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.