【作者】 周纯；

【导师】 庄惠生；

【作者基本信息】 东华大学 ， 环境工程， 2008， 博士

【摘要】 多环芳烃(PAHs)是指含有2个或2个以上苯环的碳氢化合物的统称。煤、石油、天然气、有机高分子化合物和许多碳氢化合物等在不完全燃烧后都能生成PAHs。诸如,取暖、化石燃料燃烧、公路交通、餐饮业,甚至是吸烟等人类活动都是PAHs的污染源。多环芳烃类物质数量多,分布广,对人类危害极大,有多种PAHs已被鉴定出具有致癌性,这使得近年来对于PAHs的检测研究成为一个新的热点。美国环保局将16种PAHs列为优先控制污染物并作为环境监测必检项目,本论文研究的四种PAHs均在此列。因此,非常有必要对上述的PAHs建立灵敏可靠的分析方法。目前,检测PAHs最常见的方法是气相色谱法(GC)和高效液相色谱法(HPLC)。但是这些方法复杂且耗时的前处理过程使得它们不适合于现场快速检测。而免疫PCR(IPCR)技术由于其特异性和灵敏性而无需复杂的前处理过程,因此是一种非常适用于现场快速检测的分析方法。免疫PCR是1992年由Sano首次提出的,它将体系完善的ELISA分析方法和具有强大信号放大能力的PCR体系结合起来。大量的应用研究表明,该方法与ELISA相比不仅具有很好的一致性而且灵敏度更高,线性范围更广,具有更强的检测能力。免疫PCR方法建立以来,已经在癌症、自身免疫缺陷、致病菌和细菌毒性等方向得到广泛应用,但是还没有应用在环境小分子污染物检测方面的报道。免疫PCR在技术和信号检测仪器上的进一步发展导致了实时定量免疫PCR技术(RT-IPCR)的产生。实时定量免疫PCR方法尽管建立的时间不长,但是随着它应用的推广,已经证明该方法比一般的免疫PCR具有更高的精确度,与一般免疫PCR的15-20%的实验室内部误差相比,该技术的内部误差降到了5-10%。至今,只有少量PAHs的有制备单克隆或多克隆抗体的文献报道,而本论文研究的四种PAHs的抗体制备均未见报道,更未见有文献报道应用实时定量免疫PCR分析方法来检测环境中PAHs。本论文建立了三种RT-IPCR分析方法来检测环境中痕量的PAHg。本课题主要研究内容及结论如下:(1)免疫原的制备:PAHs是小分子物质,不能直接免疫动物产生抗体,必需偶联大分子载体;且结构上不具备可与载体偶联的基团,因此本论文先通过Friedel-Crafts酰基化反应在蒽、菲和荧蒽分子上引入羧基,得到PAHs-丁酸-γ-氧作为半抗原应用于接下来的实验。萘则直接购买萘氧乙酸作为半抗原。接着用活化酯法分别将这些半抗原与牛血清蛋白(BSA)偶联,制得PAHs的免疫原;用混合酸酐法将半抗原分别与卵清蛋白(OVA)偶联,制得PAHs的包被原。产物的结构和组成经过红外光谱、紫外光谱、质谱和核磁共振鉴定分析。(2)抗体的制备:将免疫原与适当的佐剂乳化后,免疫新西兰大白兔。初次免疫后进行加强免疫,在第三次加强免疫之后,以试管凝集试验和琼脂双向免疫扩散实验测定从兔耳缘采的的抗血清的效价。试管凝集试验中出现了凝集反应,琼脂双向免疫扩散试验中也出现了明显的沉淀线。将兔子体内获得的抗血清用辛酸-硫酸铵二步沉淀法、葡聚糖凝胶除盐及DEAE-纤维素精制提纯,得到纯化的四种PAHs多克隆抗体IgG。琼脂双向扩散实验表明萘、蒽、菲和荧蒽的抗体效价分别为:1∶32,1∶32,1∶64,1∶32。(3)生物素化探针DNA的制备和纯化:探针DNA是以pUC19质粒为模板扩增得到的一段103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G TAA AAC GAC GGC CAG T,下游引物的序列从5’到3’是CAG GAA ACA GCT ATG AC。PCR扩增的程序是:94℃预变性4 min。然后是30个循环反应,每个循环的程序是94℃变性20s,55℃退火20s,72℃延伸20s。最后再72℃保持3 min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。(4)生物素化抗体的制备:按照文献报道的方法,将自制的四种PAHs的特异性抗体与活化的生物素(BNHS)进行结合反应,产物经过柱纯化后,得到四种PAHs的特异性生物素化抗体。(5)生物素化半抗原的制备:按照文献报道的方法,将自制的四种PAHs半抗原与活化生物素(BNHS)进行合成反应后,产物经过柱纯化即得到四种PAHs的生物素化半抗原。(6)直接竞争RT-IPCR:本论文首次建立了直接竞争实时定量免疫聚合酶链式反应(直接竞争RT-IPCR)分析方法,用于检测环境中四种PAHs。用制备的包被原包被经过戊二醛处理的PCR小管,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的103bp探针DNA,最后通过实时定量PCR仪,在优化的扩增程序下扩增DNA并实时检测。论文建立了直接竞争RT-IPCR分析方法的标准直线,考察了方法的特异性、回收率、检测限并应用于实际环境样品的检测,结果令人满意。(7)间接竞争RT-IPCR:本论文首次建立了间接竞争实时定量免疫聚合酶链式反应(间接竞争RT-IPCR)分析方法,用于检测环境中四种PAHs。用制备的包被原包被经过戊二醛处理的PCR小管,接着用PAHs与包被原竞争结合特异性抗体,再利用生物素化二抗与结合在包被原上的特异性抗体结合,然后通过亲和素与制备的生物素化的103bp探针DNA结合,最后通过实时定量PCR仪在优化的扩增程序下扩增DNA并实时检测。论文建立了间接竞争RT-IPCR分析方法的标准直线,考察了方法的特异性、回收率、检测限并应用于实际环境样品的检测,结果令人满意。(8)抗体包被RT-IPCR:本论文首次建立了抗体包被实时定量免疫聚合酶链式反应(抗体包被RT-IPCR)分析方法,用于检测环境中四种PAHs。用制备的特异性抗体包被经过戊二醛处理的PCR小管,然后用生物素化半抗原和PAHs竞争与包被抗体结合,再利用亲和素连接生物素化半抗原和生物素化的103bp探针DNA,最后通过实时定量PCR仪在优化的扩增程序下扩增DNA并实时检测。论文建立了抗体包被RT-IPCR分析方法的标准直线,考察了方法的特异性、回收率、检测限并应用于实际环境样品的检测,结果令人满意。(9)高效液相色谱法的优化及与四种方法的比较:在参考国家标准方法高效液相色谱法(HPLC)的基础上,为本论文的研究对象——四种PAHs专门优化了它们的HPLC分离条件。论文建立了该方法的标准直线,考察了方法的检测限并应用于实际环境样品的检测,结果令人满意。并将该方法的各项指标与本论文首次建立的三种RT-IPCR分析方法进行比较,就其优缺点进行了讨论。更多还原

【Abstract】 Polycyclic aromatic hydrocarbons(PAHs) are a large group of organic compounds with two or more fused aromatic rings.PAHs are formed mainly as a result of pyrolytic processes,especially the incomplete combustion of organic materials during industrial and other human activities,such as processing of coal and crude oil,combustion of natural gas,including for heating,combustion of refuse, vehicle traffic,cooking and tobacco smoking,as well as in natural processes such as carbonization.Due to the widespread distribution of PAHs in the environment and the carcinogenic and mutagenic nature of PAHs,there has been increased concern in their detection and monitoring in recently years.The U.S.Environmental Protection Agency(EPA) has identified 16 unsubstituted PAHs as priority pollutants,which are monitored routinely for regulatory