Development Of Electrochemical Immunoassay For Two Emerging Contaminants In The Environments

Emerging Contaminants（Ecs）are harmful to human health and the ecological environment.Due to improved technology and the discovery of new biological toxicity,much concern has been raised concerning Ecs.Tetrabromobisphenol A dihydroxyethyl ether（TBBPA-DHEE）and Escherichia coli（E.coli）are examples of Ecs.Although they are present in lower concentrations in the environment,they have negative impacts on ecology and human health.Therefore,it is necessary to establish a sensitive,efficient and simple analytical method for investigating these pollutants.Electrochemical immunoassay based on antigen-antibody specific recognition is simple,easy to operate,and requires no complicated sample preparation methods,as well as requires a small amount of sample and rapid detection and analysis of the target,which can meet the requirements of measuring trace contaminants in environmentalsamples.Basedonsilicanano-brushes,self-assembled nano-polymerized spheres,syringaldazide,four immunoassay methods were established.The details are as follows:（1）In order to detect the TBBPA-DHEE,based on functionalized self-assembled nanospheres to catalyze the redox reaction of hydrogen peroxide,an electrochemical time-current immunosensor was established.This work also uses competitive immunization methods to introduce targets and antibodies onto the electrodes,and then converts the target concentration into a current signal based on the antibody marker（gold nanoparticles and catalase）catalyzing the hydrogen peroxide to produce a redox reaction.By marking a large number of gold nanoparticles and catalase on the surface of the self-assembled nanosphere particles,the gold nanoparticles and catalase have achieved a good synergistic effect in the catalytic reduction of hydrogen peroxide,which significantly improves the catalytic performance on hydrogen peroxide and increases the current value of the electrochemical response,thereby improving the sensitivity of this method further.Under optimal experimental conditions,the detection limit（LOD）of this method is 0.12 ng mL^-1,which is higher than the traditional ELISA method(LOD,0.702 ng mL^-1).（2）In the previous work,although the time-current method is sensitive,the electrochemical workstation is likely to be affected by the substrate and cause the output signal to be unstable.Therefore,a more stable method is needed.In this work,a novel electrochemical indirect competitive immunosensor capable of detecting TBBPA-DHEE in environmental samples was constructed,based on HRP-catalyzed precipitation of 4-chloro-1-naphthol by hydrogen peroxide.Using this method of competitive immunization,the target and antibody are introduced into the surface of the electrode,and then the antibody labeled horseradish peroxidase（HRP）is used to catalyze hydrogen peroxide（H₂O₂）to oxidize 4-chloro-1-naphthalene on the surface of the electrode,forming a precipitate（benzo-4-chlorohexadienone）,which can accumulate on the surface of the electrode.By hindering the electron transfer on the surface of the electrode,the impedance of the electrode surface is increased.The target concentration output is regarded as the electrical signal for detecting TBBPA-DHEE in the environment.In this work,the silica nano-brush is used as a carrier to load a large amount of HRP,which significantly expands the electrochemical signal and improves the sensitivity of the detection method.Under optimal experimental conditions,the electrochemical impedance signal intensity has a good linear relationship with the logarithm of TBBPA-DHEE concentration when the concentration of TBBPA-DHEE is in the range of 0.21¹11.31 ng mL^-1.The detection limit（LOD）is 0.08 ng mL^-1,which is higher than the traditional ELISA method(LOD,0.702 ng mL^-1).（3）In order to detect E.coli in the environment,based on the redox reaction of syringaldazin（4-hydroxy-3,5-dimethoxybenzaldehyde,Syr）and H⁺in the solution,a sensitive pH immunosensor was established.In this work,the glassy carbon electrode was modified by syringaldazine to recognize H⁺in the solution.In the process of obtaining energy,the fermentation of E.coli can convert glucose into various products,such as succinic acid,lactic acid,formic acid,ethanol,acetic acid,these products can change the concentration of H⁺in the solution.The change of H⁺can be detected by the modified electrode,thereby converting the concentration of E.coli into an electrochemical signal.The detection limit of this method is 60 cfu mL^-1,with high accurate（recovery,85-105%;CV,2.3-8.2%）.（4）By summarizing the previous work,it is found that the electrochemical method still needs an improved operation technology,hence not suitable for popularization.Therefore,it is necessary to develop a convenient method.This work involved a simple and novel immunoassay method for detecting E.coli based on the use of the barometer to detect the oxygen generated by the redox process of hydrogen peroxide.In this study,E.coli was separated from other strains by magnetic beads labeled with anti-E.coli DH-5 polyclonal antibody in the environment,then the functionalized self-assembled nanosphere-labeled antibodies were used to establish a sandwich structure.Based on the functionalized self-assembled nanosphere,hydrogen peroxide is catalyzed to generate oxygen by converting the target concentration into a barometric pressure signal in a closed space.Under optimal experimental conditions,the method has obvious advantages:high sensitivity(LOD,80 cfu mL^-1);wide linear range(10²-10⁷ cfu mL^-1);good accuracy（recovery,86.7-107%;CV,3.2-8.1%）.