Logic-gated trimodal CRISPR/Cas12a biosensor for autoimmune hepatitis monitoring via dual miRNA profiling

A novel logic-gated D -CHA/Cas12a biosensor enables accurate dual-miRNA detection. • PS-G-triplex mediates label-free fluorescence, colorimetric, and RGB outputs. • Ultra-low femtomolar sensitivity was realized through D -CHA and Cas12a amplification. • Dynamic AIH progression and prednisone efficacy in mice were tracked successfully. An innovative trimodal optical biosensor incorporating dual catalytic hairpin assembly ( D -CHA) and CRISPR/Cas12a was fabricated to specifically and sensitively detect miR-21 and miR-122, two core biomarkers of autoimmune hepatitis (AIH). The platform employs a phosphorothioate (PS)-modified G-rich hairpin (SHG3) as the reporter, enabling fluorescence, colorimetric, and smartphone-based Red Green Blue (RGB) signal readouts. Target-driven AIH produces extended double-stranded DNAs that trigger Cas12a trans-cleavage, thereby cleaving SHG3 to release G-rich strands for PS G-triplex (SG3) formation. Benefiting from this synergistic amplification, the platform achieved ultra-low limits of detection (LOD) of 153 fM (fluorescence), 516 fM (colorimetric), and 6.17 pM (smartphone) for the dual targets, coupled with exceptional specificity capable of discriminating single-base mismatches. The platform was successfully applied to detect miR-21 and miR-122 in mouse serum from a concanavalin A (ConA)‑induced AIH model, showing significantly elevated signals in model mice that were notably reduced after prednisone treatment, consistent with liver function indices and inflammatory cytokine profiles. This sensing strategy features simple operation, low cost, and no need for sophisticated instruments, providing a reliable point-of-care tool for AIH progression monitoring and therapeutic efficacy evaluation. It also offers a new methodological reference for multiplex biomarker detection in autoimmune diseases, showing promising potential for clinical translation. Download: Download high-res image (160KB) Download: Download full-size image This work presents a trimodal biosensor integrating D -CHA with CRISPR/Cas12a for sensitive detection of autoimmune hepatitis biomarkers miR-21 and miR-122. Target-triggered D -CHA activates Cas12a to cleave a G-rich reporter probe, enabling fluorescence, colorimetric, and smartphone-based RGB detection. The platform achieves fM-level sensitivity, single-base mismatch discrimination, and successful application in a mouse AIH model, offering a simple, low-cost POC tool for disease monitoring.