Reusable Electrochemical Sensing Platform for Highly Sensitive

Reusable Electrochemical Sensing Platform for. Highly Sensitive Detection of Small Molecules. Based on Structure-Switching Signaling Aptamers. Zai-She...
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Anal. Chem. 2007, 79, 2933-2939

Reusable Electrochemical Sensing Platform for Highly Sensitive Detection of Small Molecules Based on Structure-Switching Signaling Aptamers Zai-Sheng Wu, Meng-Meng Guo, Song-Bai Zhang, Chen-Rui Chen, Jian-Hui Jiang, Guo-Li Shen,* and Ru-Qin Yu*

State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, P. R. China

Aptamers are nucleic acids that have high affinity and selectivity for their target molecules. A target may induce the structure switching from a DNA/DNA duplex to a DNA/target complex. In the present study, a reusable electrochemical sensing platform based on structureswitching signaling aptamers for highly sensitive detection of small molecules is developed using adenosine as a model analyte. A gold electrode is first modified with polytyramine and gold nanoparticles. Then, thiolated capture probe is assembled onto the modified electrode surface via sulfur-gold affinity. Ferrocene (Fc)-labeled aptamer probe, which is designed to hybridize with capture DNA sequence and specifically recognize adenosine, is immobilized on the electrode surface by hybridization reaction. The introduction of adenosine triggers structure switching of the aptamer. As a result, Fc-labeled aptamer probe is forced to dissociate from the sensing interface, resulting in a decrease in redox current. The decrement of peak current is proportional to the amount of adenosine. The present sensing system could provide both a wide linear dynamic range and a low detection limit. In addition, high selectivity, good reproducibility, stability, and reusability are achieved. The recovery test demonstrates the feasibility of the designed sensing system for an adenosine assay. Aptamers are a new class of synthetic DNA/RNA oligonucleotides with characteristic 3-D structures obtained from randomsequence nucleic acid libraries by an in vitro evolution process called SELEX (systematic evolution of ligands by exponential enrichment ). Aptamers are able to recognize their targets with high affinity and specificity and by and large undergo the targetinduced conformational changes. Moreover, aptamers exhibit a number of advantages such as simple synthesis, easy labeling, good stability, and wide applicability. Thus, aptamers are becoming promising recognition probes for protein analysis, disease diagnosis, new drug selection, biosensor and molecular switch development, etc. Despite the efforts of a few groups, attempts to manufacture a simple and convenient colorimetric sensing system * To whom correspondence should be addressed. E-mail: [email protected]. Phone: 86-731-8821355. Fax: (+86) 731-8821355. E-mail: [email protected]. Fax: +86-7318822577. 10.1021/ac0622936 CCC: $37.00 Published on Web 03/06/2007

