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Label-Free Electrochemiluminescent Aptasensor with Attomolar Mass Detection Limits Based on a Ru(phen)32+-Double-Strand DNA Composite Film Electrode ...
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Anal. Chem. 2009, 81, 9299–9305

Label-Free Electrochemiluminescent Aptasensor with Attomolar Mass Detection Limits Based on a Ru(phen)32+-Double-Strand DNA Composite Film Electrode Xue-Bo Yin,* You-Ying Xin, and Yue Zhao Research Center for Analytical Sciences, College of Chemistry, Nankai University, Tianjin 300071, P. R. China Precisely known ligand-induced conformation change and complex chemical labeling of the DNA sequence with probe molecules are often needed for the signal generation in most of the previous aptasensors. Herein, a solution to the above problems was reported by the use of the Ru(phen)32+ intercalated into double strand DNA (dsDNA) as an electrochemiluminescence (ECL) probe with thrombin as the target. After the antithrombin thiolated aptamer (27-mer) was attached to a gold electrode, ds-DNA structure was formed with its complementary 20-mer single strand DNA. Instead of the chemical modification of the aptamer or target with the probe molecule, Ru(phen)32+, as the probe, was intercalated into the ds-DNA structure. After thrombin hybridized with its aptamer, the ds-DNA dissociated and the intercalated Ru(phen)32+ released because of the higher stability of the aptamer-thrombin complex than that of the aptamer-complementary strand hybrid. The difference in ECL intensity with tripropylamine (TPA) as coreactant before and after the hybridization of thrombin and its aptamer was used to quantify thrombin. Besides the increase in the number of probe molecules over the single-site labeling, a ca. 80-fold improvement on the TPA oxidation at the dsDNA modified electrode was found over the bare gold electrode. With the two amplification factors, the mass detection limits of 0.2 attomolar for thrombin are obtained. Because of the independence of conformational changes, the present method is readily extended to the targets whose aptamers have no specific conformational changes or other DNA-related detection without the need for chemical labeling. Simple, sensitive, and specific detection of proteins is of importance in forensic science, biomedical research, and clinic diagnosis. For example, determination of thrombin, a kind of serine protease, can be used to understand thrombosis and hemostasis.1 Owing to the high affinity and specificity of aptamer, various aptasensors have been developed for detecting proteins * To whom correspondence should be addressed. E-mail: [email protected]. Fax: 86-22-23502458. (1) Holland, C. A.; Henry, A. T.; Whinna, H. C.; Church, F. C. FEBS Lett. 2000, 484, 87–91. 10.1021/ac901609g CCC: $40.75  2009 American Chemical Society Published on Web 10/14/2009

