Sample-pretreatment-free based high sensitive determination of aflatoxin M1 in raw milk using a time-resolved fluorescent competitive immunochromatographic assay

Abstract

A highly-sensitive time-resolved fluorescent immunochromatographic assay (TRFICA) was developed to detect aflatoxin M₁ (AFM₁) in raw milk samples within 6 minutes without any sample pretreatments. This method could meet the requirement for rapid and sensitive milk monitoring in dairy farms and milk industries. Based on a competitive format and the home-made monoclonal antibody 2C9 against AFM₁, this assay enhanced the sensitivity from 0.3 ng mL⁻¹ (by using nanogold-strip method previously reported) to 0.03 ng mL⁻¹ (by using this TRFICA method). The improved sensitivity could probably result from the increases in both the higher affinity of monoclonal antibody 2C9 against AFM₁ and detection signals of immunoassay probes (with each europium microbead coupled with numbers of 2C9 antibodies). The TRFICA method showed a considerable dynamic range of 0.1–2.0 ng mL⁻¹ and spiked recoveries of 80–110% for AFM₁ quantification in raw milk. The results via TRFICA method was found to be high consistency (R² = 0.988) with results via standard high-performance liquid chromatography (HPLC) method, when detecting AFM₁ in 17 blind milk samples.