An electrochemiluminescence aptasensing platform based on ferrocene-graphene nanosheets for simple and rapid detection of thrombin

Sensors and Actuators B 208 (2015) 518–524

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An electrochemiluminescence aptasensing platform based on ferrocene-graphene nanosheets for simple and rapid detection of thrombin Bangrong Zhuo a,1 , Yuqin Li b,1 , Xiang Huang a , Yuejuan Lin c , Yaowen Chen c,∗ , Wenhua Gao a,c,∗ a

Department of Chemistry, Shantou University, Shantou, Guangdong 515063, PR China Department of Pharmacy, Taishan Medicine College, Taian, Shandong 271016, PR China c Analysis & Testing Center, Shantou University, Shantou, Guangdong 515063, PR China b

a r t i c l e

i n f o

Article history: Received 26 May 2014 Received in revised form 17 July 2014 Accepted 4 November 2014 Available online 20 November 2014 Keywords: Electrochemiluminescence Ru(bpy)3 2+ Thrombin Ferrocene-graphene nanosheets Conformational transformation

a b s t r a c t In this work, we tactfully constructed an electrochemiluminescence (ECL) aptasensing platform with ferrocene-graphene nanosheets (Fc-GNs) using the ferrocene (Fc) as quench unit to tris(2.2 -bipyridyl) ruthenium (II) [Ru(bpy)3 2+ ] and the Ru(bpy)3 2+ tagged thrombin binding aptamer (Ru-TBA) to recognize the thrombin molecules. Duing to the unique ␲-␲ interaction between nucleotides and graphene, the Ru-TBA could be preferentially adsorbed on the surface of ferrocene-graphene nanosheets with the signal generator into off-state. The conformational transformation of Ru-TBA leads to the desorption of Ru-TBA from Fc-GNs after the biosensing electrode incubating with the thrombin solution and the ECL “signal-on” was triggered. With the transformation of luminescence signal from “off” to “on”, the biosensor exhibited high sensitivity for the determination of thrombin with a detection limit of 0.21 nM. Particularly, the proposed method could be widely applied to the aptamer-based determination of other target analytes. © 2014 Published by Elsevier B.V.

1. Introduction Thrombin, a kind of serine protease, is well-known major target for anticoagulation and cardiovascular disease therapy for its pivotal role in both thrombosis and hemostasis [1]. The high picomolar range of thrombin in blood was known to be associated with diseases [2,3]. Therefore, its highly sensitive detection of thrombin in blood is of great interest and importance. Bock and his coworkers [4] found some sequence-specific single-stranded DNA oligonucleotides and termed “thrombin aptamers”. The 15-nucleotide consensus sequence d (GGTTGGTGTGGTTGG) oligonucleotide also known as thrombin-binding aptamer (TBA) and is widely used as sensing element for constructing various aptamer-based sensors (aptasensors) [5,6]. To date, substantial research achievements about the development of the aptasensors with different

∗ Corresponding author at: Department of Chemistry, Shantou University, Shantou, Guangdong 515063, PR China. Tel.: +86 754 8650 2774; fax: +86 754 8290 3941. E-mail addresses: [email protected] (Y. Chen), [email protected] (W. Gao). 1 Both the authors contributed equally to the paper. http://dx.doi.org/10.1016/j.snb.2014.11.064 0925-4005/© 2014 Published by Elsevier B.V.

detection techniques were demonstrated, such as fluorescent sensors, colorimetric sensors, quartz crystal microbalance sensors, electrochemical sensors and electrochemiluminescence (ECL) sensors [7–11]. Owing to the combination of advantages of both electrochemistry and chemiluminescence, such as high sensitivity and easy of control, ECL biosensors show great promising in the application of detection of proteins. Among many organic and inorganic ECL system, ECL based on inorganic compound tris(2.2 bipyridyl) ruthenium (II) [Ru(bpy)3 2+ ] has received considerable attention due to its importance to, for example, clinical test and biomolecule detection [12]. Graphene (GN), a recently discovered form of carbon that consists of only one plain layer of atoms arranged in a honeycomb lattice, exhibits a number of intriguing properties, such as unique electrical conductivity, high specific surface area, low manufacturing cost and easy functionalization [13–17]. In particular, graphene is capable of adsorbing oligonucleotides due to the strong unique ␲–␲ interactions between graphene and the nitrogenous bases of nucleotides [18,19]. Hence, these unique properties of graphene hold great promise in constructing a platform for high-performance electrochemical sensors or biosensors. Aptamer, as a new class of single-stranded DNA or RNA oligonucleotides, is obtained by

