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Biosensors and Bioelectronics 25 (2009) 259–262

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

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mplified electrochemical aptasensor for thrombin based onio-barcode method

iaoru Zhang, Baoping Qi, Ying Li, Shusheng Zhang ∗

ey Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering,ingdao University of Science and Technology, Qingdao 266042, China

r t i c l e i n f o

rticle history:eceived 7 May 2009eceived in revised form 15 June 2009ccepted 16 June 2009

a b s t r a c t

In the present study, an electrochemical aptasensor for highly sensitive detection of thrombin was devel-oped based on bio-barcode amplification assay. For this proposed aptasensor, capture DNA aptamerI wasimmobilized on the Au electrode. The functional Au nanoparticles (DNA–AuNPs) are loaded with bar-code binding DNA and aptamerII. Through the specific recognition for thrombin, a sandwich format of

vailable online 24 June 2009

eywords:hrombinptameru nanoparticle

Au/aptamerI/thrombin/DNA–AuNPs was fabricated. After hybridization with the PbSNPs-labeled barcodeDNA, the assembled sensor was obtained. The concentration of thrombin was monitored based on the con-centration of lead ions dissolved through differential pulse anodic stripping voltammetric (DPASV). Underoptimum conditions, a detection limit of 6.2 × 10−15 mol L−1 (M) thrombin was achieved. In addition, thesensor exhibited excellent selectivity against other proteins.

© 2009 Elsevier B.V. All rights reserved.

bS nanoparticleio-barcode

. Introduction

Aptamers, single-stranded DNA/RNA oligonucleotides, haveeen selected for binding to a broad range of targets, including pro-eins, carbohydrates, lipids, or small molecules (Centi et al., 2007).hese artificial nucleic acid ligands offer several advantages overntibodies owing to their relative ease of isolation, modification,ailored binding affinity and easier storage (Hansen et al., 2006a,b).hrombin, a highly specific serine protease, plays a central role

n cardiovascular diseases, anti-clotting therapeutics, regulation ofnflammation processes, tissue repair at the vessel wall (Tasset etl., 1997; Centi et al., 2007), and acts as tumor marker in the diag-osis of pulmonary metastasis (Mir et al., 2006). Thrombin has twoNA aptamers (aptamerI, 15 bases DNA sequence, and aptamerII,9 bases DNA sequence). These two aptamers recognize the dif-

erent parts of thrombin with Kd of 26 nM and 0.5 nM, respectivelyIkebukuro et al., 2005). Until now, a lot of aptamer-based detectionystems for thrombin have been developed such as optical trans-uction (Pavlov et al., 2004), electrogenerated chemiluminescenceWang et al., 2007), fluorescence (Lettau et al., 2008), colorimetry

Wang et al., 2008) and electrochemistry (Xiao et al., 2005; Li etl., 2008; He et al., 2007). Among them, electrochemistry aptasen-or has attracted particular attention because it provides a simple,ensitive and selective platform for molecular detection.

∗ Corresponding author. Tel.: +86 532 84022750; fax: +86 532 84022750.E-mail address: shushzhang@126.com (S. Zhang).

956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2009.06.026

Nanoparticles-based materials offer excellent prospects forchemical and biological sensing because of their unique electricalproperties (Zhu et al., 2004). In recent years, metal nanoparticleshave been applied for the detection of DNA (Hansen et al., 2006a,b)and protein (Jie et al., 2007) with sensitivities in the pico- andfemtomolar range. Willner (Polsky et al., 2006) has introduced athrombin sensor by employing aptamer-functionalized PtNPs ascatalytic labels for the amplified electrochemical detection. Fang(Zheng et al., 2007) has built up an ultrasensitive electrochemicalsensor for detecting thrombin based on network-like thiocyanuricacid/gold nanoparticles. Wang (Hansen et al., 2006a,b) has intro-duced aptamer/quantum-dot-based dual-analyte biosensor for thedetection of thrombin and lysozyme.

