Cryo-Assembled AuNP/QD Probe Pairs Enabling Single-Step Ochratoxin A Detection

The extensive utilization of nanomaterials and aptamers in biosensing has positioned the construction of efficient, stable, and user-friendly nanoprobe systems as a pivotal strategy for rapid small-molecule detection. This study developed a dual-probe fluorescence detection system utilizing gold nanoparticles (AuNPs) and quantum dots (QDs) for ochratoxin A (OTA) analysis. The AuNP probes were functionalized via an innovative freeze-driven asymmetric conjugation approach, while a distinct freeze-driven labeling strategy was applied to QDs. Compared to conventional labeling protocols, the freeze-driven methodology reduced conjugation duration from several hours to merely 20 min and was successfully implemented for QD-aptamer conjugation for the first time, facilitating efficient dual-probe assembly. The complete process, spanning from probe preparation to OTA quantification, was accomplished within 1.5 h. Under optimized parameters, the biosensor demonstrated a linear detection range of 0.5-50 ng/mL, achieved a detection limit of 0.183 ng/mL, and exhibited satisfied specificity, reproducibility, and matrix interference resistance. Validation experiments across various herbal specimens confirmed its accuracy and practical applicability. This research not only establishes a rapid and efficient platform for OTA monitoring but also provides a generalized framework for developing nanomaterial-based biosensing systems.