B. Zhuo et al. / Sensors and Actuators B 208 (2015) 518–524

the method called “systematic evolution of ligands by exponential enrichment (SELEX)” from random RNA or DNA libraries [20–22]. Aptamers have advantages over traditional recognition elements such as antibodies, including ease of synthesis, thermal stability and lack of immunogenicity [23]. On account of their relative ease of isolation and modification, impressive target analytes selectivity and affinity, and resistance against denaturation, aptamers have been used as ideal recognition elements for biosensing applications [24–28]. Because of the high diversity of molecular shapes of all possible nucleotide sequences, aptamers have been selected for a wide array of targets, including proteins, carbohydrates, lipids, or small molecules [29,30]. Herein, we developed an ECL biosensor with the determination of thrombin which takes advantage of the effective quenching pattern of ferrocene (Fc) to Ru(bpy)3 2+ via unique ␲–␲ interaction between nucleotides and ferroceme-graphene nanosheets (Fc-GNs). Fc-GNs not only shows an effective quenching to Ru(bpy)3 2+ , but also provides a platform for the immobilization of the Ru(bpy)3 2+ tagged thrombin binding aptamer (Ru-TBA) and simplifies the experimental design. As shown in Scheme 1, the glassy carbon electrode (GCE) modified by Fc-GNs formed a platform for the adsorption of the Ru-TBA; then the biosensing electrode was immersed into a certain concentration of thrombin solution for a period of time; after binding with the thrombin, the conformational transition of Ru-TBA leads to the desorption from the surface of Fc-GNs, coinciding with the transformation of luminescence signal from “off” to “on”. This strategy was demonstrated as a convenient, sensitive and selective “signal-on” detection platform for a spectrum of target analytes.

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2.2. Apparatus A traditional three-electrode system composed of a bare GCE (3 mm in diameter) or biosensor as working electrode, a platinum wire as counter electrode and a Ag/AgCl (1 M KCl) as reference electrode was applied in a 10 mL glassy analytical cell. Cyclic voltammetry (CV) curves and electrochemical impedance spectroscopy (EIS) were measured with IM6ex electrochemical workstation (Zahner IM6ex, Germany). ECL detections were carried out with a MPI-B ECL Analyzer Systems (Remax, China). A PHS-3CA precision pH meter (Dapu, China) was used in the experiment. Energy-dispersive spectroscopy (EDS) was carried out on a JSM-6360LA (JEOL, Japan). The UV/vis spectrum was recorded on a Lambda 950 spectrophotometer (Perkin Elmer, USA). Raman spectra were recorded on a Jobin Yvon LABRAM-HR confocal laser micro-Raman spectrometer (Jobin Yvon, France) at room temperature and an excitation wavelength of 514.5 nm. 2.3. Electrode cleaning GCEs with the glassy carbon (GC) rod diameter of 3 mm were polished with 0.3 ␮m and 0.05 ␮m alumina slurry (Al2 O3 ) on polishing cloth sequentially. The electrodes were fully rinsed after each polishing step and finally sonicated in deionized water and anhydrous ethanol for 5 min respectively, followed by electrochemical conditioning by potential scanning from −0.2 V to 1.6 V in 0.5 M H2 SO4 for at least five complete scans at 100 mV s−1 until the reproducible cyclic voltammogram was obtained. 2.4. Preparation of Fc-GNs and synthesis of ECL reporting probe

