A modified lateral flow immunoassay for the detection of trace aflatoxin M1 based on immunomagnetic nanobeads with different antibody concentrations

A modified lateral flow immunoassay (two-step assay) was developed to detect trace aflatoxin M1 (AFM1) in raw milk. In contrast to conventional LFIA, two kinds of immunomagnetic nanobeads (IMNBs) were used. One IMNB with high antibody concentration was used to capture AFM1 in the test sample, whereas the other IMNB with low antibody concentration was used to elucidate the results of the test. Critical factors, such as antibody concentration of IMNBs and size of IMNBs, were investigated. The two-step assay exhibited an ideal sensitivity to screen trace AFM1 in milk samples without extra sample pretreatment. The cutoff value of the naked eye was 0.02 μg/L and satisfied the European Union's maximum limit of AFM1 in raw milk, heat-treated milk, and milk used to manufacture milk-based products and even in baby foods. With the same antibody, sensitivity was enhanced approximately 25 and 50 times when compared with conventional IMNB-based LFIA and gold-based LFIA, respectively. Corresponding results of 13 raw milk samples were obtained between this two-step assay and referenced enzyme-linked immunosorbent assay.