Electrochemical Aptameric Recognition System for a Sensitive Protein

Feb 15, 2010 - linear padlock probe, and primer probe were utilized to introduce a RCA process into the aptamerrtarget binding event while a new aptam...
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Anal. Chem. 2010, 82, 2282–2289

Electrochemical Aptameric Recognition System for a Sensitive Protein Assay Based on Specific Target Binding-Induced Rolling Circle Amplification Zai-Sheng Wu,* Hui Zhou, Songbai Zhang, Guoli Shen,* and Ruqin Yu State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China A reusable aptameric recognition system was described for the electrochemical detection of the protein PDGFBB based on the target binding-induced rolling circle amplification (RCA). A complementary DNA (CDNA), linear padlock probe, and primer probe were utilized to introduce a RCA process into the aptamer-target binding event while a new aptamer was elegantly designed via lengthening the original aptamer by the complement to the CDNA. The aptameric sensing system facilitates the integration of multiple functional elements into a signaling scheme: a unique electrochemical technique, an attractive RCA process, reversible DNA hybridization, and desirable aptameric target recognition. This RCA-based electrochemical recognition system not only exhibits excellent performance (e.g., a detection limit of 6.3 × 10-11 M, a linear dynamic range of 2 orders of magnitude, high specificity, and satisfactory repeatability) but also overcomes the limitations associated with conventional aptameric biosensors (e.g., dependence of signaling target binding on specific aptamer sequence or requirement of sandwich assays for two or more binding sites per target molecule). A recovery test demonstrated the feasibility of the developed target protein assay. Given the attractive characteristics, this aptameric recognition platform is expected to be a candidate for the detection of proteins and other ligands of interest in both fundamental and applied research. Proteins are functional units of all living organisms. The recognition and quantification of proteins are of particular importance in fundamental research and corresponding applications such as medical diagnosis and treatment. While antibody-based protein assay systems for specific antigens are versatile and powerful tools in these areas, they are faced with considerable challenges in some cases.1,2 As complements to antibody-based detection methodologies, molecular probes based on aptamerprotein recognition have gained increasing attention. * To whom correspondence should be addressed. Phone: 86-731-8821355. Fax: (+86) 731-8821355. E-mail: [email protected]; [email protected]. (1) Heyduk, E.; Heyduk, T. Anal. Chem. 2005, 77, 1147–1156. (2) Schweitzer, B.; Wiltshire, S.; Lambert, J.; O’Malley, S.; Kukanskis, K.; Zhu, Z.; Kingsmore, S. F.; Lizardi, P. M.; Ward, D. C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10113–10119.

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Aptamers are artificial, short, single-stranded DNA/RNA oligonucleotides isolated from random-sequence nucleic acid libraries by an in vitro evolution process called SELEX (systematic evolution of ligands by exponential enrichment). Aptamers are able to recognize a variety of different targets3 with specific recognition properties, equal to or often superior to those of antibodies.4 Besides their attractive binding performance, other significant advantages over traditional protein antibodies are achieved,5,6 endowing aptamers with great potential for numerous applications in protein analysis. Meanwhile, the aptamer-ligand recognition event can be easily amplified by nucleic acid sequencebased amplification protocols, such as the polymerase chain reaction (PCR) and rolling circle amplification (RCA), making an extraordinarily low detection limit possible.7,8 Additionally, original or adapted aptamers can undergo conformational changes upon target binding, usually offering a good starting point for the development of signaling probes and sensing designs.9-20 Via manipulation of the secondary structure of an aptamer, research(3) Famulok, M.; Hartig, J. S.; Mayer, G. Chem. Rev. 2007, 107, 3715–43. (4) So, H.; Won, K.; Kim, Y. H.; Kim, B.; Ryu, B. H.; Na, P. S.; Kim, H.; Lee, J. J. Am. Chem. Soc. 2005, 127, 11906–11907. (5) Jayasena, S. D. Clin. Chem. 1999, 45, 1628–1650. (6) Yang, C. J.; Jockusch, S.; Vicens, M.; Turro, N. J.; Tan, W. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17278–17283. (7) Fredriksson, S.; Gullberg, M.; Jarvius, J.; Olsson, C.; Pietras, K.; Gustafsdottir, S. M.; Ostman, A.; Landegren, U. Nat. Biotechnol. 2002, 20, 473–477. (8) Zhou, L.; Ou, L. J.; Chu, X.; Shen, G. L.; Yu, R. Q. Anal. Chem. 2007, 79, 7492–7500. (9) Hamaguchi, N.; Ellington, A.; Stanton, M. Anal. Biochem. 2001, 294, 126– 131. (10) He, F.; Tang, Y.; Wang, S.; Li, Y.; Zhu, D. J. Am. Chem. Soc. 2005, 127, 12343–12346. (11) Liu, J.; Lee, J. H.; Lu, Y. Anal. Chem. 2007, 79, 4120–4125. (12) Katilius, E.; Katiliene, Z.; Woodbury, N. W. Anal. Chem. 2006, 78, 6484– 6489. (13) Stojanovic, M. N.; Kolpashchikov, D. M. J. Am. Chem. Soc. 2004, 126, 9266–9270. (14) Du, Y.; Li, B.; Wei, H.; Wang, Y.; Wang, E. Anal. Chem. 2008, 80, 5110– 5117. (15) Zuo, X.; Song, S.; Zhang, J.; Pan, D.; Wang, L.; Fan, C. J. Am. Chem. Soc. 2007, 129, 1042–1043. (16) Xiao, Y.; Piorek, B. D.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2005, 127, 17990–17991. (17) Xiao, Y.; Lubin, A. A.; Heeger, A. J.; Plaxco, K. W. Angew. Chem., Int. Ed. 2005, 44, 5456–5459. (18) Lu, Y.; Li, X. C.; Zhang, L. M.; Yu, P.; Su, L.; Mao, L. Q. Anal. Chem. 2008, 80, 1883–1890. (19) Zhang, S.; Xia, J.; Li, X. Anal. Chem. 2008, 80, 8382–8388. (20) 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. 10.1021/ac902400n  2010 American Chemical Society Published on Web 02/15/2010

