Hue Recognition Competitive Fluorescent Lateral Flow Immunoassay for Aflatoxin M1 Detection with Improved Visual and Quantitative Performance

Immunological detection of small molecules in a point-of-care (POC) format is of great significance yet remains challenging for accurate visual discrimination and quantitative analysis. Here, we report a novel hue recognition competitive fluorescent lateral flow immunoassay (HCLFIA) strip that allows both visual and quantitative detection of aflatoxin M₁ (AFM₁). The HCLFIA strip works on the basis of the ratiometric change of emission, arising from the overlap of fluorescence signals of two nanocomposites tagged with probe antibodies and coated antigens. A visually discernible orange-red-to-green fluorescence color change allows the naked eye semiquantitative readout of AFM₁ around the threshold concentration (0.05 ng mL^-1), yielding a visible detection limit of 0.02 ng mL^-1. Moreover, using a custom smartphone-based device and color chart analysis, ultrasensitive quantitative detection of AFM₁ can be achieved with a low limit of detection at 0.0012 ng mL^-1, which is considerably better than those of the previously reported colorimetric and fluorescent strips. The accuracy performed in spiked milk samples ranged from 97.91 to 113.12% with a coefficient of variation below 7.8%, showing good consistency with the results from isotope dilution liquid chromatography-tandem mass spectrometry. Thanks to the unique hue recognition scheme, the HCLFIA strip holds great potential for POC detection of small molecules.