Optical Biosensor for Ochratoxin A Detection in Grains Using an Enzyme-Mediated Click Reaction and Polystyrene Nanoparticles

Herein, we develop an optical biosensor for highly sensitive and facile detection of ochratoxin A (OTA) using an enzyme-mediated click reaction for signal amplification and polystyrene nanoparticles (PNPs) for signal readout. Alkaline phosphatase was employed to hydrolyze the ascorbic acid-phosphate to generate ascorbic acid, which reduces Cu(II) to Cu(I). Cu(I) can catalyze the click reaction between alkyne-functionalized magnetic beads and azide-functionalized PNPs to form complexes, while unbound PNPs acted as the signal probe. This strategy utilized the high efficiency of click chemistry and the inherent optical absorption properties of PNPs, which effectively improved the sensitivity of conventional immunoassays and simplified the procedures using magnetic separation technology. This optical biosensor enabled OTA detection in a linear range of 0.1 to 50 ng/mL with a detection limit of 54 pg/mL. Moreover, it has been successfully challenged with OTA detection in maize samples, revealing its potential as a promising tool for mycotoxin screening.