Aptamer-Based Rolling Circle Amplification: A Platform for

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Anal. Chem. 2007, 79, 7492-7500

Aptamer-Based Rolling Circle Amplification: A Platform for Electrochemical Detection of Protein Long Zhou, Li-Juan Ou, Xia Chu,* Guo-Li Shen, and Ru-Qin Yu

State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China

Aptamer-based rolling circle amplification (aptamer-RCA) was developed as a novel versatile electrochemical platform for ultrasensitive detection of protein. This method utilized antibodies immobilized on the electrode surface to capture the protein target, and the surface-captured protein was then sandwiched by an aptamer-primer complex. The aptamer-primer sequence mediated an in situ RCA reaction that generated hundreds of copies of a circular DNA template. Detection of the amplified copies via enzymatic silver deposition then allowed enormous sensitivity enhancement in the assay of target protein. This novel aptamer-primer design circumvented time-consuming preparation of the antibody-DNA conjugate for the common immuno-RCA assay. Moreover, the detection strategy based on enzymatic silver deposition enabled a highly efficient readout of the RCA product as compared to a redox-labeled probe based procedure that might exhibit low detection efficiency due to RCA product distance from the electrode. With the platelet-derived growth factor B-chain (PDGF-BB) as a model target, it was demonstrated that the presented method was highly sensitive and specific with a wide detection range of 4 orders of magnitude and a detection limit as low as 10 fM. Because of the wide availability of aptamers for numerous proteins, this platform holds great promise in ultrasensitive immunoassay. The detection of proteins plays essential roles in basic discovery research as well as clinical practice. In general, antibodybased immunoassay systems are versatile and powerful tools for various molecular analyses.1 There are, however, many examples where important biological markers for cancer, infectious diseases, or biochemical processes are present at too low a concentration to be detected using conventional immunoassays. To resolve this limitation, alternative approaches to improving the sensitivity of conventional immunoassays have been developed, including strong fluorescent and luminescent substrates for ELISAs2 and signal amplification methods such as tyramide deposition3 and * To whom correspondence should be addressed. E-mail: [email protected]. Phone: 86-731-8821916. Fax: 86-731-8821916. (1) Gosling, J. P. Clin. Chem. 1990, 36, 1408-1427. (2) Kricka, L. J. In Immunoassay; Diamandis, E. P., Christopoulos, T. K., Eds.; Academic: San Diego, CA, 1996; pp 337-350. (3) Van Gijlswijk, R. P.; Zijlmans, H. J.; Wiegant, J.; Bobrow, M. N.; Erickson, T. J.; Adler, K. E.; Tanke, H. J.; Raap, A. K. J. Histochem. Cytochem. 1997, 45, 375-382.

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metal silver deposition.4 Although these techniques can be very powerful, greater sensitivity and specificity are frequently required, particularly when analysis is limited by the sample amount available or when antigen level is extremely low.5 A high-sensitivity nucleicacidamplificationtechnique,termedimmuno-PCRmethod,6-8 has been introduced for signal amplification in immunoassay, which typically results in 100-10 000-fold increase in sensitivity due to the enormous efficiency of nucleic acid amplification as compared with conventional enzyme-amplified immunoassay.9 Although the requirement for thermal cycling and experimental expertise in PCR technique have ultimately restricted the widespread adoption of immuno-PCR as an alternative to ELISA,9 it has presented a novel strategy for improving the sensitivity of immunoassay, that is, utilizing the nucleic acid amplification and detection technique as an ultrasensitive platform for signaling antigen-antibody recognition events. Rolling circle amplification (RCA) based immunoassay, immuno-RCA, is another ultrasensitive immunoassay approach based on a nucleic acid amplification and detection technique. In immuno-RCA, the 5′ end of an RCA primer is attached to an antibody. The presence of antigen then mediates RCA to produce a concatamer of the complement of a circular DNA template that extends from the end of the antibody-conjugated primer. Thus RCA generates a localized signal via isothermal amplification of a circle reporter sequence. Compared with immuno-PCR, immunoRCA possesses the following advantages: (1) suitability for protein microarray or sensing interface applications because RCA product remains localized at the target molecules captured on the support; (2) no need for thermal cycling because the RCA method is an isothermal amplification procedure; and (3) possibility of direct quantification without reference to multiple standard samples because the RCA reaction is performed according to the linear kinetic model. Immuno-RCA has been shown to improve the sensitivity of the immunoassay in several formats, including microarrays,10,11 fluorescent microspheres,12,13 microtiter plates, and magnetic beads.14 Nevertheless, there has been no demon(4) Chu, X.; Fu, X.; Chen, K.; Shen, G. L.; Yu, R. Q. Biosens. Bioelectron. 2005, 20, 1805-1812. (5) Cousino, M. A.; Jarbawi, T. B.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1997, 69, 544A-549A. (6) Sano, T.; Smith, C. L.; Cantor, C. R. Science 1992, 258, 120-122. (7) Ruzicka, V.; Marz, W.; Russ, A.; Gross, W. Science 1993, 260, 698-699. (8) Hendrickson, E. R.; Truby, T. M. H.; Joerger, R. D.; Majarian, W. R.; Ebersole, R. C. Nucleic Acids Res. 1995, 23, 522-529. (9) Niemeyer, C. M.; Adler, M.; Wacker, R. Trends Biotechnol. 2005, 23, 208216. 10.1021/ac071059s CCC: $37.00

