表面修饰有阳离子的磁性二氧化硅微粒的制备及其应用

【作者】 杨磊；

【导师】 崔亚丽； 李如琳；

【作者基本信息】 西北大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 磁性复合微粒是超顺磁性纳米粒子与有机或无机材料通过包覆、交联等方式形成的纳微米级复合粒子。磁性复合微粒具有超顺磁性、较高的比表面积、可修饰功能基团等特点。因此,广泛应用于免疫学检测、核酸纯化、细胞分选、药物载体和酶的固定化等生物医学领域。本研究制备了两种表面修饰有阳离子的磁性二氧化硅微粒,基于两种磁性微粒建立了200μL人全血基因组DNA纯化方法,并对其进行优化。采用微乳液法,以十六烷基三甲基溴化铵(CTAB)作为表面活性剂、甲苯为溶剂,与铁盐溶液(FeCl2和FeC13的混合溶液)剧烈搅拌形成微乳液,以氨水提供碱性环境合成Fe304磁性微粒,在原位以正硅酸乙酯(TEOS)提供硅源,直接在微乳液中包覆Fe304磁性微粒制备得到二氧化硅磁性微粒。再经修饰分别得到表面含有二乙氨基乙基(DEAE)和L-组氨酸(L-His)的两种磁性阴离子交换二氧化硅微粒。基于表面修饰有二乙氨基乙基(DEAE)和L-组氨酸(L-His)的两种磁性阴离子交换二氧化硅微粒,以人全血作为样本,分别建立适用于200μL人全血基因组DNA纯化方法。并通过对纯化过程中裂解条件、结合条件、磁性微粒用量及清洗、洗脱条件等步骤对该方法进行优化,实验结果如下：(1)对于200μL全血体系,DEAE-磁粒纯化可得到基因组DNA 3～5μg,其A260／A280值均介于1.70～1.90之间,纯化所得DNA分子结构完整,可用于PCR等分子生物学下游应用。但该方法需高盐洗脱,洗脱得到的DNA需经醇沉脱盐处理。(2)基于His-磁粒200μL人全血基因组DNA纯化方法可纯化得到基因组DNA 3～4μg,其A260／A280值均介于1.65～1.8之间,相比前者纯化得到DNA的量较少,该方法结合所需pH5.0,可通过改变溶液的pH值洗脱,所需pH为8.5,洗脱得到的基因组DNA可直接用于下游分子生物学实验。更多还原

【Abstract】 Magnetic particles is a kind of composite particles which composed of magnetite and some matrix such as organic or inorganic materials. Magnetic composite particles have the properties of superparamagnetism, surface effects and can be modified with functional groups. Therefore, they are widely used in research of immunology detection, nucleic purification, cell sorting, drug carrier and biological enzyme immobilization. Herein, two kinds of magnetic silica particles modified with cationic group (DEAE and His) were successfully prepared and used for genome DNA purification from 200μL human whole blood.First, iron oxide nano-particles were synthesized by water-in-oil microemulsion technique, and in situ formed SiO2 as a shell on Fe3O4 particle surface. Based on core-shell structure magnetic silica nanoparticles (Fe3O4/SiO2), diethylaminoethyl (DEAE) and L-histidine (L-His) was modified on the particles and formed anion exchange magnetic silica particles respectively.Using the particles modified with DEAE and L-His as carriers, genomic DNA was extracted from 200μL human whole blood, the steps including the lysis of whole blood, amount of the particles added, washing and eluting were optimized and the results show as follow:.(1) 3-5μg genome DNA was obtained using 1.5 mg of DEAE-magnetic particles from 200μL human whole blood, the ratios of obtained genomic DNA were in the range of 1.70～1.90. However, the step of elution of this method need high salt concentration and need further ethanol precipitation to get genome DNA sample without salt.(2) 3～4μg genome DNA was obtained using 1 mg of His-magnetic particles from 200μL human whole blood, the ratios of obtained genomic DNA were in the range of 1.65-1.80. The genome DNA yielded after the step of elution can be directly used in biological downstream applications. Because the elution step depends on the pH of the elution buffer using TE of pH 8.5.更多还原