ANALYTICAL CHEMISTRY, cilt.77, ss.4908-4917, 2005
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 mild 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 time. Features of this protocol are discussed and optimized.