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stration that immuno-RCA could be utilized for sensitive protein detection at the sensing interface such as electrochemical electrodes. Because of the high sensitivity, inherent simplicity, portability, and low cost derived from electrochemical techniques,15-22 the incorporation of the immuno-RCA assay in an electrochemical immunoassay might hold great promise for ultrasensitive protein determination in decentralized studies and point-of-care diagnosis. However, the distance-dependent electrontransfer efficiency presents a great challenge to the electrochemistry-based RCA assay. RCA products distant from the electrode might have very small contribution to the electrochemical readout. The implementation of the electrochemical RCA assay still calls for the development of novel techniques with high efficiency in detecting products. Both immuno-PCR and immuno-RCA involve the antibodyDNA conjugate, an essential reagent that converts ELISA into an immuno-PCR or immuno-RCA assay. In general, the antibodyDNA conjugate can be achieved through the streptavidin (STV)biotin bridge with antibody-STV fusion proteins or covalent linkage between a NH2/SH label on DNA and the antibody.9 The conjugation is time-consuming and labor-intensive because of several incubation and separation steps and, on the other hand, will influence the affinity of the antibody, i.e., the covalently linked label might occupy the binding site and affect the interaction between antibody and antigen. To circumvent the limitations, a novel approach for ultrasensitive detection of protein based on RCA has been introduced in this work, that is, utilizing aptamerprimer complexes to replace antibody-primer conjugates. Aptamers are in vitro selected short RNA or DNA oligonucleotides that exhibit high-affinity binding to given ligands as proteins.23 As an alternative to the universally used antibodies and antibody fragments, aptamers offer several considerable benefits: (1) improved temperature stability and shelf life, as aptamers are chemically produced oligonucleotides; (2) ease in conjugation to various molecules at desired locations without affecting the affinity, as labeled nucleotide can be introduced outside the recognition scaffold; and (3) adaptability to various targets including toxic or poorly immunogenic molecules and minimized batch-to-batch (10) Schweitzer, B.; Roberts, S.; Grimwade, B.; Shao, W.; Wang, M.; Fu, Q.; Shu, Q.; Laroche, I.; Zhou, Z.; Tchernev, V. T.; Christiansen, J.; Velleca, M.; Kingsmore, S. F. Nat. Biotechnol. 2002, 20, 359-365. (11) Mullenix, M. C.; Wiltshire, S.; Shao, W.; Kitos, G.; Schweitzer, B. Clin. Chem. 2001, 47 (10), 1926-1929. (12) Raghunathan, A.; Sorette, M. P.; Ferguson, H. R.; Piccoli, S. P. Clin. Chem. 2002, 48 (10), 1853-1855. (13) Sivakamasundari, R.; Feaver, W. J.; Krishna, R. M.; Piccoli, S. P. Clin. Chem. 2002, 48 (10), 1855-1858. (14) 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. (15) Daniel, M. C.; Astruc, D. Chem. Rev. 2004, 104, 293-346. (16) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042-6108. (17) Liu, G.; Wang, J.; Kim, J.; Jan, M. R.; Collins, G. E. Anal. Chem. 2004, 76, 7126-7130. (18) Wang, J.; Ibanez, A.; Chatrathi, M. P. J. Am. Chem. Soc. 2003, 125, 84448445. (19) Wang, J.; Xu, D.; Kawde, A.-N.; Polsky, R. Anal. Chem. 2001, 73, 55765581. (20) Authier, L.; Grossiord, C.; Brossier, P.; Limoges, B. Anal. Chem. 2001, 73, 4450-4456. (21) Wang, J.; Liu, G. D.; Merkoci, A. J. Am. Chem. Soc. 2003, 125, 3214-3215. (22) Wang, J.; Li, J. H.; Baca, A. J.; Hu, J. B.; Zhou, F. M.; Yan, W.; Pang, D. W. Anal. Chem. 2003, 75, 3941-3945. (23) Hermann, T.; Patel, D. J. Science 2000, 287, 820-825.

