title: Electrochemical DNA sensor-based strategy for sensitive detection of DNA demethylation and DNA demethylase activity.
abstract: 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.
authors: Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced Materials, Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing, 210023, PR China. Electronic address: iamqmshen@njupt.edu.cn.
authors: Qingming
authors: Q
authors: Shen
authors: Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced Materials, Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing, 210023, PR China.
authors: Mengxing
authors: M
authors: Fan
authors: Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, PR China.
authors: Yin
authors: Y
authors: Yang
authors: Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, PR China. Electronic address: zhangh@njnu.edu.cn.
authors: Hui
authors: H
authors: Zhang
mesh_terms: D015374:Biosensing Techniques; D004247:DNA / Q000032:analysis*; D053837:DNA Cleavage; D000073399:DNA Demethylation; D015254:DNA Modification Methylases / Q000378:metabolism*; D055664:Electrochemical Techniques; D004566:Electrodes; D006046:Gold / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties
chemical_list: D006046:Gold; D004247:DNA; D015254:DNA Modification Methylases
keywords: DNA demethylase; DNA demethylation; Electrochemical DNA sensor; Endonuclease; Redox label