Dual-responses for electrochemical and electrochemiluminescent detection based on a bifunctional probe

Article abstract of DOI:10.1039/c3cc49319a

A bifunctional probe (PTC-Tb) which acts as not only a well-defined and stable electrochemical redox molecule but also as a highly efficient co-reactant of an electrochemiluminescent oxygen-peroxydisulfate system was firstly synthesized and applied to the construction of dual-response aptasensors for thrombin detection. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c3cc49319a

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Dual-responses for electrochemical and
electrochemiluminescent detection based
on a bifunctional probe†
Cite this: Chem. Commun., 2014,
50, 3367
Received 8th December 2013,
Accepted 7th February 2014
Jing Han, Ying Zhuo,* Yaqin Chai and Ruo Yuan*
DOI: 10.1039/c3cc49319a
www.rsc.org/chemcomm
A bifunctional probe (PTC-Tb) which acts as not only a well-defined and Next, Jie and colleagues15 also designed two different biosensors but
stable electrochemical redox molecule but also as a highly efficient one bi-functional probe (quantum dot nanocluster) on the different
co-reactant of an electrochemiluminescent oxygen–peroxydisulfate sensing platform to obtain dual-responses for analysis of the targets
system was firstly synthesized and applied to the construction of (DNA and cancer cells). Furthermore, owing to the dual EC and ECL-
dual-response aptasensors for thrombin detection.
properties of Ru(bpy)32+ itself, Dennany and coworkers16 developed a
sensor using [Ru(bpy)2(PVP)10]2+ assembled layer-by-layer with DNA
Aptamer, an artificial oligonucleic acid, can bind with high affinity as a sensing platform to achieve EC and ECL detection. However,
2+
and specificity to a wide range of targets,1 such as proteins,2 small because of the inherent water-solubility of Ru(bpy)3 and its
molecules,3 cells,4 viruses,5 etc. Recently, various aptamer-based derivatives, it is difficult to overcome the leakage of the Ru complex
biosensors have been developed based on different analytical from the modified electrode, which limited the development of the
techniques, such as fluorescence,6 colorimetric analysis,7 ultraviolet Ru complex based solid-state ECL biosensors. To date, with the
absorbance,8 electrochemical (EC)9 and electrochemiluminescent exception of the Ru(bpy)3 system, there has been no report
2+
(ECL)10 methods. Among them, the EC technique owing to its focusing on one biosensor for dual-response detection based on
distinct advantages of simplicity, fast response as well as relatively other systems. The reason may be that it is difficult to search for a
cheap cost, and the ECL technique owing to its intrinsic advantages bifunctional probe with dual-responses for EC and ECL signals.
of high sensitivity, excellent selectivity as well as low background
Perylene-3,4,9,10-tetracarboxylic acid (PTCA), an archetypal
have been widely used in aptasensors.11,12 More inspiringly, the p-stacking organic perylene dye, has been widely acknowledged
dual EC and ECL detection strategy can provide an easier and more as one of the most promising and rapidly emerging research
effective way as well as a wider dynamic concentration response areas for advanced materials owing to its large specific surface
range for the analysis of targets than the single EC or ECL detection area and low manufacturing cost as well as desirable organic
alone. The development of dual-response mode could lead to more electronic and optical properties.17–20 Interestingly, PTCA could
2À
rapid clinical decision making and corresponding reduction in considerably enhance the ECL signal of peroxydisulfate (S2O8
patient stress and healthcare costs.13
)
emission, which has been demonstrated in our previous work.21
Nevertheless, it is hard to achieve dual-responses for EC and With the goal of obtaining a bifunctional EC and ECL probe,
ECL detection on the basis of one aptasensor. Usually, the EC or the toluidine blue (Tb), which is a kind of dye molecule exhibiting
ECL aptasensor is indispensable to label its signal probe, so it is reversible redox-activity, was conjugated to PTCA (the product was
difficult to search for a bifunctional probe with the dual EC and ECL abbreviated as PTC-Tb). Herein, the dual-response PTC-Tb probe
responses. Up to now, dual-response modes have been mainly was firstly synthesized and applied to biosensor construction.