ers can make the probe change its conformation from one conformation in the absence of the cognate analyte to another conformation in the presence of the analyte, generating a detectable response signal. The desirable properties make aptamers hold great promise for molecular recognition elements. Since its discovery, the aptamer as a novel molecular tool has received tremendous attention.6,9,15,17,18,21-27 Albeit substantial progress has been accomplished, a major disadvantage of aptamer probes is that their relatively low association constant to the corresponding analytes often resulted in a low detection limit. Thus, it is essential to develop amplification paths for aptamer-based sensing systems, and several impressive works have been reported.28-31 Recently, target recognitioninduced conformational changes were used to trigger the RCA32,33/PCR34 process or the machinelike operation cycles between replication and scission35 to accomplish the amplified detection of target-aptamer complexes. Although the PCR-based strategy in principle offers extremely high sensitivity and wide quantitative dynamic range, the technique is considered too complicated for the diagnostic.36 Therefore, RCA, often used for enzymatic analysis and amplification for detecting sets of gene sequences, is attracting increasing attention in aptamer-based sensing systems for protein detection as an alternative approach.8,37 Besides high selectivity and sensitivity, the RCA-based assay exhibits several distinct advantages.38 For example, the RCA method is an isothermal amplification procedure rather than thermal cycling, avoiding the requirement for the thermal circling. The RCA protocol can be readily adapted to diverse detection platforms and is suitable for parallel or high-throughput analysis. Additionally, this amplification protocol is applicable to the direct quantification of target analytes without reference to multiple standard samples because the RCA reaction proceeds according to the linear kinetic model. Because of the high sensitivity, inherent simplicity, portability, and low cost derived from elec(21) Heyduk, T.; Heyduk, E. Nat. Biotechnol. 2002, 20, 171–176. (22) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J. Am. Chem. Soc. 2001, 123, 4928–4931. (23) Takenaka, S.; Ueyama, H.; Nojima, T.; Takagi, M. Anal. Bioanal. Chem. 2003, 375, 1006–1010. (24) Su, S.; Nutiu, R.; Filipe, C. D. M.; Li, Y.; Pelton, R. Langmuir 2007, 23, 1300–1302. (25) Nutiu, R.; Li, Y. J. Am. Chem. Soc. 2003, 125, 4771–4778. (26) Zayats, M.; Huang, Y.; Gill, R.; Ma, C.; Willner, I. J. Am. Chem. Soc. 2006, 128, 13666–13667. (27) Xiao, Y.; Lai, R.; Plaxco, K. W. Nat. Protoc. 2007, 2, 2875–2880. (28) Gill, R.; Polsky, R.; Willner, I. Small 2006, 2, 1037–1041. (29) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Anal. Chem. 2006, 78, 2268– 2271. (30) Pavlov, V.; Xiao, Y.; Shlyahovsky, B.; Willner, I. J. Am. Chem. Soc. 2004, 126, 11768–11769. (31) Hansen, J. A.; Wang, J.; Kawde, A.; Xiang, Y.; Gothelf, K. V.; Collins, G. J. Am. Chem. Soc. 2006, 128, 2228–2229. (32) Yang, L.; Fung, C. W.; Cho, E. J.; Ellington, A. D. Anal. Chem. 2007, 79, 3320–3329. (33) Cho, E. J.; Yang, L.; Levy, M.; Ellington, A. D. J. Am. Chem. Soc. 2005, 127, 2022–2023. (34) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605–5620. (35) Shlyahovsky, B.; Di, L.; Weizmann, Y.; Nowarski, R.; Kotler, M.; Willner, I. J. Am. Chem. Soc. 2007, 12, 3814–3815. (36) Stro ¨mberg, M.; Go ¨ransson, J.; Gunnarsson, K.; Nilsson, M.; Svedlindh, P.; Strømme, M. Nano Lett. 2008, 8, 816–821. (37) Di Giusto, D. A.; Wlassoff, W. A.; Gooding, J. J.; Messerle, B. A.; King, G. C. Nucleic Acids Res. 2005, 33, e64. (38) Zhao, W.; Ali, M. M.; Brook, M. A.; Li, Y. Angew. Chem., Int. Ed. 2008, 47, 6330–6337.

