A zero-background electrochemical DNA sensor coupling ligase chain reaction with lambda exonuclease digestion for CYP2C192 allele genotyping in clinical samples
A zero-background electrochemical DNA sensor coupling ligase chain reaction with lambda exonuclease digestion for CYP2C192 allele genotyping in clinical samples
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零背景电化学 DNA 传感器耦合连接酶链反应与 lambda 核酸外切酶消化，用于临床样本中 CYP2C192 等位基因基因分型
DOI：
10.1016/j.snb.2022.132096
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Jin-Yuan Chen
中科院分区：
文献类型：
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作者：
Zhou-Jie Liu;Liang-Yong Yang;Tai-Cheng Lu;Yu-Qi Liang;Meng-Meng Liu;Guang-Xian Zhong;Xin-Hua Lin;Pin-Fang Huang;Jin-Yuan Chen
为了实现高精度的真实样品分析，人们迫切需要一种零本底电化学DNA传感器。为此，我们构建了一种基于波长外切酶(λ-Exo)辅助连接酶链式反应(LCR)的电化学生物传感器λ-eLCR，利用亚甲基蓝(MB)标记的探针和反向杂交技术，实现了高灵敏度的零本底基因分型。具体地说，在靶DNA存在的情况下，通过LCR扩增和λ-Exo消化以指数方式产生连接的MB标记的单链DNA，然后它们与金电极上捕获探针的上游序列反向杂交，使氧化还原报告MB足够接近电极表面。在这一点上，方波伏安法检测到的MB衍生信号与目标DNA浓度定量相关，并保证了零本底。在最佳条件下，λ-ELCR对CYP2C19*2基因具有良好的线性范围(100Fm-10)和低检测限(100Fm)。最后，本λ-ELCR成功地区分了人全血样本中的CYP2C19*2等位基因，在个体化医学中具有很大的应用前景。
A zero-background electrochemical DNA sensor is badly desired for highly accurate real-sample assay. Herein, we built a lambda exonuclease (λ-Exo)-assisted ligase chain reaction (LCR)-based electrochemical biosensor, named λ-eLCR, for highly sensitiveCYP2C19*2allele genotyping with zero-background by using the methylene blue (MB)-attached DNA probe and an inverted hybridization format. Specifically, the ligated MB-labeled single-stranded DNAs (ssDNA) were generated exponentially by LCR amplification followed by λ-Exo digestion in the presence of target DNA, then they were invertedly hybridized with the upper sequence of capture probes on the gold electrode, bringing the redox reporter-MB close enough to the surface of the electrode. In this regard, the MB-derived signal detected by square wave voltammetry technique was quantitatively related with the target DNA concentration and guaranteed the zero background. Under the optimum condition, the proposed λ-eLCR exhibited excellent performance in terms of good linear range (100 fM–10 pM) and a low limit of detection (100 fM) forCYP2C19*2gene. Finally, this λ-eLCR successfully distinguished theCYP2C19*2allele genotypes in human whole blood samples, which has a great prospect in individualized medicine.