Development of a Highly Sensitive Direct Competitive Fluorescence Enzyme Immunoassay Based on a Nanobody–Alkaline Phosphatase Fusion Protein for Detection of 3‑Phenoxybenzoic Acid in Urine

3-Phenoxybenzoic acid (3-PBA) is a human urinary metabolite of many pyrethroid insecticides and can be used as a biomarker to monitor human exposure to these pesticides. A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for detecting 3-PBA on the basis of a nanobody (Nb)–alkaline phosphatase (AP) fusion protein was developed. The anti-3-PBA Nb–AP fusion protein was expressed and purified. The 50% inhibitory concentration (IC50) and linear range of dc-FEIA were 0.082 and 0.015–0.447 ng/mL, respectively, with a detection limit of 0.011 ng/mL. The IC50 of dc-FEIA was improved by nearly ten times compared with those of one-step and three-step direct competitive enzyme-linked immunosorbent assay (dc-ELISA). Spiked urine samples were detected by both dc-FEIA and liquid chromatography–mass spectrometry (LC-MS), and the results showed good consistency between the two analysis methods, indicating the reliability of dc-FEIA based on the Nb–AP fusion protein for detecting 3-PBA in urine.