Electrochemical Genosensor Based on Peptide Nucleic Acid

Oriental countries lack ALDH activity responsible for the oxidation of acetaldehyde produced during ethanol metabolism and suffer the alcohol-flus...
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Anal. Chem. 2006, 78, 2182-2189

Electrochemical Genosensor Based on Peptide Nucleic Acid-Mediated PCR and Asymmetric PCR Techniques: Electrostatic Interactions with a Metal Cation Kagan Kerman,† Mun’delanji Vestergaard,† Naoki Nagatani,‡ Yuzuru Takamura,† and Eiichi Tamiya*,†

School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City, Ishikawa, 923-1292, Japan, and Japan Science and Technology Agency (JST), Innovation Plaza, 2-13 Asahidai, Nomi City, Ishikawa 923-1211, Japan

The unique structure of peptide nucleic acids (PNAs), linking the N-(2-aminoethyl)glycine units that create a neutral backbone, and prevent it from acting as a primer for DNA polymerase, has been utilized in an electrochemical biosensor scheme for simple and sensitive detection of hybridization. When the PNA is targeted against a single-nucleotide polymorphism (SNP) or wild-type site on the gene, PNA-mediated polymerase chain reaction (PCR) clamping method effectively blocks the formation of a PCR product. In our report, PNA probe for PCR clamping was targeted against the wild-type site of alcohol dehydrogenase. The electrostatic interactions between the negatively charged DNA and neutral PNA molecules with redox-active metal cation cobalt(III)hexamine ([Co(NH3)6]3+) were monitored using differential pulse voltammetry. The electrostatic binding of [Co(NH3)6]3+ to DNA provided the basis for the discrimination against PNA/ PNA, PNA/DNA, and DNA/DNA hybrid molecules. We have optimized the experimental conditions, such as probe concentration, [Co(NH3)6]3+ concentration, accumulation time for [Co(NH3)6]3+, and target concentration. A new pretreatment method has also been employed to allow fast and simple detection of hybridization reaction between the PCR amplicon and the probe on glassy carbon electrode (GCE) surface. This method was based on the application of a high-temperature treatment (95 °C, 5 min), followed by a 1-min incubation in the presence of DNA primers. The excess concentration of DNA primers prevented the rehybridization of the denatured strands, while enabling the target gene sequence to bind with the immobilized probe. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism in standard Roundup Ready soybean samples. The amplicons of asymmetric PCR, which were predominantly single-stranded DNA as a result of unequal primer concentration, hybridized with the DNA probe on the sensor surface efficiently. The attachment of long singlestrands on GCE surface caused the accumulation of [Co(NH3)6]3+ and a high current response. Here, we report a versatile method that would allow for simple and rapid 2182 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

analysis of nucleic acids in combination with PNAmediated PCR and asymmetric PCR techniques by using an electrochemical genosensor. The Human Genome Project has provided an extensive library of opportunities to reveal the secrets of biological events.1 The map of human genome sequence variation was reported to contain 1.42 million single-nucleotide polymorphisms (SNPs).2 The methods for in vitro analysis of SNPs include enzymatic3 or chemical4,5 probing of mismatched complexes, gradient gel electrophoresis,6 application of nucleotide analogues,7 hybridization with allelespecific oligonucleotides,8 and the oligonucleotide ligation assay.9 Furthermore, DNA biosensors have been playing an important role in the detection of SNPs for over a decade.10 Optical,11,12 quartz crystal microbalance (QCM),13 and electrochemical14,15 methods have been successfully employed with sensitive and selective results. * Corresponding author: (e-mail) [email protected]. † Japan Advanced Institute of Science and Technology (JAIST). ‡ Japan Science and Technology Agency (JST). (1) International Human Genome Sequencing Consortium. Nature 2001, 409, 860-921. (2) International SNP Map Working Group. Nature 2001, 409, 928-933. (3) Myers, R. M.; Larin, Z.; Maniatis, T. Science 1985, 230, 1242-1246. (4) Novack, D. F.; Casna, N. J.; Fisher, S. G.; Ford, J. P. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 586-590. (5) Cotton, R. G. H.; Rodrigues, N. R.; Campbell, D. R. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4397-4401. (6) Myers, R. M.; Lumelsky, N.; Lerman, L. S.; Maniatis, T. Nature 1985, 313, 495-498. (7) Kornher, S.; Livak K. J. Nucleic Acids Res. 1989, 17, 7779-7784. (8) Saiki, R. K.; Walsh, P. S.; Levenson, C. H.; Erlich, H. A. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6230-6234. (9) Landegren, U.; Kaiser, R.; Saunders: J.; Hood, L. Science 1988, 241, 10771080. (10) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317-2323. (11) Matsubara, Y.; Kerman, K.; Kobayashi, M.; Yamamura, S.; Morita, Y.; Takamura, Y.; Tamiya, E. Anal. Chem. 2004, 76, 6434-6439. (12) Matsubara, Y.; Kerman, K.; Kobayashi, M.; Yamamura, S.; Morita, Y.; Tamiya, E. Biosens. Bioelectron. 2005, 20, 1482-1490. (13) Minunni, M.; Tombelli, S.; Scielzi, R.; Mannelli, I.; Mascini, M.; Gaudiano, C. Anal. Chim. Acta 2003, 481, 55-64. (14) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 11921199. (15) Kerman, K.; Kobayashi, M.; Tamiya, E. Meas. Sci. Technol. 2004, 15, R1R11. 10.1021/ac051526a CCC: $33.50

