Label-Free Voltammetric Detection Using Individually Addressable

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Anal. Chem. 2010, 82, 9028–9033

Label-Free Voltammetric Detection Using Individually Addressable Oligonucleotide Microelectrode Arrays Roya Kalantari, Ryan Cantor, Hang Chen, George Yu, Jiri Janata, and Mira Josowicz* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, United States The utility and performance of label-free, oligonucleotide probes for reagentless detection of dilute target analytes was examined using a voltammetric transduction principle in an array format. Multistep, solid-state fabrication yielded preproduction arrays of 16 individually addressable, 30 µm diameter microelectrodes in a 30 mm × 6.5 mm × 0.5 mm dipstick disposable device. The specificity of 16 nucleotide (nt) 2′-O-methylribonucleic acid and 22 nt DNA backbone probes bound through Mg2+phosphonate bridges to polypyrrole films on the microelectrodes were studied using microbial target RNAs of various lengths. Probe-specific interactions with Escherichia coli O157 H7 23S rRNA (2907 nt) and Candida albicans 18S rRNA (1788 nt) were detected at 65 and 58 fmol/mL, respectively, in volumes as low as 0.5 mL. Specificity studies showed that, for a given probe, “nontarget” transcripts can contribute to changes in the voltammetric detection signal, though with responses that never exceed 70% of the detection signal acquired for specifically designed complementary targets. These results statistically validate the use of the voltammetric microelectrode array for obtaining a “yes-no” answer on complementary specific binding. The study also identifies challenges and pitfalls for the selection strategies of oligonucleotide probes. Genetic approaches such as nucleic acid sequencing,1-5 homogeneous probes,6-10 and arrays11-14 are important techniques for rapid identification of organisms and analytes from * Corresponding author. E-mail: [email protected]. (1) Lewis, E. K.; Haaland, W. C.; Nguyen, F.; Heller, D. A.; Allen, M. J.; MacGregor, R. R.; Berger, C. S.; Willingham, B.; Burns, L. A.; Scott, G. B. I.; Kittrell, C.; Johnson, B. R.; Curl, R. F.; Metzker, M. L. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (15), 5346–5351. (2) Ozsolak, F.; Platt, A. R.; Jones, D. R.; Reifenberger, J. G.; Sass, L. E.; McInerney, P.; Thompson, J. F.; Bowers, J.; Jarosz, M.; Milos, P. M. Nature 2009, 461 (7265), 814–U73. (3) Meller, A.; Nivon, L.; Brandin, E.; Golovchenko, J.; Branton, D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (3), 1079–1084. (4) Margulies, M.; Egholm, M.; Altman, W. E.; Attiya, S.; Bader, J. S.; Bemben, L. A.; Berka, J.; Braverman, M. S.; Chen, Y. J.; Chen, Z. T.; Dewell, S. B.; Du, L.; Fierro, J. M.; Gomes, X. V.; Godwin, B. C.; He, W.; Helgesen, S.; Ho, C. H.; Irzyk, G. P.; Jando, S. C.; Alenquer, M. L. I.; Jarvie, T. P.; Jirage, K. B.; Kim, J. B.; Knight, J. R.; Lanza, J. R.; Leamon, J. H.; Lefkowitz, S. M.; Lei, M.; Li, J.; Lohman, K. L.; Lu, H.; Makhijani, V. B.; McDade, K. E.; McKenna, M. P.; Myers, E. W.; Nickerson, E.; Nobile, J. R.; Plant, R.; Puc, B. P.; Ronan, M. T.; Roth, G. T.; Sarkis, G. J.; Simons, J. F.; Simpson, J. W.; Srinivasan, M.; Tartaro, K. R.; Tomasz, A.; Vogt, K. A.; Volkmer, G. A.; Wang, S. H.; Wang, Y.; Weiner, M. P.; Yu, P. G.; Begley, R. F.; Rothberg, J. M. Nature 2005, 437 (7057), 376–380.

