A universal immunosensing strategy based on regulation of the interaction between graphene and graphene quantum dots

Article abstract of DOI:10.1039/c2cc35503e

We provide a novel and versatile signaling transduction strategy in the fluoroimmunoassay through regulating the interaction between graphene (Gr) and graphene quantum dots (GQDs), and demonstrate its feasibility in sensitive detection of human immunoglobulin G (IgG). This journal is

Full text of DOI:10.1039/c2cc35503e

Showcasing research from Dr Huimin Zhao’s
laboratory, Dalian University of Technology, China
As featured in:
A universal immunosensing strategy based on regulation of the
interaction between graphene and graphene quantum dots
A novel and versatile signaling transduction strategy in
fluoroimmunoassay through regulating the interaction between
graphene and graphene quantum dots has been proposed,
which opens new opportunities for applications of carbon-based
nanomaterials in the immunoassay.
See Huimin Zhao et al.,
Chem. Commun., 2013, 49, 234.
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A universal immunosensing strategy based on
regulation of the interaction between graphene and
graphene quantum dots†
Cite this: Chem. Commun.,
2013, 49, 234
Received 30th July 2012,
Accepted 4th October 2012
Huimin Zhao,* Yangyang Chang, Meng Liu, Sheng Gao, Hongtao Yu and Xie Quan
DOI: 10.1039/c2cc35503e
www.rsc.org/chemcomm
We provide a novel and versatile signaling transduction strategy in the
As a kind of carbon quantum dots (CQDs), graphene quantum
fluoroimmunoassay through regulating the interaction between dots (GQDs), consisting of a single atomic layer of nano-sized
graphene (Gr) and graphene quantum dots (GQDs), and demonstrate graphite, have the excellent performances of both CQDs and Gr.8
its feasibility in sensitive detection of human immunoglobulin G (IgG). Above all, they have a unique sp2 and sp3 hybrid structure differing
from other QDs (heavy-metal-based QDs and silicon QDs). Further-
Graphene (Gr) and its derivatives are widely used in biosensors due more, this structure is similar to that of Gr, hence the p–p stacking
to their unique physical and chemical properties.1,2 In particular, between Gr and GQDs can induce them into LRET proximity, and
graphenes can act as the highly efficient luminescence quenchers then lead to the luminescence quenching of CQDs by Gr. More-
for donors (organic dyes or quantum dots) based on either an over, in contrast to other CQDs, GQDs can provide a larger contact
electron transfer mechanism or a luminescence resonance energy area owing to their two-dimensional nano-structure in a certain
transfer (LRET) mechanism, thus laying a powerful basis for the size, make GQDs be quenched by Gr more efficiently. In addition,
design of a novel graphene-based biosensing platform.3,4 Among considering the known toxicological effects of heavy-metal-based
them, immunoassay has been regarded as an effective tool QDs on humans and the environment,9 it is necessary and
for early diagnosis of diseases, environmental monitoring, important to develop some low-toxicity and eco-friendly alterna-
and biochemical studies. In our previously reported work,5 we tives, such as GQDs,10 for biological detection. Therefore, it is
developed a ‘‘turn off’’ LRET immunosensing platform, which appropriate to use GQDs as donors to establish LRET systems.
took advantage of the long-range quenching capabilities of Gr
Herein, we present a universal signaling transduction strategy in
and broke the distance limit (100 Å) of traditional fluorescent the fluoroimmunoassay based on regulation of the interaction
biosensors. Nevertheless, it is well known that the ‘‘turn off’’ between Gr and GQDs, and explore its application in detecting
luminescent model exhibits higher background and lower antigens. The principle of our strategy is illustrated in Scheme 1.
efficiency than the ‘‘turn on’’ model. Lately, Chen et al.6 Gr as an acceptor and mouse anti-human immunoglobulin G (mIgG,
proposed a ‘‘turn on’’ luminescent immunoassay method, in antibody)–conjugated GQDs as donors were chosen to fabricate a
which quantum dots–antibody conjugates were absorbed onto LRET immunosensor for detecting human immunoglobulin G (IgG,
the graphene oxide (GO) surface by p–p stacking and hydrogen antigen). When adding Gr to the GQDs covalently functionalized
bonding interactions between antibody and GO, which realized a with mIgG (mIgG–GQDs) solution, both the p–p stacking interaction
simultaneous detection of two types of viruses. Despite these between Gr and GQDs and the nonspecific binding interaction
achievements, it is not possible to apply this model to detect between mIgG and the Gr surface6 would bring Gr and GQDs into
other antigens because not all of the antibodies possess the p–p LRET proximity to facilitate the luminescence quenching of GQDs
stacking interaction with graphenes, as deduced from the
reports of Liu et al.5 and Lee et al.7 In this respect, it is highly
desirable and urgent to explore other universal signaling trans-
duction strategies in the fluoroimmunoassay to meet these
challenges.