trochemical techniques, the incorporation of the RCA strategy in the electrochemical aptasensor would offer a unique opportunity for the analysis of biological markers in decentralized studies and point-of-care diagnosis. However, RCA, as well as PCR, combined aptamer assay systems usually require two or more binding sites on each analyte to accomplish the proximity-dependent ligation or the formation of sandwich complex. This assay format is not applicable to the screening of common target molecules, for example, small analytes. Therefore, very few electrochemical RCAbased aptasensors8 are proposed at the present time. Because of the complexity of the conformational structure and ligand binding, it is very difficult to develop a general method for design of the aptameric detection system. Fortunately, regardless of whether the ligands are incorporated into their secondary structure or not, it seems that almost all aptamers (original or adapted sequences) undergo conformational change during ligand binding. In addition to the inherent conformational change, lengthening or shortening the aptamer sequences might make ligand binding induce an adscititious change in secondary structure as shown in several works reported by us39 and other groups.9,22 On the basis of the above considerations, in the present contribution, by utilizing the target binding-induced conformational change to force the release of a complementary sequence from a designed aptamer that can trigger the RCA reaction, we introduce a reusable electrochemical aptameric sensing platform as a combined paradigm of homogeneous target binding/recognition amplification and electrochemical measurements on an interface. Target recognition and the resultant RCA reaction were conducted on a small amount of samples in a homogeneous solution that consists of only simple mixing steps while the signal readout of target-aptamer binding is accomplished by electrochemical measurements on the electrode surface. The former can be implemented in a parallel or high-throughput manner, whereas the latter can be readily adapted to microarrays and the biosensing interface might be easily regenerated. This combined screening strategy offers a reusable aptamer-based biosensing interface and achieves a synergetic effect of easy operation in a homogeneous format, RCA-based signal enhancement, and high sensitivity of the electrochemical technique, achieving an efficient analysis of the protein analyte. In the present sensing scheme, except for the designed aptamer, other probes involved are not directly related to the target binding sites, avoiding the requirement for the specific base sequence. Thus, via lengthening the original aptamer sequence by the complement to the CDNA and regulating the length of the outer helix to achieve a large target bindinginduced conformational change, different aptameric sensing systems could be developed even though the same set of DNA probes are used. Namely, the present RCA-based electrochemical biosensor can be utilized to detect separately various target molecules when different aptamers are adapted using the developed strategy, lowering the cost considerably. In addition, the sensing scheme described herein circumvents the requisite of two binding sites per target protein and could be immediately adapted to almost any aptamer probe. These attractive advantages endow the developed screening platform with additional design flexibility and great operation convenience, making the proposed sensing (39) Wu, Z. S.; Zheng, F.; Shen, G. L.; Yu, R. Q. Biomaterials 2009, 30, 2950– 2955.

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Table 1. Oligonucleotides Synthesized in the Present Studya aptamer1 aptamer2 CDNA padlock probe primer capture probe a

5′-ACTGCCCTGTGGGGCTAGGTGAACGTGGATGAAGTCACACAGGCTACGGCACGTAGAGCATCACCATGATCCTGTGTG-3′ 5′-ACTGCCCTGTGGGGCTAGGTGAACGTGGATGAAGTTTTTCAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3′ 5′-CCAACTTCATCCACGTTCACCTAGCCCCACAGGGCAGTAACG-3′ 5′-P-AGGTGAACGTGGATGAAGTTGGTAGAATGAAGATAGCGC ATCGTAGGACCGTTACTGCCCTGTGGGGCT-3′ 5′-AGGTGAACGTGTTTTTTGCGCTATCTTCA-3′ 5′-SH-(CH2)6CGTTACTGCCCTGTGGGGCT-3′

P in padlock probe represents phosphate at the 5′ end.

