Anal. Chem. 2005, 77, 6320-6323
Reusable Impedimetric Aptasensor Abd-Elgawad Radi,*,† Josep Lluı´s Acero Sa´nchez,‡ Eva Baldrich,‡ and Ciara K. O’Sullivan‡,§
Department of Chemistry, Faculty of Science, Mansoura University, 34517 Dumyat, Egypt, Nanobiotechnology and Bioanalysis Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Tarragona, Spain, and Institucio´ Catalana de Recerca i Estudis Avanc¸ ats, Passeig Lluı´s Companys 23, 08010 Barcelona, Spain
A novel impedimetric aptasensor using a mixed selfassembled monolayer composed of thiol-modified thrombin binding aptamer and 2-mercaptoethanol on a gold electrode is reported. The changes of interfacial features of the electrode were probed in the presence of the reversible redox couple, Fe(CN)63-/4-, using impedance measurements. The electrode surface was partially blocked due to the self-assembly of aptamer or the formation of the aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by electrochemical impedance spectroscopy or cyclic voltammetry. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution, and the immobilized aptamer subsequently was used for repeated detection of thrombin. The aptamer-functionalized electrode showed a linear response of the charge-transfer resistance to the increase of thrombin concentration in the range of 5.0-35.0 nM and the thrombin was easily detectable to a concentration of 2.0 nM. Organized, surface-confined monolayers are commonly employed to assign desired chemical or physical properties to surfaces.1-3 With respect to biosensors, immobilization strategies for biomolecules are of paramount importance in order to preserve their biological activity. Self-assembled monolayers (SAMs), which are spontaneously formed from sulfur-containing compounds in contact with the gold surface, provide a convenient basis for biomolecule immobilization capable of specific molecular recognition for sensing applications.4-6 Electrochemical impedance spectroscopy (EIS) is a rapidly developing electrochemical technique for the investigation of bulk and interfacial electrical properties of any kind of solid or liquid material connected to or part of an appropriate electrochemical * To whom correspondence should be addressed. E-mail:
[email protected]. Tel: 34-977-558722. Fax: 34-977-559667. † Mansoura University. ‡ Universitat Rovira i Virgili. § Institucio ´ Catalana de Recerca i Estudis Avanc¸ ats. (1) Ulman, A. Chem. Rev. 1996, 96, 1533. (2) Liu, Z. Amiridis, M. D. Colloids Surf., B 2004, 35, 197-203. (3) Miura, Y.; Sasao, Y.; Dohi, H.; Nishida, Y.; Kobayashi, K. Anal. Biochem. 2002, 310, 27-35. (4) Luppa, P. B.; Sokoll, L. J.; Chan, D. W. Clin. Chim. Acta 2001, 314, 1-26. (5) Ferretti, S.; Paynter, S.; Russell, D. A.; Sapsford, K. A.; Richardson, D. J. TrAC. Trends. Anal. Chem. 2000, 19, 530-540. (6) Jiang, L.; Glidle, A.; Griffith, A.; McNeil, C. J.; Cooper, J. M. Bioelectrochem. Bioenerg. 1997, 42, 15-23.
