Signal-on electrochemical detection of DNA methylation based on the target-induced conformational change of a DNA probe and exonuclease III-assisted target recycling.

A promising electrochemical system was explored for DNA methylation detection according to the construction of a signal-on biosensor. Based on the ingenious design of probe DNA and auxiliary DNA, methylated target DNA triggered the exonuclease III (Exo III) digestion of auxiliary DNA from 3'-terminus, resulting in the conformational change of probe DNA with an electroactive methylene blue (MB) tag at 5'-terminus. Consequently, the MB tag in the probe DNA was close to the electrode surface for electron transfer, generating an increased current signal. Because of the target recycling of methylated DNA, significant signal amplification was obtained. Moreover, bisulfite conversion conferred an efficient approach for the universal analysis of any CpG sites without the restriction of specific DNA sequence. As a result, the target DNA with different methylation statuses were clearly recognized, and the fully methylated DNA was quantified in a wide range from 10 fM to 100 pM, with a detection limit of 4 fM. The present work realized the assay of methylated target DNA in serum samples with satisfactory results, illustrating the application performance of the system in complex sample matrix.