A homogeneous immunosensor for AFB1 detection based on FRET between different-sized quantum dots.

Mycotoxins are fatal threats in food safety due to their strong carcinogenesis and toxicity, thus requiring highly sensitive detections. Herein, different-sized quantum dots (QDs) were used to construct a Förster resonance energy transfer (FRET) based immunosensor for sensitive detection of aflatoxin B1 (AFB1) in rice grains. To avoid irregular aggregation between two kinds of QDs, monovalent monoclonal antibody (mAb)-labeled red QDs (~0.84 anti-AFB1 mAbs per QD) and multivalent hapten-labeled green QDs (~6.8 AFB1 per QD) were designed as acceptor and donor, respectively. The anti-AFB1 mAbs and AFB1 interactions promoted one or more acceptors bound with a multivalent AFB1-labeled donor, resulting in energy transfer from the green QDs to the red QDs. Various parameters that influence the immunoassay including reactant ratio of donor to acceptor, buffer pH value, buffer ionic strength and immunoreaction time were systematically investigated and optimized. With optimal conditions, the obtained energy transfer efficiency is proportional to the logarithm of AFB1 concentration in a range over 0.19-16 pM (0.06-5 ng/mL), while offering a limit of detection of 0.13 pM (0.04 ng/mL) in rice extracts. The recovery rates of the intra-assay for spiked samples at AFB1 concentrations of 0.1, 1.0, and 5.0 ng/mL were 83.27% ± 3.27%, 97.36% ± 4.55% and 83.04% ± 4.94%, respectively, and those for the inter-assay were 81.28% ± 6.11%, 95.97% ± 7.07%, and 82.78% ± 5.99%, respectively. Statistical analysis using t-test had no significant difference between the proposed FRET-based immunoassay and the commercial enzyme-linked immunosorbent assay kit.