variation distinctive of polyclonal antibody production, as aptamer synthesis is animal-free. Drolet reported the first immunoassay application of an aptamer, enzyme-linked aptamer assay (ELAA), which comprised a mixed ELISA/ELAA sandwich to detect human vascular endothelial growth factor on microtiter plates.24 Similar mixed assays have been reported on the surface of microbeads, using either immobilized or labeled aptamers as capture or detecting reagents.25-29 Applications of aptamers as reagentless aptasensors30-34 and aptamer molecular beacons35-39 have also been demonstrated. Herein, we present for the first time an example of the aptamerbased immuno-RCA assay as an ultrasensitive and versatile electrochemical platform for protein detection. Platelet-derived growth factor B-chain (PDGF-BB), an important protein for cell transformation and tumor growth and progression, was selected as the model protein. With appropriate design of the sequence of the aptamer-primer complex, the aptamer-primer oligonucleotide is successfully utilized instead of antibody-DNA conjugates in an immuno-RCA assay followed by an electrochemical detection of RCA product using enzymatic silver deposition.40 This novel design furnishes the electrochemical aptamer-RCA technique with three salient advantages: (1) The aptamer-primer complex can be easily achieved by standard oligonucleotide chemical synthesis, avoiding complicated and time-consuming preparation of the antibody-DNA conjugates. (2) This aptamer-primer complex can be stored without loss of sensitivity for 1∼2 years at a relatively wide temperature range as compared to the antibody-DNA conjugates that are normally stable at 4 °C for only 6 months. (3) The enzymatic silver deposition allows the accumulation of the alkaline phosphatase (ALP) product at the electrode surface, enabling full use of the RCA product for a linear sweep voltammetric (LSV) readout as compared to redox labeled detection probes that might be limited by the RCA product distance from the electrode and not detected in the electrochemical measurement. The ease of production of the aptamer-primer complex and their applicability to the electrochemical immuno-RCA assay, (24) Drolet, D. W.; Moon-McDermott, L.; Romig, T. S. Nat. Biotechnol. 1996, 14, 1021-1025. (25) Le Floch, F.; Ho, H. A.; Leclerc, M. Anal. Chem. 2006, 78, 4727-4731. (26) Radi, A.-E.; Acero Sanchez, J. L.; Baldrich, E.; O’Sullivan, C. K. Anal. Chem. 2005, 77, 6320-6323. (27) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Anal. Chem. 2006, 78, 22682271. (28) Xu, D.; Xu, D.; Yu, X.; Liu, Z.; He, W.; Ma, Z. Anal. Chem. 2005, 77, 51075113. (29) Centi, S.; Tombelli, S.; Minunni, M.; Mascini, M. Anal. Chem. 2007, 79, 1466-1473. (30) Lai, R. Y.; Plaxco, K. W.; Heeger, A. J. Anal. Chem. 2007, 79, 229-233. (31) Kleinjung, F.; Klussmann, S.; Erdmann, V. A.; Scheller, F. W.; Fu ¨ rste, J. P.; Bier, F. F. Anal. Chem. 1998, 70, 328-331. (32) Potyrailo, R. A.; Conrad, R. C.; Ellington, A. D.; Hieftje, G. M. Anal. Chem. 1998, 70, 3419-3425. (33) 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. (34) Liss, M.; Petersen, B.; Wolf, H.; Prohaska, E. Anal. Chem. 2002, 74, 44884495. (35) Li, J. J.; Fang, X.; Tan, W. Biochem. Biophys. Res. Commun. 2002, 292, 31-40. (36) Stojanovic, M. N.; de Prada, P.; Landry, D. W. J. Am. Chem. Soc. 2001, 123, 4928-4931. (37) Frauendorf, C.; Ja¨schke, A. Bioorg. Med. Chem. 2001, 9, 2521-2524. (38) Yamamoto, R.; Kumar, P. K. R. Genes Cells 2002, 5, 389-396. (39) Fang, X.; Sen, A.; Vicens, M.; Tan, W. ChemBioChem 2003, 4, 829-834. (40) Hwang, S., Kim, E., Kwak, J. Anal. Chem. 2005, 77, 579-584.

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together with their stability for longtime storage, as demonstrated in the present study, promise an ultrasensitive, simple, accurate, inexpensive platform for protein detection. EXPERIMENTAL SECTION Reagents and Materials. Recombinant human platelet-derived growth factor B-chain (PDGF-BB) was purchased from Bowling Vaccine & Pharmaceutical Inc. (California). Rabbit anti-humanPDGF-BB polyclonal antibody was purchased from Boster Biotechnology Co. Ltd. (Wuhan, China). Escherichia coli DNA ligase and deoxyribonucleoside 5′-triphosphates mixture (dNTPs) were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Phi29 DNA polymerase was purchased from Epicentre Technologies (Madison, WI ). β-Nicotinamide adenine dinucleotide (oxidized, NAD+) was from ICN (Germany). Streptavidin-alkaline phosphatase (SA-ALP) and bovine serum albumin (BSA) were from Bio Basic Inc. (Canada). Cysteamine and glutaraldehyde wereas from Sigma Aldrich Chemical Co. (St. Louis, MO). Ascorbic acid 2-phosphate was from Express Technology Co., Ltd (Japan). Tris was from Roche (Basel, Switzerland). EDTA, trisodium citrate, NaCl, MgCl2, KH2PO4, Na2HPO4, KNO3, and (NH4)2SO4 were all purchased from Amresco (Solon, OH). Ultrapure water was obtained through a Nanopure Infinity Ultrapure water system (Barnstead/Thermolyne Corp., Dubuque, IA) with an electrical resistance larger than 18.3 MΩ. The DNA oligonucleotides were obtained from Takara Biotechnology Co. Ltd. (Dalian, China) and had the following sequences. Aptamer-primer complex: 5′-TAC TCA GGG CAC TGC AAG CAA TTG TGG TCC CAA TGG GCT GAG TAT TTT TTT TGT CCG TGC TAG AAG GAA ACA GTT AC-3′. (The boldface portion is the aptamer sequence and the italicized portion is the primer sequence.) Circular template: 5′-p-TAG CAC GGA CAT ATA TGA TGG TAC CGC AGT ATG AGT ATC TCC TAT CAC TAC TAA GTG GAA GAA ATG TAA CTG TTT CCT TC-3′. (The italicized portion matches the italicized sequence of the aptamer-primer complex, p ) 5′ phosphate.) Detection probe: 5′-GTT TCC TTC TAG CACbiotin-3′ (The sequence in italic is the same as those in italic on the circular template.) Biotinylated aptamer: 5′-TAC TCA GGG CAC TGC AAG CAA TTG TGG TCC CAA TGG GCT GAG TAT TTT TTT-biotin-3′. (The boldface portion is the aptamer sequence.) Apparatus. Electrochemical experiments were performed with a model CHI 660C electrochemical workstation (Shanghai Chenhua Equipments, China). Bare gold working electrodes (99.99%, polycrystalline gold rod, 4 mm diameter), platinum wire counter electrodes, KCl saturated calomel reference electrodes (SCE), and a conventional three-electrode electrochemical cell were all purchased from Shanghai Chenhua Equipments. All potentials were referenced to the SCE reference electrode. Microgravimetric analysis was performed using a QCM analyzer (Shanghai Chenhua Equipments, China) linked to a personal computer. Quartz crystals (9 MHz, AT-cut) between two Au electrodes (5 mm diameter) were used for the QCM experiment. Before use, the quartz crystals were cleaned by immersion in piranha solution (30% H2O2/70% concentrated H2SO4), rinsed with water, and dried in nitrogen gas. Warning: piranha solution reacts violently with organics. Preparation of Antibody Modified Gold Electrode. The gold electrode was polished sequentially with 0.3 and 0.05 µm 7494

