Department of Clinical Laboratory, Nanjing Medical University Cancer Hospital & Jiangsu Cancer Hospital, 42 Baiziting Road, Nanjing 210009, Jiangsu, China; State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093, China.
State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093, China.
Biosens Bioelectron. 2014 Nov 15;61:593-7. doi: 10.1016/j.bios.2014.06.010. Epub 2014 Jun 10.
A simple, fast and sensitive colorimetric biosensing method for DNA methylation analysis was developed by combining methylation-sensitive endonuclease based digestion with hyperbranched rolling circle amplification (HRCA) induced signal enhancement. The assay was carried out on a DNA capture probe modified 96-cell microplate with four sequential steps of target recognition, endonuclease-based digestion, isothermal HRCA, and enzyme-catalyzed colorimetric readout within 3h. The semi-quantitative and precise analysis of methylated DNA could be easily achieved by naked eye and absorbance measurements, respectively. The strategy exhibited excellent detection specificity and accuracy with a log-linear response to methylated DNA from 100 fM to 10nM. As a proof of concept, the assay was applied to investigate the methylation status of p16/CDKN2 promoter of breast cancer patients. The methylated p16 concentration was not significantly associated with the clinical parameters. The proposed method allowed efficient methylation detection with simplicity, rapidness, low cost and high sensitivity, showing great promise for application in early diagnosis of methylation-related diseases.
一种简单、快速且灵敏的用于 DNA 甲基化分析的比色生物传感方法，通过将基于甲基化敏感内切酶的消化与超支化滚环扩增（HRCA）诱导的信号增强相结合而开发。该测定在经 DNA 捕获探针修饰的 96 孔微孔板上进行，通过目标识别、基于内切酶的消化、等温 HRCA 和酶催化比色读取四个连续步骤，在 3 小时内完成。通过肉眼观察和吸光度测量，可分别实现半定量和精确分析甲基化 DNA。该策略表现出优异的检测特异性和准确性，对数线性响应范围从 100 fM 到 10 nM。作为概念验证，该测定法应用于研究乳腺癌患者 p16/CDKN2 启动子的甲基化状态。甲基化 p16 的浓度与临床参数无显著相关性。该方法具有高效的甲基化检测效率，具有简单、快速、低成本和高灵敏度的特点，有望应用于甲基化相关疾病的早期诊断。
