Development Of A Highly Sensitive Phenolic Estrogen Multi-residue Electrochemical Immunosensor

Food safety has always been one of the research hotspots and the harm of drug residues in food to human health is the main issue.With the development of science and technology,a large number of artificially synthesized environmental estrogens are added into foods,and have become the focus of monitoring by the national testing department.However,many existing technologies cannot achieve rapid,efficient,and accurate detection because of the large variety of synthetic estrogens.Therefore,the urgent task in the current research is to find a rapid,efficient and on-site method for the detection of synthetic environmental estrogens.Currently,there is a method that combines electrochemical analysis technology with multi-residue immunoassay technology,which has the advantages of high-throughput detection,low cost,and high sensitivity.This is important for rapid primary screening of estradiol compounds in food and environment.Therefore,in this dissertation,a broadspectrum-characterized HEX monoclonal antibody was first prepared and applied into the electrochemical immunosensor modified with nanomaterials to construct three highly sensitive multi-residue electrochemical immunosensors for the simultaneous and rapid detection of four phenolic estrogens.The specific content is divided into the following four chapters:Chapter 1:The structure of the main four phenolic estrogens,their hazards,and the mechanism of poisoning are briefly introduced in this section.Subsequently,the advantages and disadvantages of the current methods for the detection of phenolic estrogens are described.In this study,we focused on the application of nanomaterials in electrochemical immunosensors and the immobilization of bioactive materials on the electrode surface.Chapter 2:In this chapter,we first modified the gold nanoparticles on the surface of the glassy carbon electrodes by electrochemical deposition.Then,thioglycolic acid was modified on the surface of nanogold using self-assembled molecular membrane technology.Finally,the HEX monoclonal antibody was chemically bonded to the surface of the sensor,and a HEX monoclonal antibody/thioglycolic acid/nanogold immunosensor was prepared.Four kinds of phenolic estrogens were determined by sandwich immunoassay,and a wide linear range and low detection limit were achieved.Bisphenol A was determined in the range from 0.5 to 500 ng/mL and the detection limit was determined to be 0.037 ng/mL.In addition,the recovery experminent of diethylstilbestrol added to three kinds of actual samples were determined.The recovery range between 90.72%-110.99%was satisfactory.Chapter 3:The hapten of carboxyl functional diethylstilbestrol was prepared and characterized by H1-NMR spectroscopy.Nanogold was electrochemically deposited on the surface of the glassy carbon electrodes.The thiol-ethylamine molecular membrane was later self-assembled on the surface of the nanogold and then the Monomethylene ether estrene(HEX-CME)was chemically modified.Finally,a highly sensitive multi-residue immunosensor was successfully constructed,which can simultaneously detect four estrogens.The electrodes were characterized by scanning electron microscopy(SEM),near-infrared spectroscopy(IR)and alternating current impedance(EIS).The indirect competitive immune mechanism was applied to the electrochemical immunoassay for the detection of four phenolic estrogens:hexestrol(HEX),diethylstilbestrol(DES),diethylstilbestrol(DE)and bisphenol A(BPA).The results showed that the minimum detection limit is about 0.2 ng/mL.In addition,the recovery experiments of diethylstilbestrol added in actual samples were done,and the recovery range was between 88.7%-105.7%.The results showed that the immunosensor constructed in this experiment has the advantages of high sensitivity,good stability,simple and practical,and has practical significance for rapid primary screening of estradiol compounds.Chapter 4:A gold nanowire electrode was prepared and the size and morphology were analyzed by transmission electron microscopy(TEM).The self-assembly modification of mercaptoethylamine on gold surface was achieved and then the carboxyestradiated estradiol was chemically modified to obtain the hexaestrol/mercaptoethylamine/gold nanowire electrochemical immunosensor.Using traditional indirect competitive immune mechanisms and electrochemical analysis techniques,we performed highly sensitive multiresidue assays for four phenolic estrogens.The results showed that the detection range of the four phenolic estrogens was very wide and the sensitivity was very high.The detection limit,which was as low as 0.35 pg/mL,was far lower than the other traditional electrochemical immunosensors,due to the small size of the gold nanowire electrode which has the advantages of high current density,fast mass transfer rate,etc.Therefore,it has good scientific and practical value in the future researches.