based on electrochemistry (EC),2 fluorescence,3 electrochemiluminescence (ECL),4 and colorimetry.5 Among all the aptasensors, (2) (a) Xiao, Y.; Uzawa, T.; White, R. J.; DeMartini, D.; Plaxco, K. W. Electroanalysis 2009, 21, 1267–1271. (b) Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Angew. Chem., Int. Ed. 2005, 44, 5456–5459. (c) Huang, Y. C.; Ge, B. X.; Sen, D.; Yu, H.-Z. J. Am. Chem. Soc. 2008, 130, 8023– 8029. (d) Xu, Y.; Yang, L.; Ye, X. Y.; He, P. G.; Fang, Y. Z. Electroanalysis 2006, 18, 1449–1456. (e) Deng, C. Y.; Chen, J. H.; Nie, Z.; Wang, M. D.; Chu, X. C.; Xiao, X. L.; Lei, C. Y.; Yao, S. Z. Anal. Chem. 2009, 81, 739– 745. (f) He, P. L.; Shen, L.; Cao, Y. H.; Li, D. F. Anal. Chem. 2007, 79, 8024–8029. (g) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Anal. Chem. 2006, 78, 2268–2271. (h) Lee, J. A.; Hwang, S.; Kwak, J.; Park, S. I.; Lee, S. S.; Lee, K.-C. Sens. Actuators, B 2008, 129, 372–379. (i) Kang, Y.; Feng, K. J.; Chen, J. W.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. Bioelectrochemistry 2008, 73, 76–81. (j) Radi, A.-E.; Sanchez, J. L. A.; Baldrich, E.; O’Sullivan, C. K. Anal. Chem. 2005, 77, 6320–6323. (k) Yang, H.; Ji, J.; Liu, Y.; Kong, J. L.; Liu, B. H. Electrochem. Commun. 2009, 11, 38–40. (l) Mir, M.; Vreeke, M.; Katakis, I. Electrochem. Commun. 2006, 8, 505–511. (m) Centi, S.; Messina, G.; Tombelli, S.; Palchetti, I.; Mascini, M. Biosen. Bioelectron. 2008, 23, 1602–1609. (n) Ikebukuro, K.; Kiyohara, C.; Sode, K. Biosens. Bioelectron. 2005, 20, 2168–2172. (o) Fan, H.; Chang, Z.; Xing, R.; Chen, M.; Wang, Q. J.; He, P. G.; Fang, Y. Z. Electroanalysis 2008, 20, 2113– 2117. (p) Tan, E. S. Q.; Wivanius, R.; Toh, C.-S. Electroanalysis 2009, 21, 749–754. (q) Radi, A.-E.; Snchez, J. L. A.; Baldrich, E.; O’Sullivan, C. K. J. Am. Chem. Soc. 2006, 128, 117–124. (r) Lu, Y.; Li, X. C.; Zhang, L. M.; Yu, P.; Su, L.; Mao, L. Q. Anal. Chem. 2008, 80, 1883–1890. (s) Xiao, Y.; Piorek, B. D.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2005, 127, 17990–17991. (t) Rahman, Md. A.; Ik Son, J.; Won, M.-S.; Shim, Y.-B. Anal. Chem. 2009, 81, 6604–6611. (3) (a) Potyrailo, R. A.; Conrad, R. C.; Ellington, A. D.; Hieftje, G. M. Anal. Chem. 1998, 70, 3419–3425. (b) Lin, C. X.; Katilius, E.; Liu, Y.; Zhang, J. P.; Yan, H. Angew. Chem., Int. Ed. 2006, 45, 5296–5301. (c) Lao, Y.-H.; Peck, K.; Chen, L.-C. Anal. Chem. 2009, 81, 1747–1754. (d) Li, B. L.; Wei, H.; Dong, S. J. Chem. Commun. 2007, 73–75. (e) Choi, J. H.; Chen, K. H.; Strano, M. S. J. Am. Chem. Soc. 2006, 128, 15584–15585. (f) Tang, Z. W.; Mallikaratchy, P.; Yang, R. H.; Kim, Y. M.; Zhu, Z.; Wang, H.; Tan, W. H. J. Am. Chem. Soc. 2008, 130, 11268–11269. (g) Hamaguchi, N.; Ellington, A. D.; Stanton, M. Anal. Biochem. 2001, 294, 126–131. (h) Dittmer, W. U.; Reuter, A.; Simmel, F. C. Angew. Chem., Int. Ed. 2004, 43, 3550–3553. (i) Wang, L. Q.; Li, L. Y.; Xu, Y.; Cheng, G. F.; He, P. G.; Fang, Y. Z. Talanta 2009, 79, 557–561. (4) (a) Wang, X. Y.; Zhou, J. M.; Yun, W.; Xiao, S. S.; Chang, Z.; He, P. G.; Fang, Y. Z. Anal. Chim. Acta 2007, 598, 242–248. (b) Bai, J. G.; Wei, H.; Li, B. L.; Song, L. H.; Fang, L. Y.; Lv, Z. Z.; Zhou, W. H.; Wang, E. K. Chem.-Asian J. 2008, 3, 1935–1941. (c) Li, Y.; Qi, H. L.; Peng, Y.; Gao, Q.; Zhang, C. X. Electrochem. Commun. 2008, 10, 1322–1325. (d) Guo, W. W.; Yuan, J. P.; Li, B. L.; Du, Y.; Ying, E. B.; Wang, E. K. Analyst 2008, 133, 1209–1213. (e) Fang, L. Y.; Lv, Z. Z.; Wei, H.; Wang, W. K. Anal. Chim. Acta 2008, 628, 80–86. (f) Wang, X. Y.; Dong, P.; Yun, W.; Xu, Y.; He, P. G.; Fang, Y. Z. Biosens. Bioelectron. 2009, 24, 3288–3292. (5) (a) Wei, H.; Li, B. L.; Li, J.; Wang, E. K.; Dong, S. J. Chem. Commun. 2007, 3735–3737. (b) Wang, Y. L.; Li, D.; Ren, W.; Liu, Z. J.; Dong, S. J.; Wang, E. K. Chem. Commun. 2008, 2520–2522. (c) Higuchi, A.; Siao, Y. D.; Yang, S. T.; Hsieh, P.-V.; Fukushima, H.; Chang, Y.; Ruaan, R.-C.; Chen, W.-Y. Anal. Chem. 2008, 80, 6580–6586. (d) Li, T.; Wang, E. K.; Dong, S. J. Chem. Commun. 2008, 3654–3656.