2. Experimental 2.1. Materials Oligonucleotide was purchased from Sangon Bioengineering Ltd. Company (Shanghai, China) and the sequence is shown as follows:ss-TBA: 5 -NH2 -(CH2 )6 -TAC ATG TGG TTG GTG TGG TTG G-3 Ruthenium (III) chloride hydrate [RuCl3 ·3H2 O], sodium hexafluorophosphate (NaPF6 ), 2,2 -bipyridine-4,4 -dicarboxylic acid, ethylenediamine (ED), N, N -dicyclohexyl-carbodiimide (DCC, 99%), sodium hydrogen carbonate (NaHCO3 ), N, N -dimethylformamide (DMF) and lithium chloride anhydrous (LiCl) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Tri-n-propylamine (TPrA), ferrocene-carboxaldehyde (FcCHO), Nhydroxysuccinimide (NHS) and 2,2 -dipyridyl were obtained from Aladdin Chemical Reagent Co. Ltd. (Shanghai, China). Sodium borohydride (NaBH4 ) was obtained from Beijing Chemical Factory (Beijing, China). Acetone and sulfuric acid were purchased from Guangdong Guanghua Chemical Factory Co. Ltd. (Shantou, China). Methanol and diethyl ether were obtained from Guangdong Xilong Chemical Co. Ltd. (Shantou, China). Ethanol absolute was purchased from Tianjin Damao Chemical Reagent Factory (Tianjin, China). Thrombin (1000 U), human serum albumin (HSA), bovine serum albumin (BSA), immunoglobulin G (IgG), insulin, trypsin, histidine and lysozyme were purchased from HeFei Bomei Biotechnology Co., Ltd. Fresh human whole blood was obtained from the local hospital. All other chemicals not mentioned here were of analytical reagent grade and were used as received. Millipore Milli-Q water (18 M cm) supplied by a Millipore Milli-Q water purification system (Bedford, MA, USA) was used throughout the process. The working solutions were prepared by diluting stock solution with phosphate buffer solution (PBS, pH 7.50, 0.10 M NaCl + 0.10 M NaH2 PO4 /Na2 HPO4 ).

Graphene oxide (GO) was prepared from graphite flake based on the modified Hummers method [31]. Fc-GNs were prepared in our previous experimental work [32] by taking the ED functionalized GO as the building block and reduced by NaBH4 in the FcCHO ethanol solution, and some characterizations have been completed to verify that ferrocene (Fc) was immobilized on the surface of graphene nanosheets through stable bonding, not simple physical adsorption. The Fc-GNs was also characterized by Raman spectra (Fig. S1) to verify that GO had been reduced to graphene already and Energy dispersive X-ray spectroscopy (EDS) (Fig. S2) so as to verify that Fc was indeed grafted on the graphene nanosheets. More details about the preparation and characterization of Fc-GNs can be found in the Supplementary data. Ruthenium bis(2,2 -bipyridine)(2,2 -bipyridine 4,4 -dicarboxylic acid)-N-hydroxy succinimide ester [Ru(bpy)2 (dcbpy)NHS] was synthesized according to our previous experimental work [33] with some modification and directly used to mark the ssTBA to fabricate the luminescence signal reporter probe (Ru(bpy)2 (dcbpy)NHS-TBA, abbreviated as RuTBA). The Ru-TBA was characterized by UV/vis spectroscopy (Fig. S3) to verify that Ru(bpy)2 (dcbpy)NHS tag has been attached to ss-TBA successfully and the concentration was estimated to be 6.02 × 10−4 M on the basis of UV absorbance at 257 nm. More details about the synthesis and characterization of Ru-TBA can be found in the Supplementary data. 2.5. Fabrication of the ECL biosensor Firstly, 10 ␮L 2.0 mg mL−1 Fc-GNs homogeneous solution was dropped on the clean GCE surface and then dried under ambient condition. Secondly, the modified electrode was immersed into the 1 ␮M Ru-TBA solution in order to be adsorbed on the surface of Fc-GNs. The adsorption process was kept for at least 3 h at 37 ◦ C, followed by being thoroughly washed in a stirred PBS solution for

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Scheme 1. Schematic illustration of the ECL biosensor for thrombin detection.

20 min to remove any weakly bound DNA strands, and then the ECL biosensor was obtained. 2.6. ECL measurement of thrombin Various ranges of concentration of thrombin were prepared for determining the sensitivity of this ECL biosensor by serial dilution of the thrombin stock solution. After a pre-scan to record the luminescence intensity of the “signal-off” biosensor, the biosensor was immersed into 100 ␮L PBS solution containing certain concentration of thrombin for 40 min at room temperature followed by washing for at least 5 min with 0.10 M PBS solution and deionized water to remove any unbound substances. The ECL determinations were performed at 37 ◦ C and CV mode with continuous potential scanning from 0.2 V to 1.25 V at a scanning rate of 100 mV s−1 was applied to achieve ECL signals in 0.10 M PBS containing 0.10 M TPrA. The supersaturated TPrA solution was prepared as previous protocol [34]. A negative high voltage of −800 V was supplied to the photomultiplier for luminescence intensity determination.