To further improve the sensitivity for the detection of throm-bin, we fabricated an electrochemical aptasensor based on twonanoparticles. PbSNPs, whose metal components yield well-resolved highly sensitive stripping voltammetric signals, was usedas DNA labeling tag. AuNPs bio-barcode, which is the only bio-detection method that has the PCR-like sensitivity without a needfor enzymatic amplification (Chang et al., 2007; Nam et al., 2003),was involved in this strategy. Different from the reported AuNPswith one kind of oligonucleotides, AuNPs labeled with two kinds ofDNA were used here. One can specifically recognize the target pro-

tein, while the other cannot, reducing the cross-reaction of targetswith DNA loaded on the same one AuNP. With dual-amplificationeffects of labeled PbSNPs and bio-barcode AuNPs, the thrombincould be specifically detected with a relatively low detection limitof 6.2 × 10−15 M.

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60 X. Zhang et al. / Biosensors an

. Experimental section

.1. Chemicals and materials

All of synthetic oligonucleotides were purchased from SBSenetech. Co. Ltd. (China) with the following sequences:

aptamerI: 5′-HS-(CH2)6-GGTTGGTGTGGTTGG-3′;aptamerII: 5′-HS-(CH2)6-AGTCCGTGGTAGGGCAGGTTGGGGTGA-CT-3′;barcode binding DNA: 5′-SH-(CH2)6-ATGGTCTATTAGTGA-3′;barcode DNA: 5′-NH2-TTTTCACTAATAGACCAT-3′;amino capped aptamerII: 5′-NH2-TTTAGTCCGTGGTAGGGCAGGTT-GGGGTGACT-3′.

Purified thrombin (the activity of enzyme was 10 U mg−1,reeze-dry powder) was purchased from Dingguo Biological Tech-ology Corporation (Beijing, China). Tri(2-carboxyethyl)phosphineydrochloride (TCEP), 1-ethyl-3-(3-dimethylaminopropyl) car-odiimide (EDC), mercaptoacetic acid (MAA), imidazole, bovinelasma albumin (BSA) and bovine hemoglobin (BHb) were pur-hased from Sigma (USA). Sodium citrate, AuCl3HCl·4H2O andb(NO3)2 were purchased from Shanghai Chemical Reagent

nc. (Shanghai, China). Synthesis and modification of AuNPsere described in supplementary material. 6-Mercapto-1-hexanol

MCH) was obtained from Fluka (USA). All reagents were of analyt-cal grade and used without further purification. Double distilled

ater was employed after ion exchange process and stored in plasticessel. All glassware used in the following procedure was cleanedn a bath of freshly prepared 3:1 HCl–HNO3, rinsed thoroughly withretreated water.

.2. Apparatus

DPASV measurements were performed using a CHI 832B electro-hemical analyzer (Shanghai CH Instrument Company, China). Thelectrochemical system is comprised of a glassy carbon workinglectrode (GCE, 4 mm in diameter), a Ag/AgCl reference electrode,nd a platinum wire auxiliary electrode. The electrochemical mea-urements for electrochemical impedance spectroscopy (EIS) werearried out on a CHI 660C electrochemical analyzer (ShanghaiH Instrument Company, China) using a three-electrode sys-em consisted of a platinum wire as an auxiliary electrode, ag/AgCl electrode as reference electrode, and Au electrode (2 mm

n diameter) as working electrode. UV–vis absorption spectra werearried out on a Cary 50 probe UV–vis spectrophotometer (Varian).ransmission electron microscopy (TEM) image was taken withSM-6700F instrument (JEOL, Japan).

