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

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.