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alumina slurry followed by ultrasonic cleaning in ethanol and double distilled water. Subsequently, the gold electrode was cleaned with piranha solution, rinsed with water, and then dried under nitrogen gas. The pretreated gold electrode was immersed in ethanol solution containing 10 mM cysteamine for 10 h to produce a self-assembled monolayer (SAM) of cysteamine. After reacting with 2.5% glutaraldehyde aqueous solution at 37 °C for 1 h, the electrode was incubated with 20 µL of 10 µg/mL PDGFBB antibody solution in pH 7.3 PBS at 37 °C for 1 h in a humidity chamber. The unreacted aldehyde was blocked with 5 mg/mL BSA solution at 37 °C for 1 h. The electrode was stored at 4 °C for the aptamer-RCA immunoassay after rinsing with 0.5 M NaCl solution and double distilled water. Aptamer-RCA Immunoassay. A series of 20 µL volumes of samples containing either purified PDGF-BB antigen or serum at various concentrations in Tris-HCl solution (20 mM Tris-HCl, pH 7.1, 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2) were applied to the antibody-modified electrode surfaces and incubated at 37 °C for 1 h in a humidity chamber. After washing with a 0.5 M NaCl solution and water, 20 µL of the 2.5 µM aptamer-primer complex in PBSM solution (0.1 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, pH 7.3) was applied to the electrode surface and incubated at 37 °C for 30 min. (Immediately before use, the aptamer-primer complex solution was heated to 95 °C for 10 min and chilled on ice to ensure proper intramolecular folding and to form the aptamer 3D conformation.) After the electrode was washed with PBSM three times, 20 µL of 0.5 µM circular template in 2× SSC (0.03 M C6H5Na3O7, 0.3 M NaCl, pH 7.0) and 0.05% Tween-20 was hybridized with the aptamer-primer sequences captured on the electrode surface at 37 °C for 1 h and excess circular template was removed by washing with 2× SSC and 0.05% Tween-20 twice for 2 min. Circular template on the electrode was ligated by incubating at 37 °C for 30 min with 20 µL of solution containing 2 U E. coli DNA ligase, 30 mM Tris-HCl, pH 8.0, 4 mM MgCl2, 10 mM (NH4)2SO4, 0.2 mM NAD, and 0.005% BSA. Then, RCA was performed at 37 °C for 1 h in a volume of 20 µL of solution containing 10 U φ29 DNA polymerase, 1 mM each dNTP, 50 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 20 mM (NH4)2SO4, and 0.2 mg/mL BSA. The electrode was washed twice for 2 min in 2× SSC and 0.05% Tween-20. An aliquot of 20 µL of the 0.5 µM biotinylated detection probe in 2× SSC and 0.05% Tween-20 was hybridized to the RCA products on the electrode for 30 min at 37 °C. The electrode was then washed as described above. Direct Enzyme Linked Aptamer Immunoassay. The reaction of a series of samples containing either purified PDGF-BB antigen or serum at various concentrations with antibody-modified electrode was the same as the aptamer-RCA immunoassay. After the electrode was washed with a 0.5 M NaCl solution and water, 20 µL of the 2.5 µM biotinylated aptamer in PBSM solution was applied to the electrode surface and incubated at 37 °C for 30 min. (Immediately before use, the biotinylated aptamer was also pretreated, as the aptamer-DNA complex was, to form the aptamer 3D conformation.) The electrode was then washed with PBSM three times. Enzymatic Silver Deposition and Electrochemical Detection. A 20 µL SA-ALP solution (1:50 dilution from the stock solution using 1/15 M pH 7.1 PBS buffer containing 0.2 M KNO3)

