Circle Padlock-Mediated Catalytic Hairpin Assembly Cooperating Primer Exchange Reaction for Sensitive and Label-Free MicroRNA Detection.

MicroRNAs (miRNAs), which play critical roles in regulating gene expression and cell functions, are recognized as potential biomarkers for various human diseases, including gastric ulcers. The reliable, specific, and sensitive detection of miRNA is highly recommended for the clinical diagnosis and therapy of different diseases. Herein, we depict a label-free and low-background fluorescent assay for the highly sensitive detection of miRNAs by coupling target miRNA-triggered cyclization of a padlock, circular padlock-mediated catalytic hairpin assembly (CHA), and primer exchange reaction (PER)-assisted signal generation. The padlock probe recognizes the target miRNA, forming a circular padlock that subsequently facilitates the CHA. The subsequent PER process generates substantial quantities of G-quadruplex sequences that rapidly combine with thioflavin T to create substantial fluorescence, thereby enabling the highly sensitive detection of the target miRNA. This method demonstrated significant potential for the early diagnosis of diseases such as gastric ulcers, as it could conclude the detection process in human serum samples within hours.