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transducer.7 Any intrinsic property of a material or a specific process that could affect the interfacial properties of an electrochemical system can potentially be studied by EIS.8,9 Thus, EIS is well suited to monitor the different stages necessary for biosensor fabrication, its characterization, and detection of the recognition event when the immobilized molecule and its ligand (the analyte) interact. EIS can also be utilized as an analytical tool for the measurement of electric property changes of the biosensor in the presence of increasing concentrations of the analyte. Several papers have been published based on the use of the impedance transduction on enzyme-based biosensors,10-12 immunosensors,13-19 DNA hybridization,20-22 biotin-avidin interaction,23 and cellular growing behavior,24 indicating the immense power of the technique for use in biosensors. Due to the combination of their chemical characteristics and method of production, aptamers appear as excellent biorecognition elements with great potential in the future development of analytical devices. In this context, aptamers present significant advantages over other recognition molecules, such as antibodies, (7) Katz, E.; Willner, I. Electroanalysis 2003, 15, 913-947. (8) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamental and Applications; Wiley: New York, 1980. (9) McDonald, J. R. Impedance Spectroscopy: Emphasizing Solid Materials and Systems; Wiley: New York, 1987. (10) Calvo, E. J.; Etchenique, R.; Danilowicz, C.; Diaz, L. Anal. Chem. 1996, 68, 4186-4193. (11) Mirsky, M.; Krause C.; Heckmann, K. D. Thin Solid Films 1996, 284/285, 939-941. (12) Saum, G. E.; Cumming, R. H.; Rowell, F. J. Biosens. Bioelectron. 1998, 13, 511-518. (13) DeSilva, M. S.; Zhang, Y.; Hesketh, P. J.; Maclay, G. J.; Gendel, S. M.; Stetter, J. R. Biosens. Bioelectron. 1995, 10, 675-682. (14) Rickert, J.; Gopel, W.; Beck, W. B.; Jung, G.; Heiduschka, P. Biosens. Bioelectron. 1996, 11, 757-768. (15) Maupas, H.; Soldatkin, A. P.; Martelet, C.; Jaffrezic, N.; Mandrand, B. J. Electroanal. Chem. 1997, 421, 165-171. (16) Patolsky, F.; Filanovsky, B.; Katz, E.; Willner, I. J. Phys. Chem. B 1998, 102, 10359-10367. (17) Patolsky, F.; Zayats, M.; Katz, B.; Willner, I. Anal. Chem. 1999, 71, 31713180. (18) Knichel, M.; Heiduschka, P.; Beck, W.; Jung, G.; Gopel, W. Sens. Actuators, B 1995, 28, 85-94. (19) Ameur, S.; Martelet, C.; Jaffrezic-Renault, N.; Chovelon, J. M. Appl. Biochem. Biotechnol. 2000, 89, 161-170. (20) Bardea, A.; Patolsky, R.; Dagan, A.; Willner, I. Chem. Commun. 1999, 21, 21-22. (21) Patolsky, F.; Katz, E.; Bardea, A.; Willner, I. Langmuir 1999, 15, 37033706. (22) Athey, D.; Ball, M.; McNeil, C. J.; Armstrong, Electroanalysis 1995, 7, 270273. (23) Lee, T.-Y.; Shim, Y.-B. Anal. Chem. 2001, 73, 5629-5632. (24) Ehret, R.; Baumann, W.; Brischwein, M..; Schwinde, A.; Stegbauer, K.; Wolf, B. Biosens. Bioelectron. 1997, 12, 29-41. 10.1021/ac0505775 CCC: $30.25
© 2005 American Chemical Society Published on Web 08/25/2005
for example their small size, chemical simplicity, and production reproducibility. In addition, aptamers can be easily modified in order to incorporate molecular markers or favor immobilization, can be produced and used under nonphysiological conditions, and can be reversibly denatured, allowing the optimization of reusable devices.25-27 Previously existing label-free biosensoric platforms have been successfully applied to the detection of specific aptamer-protein interactions including plasmon surface resonance, piezoelectric transduction, resonant oscillating quartz sensor, and a micromachine cantilever.28-34 In addition, optical detection of aptamer-protein interactions has also been reported using fluorescence35 or evanescent wave-induced fluorescence anisotropy,36 and it is being exploited by Pharmacon Microelectronics to develop a sensor to detect theophylline.37 The thrombin-binding aptamer (TBA, 5′-GGTTGGTGTGGTTGG-3′) was the first in vitro selected aptamer targeted toward a protein with not known physiological binding to nucleic acids38 and, due to its potential applicability for anticlotting therapeutics, has been extensively studied.39-41 This aptamer has also been used for the optimization of a variety of enzyme-linked aptamer assays42,43 and molecular beacons.44,45 In addition, the specific interaction between thrombin and its binding aptamer has been investigated in depth, and for example, the colorimetric detection of the aptamer-thrombin interactions was followed with polythiophene polyelectrolyte chromophores.46 The use of an aptamerfunctionalized Au NP as a catalytic label for the amplified detection of thrombin in solution and on surfaces has also been explored.47 Two methods for detection of thrombin-aptamer interaction (25) Jayasena, S. D. Clin. Chem. 1999, 45, 1628-1650. (26) Hesselberth, J.; Robertson, M. P.; Jhaveri, S.; Ellington, A. D. Rev. Mol. Biotechnol. 