Figure 1. Schematic outline of the electrochemical aptamer-RCA immunosensor based on enzymatic silver deposition. (a) Immobilization of PDGF-BB antibody on the Au electrode; (b) capture of the analyte PDGF-BB in sample solution; (c) association with aptamer-primer complex; (d) hybridization and ligation of the padlock probe; (e) RCA reaction in the presence of φ29 DNA polymerase and dNTPs; (f) hybridization with biotinylated detection probe, association with streptavidin-ALP, reduction of silver ion by AAP, and dissolution of silver during anodic stripping voltammetry.

was applied to the electrode surface. After incubation at 37 °C for 30 min, the electrode was thoroughly rinsed with PBS buffer containing 0.2 M KNO3. Then, the electrode was incubated with the freshly prepared 50 mM glycine buffer solution (pH 9.08) containing 1 mM AgNO3 and 1 mM ascorbic acid 2-phosphate (AA-P) at 37 °C for 40 min. After incubation, the electrode was rinsed with ultrapure water. Linear sweep voltammetric measurement was performed at a potential range from 0 to 0.8 V (vs SCE) with a 100 mV/s scanning rate. A 0.6 M KNO3 solution containing 0.1 M HNO3 was used as the supporting electrolyte for electrochemical experiments. The experimental temperature was controlled at room temperature. RESULTS AND DISCUSSION Design Strategy of Electrochemical Aptamer-RCA Immunosensor. Figure 1 depicts schematically the assay protocol of the electrochemical aptamer-RCA immunosensor based on enzymatic silver deposition. Anti-human-PDGF-BB polyclonal antibody was immobilized on the electrode surface via the SAM of cysteamine and the bifunctional linker of glutaraldehyde and then used to capture the analyte PDGF-BB in sample solution. The aptamer-primer complex oligonuceotide was subsequently bound to the surface-captured antigen, resulting in an analyte-specific

single-stranded DNA that could serve as not only the primer for the RCA reaction but also the template for padlock probe ligation. After hybridization and ligation of the padlock probe, a linear RCA reaction was initiated, producing a single-stranded tandem repeated copy of the circular template that was extended from the end of the surface-bound aptamer-primer complex. Hybridization of the RCA product with biotinylated detection probes, followed by the binding of SA-ALP conjugates, allowed a nonreductive substrate of alkaline phosphatase, ascorbic acid 2-phosphate (AAP), to be converted into reducing agent ascorbic acid (AA) at the electrode surface. Finally, silver ions were reduced and deposited on the electrode surface and the DNA backbone, as metallic silver that is determined by linear sweep voltammetry (LSV), gives the analytical signal for PDGF-BB. Note that the immunosensor utilized the enzymatic silver deposition procedure for electrochemical quantification of the RCA product. This permitted the accumulation of the enzymatic product at the electrode surface for a highly sensitive LSV readout. It was found in our preliminary experiments that ferrocene labeled detection probes gave significantly inferior sensitivity, indicating that redox labeled probes might have low electron-transfer efficiency due to the fact that most of the RCA products were distant from the electrode and could not be detected in the electrochemical readout. Analytical Chemistry, Vol. 79, No. 19, October 1, 2007

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Figure 3. LSVs of enzymatically deposited electrodes in 0.6 M KNO3/0.1 M HNO3 solution in (a) aptamer-RCA immunoassay (-‚‚-‚-); (b) RCA-free enzyme linked aptamer immunoassay (- - -); (c) reaction with BSA instead of target protein PDGF-BB (____). The concentration of PDGF-BB was 10 pM. Scan rate, 100 mV/s.

Figure 2. Structure of aptamer-primer complex.

In the present study, a PDGF B-chain aptamer was used in the aptamer-RCA assay for detecting PDGF-BB. The consensus secondary structure motif of the PDGF aptamer is a three-way helix junction with a three-nucleotide loop at the branch point, and the helix junction domain represents the core of the structural motif required for high-affinity binding. The aptamer has comparable affinities (Kd ∼ 0.1 nM) to PDGF-BB and PDGF-AB and exhibits much weaker affinity (Kd > 10 nM) to PDGF-AA.41 The aptamer-primer complex was designed to include the aptamer sequence and a primer sequence with 26 nucleotides. Introduction of a (T)6 spacer sequence between the aptamer and the primer sequence is beneficial for mitigating the steric hindrance in hybridization between the primer and the padlock probe as well as in the subsequent surface RCA reaction. Figure 2 shows the secondary structure of the aptamer-primer complex, as calculated using the Zucker DNA folding program.42 One observes that the aptamer-primer complex remains the three-way helix junction structure in the aptamer moiety and gives a single-stranded tail at the 3′ terminal, indicating that the aptamer-primer complex could function as not only the recognition element for analyte protein but also the primer for the RCA reaction, an ideal complex for the immuno-RCA assay. In contrast to the antibody-DNA conjugate used in the conventional immuno-RCA assay, the aptamer-primer complex is merely an oligonuleotide that can be prepared immediately using a standard DNA synthesis system and stored in a lyophilized or frozen state for a long time. As a matter of fact, it was observed in our experiments that the aptamer-primer complex could be used after 1 year of storage, implying that aptamer-RCA provides a simple and efficient platform for immuno-RCA assay. (41) Green, L. S.; Jellinek, D.; Jenison, R.; Ostman, A.; Heldin, C.-H.; Janjic, N. Biochemistry 1996, 35, 14413-14424. (42) Zucker, M. Nucleic Acids Res. 2003, 31, 3406-3415.