developed based on the heterogeneous biosensors. For example, We find that PTC-Tb exhibits good membrane-forming proper-
Zhang’s group14 constructed two distinct biosensors utilizing various ties for the electrode surface modification. More importantly,
probes (magnetic bead-Au-CdS and magnetic bead-Ru biocomplex) this bifunctional PTC-Tb probe acts as not only a stable redox
to achieve dual-responses for the determination of Ramos cells. molecule with a pair of well-defined EC redox peaks but also as
a highly efficient co-reactant of the O2–S2O82À system for greatly
Education Ministry Key Laboratory on Luminescence and Real-Time Analysis,
enhancing ECL emission.
In view of the advantageous features of the bifunctional
PTC-Tb probe, the dual-response EC and ECL aptasensors were
College of Chemistry and Chemical Engineering, Southwest University,
Chongqing 400715, PR China. E-mail: yingzhuo@swu.edu.cn, yuanruo@swu.edu.cn;
Fax: +86-23-68253172; Tel: +86-23-68252277
designed to detect thrombin based on one sensing platform
which was constructed by a covalently immobilized thrombin
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc49319a
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 3367--3369 | 3367

View Article Online
Communication
ChemComm
Scheme 1 Schematic illustration of the aptasensor preparation process and dual-responses for EC and ECL detection.
aptamer on the hexanethiol/nano-Au/PTC-Tb modified gold
electrode. After incubation with the target, the dual-response
EC and ECL signals were obtained and decreased with increas-
ing concentrations of thrombin.
Scheme 1 shows the schematic diagram for the fabrication
of aptasensors and dual-response detection of thrombin. First,
5 mL PTC-Tb was coated on a pretreated gold electrode (AuE) and
dried in air. Then the PTC-Tb modified electrode was immersed
into 2 mL colloidal nano-Au for 4 h to obtain the nano-Au/PTC-Tb
modified electrode via electrostatic interaction. Subsequently, the
prepared nano-Au/PTC-Tb/AuE was incubated with 15 mL of 2 mM
TBA for 16 h at room temperature. Following this, the resulting
electrode was blocked with 15 mL of 1.0 mM hexanethiol (HT) for
45 min to avoid the nonspecific adsorption. After every step, the
modified electrode was extensively rinsed with doubly distilled
water to remove nonspecific adsorption and dried under a stream
of nitrogen prior to EC and ECL characterization. The obtained
aptasensor was stored at 4 1C when not in use.
Fig. 1 (A) CV responses of different electrodes in 0.1 M HAc-NaAc buffer
(pH 5.5) at a scan rate of 50 mV sÀ1 and (B) ECL responses of different
electrodes in 0.1 M HAc-NaAc buffer (pH 5.5) containing 5 mM K2S2O8
(the voltage of the photomultiplier tube was set at 800 V) at a scan rate
of 100 mV sÀ1: (a) bare AuE; (b) PTC-Tb/AuE; (c) nano-Au/PTC-Tb/AuE;
(d) TBA/nano-Au/PTC-Tb/AuE; (e) HT/TBA/nano-Au/PTC-Tb/AuE; (f) after
incubation with 5 nM thrombin.