© 2007 American Chemical Society

based on an aptamer probe have not obtained a high sensitivity.1-3 On the other hand, as fluorescent “signaling aptamers” possess adequate transducing elements to generate physically detectable signals from the recognition events, an impressive number of inventive designs for the aptamer-based fluorescence-signaling systems have emerged.4,5 Some research activities are to covalently attach fluorophores at specific locations of aptamers.6-8 The conformational change of aptamers upon target binding causes a change in the fluorescence property of the attached fluorophores. However, a small fluorescence response to targets is usually obtained. Additionally, this method is not easy to generalize due to the difficulty in successfully designing a desirable signaling aptamer. Therefore, numerous aptamer-based analyte detections are based on “aptamer beacons” that are similar to molecular beacons. One strategy is based on the splitting of an aptamer containing the natural binding site for an analyte into half-sites, each of which is labeled with a fluorophore or a quencher.9,10 The other is to use an intact aptamer labeled with a fluorophore-quencher pair at the two ends to fabricate an aptamer beacon.11 In the two cases, the introduction of a specific analyte induces the conformational change of aptamer that leads to a change in fluorescence. Unfortunately, only the aptamer with a unique secondary structure that contains a long stretch of paired nucleotides can maintain its biochemical recognition activity after being split into two molecules; the aptamer beacon based on an intact aptamer may also lose its binding activity due to the change in the correct tertiary folding triggered by the hairpin structure (an additional folded structure).11 Therefore, to overcome these problems, not only should an intact aptamer probe be preferably (1) Liu, J.; Lu, Y. Anal. Chem. 2004, 76, 1627-1632. (2) Stojanovic, M. N.; Landry, D. W. J. Am. Chem. Soc. 2002, 124, 9678-9679. (3) Liu, J.; Lu, Y. Angew. Chem. 2006, 118, 96-100. (4) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J. Am. Chem. Soc. 2001, 123, 4928-4931. (5) Heyduk, E.; Heyduk, T. Anal. Chem. 2005, 77, 1147-1156. (6) Fang, X.; Cao, Z.; Beck, T.; Tan, W. Anal. Chem. 2001, 73, 5752-5757. (7) Jhaveri, S.; Kirby, R.; Conrad, R.; Maglott, E.; Bowser, M.; Kennedy, R. T.; Glick, G.; Ellington, A. D. J. Am. Chem. Soc. 2000, 122, 2469-2473. (8) Jhaveri, S.; Rajendran, M.; Ellington, A. D. Nat. Biotechnol. 2000, 18, 12931297. (9) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J. Am. Chem. Soc. 2000, 122, 11547-11548. (10) Yamamoto, R.; Baba, T.; Kumar, P. K. Genes Cells 2000, 5, 389-396. (11) Hamaguchi, N.; Ellington, A.; Stanton, M. Anal. Biochem. 2001, 294, 126131.

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used to prepare an aptamer-based sensing system but also its secondary structure should be retained. Although an alternative transduction strategy based on a unimolecular reverse molecular beacon has been described, the fluorophore and the quencher at the two ends of aptamer beacon do not move away from each other due to the intramolecular folded structure even in the absence of analytes.4,12 Electrochemical detection systems provide possibly an alternative detection approach to the fluorescencesignaling aptamer probe. Thus, electrochemical methods for the screening of the interaction between aptamer and specific ligands have recently been reported by many investigators.13-17 Yet, only a few aptamer-based sensing platforms have been proposed so far for the detection of small molecules.18,19 Adenosine is an endogenous nucleoside with potent vasodilator and antiarrhythmic activities. Adenosine has received much attention due to its crucial signaling functions in both the peripheral and central nervous system. In the peripheral nervous system, it is involved in the control of smooth muscle contraction, the regulation of cerebral20 and ocular blood flow,21 and is a powerful vasodilator.22 In the central nervous system, adenosine plays a well-established modulatory role of neurotransmission and serves as a neuroprotective agent against ischemic- and seizureinduced neuronal injury.23 Moreover, adenosine is the core of the cell’s energy-containing compound, ATP. Elevated levels of adenosine in the brain appear to cause sleep. There is also good evidence that it has some important function in the immune system.24 The detection of adenosine therefore is of great value. Ferrocene (Fc), an electroactive molecule, and its derivatives have been attractive candidates for developing electrochemical probes due to their good stability, convenient synthetic access, and ease of redox tuning.25-27 Nucleic acid labeled with Fc can successfully mark a hybridization event and plays a key role in the detection of DNA hybridization, point mutations, DNA lesions, gene sequencing, and DNA-analyte interaction.28-31 (12) Yang, C. J.; Jockusch, S.; Vicens, M.; Turro, N. J.; Tan W. Proc. Natl. Acad. Sci. U.S.A. 2005; 102, 17278-17283. (13) Xu, D.; Xu, D.; Yu, X.; Liu, Z.; He, W.; Ma, Z. Anal. Chem. 2005, 77, 51075113. (14) Radi, A.-E.; Acero Sanchez, J. L.; Baldrich, E.; O’Sullivan, C. K. Anal. Chem. 2005, 77, 6320-6323. (15) Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Angew. Chem., Int. Ed. 2005, 44, 5456-5459. (16) Xiao, Y.; Piorek, B. D.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2005, 127, 17990-17991. (17) Radi, A.-E.; Acero Sanchez, J. L.; Baldrich, E.; O’Sullivan, C. K. J. Am. Chem. Soc. 2006, 128, 117-124. (18) Zayats M.; Huang, Y.; Gill, R.; Ma, C.-a.; Willner, I. J. Am. Chem. Soc. 2006, 128, 13666-13667. (19) Baker, B. R.; Lai, R. Y.; Wood, M. S.; Doctor, E. H.; Heeger, A. J.; Plaxco, K. W. J. Am. Chem. Soc. 2006, 128, 3138-3139. (20) Phillis, J. W. Cerebrovasc. Brain Metab. Rev. 1989, 1, 26-54. (21) Portellos, M.; Riva, C. E.; Cranstoun, S. D.; Petrig, B. L.; Brucker, A. J. Invest. Ophthalmol. Vis. Sci. 1995, 36, 1904-1909. (22) McMillan, M. R.; Burnstock, G.; Haworth, S. G. Br. J. Pharmacol. 1999, 128, 543-548. (23) Dunwiddie, T. V.; Masino, S. A. Annu. Rev. Neurosci, 2001, 24, 31-55. (24) “Damping the flames: inflammation control mechanism determined by NIH researchers”, to be found under http://www.aarda.org/infocus_article.php, 2002. (25) Zu, X.; Rusling, J. F. Langmuir. 1997, 13, 3693-3699. (26) D’Souza, F.; Zandler, M. E.; Smith, P. M.; Deviprasad, G. R.; Arkady, K.; Fujitsuka, M.; Ito, O. J. Phys. Chem. A 2002, 106, 649-656. (27) Laforge, F. O.; Kakiuchi, T.; Shigematsu, F.; Mirkin, M. V. J. Am. Chem. Soc. 2004, 126, 15380-15381. (28) Cardona, C. M.; Kaifer, A. E. J. Am. Chem. Soc. 1998, 120, 4023-4024.