scheme a promising protocol for the development of diverse aptameric systems. The preparation and analytical characteristics of the proposed aptameric sensing scheme are detailed in this work. EXPERIMENTAL SECTION Chemicals. Oligonucleotides designed in this work were synthesized by Takara Biotechnology Co., Ltd. (Dalian, China), and their sequences are listed in Table 1. The 5′ end of the padlock probe is phosphorylated while the italicized portions are designed to match with the complementary DNA (CDNA). The underlined portions in two aptamer probes are the specific binding sequences for platelet-derived growth factor (PDGF)-BB while the italicized portions can hybridize with the CDNA sequence. Compared with aptamer2, aptamer1 is extended by four bases at the 3′ end, and “TTTT” is replaced with “CACA” in the middle of this sequence. The capture probe is a 5′-thiolated oligonucleotide. Clearly, the designed aptamer can compete with the padlock probe for CDNA. PDGF-BB was purchased from Tiancheng Biotechnology Co., Ltd. (Shanghai, China). Human IgG, bovine serum albumin (BSA) and human serum albumin (HSA) were obtained from Dingguo Biochemical Reagents Company (Changsha, China). Complement IV (C4) and immunoglobulin E (IgE) purified from human plasma were purchased from the Health Department Shanghai Institute of Biological Products (Shanghai, China) and Meridian Life Science, Inc. (Saco, ME), respectively. Escherichia coli DNA ligase set (Escherichia coli DNA ligase, 10×Escherichia coli DNA ligase buffer, and 10×BSA (0.05%)) and a deoxyribonucleoside 5′-triphosphate mixture (dNTPs) were provided by Takara Biotechnology Co., Ltd. (Dalian, China). Phi29 DNA polymerase, including the 10×Phi29 DNA polymerase buffer, was obtained from Epicenter Technologies (Madison, WI). Stock solutions of proteins and two aptamer probes were prepared with Tris-HCl buffer1 (30 mM Tris-HCl buffer (pH 7.8), 4 mM MgCl2, and 150 mM KCl). dNTP and other oligonucleotides were dissolved in water. Prior to use, capture probe, padlock probe, and CDNA were diluted with Tris-HCl buffer1 by a factor of 10, 2, and 72, respectively. DNA polymerase and ligase solutions were prepared with polymerization buffer (4 mM Tris-HCl, pH 7.5, 5 mM KCl, 1 mM MgCl2, 0.5 mM (NH4)2SO4, 0.4 mM DTT) and ligation buffer (30 mM TrisHCl, pH 7.8, 4 mM MgCl2, 10 mM (NH4)2SO4, 1.2 mM EDTA, and 0.1 mM NAD+), respectively. Methylene blue (MB) (1 mM) was prepared with Tris-HCl buffer2 (30 mM Tris-HCl buffer (pH 7.8), 4 mM MgCl2, and 300 mM KCl). All other chemicals were of analytical-reagent grade and were used without further purification. Deionized and autoclaved water (resistance > 18 MΩ · cm) was used throughout the experiments. Apparatus and Electrochemical Measurements. Alternating current (AC) voltammetric measurements were carried out 2284

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over the potential range from -0.6 to 0.1 V at a frequency of 1 Hz, a sample period of 1.1 s, and an amplitude of 50 mV using a CHI 760B electrochemical workstation (Shanghai, China). The background-subtracted peak currents were presented unless otherwise stated. Gold electrodes exposed to the final reaction solution were used as working electrodes while a platinum foil served as the auxiliary electrode and a saturated calomel electrode (SCE) as the reference. All measurements were conducted in 10 mM phosphate buffer (pH 7.4, 0.1 M KCl) (0.1 M PBS) at room temperature. Fabrication of the Interrogating Electrode. The gold electrode was cleaned according to the literature method.39 To fabricate a capture probe-functioned interface, 20 µL of 1.2 µM capture probe solution was dropped onto the surface of the inverted cleaned electrode, and the self-assembly reaction was allowed to proceed in a humidity chamber at room temperature overnight. Subsequently, the resulting interface was rinsed with copious water to remove the physically adsorbed capture probes. After the remaining bare region was blocked via incubation in 1 mM 6-mercaptohexanol solution for 10 min, the resulting electrode was ready for the analyte detection. RCA-Based Protein Assay. PDGF-BB solution (4 µL) at a certain concentration was mixed with 1 µL of 0.1 µM aptamer, 1 µL of 10 µM dNTP, and 0.6 µL of 10×Phi29 DNA polymerase reaction buffer, and then the aptamer-target binding was allowed to proceed for 40 min. To this solution was added Phi29 DNA polymerase (1 U/µL, 6 µL), and the polymerization (aptamer polymerization) was allowed to proceed for another 40 min, resulting in reaction solution 1. In the meantime, reaction solution 2 was prepared by incubating the mixture of padlock probe (0.7 µM, 1 µL), CDNA (0.1 µM, 1 µL), and 10×E. coli DNA ligase buffer (2 µL) at 67 °C for 40 min followed by cooling to 37 °C to afford the padlock probe-CDNA hybrids. Subsequently, the following steps were followed: (1) a mixture of reaction solution 1 and reaction solution 2 was prepared and was incubated for 40 min to allow competitive hybridization to reach equilibrium; (2) 10×BSA (0.05%, 2 µL) and E. coli DNA ligase (1.8 U/µL, 2 µL) were injected in succession, and the ligation was left to react for 40 min to generate circularized padlock probes. The final volume of the resulting solution (reaction solution 3) was approximately 20.6 µL; (3) 10×Phi29 DNA polymerase reaction buffer (1 µL), dNTP (2.5 µM, 4 µL), and primer probe (3.4 µM, 1 µL) were added to 4 µL of reaction solution 3 and the hybridization of primer with circular padlock probe was allowed to proceed for 60 min; (4) to this was added Phi29 DNA polymerase (1 U/µL, 6 µL), obtaining the RCA reaction solution. The RCA reaction (primer polymerization) proceeded for 40 min, generating RCA products; finally, the resulting mixture was incubated at 65 °C for 10 min to inactivate the polymerase. The volume of each species involved

