Researches On Developing Novel Immunoassays For The Determination Of TACE Brominated Flame Retardant In Environment

Brominated flame retardant?BFRs?are a class of organic flame retardants with the highest flame retardant efficiency.BFRs can meet the requirements of many flame retardant products.As a type of BFRs,polybrominated diphenyl ethers?PBDEs?have high flame retardant efficiency and good thermal stability.Thus,they have been used in many polymers.Both 2,4,4'-tribromodiphenyl ether?BDE-28?and 2,2',4,4'-tetrabromodiphenyl ether?BDE-47?are polybrominated diphenyl ether with the high detection rate,and they have strong biological toxicity.Tetrabromobisphenol A?TBBPA?is a type of widely used industrial BFRs,and it can be released into environment due to the effect of high temperature or other physical and chemical factors.So far,the methods for detecting BFRs are mainly based on gas chromatography and liquid chromatography.The stability and accuracy of the chromatographic techniques are well.However,such instrumental methods are usually time-consuming,and the process of sample pretreatment is complicated;Moreover,the maintenance of the instrument is expensive.Therefore,it is of important scientific significance and application value in the field of environmental monitoring research to develop the simple,convenient,highly sensitive and high-throughput methods for the detection of trace BFRs.In this study,several BFRs with high detection rate,including BDE-28,BDE-47and TBBPA,were taken as study objects.Based on the preparation of BDE-28,BDE-47 and TBBPA antigens and antibodies,several novel immunoassays were developed and applied to the detection of several environmental samples.It is of great significance to develope rapid,sensitive and convenient immunoassays for the detection of trace BFRs.The details are shown as follows:?1?First,BDE-28 hapten containing active functional group was prepared in this study.The prepared BDE-28 hapten was characterized by the Fourier transform infrared spectroscopy,¹H nuclear magnetic resonance and UV-visible absorption spectrum respectively.Then,BDE-28 hapten were coupled with bovine serum protein?BSA?and ovalbumin?OVA?respectively to prepare BDE-28 immunogen and coating antigen in different methods.Finally,the New Zealand white rabbits were immunized with BDE-28 immunogen BSA-BDE-28 to prepare anti BDE-28 antibody?pAb-BDE-28?.The specificity of pAb-BDE-28 was well,and the cross-reactivity between pAb-BDE-28 and the analogues was below 10%.The titer of pAb-BDE-28was above 64000 and the affinity constant of pAb-BDE-28 was 2.53×10⁷ M^-1.The obtained pAb-BDE-28 can be used for the follow-up immunoassay.?2?The gold nanoparticles?GNPs?were modified with goat anti-rabbit IgG labeled by Biotin-N-hydroxy succinimide ester?BNHS?.Then,a functionalized gold nanoparticles based indirect competitive BA-ELISA method?GNPs-BA-ELISA?was developed for detecting TBBPA.Several detection conditions of the immunoassay were optimized in the assay.The standard curve of GNPs-BA-ELISA method for detecting TBBPA was constructed by plotting inhibition values against the logarithm of TBBPA concentrations.Under the optimal reaction conditions,the linear relationship was well when TBBPA concentration was 0.009?g/L-3.126?g/L;linear regression equation:y=23.58logC₀+68.33?R²=0.9879?.IC₅₀ was 0.167?g/L,and the limit of detection?LOD?was 0.0034?g/L.The recovery rates of the spiked samples were 89.22%-107.91%,and the coefficient of variations?CVs?were6.23%-11.73%.Both the CVs of intra-assay and inter-assay of this immunoassay were less than 13%,which indicated that the immunoassay has good repeatability and high accuracy.The GNPs-BA-ELISA was applied to detect TBBPA in PM_2.5 and agricultural soil samples;the TBBPA results were consistent with those obtained by HPLC?R²=0.9887?.Compared with the BA-ELISA method,the sensitivity of