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Signal Amplification in Electrochemical Aptamer-RCA Immunosensor. Aptamer-primer sequence bound to the surfacecaptured PDGF-BB antigen could mediate a RCA reaction at the electrode surface, producing many tandem repeated copies of the circular template and resulting in enormous signal amplification in enzymatic silver deposition based electrochemical detection. Figure 3 depicts the LSV signal of the electrochemical aptamerRCA immunosensor as compared with that of the direct enzyme linked aptamer immunoassay. One observes that in the presence of PDGF-BB, well-defined peaks corresponding to silver stripping are achieved for both the aptamer-RCA assay and the direct enzyme linked aptamer immunoassay, whereas only an insignificant current is obtained in the absence of PDGF-BB, indicating that the enzymatic silver deposition based detection exhibited a high signal-to-background ratio in immunoassay. The use of the biotinylated aptamer instead of the aptamer-primer complex sequence excluded the RCA step in the assay. It is clear that such a RCA-free assay merely gives a small current peak in the LSV measurement. The total peak area during silver stripping is 19.3 µC, which amounts to 21.1 ng of silver deposited, as estimated according to Faraday’s Law. The introduction of aptamer-RCA yields a substantial enhancement in the LSV signal. The peak area for the aptamer-RCA immunosensor is about 90.2 times as much as that for the RCA-free one, implying that the RCA reaction achieves an amplification of the amount of deposited silver by 90.2fold. QCM experiments were also performed to determine the amount of enzymatic deposited silver so as to access the amplification efficiency. After a stable resonance frequency was achieved in 0.50 mL of a 50 mM glycine buffer solution (pH 9.08) containing 1 mM AgNO3, 0.1 mL of a 5 mM aqueous AA-P solution was added in the electrochemical cell for QCM monitoring of the deposition of silver. Figure 4 depicts the time-dependent QCM frequency change in aptamer-RCA and direct enzyme linked aptamer immunoassay formats. One observes that, when the reaction is performed for the RCA-aptamer immunoassay format (curve a), the crystal frequency decreases rapidly and gives a frequency decrease of 1460 Hz after 40 min deposition. In contrast, a small

Figure 4. Time-dependent frequency changes of an Au quartz crystal modified by streptavidin-ALP in different immunoassay formats: (a) aptamer-RCA immunoassay (;;); (b) RCA-free enzyme linked aptamer immunoassay (- - - ); (c) reaction with BSA instead of target protein PDGF-BB in the aptamer-RCA immunoassay (‚‚‚‚‚). The concentration of PDGF-BB was 10 pM. A stable resonance frequency was recorded in 0.5 mL of 50 mM glycine buffer solution (pH 9.08) containing 1 mM AgNO3, followed by injection of 0.1 mL of glycine buffer containing 5 mM AA-P.

Figure 5. Agarose gel (0.7%) electrophoresis experiments. The products of RCA reaction (1 h) in three replicate reactions (indicated by 1-3) were denatured at 95 °C for 5 min and quenched with icecooled water for 10 min. The marker was indicated by M. The high molecular weight RCA products are observed in lines 1-3.

frequency decrease of 19 Hz is obtained for the RCA-free enzyme linked aptamer immunoassay format (curve b), and the resonance frequency remains almost constant in the control experiments where BSA is used instead of the target protein PDGF-BB (curve c). Considering the Sauerbrey equation,43 one calculated that about 1825 ng of silver was deposited on the QCM electrode for the RCA-aptamer immunoassay format, whereas for direct enzyme linked aptamer immunoassay format, only 23.7 ng of silver was obtained at the surface, implying an amplification factor of 77.0 was achieved via the RCA reaction. Note that the calculated amount of deposited silver on the electrode surface exhibited small deviation between the electrochemical measurements and the QCM experiments, which seems to be attributed to the measurement errors. The amplification of the RCA reaction was also verified using agarose gel electrophoresis experiments. In the experiments, the RCA products in three replicate reactions were denatured at 95 °C for 5 min and quenched with ice-cooled water for 10 min, then were investigated by gel electrophoresis. Figure 5 gives the fluorescence image of the electrophoresis results. It is observed that the RCA products in three replicate reactions show extremely low mobility. Although the difference in electrophoretic behavior between single-stranded and double-stranded DNA might elude a reliable assessment of the molecular weight of the RCA products, these results give immediate evidence for the high molecular weight of these products, indicating the proposed aptamer-RCA method could provide enormous signal amplification in immunoassay. Effects of Antibody Concentration and RCA Reaction Time. With a fixed design strategy, the performance of the developed aptamer-RCA assay is still strongly influenced by the assay conditions such as immunosensor preparation, immunoreaction, and RCA reaction conditions. Different assay conditions