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However, the detection of a SNP within an immense genomic DNA is truly a difficult task. In most methods, the target nucleic acid is amplified to detectable quantities by the polymerase chain reaction (PCR) to enhance the sensitivity.16 Thus, PCR has become an important part of an integrated DNA biosensor.17 Additionally, the use of sequence-specific oligonucleotides for hybridization is an essential part of the DNA biosensors. A promising candidate for this role would be peptide nucleic acids (PNAs). PNA is a potent DNA mimic consisting of N-(2-aminoethyl)glycine units, which makes it electrically neutral and stable against nucleases and proteases.18,19 Nielsen and co-workers reported that PNA/DNA duplexes are generally 1 °C/base pair more stable thermally than the corresponding DNA/DNA duplexes at physiological ionic strength.20-22 Furthermore, SNP discrimination is greater for PNA/DNA than for the corresponding DNA/DNA duplexes,23 because they are less tolerant of SNPs than are DNA/ DNA ones.24 The neutral peptide-like backbone of PNA provides the basis for the PNA oligonucleotide (probe) to hybridize to target DNA sequences with high affinity and specificity.25,26 PNA molecular beacons27,28 have been utilized for the detection and quantification of rRNA in solution and in whole cells.29 PNA-based biosensors in connection with surface plasmon resonance,30,31 capillary electrophoresis,32 QCM,33,34 and electrochemical transducers35,36 have been successfully developed for the specific detection of SNPs. PNA microarrays have been fabricated for hybridization-based DNA screening.37 (16) Saiki, R. K.; Gelfand, D. H.; Stoffel, S.; Scharf, S. J.; Higuchi, R.; Horn, G. T.; Mullis, K. B.; Erlich, H. A. Science 1988, 239, 487-491. (17) Del Giallo, M. L.; Lucarelli, F.; Cosulich, E.; Pistarino, E.; Santamaria, B.; Marrazza, G.; Mascini, M. Anal. Chem. 2005, 77, 6324-6330. (18) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497-1500. (19) Ray, A.; Norden. B. FASEB J. 2000, 14, 1041-1060. (20) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497-1500. (21) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg, R. H. J. Am. Chem. Soc. 1992, 114, 1895-1897. (22) Giesen, U.; Kleider, W.; Berding, C.; Geiger, A.; Orum, H.; Nielsen, P. E. Nucleic Acids Res. 1998, 26, 5004-5006. (23) Wittung, P.; Nielsen, P. E.; Buchardt, O.; Egholm, M.; Norden, B. Nature 1994, 368, 561-563. (24) Demidov, V. V. Expert Rev. Mol. Diagn. 2002, 2, 89-91. (25) Ozkan, D.; Kara, P.; Kerman, K.; Meric, B.; Erdem, A.; Jelen, F.; Nielsen, P. E.; Ozsoz, M. Bioelectrochemistry 2002, 58, 119-126. (26) Kerman, K.; Ozkan, D.; Kara, P.; Erdem, A.; Meric, B.; Nielsen, P. E.; Ozsoz, M. Electroanalysis 2003, 15, 667-670. (27) Kuhn, K,; Demidov, V. V.; Coull, J. M.; Fiandaca, M. J.; Gildea, B. D.; FrankKamenetskii, M. D. J. Am. Chem. Soc. 2002, 124, 1097-1103. (28) Xi, C.; Raskin, L.; Boppart, S. A. Biomed. Microdevices 2005, 7, 7-12. (29) Xi, C.; Balberg, M.; Boppart, S. A.; Raskin, L. Appl. Environ. Mocrobiol. 2003, 69, 5673-5678. (30) Sawata, S.; Kai, E.; Ikebukuro, K.; Iida, T.; Hoda, T.; Karube, I. Biosens. Bioelectron. 1999, 14, 397-404. (31) Corradini, R.; Feriotto, G.; Sforza, S.; Marchelli, R.; Gambari, R. J. Mol. Recognit. 2004, 17, 76-84. (32) Basile, A.; Giuliani, A.; Pirri, G.; Chiari, M. Electrophoresis 2002, 23, 926929. (33) Wang, J.; Nielsen, P. E.; Jiang, M.; Cai, X.; Fernandes, J. R.; Grant, D. H.; Ozsoz, M.; Beglieter, A.; Mowat, M. Anal. Chem. 1997, 69, 5200-5202. (34) Wittung-Stafshede, P.; Rodahl, M.; Kasemo, B.; Nielsen, P. E.; Norden, B. Colloid Surf., A 2000, 174, 269-273. (35) Wang, J.; Palecek, E.; Nielsen, P. E.; Rivas, G.; Cai, X.; Shiraishi, H.; Dontha, N.; Luo, D.; Farias, P. A. M. J. Am. Chem. Soc. 1996, 118, 7667-7670. (36) Kerman, K.; Matsubara, Y.; Morita, Y.; Takamura, Y.; Tamiya, E. Sci. Technol. Adv. Mater. 2004, 5, 351-357. (37) Weiler, J.; Gausepoh, H.; Hauser, N.; Jensen, O. N.; Hoheisel, J. D. Nucleic Acids Res. 1997, 25, 2792-2799.

