Colorimetric and electrochemical quantification of global DNA methylation using a methyl cytosine-specific antibody

The Analyst
Md Hakimul HaqueMuhammad J A Shiddiky

Abstract

We report a simple colorimetric (naked-eye) and electrochemical method for the rapid, sensitive and specific quantification of global methylation levels using only 25 ng of input DNA. Our approach utilises a three-step strategy; (i) initial adsorption of the extracted, purified and denatured bisulfite-treated DNA on a screen-printed gold electrode (SPE-Au), (ii) immuno-recognition of methylated DNA using a horseradish peroxidase (HRP)-conjugated methylcytosine (HRP-5mC) antibody and (iii) subsequent colorimetric detection by the enzymatic oxidation of 3,3',5,5'-tetramethylbenzidin (TMB)/H2O2 which generated a blue-coloured product in the presence of methylated DNA and HRP-5mC immunocomplex. As TMB(ox) is electroactive, it also produces detectable amperometric current at +150 mV versus a Ag pseudo-reference electrode (electrochemical detection). The assay could successfully differentiate 5-aza-2'-deoxycytidine drug-treated and untreated Jurkat DNA samples. It showed good reproducibility (relative standard deviation (% RSD) = <5%, for n = 3) with fairly good sensitivity (as low as 5% difference in methylation levels) and specificity while analysing various levels of global DNA methylation in synthetic samples and cell lines. The ...Continue Reading

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Citations

Apr 19, 2018·The Analyst·Md Nazmul IslamMuhammad J A Shiddiky
Aug 7, 2018·Journal of Materials Chemistry. B, Materials for Biology and Medicine·Ripon BhattacharjeeMuhammad J A Shiddiky
Feb 13, 2021·ACS Applied Materials & Interfaces·Qingyu RuanChaoyong Yang
Apr 8, 2020·Analytica Chimica Acta·S CampuzanoJ M Pingarrón
Sep 5, 2019·Current Medicinal Chemistry·Shu ZhangJunsong Zheng

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