DNA methylation detection and site analysis by using an electrochemical biosensor constructed based on toehold-mediated strand displacement reaction.
DNA methylation detection and site analysis by using an electrochemical biosensor constructed based on toehold-mediated strand displacement reaction.
复制标题
DOI：
10.1016/j.talanta.2022.123603
复制
发表时间：
2022-06
期刊：
影响因子：
6.1
通讯作者：
Shu Zhang;Jiaoyan Yan;Ye Yang;F. Mo;Yan Li;Hui-fang Huang;Lichao Fang;Jian Huang;Junsong Zheng
中科院分区：
文献类型：
--
作者：
Shu Zhang;Jiaoyan Yan;Ye Yang;F. Mo;Yan Li;Hui-fang Huang;Lichao Fang;Jian Huang;Junsong Zheng
DNA甲基化已成为癌症及其他相关疾病早期诊断和预后的新靶点。准确检测特定基因的甲基化位点具有重要意义。然而，临床标本生物学性质的复杂性和低丰度靶标的检测对检测技术提出了更高的要求。研究发现，电化学传感器具有灵敏度高、响应速度快、成本低、操作简便、适用于微量分析等优点，在临床诊断过程中发挥了重要作用。本研究基于PNA链置换的高选择性和高效率，研制了一种简便、快速、高灵敏度的基于峰电流变化的电化学生物传感器，用于DNA甲基化的检测和位点分析。此外，与未甲基化的DNA序列相比，甲基化的DNA序列很容易被PNA探针侵入，从而导致链移位和显著的电信号。因此，基于电信号初步分析了胞嘧啶位点的甲基化。具有不同甲基化位点的靶DNA序列的链置换会导致链置换效率的实质性变化。因此，可以根据相应的峰电流响应关系来分析甲基化位点。该方法的检出限为0.075 pM，不涉及亚硫酸氢盐处理、酶消化和聚合酶链式反应等复杂步骤。事实上，一个检测周期可以在60分钟内完成。该技术可能在癌症及相关疾病的早期临床诊断和风险评估方面显示出巨大的潜力。
DNA methylation has become a novel target for early diagnosis and prognosis of cancer as well as other related diseases. The accurate detection of the methylation sites of specific genes proved to be of great significance. However, the complex biological nature of clinical samples and the detection of low-abundance targets led to higher requirements for the testing technology. It has been found that by virtue of high sensitivity, rapid response, low cost, facile operation and applicability to microanalysis, electrochemical sensors have greatly contributed to the process of clinical diagnosis. In this study, a facile, rapid and highly sensitive electrochemical biosensor based on the peak current change was developed on the basis of high selectivity of toehold and greater efficiency of PNA strand displacement and used for the detection and site analysis of DNA methylation. Moreover, compared with non-methylated DNA sequences, methylated DNA sequences could be readily invaded by PNA probes, thereby resulting in the strand displacement and significant electrical signals. Therefore, methylation of cytosine sites was primarily analyzed based on electrical signals. Strand displacement by the target DNA sequences with different methylated sites can lead to substantial changes of strand displacement efficiency. As a result, the methylation sites can be analyzed on the basis of corresponding peak current response relation. This method has a detection limit of 0.075 pM and does not involve various complicated steps such as bisulfite treatment, enzyme digestion and PCR amplification. Indeed, one detection cycle can be completed in 60 min. The proposed technology might exhibit great potential in early clinical diagnosis and risk assessment of cancers and related diseases.