A facile fluorescence microplate immunoassay based on an in situ fluorogenic reaction for the detection of two highly toxic anticoagulant rodenticides in food and biological matrix

Bromadiolone and brodifacoum, the most frequently used anticoagulant rodenticides, are highly toxic and pose a threat to public health by causing food poisoning incidents. Here, we developed a fluorescence microplate immunoassay for facile and sensitive detection of bromadiolone and brodifacoum by introducing three commercial chemicals (p-phenylenediamine, polyethyleneimine, H2O2) as a new substrate of horseradish peroxidase and then generating fluorescence signals based on an in situ fluorogenic reaction (detection time within 75 min). This assay exhibited higher efficiency in generating fluorescence signals, thereby exhibiting a 6-fold improvement in sensitivity compared with colorimetric ELISA. The limit of detection was 0.23–0.28 ng/mL (ng/g) for bromadiolone and 0.34–0.61 ng/mL (ng/g) for brodifacoum in corn and human serum, with recovery ratios higher than 82.3 %. These satisfactory results illustrated our proposed assay was a potential tool for food analysis and poisoning diagnosis caused by bromadiolone and brodifacoum.