Electrochemical DNA biosensor for the detection of TT and Hepatitis B virus from PCR amplified real samples by using methylene blue

Abstract

DNA biosensors based on nucleic acid hybridization processes are rapidly being developed towards the goal of rapid and inexpensive diagnosis of genetic and infectious diseases. Electrochemical transducers are often being used for detecting the DNA hybridization event, due to their high sensitivity, small dimensions, low cost, and compatibility with microfabrication technology. In this study, an electrochemical biosensor for the voltammetric detection of DNA sequences related to the Hepatitis B virus (HBV) and TT virus (TTV) from polymerase chain reaction (PCR) amplified real samples is described for the first time. The biosensor relies on the immobilization of the 21- or 24-mer single stranded oligonucleotides (probe) related to the HBV and TTV sequences and hybridization of these oligonucleotides with their complementary sequences (target) at carbon paste electrode (CPE). The extent of hybridization between the probe and target sequences was determined by using square wave voltammetry (SWV) with moving average baseline correction and methylene blue (MB) as the hybridization indicator. As a result of the interaction between MB and the bound guanine bases of hybrid at CPE surface, the MB signal decreased, when it was compared with the MB signal, which was observed with probe modified CPE. The difference between the MB signals, obtained from the hybrid modified and the probe modified CPE is used to detect the DNA sequences of the infectious diseases from PCR amplified real samples. Numerous factors affecting the target hybridization and indicator binding reactions are optimized to maximize the sensitivity.