Immunoassay Methods For The Detection Of TBBPA-DBPE And Its Preliminary Application

Tetrabromobisphenol A(TBBPA)is a type of typical brominated flame retardant(BFRs),which is widely used as an additive and added to electronic products and textile products.TBBPA enter into environment through water circulation and atmospheric circulation,and have a certain impact on human health.Tetrabromobisphenol A-bis(2,3-dibromopropyl)ether(TBBPA-DBPE)is one of important TBBPA derivatives.At present,instrumental analysis methods are applied to detect TBBPA,but there are disadvantages such as expensive testing equipment,professional testing personnel,and complex sample pre-processing procedures.It is urgent to explore new fast,simple and sensitive testing methods for pollutants distribution,which could provide technical support for pollutants ecological risk assessment.In this study,TBBPA-DBPE was used as the research object to prepare specific monoclonal antibodies;and based on monoclonal antibodies,combined with novel enzyme-like nanomaterials,several new rapid and sensitive immunoassay methods were constructed.The research content is as follows:(1)TBBPA-DBPE haptens(D₄,D₈,M₄,M₈)were synthesized and identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy.The immunogens and coatings of D₄,D₈,M₄,and M₈ were prepared respectively,and identified by UV scanning were used.Balb/C mice were immunized,and three monoclonal antibodies(A11C1,A11C2,E9D5)were screened and prepared by hybridoma technology.The heavy chain of these monoclonal antibodies obtained was of Ig G1 type and the light chain was of Kappa type.Among them,the cross-reaction rate of A11C1 was less than 0.01%with other TBBPA-DBPE analogs.(2)An ELISA method was established based on M₄-OVA and A11C1 monoclonal antibody.The reaction conditions(coating time,blocking time,competition time,buffer conditions,etc.)were optimized,and the standard curve of the ELISA detection method was constructed.The limit of detection(LOD)value was 0.026?g/m L,IC₂₀?IC₈₀ was0.056?0.49mg/m L.The recovery rate of TBBPA-DBPE in water samples was between96.4%and 106.1%.After sampling in typical areas,it was found that in all water samples collected,the detection rate of TBBPA-DBPE was 17.8%,and the detection concentration range was ND?0.091mg/m L.The sample detection rate in Taizhou,Zhejiang(46.7%)was much higher than that in Shouguang,Shandong(20%)and Liuyang,Hunan(0%).As an e-waste dismantling site,pollution in Taizhou needs attention.(3)Optimized conditions of K₂Pt Cl₆ and HAu Cl₄ were 30 m M and 20 m M.Au@Pt was synthesized and characterized by Transmission Electron Microscope,and Au@Pt had good catalytic activity of mimic enzymes.An immunochromatographic method based on double Au@Pt particles was established.After optimizing antibody concentration and working concentration of Au@Pt/Ab and Au@Pt/GAM,LOD was41 ng/m L;using the catalytic activity of Au@Pt and adding AEC for color development,an enhanced immunochromatographic method based on double Au@Pt particles was developed.After optimizing the working concentration of Au@Pt/GAM particles,the LOD was 2 ng/m L,which the sensitivity was increased 20 times.(4)Combined with multi-walled carbon nanotubes with extremely high specific surface area,the hybrid pseudo-enzyme materials Au@Pt/MWCNTs-s and Au@Pt/MWCNTs-o were prepared by the superposition method and the one-pot method respectively.It was found that Au@Pt/MWCNTs-s had higher catalytic efficiency than Au@Pt/MWCNTs-o.Based on Au@Pt/MWCNTs-s and Au@Pt/MWCNTs-o,direct competition immunomagnetic bead methods were constructed.After optimizing the reaction conditions,the LOD were 1.4 ng/m L and 6.2ng/m L,respectively.Compared with the traditional IMB method(LOD 45.1 ng/m L),the sensitivity was increased by 32 times and 7 times respectively.