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ECL-based methods are attractive, as they integrate the advantages of EC detection and chemiluminescent techniques, such as high sensitivity, wide linear ranges, and high specificity.4 The generation of the signal related to the concentration of targets in previous aptasensors is usually achieved on the basis of the interaction between the target and its aptamer due to the ligand-induced conformation change and the chemical labeling of the DNA sequence with probe molecules.2-4 However, it is difficult to know the target binding sites and conformational changes of the aptamer precisely for the optimal efficiency and the binding-induced conformational changes of some aptamers are weak. Meanwhile, the free aptamer is usually in a conformational equilibrium between its unfolded state and folded conformation. The binding from the target, such as thrombin, only drives the equilibrium toward the folded state,2a,b so the false positive possibly arises. For example, a ca. 30% signal upon the saturated target levels was observed even in the absence of the target protein.2a Moreover, the chemical labeling procedures are usually complex, time-consuming, and labor-intensive. Therefore, alternative protocols for developing aptasensor with a simplified analytical procedure are important and attractive. The helical double-strand DNA (ds-DNA) has the capacity to be intercalated with some small molecules into its grooves with high affinity.6,7 If this interaction is used to develop DNA-related sensors, more than one probe molecule can be intercalated into a DNA sequence and no chemical modification of the aptamer is needed. Some DNA sensors have been developed by the use of the intercalation of small molecule probes into the DNA structures.6 Dong and co-workers6a developed a fluorescence aptasensor using ethidium bromide as intercalator and fluorescent probe for thrombin determination. After thrombin interacted to its aptamer, the release of ethidium bromide resulted in the decreased fluorescence.6a [Ru(phen)2(dppz)]2+ intercalated into the double-stranded regions of the aptamer interacting to its target was used to develop electrochemical aptasensors for adenosine 5′-triphosphate (ATP) and immunoglobulin G (IgG) by Fang’s group.6b,c Methylene blue, as electrochemical probe, was intercalated into the aptamer.6d While Ru(phen)32+ (phen ) 1,10-phenanthroline) and its derivatives can be intercalated into the grooves of ds-DNA,7 Ru(phen)32+ has a high ECL emission efficiency.7a-d Herein, we report a (6) (a) Li, B. L.; Wei, H.; Dong, S. J. Chem. Commun. 2007, 73–75. (b) Wang, J.; Jiang, Y. X.; Zhou, C. S.; Fang, X. H. Anal. Chem. 2005, 77, 3542–3546. (c) Jiang, Y. X.; Fang, X. H.; Bai, C. L. Anal. Chem. 2004, 76, 5230–5235. (d) Huang, C. C.; Chiu, S. H.; Huang, Y. F.; Chang, H. T. Anal. Chem. 2007, 79, 4798–4804. (e) Tansil, N. C.; Xie, H.; Xie, F.; Gao, Z. Q. Anal. Chem. 2005, 77, 126–134. (f) Gao, Z. Q.; Tansil, N. C. Anal. Chim. Acta 2009, 636, 77–82. (g) Wang, J. L.; Wang, F.; Dong, S. J. J. Electroanal. Chem. 2009, 626, 1–5. (h) Lee, J.-G.; Yun, K.; Kim, G.-S.; Lee, S. E.; Kim, S.; Park, J.-K. Bioelectrochemistry 2007, 70, 228–234. (7) (a) Carter, M. T.; Bard, A. J. Bioconjugate Chem. 1990, 1, 257–263. (b) Xu, X. H.; Yang, H. C.; Bard, A. J. J. Am. Chem. Soc. 1994, 116, 8386– 8387. (c) Xu, X. H.; Bard, A. J. J. Am. Chem. Soc. 1995, 117, 2627–2631. (d) Kuwabara, T.; Noda, T.; Ohtake, H.; Ohtake, T.; Toyama, S.; Ikariyama, Y. Anal. Biochem. 2003, 314, 30–37. (e) Mihailovic, A.; Vladescu, I.; McCauley, M.; Ly, E.; Williams, M. C.; Spain, E. M.; Nenez, M. E. Langmuir 2006, 22, 4699–4709. (f) Xin, A. P.; Dong, Q. P.; Xiong, C.; Ling, L. S. Chem. Commun. 2009, 1658–1660. (g) Wang, S. J.; Milam, J.; Ohlin, A. C.; Rambaran, V. H.; Clark, E.; Ward, W.; Seymour, L.; Casey, W. H.; Holder, A. A.; Miao, W. J. Anal. Chem. 2009, 81, 4068–4075. (h) Sun, Y. J.; Lutterman, D. A.; Turro, C. Inorg. Chem. 2008, 47, 6427–6434. (i) Lutterman, D. A.; Chouai, A.; Liu, Y.; Sun, Y. J.; Stewart, C. D.; Dunbar, K. R.; Turro, C. J. Am. Chem. Soc. 2008, 130, 1163–1170.

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solution to problems of the need for conformational change and chemical labeling by the use of the Ru(phen)32+ intercalated into ds-DNA as the ECL probe. Thrombin was used as the target to demonstrate the principle of the present ECL aptasensor. MATERIALS AND METHODS Apparatus. The electrochemical measurements were carried out on a model LK98BII microcomputer-based electrochemical analyzer (Tianjin Lanlike High-Tech Company, Tianjin, China). A traditional three-electrode system was used with Ag/AgCl/KCl (sat) as reference electrode, a 2 mm diameter Au disk electrode modified with ds-DNA structures as working electrode, and Pt wire as counter electrode. The ECL emission was detected by the use of a model MPI-A electrochemiluminescence analyzer (Xi’An Remax Electronic Science & Technology Co. Ltd., Xi’An, China) at room temperature, and the voltage of the PMT was set at -900 V in the detection process. The fluorescence titrations were performed on an F-4500 spectrofluorometer (Hitachi, Japan) with a 1 cm quartz cell at room temperature. Chemicals and Materials. 27-Mer thiolated antithrombin aptamer (1), its 20-mer complementary ss-DNA (2), 42-mer thiolated antilysozyme aptamer (3), and its 30-mer complementary ss-DNA (4) were prepared by Takara Biotechnology (Dalian, China) with the following sequences. 1: 5′-SH-(CH2)6-GTCCGTGGTAGGGCAGGTTGGGGTGAC-3′ 2: 3′-CAGGCACCATCCCGTCCAAC-5′ 3: 5′-SH-(CH2)6-ATC TAC GAA TTC ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG-3′ 4: 5′-GCA CTC TTT AGC CCT GAT GAA TTC GTA GAT-3′ R-Thrombin was obtained from Haematologic Technologies Inc. (Essex Junction, VT). Lysozyme was from Solarbio Bioscience & Technology Co. LTD, Shanghai, China. Dichlorotris(1,10phenanthroline)ruthenium hydrate (Ru(phen)32+Cl2 · H2O) and tripropylamine (TPA) (Sigma-Aldrich, Shanghai, China) were used as ECL probe and coreactant. The phosphate buffer solution containing 20 mM TPA is used as detection electrolyte. 2-Mercaptoethanol (MCE) used to block the sensing interface for detection, was obtained from Yangguang Yunneng Biotechnology Company, Tianjin, China. The 2 mm diameter Au disk electrode was obtained from Tianjin Lanlike High-Tech Company (Tianjin, China). Thrombin and lysozyme were prepared in Tris-HCl buffer (4.7 mM NaCl, 0.56 mM Tris-HCl, 0.14 mM KCl, pH 6.5). All oligonucleotides were diluted to 5 µM in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). The stock solution of TPA and Ru(phen)32+ were prepared in doubly distilled water. Phosphate buffer saline (PBS; 0.2 M) was used as the electrolyte in the experiment. Procedure of the Detection of the Proteins with the ECL Aptasensor. The procedure for determination of thrombin using the ECL aptasensor was schematized in Figure 1. The new gold electrode was polished with 0.3 and 0.05 µm aluminum slurry and ultrasonicated with distilled water for 15 min. It was electrochemically cleaned in 0.1 M H2SO4 via potential scanning between -0.2 and +1.6 V until a remarkable voltammetric peak was obtained. Further, the electrode was sonicated again and thoroughly cleaned with doubly distilled water. After being dried with nitrogen airflow, the gold electrode was soaked in 5 µM thiolated aptamer (1 or 3) solution to prepare aptamer-modified electrode for 2 h at 36