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Herein, a reusable electrochemical sensing platform for highly sensitive detection of small molecules using adenosine as a model analyte was developed based on a structure-switching aptamer with a Fc terminus. Using this system, the adenosine could be specifically detected, and a relatively low detection limit as well as a wide linear dynamic range could be achieved. Other performance characteristics, for example, storage stability, were also evaluated. Furthermore, the regeneration of the used sensing interface could be performed successfully by thermal dehybridization in hot water. The optimal conditions for the detection of adenosine were investigated. EXPERIMENTAL SECTION Chemicals. Oligonucletides designed according to the literature3,32 in the present study were synthesized by Generay Biotech Co., Ltd. (Shanghai, China), and sequences of all oligonucleotides are listed in Scheme 1A. Adenosine, cytidine, uridine, and guanosine were all purchased from Generay Biotech Co., Ltd. Tyramine and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) were received from Sigma. N-Hydroxysuccinimide (NHS), cystamine dihydrochloride, and ferrocenemonocarboxylic acid were obtained from Acros Organics. Glutaraldehyde (25% aqueous solution) was supplied by Sinopharm Chemical Reagents Co., Ltd. (Shanghai, China). All other chemicals used were of analyticalreagent grade and were used without further purification. All samples at various concentrations were prepared by dissolving analytes in 0.3 M NaCl and 10 mM PBS at pH 7.4 (0.3 M PBS). Triply distilled water (resistance >18 MΩ‚cm) was used throughout the experiments. Synthesis of Gold Nanoparticles. All glassware used in the following procedures were cleaned in a bath of freshly prepared solution (HNO3/HCl, 1:3 by volume), followed by washing with distilled water, and dried prior to use. Gold nanoparticles were prepared according to the literature.33 In general, 1 mL of 1.0% trisodium citrate was immediately added to 100 mL of 0.01% HAuCl4 refluxing solution under stirring, and the mixture was kept boiling for another 10 min. The solution color turned to a deep wine red, indicating the formation of gold nanoparticles. Activation of the COOH Group of Ferrocencemonocarboxylic Acid. The COOH group of ferrocenemonocarboyxylic acid was activated according to the method described in ref 34 with a minor modification. Briefly, 1 mg of ferrocenemonocarboxylic acid was added to 2 mL of 0.3 M PBS containing EDC/ NHS (0.1 M each). The reaction mixture was maintained under stirring for 2 h. Preparation of Fc-Labeled Probe. The process of aptamer probe labeling was performed as follows: first, 50 µL of the aptamer probe solution was diluted to 500 µL with 0.3 M PBS solution; then, 500 µL of activated ferrocenemonocarboxylic acid solution was added. After reacting at room temperature overnight, the mixture was stored in refrigerator at 4 °C until use. (29) Yu, C. J.; Wang, H.; Wan, Y.; Yowanto, H.; Kim, J. C.; Donilon, L. H.; Tao, C.; Strong, M.; Chong, Y. J. Org. Chem. 2001, 66, 2937-2942. (30) Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2002, 124, 770772. (31) Gibbs, J. M.; Park, S. J.; Anderson, D. R.; Watson, K. J.; Mirkin, C. A.; Nguyen, S. T. J. Am. Chem. Soc. 2005, 127, 1170-1178. (32) Chin, H. L.; Dinshaw, J. P. Chem. Biol. 1997, 4, 817-832. (33) Huang, M. F.; Kuo, Y. C.; Huang, C. C.; Chang, H. T. Anal. Chem. 2004, 76, 192-196. (34) Dai, J.; Baker, G. L.; Bruening, M. L. Anal. Chem. 2006, 78, 135-140.