Key Laboratory of Industrial Ecology and Environmental Engineering
(Ministry of Education, China), School of Environmental Science and Technology,
Dalian University of Technology, Dalian, 116024, China.
E-mail: zhaohuim@dlut.edu.cn; Fax: +86-411-84706263; Tel: +86-411-84706263
Scheme 1 Schematic illustration of a universal immunosensing strategy based on
† Electronic supplementary information (ESI) available: Experimental details
and relevant spectra. See DOI: 10.1039/c2cc35503e
regulation of the interaction between Gr and GQDs.
c
234 Chem. Commun., 2013, 49, 234--236
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based on LRET. In the sensing process, the addition of human
IgG would bind the mIgG due to the specific antibody–antigen
interaction, which effectively increased the distance between
mIgG–GQDs and Gr surface, thus hindering the LRET process
and then producing a restoration of luminescence. In this ‘‘on’’
state, the overall luminescence response depended on the
amount of human IgG in the samples, thus providing an
effective basis for quantitative determination of targets.
Furthermore, the binding interaction between mIgG and
human IgG can ensure the specificity of this design.
In order to demonstrate the feasibility of our design, we firstly
prepared Gr according to a reported hydrothermal reduction
method,11 and the details are given in ESI.† The resultant Gr
was observed with occasional folds, crinkles, and rolled edges
from a typical TEM image (Fig. S1, ESI†). In the following, GQDs
were synthesized by the reported method.12 Initially, hydroxyl
groups on the GQD surface were converted into carboxylic acid
moieties to increase the carboxylic acid groups for antibody
conjugations.13,14 Then, mIgG was covalently conjugated to GQDs
through N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydro-
chloride (EDC) and N-hydroxysuccinimide (NHS) for specific
identification. The AFM image (Fig. 1a) showed that GQDs have
an average height of about 2 nm, indicating the formation
of GQDs and their two-dimensional structure in a certain size.
The representative TEM image of GQDs (Fig. S2, ESI†) further
confirmed the formation of GQDs.
The original GQDs exhibited the optimized excitation wave-
length at 360 nm (Fig. S3, ESI†). When GQDs were excited, an
emission peak was observed at 460 nm (Fig. 1b). The bright blue
was strong enough to be observed under UV light illumination by
naked eyes (Fig. 1b, inset). The quantum yield was estimated to be
7.2% (relative to rhodamine B in ethanol), suggesting the relative
high quality of the as-prepared GQDs. Up to now, the photolumi-
nescence (PL) mechanism of GQDs is still not clearly understood.
The origin of PL in GQDs was tentatively proposed to be from the
carbon sp2 domains/clusters, which essentially behaved as the
luminescence centers.15 Furthermore, the fluorescence intensity of
GQDs remained unchanged under continuous illumination,
implying their good photostability (Fig. S4, ESI†). Taken together,
these results suggested that the synthesized GQDs exhibited high
fluorescence intensity and high photostability. Notably, both Gr
and mIgG–GQDs showed good dispersibility in aqueous solution,
which would facilitate the antibody–antigen interaction in the
following sensing process.
Fig. 2 (a) Changes in the PL spectra of unmodified GQDs in phosphate buffer
solution (PBS, 20 mM, pH 8.0) with increasing concentration of Gr, with
concentrations of 0, 1.25, 2.5, 3.75, 5, 7.5, 8.75, 10, 11.25 mg mLÀ1 from top to
bottom. lex = 360 nm, lem = 460 nm. (b) Changes in the PL spectra of
mIgG–GQDs in PBS (20 mM, pH 8.0) with increasing concentration of Gr, with
concentrations of 0, 1.25, 2.5, 3.75, 5, 7.5, 8.75 mg mLÀ1 from top to bottom.
lex = 360 nm, lem = 460 nm.
