Researches On Developing Novel Immunoassays For The Determination Of Trace Environmental Endocrine Disruptors – Phthalic Acid Esters

Phthalic acid diesters（PAEs）are a group of compounds that are widely used as additives and plasticizers in various commercial and personal care products to improve flexibility,long durability and general handling properties.However,PAEs are ubiquitous endocrine disrupting chemicals（EDCs）that can cause birth defects and sexual dysfunction,even cancers and possibly heart disease in living species.So far,several analysis methods,such as chromatography,chromatography-mass spectrometry,spectrophotometry,etc are used to detect PAEs in all kinds of samples.Although the above instrument analysis methods are accurate,the instruments need to take much more time and higher cost to complete purification and pre-concentration of PAEs in these samples because of the requirement of higher purity and the relatively high LOD,and requiring more skill to operate.What’s more,the above methods could not achieve rapid and high-throughput field detection of batch samples.Compared with instrumental methods,immunoassay has some advantages,such as its ease-of-operation,cost-efficient,higher sensitivity,highly specificity.Hence,how to establish highly efficient,highly sensitive and high-throughput immunoassay for detecting trace PAEs has very important theory significance and extremely broad application prospect.The main research results and conclusions of these work are summarized as follows:（1）In this research,dimethyl phthalate（DMP）,diethyl phthalate（DEP）,dibutyl phthalate（DBP）and di 2-ethyl hexyl phthalate（DEHP）which get more public attention in these most commonly used PAEs,are selected as the research objects.Optimal haptens of DMP,DEP,DBP and DEHP are synthesized and characterized.In order to reduce the specific binding caused by linking arm,the artificial DMP,DEP,DBP and DEHP hapten,antigens（including immunogen and coating antigen）are prepared by conjugating hapten to carrier proteins through different coupling methods（i.e.the diazotization method and the glutaraldehyde method）.After the immunization of male New Zealand white rabbits through subcutaneous and intramuscular injections with the immunogen,the rabbit polyclonal anti-DMP antibodies（pAb-DMP）,the rabbit polyclonal anti-DEP antibodies（pAb-DEP）,the rabbit polyclonal anti-DBP antibodies（pAb-DBP）and the rabbit polyclonal anti-DEHP antibodies（pAb-DEHP）are obtained from separating rabbit serum.After purification through ammonium sulfate precipitation method,the titers of pAb-DMP,pAb-DEP,pAb-DBP and pAb-DEHP are both higher than 110,000.Moreover,the affinity constant of pAb-DMP,pAb-DEP,pAb-DBP and pAb-DEHP are 4.17×10⁷L/mol,5.89×10⁷ L/mol,3.85×10⁷ L/mol and 3.72×10⁷ L/mol,respectively.Otherwise,these polyclonal antibody have a low CRs（below 10%）between analyte and other structurally similar compounds,which indicates that these polyclonal antibody have highly specificity.（2）Biotinylated antibodies are prepared based on the obtained antibodies and biotin–n-hydroxysuccinimide esters（BNHS）,and then the indirect competitive biotin-streptavidin enzyme-linked immunosorbent assay（BA-ELISA）is established for detecting DMP,DEP,DBP and DEHP.Several physicochemical factors that influenced the performance of the BA-ELISA assay are investigated and optimized.Under optimal conditions,a wide linear range for detecting DMP is from 0.024 to 6.027μg/L;the median inhibitory concentration(IC₅₀)and the limit of detection(IC₁₀)are 0.356μg/L and 0.0082μg/L,respectively.For detecting DEP,the linear range is from 0.021 to 9.512μg/L;the IC₅₀ and IC₁₀0 are 0.443μg/L and 0.0079μg/L,respectively.For detecting DBP,the linear range is from 0.015 to 8.895μg/L;the IC₅₀ and IC₁₀0 are 0.361μg/L and 0.0053μg/L,respectively.For detecting DEHP,the linear range is from 0.021 to 12.948μg/L;the IC₅₀ and IC₁₀0 are 0.526μg/L and 0.0074μg/L,respectively.Furthermore,the sensitivity of these proposed BA-ELISA is raised 5-8 times than conventional ELISA.These established indirect competitive BA-ELISA assays are used to detect DMP,DEP,DBP and DEHP in foodstuff,water and sea sediment samples,and the results are consistent with those using gas chromatography-mass spectrometry（GC-MS）.Otherwise,the recoveries（88.1-111%）and coefficient of variation（CV）（below 15%）of these indirect competitive BA-ELISA assays are acceptable.