might be regulated when needing larger volume of final reaction solution while the molar ratio of species was kept constant. Unless otherwise stated, the above-mentioned experiments were conducted at 37 °C and the aptamer1 was used throughout. To transduce molecular recognition into a detectable electrochemical signal, the RCA products were dropped onto the interrogating electrode surface and the hybridization was allowed to proceed for 60 min. After being rinsed with Tris-HCl buffer2, the resulting electrode was incubated in 1 mM MB solution for 40 min to accumulate MB. Subsequently, the electrode was rinsed in 0.1 M PBS with gentle stirring for 0.5 min prior to the electrochemical measurements. Regeneration of Sensing Interface. After each assay, the used sensing interface can be easily regenerated by removing the surface-adsorbed molecules in water at 90 °C while the covalently attached species are left undisturbed. After incubation in hot water for 20 min, the electrode was placed in another aliquot of water at the same temperature, followed by cooling to room temperature. The resulting electrode was ready for the next measurement. RESULTS AND DISCUSSION Signaling Scheme Based on Aptamer-Target BindingActivated RCA Effect. The RCA-based aptameric biosensing system facilitates the integration of multiple functional elements into a cooperative detection device: rolling circle amplification, aptameric target recognition, and DNA hybridization specificity. Although the conformational change of aptamers upon ligand binding as a useful starting point for the development of aptamerbased biosensors were usually used to translate target recognition into a detectable electrochemical,40 colorimetric,41,42 or fluorescent20,22 signal, it remains a challenge to extend the approach to the RCA-based system for accomplishing the amplification detection and quantification of target analytes. As shown in Scheme S1 of Supporting Information, a typical RCA detection system often contains three parts:43 Complementary DNA (CDNA) (the template for padlock probe ligation), padlock probe (the template for RCA reaction), and primer probe (DNA sequence extended by polymerase on the circularized padlock probe). In a conformational change-dependent assay system, the aptamer is usually designed to be the primer probe,44 padlock probe,32 or complex sequence probe8 so that target binding can directly trigger the RCA process. Compared with the traditional interrogating strategies, to demonstrate a proof-of-concept of a novel general detection scheme, we developed a reusable electrochemical aptamer-based biosensor in which the RCA reaction is indirectly initiated for amplifying the target binding event. The detection principle and signaling procedure are shown in Scheme 1. The present sensing system contained an extended anti-PDGF-BB aptamer, CDNA, padlock probe, primer probe, and capture probe. The interrogating interface is constructed by covalently attaching the capture probe that can hybridize with the (40) Lai, R. Y.; Plaxco, K. W.; Heeger, A. J. Anal. Chem. 2007, 79, 229–233. (41) Zhao, W.; Chiuman, W.; Lam, J. C. F.; McManus, S. A.; Chen, W.; Cui, Y.; Pelton, R.; Brook, M. A.; Li, Y. J. Am. Chem. Soc. 2008, 130, 3610–3618. (42) Li, D.; Shlyahovsky, B.; Elbaz, J.; Willner, I. J. Am. Chem. Soc. 2007, 129, 5804–5805. (43) Lizardi, P. M.; Huang, X.; Zhu, Z.; Bray-Ward, P.; Thomas, D. C.; Ward, D. C. Nat. Genet. 1998, 19, 225–232. (44) Fischer, N. O.; Tarasow, T. M.; Tok, J. B. H. Anal. Biochem. 2008, 373, 121–128.