were investigated in our studies, and it revealed that of most importance were the concentration used for antibody immobilization and the RCA reaction time. Figure 6A shows the response of the aptamer-RCA based immunosensor as a function of the concentration of anti-PDGFBB polyclonal antibody. In these cases, different concentrations of anti-PDGF-BB polyclonal antibody were incubated with the aldehyde-modified electrode during immobilization. It is clear from Figure 6A that the sensor response increases substantially when the concentration of antibody changes from 0.625 to 2.5 µg mL-1. The response exhibits a gradual increase with a further increase in antibody concentration up to 10 µg mL-1. No significant increase in the sensor response is observed at higher antibody concentrations. Note that the optimal concentration for antibody immobilization is much lower than that documented in previous studies.44 This might be due to the fact that the presented aptamer-RCA method offers a high sensitivity such that the working range at low target concentration only requires a relatively small loading of immobilized antibody. The small antibody loading is also beneficial for the deposition of silver on the electrode surface, allowing a sensitive stripping determination of the deposit. As a result, the optimal concentration of antibody for immobilization was selected as 10 µg mL-1 in subsequent studies. Aptamer-primer sequence bound to the surface-captured PDGF-BB antigen mediated a RCA reaction at the electrode surface that generated tandem repeated copies of the circular template, enabling the binding of a number of ALP for enzymatic silver deposition. A long RCA reaction time is then expected to yield enhanced signal amplification. Figure 6B depicts the effect of RCA reaction time on the electrochemical readout of the immunosensor. The electrochemical response was obtained with the aptamer-RCA assay by incubating the electrode in RCA solution for varying reaction time. One observes that the LSV readout increases rapidly with the RCA reaction time up to 60

(43) Sauerbrey, G. Z. Phys. 1959, 155, 206-222.

(44) Chu, X.; Lin, Z. H.; Shen, G. L.; Yu, R. Q. Analyst 1995, 120, 2829-2832.

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Figure 6. Influences of the anti-PDGF-BB antibody concentration (A) and the RCA reaction time (B) on the electrochemical aptamerRCA immunosensor.

min, and the signal exhibits no further significant increases as the RCA reaction time increased to more than 60 min. It seems that the saturation of LSV signal after RCA reaction for 60 min is attributed to the fact that the surface-bound ALP is blocked by the silver deposition. Thus, 60 min was selected as the optimum time for the RCA reaction. Analytical Performance of Electrochemical Aptamer-RCA Immunosensor. Figure 7A shows the LSV response of the aptamer-RCA immunosensor to the target analyte of different concentrations. One observes that the stripping current increases along with a positive shift of peak potential as the target concentration increases over the range of 10 fM-100 pM. The concentration-dependent positive shift of the peak potential seems attributed to the increase in the thickness of the metallic film on an inert electrode, which causes the oxidation peak potential to shift toward more positive values.45 Figure 7B depicts the calibration curves of the electrochemical aptamer-RCA immunosensor together with the RCA-free enzyme linked aptamer immunosensor. It is clear that the aptamer-RCA immunosensor offers more than 100-fold improvement of the detection limit as compared to the RCA-free enzyme linked aptamer immunosensor. In logarithmic scales, the stripping peak current of aptamer-RCA immunosensor (45) Brainina, Kh. Z. Talanta 1971, 18, 513-539.

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Figure 7. (A) LSVs of the electrochemical aptamer-RCA immunosensing electrodes in 0.6 M KNO3/0.1 M HNO3 solution with target protein at concentrations of 3 × 10-10 M, 1 × 10-10 M, 3 × 10-11 M, 1 × 10-11 M, 3 × 10-12 M, 1 × 10-12 M, 3 × 10-13 M, 1 × 10-13 M, 3 × 10-14 M, and 1 × 10-14 M (from upper to lower). Scan rate, 100 mV/s. (B) Calibration curve of peak current as a function of target protein concentration using the electrochemical aptamer-RCA immunosensor (a) and using the RCA-free enzyme linked aptamer immunosensor (b). Peak currents are averages for four experiments.

exhibits a linear correlation to target concentration through a 4-decade range of 10 fM-100 pM, and the linear correlation coefficient was 0.994 with a detection limit of 10 fM as calculated in terms of the 3σ rule. The wide dynamic range of the presented sensor is about 3 orders of magnitude larger than those shown previously,46-48 and the low detection limit is 103∼104-fold lower than those obtained in aptamer-based fluorescence anisotropy measurements46 and high-throughput fluorescence quenching assays.47 Moreover, starting with the lowest detectable target concentration, the dose response is linear over 3 orders of magnitude in correlation to a target concentration through the 4-decade range, indicating the developed method demonstrates a significantly better dose-response slope than that of most sensitive protein assays procedures49,50 and is expected to show improved precision in target determination. (46) Fang, X.; Cao, Z.; Beck, T.; Tan, W. Anal. Chem. 2001, 73, 5752-5757. (47) Fang, X.; Sen, A.; Vicens, M.; Tan, W. ChemBioChem 2003, 4, 829-834. (48) Vicens, M. C.; Sen, A.; Vanderlaan, A.; Drake, T. J.; Tan, W. ChemBioChem 2005, 6, 900-907.

Figure 8. Specificity analysis of the electrochemical aptamer-RCA immunosensor. The concentration of PDGF-BB, IgA, IgG, IgM, AFP, thrombin, and lysozyme were 1.4 ng/mL, 100 µg/mL, 300 µg/mL, 100 µg/mL, 140 ng/mL, 15 µg/mL, and 15 µg/mL, respectively. The results were the average of three experiments.