（3）Based on biotinylated antibodies,biotinylated DNA（Bio-DNA）and the biotin-streptavidin binding system,the direct competitive biotin-streptavidin-amplified real-time immune-PCR assay（BA-rt-IPCR）is constructed for detecting DMP,DEP,DBP and DEHP.The detection sensitivity of this assay is enhanced greatly because of the amplification of a 103 base-pair DNA.After several factors are discussed and optimized,the standard curve is determined by plotting the average Ct values against the logarithm of the standard concentrations about known analyte.For detecting DMP,the linear range is from 10 pg/L to 100 ng/L with the linear regression coefficient of R²=0.9846,and the LOD is 1.98 pg/L.For detecting DEP,the linear range is from 10 pg/L to 100 ng/L with the linear regression coefficient of R²=0.9716,and the LOD is 4.85 pg/L.For detecting DBP,the linear range is from 10 pg/L to 50 ng/L with the linear regression coefficient of R²=0.9650,and the LOD is 2.27 pg/L.For detecting DEHP,the linear range is from 10pg/L to 50 ng/L with the linear regression coefficient of R²=0.9751,and the LOD is 2.05pg/L.Moreover,the limit of detection（LOD）of the proposed BA-rt-IPCR assay is reduced about 1500-4000 times than the BA-ELISA assay.The proposed direct competitive BA-rt-IPCR assay are implemented to detect DMP,DEP,DBP and DEHP in foodstuff,water and seabed sediment samples,and these BA-rt-IPCR results are consistent with those using GC-MS.Otherwise,the acceptable recovery rates（89.4-110%）and CVs（4.87-13.2%）are obtained.（4）All types of polyclonal antibody（including pAb-DMP,pAb-DEP,pAb-DBP and pAb-DEHP）are labeled on the surface of gold nanoparticles（GNPs）respectively,and then sulfydryl modified DNA are conjugated to the surface of GNPs to form Barcode DNA-GNPs-pAb complexes,i.e.bio-probes.Based on these bio-probes,the direct competitive gold nanoparticles improved real time immuno-PCR assay（GNP-rt-IPCR）are established for the detection of DMP,DEP,DBP and DEHP.Compared with the direct competitive BA-rt-iPCR assay,the major feature of the direct competitive GNP-rt-IPCR assay is the large combining ratio of of 1:N between antibody and DNA,which means the enhancement of sensitivity.After several factors are optimized,the standard curve is determined by plotting the average Ct values against the logarithm of the standard concentrations about analyte.For detecting DMP,the linear range is from 5 pg/L-50,000pg/L with the linear regression coefficient of R²=0.9807,and the LOD is 1.38 pg/L.For detecting DEP,the linear range is from 4 pg/L-40,000 pg/L with the linear regression coefficient of R²=0.9756,and the LOD is 1.06 pg/L.For detecting DBP,the linear range is from 5 pg/L-5,000 pg/L with the linear regression coefficient of R²=0.9709,and the LOD is 1.23 pg/L.For detecting DEHP,the linear range is from 6 pg/L-6,000 pg/L with the linear regression coefficient of R²=0.9662,and the LOD is 1.57 pg/L.These proposed direct competitive GNP-rt-IPCR assays are implemented to detect the trace amout of DMP,DEP,DBP and DEHP in foodstuff,water and seabed sediment samples,and these GNP-rt-IPCR results are consistent with those using GC-MS.Otherwise,the acceptable recovery rates（89.8-109%for DMP,88.8-111%for DEP,90.1-109%for DBP,87.9-109%for DEHP,respectively）and coefficients of variation（below 15%）are obtained.What’s more,the LOD of the proposed GNP-rt-IPCR assay is reduced about0.5-4 times than the BA-rt-IPCR assay.Meanwhile,the proposed direct competitive GNP-rt-IPCR assays could avoid the tedious and time-consuming sample purification and pre-concentration steps because of its lower LOD and higher specificity,and achieve high-throughput and batch mode analysis because of 96-well PCR tube used,thus the required time for monitoring large sums of samples reduces greatly.This research take only 4.5 h to complete the analysis of analyte in 15 samples by GNP-rt-IPCR actually while GC-MS spend 75 h to complete the analysis of analyte in 15 samples（50 min per test,6 repeat tests each samples）.These above results further confirm that these direct competitive GNP-rt-IPCR assays could achieve high-throughput and rapid detection of trace DMP,DEP,DBP and DEHP in the foodstuff,water and sea sediment samples with ultra-sensitivity and specificity.To summarize,the established indirect competitive BA-ELISA assay,direct competitive BA-rt-IPCR assay and direct competitive GNP-rt-IPCR assay for the determination of PAEs in practical samples have the advantage of high efficiency,high sensitivity,high specificity,convenient operation,high stability and reproducibility,etc.Using the above three kinds of immunoassays to detect PAEs in practical samples,these immunoassays show good correlation with GC-MS.Therefore,they would be a useful option for batch detection of PAEs with rapid and high throughout screening in all kinds of samples,and have good application prospect in real-time monitoring the residual level of PAEs quickly.