Bivalent aptamer-assisted CRISPR-Cas12a sensor for precise vancomycin therapeutic drug monitoring.

Therapeutic drug monitoring (TDM) of vancomycin (VAN) is critical for maximizing efficacy and minimizing toxicity, but conventional methods are constrained by high costs, slow turnaround times, and operational complexity. To address these limitations, we developed a novel Bivalent Aptamer-assisted CRISPR-Cas12a Sensor (termed BACS) for rapid and precise VAN detection. Central to this platform is a high-affinity bivalent aptamer (2AP33), engineered via molecular docking-guided truncation and rational linker design, which exhibits significantly enhanced binding avidity compared to its monovalent counterpart. This aptamer was integrated into a CRISPR-Cas12a system based on a competitive binding mechanism, where target binding modulates Cas12a trans-cleavage activity. The optimized BACS achieved a wide linear detection range (1-50 μM) with a low limit of detection (0.64 μM) in clinical serum, fully covering the clinical therapeutic window. Notably, the assay is rapid (within 10 min), cost-effective, and simple. Critically, the clinical practicality and reliability of BACS were rigorously validated with 175 clinical serum samples, showing exceptional concordance with both the gold standard method and a classical method. This work not only provides a reliable tool for VAN TDM but also offers an adaptable strategy for developing high-performance CRISPR-powered biosensors for diverse clinical analytes through a streamlined molecular engineering pipeline.