PNA probes cannot function as primers for DNA polymerases in PCR.38 Nielsen and co-workers38 showed that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA is targeted against one of the PCR primer sites. This event was named “PCR clamping”.38-40 Furthermore, they demonstrated that this blockage allowed selective amplification/ suppression of target sequences that differed by only one base pair.38 Following this pioneering work, several groups have used PNA-mediated PCR clamping and hybridization probes.41-43 Assays using wild-type-specific PNA as competitors to mutation-specific primers have been described for the detection of point mutations in Ki-ras.44,45 The somatic activating codon 816 c-kit SNPs in pediatric urticaria pigmentosa were detected by using PNAmediated PCR clamping in connection with fluorescein-labeled hybridization probes.46 The A3243G mutation of mitochondrial DNA was detected with high sensitivity by a combination of allelespecific PCR and PNA-mediated PCR clamping.47 Electrostatic interactions between metal ions and DNA have an important impact on the conformational stability of the double helix.48 The presence of mobile cations in proximity to the DNA strands neutralizes negative charges on the DNA’s phosphate groups, so that the repulsion between the phosphodiester backbones does not drive the double helix apart.48 Based on this fundamental function, electrostatic binding of metal cations to DNA has also been found useful in the development of new DNA biosensor technologies.49,50 The application of electrochemistry provided useful information about the interactions of chelated metal cations with DNA.51,52 Millan and Mikkelsen10 reported that DNA on an activated glassy carbon electrode (GCE) could be detected using redox indicators such as [Co(bpy)3]3+ and [Co(phen)3]3+.53 The binding thermodynamics between redox cations ([Co(bpy)3]3+, [Co(phen)3]3+, and benzyl viologen) and calf thymus DNA on a gold electrode were studied by Pang and Abruna.54,55 (38) Orum, H,; Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O.; Stanley, C. Nucleic Acids Res. 1993, 21, 5332-5336. (39) Murdock, D. G.; Wallace, D. C. Methods Mol. Biol. 2002, 208, 145164. (40) Shuermann, M.; Behn, M. Methods Mol. Biol. 2002, 208, 165-179. (41) Hancock, D. K.; Schwarz, F. P.; Song, F.; Wong, L.-J. C.; Levin, B. C. Clin. Chem. 2002, 48, 2155-2163. (42) Takiya, T.; Futo, S.; Tsuna, M.; Namimatsu, T.; Sakano, T.; Kawai, K.; Suzuki, T. Biosci. Biotechnol. Biochem. 2004, 68, 360-368. (43) Ohishi, W.; Shirakawa, H.; Kawakami, Y.; Kimura, S.; Kamiyasu, M.; Tazuma, S.; Nakanishi, T.; Chayama, K. J. Med. Virol. 2004, 72, 558-565. (44) Chen, C. Y.; Shiesh, S. C.; Wu, S. J. Clin. Chem. 2004, 50, 481-489. (45) Dabritz, J.; Hanfler, J.; Preston, R.; Stieler, J.; Oettle, H. Br. J. Cancer 2005, 92, 405-412. (46) Sotlar, K.; Escribano, L.; Landt, O.; Mohrle, S.; Herrero, S.; Torrelo, A.; Lass, U.; Horny, H.-P.; Bultmann, B. Am. J. Pathol. 2003, 162, 737-746. (47) Urata, M.; Wada, Y.; Kim, S. H.; Chumpia, W.; Kayamori, Y.; Hamasaki, N.; Kang, D. Clin. Chem. 2004, 50, 2045-2051. (48) Barton, J. K.; Lippard, S. J. In Nucleic Acid-Metal Ion Interactions; Spiro, T. G., Ed.; John Wiley & Sons: New York, 1980; pp 60-88. (49) K’Owino, I. O.; Agarwal, R.; Sadik, O. A. Langmuir 2003, 19, 4344-4350. (50) Del Pozo, M. V.; Alonso, C.; Pariente, F.; Lorenzo, E. Anal. Chem. 2005, 77, 2550-2557. (51) Carter, M. T.; Bard, A. J. J. Am. Chem. Soc. 1987, 109, 7528-7530. (52) Carter, M. T.; Rodriguez, M.; Bard, A. J. J. Am. Chem. Soc. 1989, 111, 8901-8911. (53) Millan, K. M.; Saraullo, A.; Mikkelsen, S. R. Anal. Chem. 1994, 66, 29432948. (54) Pang, D. W.; Abruna, H. D. Anal. Chem. 1998, 70, 3162-3169. (55) Pang, D. W.; Abruna, H. D. Anal. Chem. 2000, 72, 4700-4706.

