Au(iii)-promoted polyaniline gold nanospheres with electrocatalytic recycling of self-produced reactants for signal amplification

Article abstract of DOI:10.1039/c2cc35351b

A novel and redox-active nanocatalyst, Au(iii)-promoted polyaniline gold nanosphere (GPANG), was designed as the nanolabel for highly efficient electrochemical immunoassay of human IgG by coupling with electrocatalytic recycling of self-produced reactants.

Full text of DOI:10.1039/c2cc35351b

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COMMUNICATION
Au(III)-promoted polyaniline gold nanospheres with electrocatalytic
recycling of self-produced reactants for signal amplificationw
Yuling Cui, Huafeng Chen, Dianping Tang,* Huanghao Yang and Guonan Chen*
Received 24th July 2012, Accepted 3rd September 2012
DOI: 10.1039/c2cc35351b
A novel and redox-active nanocatalyst, Au(III)-promoted poly-
aniline gold nanosphere (GPANG), was designed as the nano-
label for highly efficient electrochemical immunoassay of human
IgG by coupling with electrocatalytic recycling of self-produced
reactants.
play an important role in the catalytic processes including low-
temperature CO oxidation, reductive catalysis of chlorinated or
6
nitrogenated hydrocarbons, and organic synthesis. Signifi-
cantly, AuNPs can catalyze the reduction of p-nitrophenol
1
a,7
4
(NP) to p-aminophenol (AP) in the presence of NaBH .
Meanwhile, the produced AP molecules can be oxidized to
p-quinone imine (QI) with the help of electron mediators,
An ultrasensitive and simple method for the detection of disease-
related biomolecules is critical for early diagnosis in biomedical
4
which can be reduced again to AP via NaBH . The catalytic
1
researches. Great effort has been made worldwide to design and
recycling of self-produced reactants resulted in the amplifica-
tion of the electrochemical signal. In these methods, however,
AuNPs and electron mediators were often separated in two
different phases, which increased the steric hindrance of
electron transfer. More importantly, the efficiency of electron
communication between AuNPs and electron mediators might
be largely decreased. Our motivation in this work is to
synthesize a novel class of redox-active nanocatalysts for the
catalytic reduction of NP to AP, and the catalytic recycling of
AP to QI without the participation of other electron media-
tors. Conductive polymer nanomaterials represent an attrac-
tive family of nanocatalysts due to their unique properties,
e.g. high conductivity, relatively environmental stability, and
develop a highly sensitive assay protocol. Electrochemical
immunoassay is one of the predominant analytical methods
2
due to its high sensitivity, low cost and low power requirements.
To acquire a high sensitivity, the amplification of the electro-
chemical signal is extremely important. Although the antigen–
antibody reaction by itself can cause the change of the
electrochemical signal to some extent, it is very limited. Hence,
labelling of the secondary antibody is essential. Enzyme labels
and nanolabels are usually employed for the amplification of a
measurable product as a result of their very fast and highly
selective catalytic reactions, which enable high signal amplifica-
3
tion in the presence of bound target biomolecules. However, we
8
simple doping–dedoping chemistry. Polyaniline, with a rather
recently found that (i) since there is, for sterical reasons, usually
a 1 : 1 ratio of enzyme and signal antibody used in the conven-
tional ELISA, the obtainable signal amplification is always
limited, and (ii) the bioactivity of the labelled enzymes is usually
weakened when enzymes are labelled onto the signal antibody or
unique structure, contains an alternating arrangement of
benzene rings and nitrogen atoms, which can be used as a
candidate for the preparation of redox-active nanocatalysts.
Herein, we synthesized a novel, redox-active nanocatalyst,
Au(III)-promoted polyaniline gold nanosphere (GPANG), for
the highly efficient electrochemical immunosensing of human
IgG (HIgG, as a model) by coupling with electrocatalytic
recycling of self-produced reactants. The GPANG was prepared
through one-pot in situ copolymerized deposition of polyaniline
on the AuNPs with the help of Au(III) (see ESIw). The as-prepared
GPANGs showed excellent adsorption properties for selective
attachment of biomolecules and strong catalytic properties. Fig. 1
illustrates GPANG-catalyzed recycling of self-produced reactants
for signal amplification of the sandwich-type electrochemical
immunosensing. In this configuration, the added NP was
initially reduced to AP via the catalysis of the doped nanogold
4
nanostructures. Therefore, exploring a newly amplified strategy
for highly sensitive electrochemical immunosensing without the
participation of enzymes would be advantageous.
