Dual-signal amplification strategy for electrochemiluminescence sandwich biosensor for detection of thrombin

Accepted Manuscript Title: Dual-signal amplification strategy for electrochemiluminescence sandwich biosensor for detection of thrombin Author: Yingjie Li Yuqin Li Ning Xu Jiahong Pan Tufeng Chen Yaowen Chen Wenhua Gao PII: DOI: Reference:

S0925-4005(16)31460-5 http://dx.doi.org/doi:10.1016/j.snb.2016.09.043 SNB 20908

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

29-4-2016 16-8-2016 8-9-2016

Please cite this article as: Yingjie Li, Yuqin Li, Ning Xu, Jiahong Pan, Tufeng Chen, Yaowen Chen, Wenhua Gao, Dual-signal amplification strategy for electrochemiluminescence sandwich biosensor for detection of thrombin, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.09.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights 1. Combing the GNPs-graphene withRu-TBA2-AuNPs to amplify the ECL biosensor response. 2. Have a low LOD 6.3 pM 3. Have a good selectivity to different interferences.

Graphical Abstract

GO

GCE

Dual-signal amplification strategy for electrochemiluminescence sandwich biosensor for detection of thrombin

Yingjie Li,a,1 Yuqin Li,b,1Ning Xuc, Jiahong Pana, Tufeng Chena, Yaowen Chena, Wenhua Gao*a

a

Department of Chemistry and Laboratory for Preparation and Application of Ordered

Structural Materials of Guangdong Province, Shantou University, Shantou, Guangdong 515063, P. R. China. E-mail: [email protected]; Fax: +86-22-82903941; Tel: +86-22-86502774 b

Department of Pharmacy, Taishan Medicine College, Taian, Shandong 271016, P. R.

China. c

National Detergent and Cosmetics Products Quality Supervision and Inspection

Center (Guangdong), Shantou, Guangdong 515041, P. R. China.

∗ 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] (W. Gao). 1

Both the authors contributed equally to the paper.

Abstract In the present study, the authors report a novel sandwich biosensor for detection of thrombin using electrochemiluminescence method based on dual-signal amplification strategy. Firstly, the gold nanoparticles graphene composite (GNPs-graphene) was obtained by simultaneously depositing the chloroauric (III) acid tetrahydrate [HAuCl4·4H2O] and graphene solution with a cyclic voltammetry. Secondly, one thiol-modified thrombin aptamer (TBA1) was immobilized the glassy carbon electrode (GCE) via Au-S bonding. And then the other thiol-modified biotinylated thrombin aptamer (TBA2) that were modified with Ru(bpy)32+ were anchored on the surface of the gold nanoparticles in solution. This proposed assay took dual signal amplification strategies. On the one hand, a single gold nanoparticle can connect many TBA2 to come into being a signal cluster (Ru-TBA2-AuNPs), thus when a little thrombin were present the system the ECL intensity will have a strong signal response; On the other hand, the GNPs-graphene can promote the electron transfer efficiency and increase the signal response. The ECL signal had a good linear relationship with thrombin concentration in range of 0.01-10 nM, and the detection limit for thrombin was determined to be as low as 6.3 pM. This biosensor also showed good selectivity for thrombin without being affected by some other proteins, such as BSA and lysozyme and so on. Moreover, this proposed method was successfully applied to thrombin analysis in diluted human serum samples.

Keywords: ECL; biosensor; Ru(bpy)32+; thrombin; aptamer

1. Introduction Thrombin, a kind of serine protease, is the fibrous matrix of blood clots and promote fibrinogen convert to fibrin and accelerate blood agglomeration [1-2]. Human thrombin is used to help control bleeding during surgery [3-6]. It is necessary to detect thrombin in blood not only for patients suffering for diseases associated with coagulation abnormalities, but also for determining the effectiveness of the therapeutic drugs after surgery or in thromboembolic disease treatments. Accordingly, sensitively and quantitatively assaying thrombin is critical and useful in disease prevention. Aptamers can bind to different small molecules, nucleic acids, and proteins and bring into new secondly and tertiary strcutrue, which are selected with a combinatorial method called systematic evolution of ligands by exponential enrichment (SELEX) [7-9]. Using the specific feature, many biosensors have been fabricatedincluding electrochemiluminescent (ECL) [10-12], electrochemical [13-14], fluorescent [15], and colorimetric [16]. To date, two thrombin aptamers have been widely used, one (TBA1) binding to the fibrinogen-recognition exosite of thrombin with a Kd around 100 nM [17], and the other one (TBA2) bound to the thrombin with higher affinity (Kd= 0.5 nM) [18]. While, most biosensor for thrombin focused on one kind of apatamer [10-11]. In this work, we take two apatamers into the biosensor. Many

