Nanobody/NanoBiT system-mediated bioluminescence immunosensor for one-step homogeneous detection of trace ochratoxin A in food

Hazardous small molecules in food and environment seriously threatens human health, which requires sensitive and rapid tools for monitoring. Using a previously identified nanobody against ochratoxin A (OTA), we herein proposed a homogeneous sensing platform "nanobody/NanoLuc Binary Technology (NanoBiT) system" and developed a nanobody/NanoBiT system-mediated bioluminescence immunosensor (NBL-Immunosens) for OTA using LgBiT (Lg) and SmBiT (Sm), two subunits of the split nanoluciferase (NanoLuc). The core elements of NBL-Immunosens include Lg-nanobody fusion (NLg) and Sm-labeled OTA-bovine serum albumin conjugate (OSm). The antigen-antibody interaction between NLg and OSm triggers the reconstitution of NanoLuc for generating luminescence signals. Moreover, free OTA can compete with OSm for binding to NLg, resulting the decrease of dose-dependent signals. NBL-Immunosens can detect OTA in a one-step assay of 5 min without washing and exhibit a limit of detection of 0.01 ng/mL with a linear range of 0.04-2.23 ng/mL. It shows high selectivity for OTA and has good accuracy and precision in the spiking-and-recovery experiments. Furthermore, its effectiveness was evaluated with real cereal samples and confirmed by liquid chromatography tandem mass spectrometry and commercial ELISA kits. Hence, the NBL-Immunosens is a very promising tool for rapid, accurate, and selective detection of trace OTA in food.