【作者】 叶其炎；

【导师】 庄惠生；

【作者基本信息】 东华大学 ， 环境科学与工程， 2010， 博士

【摘要】 持久性有毒污染物（Persistent Toxic Substances,PTS）与臭氧层破坏以及温室效应并称为21世纪影响人类生存与健康的三大环境问题。无论是《斯德哥尔摩公约》中确定的21类持久性有机污染物（Persistent Organic Pollutants,POPs）;还是美国EPA确定的12类持久性生物富集有毒化合物（Persistent Bioaccumulative& Toxic Chemicals,PBT）;以及环境内分泌干扰物（Environmental EndocrineDisruptors,EEDs）的研究都与持久性有毒化学污染物有关。多环芳烃（PAHs）和多氯联苯（PCBs）就是其中两类典型的持久性有毒污染物,它们在环境中分布广泛,但多是痕量的,并且不易分解,具有高脂溶性,水溶性低,易于生物富集,同时不仅具有“三致”毒性（致癌、致畸和致突变）,而且具有内分泌干扰作用。因此对环境生物及人类健康已经形成了不可估量的潜在威胁。加强对PTS的检测分析是进行环境风险评价及对它们实施防控的重要前提。目前,检测PAHs和PCBs常见的方法有气相色谱法（GC）、高效液相色谱法（HPLC）和气质联用技术（GC/MS）。但是这些仪器分析方法存在设备复杂、操作要求尤其是样品前处理要求苛刻、价格昂贵、测定周期长等缺陷,不适合于现场快速检测和大批量分析。因此发展有效的生物分析技术将是未来的发展方向。荧光免疫分析及荧光定量免疫PCR技术由于其高特异性和高灵敏性而无需复杂的前处理过程,是很有发展前景的检测分析方法。随着交叉学科的发展,作为在生物及医学领域广泛应用的这些免疫分析方法,目前已经被引入应用于环境监测分析领域。本论文拟选择荧光免疫分析技术和实时荧光定量免疫PCR技术对具有典型性和代表性的PTS—多氯联苯和多环芳烃,进行分析检测方法上的研究,为PTS的环境毒理研究和环境污染治理提供检测方法上的科学的参考依据。同时荧光标记物的性质是荧光法免疫分析技术的关键,本论文拟引用新型荧光探针—量子点和分子信标作为荧光免疫分析和实时荧光定量免疫PCR实验的荧光标记物质,从而改善传统荧光染料的高本底值,特异性差的特点,并提高灵敏度和准确性。本论文通过合成CdTe量子点和设计出针对pUC18具有特异性的分子信标,建立了基于CdTe量子点的荧光免疫测定技术和基于分子信标探针的实时荧光定量免疫PCR技术对几种多环芳烃和多氯联苯进行分析检测。本论文主要研究内容及结论分述如下:1、量子点CdTe的制备及条件优化:采用巯基乙酸为稳定剂,在96℃下回流4h,就可以在水相中直接合成了水溶性的碲化镉（CdTe）纳米晶,其荧光量子产率最高可达66.92%。通过TEM和XRD衍射图谱对其进行表征,说明了制备的CdTe量子点具备良好的结构特性。实验优化得到了制备量子点的各种最佳条件。CdTe经过表面羧基修饰,具备了生物标记功能,通过分子偶联作用,可链接DNA、多肽以及相关环境持久性有机污染物的抗体或抗原,为CdTe作为标记探针在环境污染物分析中的应用奠定了基础。2、生物素化探针DNA的制备和纯化:探针DNA是以pUC18质粒为模板扩增得到的一段123个碱基对的生物素化DNA。根据DNA扩增试剂盒的说明,在PCR扩增体系中加入各种反应物,包括两条生物素化了的引物。引物序列分别是:上游引物序列从5’到3’是CTGACTCCCCGTCGTGTAGA,下游引物的序列从5’到3’是GCTGGCTGGTTTATTGCTGAT。PCR扩增的程序是:94℃预变性4 min;30个循环:每个循环94℃变性20s,55℃退火20s,72℃延伸20s;最后再72℃保持3min使延伸完全。使用UNIQ-10 PCR纯化试剂盒,对PCR扩增产物进行纯化后,再用琼脂糖凝胶电泳后染色进行定性,并用紫外分光光度法定量检测分析。3、分子信标的设计与合成:依据分子信标探针设计原则,设计合成了针对pUC18 DNA具有特异性的分子信标探针,其序列为:5’-FAM-CAGCGATCTGGCCCCAGTGCTGCAATCGCTG-DABCYL-3’。通过特异性考察实验,结果表明该分子信标探针能特异识别反应体系中的DNA,为避免PCR体系的非特异扩增,降低本底值,提高灵敏度和准确性奠定了良好基础。4、直接竞争荧光免疫分析:以CdTe作为荧光标记物,在优化标记条件基础上,成功标记了抗萘、抗荧蒽和抗PCB12抗体,制成了相关的荧光免疫标记试剂。将这些标记试剂运用于对应污染物的荧光免疫检测分析（FIA）。通过考察包被介质、包被时间、离子强度、pH值、表面活性剂、抗原抗体最佳工作浓度等影响因素,得到了直接竞争FIA的优化条件,由此建立的三种污染物的直接竞争FIA抑制率标准曲线,得到线性范围为10^-2-10³ng/mL,检测萘、荧蒽和PCB12得到的检出限IC20分别为7.384pg/mL、9.046pg/mL和8.186pg/mL。该方法成功应用于环境样品的检测,并考察了方法的特异性、回收率、精密度,结果较满意。5、抗体包被荧光免疫分析:以CdTe作为荧光标记物,在优化标记条件基础上,成功标记了萘、荧蒽和PCB12完全抗原,制成了相关的荧光免疫标记试剂。将这些标记试剂运用于对应污染物的荧光免疫检测分析（FIA）。通过考察包被介质、包被时间、离子强度、pH值、表面活性剂、抗原抗体最佳工作浓度等影响因素,得到了抗体包被FIA的优化条件,由此建立的三种污染物的抗体包被FIA抑制率标准曲线,得到线性范围为10^-2-10³ ng/mL,检测萘、荧蒽和PCB12得到的IC20分别为13.95 pg/mL,15.94 pg/mL和12.94 pg/mL。该方法成功应用于环境样品的检测,并考察了方法的特异性、回收率、精密度,结果较为满意。6、直接竞争实时荧光定量免疫PCR:以分子信标作为荧光探针,运用直接竞争实时荧光定量免疫聚合酶链式反应（直接竞争RTFQ-IPCR）分析方法检测环境中多环芳烃类污染物—萘、蒽和荧蒽。用制备好的包被原包被经过戊二醛处理过的PCR管,然后用PAHs与包被原竞争结合生物素化抗体,再利用亲和素连接生物素化抗体和生物素化DNA,最后通过实时定量PCR仪,在优化的扩增程序下扩增DNA并实现实时定量检测。论文建立了直接竞争RTFQ-IPCR方法的标准曲线,对萘、蒽和荧蒽三种污染物进行检测,得到线性范围分别为1-10⁴fg/mL、10-10⁵ fg/mL和10-10⁷ fg/mL,相关性较好,检出限分别为0.81、5.94、3.84 fg/mL。该方法应用于实际环境样品的分析,并考察了方法的特异性、回收率,结果令人满意。实际样品分析结果与ELISA测定结果进行比较,具有较好的一致性,表明所建立的方法对环境持久性有毒污染物的检测分析是可行的有效的。7、间接竞争实时荧光定量免疫PCR:以分子信标作为荧光探针,运用间接竞争实时荧光定量免疫聚合酶链式反应（间接竞争RTFQ-IPCR）分析方法检测环境中多环芳烃类污染物—菲。用制备好的包被原包被经过戊二醛处理的PCR管,然后用待测物菲与包被原竞争结合一抗,接着利用生物素化二抗与结合在包被原上的特异性一抗结合,再利用亲和素连接生物素化抗体和生物素化DNA,最后通过实时定量PCR仪,在优化的扩增程序下扩增DNA并实现实时定量检测。