Koo Kevin M, Sina Abu Ali Ibn, Carrascosa Laura G, Shiddiky Muhammad J A, Trau Matt
Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Qld 4072, Australia.
Analyst. 2014 Dec 7;139(23):6178-84. doi: 10.1039/c4an01641f.
Simple, sensitive and inexpensive regional DNA methylation detection methodologies are imperative for routine patient diagnostics. Herein, we describe eMethylsorb, an electrochemical assay for quantitative detection of regional DNA methylation on a single-use and cost-effective screen-printed gold electrode (SPE-Au) platform. The eMethylsorb approach is based on the inherent differential adsorption affinity of DNA bases to gold (i.e. adenine > cytosine ≥ guanine > thymine). Through bisulfite modification and asymmetric PCR of DNA, methylated and unmethylated DNA in the sample becomes guanine-enriched and adenine-enriched respectively. Under optimized conditions, adenine-enriched unmethylated DNA (higher affinity to gold) adsorbs more onto the SPE-Au surface than methylated DNA. Higher DNA adsorption causes stronger coulombic repulsion and hinders reduction of ferricyanide Fe(CN)6ions on the SPE-Au surface to give a lower electrochemical response. Hence, the response level is directly proportional to the methylation level in the sample. The applicability of this methodology was tested by detecting the regional methylation status in a cluster of eight CpG sites within the engrailed (EN1) gene promoter of the MCF7 breast cancer cell line. A 10% methylation level sensitivity with good reproducibility (RSD = 5.8%, n = 3) was achieved rapidly in 10 min. Furthermore, eMethylsorb also has advantages over current methylation assays such as being inexpensive, rapid and does not require any electrode surface modification. We thus believe that the eMethylsorb assay could potentially be a rapid and accurate diagnostic assay for point-of-care DNA methylation analysis.
简单、灵敏且经济的区域DNA甲基化检测方法对于常规患者诊断至关重要。在此，我们描述了eMethylsorb，一种用于在一次性且经济高效的丝网印刷金电极（SPE-Au）平台上定量检测区域DNA甲基化的电化学检测方法。eMethylsorb方法基于DNA碱基对金的固有差异吸附亲和力（即腺嘌呤>胞嘧啶≥鸟嘌呤>胸腺嘧啶）。通过对DNA进行亚硫酸氢盐修饰和不对称PCR，样品中的甲基化和未甲基化DNA分别变得富含鸟嘌呤和腺嘌呤。在优化条件下，富含腺嘌呤的未甲基化DNA（对金的亲和力更高）比甲基化DNA更多地吸附到SPE-Au表面。更高的DNA吸附会导致更强的库仑排斥，并阻碍铁氰化物Fe(CN)6离子在SPE-Au表面的还原，从而产生较低的电化学响应。因此，响应水平与样品中的甲基化水平成正比。通过检测MCF7乳腺癌细胞系的engrailed（EN1）基因启动子内八个CpG位点簇中的区域甲基化状态，测试了该方法的适用性。在10分钟内快速实现了10%甲基化水平的灵敏度，且具有良好的重现性（RSD = 5.8%，n = 3）。此外，eMethylsorb相对于当前的甲基化检测方法还具有优势，如价格低廉、速度快且不需要任何电极表面修饰。因此，我们认为eMethylsorb检测方法有可能成为一种用于即时护理DNA甲基化分析的快速准确的诊断检测方法。
