Catalytic hairpin assembly-triggered endonuclease-mediated cascade signal recycling for sensitive and colorimetric miRNA detection

Abstract The catalytic hairpin assembly (CHA)-based miRNA detection methods have garnered significant attention due to their simplicity and acceptable amplification efficiency. However, these methods necessitate improved sensitivity. In this work, we present a colorimetric and ultrasensitive approach for the detection of cancer-related miRNAs which is initiated by CHA-mediated nicking endonuclease-assisted signals recycling. The target initiates the CHA process to expose the functional section in the P2 probe. This section can activate cascaded recycling cycles to produce numerous linker sequences by Nt.AlwI endonuclease-assisted cleavage of two hairpin signal probes. The 3,3’,5,5’-tetramethylbenzidine sulfate (TMB)/H 2 O 2 -based color reaction is induced by the fixation of the cDNA-HRP on the surface of magnetic beads, which is mediated by the linker sequence. The proposed method demonstrates a sensitivity that is either comparable to or superior to that of previous colorimetric miRNA detection methods, as a result of this design. Furthermore, the method demonstrated a promising potential for clinical applications and a high selectivity to target miRNA. Consequently, it provides a colorimetric assay that is both ultrasensitive and dependable for the visual detection of miRNA, which has the potential to revolutionize the early diagnosis of cancer.