A novel high-sensitivity fluorescence detection technology for zearalenone based on the PER-triggered crRNA conformational change and CHA-coordinated energy supply

Abstract

Zearalenone (ZEN), frequently encountered in corn, is a hazardous mycotoxin capable of impairing liver and kidney function, compromising immune responses, and potentially inducing carcinogenesis. Current detection methodologies are hampered by elevated costs, complex workflows, limited sensitivity, and poor specificity. There is a pressing need to develop simple, rapid, and ultrasensitive assays that combine high specificity with operational convenience, thereby facilitating precise biotoxin surveillance and control. This study developed a novel biosensing strategy for ultrasensitive detection of zearalenone (ZEN) by engineering a blocked Primer Exchange Reaction (PER) dumbbell-hairpin structure integrated with a Catalytic Hairpin Assembly (CHA)-based DNA machine. We constructed a highly specific and sensitive fluorescence biosensor for zearalenone (ZEN) by integrating a Primer Exchange Reaction (PER) with the trans-cleavage activity of CRISPR/Cas12a. This strategy significantly simplifies the operational procedure compared to conventional techniques. Furthermore, its modular design establishes a versatile and efficient platform adaptable for the detection of various trace analytes, offering a promising proof-of-concept for mycotoxin screening in agricultural products, although further extensive validation across diverse realistic matrices is warranted.