Fig. 1. Nyquist plot for electrochemical impedance measurements in 5.0 mM [Fe(CN)6 ]3−/4− solution for the bare GCE (black), the Fc-GNs modified GCE (red), the Fc-GNs/Ru-TBA modified GCE biosensor (blue) and GCE biosensor after binding with thrombin (green). Inset is the equivalent circuit used to fit the data.

3. Results and discussion 3.1. Characterization of the biosensing electrode impedance Electrochemical impedance spectroscopy (EIS) is a very attractive technique for effectively and directly probing the interfacial property changes at modified electrode, such as electron transfer resistance and capacitance. The Randles equivalent circuit was used to fit the data (inset of Fig. 1). Where Rs is electrolyte solution resistance, Rct represents the element of interfacial electron transfer resistance, constant phase element (CPE) related to double layer capacitance, Warburg impedance Zw , where Zw = Rw /(jw )1/2 and Rw is the diffusion resistance. Among these electrical parameters, we focused on the Rct value recorded after each modification step, since electron transfer process of [Fe(CN)6]3−/4− was strongly influenced by electrode modification. In electrochemical impedance spectra, the semicircle portion observed at high frequencies corresponds to the charge transfer limiting process. The charge transfer resistance Rct can be directly measured as the semicircle diameter. As shown in Fig. 1, the bare GCE gave an almost linear arc plot with a micro radian in high frequency region and behaved as an ideal conductor (curve black), indicating a very fast electron transfer process of [Fe(CN)6 ]3−/4− . The immobilization of Fc-GNs on the

surface of GCE affected the impedance feature of the electrode and showed a larger charge transfer resistance (Rct ) (curve red) than the bare GCE. Owing to the strong ␲–␲ interactions of Ru-TBA with Fc-GNs, the Ru-TBA could be preferentially adsorbed on the Fc-GNs and the insulating layer of Ru-TBA significantly hindered the electron transfer process of [Fe(CN)6 ]3−/4− , a distinct increase in electrochemical impedance can be observed (curve blue). After the biosensing electrode incubating in a certain concentration of thrombin for a period of time, the conformational transition of RuTBA induced by thrombin leads to the desorption of Ru-TBA from the surface of Fc-GNs. It is obvious that the desorption of Ru-TBA could produce an affirmative impedance performance and show a slight decrease in impedance (curve green). 3.2. Optimum conditions In order to obtain the optimal conditions, factors (e.g., reaction temperature, pH value and reaction time) affecting the sensing performance were studied systematically. To investigate the effect of pH on the ECL intensity, the test solutions at diverse pH values (5.5–9.0 in intervals of 0.5, PBS: 0.10 M NaCl + NaH2 PO4 /Na2 HPO4 ) were investigated. As shown in Fig. 2,

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Fig. 2. The effect of pH. ECL intensity was measured in PBS (0.10 M NaCl + NaH2 PO4 /Na2 HPO4 ) containing 0.10 M TPrA. Scan rate: 100 mV s−1 , scan range: 0.2–1.25 V. Temperature: 37 ◦ C. Thrombin at concentration of 20 nM. Error bars represent the standard deviation of five parallel experiments.

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Fig. 4. Incubation time of ECL biosensing electrode with thrombin at concentration of 20 nM. ECL intensity was measured in 0.10 M PBS (pH 7.50, 0.10 M NaCl + 0.10 M NaH2 PO4 /Na2 HPO4 ) containing 0.10 M TPrA. Scan rate: 100 mV s−1 , scan range: 0.2–1.25 V. Temperature: 37 ◦ C. Error bars represent the standard deviation of five parallel experiments.

