Click Chemistry Actuated Exponential Amplification Reaction Assisted CRISPR-Cas12a for the Electrochemical Detection of MicroRNAs.

MicroRNAs (miRNAs) play a very important role in biological processes and are used as biomarkers for the detection of a variety of diseases, including neurodegenerative diseases, chronic cardiovascular diseases, and cancers. A sensitive point-of-care (POC) method is crucial for detecting miRNAs. Herein, CRISPR-Cas12a combined with the click chemistry actuated exponential amplification reaction was introduced into an electrochemical biosensor for detecting miRNA-21. The target miRNA-21 initiated the click chemistry-exponential amplification reaction in the electrochemical biosensor to produce numerous nucleic acid fragments, which could stimulate the trans-cleavage ability of CRISPR-Cas12a to cleave hairpin DNA electrochemical reporters immobilized on the electrode surface. Under optimal conditions, the minimum detection limit for this electrochemical biosensor was as low as 1 fM. Thus, the proposed electrochemical biosensor allows sensitive and efficient miRNA detection and could be a potential analysis tool for POC test and field molecular diagnostics.