A label-free electrochemical DNA sensor using methylene blue as redox indicator based on an exonuclease III-aided target recycling strategy

In this work, using methylene blue (MB) as a redox marker and exonuclease III (Exo III) as an amplificatory enzyme, we developed a facile and a label-free electrochemical method for sensitive DNA detection. A double-stranded DNA (dsDNA) probe was prepared by hybridizing two single-stranded DNA (ssDNA) probes. In the ssDNA probes, one ssDNA was guanine bases free and the other one consisted of many unbound guanine bases. MB could be absorbed on the unbound guanine bases owing to the specific interaction between MB and the guanine bases. When the dsDNA probe was challenged with target DNA, it induced a simple Exo III assisted cleavage process, accompanied by the release of the unbound guanine bases. Thus, the amount of MB absorbed on the electrode was much less compared to the initial signal. The detection limit for DNA was found to be as low as 20 fM. Moreover, it could discriminate mismatched DNA from perfectly matched target DNA. This detection method is simple in design, fast in operation and can be applied to detect different DNA sequences.