works

have

been

done

on

assaying

thrombin

including

fluorescence,differential pulse voltammetry, surface-enhanced Raman spectroscopy etc.Compared with other approaches, electrochemiluminescence (ECL) owns many advantages such as low back-ground signal, being easily controlled and low detection limit [19-21]. Combination of these advantages and specific property of aptamers allows ECL to become an important and promising method in aptamer biosensors. Specially, due to their excellent stability and high luminescence efficiency, the ECL biosensors based on Ru(bpy)32+/TPrA system have been mostly studied. Particularly, the ECL biosensors based on the enhancement of Ru(bpy)32+/TPrA ECL system have been extensively investigated with

improved performance [22-24].The use of

nanomaterial as amplifiers has attracted special interesting in ECL biosensor designing such as carbon nanotubes, graphene, gold nanoparticle, nanocomposites etc. [25-27].Recently, graphene and graphene based hybrid nanoassemblies have gained considerable attention in electronic devices such as light-emitting, solar cells, which demands materials with low sheet resistivity and high charge conductive. Especially, graphene is a one-atom-thick layer of graphite with two linear bands crossing at the Dirac point. Moreover, graphene-metal nanoparticles nanocompostie with excellent physical and chemical properties, large surface area and controllable electronic properties has become a promising electrode material in constructing ECL biosensor [28]

.

In this work, the authors report a novel sandwich biosensor for detection of thrombin using electrochemiluminescence method based on two different thrombin aptamers (TBA1-thrombin-TBA2). As shown in scheme 1, Firstly, the gold nanoparticles graphene composite (GNPs-graphene) was obtained by simultaneously depositing the terachloroanric (III) acid tetrahydrate [HAuCl4·4H2O] and graphene solution with a cyclic voltammetry. Secondly, one thiol-modified thrombin aptamer (TBA1) was immobilized the glassy carbon electrode (GCE) via Au-S bonding. And then the other thoil-modified biotinylated thrombin aptamer (TBA2) that were modified with Ru(bpy)32+ were anchored on the surface of the gold nanoparticles in solution. A single gold nanoparticle can connect many TBA2 to come into being a signal cluster (Ru-TBA2-AuNPs), thus when a little thrombin were present the system the ECL intensity will have a strong signal response. In this system, on the one hand, the GNPs-graphene can promote electron transfer and amplify the ECL signal; On the other hand, the synthesized big ECL signal beacon (Ru-TBA2-AuNPs) can improve the ECL response. Under the best condition, the ECL signal had a good linear relationship with thrombin concentration in range of 0.01-10 nM, and the detection limit for thrombin was determined to be as low as 6.3 pM.The proposed sandwich biosensor platform not only exhibits its unique superiorities with respect to sensitivity, specificity, low background interference, time-efficiency and good repeatability, but also suggests its potential in drug screening and large-scale clinical examination.

2. Experimental 2.1 Reagents The DNA oligonucleotide sequences for this experiment are shown below: TBA1: 5’-SH-(CH2)6-TTTTTGGTTGGTGTGGTTGG-3’ TBA2: 5’-SH-(CH2)6-TTTTTTTTTTTTTTTAGTCCGTAGGGCAGGTGGGGGG TGACTT-(CH2)6-NH2-3’ All oligonucleotides were purchased from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai,

China).Cis-Bis-(2,2'-bipyridine)dichlororuthenium(II)