论文建立了间接竞争RTFQ-IPCR分析方法标准曲线,对污染物菲进行检测,得到线性范围为10-10⁶ fg/mL,检出限为5.34 fg/mL,相关系数为0.9747。应用于实际环境样品的分析,并考察了方法的特异性及样品回收率,结果满意。实际样品检测结果与HPLC测定结果比较发现两者相差不大,在误差范围内是可以接受的。8、抗体包被实时荧光定量免疫PCR:以分子信标作为荧光探针,运用固相抗体包被实时荧光定量免疫聚合酶链式反应（抗体包被RTFQ-IPCR）分析方法检测环境中多氯联苯类污染物—PCB77。用制备好的包被抗体包被经过戊二醛处理的PCR管,然后用待测污染物PCB77小分子和生物素化半抗原竞争结合包被抗体,再利用亲和素连接生物素化半抗原和生物素化的DNA,最后通过实时定量PCR仪,在优化的扩增程序下扩增DNA并实现实时定量检测。论文建立了抗体包被RTFQ-IPCR分析方法标准曲线,对污染物PCB77进行检测,得到线性范围为10-10⁵ fg/mL,检出限为6.63 fg/mL,相关系数为0.9972。应用于实际环境样品的分析,并考察了方法的特异性及样品回收率,结果令人满意。实际样品检测结果与GC/MS测定结果比较发现两者相关性较好,在误差范围内存在的差异是可以接受的。本论文首次成功的将CdTe量子点和分子信标探针分别作为荧光免疫分析方法和实时荧光定量免疫PCR方法的荧光标记探针对多环芳烃和多氯联苯污染物进行了检测分析,结果满意可靠,为环境中痕量的持久性有毒污染物的检测分析,提供了新的研究思路。更多还原

【Abstract】 Persistent Toxic Substances（PTS） which affecting human survival and health,are known as the three major environmental problems in 21st century,as well as the greenhouse effect and ozone depletion.Whether the 21 categories of Persistent Organic Pollutants（POPs） identified in "the Stockholm Convention",the 12 categories of Persistent Bioaccumulative & Toxic Chemicals（PBT） identified by U.S.EPA,or the study of Environmental Endocrine Disruptors（EEDs）,they are closely related with PTS.Polycyclic aromatic hydrocarbons（PAHs） and polychlorinated biphenyls（PCBs） are two types of typical persistent toxic substances.They are widely distributed in the environment,but mostly trace,and difficult to break down,with a high fat-soluble,water-insoluble,and easy to bioaccumulation.Moreover,they are not only carcinogenic,teratogenic and mutagenic,but also has the role of endocrine disruptors.Therefore they have an invaluable potential threat for biology and human health.The monitoring and analysis of PTS are the base and the important prerequisite of carrying out environmental risk assessment and implementation of prevention and control.At present,the most common methods used for analysis of PAHs and PCBs are gas chromatography（GC）,high performance liquid chromatography（HPLC） and gas chromatography-mass spectrometry （GC/MS）.However,these are known to manifest underlying disadvantages,such as the complexity of operating requirements for equipment,sample pre-treatment demanding,and they are also known to be time-consuming and expensive.Moreover,they are deemed unsuitable for detecting very low quantities of pollutants in the environment.Especially,they are not suitable for on-site rapid testing and large quantities of samples.Therefore,the development of effective bio-analysis techniques will be the direction of development in the future.Fluorescence immunoassay and fluorescence quantitative immuno-PCR technology are two very promising detection methods for its high immune specificity and high sensitivity without the need for complicated pre-treatment process.These immunoassay methods,which were known in the biomedical fields,are currently being used in the environmental monitoring field.This paper intends to select fluorescence immunoassay and real-time fluorescence quantitative immuno-PCR technique