AND-Logic Gated Electrochemical Sensor for Separate Detection of Aflatoxin B1 and Ochratoxin A via Split-Activator CRISPR/Cas12a

Abstract

To address the frequent co-occurrence of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in real food matrices, as well as the difficulty of achieving both high-specificity recognition and rapid quantification with existing detection methods, this study developed an AND logic-gated electrochemical sensor based on split-activator CRISPR/Cas12a. The sensor constructed an ordered signaling layer on the electrode interface using a DNA tetrahedron. Through DNAzyme-catalyzed reactions triggered separately by the two targets, two split DNA fragments were generated. Only when both fragments coexisted could they jointly activate the trans-cleavage activity of CRISPR/Cas12a, leading to extensive cleavage of the G-quadruplex sequence supported by the DNA tetrahedron. This cleavage caused the loss of the G-quadruplex/hemin signal-reporting group, resulting in a sharp attenuation of the electrochemical signal. By optimizing the heparin sodium concentration, a one-pot integration of the DNAzyme catalytic cycle and the CRISPR cleavage reaction was successfully achieved. The sensor achieved detection limits of 1.12 pg/mL for AFB1 and 0.26 pg/mL for OTA, which are significantly lower than international regulatory limits. For real corn flour, wheat flour, rice flour, and soybean flour samples, spike recoveries ranged from 90.99% to 107.29%, and the results were highly correlated with those obtained using a commercial enzyme-linked immunosorbent assay method (R2 > 0.996) and high-performance liquid chromatography-tandem mass spectrometry (R2 > 0.998). This work not only provides a new, ultrasensitive, and highly specific tool for copresence reporting of dual mycotoxins, but also offers an innovative paradigm for constructing logic-based biosensors designed for intelligent analysis of complex matrices.