RCA product to a gold electrode via the well-established selfassembled monolayer chemistry. Both ends of the linear padlock probe are designed to match the CDNA sequence in a head-totail fashion. The aptamer is predesigned by introducing two base segments into a 35-base sequence from both ends that specifically binds to PDGF with high affinity. This aptamer obtained was a desirable competitor of the linear padlock probe toward CDNA. For an original aptamer,45 the consensus secondary structure motif upon PDGF-BB binding is a three-way helix junction (folded conformation) whereas, in the absence of target protein, only two of the helices (free conformation) are formed under physiological conditions while the outer helix composed of the two ends is separated into two strands. By contrast, according to the experimental observation (see Design of Aptamer Probe) and the secondary structure of the adapted aptamer predicted by the program “mfold” (http://frontend.bioinfo.rpi.edu/applications/ mfold/cgi-bin/dna-form1.cgi), we presume that the designed aptamer is in a dynamic equilibrium between the free conformation and folded conformation. Target protein binding drives the conformational equilibrium to the folded structure. In this case, use of a single-stranded segment at the 5′ end as the template sequence allows the aptamer to be extended from the 3′ end by DNA polymerase, creating a considerably stable folded structure and prevents competitive hybridization from occurring. As a result, the target-aptamer binding indirectly triggers the ligation reaction by DNA ligase, forming a circular amplifying template by joining the 5′-phosphste and 3′-hydroxyl ends of a linear padlock probe. The circular padlock probe can be amplified based on the RCA mechanism using the primer probe by Phi29 DNA polymerase having exceptional strand displacement ability and great processivity, creating a long DNA strand (RCA product) composed of a large number of tandem copies of the complement to the template. The RCA products can specifically hybridize with the surfaceconfined capture probes, causing the accumulation of electroactive species on the interrogating electrode when immersing in MB solution and achieving an amplified response signal. In the nontarget protein solution, it is difficult to initiate the polymerization because of the instability of the three-way helix junction of synthesized aptamer. This aptamer can take the CDNA from the CDNA-padlock probe complex because of competitive hybridization and inhibit the subsequent circularization and RCA process, sharply suppressing the peak current. Utilizing the present electrochemical sensing platform, we accomplished highly selective detection of target protein and also achieved several distinct advantages over the existing RCA-based aptasensor, as shown in the following sections. RCA-Based Electrochemical Signaling. MB is an organic dye with a reversible redox property that belongs to the phenothiazine family.46 It is well-known that MB can accumulate on the electrode surface (called surface accumulation) via interaction with guanines of the immobilized single-stranded oligonucleotide probes or intercalation into the double strands of nucleic acids and often serves as a redox-active indicator in the development of electrochemical biosensors for the detection of diverse bioactive (45) Green, L. S.; Jellinek, D.; Jenison, R.; Ostman, A.; Heldin, C. K.; Janjic, N. Biochemistry 1996, 35, 14413–11424. (46) Pa¨ke, O.; Kirbs, A.; Lisdat, F. Biosens. Bioelectron. 2007, 22, 2656–2662.

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Scheme 1. Schematic Representation of the Target Binding-Induced RCA Reaction for the Electrochemical Detection of Proteinsa

a The configuration and operation of the present sensing system involves the following basic steps: (a) target-aptamer binding, (b) aptamerrelated polymerization, (c) DNA hybridization in which the extended aptamer probe competes with the linear padlock probe for hybridization to CDNA sequence, (d) RCA reaction, and (e) quantification of target protein on the sensing interface where the target binding event is transduced into an electrochemical signal. The details are presented in the Experimental Section. Not only is the final step compatible with microarray technologies for massive analysis but also the used screening interface can be easily regenerated by thermal denaturation. Additionally, other operations in homogeneous format are capable of being conducted in a parallel and high-throughput format.

molecules.47-50 The intensity of peak current typically reflects the length of DNA sequence or the integrity of DNA base pair stacking. A reasonable RCA design to generate a long singlestranded DNA is the prerequisite for successful amplification of the target-aptamer recognition event. In the initial stage of this work, we tested the feasibility of the used RCA scheme for the enhancement of the electrochemical signal in the absence of target and aptamer. The experimental results, including the control experiments, are shown in Figure 1. The current peak obtained was at about -0.26 V, which is a typical position for the electrochemical active label, MB.16,20,51 In a control experiment where no dissolved probe was involved, there was a very small peak current in the AC voltammetric curve as shown in line a of Figure 1, which should be attributed to the interaction of the surface-immobilized capture probe with MB. In another control experiment, in which the RCA reaction solution has DNA probes except for the circularizing padlock probe, only a slight increment (current change) in the peak current is observed in the corresponding AC voltammetric curve (seen in line b of Figure 1) because of the increased amount of accumulated MB originating (47) Huang, Y.; Nie, W. M.; Gan, S. L.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. Anal. Biochem. 2008, 382, 16–22. (48) Wain, A. J.; Zhou, F. Langmuir 2008, 24, 5155–5160. (49) Kerman, K.; Ozkan, D.; Kara, P.; Meric, B.; Gooding, J. J.; Ozsoz, M. Anal. Chim. Acta 2002, 462, 39–47, and the corresponding references. (50) Feng, K. J.; Sun, C. H.; Kang, Y.; Chen, J. W.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. Electrochem. Commun. 2008, 10, 531–535. (51) Zuo, X.; Xiao, Y.; Plaxco, K. W. J. Am. Chem. Soc. 2009, 131, 6944–6945.