to detect the typical and representative pollutants of the PTS—PCBs and PAHs,and providing a scientific frame of reference for the study of environmental toxicology and governance in PTS.As we known,the nature of the fluorescent marker is important to fluorescent immune analysis technique.So the new type of fluorescent probe—quantum dots and molecular beacons will be used as fluorescent marker substances in fluorescence immunoassay and real-time fluorescence quantitative immuno-PCR experiments,so as to lower the background values,and improve the specificity,sensitivity and accuracy. In this paper,CdTe-based fluorescence immunoassay and MB-based real-time fluorescence quantitative immuno-PCR technique were established to detect PAHs and PCBs through the synthesis of CdTe quantum dot and designing of a specific molecular beacon for the pUC18 DNA. The main content and conclusions of this thesis are as follows:1,Preparation of CdTe quantum dot and optimization of conditions:The red water-soluble cadmium telluride（CdTe） was synthesized in four refluxing hours at 96℃in water phase with mercapto-acetate acid used as stabilizing agent.The fluorescence quantum yield is 66.92%.The characters of TEM and XRD diffraction patterns demonstrated good structural properties of CdTe. Various optimal conditions were obtained for preparation of CdTe in experiment.With the modification of carboxyl group to CdTe surface,the CdTe quantum dot has the function of biological tagging,and they are able to link to DNA,peptides and antibodies or antigen of related environmental persistent organic pollutants with the role of molecular coupling.And which laid the foundation for CdTe acting as a labeled probe in the analysis of environmental pollutants.2,Preparation and purification of biotinylated reporter DNA:The reporter DNA with biotin is a 123-base pair sequence from the pUC18 plasmid.The reporter DNA was generated by PCR as follows.The forward primer,M1341/pUC forward sequencing as CTGACTCCCCGTCGTGTAGA,was biotinylated at the 5’ end to generate a biotiny group to the DNA.The reverse primer,M1342/pUC reverse sequencing as GCTGGCTGGTTTATTGCTGAT was also biotinylated at the 5’ end to generate a biotiny group to the DNA.All regents were added to PCR tube as described by the DNA PCR kit handbook.The PCR conditions were:hold 94℃for 4 min;30 cycles of 94℃for 20 s,55℃for 20 s,and 72℃for 20 s;The 72℃step was extended to 3 min in the final cycle.The reporter DNA was purified and retrieved by UNIQ-10 PCR DNA Extraction Kit.The DNA was quantified by UV absorbency and checked qualitatively using agarose gel.3,Design and synthesis of molecular beacon:Based on the design principles of molecular beacon probes,we designed and synthesized a molecular beacon probe,which has specificity for pUC18 DNA.The sequence is: 5’-FAM-CAGCGATCTGGCCCCAGTGCTGCAATCGCTG-DABCYL-3 ’.After investigating the specificity of the probe,the result showed that the molecular beacon could be specifically identified the complementary DNA during PCR.The specificity of the