Furthermore, in order to characterize the fabrication process of
The fabrication process for the dual-response aptasensor was the ECL aptasensor, ECL signals at each immobilization step were
2À
monitored by EC and ECL characterization. As shown in Fig. 1A, also recorded. In Fig. 1B, the bare AuE in the O2–S2O8 system
CVs of different modified electrodes for EC characterization were produced a weak ECL response (Fig. 1B, curve a). Upon modifying
recorded for HAc-NaAc (pH 5.5) at a scan rate of 50 mV sÀ1. No PTC-Tb on AuE, a remarkable ECL increase is observed (Fig. 1B,
obvious redox peaks are observed at bare AuE (Fig. 1A, curve a). curve b), demonstrating that PTC-Tb is a highly efficient co-reactant
2À
Nevertheless, when PTC-Tb was modified onto the AuE, a pair of well- of the ECL O2–S2O8 system. The ECL response is increased after
defined peaks could be obtained (Fig. 1A, curve b), which were adsorbing nano-Au on the PTC-Tb film via electrostatic interaction
attributed to the excellent electrochemical activity of PTC-Tb. After (Fig. 1B, curve c), because nano-Au accelerated the electron transfer
adsorbing nano-Au on the PTC-Tb membrane, the redox peak current in ECL reaction. When TBA was immobilized onto the nano-Au/
is further improved (Fig. 1A, curve c), which indicates that the nano- PTC-Tb/AuE, the ECL signal decreased apparently (Fig. 1B, curve d)
Au is similar to a conducting wire, making the electron transfer easier. for which TBA hinders the diffusion of luminescent reagents
The peak current decreased obviously, however, when TBA was toward the electrode surface. The decreased ECL response is also
assembled on the electrode surface (Fig. 1A, curve d). The reason observed after immersing HT to block the nonspecific adsorption
for this is that the aptamer assembled on the electrode can block the on the electrode surface (Fig. 1B, curve e). Finally, the ECL intensity
electron transfer of the redox probe. Subsequently, non-electroactive further decreased (Fig. 1B, curve f) after incubation with thrombin,
HT was employed to block nonspecific sites on the electrode surface due to forming the complementary pairing blocking layer. As a
and the peak current decreased further (Fig. 1A, curve e). Finally, after result, we can make a conclusion that the thrombin has been
incubation with thrombin, the CV response is decreased again owing successfully immobilized on the base electrode.
to the fact that the complementary pairing of thrombin and TBA
retards the electron transfer tunnel (Fig. 1A, curve f).
The performance of the proposed aptasensor was monitored
by incubating with 15 mL thrombin standard solutions based on
3368 | Chem. Commun., 2014, 50, 3367--3369
This journal is ©The Royal Society of Chemistry 2014

View Article Online
ChemComm
Communication
respectively, indicating that our proposed aptasensor is of
acceptable stability.
In summary, a simple dual-response aptasensor has been
designed for thrombin detection by employing a bifunctional
PTC-Tb probe to construct one sensing platform. Interestingly,
the PTC-Tb probe is easily prepared and acts as not only a well-
defined and stable EC redox molecule but also as a highly efficient
2À
co-reactant of the ECL O2–S2O8 system. More importantly, the
present assays can provide easier and more effective way as well as
a wider dynamic concentration response range for the accurate
detection of proteins. We believe that this dual-response biosensor
may open a new exciting avenue for target detection.
Fig. 2 (A) DPV and (B) ECL responses of the proposed aptasensor after
incubation with different concentrations of thrombin (insets: the corre-
sponding calibration curves).
This research was supported by the NNSF of China (21275119,
21105081, 21075100), the Research Fund for the Doctoral Program
of Higher Education (RFDP) (20110182120010), Ministry of Educa-
tion of China (Project 708073), the Specialized Research Fund for the
Doctoral Program of Higher Education (20100182110015) and the
Natural Science Foundation Project of Chongqing City (CSTC-
2010BB4121, CSTC-2009BA1003), the Fundamental Research Funds
for the Central Universities (XDJK2010C062, XDJK2012A004), China.
Notes and references
1 S. P. Song, L. H. Wang, J. Li, J. L. Zhao and C. H. Fan, TrAC, Trends
Anal. Chem., 2008, 27, 108.
Fig. 3 (A) EC and (B) ECL stabilities of aptasensor for 0 week, 1 week and
2 weeks, respectively (error bars: SD, n = 3).
2 J. Li, H. E. Fu, L. J. Wu, A. X. Zheng, G. N. Chen and H. H. Yang, Anal.
Chem., 2012, 84, 5309.
3 L. Wu, X. H. Zhang, W. Liu, E. H. Xiong and J. H. Chen, Anal. Chem.,
2013, 85, 8397.
4 H. Y. Liu, S. M. Xu, Z. M. He, A. P. Deng and J. J. Zhu, Anal. Chem.,
2013, 85, 3385.
