Electrochemistry Communications 11 (2009) 38–40

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Electrochemistry Communications

journal homepage: www.elsevier .com/locate /e lecom

An aptamer-based biosensor for sensitive thrombin detection

Hui Yang, Ji Ji, Yun Liu, Jilie Kong, Baohong Liu *

Department of Chemistry, Institute of Biomedical Sciences, Fudan University, No. 220, Handan Road, Shanghai 200433, China

a r t i c l e i n f o

Article history:Received 17 September 2008Received in revised form 13 October 2008Accepted 15 October 2008Available online 1 November 2008

Keywords:AptamerQuantum dotsThrombinElectrochemistrySensor

1388-2481/$ - see front matter � 2008 Elsevier B.V. Adoi:10.1016/j.elecom.2008.10.024

* Corresponding author.E-mail address: bhliu@fudan.edu.cn (B. Liu).

a b s t r a c t

A sensitive aptamer-based sandwich-type sensor is presented to detect human thrombin using quantumdots as electrochemical label. CdSe quantum dots were labeled to the secondary aptamer, which weredetermined by the square wave stripping voltammetric analysis after dissolution with nitric acid. Theaptasensor has a lower detection limit at 1 pM, while the sample consumption is reduced to 5 ll. The pro-posed approach shows high selectivity and minimizes the nonspecific adsorption, so that it was used forthe detection of target protein in the human serum sample. Such an aptamer-based biosensor provides apromising strategy for screening biomarkers at ultratrace levels in the complex matrices.

� 2008 Elsevier B.V. All rights reserved.

1. Introduction

Aptamers are RNAs or DNAs selected from systematic evolutionof ligands by exponential enrichment process in vitro that bind totheir target molecules with high affinity, including metal ions,small organic compounds, metabolites, proteins and even cells[1]. Aptamers offer several advantages over antibodies, owing totheir relative ease of isolation and modification, tailored bindingaffinity, and resistance against denaturizing [2]. Research focusedon aptamers has exhibited promising potentials in various fieldssuch as pharmaceutics and diagnostics [3].

Aptamer based analytical methods have been developed forprotein detection in recent years, including electrochemistry [4],fluorescence [5], binding-induced label-free detection [6] and soon. Electrochemistry has been widely employed and consideredto take a very important role in future research according to itssensitivity and simplicity [7,8]. For example, the electrochemicalthrombin aptasensor was fabricated by tethering a redox-activesubstance label, such as ferrocene and methylene blue, to theterminal of aptamer nucleic acid and immobilizing the oligonu-cleotide on an electrode, and the thrombin binding aptamerundergoes a transition to a G-quadruplex structure after bindingthe thrombin [9,10]. The aptamer-based sandwich-type electro-chemical sensors have been reported, which are labeled withenzymes including HRP [11], streptavidin-alkaline phosphatase[12] and pyrroquinoline quinone glucose dehydrogenase [13],organic or inorganic catalysts, Nanoparticles, such as gold, silver,silica, and quantum dots (QDs), are ideal encoding materials, and

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they have been extensively used for ultrasensitive optical and elec-trochemical bioassays [14–16]. Recent activity has demonstratedthe utility of QDs as encoding tracer for enhanced electrochemicaldetection of DNA hybridization and sandwich immunoassays ofproteins [17,18].

Thrombin, a kind of serine protease, plays important role in thecoagulation cascade, thrombosis and haemostasis. The high-picomolar range of thrombin in blood was known to be associatedwith diseases so that it was important to assess this protein attrace level with high sensitivity. The goal of this work was to devel-op a selective and sensitive aptamer-based sandwich-type sensorto detect the thrombin in human serum. The 15-mer ssDNA throm-bin binding aptamer is the first one selected in vitro [19]. It canbind with the thrombin strongly and selectively. Herein CdSeQDs were used as nanocrystal tracers, which can be easily dis-solved in nitric acid. The detection of Cd2+ was demonstrated insmall volumes by the stripping voltammetry with glassy carbonelectrode as the working electrode. Compared with the ion-selec-tive electrode, the glassy carbon electrode is more sensitive andconvenient [20,21]. This method not only possesses the advantagesof aptamer-based electrochemical assays on sensitivity and selec-tivity but also is specially suitable for detecting proteins in realsample with fewer sample consumption down to 5 ll.

2. Experimental

2.1. Reagent and chemicals

Thrombin (human a-Thrombin) was purchased from Sigma Al-drich (USA). DNA was purchased from Sangon (Shanghai). Their se-quences are as follows: The primary aptamer 50-SH-(CH2)6-TT TTT

H. Yang et al. / Electrochemistry Communications 11 (2009) 38–40 39

TTT TTG GTT GGT GTG GTT GG-30 and the secondary aptamer 50-biotin-GGT TGG TGT GGT TGG-30. Qdot Streptavidin Conjugateswere obtained from Invitrogen (USA). Fetal calf serum (FCS) waspurchased from Gibco (USA). BSA was obtained from Westang(Shanghai). Human serum was provided by Shanghai Thoracic Hos-pital. 2-Mercaptoethanol was purchased from Sigma Aldrich (USA).All chemicals were of analytical grade and were used without fur-ther purification. All solutions were prepared with bidistilledwater.