Fabrication of Sensing Interface. The gold electrode was polished with a 0.05-µm alumina powder and soaked in an ultrasonic bath successively with distilled water, absolute alcohol, and distilled water for 5 min each. Then, the gold electrode was dipped in piranha solution (H2SO4/H2O2, 7:3 by volume) for 20 min and electrochemically treated by cycling the potential between -1.0 and +1.55 V in 0.1 M H2SO4 for 10 min. Finally, the electrode was washed with distilled water and dried in a nitrogen stream. Electropolymerization of tyramine was carried out in an unstirred methanolic solution containing 0.2 M NaOH and 0.1 M tyramine by cycling the potential between 0 and 1.5 V at a rate of 50 mV/s.35 After rinsing thoroughly with distilled water, the polytyramine (Pty)-modified electrode was dipped in a 2.5% glutaraldehyde (GA) solution for 1 h and then was coated with 40 µL of cystamine dihydrochloride (20 mM) casting solution. After reduction of the imine group of the Schiff base and residual aldehyde group on the electrode surface with NaBH4, the gold electrode was immerged in the gold nanoparticle solution for 12 h to form a gold nanoparticle layer. Afterward, the modified electrode was covered with 40 µL of capture probe solution for 2 h followed by immersing in mercaptoethanol solution (1 mM) for 1 h to cover the nonspecific sites. Finally, a solution of Fc-labeled aptamer probe was dropped onto the surface of the electrode and allowed to react for 60 min to obtain the sensing interface. When not in use, the resulting electrode was stored in distilled water at room temperature. Electrochemical Detection of Adenosine. Various samples at a specific concentration (20 µL) were pipetted onto a sensing interface and allowed to react for 90 min. An ac voltammogram was recorded at a specific frequency in 5 mL of 1.0 M NaCl4 solution, and the peak current was used to evaluate the response characteristics of sensing interface. Cyclic voltammetry (CV) was performed at a scan rate of 100 mV/s in 5 mL of 10 mM PBS (pH 7.4) containing 5 mM Fe(CN)63-/Fe(CN)64- and 0.1 M KCl. All electrochemical measurements were carried out at room temperature in a laboratory-made measuring cell using a CHI 760B electrochemical workstation (Shanghai, China). The threeelectrode system used is consisted of the working electrode of interest, a saturated calomel reference electrode, and a platinum foil auxiliary electrode. Regeneration of Sensing Interface. After each adenosine detection, the used sensing interface could be regenerated readily by thermal dehybridization. Following incubation in hot water at 80 °C for 10 min, the electrode was transferred to fresh water heated to the same temperature, and the water was cooled slowly back to room temperature over 20 min. Subsequently, 40 µL of aptamer probe solution was placed on the electrode surface, and the hybridization reaction was allowed to proceed for 60 min. The resulting electrode cleaned with water was ready for use in another assay. RESULTS AND DISCUSSION Fabrication of Sensing System and Experimental Principle. In the present study, to fabricate a reusable electrochemical sensing platform for highly sensitive detection of small molecules, an intact aptamer labeled with Fc, a thiolated DNA sequence, and (35) Wu, Z. S.; Li, J. S.; Deng, T.; Luo, M. H.; Shen, G. L.; Yu, R. Q. Anal. Biochem. 2005, 337, 308-315.

