Anal. Chem. 2006, 78, 2710-2716
Redox Polymer and Probe DNA Tethered to Gold Electrodes for Enzyme-Amplified Amperometric Detection of DNA Hybridization Paul Kavanagh and Do´nal Leech*
Department of Chemistry, National University of Ireland, Galway, Ireland
The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2′-bipyridine)2(polyvinylimidazole)10Cl]+/2+, and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 × 10-9 to 2 × 10-6 M. The detection limit is ∼1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach. Considerable recent interest in nucleic acid biosensors has been generated due to their potential to detect nucleic acid binding events in a more rapid, simplistic, and cost-effective manner, as compared to conventional hybridization and real-time hybridization assays.1 In particular, biosensors based on electrochemical transduction are useful for DNA detection due to their high sensitivity, low cost, rapid response, small dimensions, low power requirements, and compatibility with microfabrication technology.2,3 The (1) Wang, J. Nucleic Acids Res. 2000, 28, 3011. (2) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 75A. (3) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192.
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simple design of DNA electrochemical sensors, incorporating nucleic acid layers coupled to an electrochemical transducer, could potentially deliver precise and economical platforms for clinical and environmental diagnostics. Various electrochemical approaches to nucleic acid detection have been demonstrated. DNA sensing, based on monitoring voltammetry of redox-active inorganic4-10 or organic11-15 indicators that interact preferentially with double-stranded DNA, has been reported. Labeling of DNA with metal nanoparticles16,17 and the mediated catalytic oxidation of guanine in DNA strands18,19 have been studied to amplify the electrochemical hybridization signal. Amplification of the electrochemical signal due to hybridization has also been demonstrated upon the basis of pioneering work on electrochemical enzymelinked immunosorbent assays,20 by labeling of the target DNA sequence (simple assay) or reporter DNA probe (sandwich assay) with a redox-active enzyme, which catalyzes the electrooxidation/ reduction of a substrate to an electrochemically detectable product.21-32 This was first demonstrated by the Heller group26 (4) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317. (5) Wang, J.; Cai, X.; Rivas, G.; Shiraishi, H.; Farias, P. A. M.; Dontha, N. Anal. Chem. 1996, 68, 2629. (6) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670. (7) Mishima, Y.; Motonaka, J.; Ikeda, S. Anal. Chim. Acta 1997, 345, 45. (8) Maruyama, K.; Motonaka, J.; Mishima, Y.; Matsuzaki, Y.; Nakabayashi, I.; Nakabayashi, Y. Sens. Actors, B 2001, 176, 215. (9) Maruyama, K.; Mishima, Y.; Minagawa, K.; Motonaka, J. Anal. Chem. 2002, 74, 3698. (10) del Pozo, M. V.; Alonso, C.; Pariente, F.; Lorenzo, E. Anal. Chem. 2005, 77, 2550. (11) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 3830. (12) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chim. Acta 1994, 286, 219. (13) Kelley, S. O.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Bioconjugate Chem. 1997, 8, 31. (14) Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2000, 18, 1096. (15) Ozkan, D.; Erdem, A.; Kara, P.; Kerman, K.; Gooding, J.; Nielsen, P. E.; Ozsoz, M. Electrochem. Commun. 2002, 4, 796. (16) Wang, J.; Xu, D.; Kawde, A. N.; Polsky, R. Anal. Chem. 2001, 73, 5576. (17) Zhang, Z.-L.; Pang, D.-W.; Yuan, H.; Cai, R.-X.; Abruna, H. D. Anal. Bioanal. Chem. 2005, 381, 833. (18) Thorp, H. H. Trends Biotechnol. 1998, 16, 117. (19) Yang, I. V.; Thorp, H. H. Anal. Chem. 2001, 73, 5316. (20) Doyle, M. J.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1984, 56, 2355. (21) Aguilar, Z. P.; Fritsch, I. Anal. Chem. 2003, 75, 3890. (22) Dominguez, E.; Rincon, O.; Narvaez, A. Anal. Chem. 2004, 76, 3132. (23) Carpini, G.; Lucarelli, F.; Marrazza, G.; Mascini, M. Biosens. Bioelectron. 2004, 20, 167. (24) Liu, D.; Perdue, R. K.; Sun, L.; Crooks, R. M. Langmuir 2004, 20, 5905. (25) Marchand, G.; Delattre, C.; Campagnolo, R.; Pouteau, P.; Ginot, F. Anal. Chem. 2005, 77, 5189. (26) De Lumley-Woodyear, T.; Campbell, C. N.; Heller, A. J. Am. Chem. Soc. 1996, 118, 5504. 10.1021/ac0521100 CCC: $33.50
© 2006 American Chemical Society Published on Web 03/14/2006
Scheme 1. Schematic Diagram of the Biosensor Formata
a Upon hybridization, electrical contact is established between the glucose oxidase redox center and the electrode via mediating redox polymer OsPVI.
