Determination of Aflatoxin M1 in Milk by a Magnetic Nanoparticle-Based Fluorescent Immunoassay

A sensitive and rapid magnetic nanoparticle-based fluorescent immunoassay for the determination of aflatoxin M1 in raw milk was developed. Aflatoxin M1 was converted to aflatoxin M1-ocarboxymethyl oxime. The aflatoxin M1-oxime was used for the preparation of aflatoxin M1-oxime-fluoresceinamine conjugate through the carbodiimide reaction. The aflatoxin M1-oxime-fluoresceinamine conjugate was characterized by ultraviolet-visible and infrared spectroscopy. Magnetic nanoparticles (Fe3O4) were synthesized and modified by 3-(aminopropyl) triethoxysilane. The size of initial (139 nm) and functionalized magnetic nanoparticles (147 nm) was determined by particle analysis. The optimal mass of immobilized antibody (25 mu g) and optimal concentration of aflatoxin M1-oxime-fluoresceinamine conjugate (15 mu g mL(-1)) for magnetic nanoparticle-based fluorescent immunoassay were determined. The developed immunoassay provided a linear aflatoxin M1 concentration range from 3.0 to 100 pg mL(-1) in bovine milk. The detection limit was 2.9 pg mL(-1). The results of aflatoxin M1 magnetic nanoparticle-based fluorescent immunoassay in heat-treated milk and phosphate-buffered saline at pH 6.6 were compared. The influence of the somatic cell count, pH, and fat concentration in bovine milk on the aflatoxin M1 immunoassay was investigated. The influence of the milk species on the immunoassay was also characterized. The high fat concentration ovine milk depressed the sensitivity of the aflatoxin M1 immunoassay.