Nanostructures, especially redox-active nanostructures, are
usually used as catalysts for electrochemical reactions or organic/
3 4 2
inorganic synthesis. Various nanocatalysts, such as Fe O –MnO
hybrid nanocrystals, ZnO–SiO2 nanocomposites, Pd nano-
particles and gold nanoparticles, have been reported for catalytic
5
organic synthesis and electrocatalytic reaction. Among these
nanocatalysts, gold nanoparticles (AuNPs) have been found to
Ministry of Education & Fujian Provincial Key Laboratory of
Analysis and Detection of Food Safety, Department of Chemistry,
Fuzhou University, Fuzhou 350108, P.R. China.
E-mail: dianping.tang@fzu.edu.cn, gnchen@fzu.edu.cn;
Fax: +86 591 22866135; Tel: +86 591 22866125
w Electronic supplementary information (ESI) available: Experimental
procedures, preparation and bioconjugation of the GPANGs, and
measurement method. See DOI: 10.1039/c2cc35351b
4
labels onto/into the GPANG in the presence of NaBH , then the
generated AP was electrochemically oxidized to QI by the electron
mediator (polyaniline) in the GPANG, and then the oxidized QI
4
was reduced back to AP by NaBH . The self-produced QI
reactant was catalytically recycled and thus amplified the
signal of the electrochemical response. To verify the successful
This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 10307–10309 10307

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4À
facilitate electron transfer from BH to p-nitrophenolate and
thus acted as catalysts for the purpose. Therefore, the syn-
thesized GPANGs could catalyze the reduction of p-nitrophenol
to p-aminophenol in the presence of NaBH (Note: UV-vis
4
absorption spectroscopies using pure gold nanoparticles were
monitored, and the results are shown in Fig. S2 of ESIw).
To investigate the electrocatalytic behaviours of the prepared
2
GPANG, polyclonal goat anti-human IgG (Fc specific, Ab )
antibodies were conjugated onto the nanocatalysts via the
strong interaction between gold nanoparticles and proteins,
À1
which were used for the determination of HIgG (1.0 ng mL
used in this case) on a monoclonal mouse anti-human IgG (Fab
1
specific) antibody (Ab )-modified glassy carbon electrode with a
sandwich immunoassay format. The preparation process of the
immunosensor was characterized by using electrochemical im-
pedance spectroscopy (see Fig. S3 of ESIw). For comparison,
polyaniline nanospheres (PAN, 12 nm in diameter)-labelled Ab2
Fig. 1 Schematic illustration of Au(III)-promoted polyaniline gold
nanospheres with electrocatalytic recycling of self-produced reactants
for signal amplification.
and gold nanoparticles (AuNP, 16 nm in diameter)-labelled Ab
2
synthesis of the GPANGs, we used wide-angle X-ray diffraction,
Raman spectrum, and SEM to characterize the synthesized
were also utilized for the detection of HIgG, respectively. Next,
cyclic voltammograms of the as-prepared immunosensors were
8c
nanocatalysts, as described in our recent paper. Meanwhile,
a TEM image of the as-synthesized GPANGs is also shown in
Fig. S1 (see ESIw), which exhibited a large number of gold
nanoparticles on the surface of GPANGs.
monitored in pH 8.0 NaBH
using various bionanolabels (Fig. 3). As seen from curve ‘b’ in
Fig. 3A, upon addition of 10 mM AP in pH 8.0 NaBH -PBS,
4
-PBS with and without NP or AP
4
redox peak currents of the AuNP-based immunoassay were not
almost changed in comparison with that of curve ‘a’. In
contrast, the cathodic current was gently increased toward
An initial question to be answered was whether the designed
GPANG nanocatalysts can catalyze the reduction of NP in the
presence of sodium borohydride. We used UV-vis absorp-
tion spectroscopy to investigate the catalytic process of the
nanocatalysts. As indicated in curve ‘a’ of Fig. 2A, pure
p-nitrophenol shows a distinct spectral profile with an absorp-
1
0 mM NP (curve ‘c’ in Fig. 3A). The results revealed that
AuNP could catalyze the reduction of NP to AP with the help
of NaBH , but could not further catalyze the AP. By the same
4
token, we also found that pure PAN could not reduce the NP to
AP, but could catalyze the oxidation of AP to QI (Fig. 3B).
