Label-Free Electrochemical Hybridization Genosensor for the

Publication Date (Web): June 14, 2005 ... The aim of this study was to develop a novel assay for the voltammetric detection of DNA sequences .... Labe...
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Anal. Chem. 2005, 77, 4908-4917

Label-Free Electrochemical Hybridization Genosensor for the Detection of Hepatitis B Virus Genotype on the Development of Lamivudine Resistance Dilsat Ozkan Ariksoysal,† Hakan Karadeniz,† Arzum Erdem,† Aylin Sengonul,‡ A. Arzu Sayiner,‡ and Mehmet Ozsoz*,†

Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, 35100 Bornova-Izmir, Turkey, and Department of Microbiology, Faculty of Medicine, Dokuz Eylul University, Inciralti-Izmir, Turkey

The resistance analysis related to the hepatitis B virus (HBV) genotyping and treatment procured key information for the study of infected patients. The aim of this study was to develop a novel assay for the voltammetric detection of DNA sequences related to the HBV genotype on the development of lamuvidine resistance by monitoring the oxidation signal of guanine. This new technique not only provides a rapid, cost-effective, simple analysis but also gives information concerning both genotyping and lamivudine resistance. Synthetic single-stranded oligonucleotides (“probe”) including YMDD (HBV wild type) YVDD, or YIDD (mutations in the YMDD) variants have been immobilized onto pencil graphite electrodes with the adsorption at a controlled potential. The probes were hybridized with different concentrations of their complementary (“target”) sequences such as synthetic complementary sequences, clonned PCR products, or real PCR samples. The formed synthetic hybrids on the electrode surface were evaluated by a differential pulse voltammetry technique using a label-free detection method. The oxidation signal of guanine was observed as a result of the specific hybridization between the probes and their synthetic targets and specific PCR products. The response of the hybridization of the probes with their single-base mismatch oligonucleotides at PGE was also detected. Control experiments using the noncomplementary oligonucleotides were performed to determine whether the DNA genosensor responds selectively. Numerous factors, affecting the probe immobilization, target hybridization, and nonspecific binding events, were optimized to maximize the sensitivity and reduce the assay time. Under the optimum conditions, 457 fmol/mL was found as the detection limit for target DNA. With the help of the appearance of the guanine signal, the new protocol is based on the electrochemical detection of HBV genotype for the development of lamuvidine resistance for the first * To whom correspondence should be addressed. Tel: 0090 232 388 40 001353. Fax: 0090 232 388 52 58. E-mail: [email protected]. † Ege University. ‡ Dokuz Eylul University.

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time. Features of this protocol are discussed and optimized. Hepatitis B virus (HBV) causes acute and chronic hepatitis, chirrosis, and hepatocelular carcinoma.1 Hepatitis B virus infection is the ninth cause of death in the world.2-4 According to the World Health Organization (WHO), more than 400 million people are chronically infected by HBV worldwide.5 There are two main agents used for the treatment of chronic hepatitis B: interferon R and lamivudine [(-)-β-L-2′,3′-dideoxythiacytidine, 3TC]. Longterm treatment is often needed and is associated with many problems such as low cure rate and development of lamivudine resistance.6-8 Lamivudine is a pyrimidine nucleoside analogue that inhibits viral replication by blocking viral reverse transcriptase and DNA polymerase activities.9 Continuous lamivudine therapy may lead to selection of resistant strains that generally become detectable after 6 months or more of therapy.10 The incidence increases with the duration of therapy and may reach 14-32% after 1 year and 38-58% after 2 years.11-13 Lamivudine resistance is associated with nucleotide substitutions that induce amino acid changes in codon 204 of the polymerase gene, HBV strains revealed isoleucine (I) (1) Lee, W. M. N. Engl. J. Med. 1997, 337, 1733. (2) Dusheiko, G. In Oxford Textbook of Clinical Hepatology; McIntyre, N., Ed.; Oxford University Press: New York 1991; p 571. (3) Purcell, R. H. Gastroenterelogy 1993, 104, 955. (4) Mast, E. E.; Alter, M. J.; Margolis, H. S. Vaccine 1999, 1730. (5) Regev, A.; Shiff, E. R. Adv. Intern. Med. 2001, 46, 107. (6) Fattovich, G.; Brollo, L.; Aliberti, A.; Pontisso, P.; Giustina, G.; Realdi, G. Hepatology 1998, 8, 1651. (7) Lai, C. L.; Chien, R. N.; Leung, N. W. Y.; Chang, T. T.; Guan, R.; Tai, D. I.; Ng, K. Y.; Wu, P. C.; Dent, J. C.; Barber, J.; Stephenson, S. L.; Gray, D. F. A. N. Engl. J. Med. 1998, 339, 61. (8) Dienstag, J. L.; Schiff. E. R.; Wright, T. L.; Perillo, R. P.; Hann, H. L.; Goodman, Z.; Crowther, L. D.; Woessner, M.; Rubin, M.; Brown, N. A. N. Engl. J. Med. 1999, 341, 1256. (9) Merle, P.; Trepo, C. J. Viral Hepatol. 1994, 8, 391. (10) Tipples, G. A.; Ma, M. M.; Fischer, K. P.; Bain, V. G.; Kneteman, N. M.; Tyrell, D. L. J. Hepatol. 1996, 24, 714. (11) Leung, N. W.; Lai, C. L.; Chang, T. T.; Guan, R.; Lee, C. M., NG, K. Y.; Lim, S. G.; Wu, P. C.; Dent, J. C.; Edmundson, S.; Condreay, L. D.; Chien, R. N. Hepatology 2001, 33, 1527. (12) Kao, J. H.; Chen, P. J.; Lai, M. Y.; Chen, D. S. Gastroenterology 2000, 118, 554. (13) Kidd-Ljunggren, K.; Miyakawa, Y.; Kidd, A. H. J. Gen. Virol. 2002, 83, 1267. 10.1021/ac050022+ CCC: $30.25

