基于CRISPR-Cas12a与G-四链体构建免标记电化学生物传感器检测microRNA  

A label-free electrochemical biosensor for microRNA detection based on CRISPR-Cas12a and G-quadruplex

作　　者：史铠 陈稼轩[1] 李琼[1] 杨孝容[1] 成英[1] 宋九华[1] 孙国峰 SHI Kai;CHEN Jiaxuan;LI Qiong;YANG Xiaorong;CHENG Ying;SONG Jiuhua;SUN Guofeng(College of New Energy Materials and Chemistry,Leshan Normal University,Leshan 614000,China)

机构地区：[1]乐山师范学院新能源材料与化学学院,乐山614000

出　　处：《分析试验室》2023年第11期1476-1482,共7页Chinese Journal of Analysis Laboratory

基　　金：四川省科技计划资助(2022JDRC0126);乐山师范学院引进人才启动项目(RC2022002);乐山市2021年重点科技项目(21SZD153)资助。

摘　　要：CRISPR-Cas12a是一种功能强大且可编程的分子诊断技术。本文基于CRISPR-Cas12a的附属切割活性与G-四链体/氯化血红素(Hemin)复合物,设计了一个免标记电化学生物传感器,实现对miRNA的强特异性检测。靶标miRNA-21与双链DNA探针上的Toehold区域结合并发生链置换反应,置换出双链DNA探针中较短的DNA。置换下来的DNA可以有效地激活CRISPR-Cas12a的附属切割活性。随后,具有附属切割活性的Cas12a切割电极表面上形成G-四链体/Hemin的DNA序列,导致电流信号减弱。在最优条件下,电流信号强度变化与10~100 pmol/L范围内的miRNA-21浓度呈良好的线性关系,检出限为4.2 pmol/L。该电化学生物传感器能够实现对单个碱基突变的miRNA-21或其它miRNA序列特异性识别,并可用于人血清样本(10%)中miRNA-21的检测。CRISPR-Cas12a is a powerful and programmable molecular diagnostic technology.A highly specific and label-free biosensor herein was presented for electrochemical detection of miRNA based on the transcleavage activity of CRISPR-Cas12a and the complex of the G-quadruplex/Hemin.The target miRNA-21 binds to the toehold region of the duplex DNA and initiates strand displacement reaction to replace the short DNA of the duplex DNA.The replaced DNA can effectively activate the trans-cleavage activity of CRISPR-Cas12a.Subsequently,the G-quadruplex/Hemin DNA sequence is cleaved by activated CRISPR-Cas12a,leading to the current signal decrease.Under the optimal conditions,the electrochemical biosensor can detect target miRNA-21in a linear range of 10 to 100 pmol/L with the limit of detection lower to 4.2 pmol/L.The developed biosensor has the ability to differentiate even a single-base mismatched sequence on the miRNA-21 or other miRNAs,and can be used to detect miRNA-21 in human serum samples.

关 键 词：CRISPR-Cas12a G-四链体 免标记 电化学生物传感器 MICRORNA 

分 类 号：O657.15[理学—分析化学]