1. CRISPR-empowered electrochemical biosensor for target amplification-free and sensitive detection of miRNA.
- Author
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Ma, Chihong, Zhou, Qin, Shi, Jinjin, Gao, Hua, Huang, Di, Xue, Huimin, Wang, Han, Zhang, Zhenzhong, Yang, Sen, Zhang, Junli, and Zhang, Kaixiang
- Subjects
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CRISPRS , *SINGLE-stranded DNA , *MICRORNA , *BIOSENSORS - Abstract
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 the trans -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 10−13 to 10−7 M, 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. [Display omitted] • CRISPR-empowered electrochemical biosensor with dual-signal amplification. • Nucleic acid amplification-free and sensitive detection of miRNA by PER-E-CRISPR. • Pre-synthesis of PER concatemer significantly improve the detection signal. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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