Determination of niclosamide and its two metabolites in fish by molecularly imprinted microsphere-based pseudo-ELISA

Niclosamide is usually used for the treatment of parasite infections in animals. However, niclosamide and one of its metabolites 2-chloro-4-nitroaniline are mutagenic substances, and their residues in animal-derived foods are potential risks to consumers. As far as we know, there has been no immunoassay or pseudo immunoassay reported to determine niclosamide and its metabolites in animal-derived foods. In this study, a molecularly imprinted microsphere for niclosamide was first synthesized, and a streptavidin-horseradish peroxidase labelled conjugate was also synthesized. The two reagents were used to develop a pseudo enzyme-linked immunosorbent assay on conventional microplates for the determination of niclosamide and its two metabolites (2-chloro-4-nitroaniline and 5-chlorosalicylic acid) in fish. Because biotinylated horseradish peroxidase was used to amplify the signal, the method sensitivities for the three analytes were increased fivefold to 27.5-fold (limits of detection of 0.004-0.03 ng/mL) in comparison with the use of single horseradish peroxidase labelled conjugate (limits of detection of 0.11-0.16 ng/mL). Their recoveries from the standards fortified blank fish samples were in the range of 70.6-95.5%. This is the first study reporting a molecularly imprinted polymer-based pseudo immunoassay for screening of niclosamide and its metabolites in food sample.