.3. Synthesis and modification of PbSNPs

MAA-modified PbSNPs were prepared according to the litera-ure (Milica et al., 1990). Briefly, 9.22 �L MAA was added to 50 mLf 0.4 mM Pb(NO3)2, then the pH of the mixture was adjusted to.0 with 0.5 M NaOH. After bubbled with N2 for 30 min, 50 mL of.34 × 10−3 M Na2S was added dropwise to the solution. The reac-ion was carried out for 24 h under N2 bubbled, and a brown colloidas formed gradually. The PbSNPs have an average diameter of

bout 5 nm measured by TEM as shown in Fig. S1B (supplemen-ary material).

100 �L of 0.1 M imidazole solution (pH 6.8) was added to 1.0

D of 5′-amino group capped barcode DNA. After 30 min, 50 �Lf 0.1 M EDC and 2.5 mL of already prepared PbS colloid (pH 3.5)ere added and incubated at room temperature for 24 h with stir-

ing, then the mixture was centrifuged at 15 000 rpm for 30 mino remove unbound oligonucleotides. The precipitate was washed

lectronics 25 (2009) 259–262

and separated twice with 100 mM PBS (pH 7.4). Finally, the PbSNPs-labeled barcode DNA was resuspended in PBS and stored at 4 ◦Cbefore use. The UV–vis spectrum of the PbSNPs-labeled barcodeDNA was shown in Fig. S2 (supplementary material). Similarly,PbSNPs-labeled aptamerII were prepared by the reaction of PbSNPswith amino capped aptamerII.

2.4. Fabrication of the thrombin biosensor

The process for the fabrication of the thrombin biosensor wasshown in Scheme 1. A gold electrode was polished carefully withalumina slurries (1, 0.3, 0.05 �m) and washed ultrasonically withdeionized water. Subsequently, the electrode was electrochemi-cally cleaned in 0.5 M H2SO4 solution by cyclic potential scanningbetween −0.2 and 1.5 V until a reproducible cyclic voltammogram(CV) was obtained. Then, it was sonicated and blown dry with nitro-gen before use.

The aptamerI self-assembly process was performed by treat-ing the clean Au electrode with 100 mM PBS (pH 7.4) containing1.25 �M aptamerI for 16 h. Then the modified electrode wasimmersed in 100 mM PBS (pH 7.4) containing 1.0 mM MCH for45 min to block the uncovered gold surface.

The fabrication of sandwich format consists of two steps.First, the Au/aptamerI electrode was incubated for 60 minwith various concentrations of thrombin in 100 mM PBS (pH7.4) for protein–aptamer interaction at 28 ◦C. Second, theAu/aptamerI/thrombin electrode was immersed into the preparedaptamerII and barcode binding DNA modified AuNPs solutionfor 150 min to get the Au/aptamerI/thrombin/DNA–AuNPs sysI/thrombin electrode was incubated with 100 mM PBS (pH 7.4) con-taining PbSNPs-labeled aptamerII for 90 min at 28 ◦C to form theAu/aptamerI/thrombin/aptamerII–PbS system.

The electrode surface was rinsed with 0.1 M PBS containing 1‰SDS (pH 7.4) after each step of fabrication process in order to removenonspecifically adsorbed thrombin and DNA sequences.

2.5. Electrochemical measurements

For detection, the Au electrode modified with PbSNPs wasimmersed into a plastical cell containing 100 �L of 1.0 M HNO3 for5 min to dissolve the PbSNPs. Then 1.8 mL of 0.1 M HAc–NaAc buffer(pH 5.3, containing 200 �L of 0.1 g L−1 Hg2+) was added to the cell. Todetect the released Pb2+ sensitively, DPASV was used with the in situpreparation of mercury film on the surface of the GCE in a stirringsolution with a deposition time of 500 s and deposition potentialof −1.4 V. After a 20 s rest time, the anodic stripping peak current(ip,a) located at ca. −0.520 V was measured and taken as the analyti-cal response. The conditions selected for DPASV were: increment inpotential 0.004 V, amplitude 0.05 V, pulse width 0.05 s, pulse period0.2 s.