© 2005 American Chemical Society Published on Web 06/14/2005

or valine (V) substitutions instead of methionine in the tyrosine (Y), methionine (M), aspartate (D), and aspartate (D) motif (YMDD motif) These changes are named YVDD or YIDD, which are the most general described mutations cause the of in the this motif.14-19 The detection of emergence of lamivudine resistance in HBV is clinically important, since YMDD mutants generally cause elevation of ALT and serum viral DNA levels. These strains may increase liver damage in 10-25% of the patients.5,20 Several methods have been developed for the detection and identification of the mutations causing lamivudine resistance. Commonly used direct sequencing of HBV DNA is currently the only method that enables the identification of new mutations that could be related to resistance.21,22 However, it is expensive and time-consuming for large numbers of clinical samples. It is also unable to identify minor amounts of variant viruses in a mixed viral population.23 Easy-to-use methods such as restriction fragment length polymorphism (RFLP) and InnoLipa HBV reverse hybridization assay have been developed to detect variants with known sequence mutations.24,25 Methods based on melting curve analysis of probes used in real-time polymerase chain reaction (PCR) may also be (as rapid) an alternative.26 Despite the RFLP method providing a useful analysis, the molecular detection of point mutation on HBV by DNA-like full genomic sequence analysis is a very expensive technique. PCR-based detection is suitable for clinic applications, but it needs expensive private kits and also needs a toxic agent such as ethidium bromide. Increasing interest has appeared in the development of a simple, rapid, and user-friendly method for DNA sequence analysis and mutant gene analysis to allow early and precise diagnoses of infectious agents for routine clinical tests such as detection of an inherited disease.27-29 Electrochemical DNA biosensors (genosensors) offer promise for obtaining the knowledge necessary for development in various (14) Allen, M. I.; Deslauriers, M.; Andrews, C. W.; Tipples, G. A.; Walters, K. A.; Tyrrell, D. L. J.; Brown, N.; Condreay, L. D. Hepatology 1998, 27, 1670. (15) Han, J. H.; Hwang, S. G.; Chung, K. W.; Oh, S. W.; Hong, S. P.; Park, P. W.; Rim, K. S.; Oh, D.; Kim, N. K. Korean J. Genet. 2002, 24, 219. (16) Stuyver. L. J.; Locarnini, S. A.; Lok, A.; Richman, D. D.; Carman, W. F.; Dienstag, J. L.; Schinazi, R. F. Hepatology 2001, 33, 751. (17) Kobayashi, S.; Shimada, K.; Suzuki, H.; Tanikawa, K.; Sata, M. Hepatol. Res. 2000, 17, 31. (18) Hong, S. P.; Kim, N. K.; Hwang, S. G.; Chung, H. J.; Kim, S.; Han, J. H.; Kim, H. T.; Rim, K. S.; Kang, M. S.; Yoo, W.; Kim, S. O. J. Hepatol. 2004, 40, 837. (19) Novoa, S. R.; Tato, A. G.; Guirao, A..A.; Castroagudin, J.; Quintela, A. G.; Riestra, C. G.; Regueiro, B. J. J. Virol. Methods 2004, 115, 9. (20) Maddrey, W. C. Clin. Lab. 2001, 47, 51. (21) Bozdayi, A. M.; Uzunalimoglu, O.; Turkyilmaz, A. R.; Aslan, N.; Sezgin, O.; Sahin, T.; Bozdayi, G.; Cinar, K.; Pai, S. B.; Pai, R.; Bozkaya, H.; Karayalcin, S.; Yurdaydin, C.; Schinazi, R. F. J. Viral Hepat. 2003, 10(4), 256. (22) Niesters, H. G. M.; De Man R. A.; Pas, S. D.; Fries. E.; Osterhaus, A. D. M. E. J. Med. Microbiol. 2002, 51, 695. (23) Pas, S. D.; De Man, R. A.; Fries, E.; Osterhaus, A. D. M. E.; Niesters, H. G. M. J. Clin. Virol. 2002, 25, 63. (24) Allen, M. I.; Gauthier, J.; Deslauriers, M.; Bourne, E. J.; Carrick, K. M.; Baldani, F.; Ross, L. L.; Lutz, M. W.; Condreay, L. D. J. Clin. Microbiol. 1999, 37, 3338. (25) Stuyver, L.; Van, Geyt C.; De Gendt, S.; Van Reybroeck, G.; Zoulim, F.; Leroux-Roels, G.; Rossau, R. J. Clin. Microbiol. 2000, 38, 702-7. (26) Cane, P. A.; Cook, P.; Ratcliffe, D.; Mutimer, D.; Pillay, D. Antimicrob. Agents Chemother. 1999, 43, 1600. (27) Millan, K. M.; Saraullo, A.; Mikkelsen, S. R. Anal. Chem. 1994, 66, 2943. (28) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 3830. (29) Wang, J.; Cai, X.; Rivas, G.; Shiraishi, H.; Farias, P. A. M.; Dontha,. N. Anal. Chem. 1996, 68, 2629.