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organisms since they can be both specific and sensitive (especially when preceded by amplification). From among arrays and biosensors, a variety of approaches have been reported for direct electrochemical detection of sequence-specific DNA15-17 that lead to signals that could be or already have been integrated within a microelectrode array.18-21 In each of these approaches, a known sequence of the testing DNA probe is linked to the electrode surface at a known base position defined by the type of linkage, for example, thiols for attachment to gold substrates.22 DNA microarray technology is based on the well-established and long-exploited principle of nucleic acid hybridization.23,24 It is of interest to DNA diagnostics25 and point-of-care applications.26 However, the thermodynamics and kinetics of the interface reaction of terminally anchored oligonucleotide probes may substantially differ compared to those of the same reaction in bulk (5) Shendure, J.; Porreca, G. J.; Reppas, N. B.; Lin, X. X.; McCutcheon, J. P.; Rosenbaum, A. M.; Wang, M. D.; Zhang, K.; Mitra, R. D.; Church, G. M. Science 2005, 309 (5741), 1728–1732. (6) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14 (3), 303–308. (7) Nelson, N. C.; BenCheikh, A.; Matsuda, E.; Becker, M. M. Biochemistry 1996, 35 (25), 8429–8438. (8) Storhoff, J. J.; Lucas, A. D.; Garimella, V.; Bao, Y. P.; Muller, U. R. Nat. Biotechnol. 2004, 22 (7), 883–887. (9) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (17), 10954–10957. (10) Brown, R. C.; Li, Z.; Rutter, A. J.; Mu, X.; Weeks, O. H.; Smith, K.; Weeks, I. Org. Biomol. Chem. 2009, 7 (2), 386–394. (11) Southern, E. M. J. Mol. Biol. 1975, 98 (3), 503-&. (12) Lockhart, D. J.; Dong, H. L.; Byrne, M. C.; Follettie, M. T.; Gallo, M. V.; Chee, M. S.; Mittmann, M.; Wang, C. W.; Kobayashi, M.; Horton, H.; Brown, E. L. Nat. Biotechnol. 1996, 14 (13), 1675–1680. (13) Fulton, R. J.; McDade, R. L.; Smith, P. L.; Kienker, L. J.; Kettman, J. R. Clin. Chem. 1997, 43 (9), 1749–1756. (14) Gunderson, K. L.; Steemers, F. J.; Lee, G.; Mendoza, L. G.; Chee, M. S. Nat. Genet. 2005, 37 (5), 549–554. (15) Wang, J. Anal. Chim. Acta 2002, 469 (1), 63–71. (16) Rivas, G. A.; Pedano, M. L.; Ferreyra, N. F. Anal. Lett. 2005, 38 (15), 2653– 2703. (17) Hvastkovs, E. G.; Buttry, D. A. Analyst 2010, 135 (8), 1817–1829. (18) Sassolas, A.; Leca-Bouvier, B. D.; Blum, L. J. Chem. Rev. 2008, 108 (1), 109–139. (19) Ratilainen, T.; Lincoln, P.; Norden, B. Biophys. J. 2001, 81 (5), 2876–2885. (20) He, W.; Yang, Q. J.; Liu, Z. H.; Yu, X. B.; Xu, D. K. Anal. Lett. 2005, 38 (15), 2567–2578. (21) Pejcic, B.; De Marco, R.; Parkinson, G. Analyst 2006, 131 (10), 1079– 1090. (22) Palecek, E.; Scheller, F.; Wang, J. Electrochemistry of Nucleic Acids and Proteins: Towards Electrochemical Sensors for Genomic and Proteomics; Elsevier: Amsterdam, 2005; Vol. 1, p 789. (23) Batchelor-McAuley, C.; Wildgoose, G. G.; Compton, R. G. Biosens. Bioelectron. 2009, 24 (11), 3183–3190. (24) Palecek, E. Electroanalysis 2009, 21 (3-5), 239–251. (25) Wei, F.; Lillehoj, P. B.; Ho, C. M. Pediatr. Res. 2010, 67 (5), 458–468. (26) Luo, X. T.; Hsing, I. M. Analyst 2009, 134 (10), 1957–1964. 10.1021/ac102002k  2010 American Chemical Society Published on Web 10/07/2010

