磁性磁性荧光微球的制备及在转基因食品检测中的应用

独创性声明 本人声明所呈交的学位论文是本人在导师指导下进行的研究工作和取得的研究成果，除了文中特别加以标注和致谢之处外，论文中不包含其他人已经发表或撰写过的研究成果， 也不包含为获得 天津大学天津大学 或其他教育机构的学位或证书而使用过的材料。 与我一同工作的同志对本研究所做的任何贡献均已在论文中作了明确的说明并表示了谢意。 学位论文作者签名： 签字日期： 年 月 日 学位论文版权使用授权书 本学位论文作者完全了解 天津大学天津大学 有关保留、使用学位论文的规定。特授权 天津大学天津大学 可以将学位论文的全部或部分内容编入有关数据库进行检索，并采用影印、缩印或扫描等复制手段保存、汇编以供查阅和借阅。同意学校向国家有关部门或机构送交论文的复印件和磁盘。 （保密的学位论文在解密后适用本授权说明） 学位论文作者签名： 导师签名： 签字日期： 年 月 日 签字日期： 年 月 日 摘 要 摘 要 聚合酶链式反应（PCR）和荧光定量聚合酶链式反应（qRT-PCR）是转基因食品检测的主要手段，其关键是从待检样品中快速高通量地提取到高质量的核酸，目前主要的核酸提取方法是十六烷基三甲基溴化铵（CTAB）法、十二烷基硫酸钠（SDS）法和各种试剂盒法，由于 CTAB 法和 SDS 法操作繁琐、需要使用有机溶剂并且不断离心，因而不能满足快速高通量的发展要求；而试剂盒法由于昂贵的价格也限制了它在日常检测中的应用；同时，基于核酸水平的转基因检测的发展趋势是多重检测， 其关键是制备不同种类的荧光微球和对微球的特异探针的修饰。 针对以上问题，本论文提出了以磁性分离技术为基础的核酸提取方法，该方法的关键是制备磁响应性高、单分散性良好和易于表面修饰的磁性微球；将该磁性微球用于大豆样品核酸提取进行综合评价； 并针对多重检测制备了不同荧光性能的荧光微球。本文的研究目标主要包括三个方面： （1）通过热溶剂还原法制备磁响应性高、单分散性良好、可重复性高和易于修饰的磁性微球； （2）将磁性微球用于食品核酸提取中并进行综合评价，同时对核酸的特异性提取做了基础研究； （3）制备了不同荧光性能的荧光/磁性荧光微球。具体工作如下所述： 首先采用热溶剂还原法制备了磁性微球，考察了不同的分散剂、还原剂和前躯体的种类及用量对磁性微球的形貌及分散性的影响，探讨了其反应机理。 并且通过溶胶-凝胶法法对其表面修饰以制备不同官能团的磁性微球。 其次，将此磁性微球用于大豆样品中核酸的提取，并将此方法与传统核酸提取方法和市售试剂盒对比进行综合评价；将磁性微球进行了链霉亲和素-生物素-ssDNA 的修饰，考察了其对互补以及非互补 ssDNA 的提取效率。 最后，为了进一步完善转基因食品检测体系，针对多重检测的模式的优点：（可以同时检测样品的内源基因与多个外源基因，既减少核酸模板的用量，又提高检测速度） ，通过层层自组装的方法制备了以聚苯乙烯为基质的荧光/磁性荧光微球，该荧光/磁性微球的荧光发射峰可以通过制备的荧光量子点的大小进行调节，使荧光微球具有不同的荧光性能从而得以区分。 综上所述， 本论文对基于磁分离技术的转基因食品的核酸快速提取以及多重检测方面做了初步探讨，为推动转基因食品核酸提取的自动化、高通量和核酸多重检测的发展做了基础研究工作。 关键词关键词：转基因食品检测，溶剂热还原法，磁性微球，核酸提取，核酸探针， 磁性荧光微球 ABSTRACT Nowadays, the main ways of detecting genetically modified food are polymerase chain reaction (PCR) and real-time PCR (qRT-PCR). The key problem of the detection is how to extract high-quality DNA from food samples with high-through-put and fast speed. The main DNA-extraction methods are hexadecyl trimethyl ammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and kinds of kits. But the CTAB and SDS methods need complex operations and large amount of organic solvent, so they cannot meet the demand of auto-extraction. And the kits are always expensive and cannot be used in the routine detection of the genetically modified food. Thus, we introduced magnetic separation to the extraction of DNA, and prepared magnetic microspheres with uniform particle size, well dispersion, and high magnetic susceptibility. Meanwhile, multi-detection is becoming popular in the genetically modified food detection. So it is also necessary to prepare different fluorescent microspheres. In order to solve above problems, we performed following work consisting of three-folds: (1) Preparation and characterization of magnetic microspheres; (2) Magnetic microspheres for DNA extraction from soybean foods and with comprehensive evaluation; extraction of specific complementary strand ssDNA with DNA probe modified magnetic microspheres; (3) Preparation and characterization of different fluorescent microspheres. The details of the work are as follows: A solvothermal reduction method is described for the preparation of magnetic responsive particles. The effects of dispersant, reducing agent and precursors on the magnetic microspheres were investigated. After being coated with silica by the sol-gel approach, the particles were modified with different chemical groups. Then we applied these magnetic microspheres in the extraction of DNA from soybean derived foods, and compared this method with traditional methods like CTAB and commercial kits such as Omega® and Wizard® kits. Then, we modified this amino-magnetic microsphere with avidin, which were then coupled with biotin-ssDNA. In the mean time, we studied the specific extraction efficiency of these ssDNA-biotin-avidin mangentic microspheres with complementary strand ssDNA and non- complementary strand ssDNA. Multi-detection can detect more than one target genes in genetically modified foods. This method can not only reduce the amount of DNA for the detection, but can also accelerate detecting speed. We prepared different fluorescent microspheres by layer-by-layer method using quantum dots as fluorescent labels. The emission wavelengths of microspheres can be adjusted by different sizes of quantum dots. Also, we prepared fluorescent microspheres with magnetic susceptibility. In summary, this thesis laid a basis for the rapid extraction of nucleic acid based on magnetic separation technology from genetically modified foods as well as the multiplex assay. And this basic research work can promote the automation of nucleic acid extraction of genetically modified foods, high-throughput and multi-detection. Keywords: Detection of genetically modified food, Solvothermal method, Magnetic microspheres, DNA extraction, DNA probe, Fluorencent magnetic microspheres 目 录 第一章 文献综述. 1 1.1 转基因食品的研究现状. 1 1.1.1 转基因食品.