areas such as biomedical and environmental research. In particular, electrochemical monitoring of DNA hybridization has recently been an attractive research area.30-33 Pividori et al.34 and Palecek and Fojta35 have also reviewed this advancement. Direct electrochemical DNA analysis based on a guanine signal have been done,36-38 and there has been interest mismatch analysis.39-43 Wong and Gooding39 reported on a DNA hybridization biosensor based on long-range electron transfer that is capable of detecting DNA single-base mismatch (C-A mismatched and G-A mismatched targets) on the surface of a gold electrode by using anthraquinone-2,6-disulfonic acid intercalator solution. Kelley and co-workers used an electrocatalysis mechanism employing methylene blue (MB) as an intercalator and ferricyanide as an electrocatalyst for the detection of mismatch-contained sequences.40 In a similar study performed by Ozkan et al., MB was used for the design of an electrochemical hybridization biosensor based on peptide nucleic acid probes to detect mismatch-contained DNA sequences.43 Recent activity in this direction has also focused on the production of electrochemical microchips that will be used during patient diagnosis as hand-held analysis tools. At that point, genosensors are of importance because they provide low-cost, rapid, and compatibile technology for the detection of some inherited diseases.44-48 The electrochemical detection of a hybridization event by using PCR-amplified DNA from real samples in order to obtain reliable measurement of clinical interest has been reported.49-51 Several inherited or infectious diseases, including hemochromatosis and HIV, were successfully diagnosed by using alternating current voltammetry on a gold microchip using PCR-amplified samples.52 Ozkan and co-workers38 reported a label-free protocol for the detection of factor V Leiden mutation, which employs two (30) Wang, J.; Palee`ek, E.; Nielsen, P.; Rivas, G.; Cai, X.; Shiraishi, H.; Dontha, H.; Luo, D.; Farias, P. A. M. J. Am. Chem. Soc. 1996, 118, 7667. (31) Erdem, A.; Ozsoz, M. Electroanalysis 2002, 14(14), 965. (32) Wang, J.; Kawde, A. N.; Erdem, A.; Salazar, M. Analyst 2001, 126 (11), 2020. (33) Kobayashi, M.; Mizukami, T.; Morita, Y.; Murakami, Y.; Yokoyama, K.; Tamiya, E. Electrochemistry 2001, 69, 1013. (34) Pividori, M. I.; Merkoc¸ i, A.; Alegret, S. Biosen. Bioelectron. 2000, 15, 291. (35) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 75A. (36) Wang, J.; Rivas, G.; Fernandes, J. R.; Paz, J. L. L.; Jiang, M.; Waymire, R. Anal. Chim. Acta 1998, 375, 197. (37) Erdem, A.; Pividori, M.; del Vale, M.; Alegret, S. J. Electroanal. Chem. 2004, 567, 29. (38) Ozkan, D.; Erdem, A.; Kara, P.; Kerman, K.; Meric, B.; Hassmann, J.; Ozsoz, M. Anal. Chem. 2002, 74, 5931. (39) Wong, E. L. S.; Gooding, J. J. Anal. Chem. 2003, 75, 3845. (40) Kelley, S. O.; Boon, E.M.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Nucleic Acids Res. 1999, 27, 4830. (41) Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2000, 18, 1096. (42) Long, Y.T.; Li, C. Z.; Sutherland, T.C.; Kraatz, H.B.; Lee, J. S. Anal. Chem. 2004, 76, 4059. (43) Ozkan, D.; Erdem, A.; Kara, P.; Kerman, K.; Gooding, J. J.; Nielsen, P. E.; Ozsoz, M. Electrochem. Commun. 2002, 4 (10), 796. (44) Ozsoz, M.; Erdem, A.; Kerman, K.; Ozkan, D.; Tugrul, B.; Topcuoglu, N.; Erken, H.; Taylan, M. Anal. Chem. 2003, 75, 2181. (45) Marrazza, G.; Chianella, I.; Macsini, M. Anal. Chim. Acta 1999, 387, 297. (46) Wang, J. Chem. Eur. J. 1999, 5 (6), 1681. (47) Thorp, H. H. TIBTECH 1998, 16, 117. (48) Wang, J.; Kawde, A.-N. Anal. Chim. Acta 2001, 431 (2), 219. (49) Marrazza, G.; Chiti, G.; Macsini, M.; Anichini, M. Clin. Chem. 2000, 46, 31. (50) Napier, M. E.; Thorp, H. H. Langmuir 1997, 13, 6342. (51) Minunni, M.; Tombelli, S.; Mariotti, E.; Macsini, M. Fresenius J. Anal. Chem. 2001, 369, 589.

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capture probes by using single-base mismatch analysis technique. The nonspecific binding effects were greatly suppressed by using sodium dodecyl sulfate (SDS). There are also other electrochemical studies for the detection of HBV using synthetic oligonucleotides or real PCR samples. Erdem and co-workers53,54 reported that HBV detection using synthetic oligonucleotides related to the HBV and external electrochemical redox indicator, MB and cobalt phenanthroline [Co(phen)33+], is connected with the carbon paste electrode (CPE) surface by using a differential pulse voltammetry (DPV) technique. The electrochemical DNA biosensor was described for HBV detection using real PCR samples by square wave voltammetry with MB as the hybridization indicator.55 Ye and co-workers56 designed an electrochemical HBV detection biosensor using a selfassembled monolayer-covered gold electrode and real HBV PCR products. They detected HBV with ferrocenium haxafluorophosphate as an indicator by using cyclic voltammetry, differential pulse voltammetry, ac impedance, and XPS spectra methods. A similar kind of electrochemical biosensor was developed for the detection of HBV using a novel indicator, osmium bipyridine [Os(bpy)2Cl2].57 The main advantage of our protocol over other methods is its costeffectiveness because of its lack of need for external hybridization indicators, such as carcinogenic antitumor drugs, metal complexes, and organic dyes. The indicator-based works53-57 have some extra steps for indicator labeling on DNA hybridization. In comparison to previous experimental procedures, this reported one requires less time and no indicator in order to detect of HBV selectively and sensitively. Here for the first time, we describe an electrochemical DNA genosensor for the detection of HBV (YMDD) and the discrimination of the mutation type (YVDD and YIDD) by using the oxidation signal of guanine and a disposable pencil graphite electrode in connection with DPV. There have not yet been any literature reports about the electrochemical detection of HBV variants from PCR-amplified amplicons by using the guanine signal without any external indicators. This system provides a more standardized description related to HBV genomic changes that are formed by reason of long-term lamuvidine therapy. The features of the protocol are discussed, and results are compared with the other protocols previously reported. EXPERIMENTAL SECTION Electrochemical Assay. DPV methods was performed with an Autolab PGSTAT-30 electrochemical analysis system (Eco Chemie). The three-electrode system consisted of a pencil graphite electrode (PGE) as the working electrode, Ag/AgCl as the reference electrode, and a platinum wire as the auxiliary electrode. A Noki pencil model 2000 was used as a holder for the graphite lead (Tombo HB model 0.5mm). Electrical contact with the lead (52) Umek, R. M.; Lin, S. W.; Vielmetter, J.; Terbrueggen, R. H.; Irvine, B.; Yu, C. J.; Kayyem, J. F.; Yowanto, H.; Blackburn, G. F.; Farkas, D. H.; Chen, Y.-P. J. Mol. Diagn. 2001, 3, 74. (53) Erdem, A.; Kerman, K.; Meric, B.; Akarca, U. S.; Ozsoz, M. Anal. Chim. Acta 2000, 422 (2), 139. (54) Erdem, A.; Kerman, K.; Meric, B.; Akarca, U. S.; Ozsoz, M. Electroanalysis 1999, 11 (8), 586. (55) Meric, B.; Kerman, K.; Ozkan, D.; Kara, P.; Erensoy, S.; Akarca, U. S.; Mascini, M.; Ozsoz, M. Talanta 2002, 56, 837. (56) Ye, Y. K.; Zhao, J. H.; Yan, F.; Zhu, Y. L.; Ju, X. H. Biosens. Bioelectron. 2003, 18, 1501. (57) Ju, H. X.; Ye, Y. K.; Zhao, J. H.; Zhu, Y. L. Anal. Biochem. 2003, 313, 255.