solution as a result of the species-interface interaction, interfacial concentration gradients, and steric hindrance.27,28 Different approaches for immobilization of DNA onto a surface of conducting polymers have been recently reviewed.29 The use of shorter sequences of chemically synthesized probes (generally 20 nt or less in length) is a convenient method of microarray production and has advantages in that these probes can be designed to detect multiple variant regions of a gene or transcript. The purpose of this study was to investigate the impacts of probes randomly anchored to the intrinsically conductive polymermodified electrode as a function of the length of the interacting target and the length of the polymer linker to which the probe is attached on the applied reagentless voltammetric detection method. The utility and performance of the short probes was based on a previously reported voltammetric transduction principle.30,31 The experiments presented here demonstrate the performance of microelectrodes in an array format using 16 and 22 nt probes in contact with microbial RNA targets of different strand lengths (38, 44, 107, 1788, and 2907 nt) and different complementarities. The probes are anchored to the polypyrrole bilayer through the available free phosphonic acid functional groups at the polymer surface. Divalent metal ions bound to phosphonic acid tethers of the poly[2,5-dithienyl-N-(phosphorylalkyl)pyrrole] (pTPT) film provide electrostatic anchor sites through the negatively charged phosphate groups of the probes.30,32 Voltammetric transduction neither requires labeling of probes nor uses redox mediators or redox intercalators. It is based on the fact that a polypyrrole (PPy) film acts as an electrochemically driven ion exchanger and that large charged molecules attached near the surface of this polymer exchanger change the ability of the ions to move into or out of the film.33 The presence of an immobilized short sequence of an oligonucleotide probe at the polymer/analyte interface represents a relatively small electrostatic barrier that is dominated by the space charge of the immobile negative charge of the dissociated phosphate groups on the probes and the mobile diffuse charges of the ions in the buffer.34 This electrostatic barrier height increases during specific binding interactions with complementary nucleic acids. The identification of specific binding events is obtained from the difference of the individual cyclic voltammograms (CVs) recorded after and before the specific binding events. Consequently, the relative change in the area of the recorded CVs is the measure of the degree of complementarity between the bases of the probe and target. Using this approach, we have already demonstrated identification of specific binding based on short, single-stranded DNA probes of known sequence, immobilized on a single 1.6 mm Pt macrodisk (27) Vainrub, A.; Pettitt, B. M. Chem. Phys. Lett. 2000, 323 (1-2), 160–166. (28) Halperin, A.; Buhot, A.; Zhulina, E. B. J. Phys.: Condens. Matter 2006, 18 (18), S463–S490. (29) Peng, H.; Zhang, L. J.; Soeller, C.; Travas-Sejdic, J. Biomaterials 2009, 30 (11), 2132–2148. (30) Thompson, L. A.; Kowalik, J.; Josowicz, M.; Janata, J. J. Am. Chem. Soc. 2003, 125 (2), 324–325. (31) Aiyejorun, T.; Kowalik, J.; Janata, J.; Josowicz, M. J. Chem. Educ. 2006, 83 (8), 1208–1211. (32) Hartung, J.; Kowalik, J.; Kranz, C.; Janata, J.; Josowicz, M.; Sinha, A.; McCoy, K. J. Electrochem. Soc. 2005, 152 (11), E345-E350. (33) Ramanavicius, A.; Ramanaviciene, A.; Malinauskas, A. Electrochim. Acta 2006, 51 (27), 6025–6037. (34) Genies, E. M.; Marchesiello, M.; Bidan, G. Electrochim. Acta 1992, 37 (6), 1015–1020.

