School of Environment and Science, Griffith University, Nathan Campus, Nathan, QLD 4111, Australia; Queensland Micro, and Nanotechnology Centre (QMNC), Griffith University, Nathan Campus, Nathan, QLD 4111, Australia.
Queensland Micro, and Nanotechnology Centre (QMNC), Griffith University, Nathan Campus, Nathan, QLD 4111, Australia.
Biosens Bioelectron. 2019 Feb 1;126:102-107. doi: 10.1016/j.bios.2018.10.020. Epub 2018 Oct 12.
In this paper we report on a bisulfite treatment and PCR amplification-free method for sensitive and selective quantifying of global DNA methylation. Our method utilizes a three-step strategy that involves (i) initial isolation and denaturation of global DNA using the standard isolation protocol and direct adsorption onto a bare gold electrode via gold-DNA affinity interaction, (ii) selective interrogation of methylation sites in adsorbed DNA via methylation-specific 5mC antibody, and (iii) subsequent signal enhancement using an electrochemical-enzymatic redox cycling reaction. In the redox cycling reaction, glucose oxidase (GO) is used as an enzyme label, glucose as a substrate and ruthenium complex as a redox mediator. We initially investigated the enzymatic properties of GO by varying glucose and ruthenium concentration to delineate the redox cyclic mechanism of our assay. Because of the fast electron transfer by ruthenium (Ru) complex and intrinsic signal amplification from GO label, this method could detect as low as 5% methylation level in 50 ng of total DNA input. Moreover, the use of methylation-specific 5mC antibody conjugated GO makes this assay relatively highly selective for DNA methylation analysis. The data obtained from the electrochemical response for different levels of methylation showed excellent interassay reproducibility of RSD (relative standard deviation) < 5% for n = 3. We believe that this inexpensive, rapid, and sensitive assay will find high relevance as an alternative method for DNA methylation analysis both in research and clinical platforms.
在本文中，我们报告了一种无需亚硫酸氢盐处理和 PCR 扩增即可灵敏、选择性定量检测全基因组 DNA 甲基化的方法。我们的方法采用三步策略，包括 (i) 使用标准分离方案初始分离和变性全基因组 DNA，并通过金-DNA 亲和相互作用直接吸附到裸金电极上，(ii) 通过甲基化特异性 5mC 抗体选择性检测吸附 DNA 中的甲基化位点，以及 (iii) 随后使用电化学-酶促氧化还原循环反应进行信号增强。在氧化还原循环反应中，葡萄糖氧化酶 (GO) 用作酶标记物，葡萄糖用作底物，钌配合物用作氧化还原介体。我们通过改变葡萄糖和钌浓度来初步研究 GO 的酶促特性，以阐明我们测定的氧化还原循环机制。由于钌 (Ru) 配合物的快速电子转移和 GO 标记物的固有信号放大，该方法可以在 50ng 总 DNA 输入中检测低至 5%的甲基化水平。此外，使用与甲基化特异性 5mC 抗体偶联的 GO 使该测定法在进行 DNA 甲基化分析时具有相对较高的选择性。不同甲基化水平的电化学响应获得的数据显示，n=3 时，批间重现性的相对标准偏差 (RSD) < 5%。我们相信，这种廉价、快速、灵敏的测定法将作为替代方法，在研究和临床平台中都具有很高的相关性，用于 DNA 甲基化分析。