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was obtained by soldering a metallic wire to the metallic part. The pencil lead was held vertically with 12 mm of the lead extruded outside (10 mm of which was immersed into the solution). In each measurement, the length of pencil lead was measured by ruler and the volume of blank/DNA solution was optimized.The convective transport was provided by a magnetic stirrer. Characteristics of the Synthetic Samples. A 20-mer oligonucleotide related to the portion of the human genome for HBV was designed to serve as a wild-type capture probe (WTCP), which is capture a oligonucleotide that is complementary to the YMDD sequence.16 MT-1CP (complementary toYVDD) and MT-2CP (complementary to YIDD) probes containing a single transition to WTCP. Capture probes were perfect complements to their targets that are symbolized WTT (YMDD), MT-1T (YVDD) or MT-2T (YIDD). The synthetic short oligonucleotides of the capture probes were purchased from Thermo Hybaid (Erlangen, Germany), and their complementary targets were purchased (as lyophilized powder) from TIB MOLBIOL Synhesalabor (Berlin, Germany). The base sequences used were as follows. Wild-Type Capture Probe: (It is specifically bound with wildtype HBV.) 5′-AAT ACC ACA TCA TCC ATA TA-3′. Mutant Type-1 Capture Probe: (It is specifically bound with mutant type-1 HBVgenome-YVDD.) 5′-AAT ACC ACATCA TCC AcA TA-3′. Mutant Type-2 Capture Probe: (It is specifically bound with mutant type-2 HBVgenome-YIDD. )5′-AAT ACC ACA TCA TCa ATA TA-3′. Wild-Type Target Sequence (WTT)YMDD): 5′-TAT ATG GAT GAT GTG GTA TT-3′. Mutant Type-1 Target Sequence (MT-1T)YVDD): 5′-TAT gTG GAT GAT GTG GTA TT-3′. Mutant type-2 target sequence (MT-2T)YIDD): 5′- TAT ATt GAT GAT GTG GTA TT-3′. Noncomplementary Sequence: 5′-AAT ACC TGT ATT CCT CGC CTG TC-3′. The oligonucleotide stock solutions (1000 mg/L) were prepared with 10 mmol/L Tris-HCl, 1 mmol/L EDTA (pH 8.00, TE) and kept frozen. More dilute solutions of capture probes were prepared using 0.5 M acetate buffer solution containing 20 mmol/L NaCl (pH 4.80, ABS). More dilute solutions of targets were prepared using 20 mmol/L Tris-HCl buffer solution containing 20 mmol/L NaCl (pH 7.00, TBS). Samples. YMDD mutants are caused by a point mutation from A to G at the 743nd position (YVDD) and from G to T at the 745rd position (YIDD) of the human genome. (Nucleotide sequence positions were numbered according to Ono et al.58). Cloned PCR or real-sample PCR amplicons, which included the HBV variants, were prepared by A. Sengonul and A. Sayiner in the Department of Microbiology in the Faculty of Medicine, Dokuz Eylul University, using standard PCR techniques from isolated DNA samples. The amplicons were characterized as YMDD, YVDD, or YIDD by using 1.5% agarose gel electrophoresis, and they were 447-bp DNA fragments. A total of 10 real PCR (58) Ono, Y.; Onda, H.; Sasada, R.; Igarashi, K.; Sugino, Y.; Nishioka, K. subtype adr adw Nucleic Acids Res. 1983; p 1747.

samples were analyzed in this study, and each genotype was studied. Quantitative Determination of Samples by Spectrophotometric Assay. The UV-visible spectrophotometer (Schimadzu) was used with quartz cuvettes with a volume of 1 mL and 10-mm path length (Starna). The concentrations of all synthetic oligonucleotides and PCR products were determined by following a spectrophotometric method in which one A260 unit of doublestranded DNA represented 50 µg/mL and one A260 unit of singlestranded DNA represented 33 µg/mL by using the same calculation method previously reported in the literatures.38,44 Chemicals and Solutions. Sodium dodecyl sulfate was purchased from Meck. Other chemicals (buffers, etc.) were of analytical reagent grade. Ultrapure and deionized water was used in all solutions. All experiments were performed at room temperature (25 ( 0.5 °C). Methods. Procedure for Voltammetric Assay. Our detection scheme takes ∼101 min. Experimental steps and times are explained as follows: (a) PGE pretreatment for 30 s; (b) probe immobilization for 5 min; (c) washing and drying step for 60 min; (d) target immobilization (hybridization) for 15 min; (e) electrode drying for 5 min; (f) SDS and Tris-HCl washing step for 10 min and 15 s; (g) electrode drying for 5 min; (h) measurement for 20 s. For the real-time PCR products, analysis requires 8 min more because of the denaturation step. In fact, the duration of electrode pretreatment (a), probe immobilization (b), and washing and drying step (c) can be reduced if the electrodes are prepared before the analysis. Electrodes can be prepared like a strip and kept in the refridgerator. When the analysis samples are ready, electrodes can be used so the analysis will ∼30 min for each actual analysis. This procedure illustrated in Scheme 1 was performed by following the steps for hybridization detection and synthetic short oligonucleotides; cloned PCR-amplified amplicons or real PCR amplicons were used as hybridization materials. The procedure consisted of the following steps. Probe Immobilization on the PGE Surface. The PGE was pretreated by applying +1.40 V for 30 s. in 0.50 M ABS (pH 4.8). The WT, MT-1, or MT-2 synthetic capture probe was subsequently immobilized onto the pretreated PGE by applying a potential of +0.50 V for 5 min in the ABS (pH 4.8) containing 5 µg/mL WT, MT-1, or MT-2 probe with 200 rpm stirring. After immobilization, capture probe modified PGEs (graphite leads) were rinsed with blank ABS. Then graphite leads were taken out of the pencil, and they were allowed to air-dry for 1 h in a straight position on the staff in order to form a probe layer covered on the PGE surface. Hybridization with a Synthetic Target. The dried and probe modified graphite leads were immersed in 20 mmol/L TBS (pH 7.00) containing 6 µg/mL target solution for 15 min. After hybridization, nonspecific the adsorption effect was minimized with the following washing step. The hybrid modified leads was dipped into the vials that contained 5% sodium dodecyl sulfate dissolved in TBS (TBS-SDS) 38,56 for 10 min and then immediately dipped into blank TBS for 15 s with fast stirring. The hybrid modified leads were air-dried for 5 min on the straight staff. Hybridizations with only one-base mismatch-containing synthetic and short DNA sequence and noncomplementary DNA sequence were monitored following the same method as described above.

