Engineering bioluminescent immunoassay for mix-to-measure parathion detection in foodstuffs

The growing demand for simple and efficient methods to detect pesticides has highlighted the importance of homogeneous immunoassays. In this study, a competitive bioluminescent homogeneous immunoassay was developed for detecting the highly toxic pesticide parathion, using nanoluciferase binary technology (NanoBiT). The assay design involved fusing the large subunit (11S) of nanoluciferase (NanoLuc) to the anti-parathion nanobody VHH9, while conjugating the small subunit (86) to the coating antigen H1-OVA.The interaction between VHH9 and H1-OVA brought 11S and 86 into close proximity, enabling NanoLuc reassembly and luminescence emission upon the addition of coelenterazine h (CTZ-h). In the presence of parathion, luminescence intensity decreased proportionally to parathion concentration. This one-step, 10-min assay required no washing steps and demonstrated a half-maximum inhibition concentration (IC 50 ) of 6.67 ng/mL with a linear range of 1.44–30.9 ng/mL. The NanoBiT immunoassay achieved recovery rates of 88.30 % to 103.85 % and correlated well with results from standard gas chromatography-tandem mass spectrometry (GC–MS/MS). These results indicated that the homogeneous NanoBiT immunoassay is a rapid, convenient, and reliable tool for detecting parathion in food.