CRISPR-empowered electrochemical biosensor for target amplification-free and sensitive detection of miRNA.
CRISPR-empowered electrochemical biosensor for target amplification-free and sensitive detection of miRNA.
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DOI：
10.1016/j.talanta.2023.125125
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发表时间：
2023-08
期刊：
影响因子：
6.1
通讯作者：
Chihong Ma;Qin Zhou;Jinjin Shi;Hua Gao;Di Huang;Huimin Xue;Han Wang;Zhenzhong Zhang;Sen Yang;Junli Zhang;Kaixiang Zhang
中科院分区：
文献类型：
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作者：
Chihong Ma;Qin Zhou;Jinjin Shi;Hua Gao;Di Huang;Huimin Xue;Han Wang;Zhenzhong Zhang;Sen Yang;Junli Zhang;Kaixiang Zhang
成簇规则间隔短回文重复序列（CRISPR）系统由于其高度可编程性和靶特异性而为构建生物传感器平台提供了新的分子诊断工具。在此，我们通过结合CRISPR/Cas 13 a和引物交换反应（PER）的优点，开发了一种CRISPR授权的电化学生物传感器，命名为PER-E-CRISPR，用于miR-21的无靶扩增和灵敏检测。双信号扩增程序涉及由基于CRISPR的扩增诱导的靶miR-21的结合，沿着多个短单链DNA链与PER多联体的杂交。当靶miR-21存在时，CRISPR/Cas 13 a特异性识别靶miRNA，触发CRISPR/Cas 13 a的反式切割活性。然后Cas 13 a/crRNA/miRNA切割发夹1（HP 1）中预先设计的核糖核苷酸位点，并释放触发剂以打开修饰在电极表面上的发夹2（HP 2）。然后暴露HP 2中所含的PER桥序列并与PER多联体杂交，随后多个用亚甲蓝标记的短单链DNA（ssDNA-MB）与PER多联体键合。在优化的条件下，PER-E-CRISPR检测miR-21的方法在10− 13至10− 7 M的动态范围内呈现线性，我们获得的检测限（LOD）为30.2 fM。所建立的PER-E-CRISPR生物传感器在实际血浆中表现出良好的实用性能，在miRNA检测的分子诊断领域具有广阔的应用前景。
The clustered regularly interspaced short palindromic repeats (CRISPR) system provides a new molecular diagnostic tool for construction of biosensor platforms due to its high programmability and target specificity. Herein, we developed a CRISPR-empowered electrochemical biosensor by combining the advantages of CRISPR/Cas13a and primer exchange reaction (PER), named PER-E-CRISPR, for target amplification-free and sensitive detection of miR-21. Dual-signal amplification procedures involve the binding of target miR-21 induced by CRISPR-based amplification, along with the hybridization of multiple short single-stranded DNA strands with PER concatemers. When target miR-21 is present, CRISPR/Cas13a specifically recognizes the target miRNA, triggering thetrans-cleavage activity of CRISPR/Cas13a. Then Cas13a/crRNA/miRNA cleaved the predesigned ribonucleotide site in hairpin 1 (HP1) and released trigger to open hairpin 2 (HP2) modified on the electrode surface. Then PER bridge sequence contained in HP2 is exposed and hybridized with PER concatemers, following multiple short single-stranded DNA tagged with methylene blue (ssDNA-MB) bond with the PER concatemers. Under optimized conditions, PER-E-CRISPR assay for detecting miR-21 exhibits linearity in dynamic range from 10−13to 10−7M, and we obtained a limit of detection (LOD) of 30.2 fM. The established PER-E-CRISPR biosensor shows perfect practical performance in actual plasma, which may have great promising prospects for miRNA detection in the field of molecular diagnosis.