by using redox-mediated electroreduction of peroxide by peroxidase-labeled target DNA. Subsequently, using the redox-enzyme soybean peroxidase, this approach was used to detect a singlebase mismatch in an 18-mer oligonucleotide27 and, more recently, using various other enzymes in the development of highly sensitive sandwich-type assays.28-32 However, the covalent labeling of an oligonucleotide with an enzyme is time-consuming, involving complicated synthetic procedures. Alternatively, taking advantage of the strong avidinbiotin binding affinity, it is possible to develop a more generic approach to the labeling of an oligonucleotide. The modification of a biotin-modified oligonucleotide with an avidin or streptavidinenzyme conjugate, both of which are commercially available, can be achieved in a simple one-step process.33 Such a procedure has been successfully demonstrated for DNA detection, employing a range of enzyme-avidin34-40 and ferrocene-avidin41 conjugates. Examples include the use of avidin-conjugated horseradish peroxidase,34,35 glucose dehydrogenase (PQQ dependent),36 alkaline phosphatase,37-39 and glucose oxidase40 for development of electrochemical signals from biotin-modified oligonucleotides. In this study, we present results relating to the electrochemical characterization of surfaces based on the co-immobilization of probe DNA and an osmium-based redox polymer at the electrode surface for the enzyme-amplified amperometric detection of DNA (27) Caruana, D.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769. (28) Campbell, C. N.; Gal, D.; Cristler, N.; Banditrat, C.; Heller, A. Anal. Chem. 2002, 74, 158. (29) Zhang, Y.; Kim, H. H.; Mano, N.; Dequaire, M.; Heller, A. Anal. Bioanal. Chem. 2002, 374, 1050. (30) Zhang, Y.; Kim, H. H.; Heller, A. Anal. Chem. 2003, 75, 3267. (31) Kim, H. H.; Zhang, Y.; Heller, A. Anal. Chem. 2004, 76, 2411. (32) Zhang, Y.; Pothukuchy, A.; Shin, W.; Kim, Y.; Heller, A. Anal. Chem. 2004, 76, 4093. (33) Guerasimova, A.; Ivanov, I.; Lehrach, H. Nucleic Acids Res. 1999, 27, 703. (34) Azek, F.; Grossiord, C.; Joannes, M.; Limoges, B.; Brossier, P. Anal. Biochem. 2000, 284, 107. (35) Pividori, M. I.; Merkoci, A.; Alegret, S. Biosens. Bioelectron. 2001, 16, 1133. (36) Ikebukuro, K.; Kohiki, Y.; Sode, K. Biosens. Bioelectron. 2002, 17, 1075. (37) Kim, E.; Kim, K.; Yang, H.; Kim, Y. T.; Kwak, J. Anal. Chem. 2003, 75, 5665. (38) Hwang, S.; Kim, E.; Kwak, J. Anal. Chem. 2005, 77, 579. (39) Patolsky, F.; Lichtenstein, A.; Willner, I. Nat. Biotechnol. 2001, 19, 253. (40) Xie, H.; Zhang, C.; Gao, Z. Anal. Chem. 2004, 76, 1611. (41) Liu, J.; Tian, S.; Tiefenauer, L.; Nielsen, P. E.; Knoll, W. Anal. Chem. 2005, 77, 2756.