molecular beacon probe would be avoiding non-specific amplification throughout PCR cycle,reducing the background value,and obtaining greater sensitivity and accuracy.4,Direct competition fluorescence immunoassay:With the optimum conditions of the labeling and the CdTe acting as a fluorescent marker,we coupled the antibodies of naphthalene, fluoranthene,and PCB12 with CdTe successfully,And obtained the relevant fluorescent labeling reagent.These reagents were used in fluorescence immunoassay（FIA） for analysis of the related pollutants.After examining and optimizing FIA condition of influence factors,such as coating medium,coating time,ionic strength,pH,surface-active agent and antigen-antibody optimal working concentration,we established thereby the standard curve of the inhibitory rate of three kinds of pollutants in direct competition FIA,with the linear range of 10²-10³ ng/mL,and IC20 of naphthalene,fluoranthene,and PCB12 were 7.384pg/mL,9.046 pg / mL and 8.186 pg / mL, respectively.After examining the specificity,recovery and precision in the detection of environmental samples,we obtained satisfactory results.5,Antibody-coated fluorescence immunoassay:With the optimum conditions of the labeling and CdTe acting as a fluorescence marker,we coupled the antibodies of naphthalene,fluoranthene, and PCB12 with CdTe successfully,and obtained the relevant fluorescence labeling reagents. These reagents were used in fluorescence immunoassay（FIA） for analysis of the related pollutants. After examining and optimizing FIA conditions of influence factors,including the coating medium, coating time,ionic strength,pH,surface-active agent and antigen-antibody optimal working concentration,we established thereby the standard curve of the inhibitory rate of three kinds of pollutants in antibody-coated FIA,with the linear range of 10^-2-10³ ng/mL,and IC20 of naphthalene,fluoranthene,and PCB12 were 13.95 pg/mL,15.94 pg / mL and 12.94 pg / mL, respectively.After examining the specificity,recovery and precision in the detection of environmental samples,we obtained satisfactory results.6,Direct competition real-time fluorescence quantitative immuno-PCR:We detected polycyclic aromatic hydrocarbons-naphthalene,anthracene and fluoranthene in the environment by real-time fluorescence quantitative immuno-polymerase chain reaction（direct competition RTFQ-IPCR） using a molecular beacon probe.Coating antigens adsorbed to the PCR tubes treated with glutaraldehyde,is used to competing with the PAHs in combining biotinylated antibodies.Avidin is used as a bridge between the biotinylated antibodies and the biotinylated reporter DNA.The reporter DNA is amplified and measured by RTFQ-PCR under the optimum procedure.Standard curves of the three PAHsnaphthalene, anthracene and fluoranthene were produced.And the linear range of 1-10⁴ fg/mL, 10-10⁵ fg/mL,and 10-10⁷ fg/mL were obtained,with limits of detection of 0.81,5.94,and 3.84 fg/mL,respectively,and good correlation.Some environmental samples were analyzed with satisfactory