dehydrate

(cis-Ru(bpy)2Cl2 ˑ2H2O) were bought from Precious Metal Research Institution (Yunnan, China). 2-mercaptohexanol (MCH) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Tripropylamine (TPrA), N-hydroxysuccinimide ester (NHS), tetrachloroanric ( Ⅲ ) acid tetrahydrate [HAuCl4ˑ4H2O], N,N’-dicyclohexyl carbodiimide (DCC), N,N’-dimethlformamide (DMF) Tris(2-carboxyethyl)phosphine (TCEP) were obtained from Aladdin Chemical Reagent Co. Ltd. (Shanghai, China). Thrombin, human serum albumin (HSA), bovine serum albumin (BSA), immunoglobulin G (lgG), insulin, trypsin, histidine and lysozyme were got form Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China).Graphite flakes (325 mesh) was bought from XFNANO material Tech CO. Ltd. (Nanjing, China). All other chemical not mentioned here were of analytical reagent grade and were used as received. Millipore Milli-Q water (18MΩ cm) supplied by a Millipore Milli-Q water purification system (Bedford, MA USA) was used throughout. A concentration of 0.1 M phosphate buffer saline (PBS, pH 7.4, 0.1 M NaCl+0.1 M NaH2PO4/Na2HPO4) was used as hybridization buffer, binding buffer and washing solution and diluting the blood serum.

2.2 Apparatus The ECL emission was detected with a computerized MPI-B type ultra-weak luminescence analyzer (Xi An Remax Electronic Science Tech. CO. Ltd. Xi An,China) equipped with a photomultiplier. The voltage of the photomultiplier tube (PMT) was set at -800 V. A conventional three-electrode system with a modified GCE (3 mm in

diameter) used as a working electrode, a Ag/AgCl electrode as reference electrode and a platinum wire as auxiliary electrode. CV experiments and electrochemical impedance spectroscopy (EIS) were measured with an IM6ex electrochemical workstation (Zahner IM6ex, Germany). All measurements were carried out at room temperature. EIS was performed under an oscillation potential of 0.214 V over the frequency range of 1 Hz to 100000 Hz. The electrochemical measurements were performed in the solution of 5 mM [Fe(CN)6]4-/[Fe(CN)6]3- and 0.1 M KCl.Atomic force microscopy (AFM) image was got through tapping-mode on a Nanoscope Ⅲa Digital Instruments with NSC15 tips (Veeco, CA, USA). The morphologies of GNPs-graphene and graphene oxide were characterized by a scanning electron microscope (SEM, JSM-6360LA, JEOL, Japan), field emission scanning electron microscope (FE-SEM, XL30FEG, PHILIPS, Eindhoven) and transmission electron microscope (TEM, FEI F20 G2, Tecnai, USA). Raman spectra were recorded on a Jobin Yvon LABRAM-HR confocal laser micro-Raman spectrometer (Raman, Jobin Yvon, France) at a room temperature and an excitation wavelength of 514 nm.

2.3 Synthesis of signal cluster 2.3.1 Synthesis of gold nanoparticles (AuNPs) AuNPS were prepared by citrate reduction of HAuCl4·4H2O [29-30]. A 50 mL aqueous solution consisting of 1 mM HAuCl4·4H2O was brought to a vigorous boil with stirring in a conical flask, and them 38.8 mM sodium citrate (5 mL) was added to the solution rapidly. The solution was boiled with stirring and the color changed from the yellow to red. The solution was cooled to room temperature with continuous stirring, of which the colloidal AuNPs were produced and stored at 4 °C。 2.3.2 Synthesis of Ru(bpy)32+ modifying TBA2 Ruthenium

bis(2,2’-bipyridine)-(2,2’-bipyridine-4,4’-dicarboxylic

acid)-N-hydroxysuccinimide ester ([Ru(bpy)2(debpy)NHS]) were synthesized and [Ru(bpy)2(debpy)NHS] was directly used to mark the TBA2 to obtain the ECL probe of Ru(bpy)2(dcbpy)NHS-TBA2 (abbreviated as Ru-TBA2)according to our previous published paper[31]. More details about the synthesis of Ru-TBA2 can be found in

supplementary information. 2.3.3 Synthesis of signal cluster The signal cluster was prepared according to the literature with some modifications [32]. Briefly, 9 µL of 1mM Ru-TBA2 was pretreated with 1.5 µL of 10 mM TCEP and 1 µL of 500 mM acetate buffer (pH 5.2) for 1 h. 3 mL of AuNPs was transferred to the NaOH treated glass vials, and then the TECP treated Ru-TBA2 was added, followed by incubation at room temperature in dark for 16 h. 30 µL of 500 mM Tris acetate (pH 8.2) and 300 µL of 1 M NaCl were slowly added into the AuNPs solution. The mixture was aged for an additional 24 h at room temperature in dark. Centrifuge the TBA2-AuNPs at 16000 r/min and room temperature for 25 min to remove the excess Ru-TBA2. At last, Ru-TBA2-AuNPs were dispersed in 0.1 M PBS (pH 7.4) containing 0.1 M NaCl and then stored at 4 °C.