Scheme 2. Electron Transfer Mechanism of Catalytic Oxidation of Glucose to Gluconolactone by Glucose Oxidase Mediated by the Os2+/3+ of the Redox Polymer
hybridization. The primary aim of this study is to develop stable and reproducible sensing surfaces capable of detecting specific sequences of DNA. This is achieved by cross-linking and tethering the probe DNA sequence and the osmium-based redox polymer to a gold electrode via an anchoring self-assembled monolayer of cysteamine (Scheme 1). After hybridization with the biotinconjugated target sequence, avidin-conjugated glucose oxidase is used as an enzyme amplifier, which subsequently catalyzes the oxidation of glucose by mediated electron transfer through the redox polymer (Scheme 2). To investigate the viability of this approach, we have developed a simple capture assay for the detection of sequences coding for the ssrA gene of Listeria monocytogenes, an important food pathogen.42 EXPERIMENTAL Materials and Reagents. Poly-N-vinylimidazole was prepared by bulk free radical polymerization of vacuum-distilled N-vinylimidazole (Aldrich) using azo-isobutyronitrile (AIBN, Aldrich) as initiator.43 Synthesis and characterization of the [Os(bpy)2(PVI)10Cl]Cl polymer (OsPVI), where bpy is the 2,2′-bipyridine ligand and (PVI)10 is poly-N-vinylimidazole indicating a ratio of coordinated redox sites to free pendant groups of 1:10, was carried out according to literature methods.43 Poly(ethylene glycol)bisglycidyl ether (PEG) cross-linker (average molecular weight of 3350) was purchased from Sigma-Aldrich. Avidin-conjugated glucose oxidase was purchased from Vector Laboratories. Oligonucleotide se(42) Gasanov, U.; Hughes, D.; Hansbro, P. M. FEMS Microbiol. Rev. 2005, 29, 851. (43) Forster, R. J.; Vos, J. G. Macromolecules 1990, 23, 4372.
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Table 1. Probe, Target and Control Oligonucleotide Sequences oligonucleotide sequences (5′ f 3′) probe target control
NH2-C6-ATTCGACAGGGATAGTTCGA biotin-TCGAACTATCCCTGTCGAAT biotin-ATTCGACAGGGATAGTTCGA
quences (Table 1), designed to detect the ssrA gene of L. monocytogenes44 were custom prepared by MWG-Biotech. All other chemicals were purchased from Sigma-Aldrich. Instrumentation. Electrochemical measurements were carried out using an EcoChemie Autolab PGSTAT10 potentiostat coupled to a custom-made 500-µL, single compartment, electrochemical cell containing a gold working electrode (2-mm diameter), a Ag/AgCl reference electrode, and a platinum wire counter electrode (all CH Instruments) in 0.02 M phosphate buffer solution (pH 7.0, containing described concentration of NaCl and EDTA) at room temperature. Recognition Layer Preparation. Gold electrodes were polished using a slurry of alumina (0.05-µm diameter, Buehler) on a microcloth polishing pad, rinsed with distilled water, and cycled between -0.3 and 1.5 V (vs Ag/AgCl) in 0.1 M H2SO4 until a stable voltammogram typical of a clean gold electrode45 was observed. Electrodes were then rinsed with distilled water, dried with N2, and immersed in a 10 mM ethanolic solution of cysteamine for 1 h with stirring. Electrodes were then dried, and a 6-µL drop containing the redox polymer (2 µL of a 5 mg/mL solution in water), a poly(ethylene glycol)bisglycidyl ether (PEG) cross-linker (2 µL of a 15 mg/mL solution in water), and capture probe DNA (2 µL of a 400 µg/mL solution in water) was deposited onto the electrode surface, followed by at least 24-h drying of the film. Electrochemical Film Characterization. Modified electrodes were characterized using cyclic voltammetry at various scan rates in 0.02 M phosphate buffer solution containing 0.15 M NaCl and 1 mM EDTA. Stability of cysteamine-pretreated, gold-modified electrode surfaces was compared using anodic peak currents to films prepared on untreated gold and glassy carbon (4-mm diameter) electrodes. Hybridization and Detection. Recognition surfaces were first cycled between -0.2 and 0.6 V (vs Ag/AgCl) in 0.02 M phosphate buffer solution until a stable voltammogram was obtained to remove any soluble or nonadsorbed species from the surface. The surfaces were then rinsed with distilled water, dried with N2, and a 7-µL drop of hybridization buffer solution (0.02 M phosphate buffer containing 1.0 M NaCl and 1 mM EDTA) containing the appropriate concentration of target DNA was uniformly deposited on the recognition layer surface and allowed react for 30 min. A 2-µL aliquot of glucose oxidase-avidin conjugate (50 µg/mL solution in water) was then added to the solution and allowed to react for an additional 30 min, followed by thorough rinsing of the surface with hybridization buffer solution. The current for bioelectrocatalytic glucose oxidation was measured amperometri(44) Zwieb, C.; Gorodkin, J.; Knudsen, B.; Burks, J.; Wower, J.. Nucleic Acids Res. 2003, 31, 446. See also the tmRNA database at http:// psyche.uthct.edu/dbs/tmRDB/tmRDB.html. (45) Finklea, H. O.; Avery, S.; Lynch, M.; Furtsch, T. Langmuir 1987, 3, 409.
