【作者】 申纪伟；

【导师】 徐溢；

【作者基本信息】 重庆大学 ， 药物化学， 2008， 硕士

【摘要】 以微流控芯片上集成微管道网络构建的二维芯片电泳分离分析系统为复杂样品体系的高效分离分析提供了一个有发展前景的技术平台。二维切换接口设计是微流控二维芯片电泳技术发展的瓶颈。本文在全面综述国内外研究的基础上,对二维电泳芯片切换接口的类型进行了分类,总结了分离模式选取的原则,重点研究了胶束电动色谱(Micellar electrokinetic chromatography,MEKC)模式与毛细管区带电泳(Capillary zone electrophoresis,CZE)模式相耦联的微流控二维电泳芯片切换接口设计。课题对芯片上多维分离技术的发展和应用具有重要意义,同时能为开发新型微全分析系统提供理论依据和技术支撑。本文采用玻璃材质电泳芯片,考察了芯片的伏安曲线、电渗流等电学特性;分别采用一维胶束电动色谱和毛细管区带电泳模式对荧光标记混合氨基酸样品体系进行芯片电泳分离分析,优化了缓冲条件、进样场强、分离场强等芯片电泳操作参数。由此提出了胶束电动色谱模式为第一维,毛细管区带电泳模式为第二维,以基于时间顺序的双T切换接口实现二维耦联的微流控二维电泳芯片结构,完成了微流控二维芯片电泳分离分析系统的构建。首先对精氨酸单样品进行二维分析,考察精氨酸样品在一维管道中分段迁移的情况,并成功将第一维分离管道中的样品进样到第二维分离管道,优化出第二维分离的启动时间为55s,进样时间为5s。然后,将MEKC在线推扫富集技术引入二维芯片电泳系统中,在第一维MEKC模式中实现了样品的高效富集,对赖氨酸及苯丙氨酸的富集倍数分别达到了150倍和900倍左右,并将富集后的样品引入二维分离通道,每个氨基酸样品峰被分成2-3次进样到第二维,实现了二维分离检测。最后,采用该二维芯片电泳系统完成了对精氨酸、赖氨酸等5种氨基酸样品混合物的二维分离分析,每个样品峰被分成2-3次进样到第二维分离管道进行二维分离。通过建立数学模型,计算得到MEKC模式与CZE模式之间的正交性为56.0%,表明两种分离模式之间具有良好的正交性。研究表明本文设计的具有双T切换接口的微流控二维芯片电泳分离分析系统能够实现氨基酸样品二维分离分析。开发二维芯片电泳系统的最终目标是解决复杂生化样品体系分离分析的需要,将所构建的系统用于诸如蛋白质样品体系的分析测试,将是下一步工作需要着重解决的问题。更多还原

【Abstract】 A powerful tool was presented for high efficiency separation of complex bio-chemical samples by two-dimensional separation system constructed on the micofluidic chip platform. Research work in two-dimensional microchip electrophoresis technique was summarized in this paper. Switching interface design is the bottleneck of two- dimensional microchip electrophoresis technique.Type of switching interface for two–dimensional electrophoresis microchip was classified and principle for separation mode combination was presented. This paper focused on switching interface design for the coupling of Micellar electrokinetic chromatography mode and capillary zone electrophoresis mode. Research on the design of switching interface for two-dimensional CE microchip and corresponding quantitative estimate of the testing results are of great significance for development of muti-dimensional separation technique. Meanwhile, it will supply theory basis and technique support for new miniaturized total analysis system.Firstly, electroosmotic flow (EOF) in glass micro-channel was measured by current monitoring method and voltage-current curves of the microchip were also measured. FITC-labeled mixed amino acid samples were separated by microchip MEKC and CZE modes, respectively. According to the optimized results in the one-dimensional separation experiments, MEKC mode was selected as the first dimension and CZE as the second dimension, the time based double T switching interface was proposed. Furthermore, the two-dimensional microchip CE separation system was constructed.And then, the performance and application of the two-dimensional microchip CE separation system were investigated. Two-dimensional microchip CE analysis of single FITC-labeled arginine sample was carried out. Arginine sample was successfully injected into the channel of the second dimension. Optimized startup time and injection time of the second dimension were 55s and 5s, respectively. Furthermore, MEKC on-line sweeping technique was introduced into the separation system, high efficiency enrichment of lysine and phenylalanine was realized in the first MEKC dimension. Corresponding enrichment multiple of lysine and phenylalanine were about 150 and 900, respectively. Each enriched sample was divided into 2-3 parts and introduced into the second dimension.Finally, two-dimensional analysis of 5 mixed amino acid samples were successfully realized on the constructed two-dimensional microchip CE system. Each sample was divided into 2-3 parts and introduced into the second dimension. Mathematical model was set up to evaluate orthogonality between MEKC and CZE, calculated value was 56.0%. Results showed designed switching interface could introduce sample band in the first dimension separation microchannel into the next one effectively and there was well orthogonality between the selecteded separation modes.Research showed two-dimensional separation of amino acid samples could be realized on the two-dimensional microchip electrophoresis system with time based double T switching interface. Final goal of two-dimensional microchip CE system development was to separate complex bio-chemical samples. And thus, application of the constructed system in protein separation is the key problem which should be settled in latter research work.更多还原