results,including specificity and recovery rate.Actual results were compared with the ELISA determination,with good consistency,indicating that the method for the detection of environmental persistent toxic pollutants is feasible and effective.7,Indirect competition real-time fluorescence quantitative immuno-PCR:We detected polycyclic aromatic hydrocarbons)—phenanthrene in the environment by real-time fluorescence quantitative immuno-polymerase chain reaction（indirect competition RTFQ-IPCR） using a molecular beacon probe.Coating antigens adsorbed to the PCR tubes treated with glutaraldehyde,is used to competing with the phenanthrene in combining antibodies （primary antibody）.Then the biotinylated goat anti-rabbit IgG（second antibody） were added to combine with the primary antibody,Avidin is used as a bridge between the biotinylated goat anti-rabbit IgG and the biotinylated reporter DNA.The reporter DNA is amplified and measured by RTFQ-PCR under the optimal procedure. Standard curves of phenanthrene were produced.And the linear range of 10-10⁶ fg/mL was obtained,with detection limit of 5.34 fg/mL and correlation coefficient of 0.9747.Some environmental samples were analyzed with satisfactory results,including specificity and recovery rate.The results tested by indirect competition RTFQ-IPCR were compared with that from HPLC determination,indicating good consistency.And the differences are acceptable within the margin of error.8,Antibody-coated real-time fluorescence quantitative immuno-PCR:Using a molecular beacon as fluorescence probe,we determined polychlorinated biphenyl pollutants-PCB77 in the environmental by antibody-coated real-time fluorescence quantitative immuno polymerase chain reaction（antibody-coated RTFQ-IPCR）.Coating antibody adsorbed to the PCR tubes treated with glutaraldehyde,is used to competing with the PCB77 in combining biotinylated hapten.Avidin is used as a bridge between the biotinylated hapten and the biotinylated reporter DNA.The reporter DNA is amplified and measured by RTFQ-PCR under the optimized procedure.Standard curves of PCB77 were produced. And the linear range of 10-10⁵ fg/mL was obtained,with detection limit of 6.63 fg/mL and correlation coefficient of 0.9972.The antibody-coated RTFQ-IPCR was used in analysis of real environmental samples,and we obtained satisfactory results after examining the specificity, sample recovery.The results detected by RTFQ-IPCR were compared with that from GC/MS, showing a good correlation,and the differences between tow results are acceptable within the margin of error.This paper is the first time report on detecting successfully of polycyclie aromatic hydrocarbons and polychlorinated biphenyls by fluorescence immunoassay and real-time fluorescence quantitative immuno-PCR with CdTe quantum dot and molecular beacon probe acting as fluorescence-labeled probe,respectively.The results are satisfactory and reliable.And these methods provide a new research idea for detection and analysis of trace persistent toxic substances in the environment.更多还原