Study On The Development Of Immunoassays For The Investigation And Risk Assessment Of Bisphenol Compounds In Environments

Bisphenol compounds（BPs）are a kind of emerging organic pollutants widely existing in environmental and food samples,being an interesting issue in the environmental chemistry field recently.As an exogenous chemical,BPs can enter the environment and organisms through various ways during their production,usage,aging,and disposal,having potential ecotoxicological effects and health hazards.At present,the ecological and health risks of BPs are not very clear due to the lack of available data.Thus,it is necessary to develop fast,sensitive,and high-throughput analytical methods to study the occurrence and levels of BPs by a large number of samples analysis.And the immunoassay,based on the specific reaction of antigen and antibody,meets the requirements of BPs,which is simple,fast,and has no complex pretreatment.It can be used to detect trace organic pollutants in a large number of samples at a short time.In this study,based on metal-organic frameworks as signal labels,a series of sensitive and high-throughput colorimetric or fluorescence immunoassays were developed for the detection of bisphenol A（BPA）and bisphenol S（BPS）in environmental and food samples from Zhenjiang.Under optimized conditions,these methods were applied for systematic investigation of BPA and BPS in foods and further the comprehensive assessment of ecological and health risks.The main results of this research are summarized as follows:1.Development of sensitive colorimetric immunoassays for BPA detection（1）Inspired by the natural structure of horseradish peroxidase（HRP）,a novel biomimic nanozyme（Hemin@BSA）was prepared by the co-assembly of hemin and bovine serum albumin（BSA）.In this system,hemin was served to be the activeCofactor for regulating the catalytic activity,surrounding by BSA as a blocking pocket to construct a favorable hydrophobic spatial for substrates enrichment.Compared with pure hemin,the peroxidase-like activity of Hemin@BSA was greatly increased.Meanwhile,the introduction of BSA can also improve the solubility of hemin in water.Using Hemin@BSA as an antibody label,a direct competitive colorimetric immunoassay was established for ultra-sensitive detection of BPA in environmental and food samples.Under optimal conditions,this method exhibited good analytical performance（Limit of detection:2.61μg/L;the recovery:93.02-107.9%;CV:1.6-5.6%）.In contrast to traditional ELISA,the sensitivity of this method is greatly improved,showing potential applications in the analysis of trace organic pollutants.（2）Although the Hemin@BSA has good catalytic performance,it shows poor stability and tolerance under harsh conditions owing to its similar chemical composition to natural enzymes.Thus,using ZIF-8 as the shell,a kind of biomimic nanozyme（Hemin@BSA@ZIF-8）was designed and prepared.The introduction of ZIF-8 was identified as the ideal outer layer for Hemin@BSA to enhance its peroxidase-like activity significantly.Meanwhile,the precise encapsulation of Hemin@BSA in ZIF-8 could also protect it from decomposition in harsh environments.Based on the peroxidase-like activity of Hemin@BSA@ZIF-8,a sensitive colorimetric immunoassay was successfully constructed for the detection of BPA.The LOD of this method was 0.546 ng/m L.The recovery was90.50-107.8%,and the coefficient variation was ranging from 1.866%to 6.891%.This proposed method showed good accuracy and stability for BPA detection in environmental and food samples.2.Development of sensitive fluorescence immunoassay for BPS detectionCompared with colorimetric immunoassay,the fluorescence immunoassay usually exhibits higher sensitivity and wider detection range,which is 1-2 orders as high as traditional ELISA.Based on the enhanced AIE effect of Cu NCs-CS@ZIF-8,a sensitive and high-throughput fluorescence immunoassay was developed for BPS determination in environmental and food samples.In this system,the fluorescence of Cu NCs-CS@ZIF-8 could be quenched by the oxidation of H₂O₂,playing an important role in signal amplification of FELISA.And the catalase was served as a signal label by binding with BPS antibody（CAT@Ab）in FELISA.The AIE fluorescence enhancement of Cu NCs-CS through the conformational locking of ZIF-8 provided an effective strategy to overcome the intrinsic limitations of FELISA including high background,serious fluorescence quenching in the detection of real samples from the environment.Under optimized conditions,the LOD of this method was 0.415 ng/m L.The recovery was 87.25-106.4%and the coefficient variation was ranging from 2.11%to 7.67%.These results indicated that the proposed FELISA could be used for the detection of trace organic pollutants in environmental and food samples.3.Investigation and risk assessment of BPA and BPS in environmental water and food samples from ZhenjiangThe levels of BPA and BPS in 25 environmental samples and 100 food samples from Zhenjiang were analyzed by our proposed colorimetric and fluorescence immunoassay.Then,the ecological risk and human health risk of these two pollutants were comprehensively evaluated using some parameters including risk quotients（RQ）,hazard index（HI）,hazard quotient（HQ）.The results are as follows:（1）The BPA and BPS were widely found in environmental water and food samples from Zhenjiang.The detection frequency of BPA in 25 water samples was76%with an average concentration of 1.225 ng/m L,and the detection frequency of BPS was 68%with an average concentration of 0.6495 ng/m L.The BPA highest concentration was detected the downstream of Yangtze River（S25）.The range of BPA concentrations was:Yangtze River>Beijing-Hangzhou Grand Canal>Yunliang River,corresponding to 1.367,1.326,and 0.6266 ng/m L.For BPS,the average concentration of the Yangtze River was the highest（0.86 ng/m L）,and the level of Yunliang River was the lowest（0.2605 ng/m L）.The detection frequency of BPS samples was ranging:Yangtze River>Beijing-Hangzhou Grand Canal>Yunliang River.For food samples,the detection frequency of BPA was 76%with a concentration range from ND to 17.24 ng/m L.The detection frequency of BPS was55%with an average concentration of ND-12.08 ng/m L.The BPA and BPS in dairy drinks and coffee samples were relatively higher,but they were still lower than the average level of BPs in the same drinks from other reports.（2）Based on the pollution level,the ecological and health risk of BPA and BPS in environmental and food samples were comprehensively evaluated by risk quotients（RQ）,hazard index（HI）,hazard quotient（HQ）.The results indicated that the BPA and BPS in environmental water could not cause direct harm to organisms at different trophic levels in the ecosystem.For human health,the health risk of BPA and BPS exposure in environmental water through drinking water was higher than that of the skin.Meanwhile,the health risk for children was higher than for adults.For BPA/BPS in food,the estimated daily intake（EDI）was lower than the tolerable daily intake（TDI）,and the calculated HQ was less than 1,illustrating the health risk of BPA/BPS exposure would not threaten human health.