2000, 74, 15-25. (27) James, W. Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; John Wiley and Sons Ltd.: Chichester. 2000. (28) Kawakami, J.; Hirofumi, I.; Yukie, Y.; Sugimoto, N. J. Inorg. Biochem. 2000, 82, 197-206. (29) Hartmann, R.; Norby, P. L.; Martensen, P. M.; Jorgensen, P.; James, M. C.; Jacobsen, C.; Moestrup, S. K.; Clemens, M. J.; Justesen, J. J. Biol. Chem. 1998, 273, 3236-3246. (30) Kraus, E.; James, W.; Barclay, A. N. J. Immunol. 1998, 160, 5209-5212. (31) Liss, M.; Petersen, B.; Wolf, H.; Prohaska, E. Anal. Chem. 2002, 74, 44884495. (32) http://www.mathematik.de/mde/information/forschungsprojekte/ biosensor/technbg.html. (33) http://www.caesar.de/568.0.html. (34) Savran, C. A.; Knudsen, S. M.; Ellington, A. D.; Manalis, S. R. Anal. Chem, 2004, 76 (11), 3194-3198. (35) Kleinjung, F.; Klussman, S.; Erdmann, V. A.; Scheller, F. W.; Furste, J. P.; Bier, F. F. Anal. Chem. 1998, 70, 328-331. (36) Potyrailo, R. A.; Conrad, R. C.; Ellington, A. D.; Hieftje, G. M. Anal. Chem. 1998, 70, 3419-3425. (37) http://www.pharmacom.us/machine.rna.html. (38) Bock, L. C.; Griffin, L. C.; Latham, J. A.; Vermaas, E. H.; Toole, J. J. Nature 1992, 355, 564-566. (39) Macaya, R. F.; Waldron, J. A.; Beutel, B. A.; Gao, H. T.; Joesten, M. E.; Yang, M. H.; Patel, R.; Bertelsen, A. H.; Cook, A. F. Biochemistry 1995, 34, 4478-4492. (40) Schultze, P.; Macaya, R. F.; Feigon, J. J. Mol. Biol. 1994, 235, 1532. (41) Padmanabhan, K.; Padmanabhan, K. P.; Ferrara, J. D.; Sadler, J. E.; Tulinsky, A. J. Biol. Chem. 1993, 268, 17651-17654. (42) Baldrich, E.; Restrepo, A.; O’Sullivan, C. K. Anal. Chem 2004, 76, 70537063. (43) Drolet, D. W.; Moon-McDermott, L.; Romig, T. S. Nat. Biotechnol. 1996, 14, 1021-1025. (44) Hamaguchi, N.; Ellington, A.; Stanton, M. Anal. Biochem. 2001, 294, 126131. (45) Li, J. J.; Fang, X.; Tan, W. Biochem. Biophys. Res. Commun. 2002, 292, 31-40. (46) Ho, H. A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384-1387.
based on electrochemical measurements were reported,48,49 but to date, no work on the impedimetric detection of aptamer-target has appeared in the literature. We report here the fabrication of an impedimetric aptasensor based on a SAM of thiolated TBA on a gold electrode surface for the recognition of thrombin. The immobilization of recognition layer or recognition event introduces electrical insulation and kinetic-transfer barriers at the electrode surface. The transduction principle for following the modification steps or recognition event is based on the electron-transfer resistances in the presence of [Fe(CN)6]4-/3- redox couple, measured by electrochemical impedance spectroscopy. The present study continues a recent effort of our laboratory to apply aptamers to the development of reagentless and real-time assays and sensors. EXPERIMENTAL SECTION Reagents and Solutions. The TBA (5′-GGTTGGTGTGGTTGG-3′), HPLC purified and lyophilized TBA modified with a SH at the 3′ end (TBA-SH) and DNA (5′-GGG-GTT-TTG-GGG-C3-SH 3′, DNA-SH), was provided by Cultek, S.L.(Eurogentec, S.A.). Pure Human thrombin was supplied by Haematologic Technologies Inc. (Essex Junction). Apparatus. An Autolab model PGSTAT20 potentiostat/galvanostat (Eco Chemie), controlled by GPES4 and FRA software, were used for acquisition and analysis of the electrochemical data. A C 3 stand with a three-electrode configuration, a gold electrode (BAS model MF-2014, 1.6-mm diameter, 2.0-mm2 geometrical area) working electrode, an Ag-AgCl-3 M NaCl (BAS model MF-2078) reference electrode, and a platinum wire (BAS model MW-1032) counter electrode, was used. Procedure. Prior to aptamer immobilization, the gold electrode was cleaned in piranha solution (v/v 3:1 H2SO4/H2O2) at 60 °C for 1 h, rinsed thoroughly in deionized water, and dried under flowing nitrogen gas. (Warning: piranha solution is highly reactive and may explode on coming into contact with organic solvents. Extreme precautions must be taken at all times.) For immobilization of aptamer, a 50-µL solution of 0.5 µM thiolmodified TBA in 1.0 M KH2PO4 buffer solution was placed on a gold electrode held upside-down, and the end of the electrode was fitted with a plastic cap to protect the solution from evaporation. The assembly was kept standing overnight at room temperature. The unmodified regions of the electrode were then blocked with 2-mercaptoethanol (2-ME) using 50 µL of 0.1 M solution in KH2PO4 buffer for 10 min. Fifty microliters of different concentrations of thrombin in HEPES buffer (consisting of 10 mM N-(2hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), adjusted with 1.0 M NaOH to pH 8.0) was then placed on the electrode, which was protected from drying with a plastic cap, and the assembly kept at room temperature for 10 min. After each step, the electrode was rinsed thoroughly with deionized water and then dried with a stream of nitrogen. All electrochemical measurements were performed in HEPES buffer solution as background electrolyte. Electrochemical imped(47) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768-11769. (48) Hianik, T.; Ostatna, V.; Zajacova, Z.; Stoikova, E.; Evtugyn, G. Bioorg. Med. Chem. Lett. 2005, 15, 291-295. (49) Ikebukuro, K.; Kiyohara, C.; Sode, K. Biosens. Bioelectron. 2005, 20, 21682172.
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Figure 1. Cyclic voltammograms of 20.0 mM [Fe(CN)6]4-/3- probe in 10 mM HEPES buffer solution of pH 8.0 at scan rate of 100 mV/s for bare Au electrode (a), Au/TBA-modified electrode (b), Au/TBA/ 2-ME-functionalized electrode (c), and after formation of aptamerthrombin complex at the surface of the Au/TBA/2-ME functionalized electrode from 15.0 nM thrombin solution for 10 min (d).
ance measurements were performed in the presence of equimolar concentrations, [Fe(CN)6]3- ) [Fe(CN)6]4- ) 20 mM as redox probe at the formal redox potential, using a sinusoidal ac potential perturbation of 5 mV, in the frequency range 100 kHz to 500 mHz, and readings were taken at 20 discrete frequencies per decade. The impedance spectra were plotted in the form of Nyquist plots. All electrolyte solutions were produced by means of deionized water, and nitrogen was bubbled through the solutions for 15 min. The regenerating agent used was 2.0 M NaCl, applied for 10 min, prior to washing with HEPES buffer. RESULTS AND DISCUSSION Cyclic voltammetry. Figure 1 shows superimposed cyclic voltammograms of the oxidation/reduction of a 20 mM solution of [Fe(CN)6]4-/3- couple using a scan rate of 100 mV s-1 in HEPES buffer at bare Au (a), Au/TBA (b), and Au/TBA/2-ME (c) electrodes and after the formation of the aptamer-thrombin complex at the Au/TBA/2-ME electrode surface from 15.0 nM thrombin solution for 10 min (d). At the electrode covered by TBA, the electrochemical reaction is inhibited. The peak-to-peak separation increases, and a substantial decrease in the peak current is also observed. The self-assembly of the aptamer on the electrode surface generates a negatively charged interface that repels negatively charged [Fe(CN)6]4-/3- anions. This repulsion is anticipated to retard the interfacial electron-transfer kinetics of the redox probe at the electrode interface. The peak-to-peak separation of the reaction at Au/TBA/2-ME is almost the same as that at the Au/TBA electrode with a less significant decrease in the current, indicating that the 2-ME monolayer has a negligible effect on blocking interfacial electron transfer. It is expected that 2-ME, as a short-chain alkyl molecule, forms a self-assembled monolayer that contains many pinholes and the redox probe is sufficiently small to freely penetrate through these pinholes.50 Almost complete disappearance of the Faradaic current is observed after binding of thrombin to aptamer at the electrode surface. The associated bulky thrombin molecules provide an effective barrier to electron transfer of the redox species in solution. (50) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey; C. E D. J. Am. Chem. Soc. 1987, 109 (12), 3559-3568.