RESULTS AND DISCUSSION

Figure 1. Schematic for the principle of the developed ECL aptasensor for detecting thrombin. (A) The adsorption of thiolated antithrombin aptamer on and the MCE block to the electrode. (B) The formation of the ds-DNA between aptamer and its complementary ss-DNA. (C) The intercalation of Ru(phen)32+ into the ds-DNA sequence. (D) Dissociation of ds-DNA and release of Ru(phen)32+ due to the interaction between thrombin and its aptamer, resulting in the decreased ECL emission, which was used to quantify thrombin.

°C. To block the sensing interface, 10 µL of 0.1 M MCE solutions was dropped onto the electrode for 1.5 h at 36 °C. As shown in step B in Figure 1, the ds-DNA structures between the aptamer and its complementary ss-DNA (2 or 4) was formed through being cast 6 µL of 5 µM complementary ss-DNA solution on the electrode within 1.5 h at 36 °C. After every step, the modified electrode was thoroughly cleaned with doubly distilled water and PBS buffer to remove the nonchemadsorbed species. In order to insert or intercalate Ru(phen)32+ molecules into ds-DNA structures, the electrode modified with ds-DNA was immersed in 20 mM Ru(phen)32+ solution for more than 7 h (as shown in Figure S1 in the Supporting Information). After the doubly distilled water and PBS solutions were used to clean off the unbinding Ru(phen)32+, the ECL intensity of the resulting functionalized electrode was tested, the value of the ECL intensity was recorded as E0, which was represented an ECL emission without the protein reacting with its aptamer. To determine the protein (thrombin or lysozyme), 10 µL of various concentrations of the protein was incubated on the electrode modified with ds-DNA intercalated Ru(phen)32+ for 1.5 h at 36 °C as shown in step D in Figure 1. The thrombin in fetal calf serum (obtained from Sijiqing Biotechnique Ltd., Hangzhou, China) was detected to test the possibility of the determination of thrombin in real sample. Because no thrombin was found in the serum sample, the serum 1:5 diluted with TE buffer was spiked with thrombin at different concentrations. Similar to the determination of thrombin in standard solution, 10 µL of the serum sample spiked with thrombin was dropped on the Ru(phen)32+-ds-DNA composite film electrode surface and incubated at 36 °C for 1.5 h. The electrode was washed thoroughly with 0.2 M PBS to reduce the nonspecific binding and introduced into 20 mM TPA solution in PBS. The ECL emission was obtained, and the ECL intensity was recorded as E1. E0 - E1 was related to the concentration of thrombin or lysozyme.

