Trace detection of bromadiolone and brodifacoum in biological and food samples: A label-free fluorescence immunoassay based on an in situ formation strategy.

The widespread use of bromadiolone and brodifacoum poses a threat to public health and initiates poisoning incidents. Conventional colorimetric enzyme-linked immunosorbent assays (ELISAs) suffer from poor sensitivity, making it difficult to meet the trace detection of analytes. In this work, we constructed a sensitive and label-free fluorescence immunoassay based on the in situ formation strategy, for the quantification of bromadiolone and brodifacoum in biological and food samples. Alkaline phosphatase, an essential enzyme in ELISA, catalyzed the substrate sodium ascorbyl phosphate to L -ascorbic acid, which reacted with ο-phenylenediamine and triggered an in situ fluorescence signal with excitation/emission wavelengths of 350/430 nm. To reduce the reaction time, Fe3+ was utilized for the in situ immunoassay system. The fluorescence signal system was introduced to conventional ELISA without any label procedures, thus resulting in higher sensitivity with a 3.8-fold improvement in the half-maximal inhibitory concentration for bromadiolone, compared with colorimetric ELISA. The calculated limits of detection for bromadiolone and brodifacoum were 0.45–0.76 and 0.34–0.58 ng/mL (ng/g) in the human serum and corn samples, respectively, with recovery values ranging from 82.0 % to 93.1 %, and a coefficient of variation lower than 13.4 %. Due to its advantages of high sensitivity, cost-effectiveness, and easy substrate fabrication, this immunoassay may offer promise for applications in poisoning diagnosis and food analysis of bromadiolone and brodifacoum. • The signal transfer mechanism of in situ fluorescence immunoassay is explored. • The assay is applied for the simultaneous detection of bromadiolone and brodifacoum. • Oxidation action of Fe3+ reduces reaction time by 30 min in fluorescence immunoassay. • A 3.8-fold improvement for IC 50 is observed compared with colorimetric ELISA. [ABSTRACT FROM AUTHOR]