2.4 The fabrication of GNPs-graphene nanocomposite electrode and the biosensor Graphene oxide was prepared from graphite flakes (325 mesh) with a modified Hummer’s method.GCE was polished with 0.3 and 0.05 μm α-Al2O3 slurry sequentially on a polishing cloth. The electrodes were fully rinsed after each polishing step and finally sonicated in deionized water and anhydrous ethanol for 5 min each, followed by electrochemical conditioning by potential scanning from -1 V to 1 V in 0.5 M H2SO4 for at least ten complete cycles at 100 mV s-1 until the reproducible cyclic voltammogram was obtained. Then the electrode was immediately used for deposition modification after a rinse step. The obtained GO powder was dispersed in a 0.07 M pH 8.0 phosphate buffer solution by ultrasonication to form a 1.0 mg mL-1 GO colloidal dispersion solution. The HAuCl4ˑ4H2O was dissolved in the GO solution to form 100 µM HAuCl4solution. The GO and HAuCl4ˑ4H2O can be simultaneously reduced on a GCE in 0.07 M pH 8.0 PBS solution by CV. The GNPs-graphene composite was obtained by CV from -1.5 V to 0.5 V at a scan rate of 10 mv s-1 for 10 cycles. Then, the GCE modified with GNPs-graphene composite was immersed in 10 μM TBA1 and stirred for 4 h at room temperature and then the electrode was

incubated in 0.1 M PBS containing 1.0 mM 2-mercaptohexanol (MCH) for 20 min at room temperature. After a rinsing, the electrode is stored at room temperature for next test.

2.5 ECL measurement of the biosensor The biosensor electrode was immersed in 200 μL of different concentration of thrombin (0.01, 0.05, 0.1, 0.5, 1, 5, 10 nM) for 30 min, followed by a thorough washing with 0.1 M PBS solution to remove the released uncollected TBA1. Then the modified electrode was immersed in 200 μL of 10 μM Ru-TBA2-AuNPs for 20 min. After a simple rinsing, the ECL measurements were carried out under scanning from0.2 V to 1.25 V at 100 mV s-1 in 0.1M PBS (pH 7.4, 0.1 M NaH2PO4/Na2HPO4 + 0.1 M NaCl) containing 0.1 M TPrA with a photomultiplier tube of -800 V. Quantification of target was based on the increment of ECL peak.

3 Result and discussion 3.1 Characterization of GNPs-graphene composite Graphene oxide (GO) was synthesized from graphite by Hummers method, which was comfirmed by AFM, FE-SEM respectively (Fig. S1). The GNPs-graphene composite was got by simultaneously reducing GO and HAuCl4 in PBS with CV form -1.5 V to 0.5 V at a scan rate of 10 mV s-1. Firstly, the SEM image of GNPs-graphene was given in Fig. 1A. It can be seen from the picture that the gold nanoparticles were uniformly dispersed on the reduced graphene. Secondly, to explore the structure of the GNPs-graphene, the TEM was performed. As shown in Fig. 1B, we can see some gold nanoparticle spread on the graphene sheets and the size of the gold nanoparticles is about 2-8 nm. To ensure the composites’ composition, the EDX was given in Fig. 1C and the outcome of the elements analysis is that C atom 87.18%, O atom 10.17%, Au atom 2.03% (atomic %). At last, the composites were further characterized by Raman. As shown in Fig. 1D, Raman spectra of graphene (a), GNPs-graphene (b) were given. The Raman feature at 1352 cm-1, known as the D band, arised from breathing of the hexagonal carbon ring due to the presence of defects. And another strong Raman

feature observed at 1590 cm-1 is the E2g mode (G band), assigned for the in-plane stretching of C-C bonds, which dictates the graphitic sp2 crystalline nature of the carbon. The Raman spectrum of the reduced GO (curve a) and GNPs-graphene (curve b) contain both G and D bands. Moreover, the intensity of G and D bands have a striking enhancement. It may contribute to the charge transfer from gold to graphene and strong interaction beween the gold and graphene layers [33].