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Kelley and co-workers56-58 have developed a new electrocatalytic DNA detection assay based on a reaction between ruthenium(III)hexamine, [Ru(NH3)6]3+, a DNA-binding and redox-active trivalent cation, and [Fe(CN)6]3-. [Ru(NH3)6]3+ binds to DNA nonspecifically through electrostatic interactions with the phosphate backbone, and therefore, the electrochemical reduction of this species yields a signal that reports on the increase of negatively charged groups at the electrode surface upon hybridization of a target sequence. The signal is amplified by the Fe(III) oxidant, which permits Ru(III) to be regenerated for multiple redox cycles. Yu et al.59 determined the surface densities of both single- and double-stranded oligonucleotides by integration of the peak for reduction of [Ru(NH3)6]3+ to [Ru(NH3)6]2+. In addition, the binding constant and electron-transfer rate constant of [Ru(NH3)6]3+ on DNA-modified carbon and gold60 electrodes were evaluated with the help of classical models. Taking advantage of the electrostatic interaction between DNA and redox-active metal cations, we report here the use of cobalt(III) hexamine, [Co(NH3)6]3+ as the hybridization indicator in a PNA-based biosensor design. Structural analysis of the interaction between DNA and [Co(NH3)6]3+ revealed that [Co(NH3)6]3+ could bind to the major groove of DNA along with an electrostatic attraction to the phosphate backbone.60-63 Fourier transform infrared and capillary electrophoresis studies showed that [Co(NH3)6]3+ also induced a partial B to A transition and DNA condensation upon binding.64 Inspired by the fact that PNA cannot function as a primer for DNA polymerase, we used a PNA probe to block a PCR amplification process for an electrochemical biosensor. Furthermore, the specificity of PNA-mediated PCR clamping is such that two alleles that differ by only one SNP could be discriminated for alcohol dehydrogenase (ALDH). Many people from Oriental countries lack ALDH activity responsible for the oxidation of acetaldehyde produced during ethanol metabolism and suffer the alcohol-flush reaction. Crabb et al.65 found that the mutation in the inactive enzyme was a substitution of lysine for glutamate at position 487. The allele (ALDH2) encoding the mutant (MT) subunit was dominant. In this report, we have tested the DNA samples from two Caucasian and Japanese donors for ALDH mutation in connection with PNA-mediated PCR. When a SNP exists in the gene, PCR clamping PNA probe does not bind to that region and the PCR takes place, resulting in the amplification of the double-stranded MT DNA amplicons. The double-stranded MT PCR amplicon is exposed to thermal denaturation, which forms single-stranded DNA. Then, PNA capture probe on the (56) Lapierre, M. A.; O’Keefe, M.; Taft, B. J.; Kelley, S. O. Anal. Chem. 2003, 75, 6327-6333. (57) Gasparac, R.; Taft, B. J.; Lapierre-Devlin, M. A.; Lazareck, A. D.; Xu, J. M.; Kelley, S. O. J. Am. Chem. Soc. 2004, 126, 12270-12271. (58) Lapierre-Devlin, M. A.; Asher, C. L.; Taft, B. J.; Gasparac, R.; Roberts, M. A.; Kelley, S. O. Nano Lett. 2005, 5, 1051-1055. (59) Yu, H.-Z.; Luo, C.-Y.; Sankar, C. G.; Sen, D. Anal. Chem. 2003, 75, 39023907. (60) Su, L.; Sankar, C. G.; Sen, D.; Yu, H.-Z. Anal. Chem. 2004, 76, 5953-5959. (61) Widom, J.; Baldwin, R. L. J. Mol. Biol. 1980, 144, 431-453. (62) Widom, J.; Baldwin, R. L. Biolpolymers 1983, 22, 1595-1620. (63) Widom, J.; Baldwin, R. L. Biolpolymers 1983, 22, 1621-1632. (64) Ouameur, A. A.; Tajmir-Rahi, H.-A. J. Biol. Chem. 2004, 279, 4204142054. (65) Crabb, D. W.; Edenberg, H. J.; Bosron, W. F.; Li, T. K. J. Clin. Invest. 1989, 83, 314-316.

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surface of the GCE binds to its complementary gene sequence on the amplicon, and the subsequent accumulation of [Co(NH3)6]3+ on the sensor surface results in a high current signal. On the other hand, when there is no SNP in the gene, PCR clamping PNA probe strongly attaches to its complementary WT DNA strand and effectively blocks the PCR amplification. The hybridization of these PNA-mediated WT amplicons on probemodified GCE results in less accumulation of [Co(NH3)6]3+ on the sensor surface and a low current signal. Further, we have also carried out experiments with asymmetric PCR amplicons related to genetically modified organisms (GMOs). Asymmetric PCR predominantly produces single-stranded DNA as a result of unequal primer concentrations.66,67 As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into the double-stranded DNA. The higher concentration primer continues the primer synthesis, but only of its strand. We used the asymmetric PCR to ascertain that the copy numbers of ssDNA are substantially more than the dsDNA. The single-stranded PCR amplicons of asymmetric PCR could hybridize with the probe easily and showed the highest current signals. Our electrochemical method provides the basis for simple and rapid analysis of nucleic acids in combination with PNA-mediated and asymmetric PCR systems. EXPERIMENTAL SECTION Apparatus. Electrochemical studies were performed in conjunction with differential pulse voltammetry (DPV) using an Autolab PGSTAT 12 electrochemical analysis system (Eco Chemie, The Netherlands) in connection with its General Purpose Electrochemical System (GPES) software. The three-electrode system was purchased from Bioanalytical Systems (West Lafayette, IN) and consisted of a glassy carbon electrode (i.d. 3 mm) as the working electrode, the reference electrode (Ag/AgCl), and a platinum wire as the auxiliary electrode. Reagents. The synthetic oligonucleotides were purchased from Fasmac Co. (Kanagawa, Japan) for detection of PNA-DNA, DNA-DNA, PNA/PNA hybridization and had the following sequences: PNA or DNA probe, (5′) H-ACC ACC ACT TC-NH2 (3′); PNA or DNA target, (5′) H-GGT TTC GAA GTG GTG GTC TTGNH2 (3′); SNP target, 5′-GGT TTC GAA GCG GTG GTC TTG-3′; noncomplementary (NC) target. 5′-TAG GAC CCT GGA GGC TGA ACC CCG TGC T-3′. PNA-mediated PCR amplification of ALDH fragments including the mutation site was performed using human genomic DNA. PCR amplification of a 176-bp fragment on exon 12 and upstream of intron 12 of the ALDH gene was performed as described by Chen et al.68 using the following primer pairs: DNA primer 1, 5′- CAA ATT ACA GGG TCA ACT GCT ATG-3′; DNA primer 2, 5′-GCC GCG CCC GCC GCC CCG CGC CCC CCC GCC CGC CCC GCG CTC CAC ACT CAC AGT TTT CAC-3′; PNA probe for PCR clamping, H-CCA CAC TCA CAG TTT TC-NH2; ALDH1 PNA/ DNA target, (5′) H-GCA TAC ACT GAA GTG AA-NH2 (3′); ALDH2 PNA/DNA target, (5′) H-GCA TAC ACT AAA GTG AA-NH2 (3′); ALDH1 PNA/DNA capture probe, (5′) H-TTC ACT TCA GTG TAT (66) Innis, M. A.; Myambo, K. B.; Gelfand, D. H.; Brow, M. A. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 9436-9440. (67) Williams, J. F. Biotechniques 1989, 7, 762-769. (68) Chen, W. J.; Loh, E. W.; Hsu, Y. P.; Cheng, A. T. Biol. Psychiatry 1997, 41, 703-709.

