Direct detection and discrimination of double-stranded oligonucleotide corresponding to hepatitis C virus genotype 3a using an electrochemical DNA biosensor based on peptide nucleic acid and double-stranded DNA hybridization

Development of an electrochemical DNA biosensor for the direct detection and discrimination of double-stranded oligonucleotide (dsDNA) corresponding to hepatitis C virus genotype 3a, without its denaturation, using a gold electrode is described. The electrochemical DNA sensor relies on the modification of the gold electrode with 6-mercapto-1-hexanol and a self-assembled monolayer of 14-mer peptide nucleic acid probe, related to the hepatitis C virus genotype 3a core/E1 region. The increase of differential pulse voltammetric responses of methylene blue, upon hybridization of the self-assembled probe with the target ds-DNA to form a triplex is the principle behind the detection and discrimination. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the developed DNA sensor responds selectively to the ds-DNA target. Diagnostic performance of the biosensor is described and the detection limit was found to be 1.8 × 10⁻¹² M in phosphate buffer solution, pH 7.0. The relative standard deviation of measurements of 100 pM of target ds-DNA performed with three independent probe-modified electrodes was 3.1%, indicating a remarkable reproducibility of the detection method.