Department of Clinical Laboratory Science, College of Medical Laboratory, The Third Military Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, PR China; Department of Materials and Energy, Southwest University, 2 Tiansheng Street, Beibei District, Chongqing 400715, PR China.
Department of Clinical Laboratory Science, College of Medical Laboratory, The Third Military Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, PR China.
Biosens Bioelectron. 2018 Dec 15;121:47-53. doi: 10.1016/j.bios.2018.07.055. Epub 2018 Jul 26.
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 DNA via padlock 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 HO to 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 M to 10 M. 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.
对基因组 DNA 中的 DNA 甲基化进行敏感且特异性的检测对于快速进行表观遗传学评估至关重要。在这里，我们开发了一种新的敏感电化学策略，通过发夹探针引物生成滚环扩增（RCA）来超灵敏检测基因组 DNA 中的 DNA 甲基化。通常，在用亚硫酸氢盐处理甲基化 DNA 后，只有在存在甲基化 DNA 的情况下，甲基化特异性线性发夹才能环化，并随后用作包含 DNA 四面体的模板用于 RCA。DNA 四面体可用作纳米载体，可固定在金电极上生成 RCA 产物，负载含氯的含铁卟啉血红素，形成作为辣根过氧化物酶样 DNA 酶的 G-四链体，在存在 HO 的情况下还原亚甲基蓝 (MB)，产生明显的电流信号。使用开发的 DNA 酶与 RCA 信号放大策略，该 DNA 生物传感器可以实现对甲基化 DNA 序列进行超灵敏电化学检测的低至 0.1 fM 的检测限，检测范围为 10 M 至 10 M。同时，令人满意的特异性、重现性、稳定性和回收率表明其在临床诊断方面具有令人满意的应用潜力。最重要的是，该方法还可以进一步应用于分析其他基因组 DNA。
