Development and validation of a TRFI-based rapid quantitative test strip featuring a broad quantification range for...

Zearalenone (ZEN) contamination levels in grains and feeds are highly variable, yet existing detection methods face critical trade-offs between detection range and field applicability. Conventional immunochromatographic strips typically achieve only 10-1000 μg/kg linearity, while confirmatory LC-MS/MS requires >3 h/sample. To bridge this gap, we developed a time-resolved fluorescence immunochromatographic (TRFI) test strip for rapid ZEN quantification through three key innovations: optimized 300 nm fluorescent microsphere (FM) with a 2:1 FM:ZEN-mAb ratio to suppress Hook effect, Tween 20-enhanced sample dilution (2 % v/v) reducing matrix interference (CV <4.14 %), and dynamic reaction control enabling 6-min quantification. The TRFI-ZEN strips achieved a broad quantification range (20-2500 μg/kg) and high accuracy (recovery rates 91-102 %) in maize. Accelerated stability tests confirmed <4 % signal variation after 60 days at 37 °C. Field validation across 90 field samples demonstrated 0.9987 Pearson correlation with LC-MS/MS, detecting 94.44 % contamination rate consistent with global surveys. Crucially, the 20 μg/kg LOQ complies with grain safety standard of China (GB 2761-2017; 60 μg/kg) and European Union feed regulations (EU Commission Regulation No 574/2011; 500 μg/kg), while the 2500 μg/kg upper limit covers severe contamination scenarios. This method enables rapid on-site decisions (<15 min/sample) at grain receiving stations when coupled with representative sampling, reducing LC-MS/MS confirmation costs by >80 % through high-confidence pre-screening.