Fabrication of a high-sensitivity electrochemical immunosensor by the oriented immobilization of engineered nanobody on nanofibrous membrane.

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 = 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.