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.
Wen Peng,Su Hao,Yin Wen-jia,Hu Jian-cheng,Wang Yu,Yang Jin-yi,Xiao Zhi-li,Xu Zhen-lin,Shen Yu-dong,Wang Hong,Hammock Bruce D.
A new type of label-free electrochemical immunosensor for the high-sensitivity determination of parathion was developed based on the oriented immobilization of nanobody (VHH9) on a gold nanoparticle-loaded polyvinyl alcohol/citric acid nanofiber membrane-modified electrode. The morphology characterization and assembly process of the modified materials were investigated using scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). Under the optimum conditions, the label-free electrochemical immunosensor for parathion exhibited a linear range of 0.0015–6400 ng/mL and a low detection limit of 0.48 pg/mL, the signal response of which was 10 times higher than that of the randomly immobilized VHH9. The immunosensor possessed high selectivity, good repeatability and reusability (keeping above 90% of its initial activity after repeating 8 times), and stability (remaining 90% after 9 weeks of storage). Finally, the average recoveries of parathion from food samples were 93.76–105.73% with the coefficient of variation being 2.65–6.85%, showing good correlation with UPLC ( R 2 = 0.9950). Therefore, our nanobody immobilization protocol is simple and effective and proves the potential to be utilized as a promising candidate for sensing platform. Graphical
危险品化学品经营许可证（带存储）