Homogeneous bioluminescent immunosensor for aflatoxin B1 detection via unnatural amino acid-based site-specific labeling

Aflatoxin B1 (AFB1) which seriously threatens human health has received sustained attention due to its high toxicity and wide distribution. Hence, a sensitive and facile detection method for AFB1 is essential for food safety. Herein, we present an unnatural amino acid incorporated bioluminescent immunosensor system (UAABimmunoassay) that harnesses bioluminescence resonance energy transfer (BRET) for homogeneous detection of AFB1 in cereals. A nanoluciferase-tagged nanobody (G8-Nluc) served as an energy donor, while an AFB1-labeled superfolder green fluorescent protein (sGFP-AFB1) was used as an acceptor. The sGFP-AFB1 was specifically synthesized by coupling azidylated sGFP with alkynylated AFB1. The donor and acceptor form a complex in the absence of AFB1, generating BRET signals. The presence of free AFB1 competes with sGFP-AFB1 to bind G8-Nluc, resulting in a dose-dependent decrease in signals. The signal can be quantified by a plate reader or smartphone, allowing on-site detection. The UAAB-immunoassay can detect AFB1 in a one-step assay within 10 min by simply mixing the components and sample extracts, and it exhibits a limit of detection of 0.95 ng/mL with a linear range of 0.95-25.31 ng/mL. The recovery experiments using four types of samples showed a recovery range from 71.49 % to 112.14 %. The contents of AFB1 in 10 commercial rice samples were tested by the UAABimmunoassay and validated by high-performance liquid chromatography. Our UAAB-immunoassay presents an innovative approach that not only applies to AFB1 in food but is also applicable to the development of homogeneous detection methods for other hazardous materials and offers sensitive monitoring capabilities.