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Figure 2. Impedance spectra (Zim vs Zre, Nyquist plot) of bare Au electrode (a), Au/TBA-modified electrode (b), Au/TBA/2-ME-functionalized electrode (c), and after formation of aptamer-thrombin complex at the surface of the Au/TBA/2-ME-functionalized electrode from 15.0 nM thrombin solution for 10 min (d), in the presence of 20 mM [Fe(CN)6]4-/3-probe in 10 mM HEPES buffer solution of pH 8.0. The biased potential was 0.192 V vs Ag-AgCl; the frequency was from 100 kHz to 500 mHz and the amplitude was 5.0 mV.
Electrochemical Impedance Spectroscopy. Figure 2 shows the results of ac impedance spectroscopy at bare Au (a), Au/TBA (b), and Au/TBA/2-ME (c) electrodes and after the formation of the aptamer-thrombin complex at the Au/TBA/2-ME electrode surface in the presence of equimolar [Fe(CN)6]4-/3- (d), respectively. The Randle modified equivalent circuit51 was used to fit the impedance spectroscopy and to determine electrical parameter values for each step. As shown in Figure 2 (inset), the circuit includes the electrolyte resistance between working and reference electrodes (Rs), Warburg impedance (Zw), resulting from the diffusion of ions to the interface from the bulk of the electrolyte, electron-transfer resistance (Ret), and the constant phase element Q (instead of the double layer capacitance, Cdl) to take into account the frequency dispersion often related directly to electrode roughness.52,53 The bare gold electrode shows a very small semicircle domain implying very fast electron-transfer process with a diffusional limiting step (Figure 2, curve a). The self-assembly of a negatively charged aptamer on the electrode surface effectively reduces the response of the [Fe(CN)6]4-/3- anions and thus leads to enhanced electron-transfer resistance. This is reflected by the appearance of the semicircle part of the impedance spectrum with Ret ) 465 Ω (b). The addition of the 2-ME results in a relatively small electron-transfer resistance Ret (341 Ω, c). The thiol-derivatized aptamer is adsorbed specifically through the sulfur atom of the thiol group and nonspecifically through the nitrogen-containing bases. After treatment with 2-ME, the nonspecifically adsorbed aptamer is largely removed from the surface and only one end of the aptamer is bound to the surface with all the relevant bases freely available for reaction with thrombin.54 Such conformation along with the less densely packed monolayer of 2-ME could account for the decrease in the value of electrontransfer resistance Ret of the TBA/2-ME-functionalized electrode (51) Sluyters-Rehbach, M.; Sluyters, J. H. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1970; Vol. 4. (52) Levie, R. de, J. Electroanal. Chem. 1989, 261, 1-9. (53) Levie, R. de, J. Electroanal. Chem. 1990, 281, 1-21. (54) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792.
compared with the TBA-modified electrode. After the recognition reaction d of the TBA/2-ME-functionalized electrode with 15.0 nM thrombin for 10 min, there is an increase of Ret (934 Ω), originating from the blocking of the electrode surface with the bulky thrombin molecules. The electrode coverage rate, as θ ) Ret (Au)/Ret (Au modified)55,56 gave values of 27, 19, and 54% for Au/TBA-, Au/TBA/2-ME-modified electrodes and after thrombin association to the aptamer at the Au/TBA/2-ME-functionalized electrode surface, respectively. These results are in agreement with the cyclic voltammetric data shown in Figure 1. Control experiments were performed to reveal the selectivity and specificity of the recognition reaction. In one experiment, when 20 nM bovine serum albumin instead of thrombin is used, no obvious changes occur in the impedance response of the TBA/ 2-ME-functionalized electrode. A second control experiment was performed to ensure that thrombin did not bind to other sequences of DNA. We immobilized thiol-derivatized, singlestranded DNA (ssDNA-SH), which forms a random quadruplex structure. Thereafter, the impedance was recorded for the ssDNA/ 2-ME-functionalized electrode and after exposure to 20 nM thrombin for 10 min. No significant difference in the impedance spectrum could be observed, and the probe showed a decrease in the electron-transfer resistance Ret of ∼4.0% after exposure to thrombin, implying that the ssDNA/2-ME-functionalized Au electrode did not produce a complex with thrombin and, therefore, did not lead to significant