Dual-readout digital colorimetric assay for zearalenone in corn based on alkaline phosphatase mediated growth of Prussian blue nanoparticles

Zearalenone (ZEN) poses a substantial threat to global cereal grain security. Herein, a dual-readout biotin-avidin amplified nanozyme-linked immunosorbent assay (BA-ELISA) based on absorbance and blue intensity was established for detecting ZEN residues in corn. The method utilized alkaline phosphatase (ALP), which hydrolyzes ascorbic acid 2-phosphate (AAP) to generate ascorbic acid (AA). The resulting AA reduced K₃[Fe(CN)₆] to K₄[Fe(CN)₆], subsequently reacting with FeCl₃ to form blue Prussian blue nanoparticles (PBNPs). Concurrently, AAP reacted with FeCl₃ to yield a brown AAP-Fe 3+ complex, enabling dual-readout detection via PBNPs and AAP-Fe 3+ . The assay exhibited a linear detection range of 0.625–10 ng/mL, a limit of detection (LOD) of 0.022 ng/mL via absorbance and 0.00447 ng/mL via digital colorimetry, and spiked recovery rates of 104.63 %–118.64 % in corn samples. This dual-readout digital colorimetric detection provided a convenient, sensitive platform for on-site toxin monitoring, offering new perspectives for food safety inspection.