electrode30,31 and on a single 25 µm Au or Pt microdisk electrode.35,36 It needs to be stressed that our transduction principle is based on detection of global electrostatic changes in the diffuse space charge at the PPy/electrolyte interface caused by selective binding of nucleobases from target molecules in solution to those of immobilized probes. Therefore, even with few mismatches between the probe and target, binding and modification of the space charge can result in a detectable change in the CV area and interpretation of a positive signal. However, it is expected that the association constant between the probes and the investigated targets will depend on their lengths and concentrations and the reaction conditions (e.g., temperature, ionic strength, pH).37-39 The ability to perform experiments on multiple identical electrodes within a microelectrode array platform also afforded the opportunity to address the strengths and the weaknesses of this voltammetric label-free method. Obvious challenges to successful exploration of these goals rest on (a) fabrication of the microelectrode array, (b) attachment of probes to electrodes, (c) sensitivity of the instrumentation, and (d) handling of the experiment. With particular regard to point b, the sensitivity and reproducibility of a DNA sensor are substantially determined by the surface chemistry of the DNA recognition interface.40 Major parameters affecting the surface chemistry are characteristics of the surface properties itself such as the roughness, hydrophobicity, density of chemical functionalities, and density and orientation of the probes. When nucleic acid hybridization occurs, the probe and target strands must be free to coil around each other in an energetically stable helix. Immobilization of the probe onto a transducer surface is expected to reduce the freedom of motion that is required for helix formation, i.e., hybridization.40–42 Therefore, since it is not certain that binding on the current surfaces proceeds by the same mechanism as in solution or by probes terminally conjugated onto surfaces, we describe the complementary interactions between solution nucleic acid targets and PPy-pTPT-bound probes more generally as “specific binding”. We examined this issue by attaching probes to propylphosphonic acid (C3PO3H2) and undecylphosphonic acid (C11PO3H2) of pTPT. Finally, our study allowed statistical examination of the voltammetric detection method for a “yes-no” answer for complementary and noncomplementary bacterial and fungal targets as a function of the target lengths. EXPERIMENTAL SECTION Reagents and Materials. Pyrrole (98%) from Aldrich was distilled over calcium hydride under a vacuum and stored in a refrigerator under a N2 atmosphere before use. The mono(35) Riccardi, C. D.; Yamanaka, H.; Josowicz, M.; Kowalik, J.; Mizaikoff, B.; Kranz, C. Anal. Chem. 2006, 78 (4), 1139–1145. (36) Riccardi, C. D. S.; Kranz, C.; Kowalik, J.; Yamanaka, H.; Mizaikoff, B.; Josowicz, M. Anal. Chem. 2008, 80 (1), 237–245. (37) Lubin, A. A.; Hunt, B. V. S.; White, R. J.; Plaxco, K. W. Anal. Chem. 2009, 81 (6), 2150–2158. (38) Gong, P.; Levicky, R. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (14), 5301– 5306. (39) Bishop, J.; Chagovetz, A. M.; Blair, S. Biophys. J. 2008, 94 (5), 1726–1734. (40) Gooding, J. J. Electroanalysis 2002, 14 (17), 1149–1156. (41) Barone, V.; Cacelli, I.; Ferretti, A.; Monti, S.; Prampolini, G. Phys. Chem. Chem. Phys. 2009, 11 (45), 10644–10656. (42) Hagan, M. F.; Chakraborty, A. K. J. Chem. Phys. 2004, 120 (10), 4958– 4968.

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Figure 1. (a) Layout of the PCB connector with a bonded 16microdisk electrode array chip which is used in a dipstick configuration in the experiments. (b) Photograph and micrographs of the “dipstick” microelectrode array chip with embedded electrical contacts. The 30 µm Pt microdisk electrodes are shown after the PPy bilayer was polymerized on all of them simultaneously.

mers of 2,5-dithienyl-N-[3-(diethylphosphoryl)undecyl]pyrrole (TPTC11) or 2,5-dithienyl-N-[3-(diethylphosphoryl)propyl]pyrrole (TPTC3) were synthesized according to a literature procedure.31 Tris(hydroxymethyl)aminomethane (Tris) was from Acros or Sigma-Aldrich, hydrochloric acid (HCl), sulfuric acid (H2SO4), and acetonitrile (ACN) were from VWR, tetraethylammonium perchlorate (TEAP; 98%) was from Alfa Aesar, potassium chloride (KCl), hydrogen peroxide (H2O2; 30%), and isopropyl alcohol (IPA) were from J.T. Baker, sodium chloride (NaCl) and silver nitrate (AgNO3) were from Fisher, potassium nitrate (KNO3) was from MP Biomedicals Inc., and acetone was from Mallinckrodt Chemicals. All chemicals were of analytical grade. Magnesium chloride (MgCl2 · 6H2O) from Fisher was recrystallized from alcohol solution before use. Water as a solvent and in all aqueous solutions was purified water (18 MW) from a Millipore Milli-Q system. A digital pH meter (Metrohm) that was calibrated with standard buffer solutions was used for measuring pH values of all solutions. The primary Tris-HCl buffer solution (pH 7.2) was made using 250 mL of 0.1 M Tris and 220 mL of 0.1 M HCl. All nucleic acids (see the Supporting Information, Table S1) were synthesized43 and/or purified at Gen-Probe Inc.; stock solutions were in water and kept frozen at -70 °C prior to use. Preceding the hybridization experiments, the stock solutions were diluted stepwise. Fabrication and Assembly of the Platinum Microelectrode Array Chip. The layout of the fabricated 16-microdisk electrode array chip is shown in Figure 1a. The electrode pattern was formed on a Pyrex wafer obtained from University Wafers Inc., and the 300 nm thick platinum disk electrodes of 30 µm diameter were fabricated at the Georgia Tech MiRC clean room (see Supporting Information, Figure S1). The 16-microdisk electrode array chip is connected to the printed circuit board (PCB) connector using solder bump bonding of the self-aligning connection points. The (43) Browne, K. A. J. Am. Chem. Soc. 2005, 127 (6), 1989–1994.

