Abstract

To address the need for highly sensitive and reliable detection of trace ochratoxin A (OTA) in food matrices, we developed a ratiometric fluorescent aptasensor by integrating CRISPR/Cas12a, hybridization chain reaction (HCR), and horseradish peroxidase (HRP)-induced inner filter effect (IFE). The mechanism involves OTA releasing an activator that initiates CRISPR/Cas12a trans-cleavage, blocking HCR assembly. This reduces HRP levels, limiting the conversion of o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP) (emitting at 562?nm) while maintaining strong emission from 2-amino terephthalic acid (BDC-NH₂) at 426?nm. The F₄₂₆/F₅₆₂ ratio serves as a “signal-on” indicator, enabling sensitive OTA detection over 0.1 pM to 10?nM, with a detection limit of 0.0417 pM. The method exhibits excellent reproducibility, with intra-day and inter-day relative standard deviations (RSDs) of 1.91?%–3.87?% and 1.79?%, respectively, along with recovery rates of 90.1?%–110.6?% in real samples. These advantages highlight its significant potential for CRISPR/Cas-based OTA detection.