The Research On High-throughout Screening Environmental Estrogens

Endocrine disrupting compounds(EDCs)are a defined category ofenvironmental contaminants,which interfere with endocrine system functionand persistent in the environment.A large part of EDCs have been identified thatinduce estrogen-like responses and are classified as estrogenic EDCs.Currentdetection methods for estrogenic EDCs include chemical analytical methods andbiologically based assays.The chemical analytical methods provide excellentsensitivity and precision for monitoring environmental estrogen.However,thesetechniques only measure specific estrogenic EDCs individually,so the targetcompound must have already been identified as have estrogenic properties.Thechemical analytical methods are costly and time-consuming,since they needcomplicated steps for sample preparation,expensive equipments and highlyskilled technicians,which will not be adapt for large numbers of samples toscreened.Biologically based assays always based on Cell culture.Serum used incell culture must be treated by charcoal and dextran T70 to remove the sexsteroids.Different laboratories using different cell lines and the result can not becompared.Cellular assays are also confronted with the problem of cytotoxicsubstances,which may be present in environmental samples and could lead to erroneous results unless proper controls are included.Ligand binding to the estrogen receptor is the initial step for mostenvironmental estrogen entered the body.According to this mechanism,weestablished three method to screen the environmental estrogen.PartⅠCompetitive Recombinant Estrogen Receptor-based ELISAfor Detection of Estrogen in EnvironmentObjective Environmental estrogen is a type of EDCs which can interfere withthe synthesis,secretion,transport,binding,action,or elimination of naturalhormones in vivo.This study aimed at developing a rapid and sensitive bioassayfor the detection of environmental estrogens.Methods Here we present asimple method for quantitative assessment of environmental estrogens based oncompetitive estrogen receptor(ER)mediated immunoassay in microplates,inwhich estrogen,ER,anti-ER antibody and secondary antibody labeledhorseradish peroxidase(HPR)can form a sandwich complex.Results Theassay result showed that as little as 10ng/L of 17β-estradiol could be detectedand have a linear range from 10ng/L to 100μg/L(R~2=0.9583).ConclusionThis method provides a simple way to quantification environmental estrogens insamples.PartⅡApplication of fluorescent labeling 17β-estradiolin the detection of environmental estrogenObjictive:The fluorescent labeling is one of the most important methods inmodem analysis field as a non-radioactive labeling technique.However,it hasnot been widely applied in environmental estrogen screening.This study aimedat exploring the application of fluorescent labeling technology in the field of environmental estrogens.Method:Anti-ER is immobilized on the blackmicroplates.In the second step(competition step)the environmental sample isincubated together with a limited amount of 17β-estradiol-FITC conjugate andER.After the receptor binding reaction,the supernatant,which contains17β-estradiol-FITC which did not bind with the ER is shifted into another blackmicroplate.Finally the signals are detected with a microplate reader.ResultsThe assay result showed that as little as 10ng/L of 17β-estradiol could bedetected and have a linear range from 10ng/L to 100μg/L(R~2=0.9793).Conclusion The fluorescent labeling technology is a rapid and cost-effectivemethod to screen environmental estrogen.PartⅢDensitometry Determination of Estrogenic EDCs UsingGold Nanoparticle-Modified Estrogen Response Element ProbesObjective Estrogen receptor binding assay is an important approach to screenenvironmental estrogens.It is preferred as Tier 1 Screening method forendocrine disruptors by EDSTAC.In order to rapid screen environmentalestrogens,we develop a new high-throughout screening method based onnanoparticle technology.Since estrogen receptor(ER)is a ligand-independentreceptor,we design a simple competitive binding assay in which environmentalestrogens in the ample compete with natural estrogen(17β-estradiol)for bindingto ER.Methods 17β-estradiol-BSA conjugate is immobilized on themicroplates.In the second(competition)step the environmental sample isincubated together with a limited amount of ER.After the receptor bindingreaction,the supernatant,which contains environmental estrogen-ER complex,is incubated in another microplate coated with anti-ER antibody.Because ERhomodimerization can bind to specific sites on DNA- estrogen response elements(EREs),Nanoparticle -modified ERE probes are added and incubated.Finally the signals are amplified by silver enhancement and recorded withabsorbance.The intensity of signal is proportional to the quantity ofenvironmental estrogen in the sample.Results The assay result showed that aslittle as 100pg/L of 17β-estradiol could be detected and have a linear range from100pg/L to 1μg/L(R~2=0.9764).Conclusion The gold nanoparticle -modifiedERE assay is a screening method for the detection of environmental estrogenswith reliable,low cost,rapid,high-throughout and could be performed onmicroplates or chips.Compared with the existing methods,the detection limitcan be improved 2-3 orders of magnitude in this assay.