Preparation of the ECL Aptasensor Using Ru(phen)32+DNA-Composite Film Electrode. The ECL aptasensor using the Ru(phen)32+ intercalated into ds-DNA as the ECL probe was proposed with thrombin as target model, as shown in Figure 1. To facilitate the binding of thrombin to its aptamer, one of its complementary 20-mer single strand DNA (ss-DNA) was selected. The interaction between 20-mer cDNA and the antithrombin aptamer is weaker than that between 27-mer cDNA and the aptamer. However, the interaction between thrombin and its aptamer is constant. Therefore, the 20-mer ss-DNA makes the coupling between thrombin and its aptamer more efficient than the 27-mer one. For determination of thrombin, the antithrombin thiolated aptamer (27-mer) was attached to a gold electrode by the use of self-assembling chemistry2a,b,8 and then formed a dsDNA structure with its complementary 20-mer ss-DNA. Instead of the chemical modification of the aptamer or target with the probe molecule, Ru(phen)32+, as the probe, was intercalated into the ds-DNA structure.6,7 The ECL signal from the intercalated Ru(phen)32+ using TPA as a coreactant was recorded as the basic value for thrombin determination. After thrombin hybridized with its aptamer, the ds-DNA dissociated and the intercalated Ru(phen)32+ released because of the higher stability of the aptamer-thrombin complex than that of the aptamercomplementary strand hybrid. The difference in ECL intensity before and after hybridization of thrombin and its aptamer was used to quantify thrombin. From the above procedure, we can find the signal generation is independent of the special conformational changes, and therefore, the present method not only possesses the advantages of conventional aptamer-based ECL assays but also is readily extended to the targets whose aptamers have no specific conformational changes or other DNA-related detection without the need for chemical labeling.6a The incubation time for the intercalation of Ru(phen)32+ has a significant influence on the sensitivity of the present ECL apatasensor. As shown in Figure S1 in the Supporting Information, an incubation time beyond 7 h can give a maximum ECL emission for E0. TPA Oxidation at the DNA-Modified Electrode. The oxidation of TPA plays an important role in the sensitivity of the ECL aptasensor. Cyclic voltammetry (CV) in 0.2 M phosphate buffer saline (PBS, pH 7.5) containing 20 mM TPA is investigated to learn the oxidation of TPA on the electrodes during the modification procedure. Self-assembly of ss-DNA aptamer onto the gold electrode generated an oxidation wave at ca. 0.88 V (Figure 2b). The hybridization of the aptamer with its complementary ss-DNA resulted in a further increase in the oxidation current (Figure 2c). Intercalation of Ru(phen)32+ into the ds-DNA did not affect the oxidation current but led to a slight positive peak potential shift (0.01 V) (Figure 2c cf. Figure 2d). However, the peak at 0.88 V is not observed when the above electrodes are swept cyclically in the absence of TPA (Figure S2a in the Supporting Information). Therefore, the oxidation peaks in Figure 2 at 0.88 V were attributed to the TPA oxidation on the surface of the DNA assembled electrode, although direct TPA oxidation on the bare (8) Fan, C. H.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9134–9137.

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Figure 2. Cyclic voltammograms in 0.2 M phosphate buffer solution (pH 7.5) containing 20 mM TPA solution using (a) bare gold electrode; (b) the 27-mer aptamer assemble gold electrode; (c) the gold electrode assembled with ds-DNA between aptamer and its complementary ss-DNA; (d) the Ru(phen)32+-ds-DNA composite film electrode. Scan rate: 50 mV/s.

gold electrode was considered difficult to achieve at pH 7.5.9 Because TPA existed in the form of TPAH+ at pH 7.5, due to its acidity constant (pKa) of 10.4, the deprotonation of TPA was critical to the oxidation of TPA.9 Some experimental results (Figures S2-S5 in the Supporting Information) proved that TPA was directly oxidized at the DNA assembled electrode. The TPA oxidation at the DNA assemble electrode was proposed as follows on the basis of our results and previous research.7a,9,10 The protonated TPAH+ approached the anionic phosphate backbone of DNA via the electrostatic interaction between TPAH+ and phosphate ions.7a Meanwhile, the proton was easily transferred from TPAH+ to phosphate ions, and therefore, TPA was oxidized in a mode similar to that at high pH.9,10 The backbone of DNA attached to the electrode surface acted as the substrate to preconcentrate TPA and the acceptor of protons released from TPAH+.7a,9 The good conductivity of ds-DNA2c,10a also promoted TPA oxidation, possibly resulting in the increase in the oxidation current in Figure 2c,d. TPA oxidation efficiency was calculated by integrating the area of anodic peak of the cyclic voltammograms obtained with different electrodes (Figure S6 in the Supporting Information). The enhancement factor is defined as the ratio of peak area before and after the electrode modified with DNA strands. Compared with the bare gold electrode, the aptamer- and ds-DNA-assemble electrodes give the enhancement factor of 22.9 and 79.4 for TPA oxidation, respectively, showing a significant improvement on TPA oxidation. The sensitivity of the ECL aptasensor is, therefore, improved due to the importance of TPA oxidation to the ECL emission. Characterization of the Interaction between Ru(phen)32+ and ds-DNA. Fluorescence titration of 10 µM Ru(phen)32+ with the ds-DNA at different concentrations was performed to investigate the site size and equilibrium constant between them. (9) (a) Kanoufi, F.; Zu, Y.; Bard, A. J. J. Phys. Chem. B 2001, 105, 210–216. (b) Pastore, P.; Badocco, D.; Zanon, F. Electrochim. Acta 2006, 51, 5394– 5401. (10) (a) Pittman, T. L.; Miao, W. J. J. Phys. Chem. C 2008, 112, 16999–17004. (b) Miao, W. J.; Choi, J.-P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 14478– 14485.

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Figure 3. (A) Fluorescence titration of 10 µM Ru(phen)32+ using different concentrations of ds-DNA (formed from antithrombin aptamer and its 20-mer complementary strand) in 0.2 M PBS (pH 7.5). The concentrations of ds-DNA are (a) 0, (b) 0.25, (c) 0.5, (d) 2.0, (e) 4.0, (f) 8.0, and (g) 10 µM. The maximum emission wavelengths from (a) to (d) are 585, 586, 587, and 590 nm, respectively. (B) The relationship of the peak emission intensity and the concentrations of ds-DNA.

