|کد مقاله||کد نشریه||سال انتشار||مقاله انگلیسی||ترجمه فارسی||نسخه تمام متن|
|1162730||1490898||2016||6 صفحه PDF||سفارش دهید||دانلود رایگان|
• 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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Journal: Analytica Chimica Acta - Volume 934, 31 August 2016, Pages 66–71