A novel ultrasensitive nanobody-based electrochemical immunoassay was prepared for assessing human exposure to pyrethroid insecticides. 3-Phenoxybenzoic acid (3-PBA) is a common human urinary metabolite for numerous pyrethroids, which broadly served as a biomarker for following the human exposure to this pesticide group. The 3-PBA detection was via a direct competition for binding to alkaline phosphatase-embedded nanobodies between free 3-PBA and a 3-PBA-bovine serum albumin conjugate covalently immobilized onto citric acid-decorated nylon nanofibers, which were incorporated on a screen-printed electrode (SPE). Electrochemical impedance spectroscopy (EIS) was utilized to support the advantage of the employment of nanofibrous membranes and the success of the immunosensor assembly. The coupling between the nanofiber and nanobody technologies provided an ultrasensitive and selective immunosensor for 3-PBA detection in the range of 0.8 to 1000 pg mL–1 with a detection limit of 0.64 pg mL–1. Moreover, when the test for 3-PBA was applied to real samples, the established immunosensor proved to be a viable alternative to the conventional methods for 3-PBA detection in human urine even without sample cleanup. It showed excellent properties and stability over time.

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