3.2 Characterization of the assembly electrode The GNPs-graphene composite was got by simultaneously reducing GO and HAuCl4 in PBS with CV form -1.5 V to 0.5 V at a scan rate of 10 mV s-1. The electrode was firstly characterized by CV and electrochemical impedance spectra (EIS) in 5 mM [Fe(CN)6]4-/[Fe(CN6)]3- containing 0.1 M KCl solution. Compared with the bare GCE, there is a big increment in current. In Fig. 2A, it find that the current of bare GCE is about 0.8×10-4 A (curve a). And it changes to 1.4×10-4 A (curve b) after modifying with GNPs-graphene. The current changes to 1.0×10-4 A (curve c) after modifying with TBA1. The current of the electrode continued decreasing with thrombin (curve d, 0.6×10-4 A) and Ru-TBA2-AuNPs (curve e, 0.2×10-4 A) introducing the surface of the electrode.In the Nyquist diagram, the semicircle diameters at high frequency region reflect the electron transfer resistance (Ret) which related to the electron transfer resistance kinetics of the redox probe at electrode interface, and the linear part at lower frequencies corresponds to the diffusion resistance. The EIS was given in Fig. 2B. It show that the bare GCE exhibits a small semicircle with the Ret value at about 275 Ω (curve a). The Ret is almost close to zero (curve b) after the GNPs-graphene depositing on GCE. It means that the GNPs-graphene modifying GCE has a better electron transfer efficient. When TBA1 was introduced to the surface of GCE, Ret increased again (curve c) due to the resistance of biomolecule to electron. The Ret continued increasing with thrombin (curve d) and Ru-TBA2-AuNPs (curve e). The ECL intensity of different steps of assembly electrode was explored in Fig. 2C. Form the Fig. 2C, it can be found that there is not any obvious signal before the Ru-TBA2-AuNPs introducing to the system.

When the Ru-TBA2-AuNPs appears in the surface of the biosensor, the ECL intensity increased sharply. Furthermore, each step was performed 5 continuous cycles and the ECL intensity of each step was stable, it suggests that the proposed biosensor electrode have a good stability without any beacon escaping from the system.

3.3

Incubation

time

of

the

biosensor

electrode

with

thrombin

and

Ru-TBA2-AuNPs The effect of incubation time of the biosensor electrode with thrombin and Ru-TBA2-AuNPs was performed at thrombin concentration of 0.01 nM, 0.05 nM, and 0.1 nM. As shown in Fig. 3A, with the time of incubation in thrombin increasing, the ECL intensity increase rapidly and reached a plateau after 30 min. With different concentration of thrombin, the biosensor can absorb different amount of Ru-TBA2-AuNPs. The ECL intensity increased and reach stable after 20 min (Fig. 3B) incubating in Ru-TBA2-AuNPs. Therefore, 30 min was chosen for thrombin incubation time and 20 min for Ru-TBA2-AuNPs in our further experiments.

3.4 The stability, selectivity and reproducibility of the biosensor The long-time stability of this thrombin biosensor has been carried out in 0.5 nm thrombin. It found that the ECL response of the biosensor gradually decreased to approximately 95% of its original value after being stored in dark at 4 ◦C for half a month (Fig. 4A). In addition, the ECL intensity of various concentration of thrombin were further investigated. As shown in Fig. 4B, the ECL signal intensity increased with the increasing concentration of thrombin and a stable curve of different concentration could be obtained. Theses result suggested that the proposed biosensor owned excellent stability. To explore the specificity of the biosensor, the ECL intensity was measured under same experimental condition after incubating with BSA and lysozyme. As shown in Fig. 4C, compared with the ECL response of biosensor to 0.05 nM thrombin, the biosensor has no significant response towards 100 nm BSA,lysozyme, HAS, lgG, insulin, trypsin, and histidine respectively. It suggested that the biosensor have a good

selectivity to thrombin. The reproducibility of the proposed biosensor for thrombin was assessed by the relative standard derivations (RSD),which were evaluated by measuring one thrombin level for 5 reduplicate measurements. The RSD obtained from 0.01, 0.05, 0.1, 0.5, 1, 5, 10 nM thrombin were 10.2%, 9.3%, 5.4%, 4.3%, 6.2%, 8.2%, 9.4% respectively which indicated the biosensor have a good reproducibility.

