二恶英类化合物快速检测系统的初步构建
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摘要
二恶英是具有很强毒性的持久性有机污染物之一。随着我国社会经济的快速发展，城市化进程的不断加快以及人们物质生活的提高，对二恶英的排放标准必将进一步提高，并且将逐渐普及到大多数城市。然而，现行的二恶英类化合物的检测方法存在周期长、成本高、操作烦琐等弊端，阻碍了二恶英检测的普及。因此，构建一种集实时分析、快速灵敏、使用简单、方便携带等优点于一体的新的检测技术具有十分重要的应用价值。
首先，论文对新的二恶英检测方法进行了理论分析。论文结合压电石英晶体原理、二恶英类化合物的毒性机理、免疫反应原理以及ELISA方法提出了三种新的检测方法，并对各个方法进行了可行性分析。
其次，石英晶体微天平（QCM）生物传感器检测平台的搭建是本论文的核心内容。论文通过多次实验对所搭建的检测系统进行了调试，确定了温度、电压、加样量对稳定性的影响，得出了保证系统的稳定性的实验条件，即：稳压、恒温、最大加样量50μL。最后采用浓度分别为0.9 ppm、9 ppm、90 ppm、900ppm、9000 ppm的葡萄糖溶液对系统进行测试，验证了系统的性能。同时，为了克服静态系统的弊端，在本论文中设计了手动进样器以及缓冲瓶装置，对系统进行改进与优化，实现了连续进样，效果明显。
再次，从猪肝中提取芳香烃受体是本论文的重要内容之一。本论文通过逐步离心、硫酸铵沉淀法完成了芳香烃受体的粗提取，用离子交换层析进行纯化，
最后采用SDS-PAGE电泳实验对以上各个步骤得到的芳香烃受体进行测定。压电石英金片的表面修饰及固定化是本论文的关键内容。实验采用蛋白A固定法，以pH 7.2、反应温度20℃、SPA浓度0.2 g/L、兔抗猪IgG效价1：8、猪IgG浓度10 mg/L为实验条件，完成金片上抗体的固定以及免疫反应，证明了所用固定方法的可靠性以及所搭建的静态检测系统的可行性。最后，论文对进一步完成整个二恶英快速检测系统需要继续做的工作进行了展望。
Dioxins are one of the most toxic compounds at present in the world. With the rapid development of China's society and economy, the accelerating urbanization process and the improvement of people's material life, the dioxin emission standard will be raised further, and will gradually spread to most cities. However, traditional detection methods of dioxin-like compounds at present have their disadvantages such as very high cost, long detection period and complicated operation, which limits the spread of dioxin detection in various industries. Therefore, it will be a great value to construct a novel dioxin detection device which integrates many advantages such as online real-time analysis, sensitive and fast, easy to use and portable.
Firstly, basic dioxin detection theories with quartz crystal microbalance (QCM)are discussed in this thesis. Combining QCM mechanism, toxicity mechanism of dioxin-like compounds, principles of the immune response and ELISA method, this thesis introduced three new detection methods and did the feasibility analysis for them.
Secondly, the constructing of QCM biosensor detection platform is the core of this thesis. We obtained the experimental conditions to ensure the stability of the system through many experiments on the temperature, voltage and sample volume. They are steady voltage, constant temperature and the maximum sample volume of 50μL. Finally, we tested the system performance through glucose solution of diffiernet concentrations that were 0.9 ppm, 9 ppm, 90 ppm, 900 ppm, 9000 ppm. At the same time, for overcoming the shortcome of the static system, we designed manual sampler and buffer bottles device, improved and optimized the system and achieved continuous injection in the end.
Thirdly, the extraction of AhR from pig liver is an important part of this thesis. We accomplished the crude extraction of AhR through methods of step by step centrifugation and ammonium sulfate precipitation, then we completed the purification of AhR by ion exchange chromatography. Finally, we determined the purity of AhR from each step by SDS-PAGE electrophoresis.