GC-NH2 (3′); ALDH2 PNA/DNA capture probe, (5′) H-TTC ACT TTA GTG TAT GC-NH2 (3′). The primers for the detection of Roundup Ready soybeans were determined by Feriotto et al.69 and were purchased from Fasmac Co. (Kanagawa, Japan). The amplified PCR product was 139 bp in length. RupR1(forward), 5′-TGT ATC CCT TGA GCC ATG TTG-3′; RupR2(reverse), 5′-CGC ACA ATC CCA CTA TCC TTC-3′; GMO capture probe (P35S), 5′-GGC CAT CGT TGA AGA TGC CTC TGC C-3′, The oligonucleotide stock solutions (100 µM) were prepared in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 (TE) and stored at -20 °C. [Co(NH3)6]3+ was purchased from Fluka (Tokyo, Japan). Ultrapure water (18.3 MΩ‚cm) from Milli-Q Labo (Nihon Millipore, Tokyo, Japan) was used in all preparations. Procedures. All experiments were conducted at room temperature, except otherwise stated. For statistical evaluations, the current data from five different sensors in five similarly prepared solutions were taken into consideration (n ) 5), except as otherwise stated. Pretreatment of GCE. GCEs were polished using 1-µm alumina slurry for 1 min and rinsed in water followed by sonication for 3 min. Then, the electrodes were conditioned by applying 0.70 V for 2 min in 20 mM Tris-HCl with 25 mM NaCl and 100 mM MgCl2 at pH 7.0 (TBS-Salt) and rinsed with water. TBS-Salt was used all through the experiments. Detection of the Indicator Signal. [Co(NH3)6]3+ solution was prepared at a desired concentration in TBS-Salt. GCE was suspended in this solution while stirring at 200 rpm for 10 min at open circuit potential (0.10-0.25 V) to allow deposition of the indicator onto the sensor surface. Immediately after, DPV was employed while scanning from +0.10 to +1.20 V with an amplitude of 25 mV and a step potential of 5 mV. The raw voltammograms obtained were treated using Savitzky-Golay smoothing (level 4). Detection of the Probe Signal. Adsorption process was employed for the immobilization of the probe on GCE surface. A probe solution was prepared at a desired concentration in TBS-Salt. A drop of the probe solution (20 µL) was cast onto the surface of an inverted electrode, allowed to adsorb for 5 min at room temperature, and then rinsed with TBS-Salt. The probe-immobilized GCE was subsequently exposed to [Co(NH3)6]3+ indicator in TBS-Salt for 10 min, with stirring at 200 rpm. After the indicator accumulation, DPV was applied under similar conditions as described above and data were similarly treated. Detection of the Hybrid Signal. Hybridization between the probe and target molecules took place in TBS-Salt for 10 min without stirring at room temperature. Then, 20 µL of the hybrid solution was pipetted onto the surface of the inverted GCE and left to adsorb for 5 min. After the adsorption, the sensor was rinsed briefly with TBS-Salt. The accumulation of [Co(NH3)6]3+ and the subsequent electrochemical measurement and data treatment were performed as described above. PNA-Mediated ALDH Amplification. Human genomic DNA was extracted from human hair samples of consenting Caucasian and Japanese donors according to the standard protocols of ISO-HAIR (Nippon Gene Co., Ltd., Toyama, Japan). PCR was performed using a thermal cycler system (Takara Inc, Tokyo, Japan) as (69) Feriotto, G.; Borgatti, M.; Mischiati, C.; Bianchi, N.; Gambari, R. J. Agric. Food Chem. 2002, 50, 955-962.

