【作者】 林清湖；

【导师】 方群；

【作者基本信息】 浙江大学 ， 化学， 2011， 硕士

【摘要】 常规毛细管电泳(Capillary Electrophoresis, CE)系统通常采用数十厘米的石英毛细管做为分离通道,在数十分钟内实现分离,具有样品消耗量少、分析速度快、分离效率高、易自动化等优点。高速毛细管电泳(High-speed Capillary Electrophoresis, HSCE)系统通过采用短毛细管、高分离场强和窄进样区带,实现高速和高效率的毛细管电泳分离。第一章介绍了用于蛋白质分离的常规毛细管电泳、微流控芯片毛细管电泳和基于短毛细管的快速毛细管电泳系统的研究进展。第二章建立了一种基于缺口管阵列及短毛细管的高速毛细管SDS-凝胶电泳蛋白质分离系统。提出了一种适用于粘度较大的蛋白质分离缓冲体系的部分自发进样方法,可满足HSCE系统对进样的要求。实验考察了影响试样引入过程的主要因素,如毛细管进样端形状、平移台移动速度等。在优化实验条件下,进样区带长度达到65μm,相应的进样体积约500 pL。使用一根分离长度为1.5 cm的涂层毛细管,在凝胶电泳分离模式下,在60 s内实现了FITC标记的肌红蛋白、卵清蛋白、牛血清蛋白、磷酸化酶b和肌球蛋白的分离,各个电泳峰的每米理论塔板数在258,000-1,280,000,相应的塔板高度为0.78μm-3.88μm,且蛋白质分子量的对数与迁移时间呈现良好的线性关系,线性相关系数达0.9989,说明该系统还可应用于蛋白质分子量的测定。该系统的分离速度和分离效率与多数文献报道的基于微流控芯片的电泳系统相当。第三章研制了一种面向航天空间实验的微型毛细管电泳分析仪。仪器基于顺序注射分析和“T”型通道结构构建具有全封闭液路的毛细管电泳分离系统。仪器集成了全封闭液路系统、全自动试样引入、分离系统、高压电源控制系统、激光诱导荧光检测系统、数据采集存储记录系统和电子控制系统。仪器具有自动化程度高、体积小、功耗小、集成化程度高的特点,可在全封闭环境下实现试样引入和分离等操作。实验考察了影响分析仪性能的主要因素,包括分离场强、顺序注射系统进样体积、缓冲液流速和毛细管两端储液袋的液位差。在最优分离条件下,将该毛细管电泳分析仪应用于手性氨基酸对映体的分离分析,使用一根有效分离长度为13 cm的熔融石英毛细管,采用MEKC分离模式,在320 s内实现了FITC标记的精氨酸、丙氨酸和天冬氨酸三组氨基酸对映体的手性分离。连续8次测定,各组分的迁移时间RSD在0.70%-0.86%之间,峰高RSD在3.23%-4.33%之间,峰面积RSD在3.82%-5.39%之间。仪器连续工作19 h均能正常工作,表明分析仪具有良好的分析性能和工作稳定性,有望用于未来航天空间实验。更多还原

【Abstract】 In capillary electrophoresis (CE) systems, the separation is usually performed in capillaries with separation lengths of several tens centimeters, separation times of several tens minutes, with the advantages of low sample consumption, high analysis speed, high separation efficiency and ease of automation. The high-speed capillary electrophoresis (HSCE) systems using short capillaries, high separation field strengths and narrow sample plugs, can achieve the CE separation with high speed and high separation efficiency.In chapter 1, the developments of conventional CE, microfluidic chip-based CE and short capillary-based CE systems for protein separation in the recent years are reviewed.In chapter 2, we developed a high-speed sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) system for protein separation based on a short capillary and slotted-vial array. A partial translational spontaneous injection approach was proposed for high-viscosity buffer solution system used in protein separation under CGE mode, to obtain narrow injection volume in the picoliter range. Several factors affected the sample injection process were investigated, including the shape of the capillary inlet and the moving speed of the platform. A sample injection plug length of～65μm (corresponding to～500 pL) was obtained under the optimized conditions. Five fluorescein isothiocyanate (FITC)-labled proteins were separated within 60 s with an effective separation length of 1.5 cm. The theoretical plates per meter ranged from 258,000 to 1,280,000 (corresponding to 0.78μm-3.88μm plate heights). The RSD of the migration time was less than 1.3%(n=5). A good linear correlation with the logarithm molecular weight versus migration time was obtained in the molecular weight range of 17,200 to 500,000, which demonstrated the present system could be applied in protein molecular mass determination. The separation speed and separation efficiency of the system was comparable to most of the reported microfluidic chip-based on HSCE systems for protein separation.In chapter 3, we developed a capillary electrophoresis analyzer toward space experiment based on sequential injection analysis (SIA) and "T" configuration channel sample introduceion system. The analyzer integrated closed liquid manipulation system, automated injection and separation system, high voltage power supply, laser-induced fluorescence (LIF) detection system, data collection, recording, storage system and control system. The factors affected the performance of the analyzer were investigated, including separation field strength, SI injection sample volume, the carrier flow-rate and the liquid difference. Under the MEKC separation mode, three fluorescein isothiocyanate (FITC)-labled chiral animo acids were separated within 320 s with an effective separation length of 13 cm. The repeatabilities for the migration time, peak height and peak area were 0.70%-0.86%, 3.23%-4.33% and 3.82%-5.39%(n=8), respectively. In the test for working stablility, the analyzer continuously worked for 19 h. We expect the capillary electrophoresis analyzer has good application potential in space experiment.更多还原