Detection of a Pyrethroid Metabolite 3-Phenoxybenzoic Acid Based on a Biotinylated 3-Phenoxybenzoic Acid Nanobody Immunoassay

KeyWord:3-phenoxybenzoic acid£¨3-PBA£©  pyrethroid pesticides  nanobody£¨Nb£©  biotinylation  signal amplification

Abstract:

      3-Phenoxybenzoic acid£¨3-PBA£©£¬as the most common metabolite of pyrethroid pesticides£¬is typically employed as the risk indicator for pyrethroid pesticides exposure. Antibody-based immunoassay has the advantages of rapidity£¬high sensitivity and easy operation£¬which is considered as a promising tool for the risk exposure assessment on hazardous chemicals in environment£¬food stuffs and human body fluid samples. Nanobody£¨Nbs£© is the variable domain of heavy chain-only antibody£¨VHH£© derived from heavy chain antibodies£¨HCAbs£© of camelidae or new antigen receptor£¨NAR£© of shark£¬which are naturally lack of light chain. Unlike conventional antibodies£¬Nbs have high binding affinity with only a single domain£¬avoiding the complex combination of heavy and light chains during recombinant expression£¬which would sometime lead to the low solubility and stability of genetically engineered antibody fragments. Therefore£¬Nbs are easily expressed in soluble form£¬and have similar affinity with its natural structure. And Nbs have been reported to have excellent thermostability and organic solvent tolerance£¬which is benefit to simplify the pretreatment step. Thus£¬Nbs are considered as an ideal tool for developing rapid immunoassays. In this work£¬with the aim to develop a sensitive immunoassay for monitoring the metabolite of pyrethroid pesticides 3-PBA in water and human urine samples£¬the biotinylated 3-PBA Nbs using biotin N-hydroxysuccinimide ester£¨biotin£­NHS£© were prepared. To further amplify the signal of immunoassay£¬a polymeric horseradishperoxidase conjugated streptavidin£¨polyHRP£­SA£© was also introduced to develop a biotin£­avidin system based indirect competitive enzyme-linked immunosorbent assay£¨icELISA£©. The assay conditions were carefully optimized as follows£ºthe coating antigen concentration was 125 ng/mL£¬the dilution ratio of biotinylated 3-PBA to Nbs was 1¡Ã2 000£¬PBST with an ion concentration of 40 mmol/L£¬a pH 6.4 and a Tween£­20 concentration of 0.005% £¨by volume£© was optimal working buffer system£¬the dilution ratio of polyHRP£­SA was 1¡Ã4 000. The developed icELISA showed a half maximal inhibitory concentration£¨IC50£© of 1.7 ng/mL£¬a linear range of 0.37£­7.4 ng/mL and a limit of detection£¨LOD£© of 0.15 ng/mL. The proposed Nbs-based icELISA was applied in the detection of 3-PBA in water and human urine samples. For water samples such as lake water£¬ reservoir water£¬tap water and drinking water£¬the matrix effects could be easily eliminated by simply filtering and then used for icELISA without further dilution£¬while for the human urine samples£¬high temperature acid hydrolysis and solid phase extraction£¨SPE£© purification are benefit to reduce the matrix effects. The spiked recoveries for human urine and water samples were in the range of 87.0%£­127% and 78.0%£­113%£¬respectively£¬with relative standard deviations£¨RSD£© not more than 10%. Results indicated that the proposed method was sensitive and easy to operate£¬which could be used as an ideal tool for screening 3-PBA residues in environmental and biological samples.