Europium nanospheres based ultrasensitive fluorescence immunosensor for aflatoxin B1 determination in feed

Abstract

In this work, a new competitive immunosensor for aflatoxin B1 (AFB1) detection was developed using europium (Eu) fluorescent nanospheres and magnetic beads. Firstly, Eu nanospheres were synthesized through two steps including carboxylated polystyrene nanospheres and Eu-doped polystyrene nanospheres preparation. Then Eu nanospheres were covalently tagged to anti-AFB1 monoclonal antibody (anti-AFB1 mAb) through an EDC coupling method. Carboxylated Fe3O4 magnetic beads were conjugated to AFB1-BSA through EDC/NHS crosslinking to obtain AFB1-BSA-Fe3O4. In the absence of AFB1, Eu-anti-AFB1 mAb were incubated with AFB1-BSA-Fe3O4 to form Eu-anti-AFB1 mAb-AFB1-BSA-Fe3O4 in PBS buffer. However, in the presence of AFB1, the competitive interaction of AFB1 and AFB1-BSA-Fe3O4 to bind with Eu-anti-AFB1 mAb occurred. With the increasing concentration of AFB1, less Eu-anti-AFB1 mAb-AFB1-BSA-Fe3O4 formed. So the fluorescence intensity of Eu-anti-AFB1 mAb-AFB1-BSA-Fe3O4 was gradually decreased after magnetic separation. The degree of fluorescence decrease was linear with respect to the logarithm of AFB1 concentration in the range of 0.01–2 ng/mL in both buffer solution and feed samples and the detection limit was 0.003 ng/mL. What's more, the immunosensor showed excellent specificity for AFB1 without being interfered by other mycotoxins. In consideration of the excellent performance of this immunosensor, we can speculate that the proposed method could be widely used in detecting food contaminants.