Hybridization with Pure HBV-DNA Fragments Obtained from Cloned PCR Amplification. The amplicon obtained from the cloned PCR amplification was diluted with TBS; the 6 µg/mL concentration of target-containing diluted sample was then placed in a vial and denatured by heating in a water bath at 95 °C for 6 min and subsequent freezing in an ice bath for 2 min. After the immobilization of the probe, the graphite leads were dipped into vials containing denatured and pure YMDD, YVDD, or YIDD amplicon in order to hybridize with capture probe modified lead surfaces. The hybridization was allowed to proceed for 15 min. The hybrid modified leads were dried for 5 min, then dipped into the mixture of TBS-SDS (5% SDS) contained in vials for 10 min, and then immediately dipped into blank TBS for 15 s with stirring. Graphite leads were put on the staff in straight position to air-dry for 5 min. The same protocol was also applied to one-base mismatch containing cloned PCR products, noncomplementary PCR products, and a mixture PCR products, which are included YMDDand YIDD-type mutation at the same time in order to control selectivity of this biosensor. All of the probes also interacted with the PCR blank solution, which contained the primers and polymerase without the target amplified DNA in order to control of biosensor sensitivity. Hybridization with DNA Fragments Obtained from Real Sample PCR Amplification. The amplicons obtained from the real PCR amplification products were diluted with TBS according to the same protocols explained above. After the dilution step, the sample was placed in a vial and denatured by heating in a water bath at 95 °C for 6 min, and probe modified leads were immediately dipped into the hot and denatured solutions in vials, which included denatured YMDD, YVDD, or YIDD real samples, for 15 min in order to hybridize with their probes. The hybrid modified leads were dried for 5 min, dipped into the mixture of TBS-SDS (1% SDS) contained vials for 3 min, and then immediately dipped into blank TBS for 5 s. with stirring. Graphite leads were dried for 5 min in the straight position. The same protocol was also applied to one-base mismatch containing PCR products, noncomplementary PCR products, and the mixture of PCR samples in order to control selectivity of this biosensor. The mixture PCR samples can contain target and one-base mismatch amplicons together or two different amplicons that have one-base mismatches, for example, a YVDD (50%) and YIDD (50%) mixture. Voltammetric Transduction. The oxidation signal of guanine was measured by using DPV in blank ABS by scanning from +0.75 to +1.40 V. DPV measurements, 50-mV modulation amplitude, 8-mV step potential, and 15 mV/s scan rate versus the Ag/AgCl reference electrode, were performed. The raw data were treated using the Savitzky-Golay filter (level 2) of the General Purpose Electrochemical Software of Eco Chemie with moving average baseline correction, using a “peak width” of 0.01 V. Three of the probes were tested with these negative targets and the PCR blank solution, which contained the primers and polymerase without the target amplified DNA, in order to control biosensor selectivity. The results obtained from the electrochemical biosensor were confirmed with those obtained by classical gel electrophoresis method. Analytical Chemistry, Vol. 77, No. 15, August 1, 2005

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Scheme 1. Procedure for PGE Hybridizationa

a The steps are as follows: (1) activation of electrode surface; (2) probe immobilization on electrode surface; (3) hybridization between probe and target; (4) washing with SDS; (M) measurement.

RESULTS AND DISCUSSION The label-free detection of HBV and lamuvidine-resistant HBV genomes was performed using DPV transduction of the hybridization reaction based on the guanine signal between three different types of capture probes and their target DNA sequences, which are existent in the PCR amplified amplicons. An aliquot of the amplified amplicon is simply diluted in the hybridization buffer solution and then interacted into the probe immobilized electrode 4912

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which was in vials. Hybridization is determined with the appearance of the guanine oxidation signal at about +1.00 V,59 whereas no guanine signal can be observed from capture probes that does not contain guanine in the base sequences. The changes in the magnitude of the guanine oxidation signal thus show the HBV specific or lamivudine-resistant types of the targets immobilized on the surface. DPV provides lower detection limits than the ones (59) Wang, J. Anal. Chim. Acta 2002, 469, 63.

Figure 2. Histograms for the electrochemical guanine signals obtained from cloned PCR products (pure products). The numbers 1, 2, and 3 represent each type of guanine-free probe modified electrodes, (1) WTCP, (2) MT-1CP, and (3) WTCP. S represents different kinds of hybridization reactions: (S1) only guanine-free probe signal, (S2) probe + PCR blank signal, (S3) probe + non complementary signal, (S4) probe + mismatch signal, and (S5) probe + target (full match) signal. Before the target immobilization, cloned PCR products were denatured. Other conditions are as in Figure 1.