Under test conditions, Ru(phen)32+ has a natural fluorescence but the ds-DNAs have none (data not shown). Moreover, Ru(phen)32+ luminescence intensity is enhanced in the presence of ds-DNA due to the strong intercalation between Ru(phen)32+ and the adjacent DNA base pairs.7a Figure 3 shows the profiles and the plots of fluorescence titration results for Ru(phen)32+ in the presence of different concentrations of the ds-DNA. As the ds-DNA concentration increased, the fluorescent emission of Ru(phen)32+ increased until a plateau was reached. Meanwhile, a red shift from 585 to 590 nm was observed for the maximum emission wavelength. Carter and Bard7a suggested that the interaction originated from the intercalation of Ru(phen)32+ into the ds-DNA. The enhanced luminescence was attributed to the greater rigidity and lower collisional frequency of the molecule after being stacked within the helix.7a The binding constant K and the site size s were calculated from the fluorescence data using eqs 1 and 2:7a 1 (Ia - If) b - (b2 - 2K2C[DNA]/s) /2 ) (Ib - If) 2KC

(1)

b ) 1 + KC + K[DNA]/2s

(2)

Where C is the constant total concentration of Ru(phen)32+, i.e., 10 µM. [DNA] is the total concentration of the added ds-DNA as M base pairs, which is 20 times the concentration of dsDNA used in Figure 3. Ia is the apparent fluorescence of Ru(phen)32+ in the presence of DNA. If is the fluorescence of the free Ru(phen)32+ in the buffer. Ib is the extinction fluorescence of the DNA-bound Ru(phen)32+. The value of Ib is obtained from the saturated DNA-bound Ru(phen)32+. Because the thrombin aptamer used in this work is 27-mer ssDNA and one of its 20-mer complementary ss-DNAs is used, the so-called “ds-DNA” contains 20 base pairs (bps) double strand part and a part of the 7-mer ss-DNA in fact. The interaction between this ds-DNA and Ru(phen)32+ should include the interaction of Ru(phen)32+ into the double strand part and the electrostatic interaction between Ru(phen)32+ and the single strand part. The increased fluorescence intensity and the red shift of the maximum emission wavelength with the increase in the concentration of dsDNA indicated a strong interaction between ds-DNA and

2+

Figure 4. (A) Cyclic voltammograms of Ru(phen)3 -ds-DNA composite film electrode in 0.2 M phosphate buffer solution (pH 7.5) at different scan rates: (a) 0.01; (b) 0.05; (c) 0.1; (d) 0.15; (e) 0.2; (f) 0.25; (g) 0.3 V/s. (B) The relationship between the anodic peak currents and the scan rate.

Ru(phen)32+. Carter and Bard7a suggested that the interaction was from the intercalation of Ru(phen)32+ into the double strand part. Using eqs 1 and 2, the calculated ratio between base pair of double strand part and Ru(phen)32+, i.e., the site size, was 4:1. After Ru(phen)32+ was totally intercalated into the double strand part of the DNA structure, no increase in fluorescence emission and a red shift in maximum emission wavelength were observed even when DNA concentration was increased after the concentration of ds-DNA reached 4 µM. The phenomena shows that the electrostatic interaction between Ru(phen)32+ and ss-DNA is weak and has no obvious affect on fluorescence. The fluorescence titration results indicated that the ds-DNA containing 20 bps could intercalate five Ru(phen)32+ molecules. From Figure 3, we also find the shift point in the profile of fluorescence-Cds-DNA is at the ratio of [Ru(phen)32+]/[ds-DNA] of 10:2. This also confirmed that one ds-DNA strand could intercalate with five Ru(phen)32+ molecules. The calculated binding constant K between Ru(phen)32+ and the ds-DNA is 1.24 × 104 M-1, similar to that obtained by Carter and Bard.7a Compared with the previous single-site labeling,2,3,4b-e the intercalation of Ru(phen)32+ into dsDNA can increase the number of the probe molecule to five, which can be used to improve sensitivity of the ECL aptasensor. The CVs of the Ru(phen)32+-ds-DNA composite film electrode in PBS at different scan rates is used to investigate the redox behavior of Ru(phen)32+. Ru(phen)32+ oxidation current increases when the scan rate is increased, but its oxidation potentials are almost constant as shown in Figure 4. The oxidation current is linearly proportional to the potential scan rate ranging from 10 to 300 mV s-1. The results indicated that the Ru(phen)32+ electrochemical reaction is a surface-controlled process and Ru(phen)32+ is stably intercalated to the DNA film. The hybrid ds-DNA structure was considered to transfer electrons at a large rate constant,2c,10a which also facilitated Ru(phen)32+ oxidation. ECL and the Analytical Performance of the Aptasensor. Typical examples of CVs and ECL of the Ru(phen)32+-ds-DNA composite film electrode before and after reacting with 5 pM thrombin are shown in Figure 5. The decreased anodic peak currents achieved the difference of ca. 1850 counts in ECL emission. Figures S7 and S8 in the Supporting Information presented the CV responses to scan rate before and after Ru(phen)32+-ds-DNA composite film electrode is reacted to 5 pM thrombin. The results indicated that once TPA was added into the electrolyte, the electrochemical process was diffusion-controlled. In comparison with the results from Figure S2a in the Supporting Information,

Figure 5. CV (A) and ECL profiles (B) of the Ru(phen)32+-ds-DNA composite film electrode before (a) and after (b) reacting with 5 pM thrombin. Scan rate: 50 mV/s.

