Electrochemical DNA sensor-based strategy for sensitive detection of DNA demethylation and DNA demethylase activity

•Detect DNA demethylase activity by electrochemical DNA sensor strategy.•The principle relies on electrochemical signal changes of FcA redox label.•Combine the DNA demethylation and the BstUI endonuclease digestion.•A high sensitivity with low detection limit of 0.17 ng/mL of DNA demethylase.•The assay can be used for the related molecular diagnostics and drug screening.

DNA demethylation and demethylase activity play important roles in DNA self-repair, and their detection is key to early diagnosis of fatal diseases. Herein, a facile electrochemical DNA (E-DNA) sensor was developed for the sensitive detection of DNA demethylation and demethylase activity based on an enzyme cleavage strategy. The thiol modified hemi-methylated hairpin probe DNA (pDNA) was self-assembled on a Au electrode surface through the formation of AuS bonds. The hemi-methylated pDNA served as the substrate of DNA demethylase (using methyl-CpG-binding domain protein 2 (MBD2) as an example). Following demethylation, the hairpin stem was then recognized and cleaved by BstUI endonuclease. The ferrocene carboxylic acid (FcA)-tagged pDNA strands were released into the buffer solution from the electrode surface, resulting in a significant decrease of electrochemical signal and providing a means to observe DNA demethylation. The activity of DNA demethylase was analyzed in the concentration ranging from 0.5 to 500 ng mL−1 with a limit of detection as low as 0.17 ng mL−1. With high specificity and sensitivity, rapid response, and low cost, this simple E-DNA sensor provides a unique platform for the sensitive detection of DNA demethylation, DNA demethylase activity, and related molecular diagnostics and drug screening.

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