3. Results and discussion

3.1. The principle of the bioelectrochemical protocol

Scheme 1 shows the method used for the amplified sensing oftarget protein. Thiol-functionalized aptamerI was immobilized onAu electrode to capture thrombin. AptamerII immobilized on AuNPscould sandwich the target with aptamerI, both of the two aptamerscould bind to different sites of thrombin. Barcode binding DNA

was also immobilized on AuNPs, which can hybridize with PbSNPs-labeled DNA probe. Since a single Au nanoparticle could be loadedwith hundreds of barcode binding DNA (Zhang et al., 2008; Hu et al.,2008; Fan et al., 2005), and each barcode binding DNA could furtherhybridize with PbSNPs-labeled barcode DNA. The amount of PbSNP

X. Zhang et al. / Biosensors and Bioelectronics 25 (2009) 259–262 261

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For Au/aptamerI/thrombin/bio-barcode system, the DPASV peakcurrent increased with the increasing thrombin concentration inthe incubation solution (Fig. 1, curves a–g). The calibration plot(Fig. 1 Inset B) shows a good linear relationship between the peak

Fig. 1. DPASV responses for increasing levels (a–g) of the target thrombin in PBS(a) 0, (b) 4.0, (c) 8.0, (d) 12, (e) 20, (f) 40, and (g) 75 (×10−14 M); Inset A is the

Scheme 1. Schematic diagram for the fabrication of thrombin biosensor

n per sandwich format could be amplified. Then lead ions were dis-olved by acid treatment and DPASV technique was employed forhe measurement of lead ion. The concentration of thrombin was

onitored based upon the concentration of dissolved lead ions.

.2. EIS analysis of the developed biosensor

EIS analysis was performed in 100 mM PBS (pH 7.4) containing.1 M KCl and 1.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] to provide the evi-ence for interface assembly on electrode. As seen from Fig. S3,ignificant differences in the electron transfer resistances (Ret)ere observed upon the step-wise formation of the electrode.

he bare gold electrode almost exhibited a straight line (Fig. S3a),hich was characteristic of a diffusion limited electrochemical

rocess. When the electrode was assembled with aptamerI, thealue of Ret was found to be 550.7 � (Fig. S3b). After treatedith MCH, the value of Ret was found to be 987.0 � (Fig. S3c)

mplying remarkable increase of the resistance to electron trans-er of the electrochemical probe. After the recognition of targetrotein thrombin, the Ret of Au/aptamerI/thrombin electrode was782.9 � (Fig. S3d). This is consistent with the fact that the layerf protein molecules can insulate the conductive support anderturb the interfacial electron transfer between the electrodend the electroactive species in solution (Wang et al., 2007). Theet of Au/aptamerI/thrombin/DNA–AuNPs electrode enhanced to376.1 � after interacted with aptamerII and barcode binding DNAodified AuNPs (Fig. S3e). It is reasonable that ssDNA assembled on

he electrode can block the electron transfer of the redox indictor.fter hybridization with PbSNPs-labeled barcode DNA, a lager Retf 2914.6 � was found for more negative charge after duplex DNAormation (Fig. S3f). These results were consistent with the fact thathe electrode was fabricated as expected.

.3. Bio-barcode amplified thrombin detection

The ratio of barcode binding DNA to aptamerII should be opti-

ized in order to provide maximum signals amplification by

lectrochemical detection. Fig. S4 (in supplementary material)hows the effect of barcode binding DNA to aptamerII ratio on peakurrents. The optimal ratio of barcode binding DNA to aptamerIIas 90:1.

lectrochemical stripping detection based on bio-barcode amplification.