3.5 Analytical performance of the biosensor to thrombin Under the optimal conditions, the analytical performance of biosensor to thrombin was assessed by measuring the dependence of ECL intensity (IECL) on the concentration of thrombin. As the Fig. 5 shown, the ECL intensity was enhanced with increasing concentration of thrombin and the IECL was found to be logarithmically related to the concentration of thrombin in the range of 0.01 to 10 nM (inset of Fig. 5). The regression equation is IECL=1725.7lgCTB+3821.2 with a regression coefficient of 0.9984 and limit of detection (LOD) 6.3 pM, which is defined as the concentration corresponding to the mean blank value plus 3 standard deviations. And the LOD is lower than other methods (Table 1). This result suggested the potential use of the proposed ECL biosensor for studies of drug-development process and disease prevention. Table 1 Performance comparison of different methods Methods

Linear range

LOD

Reference

Microscale thermophoresis

18.37-554.31 nM

5.4 nM

[34]

Fluorescence

0.5-20 nM

0.18 nM

[35]

Differential pulse voltammetry

5-35 nM

0.5 nM

[36]

20 pM

[37]

6.3 pM

This work

Surface-enhanced

Raman 0.1-10 nM

spectroscopy ECL sandwich biosensor

0.01-10 nM

3.6 Thrombin detection in human blood samples

Serum has the chemical composition similar to plasma, but without coagulation proteins such as thrombin or other factors. For the real sample application, serum was used to evaluate the efficiency of the thrombin detecting platform proposed in this work. To demonstrate the feasibility of biosensor in practical sample, the recovery experiments were performed in 100-fold diluted serum and show acceptable data of the recoveries between 94.0%-102.0% (Table 2). The result suggests that the biosensor could be used in real sample application. Table 2 the recovery experiments in 100-fold diluted serum Sample Added (nM)

Found (nM)a

NO.

Recovery

RSD (%, n=3)

(%)

1

0.05

0.0047

94.0

2.7

2

0.2

0.1966

98.3

2.2

3

8

8.160

102.0

3.1

a. Each data was given as average value obtained from three successive determinations.

4 Conclusions In this work, we successfully fabricated a sandwich aptamer ECL biosensor for thrombin detection. We took dual-signal amplification strategy to amplify the ECL signal. The GNPs-graphene improves the electron transfer efficient and the Ru-TBA2-AuNPs provide enough signal source. With the GNPs-graphene composites and big signal source (Ru-TBA2-AuNPs), the biosensor has good sensitivity and gets low LOD 6.3 pM. Therefore, such novel biosensor has a potential to be applied in drug-development process and disease prevention.

Acknowledgements We are grateful for the financial support from the Natural Science Foundation of Guangdong Province (No. 2014A030313480), the Guangdong High Education Fund

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for

electrochemiluminescence (ECL)-based detection methods: recent advances and future perspectives, Biosens. Bioelectron., 24 (2009) 3191-3200. [26] S. Xu, Y. Liu, T. Wang, J. Li, Positive potential operation of a cathodic electrogenerated chemiluminescence immunosensor based on luminol and graphene for cancer biomarker detection, Anal. Chem., 83 (2011) 3817-3823. [27] J. Wang, Y. Shan, W. W. Zhao, J. J. Xu, H. Y. Chen, Gold nanoparticle enhanced electrochemiluminescnece of CdS thin films for ultrasensitive thrombin detection, Anal. Chem., 83 (2011) 4004-4011. [28] J. Wang, H. Han, X. Jiang, L. Huang, L. Chen, Quantum dot-based near-infrared electrochemiluminesent immunsensor with gold nanaoparticle-graphene nanosheet hybrids and silica nanospheres dpuble-assisted signal amplification, Anal. Chem., 84 (2012) 4893-4899.

