DNA methylation detection and site analysis by using an electrochemical biosensor constructed based on toehold-mediated strand displacement reaction

DNA methylation has become a novel target for early diagnosis and prognosis of cancer as well as other related diseases. The accurate detection of the methylation sites of specific genes proved to be of great significance. However, the complex biological nature of clinical samples and the detection of low-abundance targets led to higher requirements for the testing technology. It has been found that by virtue of high sensitivity, rapid response, low cost, facile operation and applicability to microanalysis, electrochemical sensors have greatly contributed to the process of clinical diagnosis. In this study, a facile, rapid and highly sensitive electrochemical biosensor based on the peak current change was developed on the basis of high selectivity of toehold and greater efficiency of PNA strand displacement and used for the detection and site analysis of DNA methylation. Moreover, compared with non-methylated DNA sequences, methylated DNA sequences could be readily invaded by PNA probes, thereby resulting in the strand displacement and significant electrical signals. Therefore, methylation of cytosine sites was primarily analyzed based on electrical signals. Strand displacement by the target DNA sequences with different methylated sites can lead to substantial changes of strand displacement efficiency. As a result, the methylation sites can be analyzed on the basis of corresponding peak current response relation. This method has a detection limit of 0.075 pM and does not involve various complicated steps such as bisulfite treatment, enzyme digestion and PCR amplification. Indeed, one detection cycle can be completed in 60 min. The proposed technology might exhibit great potential in early clinical diagnosis and risk assessment of cancers and related diseases.