the oxidation current in Figure 2d was mainly from the TPA oxidation although all compounds attached on the electrode surface were possibly oxidized.11 The ECL signal was obtained from the Ru(phen)32+ being catalytical oxidized by TPA. The ECL change, rapid and direct electron transfer, and the relevance of the redox reaction to Ru(phen)32+ and TPA form a strong basis for the development of ECL aptasensors. On the basis of the above discussion, the possible ECL mechanisms at the Ru(phen)32+-double-strand DNA composite film electrode were proposed as followed. The protonated TPAH+ was adsorbed on the anionic phosphate backbone of DNA via electrostatic interaction between TPAH+ and phosphate ions. The proton was then transferred from TPAH+ to phosphate ions in the backbone of DNA sequence. The backbone of DNA provides alkaline conditions and makes the TPA oxidization similar to that at high pH.9b Hence, the DNA molecule acts as the substrate to preconcentrate TPA and the acceptor of proton released from TPAH+.7a,9,10 The ECL mechanism on the composite film electrode was proposed as follows on the basis of the above results and previous research.7a,9,10 TPAH+(DNA) f TPA•+ + H+(DNA) + e-

(3)

TPA•+ f (C3H7)2N(CHC2H5•) + H+

(4)

(Ru(phen)32+ - DNA) - e- f (Ru(phen)33+ - DNA) (5) (Ru(phen)33+ - DNA) + (C3H7)2N(CHC2H5•) f (Ru(phen)32+* - DNA) + [(C3H7)2N ) CHC2H5]+ (6) (Ru(phen)32+ - DNA) + (C3H7)2N(CHC2H5•) f (Ru(phen)3+ - DNA) + products

(6a)

(Ru(phen)3+ - DNA) + TPA•+ f TPA + (Ru(phen)32+* - DNA)

(6b)

(Ru(phen)32+* - DNA) f (Ru(phen)32+ - DNA) + hv (7) (11) (a) Cao, W. D.; Ferrance, J. P.; Demas, J.; Landers, J. P. J. Am. Chem. Soc. 2006, 128, 7572–7578. (b) Wang, J.; Kawde, A.-N. Analyst 2002, 127, 383– 386.

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Figure 6. (A) ECL profiles of thrombin at different concentrations in the present ECL aptasensor: (a) 0; (b) 0.05; (c) 0.5; (d) 5; (e) 10; (f) 50 pM. (B) the calibration curve of the difference of ECL intensity (∆IECL) and logarithm of concentrations of thrombin using the present ECL aptasensor. The error bars show the standard deviation of four replicate determinations. Scan rate: 50 mV/s.

Using the Ru(phen)32+-ds-DNA composite film electrode, the proton transfer from protonated TPAH+ to the phosphate ions in the backbone of DNA makes the direct oxidation of TPA possible at 0.88 V, as shown in eqs 3 and 4. The above reaction results in the formation of an active radical. Following eq 5, Ru(phen)32+ intercalated into the ds-DNA is oxidized to Ru(phen)33+. Furthermore, the active radical of TPA• reacts with Ru(phen)33+ to generate the excited-state Ru(phen)32+*. For the low potential ECL, TPA• active radical reduces Ru(phen)32+ into Ru(phen)3+, which reacts with TPA+• to produce excited-state Ru(bpy)32+* as shown in eqs 6a and 6b. The excited-state Ru(phen)32+* emits photons. The ECL responses of the aptasensor associated with different concentrations of thrombin were measured after the DNAmodified electrode were immersed in 20 mM TPA containing PBS (pH 7.5) and exerted a sweep potential from 0 to 1.25 V. The decrease in ECL emissions was linear with the logarithm of thrombin concentration ranging from 0.05 to 50 pM with a concentration detection limit (3σ) of 0.02 pM as shown in Figure 6. A mass detection limit of 0.2 attomole is achieved with 10 µL of sample. Figure S9 in the Supporting Information shows the ECL profiles of the aptasensor for the determination of 50 pM thrombin under continuous cyclic voltammeter. Although a good reproducibility (RSD, n ) 5:3.4%) is presented, it is difficult for regeneration of the electrode because of the oxidation of thio group for immobilization of the aptamer on the electrode surface. The relative standard deviation for five replicate determinations of thrombin with different electrodes from the same batch at 0.5 pM level was 4.3%, suggesting good reproducibility of the ECL aptasensor. As shown in Table S1 in the Supporting Information, the detection limits of the ECL aptasensor were a few orders of magnitude lower than those of the previous methods for thrombin detection except Fang et al.’s works.2d,4a To improve the sensitivity of aptasensors, some amplification techniques were used previously, such as the use of Pt- or Au-nanoparticles and guanindine denaturing protein for electrochemical detection.2d-g Ru(bpy)32+doped silica nanoparticle was used as the ECL probe to increase the number of Ru(bpy)32+ molecules.4a Compared with the above extra-amplification procedures,2d-g,4a the increase in the number of probe molecules and the obvious improvement on the TPA oxidation were self-integrated in the system, making the analytical procedure simple. 9304