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Figure 1. AC voltammograms for the interrogating interface prepared with a single electrode used to investigate the RCA effect: the interrogating electrode before (a) and after exposure to RCA products in the absence (b) or presence (c) of padlock probe. Note that TrisHCl buffer1 was substituted for reaction solution 1 (described in the Experimental Section) in this section, and the operations were carried out under the same conditions as those involved in the target assay. The experimental errors for current values are not more than 6%. The electrochemical measurements were carried out in 10 mM phosphate buffer (pH 7.4, 0.1 M KCl) at room temperature.

Figure 2. (A) Influence of the outer helix length of aptamer on the current response to target protein by AC voltammetric measurements. Lines a and b represent the current response for aptamer1 and aptamer2, respectively. The AC voltammograms have been corrected from the background current, and the target detections were conducted under identical conditions. (B) A direct comparison of the current responses achieved by two different signaling strategies. Line a indicates the relative current response for the competitive hybridization-based protocol while line b indicates the displacement-based protocol. For the signaling scheme b, addition of padlock probe was followed by the thermal treatment to form the CDNA-padlock probe complex. Other experimental conditions are identical to those involved for the competitive hybridizationbased protocol. A concentration of PDGF at 8.4 × 10-10 M was used for the target-aptamer binding.

from the specific adsorption of CDNA via hybridization to the capture DNA. By contrast, the introduction of the padlock probe leads to a dramatic increase in the peak current as described in line c of Figure 1, suggesting the remarkable accumulation of MB on the interrogating electrode closely related to a long RCA product containing multiple binding sequences for the capture probes. The data herein validated the rationality of probe sequences involved in the RCA reaction and the signaling scheme. To increase the RCA efficiency, the experimental conditions, including the aptamer sequence, were optimized prior to the target assay. The details are presented in the following several sections. Design of Aptamer Probe. Despite the success of the RCA reaction/signaling, it is not easy to elegantly translate the recognition of target species by the aptamer into the RCA process through the electrochemical technique. The aptamer polymerization efficiency strongly depends on the stability of its folded conformation. The lengthening of the outer helix of aptamer facilitates the formation of folded structure and in turn promotes the RCA-based signal enhancement. Figure 2A clearly depicts the AC voltammetric measurements collected for the aptamer1- and aptamer2-based sensing system. The current intensity obtained for the aptamer1-based sensing system increases by about 67% compared with the aptamer2-based system, indicating that the longer outer helix of the aptamer promotes the RCA reaction. However, when the outer helix of the aptamer is long enough so that the folded conformation of the free aptamer is stable, the aptamer polymerization could occur even in the absence of target protein, and the subsequent RCA reaction was initiated, increasing the background current. So there is a compromise between the background current and the target binding-induced enhanced current. Although the length of the aptamer’s outer helix requires further optimization to improve the analytical performance, in this contribution, the aptamer1 was used as the target recognition element for development of a conceptually new RCA-based aptameric biosensing system.

Optimization of Signaling Scheme. The signaling strategy described in Scheme 1 is called the competitive hybridizationbased protocol. If the CDNA was prehybridized with the aptamer rather than the padlock probe, in the presence of target protein, this sequence probe was inevitably stripped away from the target-aptamer complex resulting from the replication of the single-stranded domain by high performance Phi29 DNA polymerase. The free CDNA might facilitate the circularization of the padlock probe because the hybridization with the padlock probe brings together the two ends, initiating the subsequent RCA process. This signaling scheme is designated as the displacementbased protocol. In this contribution, the current response to target protein at the same concentration obtained by the two signaling schemes was evaluated. The experimental data are shown in Figure 2B. One can see that there is no substantial difference in peak current between the two signaling schemes. The desirable electrochemical signal observed demonstrates that the targetaptamer binding can be transferred into the RCA process as expected, making the amplified detection of target protein possible. In our work, the competitive hybridization-based protocol was adopted for the target assay because of the slightly higher peak current. Interestingly, utilizing this protocol, we separated the thermal treatment from the other experimental steps, offering high flexibility in biosensor designs to some extent because the biological activity of DNA polymerase in reaction solution 1 is not affected by the subsequent steps except for the deliberately designed heat inactivation process. Other optimized experimental conditions are shown in Supporting Information. Analytical Characteristics Performance of Sensing System. To demonstrate that the present electrochemical detection system can be used for accurate quantification of target protein, the current responses to PDGF-BB at various concentrations were evaluated by AC voltammetry. As the target concentration increases, the amount of circularized padlock probe gradually increases because the competitive hybridization is inhibited by Analytical Chemistry, Vol. 82, No. 6, March 15, 2010