In the following, we devoted to investigate the interaction
between Gr and GQDs. As shown in Fig. 2a, the PL intensity of pure
GQDs decreased with increasing concentration of Gr, indicating that
the PL quenching effect of Gr was directly related to the p–p stacking
interaction between Gr and GQDs, which could induce energy
transfer or electron transfer from GQDs to Gr, hence resulting in
the luminescence quenching of GQDs. The formation of GQDs–Gr
complex by p–p stacking between Gr and GQDs was further verified
with TEM measurement (Fig. S5, ESI†). To realize the specificity of
our design, unmodified GQDs were replaced by mIgG–GQDs. Fig. 2b
shows that the PL intensity of mIgG–GQDs has the same decreasing
tendency as that of pure GQDs under different concentrations of Gr,
suggesting the formation of mIgG–GQDs–Gr complexes. The red
line in Fig. 3 shows that the PL quenching efficiency of mIgG–GQDs
reached a plateau (80.7%) after the Gr concentration exceeded
7.5 mg mLÀ1, indicating the complete reaction between Gr and
mIgG–GQDs. Thus, an excess concentration was unnecessary,
and 7.5 mg mLÀ1 was used as the optimized condition for this
research. Furthermore, the quenching reaction reached equili-
brium only after several minutes (Fig. S6, ESI†), implying the
high quenching ability of Gr and the robust interaction between
Gr and GQDs. As noted, a slightly higher PL quenching efficiency
(Fig. 3) was obtained for mIgG–GQDs, compared with that for
unmodified GQDs, which may have resulted from the non-
specific binding interaction between mIgG and the Gr surface.6
All together, the quenching effect for GQDs and mIgG–GQDs
induced by Gr could be mainly attributed to p–p stacking
interaction between the two carbon-based materials, which
provided a possibility to construct the GQDs/Gr-based sensing
platform.
Fig. 1 (a) Atomic force microscopy (AFM) image and height profile of as-prepared
GQDs on freshly cleaved mica. (b) PL intensity of GQDs with the maximum emission
wavelength. lex = 360 nm, lem = 460 nm. The inset shows the photographs of GO
and GQDs dispersion in ultrapure water with UV illumination, respectively.
Fig. 3 Comparisons of the quenching efficiency induced by mIgG–GQDs (red line)
and unmodified GQDs (black line).
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practicality of this immunoassay. Furthermore, a relative stan-
dard deviation of 4.6% (n = 5, 10 mg mLÀ1 human IgG) was
obtained for this method, indicating the good reproducibility of
this immunoassay.
In conclusion, a new and versatile signaling transduction
strategy in the fluoroimmunoassay through regulating the
interaction between Gr and GQDs was proposed, which was
applied to an immunosensor based on LRET for determination
of human IgG. The results showed that the presented sensor
exhibited high sensitivity and good reproducibility in human IgG
detection. What is more, it can be used for determination of other
antigens by simple replacement of antibodies (Fig. S11, ESI†). It is
expected that this novel strategy will open new opportunities for
development of the LRET technique and promote the application
of carbon-based nanomaterials in immunoassays.
Fig. 4 (a) Changes in the PL spectra of mIgG–GQDs upon increasing the
concentration of human IgG, with concentrations of 0, 0.2, 0.4, 2, 3, 10, 12, 20,
30, 40 mg mLÀ1 from bottom to top. (b) Plot of the PL intensity against the
concentration of human IgG. lex = 360 nm, lem = 460 nm.
Under the optimal conditions, the sensing performance of
this system was evaluated by adding various concentrations of
human IgG into the above mIgG–GQDs and Gr (7.5 mg mLÀ1
)
This work was financially supported by the National Basic
Research Program of China (No. 2011CB936002), National
Nature Science Foundation of China (No. 21277016 and No.
21007007).
mixture. As indicated in Fig. 4a, the range of PL intensity
increased. The concentration dependency of the PL intensity
is shown in Fig. 4b. It shows that the PL intensity increased
with increasing concentration of human IgG in the range from
0.2 mg mLÀ1 to 12 mg mLÀ1, and reached a plateau after
the human IgG concentration exceeded 20 mg mLÀ1. The PL
recovery of this system could be ascribed to the binding
interaction between human IgG and mIgG fixed on the GQD
surface, thus increasing the GQDs–Gr distance, and then
hindering the LRET process. To confirm our hypothesis,
human Immunoglobulin M (IgM) coexisting in human serum
and bovine serum albumin (BSA) were chosen as interferents
for the control experiment. When 10 mg mLÀ1 human IgM and
BSA were added into the mIgG–GQDs–Gr mixture, the PL
intensity of mIgG–GQDs had a little recover (Fig. S7, ESI†), in
contrast to that of human IgG under the same conditions.
Note that the false positive response could be ascribed to the
nonspecific adsorption between mIgG–GQDs and BSA. In order
to eliminate this effect, the mIgG–GQDs–Gr mixture was first
incubated with BSA. It was observed that the recovered PL
reached the maximum in the presence of 10 mg mLÀ1 BSA
(Fig. S8, ESI†). However, the PL intensity will continue to
Notes and references
1 W. Yang, K. R. Ratinac, S. P. Ringer, P. Thordarson, J. J. Gooding and
F. Braet, Angew. Chem., Int. Ed., 2010, 49, 2114–2138.
2 Y. Zhu, S. Murali, W. Cai, X. Li, J. W. Suk, J. R. Potts and R. S. Ruoff,
Adv. Mater., 2010, 22, 3906–3924.
