CHA-based microarray with Cas12a universal readout for multiple microRNA detection.

Hirschsprung's disease (HSCR), a congenital condition characterized by the absence of nerve cells in the intestinal wall, often requires early and accurate diagnosis for optimal patient outcomes. In this study, we developed a novel and ultrasensitive biosensing strategy for the detection of HSCR-related microRNAs (miRNAs) by integrating catalytic hairpin assembly (CHA) with CRISPR-Cas12a technology. A two-stage process consists of array recognition, and a universal readout is designed. In the first stage, target miRNAs are recognized and amplified on a solid-phase microarray, while in the second stage, the accumulated conversion chains which are not related to target sequences, activate Cas12a, leading to the cleavage of reporter DNA and the generation of a fluorescence signal spatially separated from the first stage. The proposed method was validated for the comprehensive detection of HSCR-related miRNAs and demonstrated high sensitivity and specificity. This work represents a significant advancement in miRNA diagnostics and holds potential for broader clinical applications.