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Figure 3. The linear relationship between the peak current response and logarithm of the target concentration. The regression equation was Y ) 45.60 log X + 465.5 with a correlation coefficient of 0.9970, where Y and X represented the relative current response and the PDGF-BB concentration, respectively.

the aptamer polymerization, promoting the RCA-based electrochemical signal gain. As expected, we observed that the peak current increased monotonically with the increase of target concentration, indicating the signal-on mechanism that is a promising signaling strategy. As presented in Figure 3, when the value of peak current recorded in AC voltammograms is plotted versus the logarithm of the target concentration, the present electrochemical aptameric biosensor can offer a linear response to target analyte over a concentration range from of 8.4 × 10-11 to 8.4 × 10-9 M. Further increasing or decreasing the target concentration could not cause a substantial change in peak current that was beyond the linear response range. The detection limit was 6.3 × 10-11 M, at which target protein binding can trigger a peak current slightly higher than the blank, indicating improvement by a factor of 280, 6, 15, and 1000 when compared with other aptameric sensing systems for the same target protein assay based on colorimetric,52 fluorescent,32 luminescent,53 and electrochemical54 techniques, respectively, even though the RCA process was involved.32 A linear dynamic range of 2 orders of magnitude obtained is considerably or slightly wider than the values reported in those works. Noteworthy is that the performance could be further improved via optimization of the probe sequences and the experimental conditions. For example, the outer stem of designed aptamer could be regulated to achieve a higher response current via amplification of the difference between the molecular configuration in the absence and presence of target protein as mentioned above or by optimization of the volume of reagents involved in reaction solution 1 (e.g., reducing the volume of polymerase solution and increasing the volume of target solution) even though their final concentrations were kept constant. (52) Huang, C.-C.; Huang, Y.-F.; Cao, Z.; Tan, W. H.; Chang, H.-T. Anal. Chem. 2005, 77, 5735–5741. (53) Jiang, Y.; Fang, X.; Bai, C. Anal. Chem. 2004, 76, 5230–5235. (54) Liao, W.; Cui, X. T. Biosens. Bioelectron. 2007, 23, 218–224.

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Table 2. Recovery of PDGF Assay at Different Concentrations sample added (mol.L-1) found (mol.L-1) recovery (%) RSD (%) 1 2 3

8.4 × 10-11 1.7 × 10-10 1.2 × 10-9

8.22 × 10-11 1.84 × 10-10 1.25 × 10-9

97.6 108.2 104.2

4.8 3.6 7.9

To test the repeatability of the present electrochemical biosensing platform, a series of three repetitive measurements of target samples were carried out. The relative standard deviations achieved for the target sample at 8.4 × 10-11 M, 1.7 × 10-10 M, or 1.2 × 10-9 M was not more than 8.0%. To evaluate the applicability and reliability of the proposed biosensor, the recovery experiments for several samples at various concentrations were carried out, where each sample was detected three times. The data are given in Table 2. The recovery was 97.6-108.2% with an average relative standard derivation of 5.4%. Satisfactory recovery test values were achieved within the linear concentration range. Detection Specificity. Because RCA as a promising amplification method possesses not only high sensitivity but also high specificity owing to the stringent strand matching requirement for the ligation reaction,55 the detection specificity of the present amplified sensing system is entirely determined by the intrinsic characteristics of the used aptamer. The excellent specificity of the original anti-PDGF aptamer sequence has been already confirmed via investigation of its anti-interference ability toward various nontarget proteins.56 Moreover, extending the stem of aptamer might offer more or less better target binding characteristics demonstrated by a direct comparison of the original aptamer and the extended one.57 Thus, the developed RCA-based (55) Konry, T.; Hayman, R. B.; Walt, D. R. Anal. Chem. 2009, 81, 5777–5782. (56) Fang, X. H.; Sen, A.; Vicens, M.; Tan, W. H. ChemBioChem 2003, 4, 829– 834. (57) Liss, M.; Petersen, B.; Wolf, H.; Prohaska, E. Anal. Chem. 2002, 74, 4488– 4495.

aptameric sensing system in which a designed aptamer with an extended outer stem was involved should display a desirable specificity. To investigate the detection specificity offered, the nonspecific current changes induced by several nontarget proteins were studied. The concentrations of C4, IgE, HSA, and BSA were 289, 37.9, 149, and 334 nM, respectively. All the detections were carried out under identical conditions. The current response to 8.4 nM target PDGF is defined as 100%. The experimental results indicated that those nontarget proteins cause a decrease in peak current rather than an increase even though the concentration of those proteins is much higher than that of target analyte. Namely, the nonspecific current response is