An exonuclease III-assisted CHA dual-cycle amplification strategy for quencher-free fluorescent detection of miRNA

MicroRNAs (miRNAs) are crucial biomarkers for cancer diagnosis, but their detection is challenged by low abundance and sequence homology. Herein, we report a simple, sensitive, and quencher-free fluorescent method for miRNA detection by combining catalytic hairpin assembly (CHA) with exonuclease III (Exo III)-assisted signal amplification. In this strategy, the target miRNA initiates the CHA reaction to form a "Y"-shaped three-branch duplex. This duplex structure is specifically recognized and cleaved by Exo III, releasing the target for the next CHA cycle and the fluorescent probe (2-aminopurine, 2-AP) into solution. The released 2-AP exhibits a significant fluorescence enhancement due to the disruption of base stacking, without requiring a separate quencher molecule. Under optimal conditions, this method achieved a wide linear range from 50.0 fmol L^-1 to 1.00 nmol L^-1 for DNA-21 (a mimic target), with a low detection limit of 2 fmol L^-1, and 500 fmol L^-1 to 10.0 nmol L^-1 for miRNA-21, with a low detection limit of 13 fmol L^-1. Furthermore, the method demonstrated excellent single-base mismatch discrimination and was successfully applied to the recovery test of miRNA-21 in human serum and the detection of miRNA-21 incancer cell lysates. Therefore, this work is expected to become a simple, sensitive and specific method for detecting miRNAs, which will contribute to the clinical detection of tumor markers.