2.2. Preparation of oligonucleotide aptamer on gold electrode

The gold electrode was polished with 1.0, 0.3, 0.05 lm aluminapowders respectively, and rinsed with bidistilled water and etha-nol. The electrode was then sonicated in bidistilled water for5 min to remove bound particles, then rinsed thoroughly withbidistilled water and dried in nitrogen stream. 1 lM primaryaptamer was diluted in 50 mM Tris–HCl buffer (140 mM NaCl,1 mM MgCl2, pH 7.4). The Au electrode was incubated with pri-mary aptamer overnight at room temperature. Then the blockingreagent 2-mercaptoethanol was added to the surface of the goldelectrode to reduce nonspecific adsorption.

2.3. Sandwich aptamer-protein assay

The CdSe QDs labeled secondary aptamer (0.5 lM) was synthe-sized by exposing the Qdot Streptavidin Conjugates to the biotinlabeled secondary aptamer for 30 min at 4 �C. Because of the inter-action between the biotin and streptavidin, the CdSe QDs can linkto the secondary aptamer. Then, the sample of 5 ll thrombin (withconcentration ranged from 0 nM to 25 nM) was added to the sur-face of the primary aptamer modified gold electrode, followed bythe addition of CdSe QDs-aptamer conjugates to the surface ofthe electrode for another 1 h.

2.4. Electrochemical detection of proteins

The CdSe QDs remaining at the gold electrode surface weredissolved by the addition of 100 ll of 0.1 M HNO3 solution. The

Fig. 1. A schematic of this experiment protocol. Firstly, immobilization of theprimary anti-thrombin aptamer to the Au electrode surface; secondly, addition ofhuman thrombin to the system; thirdly, secondary binding with QDs-labeledaptamer, and finally, dissolution of QDs label and detection by the square wavestripping voltammetric analysis.

Fig. 2. (a) Square wave voltammograms of aptamer-based electrochemical assaywith increasing thrombin concentration from 0 pM to 25 nM. (b) Dose–responsecurve for thrombin. The current peaks for the different concentrations of thrombinare included. (c) The peak currents versus logarithm of thrombin concentration islinear over the range from 1 pM to 1 nM.

solution was transferred into 900 ll of 0.1 M acetate buffer atpH 4.6; the detection of Cd2+ was performed by glassy carbonelectrode using square wave stripping voltammetric analysis(SWSV). SWSV was conducted using a CHI1030 electrochemicalworkstation equipped with a stirring machine. The detection stepwas performed in a three-electrode system comprising a KCl sat-urated calomel reference electrode (SCE), a platinum counterelectrode and the working electrode. The electrochemical proce-dure involved a 1-min pre-treatment at +0.6 V, 2-min electrode-position at �1.00 V, and stripping from �1.20 to �0.2 V usinga square wave voltammetric waveform, with 4 mV potentialsteps, 25 Hz frequency, and 25 mV amplitude. Fig. 1 displayedthe schematic model for the aptamer-based thrombin detectionsystem.

Fig. 3. Response obtained with crude human serum samples, and comparison withcurrent peaks obtained with BSA and fetal calf serum samples.

40 H. Yang et al. / Electrochemistry Communications 11 (2009) 38–40

3. Results and discussion

3.1. Amperometric response characteristics of the aptamer/thrombin/aptamer sensor

The target protein–thrombin was detected by electrochemicalmeasurement as shown in Fig. 2a. Obviously, in the presence ofthrombin, the CdSe QDs labeled secondary aptamer could be cap-tured onto the electrode surface. Then the CdSe QDs was dissolvedinto cadmium ions by HNO3, which was determined by SWSV. Theelectrochemical signal was directly proportional to the amount ofanalyte, i.e. human thrombin. When the blank buffer was addedto the surface of Au electrode, there was no obvious cadmium peak.While the concentration of target protein increased gradually, theintensity of the peak current rose accordingly.

Using this device, we accessed a detection limit of 1 pM targetconcentration (RSD = 9.3%), which was a few orders of magnitudelower than the previous methods for thrombin detection(100 pM–2.8 nM) [6,20], and was comparable to the most advancedaptamer biosensor at attomole [22]. The peak currents for differentconcentration of thrombin were shown in Fig. 2b, and the detectionrange from 1 pM to 25 nM just covered the critical concentrationbetween testing blood and the situation when the clotting cascadewas activated. As shown in Fig. 2c, the corresponding calibrationplot of peak currents versus logarithm of thrombin concentrationwas linear over the range from 1 pM to 1 nM, which was suitablefor quantitative analysis of low-level proteins. Additionally, thepeak currents were linear versus thrombin concentration from1 nM to 25 nM. When the concentrations of thrombin increased,the relative excess thrombin would impair the aptamer-thrombinbinding, resulting in the slower increase of currents. This phenom-enon was observed in the other immunoassay reports [12,20].