Figure 1. Voltammograms for the magnitude of guanine oxidation signals before and after hybridization using synthetic oligonucleotides, guanine-free wild-type probe signal (A-d), guanine-free mutant type-1 probe signal (B-d), and guanine-free mutant type-2 probe signal (Cd). Hybrid signals obtained after hybridization between WTCP and WTT (A-a), MT-1CP and MT-1T (B-a), and MT-2CP and MT-2T (Ca); after hybridization between all types of probes and noncomplementary oligonucleotides (A-c, B-c, C-c) and after the hybridization between probes and their one-base mismatch sequences (A-b, B-b, C-b). 5 µg/mL capture probe immobilization on PGE surface by applying at +0.5 V potential for 5 min with stirring; 6 µg/mL target immobilization on probe modified PGE surface in vial for 15 min; DPV measurement, scanning between +0.75 and 1.40 V in ABS with 20 mmol/L NaCl.

obtained by using potentiometric stripping analysis and square wave voltammetry.60 Figure 1 shows the guanine signals obtained when the 5 µg/ mL synthetic capture probes such as WTCP (Figure 1A-d)/MT1CP (Figure 1B-d)/MT-2CP (Figure 1C-d) were modified on the PGE surface. Almost no signal was observed from the guaninefree probe modified PGEs (Figure 1A-d, Figure 1B-d, Figure 1C(60) Palecek, E.; Fojta, M. Anal. Chem. 1994, 66, 1566.

d, respectively). The highest guanine signals were observed after hybridization with a 6 µg/mL concentration of synthetic targets because these sequences contain their complementary oligonucleotides (Figure 1A-a, Figure 1B-a, Figure 1C-a, respectively). When the MT-1T interacted with WTCP covered PGE, a decrease of approximately half was observed on the guanine signal (Figure 1A-b). Similar results were obtained when the MT-1CP and MT2CP covered PGEs were interacted with their one-base mismatch synthetic oligonucleotides including samples Figure 1B-b, Figure 1C-b). Noncomplementary synthetic oligonucleotides were hybridized with all kinds of capture probe modified PGEs and the low guanine peaks were observed (Figure 1A-c, Figure 1B-c, Figure 1C-c, respectively). A series of three repetitive measurements of the oxidation of guanine gave reproducible results with relative standard deviations (RSD) of 11.8% for the hybrid (Figure 1A-a), 11.4% for the mismatch (Figure 1A-b), and 10.2% for the noncomplementary (Figure 1A-c). The guanine signal obtained from the hybridization between MT-1CP and MT-1T gave a RSD value of 9.4% and the hybridization between MT-1CP and MT-2T gave a RSD value of 10.7%. (Figure 1B-a and Figure 1B-b, respectively). The guanine signal gave a RSD value of 12.3% after the hybridization with MT2T for MT-2CP modified PGE (Figure 1C-a). When the synthetic short oligonucleotides were used, a higher RSD value was calculated. As the mismatch oligonucleotides have a short length and they also can hybridize strongly, the removal of these oligonucleotides from the surface of the PGE was difficult. When the experiments were performed by using denatured, cloned PCR product or real PCR product, the discrimination of hybrid signal from mismatch signal was observed clearly and in good reproduciblity. Figure 2 represents the hybridization studies by using cloned PCR amplicons. There are very small signals lower than 10 nA observed with only capture probe immobilized PGEs because no hybridization occurred (S1 series, 1, 2, and 3). The appearance of the high guanine signals was obtained with positive cloned PCR amplicons containing the probes with their target confirmed DNA hybridization. Hybrid couples were WTCP-YMDD clone (S5 Analytical Chemistry, Vol. 77, No. 15, August 1, 2005

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Figure 4. Effect of hybridization time on guanine signal obtained from synthetic hybrid (WTT) and mismatch (MT-1TT) at WTCP modified PGE surface. Other conditions are as in Figure 1.

Figure 3. (A) Effect of target YMDD and (B) target YVDD concentrations upon the DPV response based on the oxidation signal of the guanine at WTCP (A) and MT-1CP (B) modified PGE surfaces. Before the target immobilizations cloned PCR products were denatured. Other conditions are as in Figure 1.

series, 1), MT-1CP-YVDD clone (S5 series, 2), and WTCPYMDD clone (S5 series, 3). These guanine signals were lower than the ones obtained with short synthetic oligonucleotides in Figure 1. In our study, it can be explained that synthetic and short oligonucleotides provide better hybridization with a washing step using SDS; so the guanine signal was higher than the ones in the presence of cloned PCR products. When the 5 µg/mL WT/MT-1 type of capture probe immobilized PGEs were immersed into vials that contained one-base mismatch denatured amplicons, the guanine signal decreased up to 50% (WTCP-YIDD (in S4 series, 1), MT-1CP-YMDD (in S4 series, 2), WTCP-YVDD (in S4 series, 3)). Noncomplementary amplicon (S3 series, 1 and 3) that belonged to the factor V Leiden mutation gene did not show high guanine signals either. Hybridization of WT probe with PCR blank solution did not show any guanine peaks (S2 series, 1 and 3). A series of three repetitive measurements of hybridization of the 5 µg/mL probe with the 6 µg/mL 20-mer target and single-base mismatch gave reproducible results. A series of three repetitive measurements of the oxidation of guanine gave reproducible results with a RSD of 4.6% in Figure 2 for hybrid (S5 series, 1) and 5.3% for mismatch (S4 series, 1). Similar results were obtained with a RSD of 7.8% for hybrid (S5 series, 2) using MT-1CP modified electrode and 6.2% for mismatch (S4 series, 2) and WTCP modified electrode(3), RSD of 5.8% for hybrid (S5 series, 3) and 7.9% for mismatch (S4 series, 3). The effects of target concentration on the guanine (Figure 3) signals were observed. The probe concentration (WTCP and MT1CP) was kept constant at 5 µg/mL, and the target contained WT cloned amplicon (YMDD) (Figure 3A) and YVDD type amplicon (Figure 3B) concentrations were increased from 2 to 8 µg/mL. After the hybridization, there was an increase of guanine signals 4914 Analytical Chemistry, Vol. 77, No. 15, August 1, 2005

Figure 5. Effect of different washing times, 0, 10, 20, 40, and 60 min, for SDS at synthetic hybrid (MT-1T) and mismatch (WTT) modified PGE after DNA interaction with MC at CPE surface. Other conditions are as in Figure 1.

