Dual-enzyme cascade colorimetric biosensor for safe, rapid on-site detection and detoxification of ochratoxins

Ochratoxins, toxic mycotoxins present in food and feed, pose a threat to human health due to their various toxic effects. Developing a safe, convenient, and rapid on-site detection tool for monitoring ochratoxins is essential for food and environmental safety, especially in developing countries. Although numerous effective ochratoxin sensing and detection systems are available, most do not combine detoxification with detection capabilities. In this work, an ochratoxin-detoxifying enzyme was cascaded with phenylalanine dehydrogenase to construct a colorimetric biosensor capable of simultaneously detecting and removing Ochratoxin A (OTA) and Ochratoxin B (OTB) contamination. Once the ochratoxins are detoxified into non-toxic OTα/OTβ and phenylalanine, 1-methoxy-5-methylphenazinium methyl sulfate (MPMS) functions as a signal reporter, converting the ochratoxin concentration into a colorimetric signal that gradually shifts from red to green. This allows for real-time visual observation with the naked eye. Additionally, OTA/OTB within a concentration range of 0-50 μg/mL can be quantitatively analyzed using a microplate reader or a smartphone. Under the optimized conditions, the colorimetric biosensor effectively eliminated and detected OTA/OTB at a spiked concentration of 5 μg/L in Chinese liquor samples within 1 h. Hence, the dual-enzyme cascade colorimetric biosensor has been demonstrated to be a safe, portable, rapid, and accurate tool for the detection and elimination of OTA/OTB in food.