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Figure 1. Cyclic voltammograms of the recognition surface prepared using OsPVI redox polymer cross-linked with DNA probe on a SAMmodified gold electrode in 20 mM phosphate buffer, pH 7.0, 0.15 M NaCl. Scan rates are (a) 100, (b) 200, (c) 300, (d) 400, and (e) 500 mV s-1. (Inset: Plot of the square root of scan rate versus anodic peak currents).
cally in 200 µL of phosphate buffer (0.15 M NaCl) containing 20 mM glucose with the electrode poised at 0.35 V (vs Ag/AgCl). RESULTS AND DISCUSSION Detection Platform Design. A major challenge involved in the design of redox polymer film-modified electrodes for biosensing on a laboratory scale is the production of stable and reproducible recognition layer surfaces. We present results relating to the production of recognition surfaces, which satisfy these requirements, for the detection of DNA/DNA binding based upon the co-immobilization of single-stranded DNA (ssDNA) probe sequences with an osmium-based redox polymer mediator on gold electrodes. The format consists of an amino-terminated 20-base sequence probe DNA cross-linked with OsPVI using a poly(ethylene glycol)bisglycidyl ether reagent that also tethers these films to the electrode via an anchoring self-assembled monolayer (SAM) of cysteamine (Scheme 1). The probe sequence is designed to detect the ssrA gene of L. monocytogenes, an important food pathogen. Our initial studies focus on monitoring the ability of the recognition surface to detect DNA target sequences that are conjugated to a biotin label to establish the method format. Following hybridization and interaction of the biotin-conjugated DNA target sequence with glucose oxidase-avidin conjugate, a current is generated due to the biocatalytic oxidation of glucose to gluconolactone in glucose-containing buffer solutions (Scheme 2). Voltammetry and amperometry are used to examine changes in recognition surface current signals, due to DNA hybridization, and a response is obtained that is binding-dependent. In addition, the fact that the redox polymer and DNA probe of the recognition surface are co-immobilized and tethered to the electrode allows regeneration of the sensing surface. Electrochemical Characterization of Films. The cyclic voltammograms (CVs) of the recognition surface (Figure 1), recorded at scan rates from 100 to 500 mV s-1, exhibit well-defined oxidation and reduction peaks corresponding to the Os2+/3+ redox couple. The formal potential of the redox polymer (E°′ of 0.208 V vs Ag/AgCl), evaluated from the mean of the oxidation and
Figure 2. Cyclic voltammogram of recognition surface in 20 mM phosphate buffer, pH 7.0, 0.15 M NaCl at a scan rate of 5 mV s-1.
reduction peak potentials, is sufficiently negative to avoid damaging the DNA probe immobilized at the electrode surface through oxidation of guanine and adenine bases.2 The CV redox peak currents scale linearly with the square root of the scan rate (Figure 1, inset), indicating semi-infinite diffusional charge transport within the redox film at these scan rates.43,46 For scan rates 95% of the magnitude of the initial signal, indicating that the redox polymer and probe DNA are still bound to the electrode surface (Figure 8). Analytical Chemistry, Vol. 78, No. 8, April 15, 2006
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CONCLUSION In this study, a simple capture assay based on recognition surfaces comprising a gold electrode modified with a SAM of cysteamine, cross-linked with redox polymer [Os(bpy)2(PVI)Cl]+/2+ and a probe sequence of DNA, is used to detect a specific target sequence of DNA. The assay displays a linear increase in current response with respect to concentration of target DNA in the range of 10-9 to 10-6 M, with a detection limit of ∼1.4 fmol in a 7-µL droplet, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible, allowing reusability of the sensor. Based on the results of this preliminary study, we intend to examine the performance of this format in a sandwich-type assay for the detection of DNA coding for L. monocytogenes. With further studies, it is anticipated that the sensitivity of the sensor
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can be improved upon, and evaluation of signals in the presence of single or multiple base mismatches will be undertaken. ACKNOWLEDGMENT This research was supported by funding from the Irish Environmental Protection Agency under the Doctoral Scholarship Scheme. Dr. Tom Barry is thanked for helpful discussions. Access to facilities provided by the Environmental Change Institute, NUI, Galway, is acknowledged.
Received for review December 1, 2005. Accepted February 17, 2006. AC0521100
