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

Md Hakimul Haque, Ripon Bhattacharjee, Md Nazmul Islam, Vinod Gopalan, Nam-Trung Nguyen, Alfred K. Lam, Muhammad J. A. Shiddiky

    Research output: Contribution to journalArticle

    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 method has also been tested for analysing the methylation level in fresh tissue samples collected from eight patients with oesophageal squamous cell carcinoma. We believe that this assay could be potentially useful as a low-cost alternative for genome-wide DNA methylation analysis in point-of-care applications.
    Original languageEnglish
    Pages (from-to)1900-1908
    JournalAnalyst
    Volume142
    DOIs
    Publication statusPublished - 26 Apr 2017

    Fingerprint Dive into the research topics of 'Colorimetric and electrochemical quantification of global DNA methylation using a methyl cytosine-specific antibody'. Together they form a unique fingerprint.

  • Cite this

    Haque, M. H., Bhattacharjee, R., Islam, M. N., Gopalan, V., Nguyen, N-T., Lam, A. K., & Shiddiky, M. J. A. (2017). Colorimetric and electrochemical quantification of global DNA methylation using a methyl cytosine-specific antibody. Analyst, 142, 1900-1908. https://doi.org/10.1039/c7an00526a