Rationally engineered aptamer-enabled nanozyme colorimetric sensor for patulin detection in fruit juices

Rational truncation enhances aptamer affinity toward patulin. • Fe 3 O 4 nanozyme enables signal amplification via catalytic activity. • Target-induced “off–on” mechanism improves detection sensitivity. • Wide linear range (2.5–160 ng/mL) and low LOD (1.02 ng/mL). • Successful application in fruit juice with high recovery and accuracy. Patulin (PAT) contamination in fruit-derived products poses a significant threat to food safety, necessitating the development of rapid, sensitive, and cost-effective detection strategies. In this study, a novel colorimetric aptasensor was constructed based on Fe 3 O 4 nanozymes and a rationally engineered high-affinity aptamer for PAT detection. A structure-guided strategy combining molecular docking and molecular dynamics simulations (MD) was employed to optimize the aptamer sequence, yielding a truncated variant (APT-63) with enhanced binding affinity (Kd = 15.68 nmol/L) compared to the parental sequence (Kd = 31.94 nmol/L). The sensing mechanism relies on a target-induced “shielding–restoration” effect, where aptamer adsorption inhibits nanozyme activity and subsequent PAT binding restores catalytic activity toward 3,3′,5,5′-Tetramethylbenzidine (TMB) oxidation, generating a measurable signal at 652 nm. Under optimized conditions, the sensor exhibited a wide linear detection range of 2.5–160 ng/mL with a low limit of detection (LOD) of 1.02 ng/mL. The platform demonstrated excellent selectivity against common mycotoxins. In spiked apple and grape juice samples, satisfactory recoveries (93.7–103.8%) and strong agreement with high-performance liquid chromatography (HPLC) results (R 2  ≥ 0.9736) were achieved. Compared with existing methods, the proposed sensor offers advantages including simple operation, low cost, and high stability, highlighting its potential for rapid on-site screening of PAT in food safety monitoring.