3.2. Application of the aptamer-based system in human serum

The high sensitivity was coupled with good selectivity and theminimized nonspecific adsorption. As shown in Fig. 3, thrombinwas further tested in the healthy human serum samples withoutany pre-treatment to demonstrate the feasibility of the system.The concentration of thrombin tested in the real sample was mea-sured about 3.1 nM, which was in the range of normal thrombin le-vel (low nM). While the high-picomolar range of thrombin in bloodwas known to be associated with diseases. Furthermore, the samples

of bovine serum albumin (BSA) and fetal calf serum (FCS) weretested as the control experiments. The very lower currents were ob-served with respect to BSA and FCS samples. These experiment re-sults demonstrated the high selectivity of the biosensor since ahigh signal was obtained only with the human serum usually con-taining thrombin at picomolar. The signal of human serum wasabout seven times higher than that obtained by BSA and FCS. Theelimination of nonspecific adsorption was mainly attributed to theaddition of the blocking reagent 2-mercaptoethanol after the immo-bilization of the primary aptamer on the Au electrode. Therefore, thisprotocol exhibited anti-nonspecific adsorption and was suitable forthe detection of thrombin in a real sample. However, it has to be no-ticed that this aptasensor could not be reused, for the assemblywould be destroyed during the dissolution step, which needs to beresolved for further application.

4. Conclusion

A sensitive aptamer-based sandwich-type sensor by usingquantum dots as electrochemical label was developed for thedetection of thrombin in human serum. The detection limit wasobtained as low as 1 pM. This aptamer-based assay demonstratedseveral advantages. On the one hand, with CdSe QDs, the protocolexhibited an extreme low detection limits with little sample con-sumptions. On the other hand, it is fit for the detection of real sam-ple, as a result of the high specificity to human thrombin, even in ablood serum. Coupled with high-throughput formats, this methodcan provide promising potential for detecting and screening ultra-trace levels of biomarkers in the complex matrices.

Acknowledgment

This work is supported by National Natural Science Foundationof China (20775016, 20575013), Shanghai Leading Academic Disci-pline B109 and Shuguang 06SG02.

References

[1] T. Hermann, D.J. Patel, Science 287 (2000) 820.[2] C.K. O’Sullivan, Anal. Bioanal. Chem. 372 (2002) 44.[3] S.E. Osborne, A.D. Ellington, Chem. Rev. 97 (1997) 349.[4] Y. Xiao, A.A. Lubin, A.J. Heeger, K.W. Plaxco, Angew. Chem. Int. Ed. 44 (2005)

5456.[5] R. Nutiu, Y.F. Li, J. Am. Chem. Soc. 125 (2003) 4771.[6] B.L. Li, H. Wei, S.J. Dong, Chem. Commun. (2007) 73.[7] G.S. Bang, S. Cho, B.G. Kim, Biosens. Bioelectron. 21 (2005) 863.[8] X.L. Zuo, S.P. Song, J. Zhang, D. Pan, L.H. Wang, C.H. Fan, J. Am. Chem. Soc. 129

(2007) 1042.[9] A.E. Radi, J.L.A. Sanchez, E. Baldrich, C.K. O’Sullivan, J. Am. Chem. Soc. 128

(2006) 117.[10] Y. Xiao, B.D. Piorek, K.W. Plaxco, A.J. Heeger, J. Am. Chem. Soc. 127 (2005)

17990.[11] M. Mir, M. Vreeke, L. Katakis, Electrochem. Commun. 8 (2006) 505.[12] S. Centi, S. Tombelli, M. Minunni, M. Mascini, Anal. Chem. 79 (2007) 1466.[13] K. Ikebukuro, C. Kiyohara, K. Sode, Biosens. Bioelectron. 20 (2005) 2168.[14] R. Polsky, R. Gill, L. Kaganovsky, I. Willner, Anal. Chem. 78 (2006) 2268.[15] S.J. Park, T.A. Taton, C.A. Mirkin, Science 295 (2002) 1503.[16] R. Cui, H.C. Pan, J.J. Zhu, H.Y. Chen, Anal. Chem. 79 (2007) 8494.[17] J. Wang, G.D. Liu, A. Merkoci, J. Am. Chem. Soc. 125 (2003) 3214.[18] G.D. Liu, T.M.H. Lee, J.S. Wang, J. Am. Chem. Soc. 127 (2005) 38.[19] L.C. Bock, L.C. Griffin, J.A. Latham, E.H. Vermaas, J.J. Toole, Nature 355 (1992)

564.[20] A. Numnuam, K.Y. Chumbimuni-Torres, Y. Xiang, R. Bash, P. Thavarungkul, P.

Kanatharana, E. Pretsch, J. Wang, E. Bakker, Anal. Chem. 80 (2008) 707.[21] R. Thurer, T. Vigassy, M. Hirayama, J. Wang, E. Bakker, E. Pretsch, Anal. Chem.

79 (2007) 5107.[22] J.A. Hansen, J. Wang, A.N. Kawde, Y. Xiang, K.W. Gothelf, G. Collins, J. Am.

Chem. Soc. 128 (2006) 2228.