Development of a magnetic separation immunoassay with high sensitivity and time-saving for detecting aflatoxin B1 in agricultural crops using nanobody

Detection methods with high sensitivity and short assay time are urgently required for quantitative analysis of small-molecule hazardous substances in food monitoring. In this work, a new anti-aflatoxin B1 (AFB1) nanobody was screened from an immunized nanobody library, and an ultrafast one-step detection of AFB1 without immobilization and multi-step washing was developed based on magnetic separation technology and nanobody (Nb)-alkaline phosphatase (ALP) fusion protein. Compared to conventional one-step chemiluminescent enzyme-linked immunosorbent assay (CLEIA) based on Nb-ALP, it was surprising to find the sensitivity and lowest limit of detection (LOD) of this method was significantly improved about threefold and fivefold separately, and the total assay time could be reduced to 30 from 120 min. Under optimal conditions, the developed method achieved the sensitive detection of AFB1 with LOD with 0.743 pg mL−1, IC50 = 0.33 ng mL−1, the linear range was 7.23 pg mL−1 ~ 12.38 ng mL−1, and showed powerful tolerance and utility for complex matrix environments in sample detection. It is believed this method could provide a newly way for the quick and sensitive detection of AFB1 and could expand the application of Nbs.