changes in the impedance spectrum. Aptasensor Regeneration. The impedance spectra of the Au/ TBA/2-ME-functionalized electrode shows that, as the concentrations of thrombin are increased, the interfacial electron-transfer resistances Ret are enhanced. Thus, the electron-transfer resistance could be used as the detectable signal to quantify analyte presented to the electrode surface. Figure 3 shows that the electron-transfer resistance increased as the concentration of thrombin increased in the analyzed sample and then plateaued at a constant value at higher concentrations. The sensor exhibits a linear dependence on the concentration ranging from 5.0 to 35.0 nM, and the thrombin was easily detectable to a concentration of 2.0 nM. With respect to clinical application, during the initial phase of coagulation, thrombin is produced in nanomolar concentrations; thus the linear range of the aptasensor is clinically relevant.57,58 To break the aptamer-thrombin complex and to regenerate the electrode after each addition of thrombin, 2.0 M NaCl was applied for 10 min. The interfacial electron-transfer resistances Ret observed for the TBA/2-ME-functionalized electrode was diminished after regeneration and the main EIS features being reversed after the process. Reassociation has restored the increased Ret. The cycle of capture, release, and analysis can be repeated at least 15 times at the same aptamer-functionalized electrode with no significant change in the electrode behavior. (55) Blank, M.; Voddyanoy, I. Biomembr. Electrochem. 1994, 235, 491-510. (56) Sabatini, E.; Cohen-Boulakia, J.; Bruening, M.; Rubinstein, I. Langmuir 1993, 9, 2974-2981. (57) Butenas, S.; Mann, K. G. Biochemistry (Moscow) 2002, 67, 3-12 (translated from Biokhimiya 2002, 67, 5-15). (58) Spiridonova, V.A and Kopylov, A. M. Biochemistry (Moscow) 2002, 67, 850854 (translated from Biokhimiya 2002, 67, 706-709). (59) Baldrich, E.; Acero, J. L.; Reekmans, G.; Laureyn, W.; O’Sullivan, C. K. Anal. Chem. 2005, 77, 4774-4784.
Figure 3. Impedance measurements (Zim vs Zre, Nyquist plot) corresponding to sensing of the Au/TBA/2-ME-functionalized electrode to variable concentration of thrombin: (a) 5.0, (b) 15.0, (c) 25.0, and (d) 35.0 nM. Inset: calibration plot corresponding to the electrontransfer resistance (Ret) of the modified electrode with the concentrations of thrombin in 20 mM Fe(CN)64-/3- redox probe solution in 10 mM HEPES of pH 8.0. The other conditions as in Figure 2.
This behavior clearly indicated the potential for repeated use of the interface as a practical sensor. Stability studies of immobilized aptamer on solid support previously carried out by our group59 have indicated that, in the presence of the stabilizing agent trehalose (1% w/v), the immobilized thrombin-binding aptamer was stable for at least 90 days when stored at 4 °C. CONCLUSION We have described a reusable aptamer based on the SAM of thiolated aptamer on a gold electrode surface. The immobilized aptamer retains its bioactivity and could be recognized by the thrombin. The aptamer immobilization step or the recognition event retards the electron-transfer kinetics of the redox probe at the electrode interface, which have been examined by cyclic voltammetry and impedance spectroscopy. The developed aptasensor proved to be specific, reproducible, and reusable in the detection of thrombin. The work described here, to the best of the authors’ knowledge is the first demonstration of the powerful coupling of the molecular recognition property of aptamers with transduction based on impedance measurement, and current work is focused on the development of an impedance-based reusable electrochemical molecular aptamer beacon. ACKNOWLEDGMENT This work has been carried out with financial support from the Commission of the European Communities, specific RTD program ‘Quality of Life and Management of Living Resources’, project QLK6-CT-2002-02583, ‘Rapid Stroke Marker Detection via immunosensors utilizing Labeless Electrochemical and Resonant Mass Detection’. It does not necessarily reflect its views and in no way anticipates the Commission’s future policy in this area. A.-E.R. is supported by a Fellowship from the Ministerio de Educacion, Cultura y Deporte (Spain). E.B. is supported by a RED fellowship from the Generalitat de Catalunya (Spain). Received for review April 5, 2005. Accepted July 27, 2005. AC0505775
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