adenosine were used as the detection probe, the capture probe, and the model analyte, respectively. Fc-labeled aptamer probe had the capacity to recognize adenosine with high affinity and specificity. Scheme 1A displays the design concept of Fc-labeled aptamer probe for adenosine assay. The aptamer probe comprises three segments: the first segment (in black) is a five-base segment close to the 5′-terminal that could hybridize with capture DNA sequence close to the 3′-terminal; the second segment (in gray) is the aptamer sequence for adenosine, whose seven-base segment could hybridize with the 5′-terminal seven-base segment of capture sequence; the third segment is a Fc label, which is coupled at 3′-terminus and employed as the electrochemical signal producer. The preparation of sensing interface and the adenosine detection are shown in Scheme 1B. The surface of a gold electrode was first modified with Pty film using voltammetry, followed by attaching cystamine to Pty film using GA as cross-linker. Then, after freshly prepared NaBH4 solution was used to reduce the imine group of Schiff base, the gold nanoparticle (GNP) layer was assembled via sulfur-gold affinity. Subsequently, a 3′-thiolmodified DNA strand (capture sequence) was immobilized on the resulting electrode surface. Finally, the hybridization of Fc-labeled aptamer probe with capture sequence was carried out, and the sensing interface was ready for adenosine assay. When the sensing interface was immersed into an analyte solution, the interaction between adenosine and detection probe resulted in the displacement of detection probe from the electrode surface while capture probe was still bound to electrode surface (see also the bottom part of Scheme 1B). The sensing interface was obtained again by hybridizing the detection probe with the capture probe that could still “catch” the regenerated electrode surface. An aptamer can bind tightly and specifically to a variety of small molecules to form a tertiary complex with a binding constant greater than that of an ordinary DNA duplex.36 We made use of the structure switching between DNA/DNA duplex and DNA/ target complex to design the aptamer probe. In the absence of adenosine, the aptamer probe was naturally bound to the surfaceimmobilized capture strand and electroactive Fc was brought close to the electrode surface. When an appropriate voltage was applied, Fc label was oxidized from Fe(II) to Fe(III) as it transferred electrons to the electrode surface. Therefore, a high peak current was acquired. In contrast, when adenosine as a specific competitor appeared, the aptamer preferred to form an adenosine-aptamer complex rather than an aptamer-DNA duplex. The aptamer probe would leave the capture probe and went off, which caused a peak current decrease. Namely, a current response was obtained. Accordingly, a novel adenosine electrochemical sensing system was successfully engineered. Eletropolymerization of Tyramine and Signal Amplification. To acquire a reproducible and stable immobilization matrix for the fabrication of a sensing interface, the electropolymerization of tyramine on an electrode surface was carried out using potential scanning.37 Cyclic voltammograms of a gold electrode in tyramine solution are described in Supporting Information Figure 1. A high and irreversible oxidation peak of tyramine was observed in the first scan, whereas a remarkable decrease appeared in the next (36) Green, L. S.; Jellinek, D.; Jenison, R.; Ostman, A.; Heldin, C. H.; Janjic, N. Biochemistry 1996, 35, 14413-14424. (37) Situmorang, M.; Gooding, J. J.; Hibber, D. B.; Barnett, D. Biosens. Bioelectron. 1998, 13, 953-962.