The amplification effect of bio-barcode was shown in theinset A of Fig. 1, without thrombin for incubation, the sand-wich format could not be formed, and the background currentof DPASV at −0.520 V was 0.27 �A, which may caused by tracelead ions in acid solution (curve a). When 120 pM of thrombinwas added, for Au/aptamerI/thrombin/aptamerII–PbS system, onlyslight increase in the peak current of DPASV (2.71 �A) was observedwithout the amplification of bio-barcode (curve b). In contrast,for Au/aptamerI/thrombin/bio-barcode system, the peak current ofDPASV is 22.38 �A (curve c), indicating that the presence of the bio-barcode provides a significant amplification for the electrochemicalsignal.

responses of the aptasensor (a) in the absence of thrombin as a control experi-ment; (b) for Au/aptamerI/thrombin/PbS–aptamerII system, the concentration ofthrombin is 7.5 × 10−13 M; (c) for Au/aptamerI/thrombin/bio-barcode system, theconcentration of thrombin is 7.5 × 10−13 M. Inset B is the calibration plot of peak cur-rent versus thrombin concentration in the range of 4.0 × 10−14 up to 7.5 × 10−13 M inthe bio-barcode amplification assay.

262 X. Zhang et al. / Biosensors and Bioe

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ig. 2. Specificity of the aptasensor (a) without thrombin; (b) with 1 �M of BSA;c) with 1 �M of BHb; (d) with 2.0 × 10−13 M of thrombin; (e) with 2.0 × 10−13 M ofhrombin when 1 �M of BSA and 1 �M of BHb were coexisted. The error bar indicateshe R.S.D. of five independent experiments.

urrent and the thrombin concentration in the range of 4.0 × 10−14

o 7.5 × 10−13 M with a correlation coefficient of 0.9996. The regres-ion equation is I(�A) = 0.1347 + 0.29526× (thrombin) (10−14 M).he detection limit of 6.2 × 10−15 M could be estimated using 3�where � is the relative standard deviation of a blank solution, n = 6).

ith respect to sensitivity, this method was better than ampero-etric detection using nucleic acid functionalized Pt nanoparticles

s catalytic labels (Polsky et al., 2006), electrochemical moleculareacon aptasensor (Radi et al., 2006) and impedimetric aptasensorased on the functionalized AuNPs (Cai et al., 2006).

.4. Control experiment

In a further set of experiments, the specific recognition tohrombin of our system has been evaluated. As shown in Fig. 2,

significant increase induced by the interaction of the aptamerrobe with 2.0 × 10−13 M thrombin was observed ((Fig. 2, curve) compared to 1 �M BSA and 1 �M BHb (Fig. 2, curves b and c),hich indicated that the aptasensor has good specificity toward

arget protein. The cross-sensitivity of the aptasensor in a mixturef three different proteins was also examined. Even though muchigher concentration of BSA (1 �M) and BHb (1 �M) were coexisted

n detecting 2.0 × 10−13 M of thrombin, the signal had no appar-nt difference (Fig. 2, curve e), indicating BSA and BHb had almostegligible influence.

. Conclusions

In conclusion, we have developed an electrochemical aptasensoror the detection of a protein target-thrombin. A sandwich for-

at was fabricated including aptamerI, thrombin and DNA–AuNPsoaded with barcode binding DNA and aptamerII. After further

lectronics 25 (2009) 259–262

hybridization with PbSNPs-labeled barcode DNA, high sensitiv-ity of the electrochemical aptasensor was achieved. Due to theamplification effect of bio-barcode and the sensitivity of DPASVfor the detection of dissolved lead ions in the hybrids, as littleas 6.2 × 10−15 M of thrombin could be specifically recognized, andother proteins such as BSA and BHb did not affect the detection. Forall, the aptamer-nanoparticle assay has been demonstrated to offera sensitive, specific and convenient method of detecting throm-bin. Using different encoding nanoparticles, it could be readilyexpanded for the simultaneous measurement of different proteins,thus opening new opportunities for protein diagnostics in clinicalas well as for bioanalysis in general.

Acknowledgements

This work was supported by the National Natural Science Foun-dation of China (No. 20775038), and the Scientific and TechnicalDevelopment Project of Qingdao (06-3-1-4-yx).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bios.2009.06.026.

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