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chip mounted in a dipstick configuration shown in Figure 1b requires less than 500 µL for contact with solution. It allows an easy interchange and a rapid electrical connection to the 16channel multipotentiostat. All of the microelectrodes are addressed individually. Electrochemical Measurements. All experiments were conducted with a custom-designed multichannel potentiostat. The instrument provides analog circuitry and digitally controlled gain and multiplexing functions. It is controlled and read by a National Instruments data acquisition card. The data acquisition card applies the cell potential, and the potentiostat mirrors the potential for each of the 16 electrodes. The current for the 16 individual electrodes is read using an operational amplifier to convert to voltage reading. The 16-channel potentiostat can apply potential between +5 and -5 V and read the current between 10 pA and 100 mA. The instrument is controlled by a custom-created LabView-based software allowing the use of a variety of electrochemical methods including cyclic voltammetry for electrochemical cleaning and sensor readout and charge-limited amperometry for electrochemical film depositions. All electrochemical methods were conducted on the individually addressed 16 channels, which allowed reduced experiment times and statistical evaluation of collected data on all electrodes within one chip. All experiments were performed in a single-compartment cell (1 cm3) using a three-electrode cell arrangement at room temperature. Two external reference electrodes were used: Ag/ AgCl in 0.1 M KCl//0.1 M KNO3 in H2O for aqueous solutions and Ag/0.1 M AgNO3 in ACN//0.1 M TEAP in ACN for measurements in ACN. A Pt wire was used as the auxiliary electrode. Methods used for functionalization of the surface of the microelectrode array are given in the Supporting Information. Measurement Procedure. The nucleic acid sequences for the two sets of probes based on previous pan-microbial44 designs and two or three targets used in the experiments are referenced in the Supporting Information, Table S1). (a) 16 nt EcoB1932-1947(-) 2′-O-methylribonucleotide probe (PR10) is complementary to 38 nt EcoB1921-1958(+) RNA target, 107 nt EcoB1885-1991(+) DNA target, and 2907 nt Escherichia coli (Eco) 23S rRNA; PR10 and the target regions complementary to PR10 share 50% G + C content. (b) 22 nt CalA1185-1206(-) DNA probe (PR80) is complementary to 44 nt CalA1174-1217(+) RNA target and 1788 nt Candida albicans (Cal) 18S rRNA; PR80 and the target regions complementary to PR80 are 41% G + C. As a quantification of the solution hybridization strength, the thermal stabilities (i.e., melting temperatures, Ts) between the probes and the short synthetic targets were measured in solution as previously described43 except in 20 mM Tris-HCl/ 0.2 M NaCl, pH 7.2. The Tm of PR10 from TAR15 was 73 °C, and the Tm of PR80 from TAR75 was 74 °C; no hyperchromic shifts demonstrating nonspecific interactions were observed between PR10 and TAR75 nor between PR80 and TAR15. Attachment of Probes. The stock solutions of the probes were diluted to 0.1 µM with 0.1 M Tris-HCl, briefly centrifuged and kept in a heating block at 45 °C for 10 min. The generic (44) Hogan; J. J. Polynucleotide Matrix-Based Method of Identifying Microorganisms. US Patent 6.821,770, 2004.