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Scheme 1. Design of Adenosine Sensing Interfacea

a (A) Function of Fc-labeled aptamer probe. (a) Detection probe sequence; (b) capture probe sequence. (B) Preparation PrLLLLocess and Operating Principle.

scan, and then the oxidation peak tended to alter only slightly. The results suggested that the electrode surface was almost completely passivated by the polymer film and further electropolymerization was hindered. Hence, the surface was entirely coated with a compact polymer layer.35,37 Since the performance of a sensing interface is related to specific surface modification, the current response of two sensing interfaces obtained using different fabricating procedures was investigated. One sensing interface was gold electrode/Pty/ cystamine/GNP/capture probe/Fc-labeled aptamer probe (electrode a), and the other was gold electrode/capture probe/Fclabeled aptamer probe (electrode b). The experimental results are shown in Figure 1. Compared with the binding approach based 2936

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on the combined use of the electropolymerized film of tyramine and gold nanoparticles, the method of assembling a capture probe directly on the gold electrode surface presented only 32% of peak current. Namely, the combined use of an electropolymerized film of tyramine and gold nanoparticles might induce a peak current enhancement of more than 300%. The amount of capture probe sequence on the electrode surface might be estimated from the peak current in ac voltammogram using eq 1,38

Iavg(Eo) ) 2nf FNtot[sinh(nFEac/RT)]/[cosh(nFEac/RT) + 1] (1) where Iav(Eo) is the average ac peak current in the voltammogram,

Figure 1. The ac voltammograms recorded with different sensing interfaces after being exposed to 1 mM adenosine in 0.3 M PBS. (a) Capture sequence was immobilized on the gold electrode surface modified with Pty/cystamine/GNPs; (b) the same as (a) without Pty/ cystamine/GNPs.

n is the number of electrons transferred per redox event, f is the frequency of the applied ac voltage perturbation, F is the Faraday constant, Ntot is the total number of moles of redox-active species giving rise to the peak, Eac is the peak amplitude, R is the universal gas constant, and T is the temperature. Assuming that the hybridization efficiency was 100% and all of the Fc labels adsorbed on the electrode surface were electrochemically active, the capture sequence surface coverage for electrode a is 3.1 × 1013 molecules/ cm2, which exceeded the general range of DNA surface coverage reported in the literature,39 and that for electrode b is 9.8 × 1012 molecules/cm2. The real surface of the gold electrode used in our experiment was calculated as ∼62.8 × 10-3 cm2 from the corresponding charge for the reduction of the oxide monolayer. Thus, gold electrode/Pty/cystamine/GNP/capture sequence was selected to fabricate a highly sensitive sensing system. Cyclic Voltammetric Characterization. Cyclic voltammetry of electroactive species in a conducting aqueous solution is a valuable mean of probing the electrochemical characterization of the modified gold electrode.40,41 As shown in Figure 2, well-defined CV (Figure 2a) could be observed even though GNPs, which had negative surface charges and exhibited electrostatic repulsion of ferri/ferrocyanides with negative charges, were chemisorbed on the surface of the modified electrode via the strong affinity of thiol groups and gold. A current decrease (Figure 2b) appeared after exposing to capture DNA strand solution. It is well-known that a self-assembled monolayer of thiolated DNA sequence as an electron-transfer blocking layer can hinder the diffusion of ferricyanide toward the electrode surface.42 Apparently, a selfassembled layer of capture probe was obtained. The hybridization (38) Sumner, J. J.; Weber, K. S.; Hockett, L. A.; Creager, S. E. J. Phys. Chem. B 2000, 104, 7449-7454. (39) Jordan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939-4947. (40) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (41) Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1990, 6, 682-691. (42) Cho, M.; Lee, S.; Han, S.-Y.; Park, J.-Y.; Rahmen, M. A.; Shim, Y.-B.; Ban. C. Nucleic Acids Res. 2006, 34, e75.

Figure 2. Cyclic voltammograms of the modified electrode at different stages in 10 mM PBS (pH 7.4) containing 5 mM Fe(CN)63-/ Fe(CN)64- and 0.1 M KCl. (a) GNP-modified electrode; (b) Capture sequence-immobilized electrode; (c) Fc-labeled aptamer probe sequence-immobilized electrode.