3.3. Stability and reproducibility of the biosensor The ECL behavior of the biosensor was studied in details using TPrA as the model. The ECL mechanism using TPrA as co-reactant had been widely studied [35,36]. The ECL emission of the Ru(bpy)3 2+ -TPrA system resulted from the reaction between the deprotonated TPrA radical (TPrA• ) and electrogenerated Ru(bpy)3 3+ to form [Ru(bpy)3 2+ ]* , which then decays to produce emission (Eqs. (1)–(4)). TPrA• was formed via catalytic oxidation by Ru(bpy)3 3+ (Eq. (2-1)) and direct electrode oxidation (Eq. (2-2)). The ECL reaction mechanism could be described as follows [37,38]: Ru(bpy)3 2+ − e− → Ru(bpy)3 3+ Ru(bpy)3

3+

+ TPrA → Ru(bpy)3

−

TPrA − e → TPrA Fig. 3. The effect of temperature. ECL intensity was measured in PBS (pH 7.50, 0.10 M NaCl + 0.10 M NaH2 PO4 /Na2 HPO4 ) containing 0.10 M TPrA. Scan rate: 100 mV s−1 , scan range: 0.2–1.25 V. Thrombin at concentration of 20 nM. Error bars represent the standard deviation of five parallel experiments.

the ECL intensity increased with the increasing pH value from 5.5 to 7.5, and then decreased when pH was higher than 7.5. This was probably due to the fact that the thrombin and DNA exhibit the best activity under the neutral condition. Therefore, pH 7.5 was chosen as the optimal pH value. The reaction temperature in the experiment was very important to the activity of DNA, while the activity of DNA could affect the performance of the biosensing electrode. In our study, we have investigated the reaction temperature range from 30 ◦ C to 40 ◦ C. As shown in Fig. 3, the ECL intensity was enhanced as the increased reaction temperature and the highest ECL intensity was obtained at 37 ◦ C, the ECL intensity decreased rapidly after the temperature exceeds 37 ◦ C. This phenomenon can be explained by the fact that the normal temperature of the human body is 37 ◦ C. Thus, 37 ◦ C was selected as the optimal reaction temperature for further experiments. The effect of the incubation time on the performance of the biosensing electrode at thrombin concentration of 20 nM is shown in Fig. 4. As the incubation time increased, the ECL intensity increased rapidly and reached a plateau after 40 min Therefore, 40 min was chosen for further experiments.

+•

+ TPrA•

Ru(bpy)3

3+

Ru(bpy)3

2+∗

→

TPrA•

(1) 2+

+H

→ Ru(bpy)3

→ Ru(bpy)3

2+

+ h␯

+ TPrA•

(2-1)

+

(2-2)

2+∗

+ Productions

(3) (4)

Fc-GNs could strongly adsorb the Ru-TBA due to strong ␲–␲ interactions, resulting in effective quenching of Fc to the ECL intensity of the Ru(bpy)3 2+ . To explore the stability of the quenching efficiency of Fc to Ru(bpy)3 2+ , the ECL method was used to record the luminescence intensity of the biosensing electrode in the 0.10 M PBS containing 0.10 M TPrA using a linear potential scan technique. The corresponding ECL intensity-potential curves of the biosensing electrode are presented in Fig. 5. As Fig. 5 shows, there is no obvious ECL signal response can be found after the Fc-GNs/Ru-TBA modified GCE (curve a), indicated that Fc-GNs strongly adsorbed the Ru-TBA and the Fc showed an effective quenching to Ru(bpy)3 2+ . The biosensor could give an obvious enhanced ECL signal response (curve b) after incubating in a certain concentration of thrombin, it can be explained as that the thrombin induced the conformational transition of Ru-TBA, causing the desorption of Ru-TBA from the surface of Fc-GNs, and then “signal-on” status was achieved. In addition, as shown in the inset of Fig. 5, continuous CV scanning of the electrodes can give a balanced ECL intensity, indicating that the biosensor has acceptable reliability and stability. The reproducibility of the biosensor was evaluated by analysis of the same concentration of thrombin (20 nM) using five biosensors under the same conditions. All biosensors exhibited close ECL intensity, and a relative standard deviation (RSD) of 4.67% was obtained, which

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Table 1 Performance comparison of different sensors. Detection technology

Linear range

Detection limit

Reference

Fluorescence Electrochemical impedance spectroscopy Differential pulse voltammetry Differential pulse voltammetry Differential pulse voltammetry Electrochemiluminescence

