Multi-DNAzymes cascade reaction mediated aptasensors for OTA detection based on the integration of autocatalytic Mg²⁺-dependent DNAzyme cleavage and entropy-driven circuit

Ochratoxin A (OTA) is a compound of concern due to its potential health effects on humans. Detecting OTA in food is crucial for safeguarding public health. In this study, we fabricated a multi-DNAzyme cascade reaction-mediated colorimetric aptasensors for OTA detection, integrating autocatalytic Mg²⁺-dependent DNAzyme cleavage (MNAzyme) and an entropy-driven circuit. In brief, the recognition between the aptamer and target OTA led to the release of DNA1. Subsequently, DNA1 hybridized with DNA2, generating an upstream MNAzyme that facilitated the production of a downstream MNAzyme. These MNAzymes possess similar substrate binding arms, enabling them to catalyze the same substrate. The catalytic efficiency of MNAzymes towards the substrates was enhanced due to the increasing concentration of MNAzymes. The cleavage products then triggered an entropy-driven cycle to generate a signal. Under optimal conditions, the sensing system exhibited low detection limits of 48.97 fM for OTA. Additionally, the proposed aptasensor was successfully applied to quantitatively analyze OTA in food samples. Thus, the multi-DNAzyme cascade reaction-mediated colorimetric aptasensors offer an adaptable platform for detecting traces of OTA contaminant in food.