Wang Xiaokun, Ding Hao, Sun Yinghui, Ma Yanling, Wang Guoqing, Chen Jiadong, Choo Jaebum, Chen Lingxin
Shandong Provincial Key Laboratory of Biochemical Engineering, College of Biological Engineering, Qingdao University of Science and Technology, Qingdao 266042, China.
Qingdao Academy of Agricultural Sciences, Qingdao 266100, China.
Food Chem. 2025 Mar 15;468:142437. doi: 10.1016/j.foodchem.2024.142437. Epub 2024 Dec 12.
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-NH) at 426 nm. The F/F 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.