“activated” microelectrode array was immersed in 0.5 mL of 0.1 µM diluted probe for 30-60 min at room temperature. The array was washed by flowing Tris-HCl buffer over the chip and stored in a solution of 10 mM Tris-HCl/0.2 M NaCl until the CVs were recorded (ca. up to 30 min). Target Binding. The solutions of Eco or Cal rRNA targets were prepared by adding 990 µL of 10 mM Tris-HCl/0.2 M NaCl to 10 µL of the stock solution. The dilutions were heated for 10 min at 90 °C and rapidly cooled to 50 °C using an ice bath before a microelectrode array with attached probes was dipped into this solution. All other target stock solutions were also diluted to 10 nM, but they were heated only to 50 °C for 10 min following the protocol above. The binding reactions were then cooled slowly from 50 °C to room temperature. After 60 min, if not described otherwise, the microelectrode array was removed from the solution, washed with 10 mM Tris-HCl/0.2 M NaCl to remove nonbound strands, and left in this solution until the CVs were recorded (e30 min). The choice of 0.2 M NaCl with 10 mM Tris-HCl was based on preliminary binding experiments conducted in 10 mM Tris-HCl and supplemented with 0.8, 0.4, and 0.2 M NaCl solutions. Concentrations higher that 0.2 M NaCl led to shrinkage of the barrier layer and to reduced differentiation between complementary and noncomplementary interactions between the probe alone and the probe plus target conditions. Data Analysis. Three CVs were always recorded after immobilization of the probe and repeated again after the probe was in contact with the target solution as outlined above. The CVs reached complete stabilization by the second CV, but the last CV (from the series of three) was always used for the subtraction of the CVs. The detection signal was evaluated from the area of the recorded CVs as the “change in area (%)” following the equation [(Atarget - Aprobe)/Aprobe] × 100. The mean and relative standard deviation for the responses of all of the microelectrodes in each array were calculated. RESULTS The label-free voltammetric biosensor array and system used in this study includes three parts: (1) an array of 16 microelectrodes integrated onto a single chip used in a dipstick configuration; (2) a dedicated, multiplexed potentiostat and signal processing circuitry serving the microelectrode array; (3) data transfer and evaluation software capable of transforming the raw data from the sensor into useful diagnostic information, preferably capable of being loaded onto a portable computer system, such as a laptop computer or PDA. To ensure a high electrochemical performance of the chip, special attention has been paid to good electrical insulation between the metal electrodes and solution and the electrochemical quality of the Pt surface. That has been accomplished by replacing the doped silicon substrate with glass (Figure S-1 in the Supporting Information) and testing the Pt surface in a solution of 2 mM K4Fe(CN)6/2 mM K3Fe(CN)6 in 1 M KCl (1:1) (Figure S-2 in the Supporting Information). After attachment of PR10 or PR80 (see the Experimental Section) to the Pt/PPy/TPT-Mg2+-modified electrode surface, the dipstick electrode array was in contact in solution either with complementary (C) or with noncomplementary (NC) targets. A series of cyclic voltammetric experiments were performed to quantify these interactions (see Table S-2 in the Supporting Information). On the basis of this result, we selected 10 mM Tris-HCl/0.2 M

Figure 2. Comparison between normalized detection signals from duplicate arrays recorded after binding of PR80 to 10 nM complementary TAR75 (marked as PR80C) or to 10 nM noncomplementary TAR15 (marked as PR80NC). The change in signal from PR10 electrodes after being in contact with 10 nM noncomplementary TAR75 (marked as PR10NC) is also shown. All values are means for 16 electrodes, and the error bars are standard deviations.

Figure 3. Comparison between normalized detection signals on duplicate arrays recorded after binding of PR80 to 10 nM complementary target Cal rRNA (marked as PR80C) or to 10 nM noncomplementary target Eco rRNA (marked as PR80NC). The change in signal from PR10 after being in contact with 10 nM noncomplementary target Cal rRNA (marked as PR10NC) is also shown. All values are means for 16 electrodes, and the error bars are standard deviations.

NaCl as the optimal background electrolyte and the -0.7 to +0.6 V potential range for further analytical characterization of the performance of the microelectrode array. First, we tested the recognition of the short RNA TAR75 (44 nt) by cDNA PR80 (22 nt) attached to the phophorylundecyltethered linker of the poly(TPTC11), Figure 2. The two replicate chips demonstrate similar mean responses of ca. 17% decreases in mean CV areas for TAR75 binding to PR80. Changes in detection signals that originated from the two replicate chips of PR80-attached electrodes in the 38 nt noncomplementary RNA TAR15 solution were much less (6-9% decreases) and distinctly different from the complementary target. The differences in responses of PR80 between the complementary and noncomplementary targets support detection of specific binding. The ca. -10% change of the CV area response of 2′-O-methyl-RNA PR10 (16 nt) with noncomplementary RNA TAR75 was also less than the specific binding response of PR80 for TAR75. The ability of PR80 to distinguish longer targets is shown in Figure 3. Addition of chips derivatized with PR80 to a solution containing the complementary target Cal rRNA (1788 nt) resulted in a ca. 14% decrease in CV area. The effect of noncomplementary target Eco rRNA (2907 nt) on electrodes with attached PR80 was less than Analytical Chemistry, Vol. 82, No. 21, November 1, 2010

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Figure 4. Comparison between normalized detection signals on duplicate arrays recorded after binding of 10 nM TAR15 to complementary PR10 (marked as PR10C) and to noncomplementary PR80 (marked as PR80NC) on the TCPC3 bilayer. All values are means for 16 electrodes, and the error bars are standard deviations. All data were obtained under the same conditions as in Figure 2.

