Development of a peptide-based bioassay for zearalenone detection via phage display and in silico analysis

Zearalenone (ZEN) is a prevalent mycotoxin threatening food and feed safety, necessitating rapid and reliable detection methods. A ZEN-binding peptide was isolated from a 12-mer phage display library and characterized by molecular docking, synthesis, HPLC purification, and streptavidin-HRP-based ELISA. Specificity, storage stability, and actual grain sample recovery rates were evaluated. The predicted binding free energy between the peptide G-W-H-V-H-S-H-R-F-A-S-I and ZEN was −4.95 kcal/mol. The biotinylated probe reached 98.77% purity and detected ZEN with a limit of detection of approximately 5 ng/mL. The cross-reactivity with other mycotoxins and ZEN analogues was below 10%. The detection responses were highly consistent between mixed and single-ZEN mycotoxin systems. The probe remained stable at 4 and 25 °C for 28 days. With recoveries of 87.43%–108.10% in spiked grains and 80.0%–89.5% in reference materials. This peptide provides a stable, synthesis-friendly recognition element for ZEN detection and future bioassay applications.