An electrochemical approach for direct detection and discrimination of fully match and single base mismatch double-stranded oligonucleotides corresponding to universal region of hepatitis C virus

Abstract

Development of an electrochemical DNA biosensor for direct detection and discrimination of HCV core/E1 region corresponding double-stranded DNA (ds-DNA) using a peptide nucleic acid (PNA) oligomer as the probe is described. The PNA probe is a cysteine conjugated 20-mer PNA oligomer, complementary to HCV core/E1 universal region, which is a consensus sequence in almost all HCV genotypes and is not present in other organisms. The significant variation in differential pulse voltammetric response of methylene blue (MB) on the probe modified gold electrode (AuE) upon hybridization with complementary double-stranded oligonucleotide (ds-oligonucleotide) following PNA/ds-DNA hybrid formation is the principle of target ds-DNA detection. No significant variations in MB signal following interaction of the probe with non-complementary and single-base mismatch (SBM) ds-DNAs was observed. This is due to the lack of hybridization between the probe and the non-complementary and SBM ds-DNA samples. Diagnostic performance of the biosensor is described and the detection limit of fully match target ds-DNA was found to be 9.63 × 10⁻¹² M and 4.97 × 10⁻¹² M for 2 h and 20 h hybridization times, respectively. The relative standard deviation over three independently probe modified electrodes measured at 100 pM of target ds-DNA was 2.9% and 2.4% for 2 h and 20 h hybridization times indicating a remarkable reproducibility of the detection method.