A Solid Phase Extraction Method For Estrogenic Disrupting Compounds Based On Estrogen Receptor Response Element

Estrogenic disrupting compounds(EDCs)is a kind of chemical that mimics and antagonizes the biological function of natural estrogen,which can interfere with the function of the endocrine system of organisms,and affect reproductive development or destroy the normal hormone metabolism of the body.It widely exists in the environment,and can be enriched through the food chain,so some substances may be residue in food,which will harm human health.Therefore,it is of great significance to establish an efficient,sensitive and rapid detection method for EDCs.At present,the detection methods of EDCs are mainly biological detection methods and chemical detection methods.Biological detection methods are mainly used for preliminary screening.Chemical detection methods were using high performance liquid chromatography(HPLC),gas chromatography and other large-scale instruments to identify and quantify the EDCs.Currently,many instrumental methods are used to detect low concentrate EDCs.Because of the complexity of food matrix,the accuracy of test results will be affected by the existence of a large number of interferences in the matrix.Therefore,it is necessary to adopt appropriate pretreatment methods when using instrumental methods.Solid phase extraction(SPE)is the most widely used sample pretreatment method.Silica gel column is the most widely used SPE column which has non-specific adsorption of the non-target.The specific binding methods based on antibodys and molecularly imprinted polymers are limited to detecting a little number of EDCs,which limits the application of the method.EDCs are mainly through binding with estrogen receptor(ER)to interfer with the endocrine system.Therefore,SPE packing material linking with ER can not only effectively avoid non-specific binding with other impurities in the matrix,but also achieve the specific recognition of all EDCs.However,ER is unstable and easy to be deactivated.In the preparation of SPE column,if ER was crosslinked on the SPE packing material through chemical method,most of the activity of ER will be lost,and the enrichment and purification effect of EDCs is poor.In this paper,a solid phase extraction method of EDCs based on estrogen receptor response element(ERE)was established.ERE was used to maintain the biological activity of ER.The concentration and purification effect of EDCs can be significantly improved by using ER.Finally,HPLC was used to identify and quantify the EDCs.The SPE method for EDCs based on ERE exhibite high selectivity and sensitivity.The paper mainly consisted of four parts,as follows:In the chapter one,the EDCs,ER,ERE and the mechanism of action among EDCs,ER and ERE,the detection method of EDCs and its pretreatment method,SPE method and its application in combination with HPLC,application and research progress of ER as a detection method for identifying materials were summarized.The emphasis was the mechanism of action between EDCs,ER and ERE and the application of ER as identification material and its detection method for EDCs.In the chapter two,a SPE method of EDCs based on ERE was established.The specific adsorption of EDCs was realized by using the ERE,and HPLC was used to identify and quantify the EDCs.Bovine serum protein(BSA)was used to block the macromolecular sites of silica gel and eliminate the non-specific adsorption of ER,so that ER could be specifically combined with ERE.Standard curve was established for calculating the adsorption amount of ER by coomassie brilliant blue assay.The specific adsorptions of EDCs and ER were achieved by blocking the carboxyl and other small molecular sites of silica gel with triphenylamine.In this paper,the elimination of non-specific adsorption of ER,the incubation time of ER and ERE,and the elimination of non-specific adsorption of EDCs were optimized.The optimal condition was carboxylated silica gel combined with ERE.Then 3%BSA was used to block the macromolecular sites.The optimal time of ER incubated with the ERE was 2 h.The optimal concentration of triphenylamine to block the carboxyl and other small molecular sites of silica gel was 0.03 mg·m L^-1.The silica gel coupled with ERE and ER was transferred to the SPE column and HPLC was used to identify and quantify the EDCs.In this chapter,the detection methods of Bisphenol A(BPA),Estradiol(E₂)and Diethylstilbestrol(DES)were established.The accuracy of the method was investigated in liquid milk,and the recovery rate can reach84.1?113.6%.In the chapter three,the SPE method of EDCs based on ERE in the chapter two was applied to detect EDCs in liquid milk,fermented milk and milk powder of different brands and batches.In the exprement,no residues of E2 and DES were detected,and BPA was detected from one batch of liquid milk.But the amount of residue is below the daily tolerable level(4?g·kg^-1·bw·day^-1)and the intake was safe for consumers.In chapter four,the SPE method of EDCs based on ERE was summarized,and the detection results of EDCs in liquid milk,fermented milk and milk powder were analyzed.Compared with the traditional method,the SPE method for EDCs based on ERE exhibite high selectivity and sensitivity.