Electrochemical approach for the specific detection of hepatitis C virus based on site-specific DNA cleavage of BamHI endonuclease

Abstract

This work proposes a new electrochemical approach for specific detection of hepatitis C virus (HCV) based on the site-specific cleavage of BamHI endonuclease. The method was developed by immobilizing a synthetic probe DNA (a short thiol-capped single strand oligonucleotide) on the surface of a gold electrode via the –SH group at the 5′-terminus of the probe, and conjugating the electroactive label of ferroceneacetic acid (FcA) moiety to the 3′-terminus of the probe via formation of a covalent bond between the –NH₂ and –COOH groups. The FcA-labeled probe was then hybridized with target cDNA (an oligonucleotide related to HCV) and cleaved by BamHI endonuclease (a site-specific endonuclease recognizing the duplex symmetrical sequence 5′–GGATCC–3′ and catalyzing double-stranded cleavage between the guanines). After digestion by BamHI, the DNA hybrid was cleaved at a specific site, and the FcA label was removed from the gold electrode surface leading to a decrease or disappearance of the electrochemical signal of the label. The extent of decrease was related to the concentration of target cDNA in solution, which forms the basis of quantitative detection of target cDNA. The detection is based on the variation of voltammetric signal (Δi) of FcA label before and after digestion with BamHI. It was demonstrated that the value of Δi has a linear relationship with the concentration of the HCV DNA (cDNA) ranging from 0.05 to 4.0 μM with a detection limit of (0.5 ± 0.2) nM at a signal/noise of 3. Moreover, the developed method has a high selectivity with ability to discriminate the complementary target DNA sequence from single-base mismatched DNA sequence, and can also be used for detection of HCV in real clinical samples. The major advantages of this enzymatic cleavage assay are its good specificity, ease of performance, and the ability to perform real-time monitoring. The proposed protocol can be taken as a general method of DNA detection and is expected to be applicable to other types of DNA analysis.