Development Of Immunoassay Methods For Two Typical And Emerging Organic Pollutants In The Environment

Recently,emerging contaminants have been research topics in the field of environmental chemistry,including DBP and TBBPA-DHEE,two typical emerging organic pollutants.During the production and usage,both of them can be released into the environment through various ways intentionally or unintentionally,causing potential ecotoxicological effects and even health effects.At present,research shows that DBP and TBBPA-DHEE may exist widely in the environment,the pollution level or distribution characteristics of which are completely unclear.Therefore,it is necessary to develop efficient,sensitive and simple methods to investigate pollution situations of the two pollutants.Compared with instrumental analysis,the immunoassay based on antibody/antigen has distinct advantages of simple pretreatment,fast analysis and high throughput,which is suitable for the analysis of organic pollutants in complex media.In this paper,several sensitive methods using novel materials are developed in the application of the analysis for environmental or food samples.The details are as follows:（1）Based on the catalysis of AuNCs@BSA,a novel enzyme-linked immunosorbent assay（ELISA）was developed for the detection of dibutyl phthalate（DBP）.It is reported that AuNCs@BSA shows totally different properties from ordinary gold nanoparticles due to its smaller size,including the catalytic capability to 3,3’,5,5’-tetramethylbenzidine（TMB）solution.Meanwhile,Ag⁺can prevent AuNCs@BSA from inducing a color change of TMB to blue by destroying its structure.In this system,AgNPs was labelled on the second antibody（Ab₂）to provide numerous Ag⁺after the competition step.Due to the signal amplification by the principle,the sensitivity of the modified ELISA was improved with the low limit of detection（LOD）of 4.017μg/L for DBP,which was decreased 16 times relative to that using conventional ELISA with the same antibody.And our method shows great potential in the detection of trace DBP from environmental and food samples.（2）A sensitive colorimetric immunoassay was designed and established by synthesizing a novel antibody marker（Cu-MOFs@Ab₂）as signal amplification for the DBP detection.In this assay,Cu⁺can react with the amino acid residues of HRP,causing that HRP fails to catalyse the colorless TMB into blue solution.Based on this principle,Cu-MOFs is labelled on Ab₂,which can release massive Cu²⁺in the presence of nitric acid（HNO₃）and Cu²⁺will be further reduced to Cu⁺with the adding of sodium ascorbate（SA）.Under the optimized conditions,the LOD of this method was 1μg/L,indicating great potential for trace DBP detection of environmental and food samples.（3）A novel plasmonic enzyme-linked immunosorbent assay（pELISA）based on H₂O₂-mediated growth of gold nanoparticle（AuNPs）was developed for the ultrasensitive simultaneous naked-eye detection of TBBPA DHEE and TBBPA MHEE,respectively.In this paper,glucose oxidase（GOx）is of vital importance to the reaction between GOx and glucose to produce H₂O₂,determining the color of the solution.In addition,further signal amplification was achieved via a large number of GOx molecules,which were immobilized on silica nanoparticles carrying poly brushes（SiO₂@PAA）to increase the enzyme load,and the whole complex was conjugated on the second antibody.Under the optimized conditions,the LOD of this method using a microplate reader is 3.3×10^-4μg/L,improving 3 orders of magnitude than that using conventional colorimetric ELISA with the same antibody.Furthermore,the proposed approach showed good repeatability and reliability after a recovery test fortified with a variety of targets was performed（recoveries,78.00%-102.79%;coefficient of variation（CV）,4.38%-9.87%）.The method will be widely used for the investigation of TBBPA DHEE and TBBPA MHEE in real environments.（4）A novel chemiluminescence immunoassay based on luminol-modified gold nanoclusters（AuNCs@Peps@luminol）was developed for simultaneous detection of TBBPA-DHEE and TBBPA-MHEE,respectively.In the system,alkaline phosphatase（ALP）was labelled on the second antibody（Ab₂）for signal amplification.When ALP-Ab₂ was captured by antigen-antibody（Ab₁）complex,disodium phenyl phosphate（PPNa）generated massive phenol under the catalysis of ALP,markedly inhibiting the chemiluminescence intensity of AuNCs@Peps@luminol.Under the optimized conditions,the calculated detection of limit（LOD,90%inhibition）was 0.078μg/L for TBBPA-DHEE with a linear range of 0.23-9.32μg/L,which was lower 9 times in comparison with conventional ELISA using the same antibody.In addition,our method showed satisfactory accuracy and precision（recoveries,88.00-113.4%;CV,2.75-8.14%）,it can be applied to systematically investigate the concentration of the trace TBBPA-DHEE and TBBPA-MHEE in environmental and food samples.