| Electrochemiluminescence immunoassay?ECLIA?is a new immunoassay method that combines highly selective immunoreaction with highly sensitive electrochemiluminescence detection technology.Combining the surface effects of metal nanoparticles with electrochemiluminescence analysis is expected to further increase the sensitivity of the analytical method.The purpose of this thesis is to apply metal nanoparticles to electrochemiluminescence immunoassay to detect the estrogen of environmental hormones.At the same time,on the basis of not affecting the characteristics of traditional electrochemiluminescence immunoassay,combined with surface enhancement effect to further improve the electrochemiluminescence immunoassay.The sensitivity of the analytical method is also studied together with the interaction between the surface effects of the metal nanoparticles and the electrochem-iluminescence radiation and the mechanism of action thereof is revealed.This paper mainly includes two parts:The first part summarizes the development process,principle and main characteristics of electrochemiluminescence immunoassay,and then systematically introduces the application of nanogold in electrochem-iluminescence analysis.The second part is the research report.The main research contents include the following three parts:1.Synthesis and characterization of bovine serum albumin-estradiol?BSA-E2?hapten:First,a small molecule of estradiol was derivatized to make it successfully coupled with bovine serum albumin,and its synthesis was characterized by UV-Vis absorption spectroscopy.The BSA-E2 hapten was found to have a BSA-E2concentration of 2.965×10-4 mol/L and an average binding ratio of E2 to BSA of 15.8.Next,the estradiol antibody(AbRu)was labeled with the terpyridinium derivative?bipyridyl-4'-methyl-4-carbonylpyridinium-N-succinimidyl ester dihexafluoropho-sphate,RuNHS?,and passed through the ultraviolet.Visible absorption spectroscopy characterized the AbRu complex with a labeling ratio of 5.4 and a concentration of3.5×10-6 mol/L.Finally,the synthesized BSA-E2 was modified on the gold electrode and the electrochemiluminescence signal was measured in the tripropylamine solution after the immune reaction.It was proved that the synthesized BSA-E2 could be recognized by the estradiol antibody and could be used for the subsequent immune response.2.Electrochemiluminescence immunoassay for detection of estradiol by glod nanoparticles quenching:glod nanoparticles with an average particle size of 15 nm was first synthesized by Frens method and characterized by scanning electron microscopy?SEM?and ultraviolet-visible absorption spectroscopy.The prepared glod nano-particles was labeled with estradiol antibody,and a red-shift of 8 cm-1 was found by UV characterization of AuNPs-Ab.Finally,the BSARu-E2 was immobilized on the working gold electrode,and the gold nanoparticles were then bound to the electrode surface through the immune reaction.The effect of gold nanoparticles on the electrochemiluminescence of the triple bipyridylium system was investigated.The study found that nanogold did not promote the electrochemical reaction process,but exhibited obvious quenching of luminescent radiation.Based on the quenching effect of nanogold on electrochemiluminescence,a signal-evolving electrochemilumine-scence competition immunoassay was constructed to achieve quantitative analysis of estradiol with a linear detection range of 0.1 to 1 ng/mL.The limit of release is 38pg/mL.3.The surface enhancement effect of glod nanoparticles:The surface enhance-ment and quenching of metallic nanomaterials is a competitive process,both related to distance and both weakened with increasing distance.In this work,we used the immune response to regulate the spatial distance between glod nanoparticles and light-emitting molecules,and investigated the surface enhancement effects and influencing factors of nanogold.Firstly,the immune response of estradiol was used as a model,and the primary antibody?Ab1?and secondary antibody?Ab2?of estradiol were labeled with gold nanoparticles respectively.One and two immune reactions occurred on the surface of the electrode,respectively,to regulate the luminescent molecules and glod nanoparticles.From the distance,it was found that the luminescence signal was reduced after using the gold-labeled antibodies.On this basis,we chose microemulsion method to prepare silica nanoparticle labeled antibody as a control of glod nanoparticles effect experiment.As a result,it was found that when a single immunization occurs?the distance between the luminescence molecule and the gold nanoparticle is Ab1?,the nanogold still annihilates the luminescence,quenching60%,and when two immune reactions occur?luminescence molecule and glod nanoparticles?The separation distance is Ab1+Ab2,and the distance increases.)Glod nanoparticles has a certain enhan-cement effect on the luminescence and is enhanced by 90%. |
PDF Full Text Request