the GNPs-BA-ELISA method was 3-4 times higher.?3?The graphene oxide carrier was modified with goat anti-rabbit IgG labeled by BNHS to prepare functionalized graphene oxide.Then,BNHS was used as derivative reagent to combine with target DNA to prepare biotinylated DNA.Then,a functionalized graphene oxide based indirect competitive BA-iPCR method?GO-BA-iPCR?was developed for detecting BDE-28,BDE-47.Several detection conditions of the immunoassay were optimized.The standard curves of the GO-BA-iPCR assay for detecting BDE-28?BDE-47?were constructed by plotting the Ct values against the logarithm of the BDE-28?BDE-47?concentrations.Under the optimal conditions,the linear relationship was well when BDE-28 concentration was5 pg/L-50000 pg/L;linear regression equation:Ct=0.32logC₀+12.63?R²=0.9756?,and the limit of detection was 1.27 pg/L.The recovery rates of the spiked samples were 90.75%-107.74%,and the coefficient of variations were 3.65%-9.73%.The linear relationship was well when BDE-47 concentration was 1 pg/L-10000 pg/L;linear regression equation:Ct=0.24logC₀+14.05?R²=0.9839?.The limit of detection was 0.72 pg/L.The recovery rates of the spiked samples were 91.67%-109.06%,and the coefficient of variations were 3.82%-11.06%.Both the CVs of intra-assay and inter-assay of the GO-BA-iPCR assay for detecting BDE-28 and BDE-47 were below 13%,which indicated that the repeatability of this immunoassay were well.The GO-BA-iPCR method was applied to detect BDE-28,BDE-47 in PM_2.5 samples;the detection results were consistent with those obtained by GC-MS?BDE-28:R²=0.9868,BDE-47:R²=0.9877?.?4?The gold nanoparticles?GNPs?modified with polyclonal antibody and signal DNA was used as biological probe.Then,a direct competitive immuno-PCR assay based on bio-functionalized gold nanoprobes?GNPs-iPCR?was established to detect BDE-28,BDE-47.The concentrations of the coating antigen and the biological probe were optimized.The standard curves of the GNPs-iPCR assay for detecting BDE-28?BDE-47?were constructed by plotting the Ct values against the logarithm of the BDE-28?BDE-47?concentrations.Under the optimal conditions,the linear relationship was well when BDE-28 concentration was 6 pg/L-60000 pg/L;linear regression equation:Ct=0.54logC₀+10.92?R²=0.9881?.The limit of detection was1.67 pg/L.The recovery rates of the spiked samples were 89.60%-109.91%,and the coefficient of variations were 4.25%-11.71%.The linear relationship was well when BDE-47 concentration was 5 pg/L-50000 pg/L;linear regression equation:Ct=0.43logC₀+12.57?R²=0.9865?.The limit of detection was 1.32 pg/L.the recovery rates of the spiked samples were 87.97%-107.90%,and the coefficient of variations were 3.70%-11.22%.Both the CVs of intra-assay and inter-assay of the GNPs-iPCR assay for detecting BDE-28 and BDE-47 were less than 10%.The sensitivity of the GNPs-iPCR was close to that of GO-BA-iPCR method.However,the GNPs-iPCR was more convenient and the detection period was less;moreover,the repeatability of the method was better and the accuracy was higher.The GNPs-iPCR method was applied to detect BDE-28,BDE-47 in different environmental samples(PM_2.5,sediment and agricultural soil samples);the detection results were consistent with those obtained by GC-MS?BDE-28:R²=0.9879,BDE-47:R²=0.9897?.For the novel immunoassays developed in this study,there is no need for preconcentration or even purification after the extraction of environmental samples.Nearly a hundred samples can be tested within 8 h.The detection level of the new immunoassays reached the level of ng/L or pg/L.The developed GNPs-BA-ELISA,GO-BA-iPCR and GNPs-iPCR methods were sensitive,simple and convenient.All the immunoassays showed good stability,and the detection results agreed with those of chromatographic analysis.These new immunoassays can be used for rapid and high throughput detection of trace BFRs in the environment.