follows: a preheating (95 °C for 5 min) step followed by 35 cycles (at 95 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min), and a final extension at 72 °C for 15 min in 50 µL of reaction mixture containing 50 ng of genomic DNA, 1× buffer, 1.5 mM MgCl2, 10 pmol of each primer and PNA probe for PCR clamping, 0.25 mM dNTPs, and 1.25 units of AmpliTaq (Perkin-Elmer, Norwalk, CT). PCR products of 176 bp were run on 4% (w/v) agarose gels prestained with ethidium bromide (0.5 µg/mL). Gels were run at 100 V in 1× TE buffer (100 mM Tris, 2 mM EDTA) for 180 min, examined under ultraviolet illumination, and gel electrophoresis images taken. PCR-amplified samples were exposed to thermal denaturation to obtain their single-stranded forms. This method has recently been optimized by Mascini and co-workers70 and found to be a simple and useful way to obtain single-stranded DNA for hybridization coupled with surface plasmon resonance70,71 and quartz crystal microbalance72 systems. The principle of this method relies on the use of oligonucleotides, which are complementary to some sequences on the amplified strand. When added to the denaturating mixture, they hybridize with the single-stranded PCR strand and suppress the reannealing process. The complementary base sequence location of these oligonucleotides on the PCR strand should not overlap with the portion forming the complex with the PNA/DNA probe on the surface. This denaturation procedure involved a 5-min incubation at 95 °C and then 1-min incubation at 55 °C, which was determined as suitable for binding of the primers. In this report, we have also made a slight modification to this procedure. We added DNA primers together at a high concentration of 1 µM for prevention of the duplex formation. After the binding of these oligonucleotides, the mutant amplicons were applied to the PNA or DNA probe-modified GCE surface (20 µL) and kept for 10 min for hybridization. In addition, PCR blank solution and another amplicon obtained from a PCR of GMO were used as the negative controls. After a stringent washing process with blank TBS-Salt, the changes in current responses were monitored. Detection of ALDH SNPs. PCR products were diluted 20 times with TBS-Salt and then used as the target molecule. First, the PNA capture probes were hybridized with the PCR product on the sensor surface for 10 min. After the hybridization, the sensor was rinsed briefly with TBS-Salt. The accumulation of [Co(NH3)6]3+ and the following electrochemical measurement and data treatment were performed as described above. Asymmetric PCR for GMOs. The GMO detection was carried out following the PCR conditions reported by Feriotto et al.69 The protocols were adjusted to AmpliTaq Gold (Applied Biosystems). Instead of genomic DNA, the amplicon from the standard PCR was used. As genomic DNA, a standard sample from Roundup Ready soybeans including 5% GMO was used. Standard PCR was performed in a final volume of 50 µL containing 1× Gold buffer and 75 mM MgCl2 by using 1.25 units/reaction of AmpliTaq Gold DNA polymerase (Applied Biosystems), 8 mM dNTPs, 25 µM (70) Wang, R,; Minunni, M.; Tombelli, S.; Mascini, M. Biosens. Bioelectron. 2004, 20, 967-974. (71) Jiang, T.; Minunni, M.; Wilson, P.; Zhang, J.; Turner, A. P.; Mascini, M. Biosens. Bioelectron. 2005, 20, 1939-1945. (72) Minunni, M.; Tombelli, S.; Fonti, J.; Spiriti, M. M.; Mascini, M.; Bogani, P.; Buiatti, M. J. Am. Chem. Soc. 2005, 127, 7966-7967.

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forward and reverse primers, and 50 ng of genomic DNA. Fifty PCR cycles involved the following conditions: denaturation, 30 s, 95 °C; annealing, 30 s, 60 °C; elongation, 30 s, 72 °C. The length of the Roundup Ready PCR product was 139 bp. Asymmetric PCR was also carried out using the product from the standard PCR with the same procedure as described above except that the reverse PCR primer concentration used was 5 µM. Since a forward to reverse primer ratio of 5 to 1 was used, the majority of the final PCR product was ssDNA. Agarose Gel Electrophoresis for GMOs. Gel electrophoresis was carried out in 3% Agarose (Takara, Japan). The amplicon was diluted with loading buffer (Bromphenol blue). Running buffer contained 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA (TAE, pH 8.0). Electrophoresis was carried out at 100 V constant for 20 min. As ladder, Amplisize (Bio-Rad) was used. The gel was stained afterward with incubation in 200 mL of 1× TAE buffer with 6 µL of ethidium bromide. The gel was visualized using a Printograph (Atto, Japan). Electrochemical Detection of GMOs. Asymmetric PCR products were diluted 20 times with TBS-Salt and then used as the target molecule. First, the DNA capture probe was hybridized with the PCR product on the sensor surface for 10 min. After the hybridization, the sensor was rinsed briefly with TBS-Salt. In addition, PCR blank solution and another amplicon obtained from the PCR of ALDH were used as the negative controls. After the stringent washing process with blank TBS-Salt, the changes in current responses were monitored. RESULTS AND DISCUSSION Since [Co(NH3)6]3+ lacks aromatic and planar ligands that can intercalate with DNA, it only associates with the negatively charged phosphate backbone electrostatically. It is therefore a sequence-neutral binder. This, coupled with the fact that it is electrochemically active, makes it an ideal probe for the detection of hybridization on the sensor surface. The electrochemical reduction signal of [Co(NH3)6]3+ at ∼-0.4 V (vs Ag/AgCl) was monitored on a GCE in 20 mM Tris HCl with 25 mM NaCl and 100 mM MgCl2 at pH 7.0 (TBS-Salt). Chart 1 illustrates the affinity of [Co(NH3)6]3+ toward the backbone of DNA. A PNA/ DNA hybrid accumulates fewer numbers of [Co(NH3)6]3+ molecules than a DNA/DNA one. On the other hand, a PNA/PNA hybrid has no charge and thus accumulates the least amount of [Co(NH3)6]3+. We have first explored the coverage of the electrode surface with DNA or PNA probes (Figure 1). We monitored the reduction signals of [Co(NH3)6]3+ upon interaction with the DNA (Figure 1, black line) and PNA (Figure 1, gray line) probes. The negative charges on the backbone of DNA enhanced the accumulation of [Co(NH3)6]3+, and the signal increased, reaching a plateau at 10 µM. On the other hand, the PNA probe does not contain any charge; hence, it has no electrostatic interaction with [Co(NH3)6]3+. Increasing the concentration of PNA led to a decrease in the current signal of [Co(NH3)6]3+, reaching its lowest output at 10 µM PNA concentration. Thereafter, the signal remained almost constant. A significant drop of current response of ∼1 µA with PNA probe immobilized sensors was attributed to the blocking effect of the PNA layer for the metal cations. For further experiments, we employed a probe concentration of 10 µM on the sensor surface. 2186 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

Chart 1. Schematic Representation of the Electrostatic Interactions of DNA and PNA Molecules with a Redox-Active Metal Cation, [Co(NH3)6]3+ (Charge (+) in Yellow)a

a [Co(NH ) ]3+ associates electrostatically with the negatively 3 6 charged backbone of DNA molecules, but it has significantly less affinity toward the neutral backbone of PNA. This differential electrostatic attraction toward DNA and PNA molecules provides the basis for the detection of hybridization between different types of nucleic acids.