purposes.Four PAHs studied in the paper are all in the list.So,sensitive and reliable analytical methods are needed to evaluate the presence of PAHs at very low concentration in environmental sample,especially these mentioned above.At present,the most common methods used for analysis of PAH are gas chromatography(GC) and high-performance liquid chromatography (HPLC).But these assays require preconcentration of analyte and are relatively time consuming and difficult to perform on-site.In contrast,immunoassays are typically very sensitive and readily adapted to analytes for which an appropriate antibody is available.The IPCR method,first described by Sano et al.in 1992,combines the well-established ELISA methodology with the signal amplification power of the PCR.A number of research applications describe the advantages of the method,that is,in particular,its high sensitivity and good quantification capabilities due to the great linearity and compatibility with established ELISA protocols.The detection was shown to be 100 to 1000 fold more sensitive than conventional Enzyme-linked immunosorbent assay(ELISA).Since its development,the method has been applied to detect antigens associated with cancer,autoimmune diseases,pathogenic bacteria and bacterial toxin.But no environmental pollutants have been detected by IPCR.Further development in the technology and instrumentation used for the signal detection of IPCR has resulted in the development of Real-Time IPCR(RT-IPCR). However,increased use of RT-IPCR has shown that the method displays improved statistical validation of accuracy over IPCR.Inter-assay error is typically 5-10%vs 15-20%for IPCR.To date,only a few mono-or polyclonal antibody-based immunoassays for PAHs have been reported.And no mono-or polyclonal antibody-based immunoassays for PAHs have been reported.While no paper have reported about the determination of PAHs using RT-IPCR.In this paper,three methods were developed for the determination of trace PAHs in the environment by RT-IPCR assay.The work is summarized as follows:(1) Synthesis of artificial antigens:PAHs are small molecules,which can not be directly used for rabbit immunization.They have to combine with carriers.Haptens should be synthesized and conjugated to protein for rabbit immunization.A carboxyl was inducted to the PAHs(Anthracene,Phenanthrene,Fluoranthene,) molecule by Friedel-Crafts acylation reaction and PAHs butanoic acid,y-oxo-was obtained as the hapten.As to Naphthalene,2-naphthoxy acetic acid was bought as the hapten.The haptens were conjugated to the carrier proteins(BSA,OVA) with the modified active ester method or with the mixed acid anhydride method to form immune antigens and coating antigens.In this way the artificial antigen of PAHs were obtained.Structures of the products were characterized by IR,UV spectra,Mass spectra and 1H NMR.(2) Preparation of polyclonal antibodies:Male New Zealand white rabbits were immunized with the mixture of immune antigens and Freund adjuvant.The immune response consisted of an initial primary response followed by a secondary immune response.After the third immune response,the rabbits were bled from the ear vein and the sera were tested for titer by the agar diffusion test and the tube agglutination reaction test.In tube agglutination reaction test,there were visible precipitation reaction;in agar double-diffusion experiment,there were clear deposit lines.The antiserum from the rabbit was purified by the method of octanoic acid ammonium sulfate two-step precipitation, Sephadex G-25 and DEAE cellulose.In this way the specific and affintive pAbs of PAHs have been yielded.