The combination of CRISPR-Cas12a with aptamers can potentially improve the sensitivity, specificity, and speed of non-nucleic acid target detection. Nevertheless, current CRISPR-Cas12a aptasensors, solely dependent on aptamer affinity and overlooking the enzymatic regulation of CRISPR-Cas12a, may produce false-positive signals. We proposed a CRISPR-Cas12a aptasensor synergistically regulated by an aptamer and RNA, which incorporated aptamer-mediated drug recognition and CRISPR-powered signal amplification in a one-pot format. Herein, a regulatory RNA probe synergized with the conformational switching of a drug-induced aptamer, enabling precise regulation of Cas12a trans-cleavage activity via toehold-mediated strand displacement (TMSD). This dual regulatory mechanism transformed the aptamer-activated CRISPR-Cas12a sensing process into a TMSD-driven conditional reaction, avoiding false-positive signals and thus achieving better detection performance with a lower detection limit. With the vancomycin (VCM) aptamer as a model, the aptasensor can detect VCM within 30 min from 2% serum, 1% synovial fluid, and 1% cerebrospinal fluid, with a detection limit of 13.62, 7.56, and 6.75 nM, respectively. The proposed aptasensor was incorporated into a custom 3D-printed portable fluorometer and underwent clinical validation in 22 VCM serum samples, reporting no significant difference when compared with the enzyme-multiplied immunoassay technique, confirming the reliability for point-of-care quantification. It further received cross-validation with a quinine aptamer, suggesting universality [a linear range of 10-250 nM (= 0.985) and a detection limit of 0.42 nM]. By integration of aptamer selectivity with CRISPR programmability, this work presents a novel robust biosensing paradigm for point-of-care drug concentration detection. R2