until 6 µg/mL, but it levelled off at 8 µg/mL. The high and reproducible hybrid signal was obtained from a 5 µg/mL probe with 6 µg/mL target, which practically indicated full surface coverage of the probe modified PGE. Figure 4 displays the hybridization time study using synthetic oligonucleotides. A 5 µg/mL concentration of WT probe and 6 µg/mL concentration level of WT target or MT-1 type target (mismatch) were immobilized PGE surfaces, and the changes in the guanine signals were examined after the hybridization. Different hybridization times were applied in the range between 15 and 60 min. There was obtained better and more reproducible discrimination based on the guanine signal applying 15-min hybridization time. Figure 5 shows the effect of the SDS washing time when the synthetic 5 µg/mL WT capture probe and 6 µg/mL target/ mismatch oligonucleotides were used for the hybridization. Ten minutes was chosen as the optimum time in order to obtain better discrimination between target and mismatch oligonucleotides after the hybridization with WTCP immobilized PGEs. The effect of the mixture sample (in the amount of 50% for each target) upon the hybridization signal was also investigated (Figure 6). In Figure 6A, synthetic target oligonucleotides were used for preparing mixture samples. When the WTCP modified sensor was exposed to a mixture containing 6 µg/mL WTT and 6 µg/mL MT-1T, high guanine signals were observed because of the hybridization between WTCP and WTT (Figure 6A-c). At the same time, when the MT-1CP modified PGEs were immersed into the same mixture, the guanine signal increased dramatically due to hybridization between MT-1CP and MT-1T (Figure 6A-d). Another mixture sample containing MT-1T and noncomplementary oligonucleotide was prepared and MT-1CP immobilized

Figure 6. (A) Synthetic mixture study. (a) WTCP modified PGE, (b) MT-1CP modified PGE, (c) WTCP modified PGE after hybridization with WWT/MT-1T oligonucleotide containing mixture, (d) MT-1CP modified PGE after hybridization with WWT/MT-1T oligonucleotide containing mixture, (e) WTCP modified PGE after hybridization with MT-1T/NC oligonucleotide containing mixture, and (f) MT-1CP modified PGE after hybridization with MT-1T/NC containing mixture. (B) Cloned PCR mixture study. (a) MT-1CP modified PGE, (b) MT-1CP modified PGE after hybridization with denatured YVDD(MT-1T)/NC, (c) MT-1CP modified PGE after hybridization with denatured YMDD(WTT)/YVDD(MT-1T) oligonucleotide containing mixture, (d) MT-1CP modified PGE after hybridization with denatured double concentratioon of YVDD(MT-1T), (e) MT-1CP modified PGE after hybridization with denatured YMDD(WTT)/YIDD(MT-2T), and (f) MT-1CP modified PGE after hybridization with denatured YIDD(MT-2T)/NC oligonucleotide containing mixture. Other conditions are as in Fig. 1.

sensor dipped into this mixture. Similar results were obtained because there was a hybridization between MT-1CP and MT-1T (Figure 6A-f). However, when WTCP modified electrodes were immersed into a mixture that contained MT-1TT and noncomplementary oligonucleotides, low guanine signals were obtained after the hybridization (Figure 6A-e). In Figure 6B, cloned PCR target samples were used for preparing mixture samples. No signal was observed with only MT1CP immobilized electrodes (Figure 6B-a and -b). After hybridization between probes and mixtures containing 50% MT-1T/ noncomplementary cloned PCR products (Figure 6B-b), MT-1T/ WTT cloned PCR products (Figure 6B-c), and a double amount concentration of MT-1T (Figure 6B-d), high guanine signal was obtained from each of hybrids because MT-1CP selectively binds to MT-1TT containing these mixtures. When MT-1CP immobilized electrode was immersed into the mixture sample containing 50% MT-2T/WTT cloned PCR products (Figure 6B-e) or MT-2T/ noncomplementary cloned PCR products (Figure 6B-f), low guanine signals were observed because MT-1CP modified PGEs could not hybridize completely with only one-base mismatch including mixture sample (Figure 6B-e) and one-base mismatch/ noncomplementary procducts including samples (Figure 6B-f). After the hybridization, the guanine signal obtained from the double amount of mismatch containing mixture sample (Figure 6B-e) was higher than one-base mismatch/noncomplementary product containing mixtures (Figure 6B-f).

To control our procedure based on denaturation of PCR products without cooling, the highest and the most reproducible guanine signals (mean response as 900 nA) were observed in comparison to denaturation of PCR products with a cooling step in an ice bath. Thus, this procedure was followed for hybridization studies with the real PCR samples. Figure 7 represents the hybridization detection studies with the real PCR samples. Hybridization detection was confirmed with the appearance of the guanine signal. In Figure 7A, WT, MT-1, or MT-2 capture probes were modified on the PGE surface and there is no signal with only probe modified PGE. After the hybridization with denatured PCR samples, the high guanine signal was observed with WT capture probe immobilized PGEs (samples 1-a, 2-a, 3-a). When the MT-1 or MT-2 capture probe immobilized PGEs were immersed into the same samples, the guanine oxidation signals decreased dramatically (samples 1-b, 2-b, 3-b) because these probes contain one-base mismatch sequences. When the WT capture probe immobilized PGE dipped into PCR sample as NC, the guanine signal showed more decrease (sample 4) and the lowest guanine signal were obtained from the negative PCR sample (sample 5). Figure 7B showed patient samples that have included YVDD DNA. There are very low signals (like no signal) obtained from only probe modified PGEs (MT-1CP, WTCP, MT-2CP; first three columns). When hybridization occurred on the PGE surface, high guanine signals were observed using MT-1 probe modified electrodes with sample 6 and sample 1(sample 6-a, sample 1-a). The WT or MT-2 capture probe immobilized electrodes showed half of the decrease with sample 6 and sample 1 (samples 6-b, 1-b) because these probes have a one-base difference in comparison to MT-1CP. Negative sample did not give any guanine signal (sample 7). The high guanine signal was obtained not only with MT-1CP immobilized PGEs but also with WTCP immobilized PGEs for sample 1. It was found that sample 1 was a mixture amplicon (YMDD-YVDD ratio 50%) because the magnitudes of the guanine signals obtained from MT-1 and WT probe modified electrodes were almost equal (not shown). Figure 7C represents PCR samples that have included YIDD DNA. There was no signal obtained only with MT-2 or WT capture probe modified electrode. When the MT-2CP modified PGEs were immersed in sample 8 and sample 9, the guanine signal increased sharply because these samples contained YIDD DNA (samples 8-a, 9-a). A half of a decrease was obtained after the hybridization between WT probe immobilized electrodes and these PCR samples because WT capture probe includes one-base mismatch according to MT-2 capture probe. (samples 8-b, 9-b). Noncomplementary amplicon (sample 10) did give the lowest guanine signal in sample 10. A series of three repetitive DPV measurements based on the guanine signal for the detection of hybridization between the WT and MT capture probes and the target DNA from 10 amplicons gave reproducible results as a mean guanine respose of ∼50 nA for amplicons. The guanine signal obtained from the WT probe modified PGE after hybridization with samples 1, 2 and 3 gave RSD values of 9.4, 7.6, and 10.2%, respectively. The guanine signal obtained from the hybridization of the MT-1 probe with the amplicons (6 and 1) gave RSD values of 10.4 and 11.8%, Analytical Chemistry, Vol. 77, No. 15, August 1, 2005