To estimate the reproducibility of the aptamer-RCA immunosensor, six assays were performed following identical processing steps. Their responses toward 10 pM target protein gave an average stripping peak current of 1206 µA with a relative standard deviation of 5.4%, indicators that the immunosensor could be constructed and used for analysis with excellent reproducibility. The specificity of the aptamer-RCA immunosensor was also examined using other proteins commonly present in serum. About 102∼105-fold concentration of IgA, IgG, IgM, lysozyme, thrombin, and AFP were incubated and detected individually with the immunosensor. The stripping peak currents were shown in Figure 8. It can be seen that no significant LSV readouts are obtained for these interfering proteins. Thus, no significant cross-reactivity was detected for these proteins. Since the aptamer-RCA immunosensor exploited the specificity of both antibody and aptamer, it was expected that the developed immunosensor could exhibit a high degree of selectivity for the PDGF-BB assay. Applications of Electrochemical Aptamer-RCA Immunosensor. A series of samples were prepared by adding PDGFBB of different concentrations to fetal calf serum. (The target concentration in the serum was not detectable as analyzed using a human PDGF-BB EIA Kit). These samples were determined by the aptamer-RCA immunosensor to demonstrate its applicability in complex biological samples. The analytical results are shown in Figure 9. One sees that the response curve obtained in fetal calf serum is in good consistency with that obtained in buffer (Figure 7B). This implies that the aptamer-RCA immunosensor holds potential for PDGF-BB detection in realistic biological samples. The proposed method was also implemented in the analysis of human serum specimens. As PDGF-BB is a valuable biomarker for hepatic fibrosis, the determination of PDGF-BB is useful to evaluate the state of this disease. Ten human serum specimens, four from healthy individuals and six from patients with hepatic fibrosis, were obtained from Xiangya Hospital and tested by the (49) Grubisha, D. S.; Lipert, R. J.; Park, H. Y.; Driskell, J.; Porter, M. D. Anal. Chem. 2003, 75, 5936-5943. (50) Wang, Z.; Lee, J.; Cossins, A. R.; Brust, M. Anal. Chem. 2005, 77, 57705774.

Figure 9. Detection of PDGF-BB in the complex biological fluid of fetal calf serum by the electrochemical aptamer-RCA immunosensor. Peak currents are averages for three experiments. Table 1. PDGF-BB Concentration in Human Serum Samples Detected by Proposed Electrochemical Aptamer-RCA Immunosensora and ELISA Method PDGF-BB concentration (pM) serum sampleb

electrochemical aptamer-RCA immunosensor

ELISA

relative deviation (%)

1 2 3 4 5 6 7 8 9 10

4.5 ( 0.2 5.0 ( 0.3 4.4 ( 0.2 5.2 ( 0.3 9.9 ( 0.5 7.6 ( 0.4 17.7 ( 0.9 7.1 ( 0.3 11.6 ( 0.6 9.4 ( 0.5

4.3 5.3 4.6 4.9 9.4 8.0 16.9 7.4 11.0 9.9

4.7 -5.7 -4.3 6.1 5.3 -5.0 4.7 -4.1 5.5 -5.1

a The data are given as average value ( SD obtained from three independent experiments. (n ) 3). b Samples from 1 to 4 were from healthy individuals and those from 5 to 10 were from patients.

electrochemical aptamer-RCA immunosensor as well as a human PDGF-BB EIA Kit as a reference method. The results are shown in Table 1. As can be seen from Table 1, the target concentrations obtained by the presented method are in good agreement with those determined by the ELISA method and the relative deviation are not more than 6.1%, indicating that it is feasible to apply the developed aptamer-RCA immunosensor to detecting PDGF-BB in human serum samples. It is important to note that, though ELISA gives similar results as compared to the aptamer-RCA method, the developed immunosensor offers a wider linear range, a larger dose-response slope, and a much lower detection limit (2 orders of magnitude) than ELISA. On the other hand, because aptamerRCA includes a RCA step for signal amplification, the presented technique requires one more hour than ELISA in an assay. CONCLUSION A versatile and ultrasensitive platform for electrochemical detection of protein based on aptamer-RCA immunoassay was for the first time constructed here. This method utilized aptamers with high affinity and specificity for proteins to bridge the communication gap between oligonucleotides and proteins such Analytical Chemistry, Vol. 79, No. 19, October 1, 2007

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that ultrasensitive techniques for nucleic acid amplification and detection could be available for signaling protein recognition events. Because of the wide availability of aptamers for numerous proteins,51 this platform holds great promise in ultrasensitive immunoassay. Compared with the antibody-DNA conjugate used in conventional immuno-RCA assay, the aptamer-primer complex could be prepared easily and stored conveniently for a long time. It was demonstrated that the presented method was highly sensitive and specific and allowed accurate quantification through a wide detection range of 4 orders of magnitude with a low detection limit of 10 fM. Because the experimental conditions, (51) Brody, E. N.; Willis, M. C.; Smith, J. D.; Jayasena, S.; Zichi, D.; Gold, L. Mol. Diagn. 1999, 4, 381-388.

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such as reagent concentrations and reaction time, for varying proteins are almost the same in the assay, there is no need to optimize the reaction conditions for each specific protein, enabling the method to be integrated in an electrochemical sensor array for high throughput detection of proteins. ACKNOWLEDGMENT This work was supported by the National Nature Science Foundation of China (Grant No. 20575020, U0632005). Received for review May 22, 2007. Accepted July 21, 2007. AC071059S