An electrochemical strategy with tetrahedron rolling circle amplification for ultrasensitive detection of DNA methylation.
An electrochemical strategy with tetrahedron rolling circle amplification for ultrasensitive detection of DNA methylation.
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DOI：
10.1016/j.bios.2018.07.055
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发表时间：
2018-12
影响因子：
12.6
通讯作者：
Huamin Liu;Jing Luo;Lichao Fang;Hui-fang Huang;Jun Deng;Jian Huang;Shu Zhang;Yan Li;
中科院分区：
文献类型：
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作者：
Huamin Liu;Jing Luo;Lichao Fang;Hui-fang Huang;Jun Deng;Jian Huang;Shu Zhang;Yan Li;
基因组DNA中DNA甲基化的灵敏和特异性检测对于快速表观遗传学评估是必要的。在这里，开发了一种新的灵敏的电化学策略，用于超灵敏地检测基因组DNA中的DNA甲基化通过apadlock探针引物产生滚环扩增（RCA）。通常，在甲基化DNA的亚硫酸氢盐处理后，甲基化特异性线性挂锁仅在甲基化DNA存在下环化，随后用作RCA的含有DNA四面体的模板。利用DNA四面体作为纳米载体，其可以固定在金电极上以产生RCA产物，以负载氯化血红素，一种含铁卟啉与氯，形成G-四链体作为辣根过氧化物酶，如DNAzyme，其在H2 O2存在下还原亚甲基蓝（MB）以产生独特的电流信号。使用开发的DNAzyme与RCA信号放大策略，DNA生物传感器可以实现低至0.1 fM的检测限，用于甲基化DNA序列的超灵敏电化学检测，检测范围为10- 15 M至10 - 9 M。同时，该方法具有良好的特异性、重现性、稳定性和回收率，具有良好的临床应用前景。最重要的是，该方法还可以进一步应用于其他基因组DNA的分析。
Sensitive and specific detection of DNA methylation in genomic DNA is imperative for rapid epigenetic evaluations. Here, a novel sensitive electrochemical strategy was developed for ultrasensitive detection of DNA methylation in genomic DNAviapadlock probe primer generating rolling circle amplification (RCA). Typically, after bisulfite treatment of methylated DNA, the methylation-specific linear padlock is only circularized in the presence of methylated DNA and subsequently serves as a template containing a DNA tetrahedron for RCA. The DNA tetrahedron is utilized as a nanocarrier that can be immobilized on a gold electrode to generate RCA product to load hemin, an iron-containing porphyrin with chlorine, forming the G-quadruplex as a horseradish peroxidase like DNAzyme, which reduces methylene blue (MB) in the presence of H2O2to yield a distinct current signal. Using the developed DNAzyme with the RCA signal amplification strategy, the DNA biosensor can achieve a detection limit as low as 0.1 fM for the ultrasensitive electrochemical detection of methylated DNA sequence with a detection range from 10–15M to 10−9M. At the same time, the satisfactory specificity, reproducibility, stability and recovery performances indicated its satisfied potentials for clinical diagnosis. Most importantly, this method can be further applied to analyse other genomic DNA also.