Aptamer-Amplified and CRISPR-Cas12a-Assisted Dual-Mode PEC-SERS Biosensor for Ultrasensitive Detection of Inflammatory Bowel Disease Biomarkers

Inflammatory bowel disease (IBD) has proven to be a critical global health problem characterized by severe life-threatening complications; thus, the development of noninvasive, reliable, and cost-effective diagnostic methods remains an urgent clinical need. Herein, a novel photoelectrochemical (PEC) and surface-enhanced Raman scattering (SERS) dual-mode platform was successfully developed for ultrasensitive detection of IBD-associated biomarkers, matrix metalloproteinases-9 (MMP-9), and intestinal alkaline phosphatase (IAP). A bifunctional covalent organic frameworks/MXene-Au substrate was synthesized with excellent PEC and SERS properties. An aptamer-based amplification strategy was first employed for MMP-9 detection, which was also the basis for the detection of IAP. The magnetic bead-conjugated double-stranded DNA was then designed to generate activator DNA in the presence of IAP, which activated the <i>trans</i>-cleavage activity of the CRISPR-Cas12a system. The resultant Cas12a specifically cleaved the electrode-immobilized single-stranded DNA (ssDNA), triggering the release of methylene blue as a dual-signal reporter, thereby enabling synchronized PEC-SERS detection for MMP-9 and IAP. The biosensor exhibited a wide linear range with detection limits of 0.074 pg/mL (PEC) and 0.016 pg/mL (SERS) for MMP-9, and 0.38 pg/mL (PEC) and 0.16 pg/mL (SERS) for IAP, respectively. Significantly, clinical validation was performed using a murine IBD model and human intestinal inflammation specimens, confirming the practical utility of the PEC-SERS platform. This study establishes a robust dual-mode biosensing strategy with multicomponent detection, enabling advanced biological analysis and precision health monitoring.