that of the complementary target, averaging ca. -5%. A noncomplementary target Cal rRNA similarly had little effect on immobilized PR10 (ca. -7.5%). The response of PR10 targets after 90 min of interaction with complementary targets 107 nt DNA TAR17 and 2907 nt Eco 23S rRNA resulted in -36.1 ± 12.1% and -19.6 ± 7.7% changes in CV areas, respectively. The reduction of the chloride ion current after interaction of PR80 with complementary targets was always higher compared to the signals resulting from interaction of PR80 or PR10 with the noncomplementary targets. These observations confirm that it is possible to distinguish the specific from the nonspecific binding interaction. The specific binding of the longer probe PR80 with long complementary target Cal rRNA generates signals that are almost at the same level as for the shorter targets but with slightly lower variability. Surprisingly, the size of the target has only a small effect on the normalized signals despite large differences in their anticipated space charge. As expected, probes of different lengths in contact with noncomplementary microbial targets generate a lower response compared to the complementary targets. This experiment let us set a threshold for the detection signal of 10% identified as nonspecific interaction. In other words, the size of the target or of the probe appears to play only a minor role in the detection scheme when the mean response from the 16 electrodes is normalized. To determine how the distance of the immobilized probe from the electrode surface affects the binding results, we tested the effect of poly(2,5-dithienylpyrrole) with propylphosphonic acid tether groups of the poly(TPTC3) layer, Figure 4. Complementary binding of TAR15 to PR10 resulted in a ca. -21% change in CV area but only a ca. -14% change for noncomplementary binding of TAR15 to PR80. The shorter length of the linker led to slightly lower resolution between complementary and noncomplementary interaction than found with the longer linker (see Figure 2). Given the large size of the rRNA targets, it was expected that the kinetics of specific binding might play an important role in the evolution of the negatively charged barrier responsible for the transport of the predominantly negative charge (anions) through the interface of the electrode. That idea was tested by immobilizing PR10 (complementary) and PR80 (noncomplementary) to the surface of separate dipstick electrode arrays. After heat treatment of the 65 pM Eco 23S rRNA target in 10 mM Tris-HCl/0.2 M NaCl, both arrays were immersed simultaneously 9032

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Figure 5. Time course of the response of the label-free voltammetric detection method. PR10 (complementary) and PR80 (noncomplementary) are interacting with target 65 pM Eco rRNA in a solution of 10 mM Tris-HCl/0.2 M NaCl. (a) Raw data showing normalized decreases of the absolute CV areas, DA/APR, plotted as a function of the time course of the experiment. (b) Same data as in (a) but plotted as log(DA/APR) and log(time, s). All values are means for 16 electrodes. Regressions through the points are shown as solid lines, and extrapolations from the regressions are shown as dashed lines. The conditions of the two experiments conducted in parallel were identical.

in 1 mL of 10 mM Tris-HCl/0.2 M NaCl, and cyclic voltammograms were recorded at periodic intervals over 64 h. The area changes (%) in the recorded CVs were evaluated statistically on the basis of the average response of all 16 electrodes within the arrays. For the global analysis, the changes were quantified at specific time intervals of the experiment as (Atargett - Aprobe)/ Aprobe ) ∆A/APR, where Aprobe is the recorded CV area for a given microelectrode within the array with the attached PR before placement in the target solution and Atargett is the area of the CV taken in target and targetless solutions at different times t. Figure 5a compares the kinetic responses in the recorded CVs. The higher rate of shrinkage for the “complementary” PR10 probe and the nonzero rate of shrinkage for the “noncomplementary” PR80 probe show that the idea of “complementarity” is relative. In conventional hybridization kinetics, such differences would be manifested by the shift of the melting curves on the temperature axis. The binding rate for a given solution formulation and polynucleic acid concentration is a function of the difference between the melting constant (Tm) and the temperature of the hybridization reaction; the rate decreases above or below an optimal melting temperature (e.g., Tm - 25 °C for long