0.5–20 nM 0.12–30 nM 1–60 nM 6–60 nM 5–35 nM 0.5–25 nM

0.18 nM 0.03 nM 0.5 nM 3 nM 0.5 nM 0.21 nM

[39] [40] [41] [42] [43] This work

Fig. 5. ECL intensity vs. potential curves for the Fc-GNs/Ru-TBA modified GCE (a) and the Fc-GNs/Ru-TBA modified GCE biosensor after binding with thrombin (b). Inset: ECL intensity vs. time curves for the biosensor under continuous CV for six cycles. ECL curves were measured in 0.10 M PBS (pH 7.50, 0.10 M NaCl + 0.10 M NaH2 PO4 /Na2 HPO4 ) containing 0.10 M TPrA. Scan rate: 100 mV s−1 , scan range: 0.2–1.25 V. Temperature: 37 ◦ C. Thrombin at concentration of 20 nM.

Fig. 6. ECL intensity vs. potential curves for the biosensor binding with various concentrations of thrombin. The concentrations of thrombin were 0.5 nM (a), 1 nM (b), 2 nM (c), 5 nM (d) 10 nM (e), 20 nM (f) and 25 nM (g). Inset: the calibration curve of the ECL response as a function of the concentration of thrombin. ECL curves were measured in 0.10 M PBS (pH 7.50, 0.10 M NaCl + 0.10 M NaH2 PO4 /Na2 HPO4 ) containing 0.10 M TPrA. Scan rate: 100 mV s−1 , scan range: 0.2–1.25 V. Temperature: 37 ◦ C. Error bars represent the standard deviation of five parallel experiments.

indicated that the reproducibility of the proposed biosensor was acceptable. 3.4. Sensitivity and selectivity of the biosensor Under the optimized test conditions, the sensitivity of ECL biosensors was assessed by measuring the dependence of increased IECL upon the concentration of thrombin. As shown in Fig. 6, the

Fig. 7. Selectivity of the ECL biosensor to thrombin with HSA, BSA, IgG, insulin, trypsin, histidine and lysozyme as competitive species. Concentration of thrombin is 10 nM and the others are 250 nM. Error bars represent the standard deviation of five parallel experiments.

ECL intensity was enhanced when a higher concentration of thrombin was used for binding with the Ru-TBA, and the inset of Fig. 6 showed that the IECL was found to be related to the concentration of thrombin in a range from 0.5 nM to 25 nM. The regression equation was IECL = 61.7 Cthrombin + 307.2 with a regression coefficient of 0.9939 and a detection limit of 0.21 nM, which is defined as the concentration corresponding to the mean blank value plus 3 standard deviations. The performance of different thrombin sensors [39–43] is shown in Table 1, which demonstrates that our proposed biosensor has good sensitivity for thrombin. The selectivity of the biosensor was examined by incubating the biosensor in the aqueous solutions containing thrombin at the concentration of 10 nM, while HSA, BSA, IgG, insulin, trypsin, histidine and lysozyme were employed undergo the same test condition at the concentration of 250 nM. As shown in Fig. 7, the biosensor showed significant ECL response to thrombin, but hardly exhibited substantial responses to these competitive species, suggesting that the biosensor possessed an excellent selective response to thrombin, which was attributed to the high selectivity of aptamer to its target. 3.5. Detection of thrombin in human plasma samples In order to investigate the possible application of this biosensor in clinical analysis, the biosensing electrode was also tested in healthy human blood serum samples under optimal experimental conditions. The blood samples were diluted 100 times by 0.10 M PBS solution. We performed an experiment with human blood serum samples, and the determination of thrombin concentration was performed by the standard addition method. As shown in Table 2, the results showed good recovery values (93.5–103.6%) and the relative standard deviation is between 1.12% and 2.36%. The results indicated that our proposed biosensor has a great potential application in real biological samples.