The surface modification and immobilization of the gold film is the key elements of this thesis. In the experiment, we chose protein A fixation as our experimental method and the experimental conditions are pH 7.2, reaction temperature 20℃, SPA concentration of 0.2 g/L, rabbit anti-swine IgG titer of 1: 8, pig IgG concentration of 10 mg/L. We completed the fixation of antibody on gold film and immune responses in detection cell, and proved the reliability of the method and the feasibility of the static detection system by measuring the△F of quartz crystal in each step.
Finally, this thesis gave the outlook for further constructing the whole dioxin rapid detection system.
首先，论文对新的二恶英检测方法进行了理论分析。论文结合压电石英晶体原理、二恶英类化合物的毒性机理、免疫反应原理以及ELISA方法提出了三种新的检测方法，并对各个方法进行了可行性分析。
其次，石英晶体微天平（QCM）生物传感器检测平台的搭建是本论文的核心内容。论文通过多次实验对所搭建的检测系统进行了调试，确定了温度、电压、加样量对稳定性的影响，得出了保证系统的稳定性的实验条件，即：稳压、恒温、最大加样量50μL。最后采用浓度分别为0.9 ppm、9 ppm、90 ppm、900ppm、9000 ppm的葡萄糖溶液对系统进行测试，验证了系统的性能。同时，为了克服静态系统的弊端，在本论文中设计了手动进样器以及缓冲瓶装置，对系统进行改进与优化，实现了连续进样，效果明显。
再次，从猪肝中提取芳香烃受体是本论文的重要内容之一。本论文通过逐步离心、硫酸铵沉淀法完成了芳香烃受体的粗提取，用离子交换层析进行纯化，
最后采用SDS-PAGE电泳实验对以上各个步骤得到的芳香烃受体进行测定。压电石英金片的表面修饰及固定化是本论文的关键内容。实验采用蛋白A固定法，以pH 7.2、反应温度20℃、SPA浓度0.2 g/L、兔抗猪IgG效价1：8、猪IgG浓度10 mg/L为实验条件，完成金片上抗体的固定以及免疫反应，证明了所用固定方法的可靠性以及所搭建的静态检测系统的可行性。最后，论文对进一步完成整个二恶英快速检测系统需要继续做的工作进行了展望。
Dioxins are one of the most toxic compounds at present in the world. With the rapid development of China's society and economy, the accelerating urbanization process and the improvement of people's material life, the dioxin emission standard will be raised further, and will gradually spread to most cities. However, traditional detection methods of dioxin-like compounds at present have their disadvantages such as very high cost, long detection period and complicated operation, which limits the spread of dioxin detection in various industries. Therefore, it will be a great value to construct a novel dioxin detection device which integrates many advantages such as online real-time analysis, sensitive and fast, easy to use and portable.
Firstly, basic dioxin detection theories with quartz crystal microbalance (QCM)are discussed in this thesis. Combining QCM mechanism, toxicity mechanism of dioxin-like compounds, principles of the immune response and ELISA method, this thesis introduced three new detection methods and did the feasibility analysis for them.
Secondly, the constructing of QCM biosensor detection platform is the core of this thesis. We obtained the experimental conditions to ensure the stability of the system through many experiments on the temperature, voltage and sample volume. They are steady voltage, constant temperature and the maximum sample volume of 50μL. Finally, we tested the system performance through glucose solution of diffiernet concentrations that were 0.9 ppm, 9 ppm, 90 ppm, 900 ppm, 9000 ppm. At the same time, for overcoming the shortcome of the static system, we designed manual sampler and buffer bottles device, improved and optimized the system and achieved continuous injection in the end.
Thirdly, the extraction of AhR from pig liver is an important part of this thesis. We accomplished the crude extraction of AhR through methods of step by step centrifugation and ammonium sulfate precipitation, then we completed the purification of AhR by ion exchange chromatography. Finally, we determined the purity of AhR from each step by SDS-PAGE electrophoresis.
The surface modification and immobilization of the gold film is the key elements of this thesis. In the experiment, we chose protein A fixation as our experimental method and the experimental conditions are pH 7.2, reaction temperature 20℃, SPA concentration of 0.2 g/L, rabbit anti-swine IgG titer of 1: 8, pig IgG concentration of 10 mg/L. We completed the fixation of antibody on gold film and immune responses in detection cell, and proved the reliability of the method and the feasibility of the static detection system by measuring the△F of quartz crystal in each step.
