【作者】 汪友宝；

【导师】 沈鹤柏；

【作者基本信息】 上海师范大学 ， 物理化学， 2005， 硕士

【摘要】 研究基于纳米粒子表面的聚合酶链反应,不仅为PCR 产物的检测分离和纯化带来了方便,而且也为DNA 生物芯片和纳米粒子/DNA 纳米结构的制备提供了一种新的方法。目前,国外有一些文献报道了基于固相表面的聚合酶链反应,但是还有许多地方值得深入地探讨。本文系统研究了基于纳米粒子表面的聚合酶链反应。(1)首先采用反相微乳液的方法,利用正硅酸乙酯在碱性条件下水解,在γ-Fe2O3 的表面包裹一层SiO2。实验结果表明在磁核γ-Fe2O3 的表面成功地包裹了SiO2 层,形成了核壳型磁性纳米粒子;而且在合适的条件下,合成的核壳型磁性纳米粒子具有均匀的粒径和良好的单分散性。(2)通过共价键合的方法将oligoDNA 连接到MNP 表面,并首次利用表面增强拉曼光谱(SERS)方法对oligoDNA 在MNP 表面的键合进行了检测。实验结果表明巯基在银基底上有很好的SRES 效应,经由过硫键能够将oligoDNA 成功地键合到磁性纳米粒子的表面,并且通过其与互补单链DNA 的杂交验证了其仍然有很好的生物活性。这为基于磁性纳米粒子表面的聚合酶反应奠定了基础。(3)进一步系统地研究了基于磁性纳米粒子表面的聚合酶链反应(PCR)的规律。实验结果表明,在合适的条件下,连接在磁性纳米粒子表面的引物能够顺利地进行PCR。当一条引物连接在磁性纳米粒子表面,另一条引物没有连接在磁性纳米粒子表面时,PCR 受退火温度的影响较小。两条引物都连接在磁性纳米粒子表面时,PCR 受退火温度的影响较大;并且在一定温度范围里,退火温度越高,PCR 产物特异性越好。同时实验结果还表明通过两条引物连接在磁性纳米粒子表面的PCR,可以制得MNP/DNA 聚合体。(4)树枝状化合物大分子是一类新型合成高分子,它分子的大小、形状和功能基团可以在合成过程中进行严格设计和控制,因此引起了人们的极大兴趣。其中,聚酰胺-胺(PAMAM)是第一个合成的树枝状化合物大分子。目前,已经对PAMAM 在基因和药物载体以及磁共振成像等方面的应用进行了研究。本文还研究了基于更多还原

【Abstract】 The study of polymerase chain reaction (PCR) based on magnetic nanoparticlewould not only bring the convenience to the detection, isolation and purification ofPCR products but also provide a new method for preparing DNA chip and thenanostructure contained DNA and nanoparticles. In recent year, some literaturesreported that PCR based on solid phase could proceed. But it was an initial study, andstill needed to do a lot of work. In this thesis, we had systematically studied PCR basedon nanoparticle.(1) A thin silica-coating layer encapsulating bare γ-Fe2O3 nanoparticles wasformed by a base-catalyzed hydrolysis and polymerization reaction of tetraethoxysilane(TEOS) in reverse microemulsion. The experimental results show that the structure ofcore-shell is successfully synthesized and the synthesized MNP holds uniform size indiameter and very fine monodispersion in suitable condition.(2) The oligoDNA was covalently immobilized on the surface of MNP bychemical method and a technique of SERS was developed to detect it. Theexperimental results show that thiols attached on Ag substrate have excellent SERS andoligoDNA was covalently conjugated to the surface of MNP by disulfide bond. At thesame time, the fact was also demonstrated that oligoDNA immobilized on the surfaceof MNP still maintain very fine biological activity by hybridization withcomplementary single strand DNA, which is prepared for PCR based on magneticnanoparticles.(3) The law of PCR based on MNP was systematically studied. The experimentalresults show that the primers anchored on MNP can start PCR in suitable condition. Theannealing temperature affects hardly PCR when the forward primer was immobilizedand the reverse one is free, but the annealing temperature affects seriously PCR whenboth primers were immobilized and the purity of PCR products was enhanced with theincrease of annealing temperature in a certain temperature range. At the same time, theexperimental results also show that the MNP/DNA aggregates were constructed byPCR in which both primers were immobilized on the surface of MNP.更多还原