【作者】 郭玉高；

【导师】 包建民；

【作者基本信息】 天津大学 ， 应用化学， 2007， 博士

【摘要】 毛细管电泳技术(一般采用内径50-100μm的石英毛细管)在人类基因组研究中扮演了举足轻重的角色,使整个人类基因组计划的完成大大提前,但是,伴随着科学技术的飞速发展,对目标产品进行更加深入的研究成为大势所趋。因此,毛细管电泳技术样品用量小的显著特性却限制了自身的应用范围,于是增大毛细管电泳的载样量成为拓宽其应用领域的技术瓶颈。为了保持毛细管电泳技术分离效率高的优点,又能弥补其载样量小、检测灵敏度低等不足,本论文将壳管换热器结构引入到大管电泳系统中,借此强化电泳系统的散热能力,使得在毫米级的分离管路中进行电泳成为可能。本文采用不同规格的内部制冷管对大管电泳系统进行内部降温处理,使电泳过程中产生的焦耳热能及时地被制冷毛细管中的冷却液带走。实验结果表明,内部制冷的存在确实起到了很好的制冷功效,使得大管电泳的电流-电压关系在较大范围内符合欧姆曲线;分别选择酸性、碱性物质为模型化合物测试了带有内部制冷的大管电泳系统的分离效果,部分化合物的分离效率达到了60, 000 plates/m以上;实验中采用的大管电泳能够承载1.0μL左右的样品,这样大的载样能力使得其能够作为半制备或制备电泳系统。高的载样能力预示着大管电泳可以实现多维操作,于是目标组分的转移也就成为进一步开发大管电泳的任务之一。因此,采用丽春红为探针化合物测试了系统的电转移性能,证实了在该装置上进行有效组分转移的可行性。建立大管电泳系统的目的之一就是搭建高通量药物筛选平台,而酶分析技术是药物筛选中的一项关键技术,因此,本文结合电泳介导酶分析方法,开发了一步法快速酶分析技术,并用该方法成功测定了碱性磷酸酶的反应动力学参数。实验结果表明,快速酶分析技术可以通过一次实验计算得出酶的反应动力学参数,而且计算结果准确、生物样品用量小、人为因素少,值得深入研究和推广。基于此理念,将一步酶分析技术与大管电泳体系相结合并实现多维、高通量筛选平台的初步设想是值得探索和深入研究的。更多还原

【Abstract】 With the rapid development of science and technology, the trend for in-depth research on object compounds has been finality in the post-genome era. Capillary electrophoresis (CE), which played an important role in Human Genome Project, exposed its limitations in dealing with large volume of samples. Therefore, the tiny sample loading capacity becomes the major obstacle to extending the application scope of CE.In order to increase the sample loading capacity while maintaining the high separation efficiency in column electrophoresis, a new system called wide-bore electrophoresis (WE) was adopted in the thesis based on the principle of shell and tube heat exchanger. The Joule heat generated during the electrophoresis process could be removed in situ in real time thoroughly by the inner cooling water. The experimental data indicated that the heat sink was effective and could make the function of the applied potential and current in the WE system matches Ohm plots well. Some acidic or alkaline model compounds were selected to evaluate the separation efficiency of the novel system and a separation efficiency as high as over 60, 000 plates/m was achieved for some compounds. The biggest advantage of this WE system is its potential as a preparative or semi-preparative electrophoretic separation technique due to the maximum sample loading capacity of 1.0μL or more.The larger sample loading capacity entitled the WE system for multi-dimensional operations. Thus, the delivery properties of object compounds were tested by using ponceau 4R as the model. The feasibility of the compound’s delivery was validated by the experiment.In future work, one intention of establishing WE system is to develop a multi-dimensional high-throughput medical screening platform. Because enzyme analysis is a vital unit in this medical screening, a one-step enzyme microanalysis technique was developed on the basis of Electrophoretically Mediated Microanalysis (EMMA) method. Furthermore, the enzyme kinetics constant of alkaline phosphatase (ALP) was determined using the traditional method and the one-step method. The experimental results demonstrated that the one-step determination method for enzyme kinetics constant was accurate and sample saving. Therefore, the one-step enzyme analysis method proposed in this thesis combined with WE system to perform multi-dimensional screening is worth of in-depth research and further development.更多还原