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Figure 7. Specificity of the ECL aptasensor to thrombin (1pM) by comparing it to the interfering agents, including four proteins and two amino acids at the 100 pM level: bovine hemoglobin (BHB), bovine serum albumin (BSA), lysozyme (Lyso), ovalbuin (OB), arginine (arg), and histidine (his). The error bars show the standard deviation of four replicate determinations. Table 1. Analytical Results of Thrombin Added in 1:5 Diluted Fetal Calf Serum added thrombin concn/nM

concn found/nM

RSD/%, n ) 3

recovery/%

0.0500 0.500 10.0 50.0

0.0450 0.462 9.20 50.4

6.6 5.7 5.3 2.0

90.0 92.4 92.0 100.8

Anti-Interference and Application to the Real Sample of the ECL Aptasensor. To show the binding specificity of the ECL aptasensor to thrombin, a series of comparative studies between thrombin and other interfering agents, such as bovine hemoglobin (BHB), bovine serum albumin (BSA), lysozyme (Lyso), ovalbuin (OB), arginine (arg), and histidine (his), were carried out. Thrombin (1 pM) shows a much stronger response, while the other interfering agents (at 100 pM level) have almost negligible ECL changes (Figure 7), validating that the present aptasensor responds to its target with high specificity. No evidence for the binding of those interferences to the sensor shows its good selectivity for thrombin array. The results in Figure 7 and Figure S10 in the Supporting Information suggest that the apatmers are a good recognition element for the development of biosensor as their excellent specificity to its targets.

Figure 8. CV (A) and ECL profiles (B) of the Ru(phen)32+-ds-DNA composite film electrode before (a) and after (b) reacting with 1 pM lysozyme. Scan rate: 50 mV/s.

There are two types of potential interferences in the present ECL aptasensor. One is the interference to the destruction of dsDNA construction, resulting in the release of the intercalated Ru(phen)22+. Because the foreign species tested do not interact to the antithrombin aptamer, the interference of the foreign species on the dissociation of the ds-DNA structure is negligible. However, nucleases should be removed from the sample before determination using the aptasensor because they can destroy the DNA structure of the aptamer. Figure S10 in the Supporting Information presents the response results of various interfering species on the ECL aptasensor constructed by the use of antilysozyme aptamer. The results shown in Figure 7 and Figure S10 in the Supporting Information validate that the interference from the dissociation of ds-DNA can be eliminated as the excellent specificity of aptamer to its target. The other is interference to the interaction between TPA and phosphate backbone of ds-DNA, which affects significantly the TPA oxidation and, therefore, ECL emission. We find that, if the electrode is not cleaned thoroughly after being incubated in protein solutions, a poor reproducibility of ECL emission is observed. The material adsorbed on the electrode surface may influence the environment of phosphate backbone of ds-DNA for TPA oxidation. However, this interference can be simply eliminated via soaking the electrode in PBS and cleaning it to decrease the adsorbed foreign species because of the weak interaction between the nonspecific species and the 2-mercaptoethanol single layer. The thrombin in fetal calf serum was detected to test the possibility of the determination of thrombin in real sample. Because no thrombin was found in the serum sample, the serum 1:5 diluted with TE buffer was spiked with thrombin at different concentrations. After 10 µL of the serum sample spiked with thrombin was dropped on the Ru(phen)32+-ds-DNA composite film electrode surface and incubated at 36 °C for 1.5 h, the electrode was washed thoroughly with 0.2 M PBS to reduce the nonspecific binding. The analytical results (Table 1 and Figure S11 in the Supporting Information) show the acceptable relative standard deviation and quantitative recoveries, implying that the present aptasensor has a promising feature for the analytical application in complex biological samples. Generalization of the Protocol Using Ru(phen)32+ Interaction into ds-DNA. The present sensing protocol can be easily extended to the determination of other proteins, and the gener-

alization of the protocol was validated by analysis of lysozyme. Once the antilysozyme aptamer and its complementary ss-DNA, instead of those for thrombin, were used, the analysis of lysozyme was achieved. The CVs and ECL of lysozyme aptasensor in 0.2 M PBS containing 20 mM TPA (pH 7.5) before and after reacting to 5 pM lysozyme are shown in Figure 8. The calibration curve for lysozyme determination using the present ECL-based protocol is presented in Figure S12 in the Supporting Information. CONCLUSION In conclusion, we present a simple, selective, and sensitive label-free ECL aptasensor using the intercalation of Ru(phen)32+ into ds-DNA. Similar to the electrochemical intercalator-based DNA sensors,6 the present ECL aptasensor does not reasonably need the labeling of aptamer or the target with the probe. The present ECL-based protocol has a number of advantages: (a) low detection limits, with 0.05 pM thrombin being detected; (b) simple experimental procedures using the intercalation of Ru(phen)32+ into ds-DNA, with no chemical labeling to DNA or the target and the modifying steps needed; (c) broadspectrum practicability, with the intercalation of Ru(phen)32+ into the ds-DNA sequence used to develop other sensitive DNA sensors for a wide range of analytes. The method can be easily extended to those aptamers without specific conformational change during the hybridization between the aptamer and its target because this protocol is independent of the conformation change of the aptamer. ACKNOWLEDGMENT This work is supported by National Nature Science Funding of China (No. 90717104) and the Program for New Century Excellent Talents in University (NCET-06-0214), the Chinese Ministry of Education. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text (the evidence of the direct oxidation of TPA at the ds-DNA-assembled electrode, Figures S1-S12 and Table S1). This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 19, 2009. Accepted September 29, 2009. AC901609G

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