Figure 1. Dependence of [Co(NH3)6]3+ current signals on the concentration of DNA (black line) and PNA (gray line) probes immobilized on a GCE surface. Error bars represent the standard deviation of current signals obtained in five experiments (n ) 5).

The effect of target DNA concentration on the signal of [Co(NH3)6]3+ was monitored as shown in Figure 2. As the concentration of target DNA increased, the signal also increased because hybridization of the probes with complementary DNA molecules provided more negative charge for interaction with [Co(NH3)6]3+ (Figure 2, black line). The hybridization between a PNA probe and a DNA target also provided a negative charge, but the signals were lower than the DNA/DNA duplex, especially at high concentrations (Figure 2, gray line). As expected, a PNA/PNA duplex formation on the surface caused a decrease of [Co(NH3)6]3+ signal at all concentrations of the target PNA molecule (Figure 2, light gray line). We further monitored the effect of accumulation time on the signal of [Co(NH3)6]3+ on DNA/DNA (Supporting Information Figure 1, black line), PNA/DNA (Supporting Information Figure 1, gray line), and PNA/PNA (Supporting Information Figure 1, light gray line) hybrid modified sensors. No significant increase in the reduction signal of [Co(NH3)6]3+ was observed for PNA/ PNA hybrid-modified GCE. On the other hand, there was a substantial increase in the signal peaking after 10 min of accumulation on DNA/DNA hybrid-modified GCE. The lower negative charge on PNA/DNA hybrid-modified sensors in com-

Figure 2. Dependence of [Co(NH3)6]3+ current signals on the concentration of target molecules for the hybridization of a DNA probe with a DNA target (DNA/DNA, black line), a PNA probe with a DNA target (PNA/DNA, gray line), and a PNA probe with a PNA target (PNA/PNA, light gray) immobilized on a GCE surface. Error bars represent the standard deviation of current signals obtained in five experiments (n ) 5).

parison with DNA/DNA ones gave almost half of the current signal at all accumulation time periods. We chose 10 min of accumulation time for further experiments, since the signal on all the sensors reached saturation at this concentration. The concentration of [Co(NH3)6]3+ also affected different nucleic acid hybrids to varying extents enabling a clear distinction to be made between the different types of nucleic acids. There was a difference between the interaction of DNA/DNA (Supporting Information Figure 2, black line), PNA/DNA (Supporting Information Figure 2, gray line), and PNA/PNA (Supporting Information Figure 2, light gray line) with the metal cation at all concentrations. The phosphate backbone of the DNA/DNA hybrid clearly attracted more of the cation and the signals were higher than the other nucleic acid couples. The concentration of [Co(NH3)6]3+ was chosen as 1 mM for further experiments. We utilized DPV with the high concentration of the metal cation, and the current signals, we observed, were useful for the discrimination against SNPs and the different types of nucleic acid hybrids. Control experiments were performed to assess whether the biosensor responded selectively, via hybridization, to the targets. Figure 3 shows the current signals obtained from the exposures of the PNA probe-modified GCE to different target DNA molecules. Following the exposure of the PNA probe-modified GCE (Figure 3, magenta line) to NC DNA (Figure 3, yellow line), almost no change in the reduction signal of [Co(NH3)6]3+ was observed. PNA probes did not form any hybrid with the NC DNA, because only a complementary sequence could have formed a hybrid as shown in Figure 3, red line. No significant increase in the [Co(NH3)6]3+ signal (Figure 3, blue line) was also observed upon hybridization with the SNP DNA. Our results are in good agreement with the previous reports,33-36 in which the selective hybridization of PNA probes with only the full-complementary gene sequences were explored in various DNA biosensor schemes. In this report, the signal obtained from PNA/DNA hybrid (Figure 3, red line) was significantly higher (P < 0.05) than the other ones obtained from PNA/NC and PNA/SNP interactions, because the formation of PNA/DNA hybrid with negative charge accumulated the cations on the surface. The peak potential for the reduction signal of [Co(NH3)6]3+ at a bare GCE (Figure 3, green

Figure 3. Voltammograms of the electrochemical detection of hybridization between PNA probe and DNA target, PNA probe and SNP containing DNA, PNA probe and NC DNA, and PNA probe alone on GCE surface in the presence of 1 mM [Co(NH3)6]3+ after 10-min accumulation with stirring at 200 rpm. The signal of 1 mM [Co(NH3)6]3+ alone is at a bare GCE is also shown.

Figure 4. Current signals obtained after the hybridization of ALDH1 PNA capture probe with DNA and PNA target molecules. Error bars represent the standard deviation of current signals obtained in five experiments (n ) 5).

line) also shifted to more negative values as the interaction between PNA probes and target molecules took place on the surface. Our detection limit for target DNA was 100 nM at a PNA probe-modified GCE, and 250 nM at a DNA probe-modified GCE. We have applied our PNA biosensor for the detection of SNP related to the ALDH. We used two PNA capture probes complementary to the wild-type alleles of ALDH. The gel electrophoresis image for the PNA-mediated PCR amplifications is shown in Supporting Information Figure 4A. First, we checked the performance of these PNA capture probes for hybridization with full-complementary DNA and PNA target molecules. Figure 4 shows the current responses obtained with the PNA capture probe, ALDH1. The current signal responses were plotted for ALDH2-modified GCEs before and after hybridization with PNA and DNA targets (Supporting Information Figure 3). The formation of PNA/PNA duplex did not cause a significant increase in the signal of [Co(NH3)6]3+, whereas the formation of PNA/DNA duplex caused a clear increase. According to a one-sample t-test, we obtained a significant difference (P < 0.05) between the [Co(NH3)6]3+ signals obtained at a bare surface and the probemodified one. There was no statistical difference between PNA capture probe-modified sensor signals and the one of the PNA/ PNA hybrid-modified sensor. We also found significant difference (P < 0.05) between the signals of PNA probes and their hybrid Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

