Aptamer-Based Fluorescence Quenching Approach for Detection of Aflatoxin M₁ in Milk

Aflatoxin M₁ (AFM₁), one of the most toxic mycotoxins, is a feed and food contaminant of global concern. In this study, we developed a fast and simple method for detection of AFM₁ based on a structure-switching signaling aptamer. This aptasensor is based on the change in fluorescence signal due to formation of an AFM₁/aptamer complex. To generate the aptasensor, the specific aptamer was modified with FAM (carboxyfluorescein), and their complementary DNAs (cDNA) were modified with a carboxytetramethylrhodamine (TAMRA) quenching group. In the absence of AFM₁, the aptamers were hybridized with cDNA, resulting in quenching of the aptamer fluorescence due to the proximity of the aptamer's fluorophore to the quenching group on the cDNA. On the other hand, in the presence of AFM₁, a structural switch in the aptamer was induced by formation of an AFM₁/aptamer complex. Changes in the structure of the aptamer led to the release of the cDNA, causing the generation of a fluorescence signal. Thus, AFM₁ concentrations could be quantitatively monitored based on the changes in fluorescences. Under optimized conditions, this assay exhibited a linear response to AFM₁ in the range of 1-100 ng/mL and a limit of detection of 0.5 ng/mL was calculated. This proposed aptasensor was applied to milk samples spiked with a dilution series of AFM₁, yielding satisfactory recoveries from 93.4 to 101.3%. These results demonstrated that this detection technique could be useful for high-throughput and quantitative determination of mycotoxin levels in milk and dairy products.