Development of a fluorescence immunoassay for highly sensitive detection of amantadine using the nanoassembly of carbon dots and MnO2 nanosheets as the signal probe

Fluorescence immunoassays are rapid, convenient and cost-effective for the sensitive quantitation of chemical contaminants in foodstuff. In this study, a competitive fluorescence ELISA was developed for the sensitive detection of amantadine (AMD) based on the alkaline phosphatase (ALP)-triggered fluorescence “turn-on” signals. As a fluorescence substrate, carbon dots (CDs) were adsorbed onto the surface of the MnO₂ nanosheets (NSs) and formed a nanoassembly of p-CDs@MnO₂ NSs which results in the fluorescence quench of CDs. The ALP labelled on antibody could catalyze the hydrolysis of the 2-phosphoascorbic acid into ascorbic acid. The latter could then reduce and decompose the MnO₂ NSs, which was accompanied by the release of CDs from the surface of MnO₂ NSs and led to the fluorescence recovery of CDs. The change of the fluorescence intensity is related to the concentration of AMD in solution and thus could be applied to detect AMD in an ALP-based ELISA system. The fluorescent ELISA showed a linear detection for AMD in the range of 0.048 ng mL⁻¹ to 1.1 ng mL⁻¹ with a detection limit (LOD) of 0.035 ng mL⁻¹. The novel fluorescent ELISA shows potential for the highly sensitive detection of AMD and other analytes in food analysis.