A ratiometric fluorescence immunoassay based on Ce4+ oxidized o-phthalylenediamine and polyvinylpyrrolidone protected copper nanoclusters for the detection of aflatoxin B1

Abstract

As the most toxic mycotoxin, aflatoxin B1 (AFB1) can cause a variety of health problems in humans and animals. Therefore, it is very important and urgent to establish a sensitive and convenient method for the detection of AFB1. In this study, a ratiometric fluorescence method is established to detect AFB1 based on Ce4+ oxidized o-phthalylenediamine (OPD) and polyvinylpyrrolidone protected copper nanoclusters (PVP-CuNCs). The free Ce4+ with strong oxidative activity can oxidize OPD to 2,3-diaminophenazine (DAP) and emit intense fluorescence. Meanwhile, the fluorescence of PVP-CuNCs can be quenched by DAP. In the catalysis of alkaline phosphatase (ALP), adenosine triphosphate (ATP) is hydrolyzed and releases phosphate ions (PO43-). PO43- has a strong affinity for Ce4+ which reduces the free Ce4+ in solution. Thus, the OPD cannot be oxidized to DAP, and the fluorescence of PVP-CuNCs at 430 nm cannot be quenched. The limit of detection (LOD) of the new immunoassay is 26.79 pg/mL, and the linear detection range is 50–250 pg/mL. Besides, the recovery of AFB1 ranges from 84.66 % to 105.21 % and coefficient of variation (CV) is in the range of 1.18 %–4.88 %. Meanwhile, the new immunoassay exhibits satisfactory selectivity. These results indicate the ratiometric fluorescence immunoassay is sensitive and reliable for detection of AFB1 in actual samples.