More inspiringly, when using the GPANG as nanolabels, the
peak currents could be greatly improved (Fig. 3C). Further-
tion maximum at 317 nm. When a NaBH
into p-nitrophenol, a new absorption peak was observed at 402 nm
curve ‘b’ of Fig. 2A). Moreover, the absorbance at 402 nm
4
solution was added
(
was gradually increased upon the addition of NaBH , while
4
more, addition of NP in pH 8.0 NaBH -PBS could result in a
4
that of 317 nm was gradually decreased (curve ‘b–e’ of Fig. 2A).
higher anodic peak current than that of AP (curve ‘c’ versus
The reason might be the formation of the p-nitrophenolate ions
9
caused by the addition of NaBH . When the added p-nitrophenol
curve ‘b’ in Fig. 3C). This is most likely a consequence of the
fact that (i) the doped gold nanoparticles in the GPANG could
catalyze the reduction of NP to AP in the presence of NaBH4,
4
molecules were completely converted to the p-nitrophenolate ions,
the GPANG nanocatalysts with various concentrations were
injected into the mixture. As shown in Fig. 2B, the absorbance
at 402 nm was gradually decreased with the increasing
GPANG nanocatalysts, while the absorbance at 315 nm was
increased. The increased peak derived from the formation of
p-aminophenol due to the catalytic reduction of p-nitrophenol
by GPANG nanocatalysts. The gradual development of the
peak at 315 nm confirmed the increase in p-aminophenol concen-
tration in the reaction mixture. The GPANG nanocatalysts could
(
(
ii) the formed AP was oxidized to QI by polyaniline, and
iii) the generated QI was reduced back to AP by NaBH . Such
4
a catalytic recycling of self-produced reactants could result in
the amplification of the electrochemical signal.
2
By using GPANG-labelled Ab secondary antibodies, the
sensitivity and dynamic range of GPANG-based electrochemical
immunoassay were evaluated toward HIgG standards in pH 8.0
4
NaBH -PBS containing 10 mM NP by using differential pulse
Fig. 3 Cyclic voltammograms of the developed immunoassays
À1
toward 1.0 ng mL HIgG in (a) pH 8.0 NaBH
4
-PBS, (b) pH 8.0
-PBS + 10 mM NP
by using various bionanolabels: (A) AuNP-labelled Ab antibodies,
4 4
NaBH -PBS + 10 mM AP, and (c) pH 8.0 NaBH
Fig. 2 UV-vis absorption spectra of (a) p-nitrophenol, (b–e) p-nitrophenol
2
after reaction with various concentrations of NaBH , and (f–h) the mixture
4
(B) PAN-labelled Ab
2
antibodies, and (C) GPANG-labelled Ab
À1
2
‘e’ upon addition of GPANG nanocatalysts with various concentrations.
antibodies. Scanning rate: 50 mV s
.
1
0308 Chem. Commun., 2012, 48, 10307–10309
This journal is c The Royal Society of Chemistry 2012

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immunoassays are capable of detecting target molecules in
real biological samples.
In conclusion, this communication designs a novel sandwich-
type electrochemical immunoassay for ultrasensitive determina-
tion of human IgG by using redox-active nanocatalysts as labels
with electrocatalytic recycling of self-produced reactants. High-
lights of this work are to explore a newly multifunctional
conductive polymer nanostructure with redox activity for the
signal amplification of electrochemical immunoassay by redox
recycling of self-produced reactants during organic synthesis
(AP 2 QI). More importantly, the methodology can avoid the
use of two working electrodes or multiple enzymes for the
amplification of the electrochemical signal. Compared with
conventional immuno-PCR assays, the nanocatalyte-based
immunoassay is of low-cost, simple, and sensitive.
