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 (UAAB-immunoassay) 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 UAAB-immunoassay 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.