3 (a) H. Dong, W. Gao, F. Yan, H. Ji and H. Ju, Anal. Chem., 2010, 82,
5511–5517; (b) H. Chang, L. Tang, Y. Wang, J. Jiang and J. Li, Anal.
Chem., 2010, 82, 2341–2346; (c) C. Zhang, Y. Yuan, S. Zhang, Y. Wang
and Z. Liu, Angew. Chem., Int. Ed., 2011, 50, 6851–6854.
´
4 E. Morales-Narvaez and A. Merkoçi, Adv. Mater., 2012, 24, 3298–3308.
5 M. Liu, H. Zhao, X. Quan, S. Chen and X. Fan, Chem. Commun.,
2010, 46, 7909–7911.
6 L. Chen, X. Zhang, G. Zhou, X. Xiang, X. Ji, Z. Zheng, Z. He and
H. Wang, Anal. Chem., 2012, 84, 3200–3207.
7 J. S. Lee, H.-A. Joung, M.-G. Kim and C. B. Park, ACS Nano, 2012, 6,
2978–2983.
8 J. Shen, Y. Zhu, X. Yang and C. Li, Chem. Commun., 2012, 48, 3686–3699.
9 R. Hardman, Environ. Health Perspect., 2006, 114.
10 S. Zhu, J. Zhang, C. Qiao, S. Tang, Y. Li, W. Yuan, B. Li, L. Tian,
F. Liu, R. Hu, H. Gao, H. Wei, H. Zhang, H. Sun and B. Yang, Chem.
Commun., 2011, 47, 6858–6860.
11 Y. Zhou, Q. Bao, L. A. L. Tang, Y. Zhong and K. P. Loh, Chem. Mater.,
2009, 21, 2950–2956.
recover after the addition of different human IgG levels 12 Q. Mei, K. Zhang, G. Guan, B. Liu, S. Wang and Z. Zhang, Chem.
Commun., 2010, 46, 7319–7321.
13 K. Yang, S. Zhang, G. Zhang, X. Sun, S.-T. Lee and Z. Liu, Nano Lett.,
(Fig. S9, ESI†), further indicating the validity of our design.
The detection limit of 10 ng mLÀ1 was estimated based on
2010, 10, 3318–3323.
3d/slope criterion (d, standard deviation of the blank samples). 14 X. Sun, Z. Liu, K. Welsher, J. Robinson, A. Goodwin, S. Zaric and
H. Dai, Nano Res., 2008, 1, 203–212.
15 G. Eda, Y.-Y. Lin, C. Mattevi, H. Yamaguchi, H.-A. Chen, I. S. Chen,
It was comparable and even better than those of many reported
immunoassay methods for human IgG, such as LRET-based
C.-W. Chen and M. Chhowalla, Adv. Mater., 2010, 22, 505–509.
immunoassay,16 flow immunoassay,17 electrochemical immuno- 16 (a) M. Wang, W. Hou, C.-C. Mi, W.-X. Wang, Z.-R. Xu, H.-H. Teng,
assay,18 time-resolved fluoroimmunoassay19 and chemilumines-
C.-B. Mao and S.-K. Xu, Anal. Chem., 2009, 81, 8783–8789;
(b) J. Wang, T. Liu, Y. Cao, X. Hua, H. Wang, H. Zhang, X. Li and
Y. Zhao, Colloids Surf., A, 2007, 302, 168–173.
cence resonance energy transfer-based immunoassay,20 which
was promising for determination of human IgG in clinical applica- 17 D. Tang, R. Niessner and D. Knopp, Biosens. Bioelectron., 2009, 24,
2125–2130.
tion. To further investigate the utility of this sensing strategy for
real biological sample analysis, the matrix effect was examined. The
18 (a) H. Zarei, H. Ghourchian, K. Eskandari and M. Zeinali, Anal. Biochem.,
2012, 421, 446–453; (b) Z. Zhong, M. Li, D. Xiang, N. Dai, Y. Qing,
variations in PL intensity of mIgG–GQDs induced by human IgG in
PBS, human serum and cell culture fluid did not show significant
difference (Fig. S10, ESI†), indicating very little interferences of
complex matrices in the proposed strategy, which could ensure the
D. Wang and D. Tang, Biosens. Bioelectron., 2009, 24, 2246–2249.
19 C. Niu, J. Liu, P. Qin, G. Zeng, M. Ruan and H. He, Anal. Biochem.,
2011, 409, 244–248.
20 G. Qin, S. Zhao, Y. Huang, J. Jiang and F. Ye, Anal. Chem., 2012, 84,
2708–2712.
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