polynucleotide complexes).45 Approximating Td, the dissociation temperature of an oligonucleotide bound to a polynucleotide on a surface, of the present nucleic acid complementary pairs bound on the microelectrode surfaces to their measured Tm (73-74 °C), the current experiments may have been performed at Td criteria of ca. 53-54 °C. If the binding rates on the microelectrode surfaces follow those of hybridization in solution, specific binding for the given target solution formulation and probe density on the electrodes may have been less than optimal and nonspecific binding may have been enhanced, resulting in the observed slow specific binding and substantial nonspecific binding. Binding at temperatures below optimal Td criteria may also have contributed to the greater changes in the CV (%) observed for shorter oligonucleotides than rRNAs (vide supra). Nonetheless, the complementary signal was again significantly higher than the noncomplementary signal. Thus, the yes-no character of information was preserved for all the lengths of the observation time. Similar behavior was observed when the CVs of the electrode arrays were recorded intermittently in 10 mM Tris-HCl/0.2 M NaCl, after the arrays were washed with the buffer solution before the CVs were recorded (data not shown). Excellent logarithmic correlation in Figure 5b indicates a common, robust cause of the CV decreases. Extrapolation of the two lines to the intersection (point X) gives the initial, completely nonspecific response of the CV to the interaction with targets, which happens at approximately 10 s. After this, the shrinkage proceeds by the same mechanism, but at different rates for the two probes. Note that a truly noncomplementary response would correspond to the horizontal line through point X. Since the experiments were performed under identical conditions, the only difference being the degree of noncomplementarity of PR80, the result can be interpreted as follows. As the target molecule is introduced to the solution, it rapidly and nonspecifically associates with both probes to a small extent (1.5% shrinkage at point X, 10 s). This step is followed by slow kinetics at the surface of the electrode, resulting in a logarithmic increase of the height of the electrostatic barrier. This increase is markedly faster for the complementary probe, but it is also present for the noncomplementary probe. This experiment shows that the dynamics of the development of the charge barrier depends on a number of factors including the size of the target molecule, the size and position of the complementary binding region, the temperature, and the ionic strength of the electrolyte. It also shows that the observation of a complementary versus noncomplementary response within the scope of this experiment is only relative. Thus, we have not obtained a purely complementary response because, to some extent, some nonspecific association always occurs. DISCUSSION AND CONCLUSIONS The goal of this work has been to demonstrate the label-free, voltammetric electrochemical detection of specific nucleic acid binding in an array format by adopting the basic concepts and operating procedures originally developed for individual microelectrodes.35 By scaling up to a microelectrode array, an unpre(45) Wetmur, J. G. Crit. Rev. Biochem. Mol. Biol. 1991, 26 (3-4), 227–259.

dictable, parasitic leakage developed between the microdisk electrodes when doped silicon was used as the substrate. Replacing the silicon substrate with glass solved this problem; however, this change of the substrate material necessitated some changes in the fabrication sequence (Figure S-1, Supporting Information) and in the final package. The significance of the array lies in the fact that statistics, namely, the confidence limits of the assay, can be significantly improved by normalizing individual responses to the mean of the 16-channel array. The current approach additionally provides future opportunities for developing mixed-probe arrays for detection of multiple analytes in a sample. The optimized dipstick probe has been used successfully in tens of assays using short, medium, and gene product size polynucleic acids. The effects of the spacer in the grafting layer of TPT, types of the buffer, ionic strength, and incubation time have been evaluated. The differences have been found to be relatively minor, and the procedure, as outlined in the Experimental Section, has been adopted as the final procedure. Even the noncomplementary targets resulted in some positive signal (i.e., reduction of the ionic current). This can be explained by nonspecific attachment of such polynucleotides to the probe on the modified electrode surface. It is possible that such nonspecific attachment could be resolved by temperature programming, but we have not examined that approach at this time. The term “hybridization” in the context of our method does not imply formation of the secondary helical structure. The probes used in our method are randomly attached to the rough surface of the polymer via one or more phosphonate-Mg2+-phosphate bridges. Such a mode of attachment likely precludes coiling (though some structural rearrangements can be envisioned), yet the specific sequences in the probe provide a higher affinity for the matching nucleotide sequence in the target molecule than for the less complementary one. However, weaker bonding through fewer base pairs in the case of a noncomplementary target is not excluded. Therefore, the strength of the voltammetric response should be seen in terms of higher binding probability for the complementary than for the noncomplementary target. ACKNOWLEDGMENT We thank Gen-Probe Inc. for providing nucleic acid materials and Dr. Janusz Kowalik for providing the precursors of TPTC3 and TPTC11. We are also grateful to Dr. Kenneth A. Browne (GenProbe) for his helpful discussions and to Dr. Josh Joshi (Georgia Tech) and to Dr. William Brogdon (Centers for Disease Control and Prevention, CDC) for their useful comments. This project was partially supported by the CDC and by the Georgia Research Alliance. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review July 28, 2010. Accepted September 19, 2010. AC102002K

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