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Chart 2. Illustration for the Electrochemical Detection Principle by Using Asymmetric or Thermally Denaturated PNA-Mediated PCR Ampliconsa

a When PNA capture probe on the surface of GCE binds to its complementary gene sequence on the amplicon, the subsequent accumulation of [Co(NH3)6]3+ on the sensor surface results in a high current signal.

signals with DNA target. An independent t-test for the PNA/PNA hybrid and PNA/DNA hybrid showed a significant difference (P < 0.05) for both of the PNA capture probes. This result was in agreement with our previous results reported above. We had also shown that [Co(NH3)6]3+ is not a sequence-specific indicator, and this method could be used for the hybridization detection of different sequences. The application of thermally denaturated and PNA-clamped amplicons as well as the asymmetric PCR amplicons to our method is illustrated in Chart 2. The gel electrophoresis image is shown in the Supporting Information Figure 4A. The long ssDNA of the amplified real samples could hybridize with the probe on the sensor surface and resulted in the accumulation of [Co(NH3)6]3+ and a high current response. After the PNA-mediated PCR amplification, the amplicons were exposed to thermal denaturation, and reannealing was blocked with the primers as described in the Experimental Section. Thus, the amplicon kept its ssDNA form, which made it easy to hybridize with the probe on the surface. We observed an increase in the current signal of [Co(NH3)6]3+ after hybridization with the homozygote mutant (hoMT) sample in both of the probes (Figure 5A, red line; B, f). This dramatic increase in the signal was attributed to the binding of the 176-bp-long amplicon strand creating more negative charge on the surface for the electrostatic binding of [Co(NH3)6]3+. There was a significant difference (P < 0.05) between the signals obtained with the PNA probe (Figure 5B, b) and the ones obtained with hoMT (Figure 5B, f). On the other hand, there was no significant difference between the signals obtained with the hybridization between MT PNA probe and the denaturated WT amplicon (Figure 5B, d). The asymmetric PCR amplicon obtained from Roundup Ready soybean samples was also applied to our electrochemical genosensor. The gel electrophoresis image is shown in the Supporting Information Figure 4B with the band of the asymmetric PCR amplicon in lane 2. The ssDNA amplicon that was obtained from the asymmetric PCR hybridized with the GMO capture probe on the sensor surface efficiently (Figure 5A, green line; B, g). Since there was an increase in the negative charge on the surface, [Co(NH3)6]3+ could accumulate and resulted in a high current response. The nonspecific adsorption of amplicons was effectively suppressed as shown in the results of our method, in which the hybridization reactions between the MT probe and the asymmetric PCR amplicon (Figure 5A, gray line; B, e) and between 2188 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

Figure 5. (A) Voltammograms of the electrochemical detection of hybridization between PCR amplified samples and DNA probes on GCE surface. After the hybridization reaction between GMO capture probe and the asymmetric GMO PCR amplicon, between the DNA mutant capture probe and the denaturated hoMT amplicon, between the GMO probe the denaturated hoMT amplicon, and between the DNA mutant capture probe and the asymmetyric GMO PCR amplicon. Other conditions were as decribed in the Experimental Section. (B) Plot of the current signals obtained (a) with only GMO DNA capture probe, (b) with only MT PNA capture probe, (c) after the hybridization between the GMO DNA capture probe and the denaturated hoMT amplicon, (d) after the hybridization between the MT PNA capture probe and the denatured WT amplicon, (e) after the hybridization between the MT PNA capture probe and the asymmetric PCR GMO amplicon, (f) after the hybridization between the MT PNA capture probe and the denaturated hoMT amplicon, and (g) after the hybridization between GMO capture probe and the asymmetric GMO PCR amplicon. Error bars represent the standard deviation of current signals obtained in five experiments (n ) 5).

the GMO capture probe and the thermally denaturated hoMT amplicon (Figure 5A, black line; B, c) resulted in low current signals.

CONCLUSIONS In this report, the PNA-mediated and asymmetric PCR systems were employed with an electrochemical hybridization biosensor. We have shown that the electrostatic interactions of [Co(NH3)6]3+ with DNA and PNA are suitable for detecting DNA hybridization. The reduction signal is found efficient for the discrimination of PNA/PNA, PNA/DNA, and DNA/DNA hybrids. The large signal enhancements obtained for DNA/DNA and PNA/DNA hybrids provided excellent selectivity and reproducibility. Indeed, [Co(NH3)6]3+ proves to be a promising electrochemical hybridization indicator. However, with respect to detection levels, further improvements are required in order to provide a more viable alternative, to other previously reported electrochemical biosensors.33-36 Nonetheless, the significant discrimination against a SNP at the sensor surface indicates that PNA-mediated PCR clamping in

connection with PNA capture probes is feasible, is simple, and provides a rapid genetic analysis. Future work in this laboratory will focus on employing [Co(NH3)6]3+ as an indicator for the detection of clinically important gene sequences by using PNAmediated and asymmetric PCR systems in connection with an electrochemical biosensor.

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review August 26, 2005. Accepted February 1, 2006. AC051526A

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