. Jia, Electrochem.
Commun., 2008, 10, 564; (b) X. M. Zhou, D. Xing, D. B. Zhu and
L. Jia, Anal. Chem., 2009, 81, 255.
8 (a) D. Matschulat, A. Deng, R. Niessner and D. Knopp, Analyst,
2005, 130, 1078; (b) T. Letilovic, R. Vrhovac, S. Verstovsek,
B. Jaksic and A. Ferrajoli, Cancer, 2006, 107, 925.
9 W. H. Yu, E. T. Kang and K. G. Neoh, Langmuir, 2004, 20, 8294.
10 Y. Zhang, K. L. Tan, B. Y. Liaw, D. J. Liaw, E. T. Kang and
K. G. Neoh, J. Vac. Sci. Technol., A, 2001, 19, 547.
11 D. Du, Z. X. Zou, Y. Shin, J. Wang, H. Wu, M. H. Engelhard,
J. Liu, I. A. Aksay and Y. H. Lin, Anal. Chem., 2010, 82, 2989.
12 D. P. Tang, R. Yuan and Y. Q. Chai, Anal. Chem., 2008, 80, 1582.
CEA containing
a
different interfering agent, such as
(IgG); no
a-1-fetoprotein (AFP) and immunoglobin
G
remarkable change of ECL intensity was observed in comparison
with the result obtained in the presence of CEA only. The
immunosensor was also incubated in 1 ng mLꢀ1 AFP solution;
almost no signal change was obtained compared with the
background (see ESI). All these results indicated a good
selectivity of the proposed CEA immunosensor and that
the signal did not originate from the cross reaction. The
reproducibility of the proposed immunosensor was also
evaluated through analysis of three independently made
immunosensors. The ECL response gave only 1.08% deviation
at 10 ng mLꢀ1 CEA (see ESI), which indicated an acceptable
reproducibility and precision.
To monitor the feasibility of the developed immunosensor,
17 serum specimens, which were collected from Jiangsu
Institute of Cancer Prevention and Cure (Nanjing, China),
were examined by the proposed method and compared with
the clinical electrochemiluminescent method. Experimental
results are described in Fig. 4. The regression equation for
these data is as follows: y = 5.485 + 0.974x (R2 = 0.997)
(x-axis, by the as-prepared immunosensor; y-axis, by clinical
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 7763–7765 7765