Figure 3. Effect of working frequency on the electrochemical response of Fc-labeled aptamer probe-modified electrode in 1.0 M NaClO4.

of Fc-labeled aptamer probe with capture probe on the electrode surface led to the further decrease of electron-transfer efficiency (seen in Figure 2c) due to the introduction of additional negative surface charges. These results demonstrated that aptamer probe was successfully fixed on the electrode and a sensing interface was obtained. Experimental Conditions for Adenosine Detection. The influence of working frequency on the electrochemical response of the present sensing system was studied over the range of 0.5500 Hz. The Fc-aptamer probe-modified electrode was dipped in 1.0 M NaClO4 solution to monitor its ac voltammetry behavior. The plot of Ipeak/Ibackground (the peak/background current ratio) versus the logarithm of the frequency was used to determine the optimum frequency, where Ipeak and Ibackground are peak current of ferrocene redox reaction and background current, respectively. Figure 3 displays that the peak/background current ratio changed slightly with the increasing working frequency from 0.5 to 1.0 Hz, while it decreased sharply at higher frequency. Finally, the value of current ratio tended to 1, showing that the peak current nearly Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

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Figure 4. Current response of sensing system to adenosine at various concentrations. Inset: the linear relationship between the difference of peak current and adenosine concentration.

disappeared. It was possibly attributed to the fact that the redox reaction eventually could not keep pace with the rapidly fluctuating potential at the high frequency.43 So, a frequency of 1 Hz was selected throughout this work. The incubation time for hybridization between capture probe strands and Fc-labeled aptamer probe strands was investigated as it may influence the hybridization efficiency and the succeeding response to adenosine. Supporting Information Figure 2 shows the current response to 1 mM adenosine for the sensing interface obtained by incubating gold electrode/Pty/cystamine/GNP/ capture probe in the aptamer probe solution for different periods of time. The decrement of peak current (∆Ipeak) increased with increasing the hybridization time and reached equilibrium over ∼40 min. To ensure the completeness of hybridization between capture probe sequences and Fc-labeled aptamer probe sequences, it is rational that 60 min is set as the hybridization time in whole experiments. The introduction of adenosine onto the sensing interface would arouse a decrease in peak current. It was found that a different assembling time of adenosine might cause a visible difference in the decrease of peak current. Therefore, the dependence of time on the adenosine-induced peak current decrease was studied to determine the optimum assembling time of adenosine. The electrochemical signal response was recorded by ac voltammetry and the results were shown in Figure 3 in Supporting Information. A peak current decreased immediately when the adenosine was introduced and then tended to stabilize after more than 80 min. Considering the fact that the specific interaction of aptamer with adenosine at a lower concentration needs more time, 90 min was chosen as the adenosine assembling time. Detection of Adenosine. The introduction of adenosine at different concentrations to the sensing interface induced different decreases in peak current associated with the amount of the released Fc-labeled aptamer probe. Series samples of 1.0 × 10-81.0 × 10-4 M adenosine were determinated by the present sensing system, and the difference of peak current (∆Ipeak) was utilized (43) Sumner, J. J.; Creager, S. E. J. Phys. Chem. B 2001, 105, 8739-8745.