Fig. 4 (a) Calibration plots and (b) specificity of the electrochemical
immunoassay toward HIgG standards in pH 8.0 NaBH -PBS containing
0.0 mM NP (inset: the corresponding DPV curves).
4
1
voltammetry (DPV) with a sandwich-type immunoassay format.
As shown from the inset of Fig. 4a, DPV peak currents increased
with the increasing HIgG concentration. A linear dependence
between the peak currents and the logarithm of HIgG was
Support by the National Natural Science Foundation of
China (no. 21075019 and 41176079), the ‘‘973’’ National Basic
Research Program of China (no. 2010CB732403), the Research
Fund for the Doctoral Program of Higher Education of China
(no. 20103514120003), and the National Science Foundation of
Fujian Province (no. 2011J06003) is gratefully acknowledged.
À1
À1
À1
obtained in the range from 0.01 pg mL to 100 ng mL with
a detection limit (LOD) of 1.0 fg mL estimated at the 3sblank
level (n = 11) (Fig. 4a). In addition, we also compared the
properties of the electrochemical immunoassay with other HIgG
assay methods reported previously. As seen from Table S1 (see
ESIw), the wide linear range and a low detection limit of the
developed immunoassay were acceptable. Although the system
has not yet been optimized for maximum efficiency, the assay
sensitivity of using GPANGs as nanocatalysts was 100-fold
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The specificity of the electrochemical immunoassay was
monitored by challenging the system with other biomolecules
such as alpha-fetoprotein (AFP), carcinoembryonic antigen
2
2
009, 81, 9730.
(
CEA), prolactin (PRL), and rabbit IgG (RIgG). Significantly
À1
3 (a) T. Selvaraju, J. Das, K. Jo, K. Kwon, C. Huh, T. K. Kim and
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À1
an example) than those with other biomolecules (10 ng mL in
4
8, 3503; (c) W. Zhao, X. Dong, J. Wang, F. Kong, J. Xu and
this case) (Fig. 4b). These results clearly manifested the high
specificity of the electrochemical immunoassay.
H. Chen, Chem. Commun., 2012, 48, 5253; (d) M. Akanda, Y. Choe
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chemical immunoassay were evaluated by calculating intra-
and inter-assay coefficients of variation (CVs). We repeatedly
(
g) G. Lai, F. Yan and H. Ju, Anal. Chem., 2009, 81, 9730.
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À1
assayed three different HIgG levels containing 1.0 pg mL
À1
,
À1
1
00 pg mL , and 10 ng mL under the same circumstances.
Experimental results revealed that the coefficients of variation
5
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2
(
CVs) of the inter-assay were 7.1%, 9.8%, and 9.5% for
À1 À1 À1
Chem. Commun., 2011, 47, 11624; (c) K. Lee, R. Anisur, K. Kim,
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1
.0 pg mL , 100 pg mL , and 10 ng mL HIgG, respec-
(
d) S. Polarz, F. Neues, M. van den Berg, W. Grunert and
tively, whereas the CVs of the intra-assay were 8.8%, 8.1%
and 9.7% towards the above-mentioned analytes. Therefore, the
precision and reproducibility of the developed immunosensor
was acceptable. In addition, the electrochemical immunosensor
exhibited satisfactory stability, and B90% of the initial peak
current was preserved after storage of the immunosensor and
nanolabels at 4 1C for 28 days.
L. Khodeir, J. Am. Chem. Soc., 2005, 127, 12028; (e) W. Yan,
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7
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application of the newly developed technique to be applied for
the testing of the real sample, it was applied for the analysis
of 3 HIgG samples of various concentrations including
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À1 À1 À1
0
.1 pg mL , 1.0 ng mL and 50 ng mL , which were
obtained by spiking HIgG standards into blank fetal calf
serum. The assayed results for the above-mentioned 3 HIgG
8 (a) M. Anderson, B. Mattes, H. Reiss and R. Kaner, Science, 1991,
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À1
À1
À1
analytes were 0.12 pg mL , 0.96 ng mL and 47.5 ng mL ,
respectively. The recoveries were 120%, 96% and 95%,
respectively. The results revealed that the newly proposed
9
S. Praharaj, S. Nath, S. Ghosh, S. Kundu and T. Pal, Langmuir,
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This journal is c The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 10307–10309 10309