5 M. Labib, A. S. Zamay, D. Muharemagic, A. V. Chechik, J. C. Bell and
M. V. Berezovski, Anal. Chem., 2012, 84, 1813.
6 L. Zhang, J. P. Lei, L. Liu, C. F. Li and H. X. Ju, Anal. Chem., 2013,
85, 11077.
7 Y. Huang, J. Chen, S. L. Zhao, M. Shi, Z. F. Chen and H. Liang, Anal.
Chem., 2013, 85, 4423.
8 D. Li, B. Shlyahovsky, J. Elbaz and I. Willner, J. Am. Chem. Soc., 2007,
129, 5804.
9 J. J. Zhao, C. F. Chen, L. L. Zhang, J. H. Jiang and R. Q. Yu, Biosens.
Bioelectron., 2012, 36, 129.
the developed protocol under the optimal conditions. The DPV
current (Fig. 2A) and ECL intensity (Fig. 2B) of the aptasensor
decreased with increasing concentration of thrombin. The
calibration plot shows a good linear relationship between DPV
responses and the logarithmic value of thrombin concentrations
ranging from 0.5 nM to 50 nM with a correlation coefficient of
0.991 (Fig. 2A, inset). The regression equation is I (mA) = À6.57 log c
(nM) À 17.91 with a detection limit of 0.2 nM (defined as 3s, in
which s is the relative standard deviation of a blank solution, n =
15). Furthermore, the calibration plot exhibits a good linear
relationship between the ECL peak intensities and the logarithmic
value of thrombin concentrations ranging from 0.005 nM to 10 nM 10 G. M. Nie, Z. M. Bai, W. Y. Yu and J. Chen, Biomacromolecules, 2013,
14, 834.
with a correlation coefficient of 0.997 (Fig. 2B, inset). The regression
11 D. W. Zhang, J. Nie, F. T. Zhang, L. Xu, Y. L. Zhou and X. X. Zhang,
equation is I (mA) = À1742.46 log c (nM) + 3254.97 (where I is the
Anal. Chem., 2013, 85, 9378.
ECL intensity and c stands for the concentration of thrombin) with 12 C. Y. Tian, J. J. Xu and H. Y. Chen, Chem. Commun., 2012, 48, 8234.
13 B. V. Chikkaveeraiah, A. A. Bhirde, N. Y. Morgan, H. S. Eden and
a detection limit of 1.7 pM (3s). With the mutual calibration of EC
and ECL curves, a wide dynamic concentration response range of
X. Y. Chen, ACS Nano, 2012, 6, 6546.
14 C. F. Ding, Y. Ge and S. S. Zhang, Chem.–Eur. J., 2010, 16, 10707.
0.005–50 nM is obtained. This dual-response aptasensor provides 15 G. F. Jie, J. Zhang, G. X. Jie and L. Wang, Biosens. Bioelectron., 2014,
52, 69.
an easier and more effective way as well as a wider dynamic
concentration response range to accurately detect thrombin.
16 L. Dennany, R. J. Forster and J. F. Rusling, J. Am. Chem. Soc., 2003,
125, 5213.
When the aptasensor was not in use, it was stored at 4 1C to 17 M. Eremtchenko, J. A. Schaefer and F. S. Tautz, Nature, 2003,
425, 602.
keep the temperature constant. The stability of the proposed
aptasensor was measured by a long-term storage assay after
18 S. R. Forrest, Chem. Rev., 1997, 97, 1793.
19 J. E. Weaver, M. R. Dasari, A. Datar, S. Talapatra and P. Kohli, ACS
incubation with 1 nM thrombin, as shown in Fig. 3. It is found
that about 96.7% and 93.8% of the original EC and ECL
responses were retained after 1 week, and about 89.7% and
87.2% of the initial EC and ECL signals were noticed after 2 weeks,
Nano, 2010, 4, 6883.
20 B. H. Wu, D. Hu, Y. J. Kuang, Y. M. Yu, X. H. Zhang and J. H. Chen,
Chem. Commun., 2011, 47, 5253.
21 X. X. Gan, R. Yuan, Y. Q. Chai, Y. L. Yuan, Y. L. Cao, Y. H. Liao and
H. J. Liu, Anal. Chim. Acta, 2012, 726, 67.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 3367--3369 | 3369