B. Zhuo et al. / Sensors and Actuators B 208 (2015) 518–524 Table 2 The recoveries of thrombin from human plasma samples. Samples

Added (nM)

Biosensing method (nM)

RSD (%)

Recovery (%)

1 2 3 4

0.50 2.00 5.00 10.00

0.51 1.87 4.93 10.36

1.69 2.36 1.12 2.04

102.0 93.5 98.6 103.6

4. Conclusions In conclusion, an ECL aptasensing platform based on ferrocenegraphene nanosheets for simple and rapid determination of proteins which taking advantage of the effective quenching pattern of Fc to Ru(bpy)3 2+ via unique ␲–␲ interaction between nucleotides and Fc-GNs was demonstrated. By virtue of easy functionalization, high ␲-conjunction and specific surface area, Fc-GNs not only provides a platform for the immobilization of Ru-TBA, but also shows an effective quenching to Ru(bpy)3 2+ . Therefore, it is possible for us to detect thrombin by monitoring the transformation of ECL signal before and after the Ru-TBA binding of target analytes. In our strategy, the biosensor presented a detection range from 0.5 nM to 25 nM with a linear coefficiency of 0.9939, and the detection limit was 0.21 nM. Furthermore, the proposed biosensor may have great potential applications in the determination of a spectrum of targets with different types of aptamers to recognize their respective target molecules. Acknowledgements We acknowledge financial support of this work by Research Start-up Funding of Shantou University (No. NTF10002), the Natural Science Foundation of Guangdong Province (No. S2011010005208) and the Guangdong High Education Fund of Science and Technology Innovation (No. 2013KJCX0078). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2014.11.064. References [1] C.A. Holland, A.T. Henry, H.C. Whinna, F.C. Church, Effect of oligodeoxynucleotide thrombin aptamer on thrombin inhibition by heparin cofactor II and antithrombin, FEBS Lett. 484 (2000) 87–91. [2] T. Arai, J. Miklossy, A. Klegeris, J.P. Guo, P.L. McGeer, Thrombin and prothrombin are expressed by neurons and glial cells and accumulate in neurofibrillary tangles in alzheimer disease brain, J. Neuropathol. Exp. Neurol. 65 (2006) 19–25. [3] P.W. Serruys, P. Vranckx, K. Allikmets, Clinical development of bivalirudin (Angiox® ): rationale for thrombin-specific anticoagulation in percutaneous coronary intervention and acute coronary syndromes, Int. J. Clin. Pract. 60 (2006) 344–350. [4] L.C. Bock, L.C. Griffin, J.A. Latham, E.H. Vermaas, J.J. Toole, Selection of singlestranded DNA molecules that bind and inhibit human thrombin, Nature 355 (1992) 564–566. [5] V. Pavlov, Y. Xiao, B. Shlyahovsky, I. Willner, Aptamer-functionalized Au nanoparticles for the amplified optical detection of thrombin, J. Am. Chem. Soc. 126 (2004) 11768–11769. [6] A.E. Radi, J.L.A. Sanchez, E. Baldrich, C.K. O’Sullivan, Reagentless, reusable, ultrasensitive electrochemical molecular beacon aptasensor, J. Am. Chem. Soc. 128 (2006) 117–124. [7] Y.J. Guo, Y.J. Han, Y.X. Guo, C. Dong, Graphene-orange II composite nanosheets with electroactive functions as label-free aptasensing platform for signal-on detection of protein, Biosens. Bioelectron. 45 (2013) 95–101. [8] L. Yang, C.W. Fung, J.C. Eun, A.D. Ellington, Real-time rolling circle amplification for protein detection, Anal. Chem. 79 (2007) 3320–3329. [9] M. Mir, M. Vreeke, I. Katakis, Different strategies to develop an electrochemical thrombin aptasensor, Electrochem. Commun. 8 (2006) 505–511. [10] A. Bini, M. Minunni, S. Tombelli, S. Centi, M. Mascini, Analytical performances of aptamer-based sensing for thrombin detection, Anal. Chem. 79 (2007) 3016–3019.

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Biographies Bangrong Zhuo is studying for MS degree at Shantou University. His research focuses on the applications of ECL biosensor.

Yuqin Li received her PhD in Chemistry from Lanzhou University in 2006. Her current research interests are the analysis of drugs. Xiang Huang is studying for MS degree at Shantou University. His research focuses on the ECL detectors and applications. Yuejuan Lin received her MS degree in Electronics and Communication Engineering from Huazhong University of Science and Technology in 2007. Her current research interests are the analysis and applications of nanomaterials. Yaowen Chen received his PhD in Medicine from Shantou University in 2006. His current research interests are biology detections. Wenhua Gao received his PhD in Chemistry from Lanzhou University in 2006. His current research interests are the applications of function materials in analytical area.