(3) Preparation and purification of biotinylated reporter DNA:The reporter DNA with biotin was a 103-base pair sequence from the pUC19 Vectors.The reporter DNA was generated by PCR as follows.The forward primer, M13/pUC sequencing as G TAA AAC GAC GGC CAG T,was biotinylated at the 5’ end to generate a biotiny group to the DNA.The reverse primer,M13/pUC reverse sequencing as CAG GAA ACA GCT ATG AC 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 20s.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.(4) Preparation of biotinylated polyclonal antibodies:The specific and affmitive pAbs yielded in our own laboratory were biotinylated using biotinamido-caproate-N-hydroxysuccinimide ester(BNHS) as reported.The biotinylated antibody was then purified on a PD-10 gel filtration column(Amersham Biosciences).(5) Preparation of biotinylated PAHs hapten:Haptens synthesized by Friedel-Crafts acylation reaction from four PAHs were biotinylated using biotinamido-caproate-N-hydroxysuccinimide ester(BNHS) as reported.The biotinylated haptens were then purified on a PD-10 gel filtration column(Amersham Biosciences).(6) Direct competitive Real-time Immuno-PCR(RT-IPCR) assay:Direct competitive Real-time Immuno-PCR(RT-IPCR) assay for the determination of four PAHs in environment was first developed.Coating antigens adsorbed to the PCR tubes treated with glutaraldehyde,is used to competing with the PAHs in combining biotinylated pAbs.Avidin is used as a bridge between the biotinylated pAbs and the biotinylated 103 bp reporter DNA.The reporter DNA is amplified and measured by RT-PCR under the optimize procedure.Standard curves of the four PAHs were produced.Cross-reactivity,recovery rates and detection limit were observed.Some environmental samples were analyzed with satisfactory results.(7) Indirect competitive Real-time Immuno-PCR(RT-IPCR) assay:Indirect competitive Real-time Immuno-PCR(RT-IPCR) assay for the determination of four PAHs in environment was first developed.Coating antigens adsorbed to the PCR tubes treated with glutaraldehyde,is used to competing with the PAHs in combining pAbs.Biotinylated goat anti-rabbit IgG were added in combining pAbs.Avidin is used as a bridge between the biotinylated goat anti-rabbit IgG and the biotinylated 103 bp reporter DNA.The reporter DNA is amplified and measured by RT-PCR under the optimize procedure.Standard curves of the four PAHs were produced.Cross-reactivity,recovery rates and detection limit were observed.Some environmental samples were analyzed with satisfactory results.(8) Antibody-Coated Real-time Immuno-PCR(RT-IPCR) assay:Antibody-Coated Real-time Immuno-PCR(RT-IPCR) assay for the determination of four PAHs in environment was first developed.Glutaraldehyde treated PCR tubes were adsorbed by Coating pAbs specific for PAHs,with which biotinylated PAHs hapten and antigen(Ag) PAHs are competing in combining. Avidin is used as a bridge between the biotinylated PAHs hapten and the biotinylated 103 bp reporter DNA.The reporter DNA is amplified and measured by RT-PCR under the optimize procedure.Standard curves of the four PAHs were produced. Cross-reactivity,recovery rates and detection limit were observed.Some environmental samples were analyzed with satisfactory results.(9) HPLC optimize and Comparison of the four methods:The chromatographic fractionation conditions for the separation of the four PAHs were optimized according to national Standard method of HPLC.Standard curves of the four PAHs were produced.Detection limit were observed.Some environmental samples were analyzed with satisfactory results.Comparison of the four methods was taken and their advantages and disadvantages were discussed.更多还原