Establishment Of Nanobody-Based Immunoassay For Visual Detection Of 3-Phenoxybenzoic Acid

3-Phenoxybenzoic acid(3-PBA)is the main metabolite of pyrethroid pesticides,which can enter human body through environmental contact and food chain.Researchers have found that it can affect the secretion of biological estrogen,lead to endocrine and metabolic disorders.3-PBA easily exists in dairy products and lake water through the accumulation of biological chain,which poses a serious threat to food safety and human health.Based on 3-PBA Nbs,the rapid and visual immunoassay methods were established using NCM as solid-phase reaction carrier for the detection of 3-PBA in actual samples.The main results are described in the following:1.Establishment of dot ELISA based Nbs for 3-PBA detection:The 3-PBA Nbs were expressed by Pcomb3xss-3-PBA-VHH-TOP 10F'engineering bacteria.The effects of expression temperature,IPTG concentration and expression time were explored,and the optimal expression conditions were determined as follows:expression temperature was 37?,IPTG concentration was 0.5 mmol/L and expression time was 12 h.The purified Nbs were identified and the ELISA conditions were optimized.The standard curve was established under the optimum conditions.The results showed that the concentration of 3-PBA had a good linear relationship in the range of 0.311?3.09 ng/m L,and the IC₅₀ was 0.981 ng/m L.Based on Nbs,dot ELISA was established for the visual detection of 3-PBA using NCM as solid-phase reaction carrier.The visual results were quantified by software,and standard curve was established.The detection limit(IC₁₀)was 0.01 ng/m L.And the detection was completed within 30min.Taking milk and lake water for samples analysis,the recoveries were 101?112%and 95?104%,respectively.Meanwhile,HPLC was used for validation and correlation analysis(R²=0.923),which showed that the results obtained by the two methods(dot ELISA and HPLC)were in good consistency.2.Establishment of AuNPs-Nbs lateral-flow immunoassay for 3-PBA detection:Gold nanoparticles(AuNPs)were prepared by the method of reducing chloroauric acid with trisodium citrate.The Nbs were labeled with AuNPs by electrostatic adsorption.The synthesized AuNPs and AuNPs-Nbs were characterized by wavelength scanning,transmission electron microscopy(TEM)and particle size analysis.The results showed that size of bare AuNP was about 20 nm,and the size of AuNP-Nbs was about 26 nm.The AuNPs-Nbs lateral-flow immunoassay was established,which used NCM with backing as solid-phase reaction carrier.The visual results were quantitatively analyzed by software,and IC₁₀ was 0.1 ng/m L.And the detection was completed within 15 min.Taking milk and lake water as analysis samples,the recoveries were 101?105%and92?106%,which were in good agreement with HPLC results(R²=0.974).Without the participation of substrate,the developed AuNPs-Nbs lateral-flow immunoassay could be simplified effectively.3.Establishment of Au@Pt-Nbs lateral-flow nanozyme immunoassay for 3-PBA detection:The core/shell Au@Pt nanozymes were prepared by reducing K₂Pt Cl₆ with AuNPs as the core.The size of Au@Pt nanozyme was determined to be 24 nm by TEM,and its peroxidase-simulating activity was optimized.The optimal Au@Pt nanozymes were labeled with Nbs.The Au@Pt-Nbs lateral-flow nanozyme immunoassay was established by using NCM with backing as reaction carrier.Au@Pt markers could catalyze the color development of substrate in the presence of H₂O₂,and achieve the visual detection of 3-PBA.The visual results were quantitatively analyzed by software,and IC₁₀ was 0.005 ng/m L.And the detection was completed within 30 min.Taking milk and lake water for sample analysis,the recoveries were 102?108%and 95?112%,respectively.The correlation analysis was carried out by HPLC and get good correlation(R²=0.984).The sensitivity of Au@Pt nanozymes lateral-flow immunoassay was 20-fold higher than that of AuNPs-Nbs lateral-flow immunoassay,which indicated that nanozymes had great potential in immunoassay.