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to evaluate the current response to adenosine. As given in Figure 4, ∆Ipeak decreased monotonously as the concentration of adenosine increased, and the adenosine could be quantified perfectly over a concentration range of 1.0 × 10-7-1.0 × 10-5 M. Such linear range is 10 times wider than those published in previous literature.1,3 The calibration equation was ∆Ipeak ) -0.0267C 0.1103 with a correlation coefficient of 0.9965 (shown in the inset of Figure 4). The detection limit was 2.0 × 10-8 M based on a signal-to-noise ratio of 3, indicating a high sensitivity. For example, the detection limit obtained using the present sensing system is 500-fold lower than the literature value achieved using label-free and reagentless aptamer-based sensor18 and more 3 orders of magnitude lower than the detection limit reported using a colorimatric method based on gold nanoparticle aggregation.1 Selectivity of Sensing System. Guanosine, cytidine, and uridine, which belong to the nucleosides family, were examined for their interference effect. Figure 5 exhibits different current response signals of the proposed sensing system after the addition of 1 mM adenosine, guanosine, cytidine, or uridine under the same experimental conditions. A significant decrease induced by the interaction of the aptamer probe and adenosine was observed compared to the other three nucleoside samples, indicating that the developed strategy had a sufficient specificity and adenosine could be unequivocally identified. This may be explained by the fact that the Fc label has not initiated the biodegradation and denaturation of aptamer and the splitting of the aptamer into two segments as well as an additional folded structure is eliminated in the present strategy. As a result, the recognition activities (including selectivity) of aptamer might be maintained. Reusability and Stability of Sensing Interface. The designed sensing interface could be regenerated with ease by immerging in hot water. The signal response recorded by ac voltammetry recovered up to ∼94, 79, and 63% of the original signal response under identical conditions after washing 30, 60, and 90 times, respectively. Apparently, the current response to adenosine lost ∼0.2% after each regeneration of the electrode; a relatively rapid loss of current response was observed when the sensing interface

Table 1. Recovery of Adenosine Detection at Different Concentrations

Figure 5. The ac voltammetry of the sensing system after being exposed to four nucleosides at 1 mM in 0.3 M PBS. A, adenosine; C, cytidine; U, uridine; G, guanosine.

was regenerated more than 30 times; 90% of the original signal response, which was generally acceptable, was retained even though the regeneration of the sensing interface was carried out 40 times. The long-term stability of the sensing interface is an important issue for the development and practical implementation of adenosine detection. Therefore, the storage stability of the proposed system has been investigated. The modified electrode, which was stored in distilled water at 4 °C over 30 days, could still be used for the measurement of adenosine without significant change of current response. Thus, the developed biosensor would attain a sufficient stability. Recovery Test. The recovery experiment of different adenosine concentrations was carried out to evaluate applicability and reliability of the developed sensing system. All the measurements were performed four times, and the results are shown in Table 1. It was seen that the recoveries of the added samples and the relative standard derivations were in the range of 91-106 and 4.16.7%, respectively. Satisfactory recovery test values were realized within the linear concentration range. The experimental results confirm that the proposed sensing system is applicable for adenosine detection.

sample

added

found

recovery (%)

RSD (%)

1 2 3 4

4.0 × 10-7 1.6 × 10-6 3.2 × 10-6 6.4 × 10-6

3.6 × 10-7 1.7 × 10-6 3.0 × 10-6 6.2 × 10-6

91 106 94 97

5.4 6.7 3.5 4.1

CONCLUSIONS A novel strategy for the construction of an electrochemical sensing system for highly sensitive adenosine detection is described. The introduction of adenosine results in the dissociation of the Fc-labled aptamer probe from the sensing interface, and the redox current decreases. The decrement of peak current linearly correlated with the concentration of adenosine over a range of 1.0 × 10-7-1.0 × 10-5 M with a detection limit of 2.0 × 10-8 M. The sensing platform may offer several advantages: the high reproducible and stable coating associated with the electropolymerized Pty film; a relatively large surface area resulting from the gold nanoparticle layer; the availability of ferrocene moieties serving as electroactive labels; the substantial decrease in residual peak current due to the target-induced detection probe displacement. Therefore, excellent characteristics, including sensitivity, selectivity, reproducibility, and stability, could be achieved. Additionally, the present sensing system exhibits other distinct advantages, such as easy to operate, a low dosage of material, and low-cost electrochemical measurement. The recovery test also demonstrated the feasibility of the designed sensing system for adenosine assay. The proposed technique provides a promising method for aptamer-based small-molecule detection due to its simplicity, rapidity, low cost, and excellent response characteristics. ACKNOWLEDGMENT The work was financially supported by the National Natural Science Foundation of China (Grants 20675028, 20435010, 20205005, 20375012), and the Science Commission of Hunan Province. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 2, 2006. Accepted February 4, 2007. AC0622936

Analytical Chemistry, Vol. 79, No. 7, April 1, 2007

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