[29] Y. C. Chuang, J. C. Li, S. H. Chen, T. Y. Liu, C. H. Kuo, An optical biosensing platform for proteinase activity using gold nanoparticles, Biomaterials, 31 (2010) 6087-6095. [30] Y. H. Lin, S. H. Chen, Y. C. Chuang, Y. C. Lu, T. Y. Shen, Disposable amperometric immunosensing trips fabricated by Au nanoparticles-modified screen-printed carbon electrodes for the detection of foodborne pathogen Escherichia coli O157: H7, Biosens. Bioelectron., 23 (2008) 1832-1837. [31] W. Gao, A. Zhang, Y. Chen, Z. Chen, Y. Chen, F. Lu, Z. Chen, A novel probe density controllable electrochemiluminescence biosensor for ultra-sensitive detection of Hg2+ based on DNA hybridization optimization with gold nanoparticles array patterned self-assembly platform, Biosens. Bioelecton., 49 (2013) 139-145. [32] J. Liu, Y. Lu, Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes, Nat. protoc. 1 (2006) 246-252. [33] R. K. Biroju, P. K. Giri, Defect enhanced efficient physical functionalization of graphene with gold nanoparticles probed by resonance raman spectroscopy, J. Phy. Chem. C, 118 (2014) 13833-13843. [34] Y. Liu, N. Liu, X. Ma, X. Li, J. Ma, Y. Li, Z. Zhou, Z. Gao, Highly specific detection of thrombin using an aptamer-based suspension array and the interaction analysis via microscale thermophoresis, Analyst, 140 (2015) 2762-2770. [35] Y. H. Wang, L. Bao, Z. H. Liu, D. W. Pang, Aptamer biosensor based on fluorescence resonance energy transfer from upconverting phosphors to carbon nanoparticles for thrombin detection in human plasma, Anal. Chem. 83 (2011) 8130-8137. [36] A. 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. [37] J. Hu, P. Zheng, J. Jiang, G. Shen, R. Yu, G. Liu, Electrostatic interaction based approach to thrombin detection by surface-enhanced Raman spectroscopy, Anal. Chem. 81 (2009) 87-93.

Author Biographies Yingjie Li is studying for MS degree at Shantou University. His research focuses on the application of ECL biosensor. Yuqin Li received her PhD in Chemistry form Lanzhou University in 2006. Her current research interests are the analysis of drugs. Ning Xu graduated from Shantou University in 1997. His current research interests are analysis of drugs. Jiahong Pan is studying for MS degree at Shantou University. His research focuses on the application of ECL biosensor. Tufeng Chen is studying for MS degree at Shantou University. His research focuses on the application of ECL biosensor. Yaowen Chen received his PhD in Medicine from Shantou University in 2006. His current research interests are biology detection. Wenhua Gao received his PhD in Chemistry from Lanzhou University in 2006. His current research interests are application of function materials in analytical area.

Scheme 1 GO

GCE

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

Fig. 1 A

B

C

D

Fig. 1 (A) the SEM image, (B) the TEM, and (C) the EDX of GNPs-graphene; (D) the Raman spectrum of (a) graphene, (b) GNPs-graphene

Fig. 2

B

A

C

Fig. 2 (A) Cyclic voltammograms, (B) Nyquist diagram of electrochemical impedance spectra and (C) the ECL intensity of each step of the biosensor of different modified electrode:

(a)

bare

TBA1/GNP-graphene

GCE, modifying

(b)

GNPs-graphene GCE,

(d)

modifying

GCE,

(c)

thrombin/TBA1/GNPs-graphene

modifying GCE, (e) Ru-TBA2-AuNPs/thrombin/TBA1/GNPs-graphene modifying GCE. CV and EIS was tested in solution of 5 mM [Fe(CN)6]4-/[Fe(CN)6]3- containing 0.1 M KCl. Scan rate: 100 mV s-1; The ECL was measured in 0.1 M TPrA.

Fig. 3

A

B

Fig. 3 (A) Incubation time of biosensor electrode with thrombin at concentration of 0.01 nM (a), 0.05 nM (b), 0.1 nM (c); (B) Incubation time of biosensor electrode with Ru-TBA2-AuNPs. The error bars represent the standard deviation of three parallel measurements and ECL intensity was measured in 0.1 M PBS containing 0.1 M TPrA, scan rate: 100 mV s-1, scan range: 0.2-1.25 V.

Fig. 4

A

B

C

Fig. 4 (A) ECL intensity of the biosensor before and after half a month in presence of 0.5 nM thrombin, a) before half a month, b) after half a month; (B) ECL stability of the biosensor to various concentration of thrombin; (C) ECL of the biosensor to 0.05 nM thrombin, 100 nM BSA, lysozyme, HSA, lgG, insulin, trypsin, histidine. ECL were measured in 0.1 M PBS containing 0.1 M TPrA at 100 mV s-1.

Fig. 5

Fig. 5 ECL intensity of the biosensor with different concentration of thrombin: (a) 0.01 nM, (b) 0.05 nM, (c) 0.1 nM, (d) 0.5 nM, (e) 1 nM, (f) 5 nM, (g) 10 nM. Inset: linear relationship between ECL intensity (IECL) and logarithm of the thrombin concentration. The error bars represent the standard deviation of three parallel measurements and ECL were measured in 0.1 M PBS containing 0.1 M TPrA at 100 mV s-1