Finally, this thesis gave the outlook for further constructing the whole dioxin rapid detection system.
引文
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[2]黄文章,胡小燕.二恶英的特性及其危害[J].重庆科技学院学报（自然科学版）, 2009, 11（1）: 72-74.
[3]黄汝广,李莎,于萍萍.二恶英污染及其防控措施的研究进展[J].安徽农业科学, 2007, 35（30）: 9670-9671.
[4]杜泽.二恶英的种类、产生机理及消除方法[J].科技信息（科学教研）, 2007,（24）: 41.
[5]王爱香,张文旭.国内外二恶英研究进展[J].临沂师范学院学报, 2006, 28（3）:75-77.
[6]姚玉红,刘格林.二恶英的健康危害研究进展[J].环境与健康杂志, 2007, 24（7）: 560-562.
[7]张秀敏,刘敏.浅析二恶英的危害与防治[J].环境, 2006, S2: 112-113.
[8]张智平,倪余文,杨志军,等.二恶英样品制备与检测技术进展[J].环境污染治理技术与设备, 2005, 6（3）: 13-17.
[9]葛宝坤.二恶英的分离及检测技术[J].口岸卫生控制, 1999, 4（3）: 38-40.
[10]章骅,何品晶,李国建,等.生活垃圾焚烧飞灰中的二恶英检测[J].同济大学学报（自然科学版）, 2004, 32（12）: 1655-1659.
[11]赵毅,张秉建,贺鹏.二恶英类化合物检测方法的研究现状及展望[J].电力环境保护, 2008, 24（6）: 44-46.
[12]王承智,胡筱敏,石荣,等.二恶英类物质的生物检测方法[J].中国安全科学学报, 2006, 16（5）: 136-138.
[13]宋春莲.多氯联苯/二恶英类化学污染物的环境行为及对人体健康的危害[J].佳木斯大学学报（自然科学版）, 2007, 25（2）: 208-209.
[14] Ohbayashi H, Sasaki T, Matsumoto M, et al. Dose and time dependent effects of2, 3, 7, 8-tetrabromodibenzo-p-dioxin on rat liver[J]. Journal of ToxicologyScience , 2007, 32（1）: 56-47.
[15] Korenaga T, Fukusato T, Ohta M, et al. Long-term effects of subcutaneouslyinjected 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin on the liver of rhesus monkeys[J].Chemosphere, 2007, 67（9）: S399-404.
[16]杨永滨,郑明辉,刘征涛.二恶英类毒理学研究新进展[J].生态毒理学报,2006, 1（2）: 106-107.
[17]张波,府伟灵.压电石英晶体生物传感器及其应用研究进展[J].生物医学工程学杂志, 2004, 21（2）: 316-320.
[18]吴永强,李可彬,周建.压电免疫传感器的研制[J].压电与声光, 2004, 26（5）: 371-373.
[19]刘虹,王源升,刘清山,等.压电免疫传感器与分子探针固定化技术[J].化学与生物工程, 2004, 21（5）: 7-9.
[20] Pandini A, Song Y, Zhao J, et al. Detection of the TCDD binding-fingerprintwithin the Ah receptor ligand binding domain by structurally driven mutagenesisand functional analysis[J]. Biochemistry, 2009, 48（25）: 5972-5983.
[21] Levin M, Morsey B, Mori C, et al. Non-coplanar PCB-mediated modulation ofhuman leukocyte phagocytosis: a new mechanism for immunotoxicity[J].Journal of Toxicology and Environment Health A, 2005, 68（22）: 1973-1997.
[22]彭洁,高洪.二恶英中毒对AhR基因表达和自由基水平的影响[J].中国畜牧兽医, 2009, 36（12）: 36-41.
[23]易金娥,袁慧,袁莉芸.二恶英免疫毒性研究进展[J].动物医学进展, 2008,29（1）: 80-83.
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