Highly sensitive visual detection of amantadine residues in poultry at the ppb level: A colorimetric immunoassay based on a Fenton reaction and gold nanoparticles aggregation

Highlights: • Combining ELISA with Fenton reaction and AuNPs aggregation. • Assay sensitivity remarkably enhanced by a cascade reaction. • Visual and quantitative detection of amantadine in poultry at the ppb level. - Abstract: Colorimetric biosensors for the on-site visual detection of veterinary drug residues are required for food control in developing countries and other resource-constrained areas, where sophisticated instruments may not be available. In this study, we developed a highly sensitive immunoassay for amantadine residues in poultry. By introducing a novel signal generation strategy into an indirect competitive immunoassay, a highly sensitive assay for amantadine residues in chicken was achieved for naked eye readout at the part per billion (ppb) level. Signal amplification was achieved in the designed immunoassay by combining conventional indirect competitive enzyme-linked immunosorbent assay, Fenton reaction-regulated oxidation of cysteine, and gold nanoparticle aggregation. Therefore, the cascade reaction remarkably enhanced the assay sensitivity and led to a pronounced color change from red to dark purple in the solution, which could be easily distinguished with the naked eye even at approximately 1 µg kg^-1 in poultry muscle. Moreover, the color change can be quantitatively assayed with a classic high-throughput plate reader for contaminated poultry samples. The limit of detection (LOD) was 0.51 nM (0.095 ng mL^-1). The recovery rates for spiked chicken samples ranged from 78% to 84% with relative standard deviations <15%. Therefore, we propose that this immunoassay could be generally applicable for on-site detection in the field of food control.