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Figure 7. Histograms with error bars of the electrochemical guanine signals obtained from real PCR products at (A) 5 µg/mL WTCP-modified PGE (first column), MT-1CP-modified PGE (second column), MT-2CP-modified PGE (third column), after hybridization with 6 µg/mL YMDD type (wild type) samples (samples 1-a, 2-a, 3-a), with 5 µg/mL MT-2CP-modified PGE after hybridization with samples 1 and 3 (mismatch) (samples 1-b, 3-b), with 5 µg/mL MT-1CP-modified PGE after hybridization with sample 2 (sample 2-b), WTCP modified PGE hybridization with NC PCR amplicon (sample 4), WTCP modified PGE hybridization with negative PCR amplicon (sample 5). (B) 5 µg/mL MT-1CP-modified PGE (first column), WTCP-modified PGE (second column), MT-2CP-modified PGE (third column), after hybridization with 6 µg/mL YVDD type (mutant type-1) sample amplicons (samples 6-a and 1-a), WTCP modified PGE after the hybridization with sample 6 (sample 6-b), MT-2CP modified PGE after the hybridization with sample 1 (sample 1-b), MT-1CP-modified PGE after hybridization with negative PCR amplicon (sample 7). (C) 5 µg/mL MT-2CP-modified PGE (first column), WTCP-modified PGE (second column) after hybridization with 6 µg/mL YIDD type (MT-2) samples amplicons (samples 8-a and 9-a), WTCP modified PGE after the hybridization with samples 8 and 9 (samples 8-b and 9-b), MT-2CP modified PGE after the hybridization with sample 10 (noncomplementary). 5 µg/mL capture probe immobilization on PGE surface by applying at +0.5 V potential during 5 min with stirring; 6 µg/mL PCR amplicon denatured 95 °C in water bath, immobilization on probe modified PGE surface in vial for 15 min; DPV measurement, scanning between +0.75 and 1.40 V in ABS with 20 mmol/L NaCl. 4916

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respectively. The guanine signal obtained from the hybridization of the MT-2 probe with samples 8 and 9 gave RSD value of 7.4 and 6.0%, respectively. The detection limits, estimated from S/N ) 3, correspond to 457 fmol/mL using the 6 µg/mL concentration level of target at PGE. Data are given in vertical bar charts, where each bar shows the mean signal values obtained with different replicates of the genosensor in three consecutive experiments (n ) 3). In comparison to the reproducibility and detection limit reported in the study by Wong and Gooding as 1.3-7.2% (n ) 6) and 0.5 µM, our method has an advantage due to its better sensitivity and selectivity to detect one-base mismatch sequence.39 The technique of Kelley et al. requires extra steps such as modification of the surface on the gold electrode and also using indicator MB to detect DNA hybridization;40 this was also seen in a similar study performed by Ozkan et al.43 In comparison with these reported studies,40,43 no indicators have been used without any modification of electrode surface in our study. CONCLUSIONS The new genotyping method that uses an electrochemical genosensor (DNA biosensor) provides simple, cost-effective, and rapid detection of variations in HBV patients. The biosensor is able to detect the complementary sequence in the PCR-amplified amplicons by using the oxidation signal of guanine. This method eliminates the need for traditional hybridization indicators50,53-57 and the use of an expensive kit24,25 or of toxic chemicals,26 such as ethidium bromide, which was used in the gel electrophoresis step of the reference method in HBV analyses. The experimental conditions were optimized by using disposable pencil graphite electrodes that have a special compatibility to microfabrication technology, and these electrodes improved the reproducibility of the results. The success of PGE over existing carbon electrodes is its commercial availability. Additionally, the PGE genosensor scheme is easy to use and portable. Early diagnosis for Lamivudine-resistant HBV genome in the human is important to change the teraphy style. The detection

limit is very low, and it provides early diagnosis for detection of HBV genome variants (YVDD or YIDD). In order for these systems to become useful for point-of-care tests, DNA biosensors should discriminate oligonucleotides with one or more mismatches. In terms of the detection of Lamuvidine resistance, this study is of importance because fast and sensitive electrochemical detection of mismatch analysis was performed. With the help of the experimental results reported here, a microarray device may be produced. Additionally, Lamuvidine resistance can be detected from patient samples using this device. Future work for this laboratory will focus on the design of electrochemical microarrays for detection of target sequences that related to the YMDD, YVDD, and YIDD HBV genomes using these three probes, in connection with simultaneous multielectrode array and multiple hybridization events. Such point-of-care HBV DNA testing would benefit from the introduction of compact, user-friendly, hand-held instruments. ACKNOWLEDGMENT The experimental work includes the doctoral thesis of D.O.A. supported by Ege University, Faculty of Pharmacy, project coordination (Project 04 ECZ 013). A.E. acknowledges the Turkish Academy of Sciences, in the framework of the Young Scientist Award Program (KAE/TUBA-GEBIP/2001-2-8). H.K. acknowledges a scholarship for Ph.D. students from the Technical Research Council of Turkey (TUBITAK). The authors thank TUBITAK for their financial support (Project TBAG-2161). The authors also acknowledge financial support from Ege University, Science and Technology Research Center (EBILTEM) (Project 2004/BIL/008).

Received for review January 6, 2005. Accepted May 13, 2005. AC050022+

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