Cancer Molecular Pathology laboratory in Menzies Health Institute Queensland, Griffith University and School of Medicine, Gold Coast, QLD 4222, Australia.
Analyst. 2017 May 30;142(11):1900-1908. doi: 10.1039/c7an00526a.
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)/HO which generated a blue-coloured product in the presence of methylated DNA and HRP-5mC immunocomplex. As TMB 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.
我们报告了一种简单的比色（肉眼）和电化学方法，仅使用 25ng 输入 DNA 即可快速、灵敏和特异性地定量全球甲基化水平。我们的方法利用了三步策略; (i) 提取、纯化和变性后的亚硫酸氢盐处理的 DNA 最初吸附在丝网印刷金电极（SPE-Au）上，(ii) 使用辣根过氧化物酶（HRP）-标记的 5-甲基胞嘧啶（HRP-5mC）抗体识别甲基化 DNA，(iii) 随后通过 3,3',5,5'-四甲基联苯胺（TMB）/HO 的酶促氧化进行比色检测，在存在甲基化 DNA 和 HRP-5mC 免疫复合物的情况下生成蓝色产物。由于 TMB 是电化学活性的，它在 +150 mV 相对于 Ag 伪参比电极（电化学检测）产生可检测的安培电流。该测定法能够成功区分 5-氮杂-2'-脱氧胞苷药物处理和未处理的 Jurkat DNA 样品。它表现出良好的重现性（相对标准偏差（%RSD）<5%，n=3），具有相当好的灵敏度（甲基化水平差异低至 5%）和特异性，同时分析了合成样品和细胞系中各种水平的全基因组甲基化。该方法还已用于分析从 8 名食管鳞状细胞癌患者采集的新鲜组织样本中的甲基化水平。我们相信，该测定法可能作为一种低成本替代方案，用于即时护理应用中的全基因组 DNA 甲基化分析。
