TY - JOUR
T1 - Colorimetric and electrochemical quantification of global DNA methylation using a methyl cytosine-specific antibody
AU - Haque, Md Hakimul
AU - Bhattacharjee, Ripon
AU - Islam, Md Nazmul
AU - Gopalan, Vinod
AU - Nguyen, Nam Trung
AU - Lam, Alfred K.
AU - Shiddiky, Muhammad J.A.
N1 - Publisher Copyright:
© The Royal Society of Chemistry 2017.
PY - 2017/6/7
Y1 - 2017/6/7
N2 - 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.
AB - 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.
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U2 - 10.1039/c7an00526a
DO - 10.1039/c7an00526a
M3 - Article
C2 - 28516982
AN - SCOPUS:85021729679
SN - 0003-2654
VL - 142
SP - 1900
EP - 1908
JO - Analyst
JF - Analyst
IS - 11
ER -