微流控液相色谱分析系统的研究
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摘要
微流控学是在微米尺度通道中操纵微量流体的科学和技术。其目标是在只有几个平方厘米甚至更小的微流控芯片上集成化学生物分析所需要的样品预处理、反应、分离、检测等基本操作单元,最终建立微全分析系统。高分辨的化学分析技术在化学和生物医学分析中发挥着非常重要的作用,发展适用于微流控系统的液相色谱系统具有重要意义。
第一章首先对基于微流控芯片的液相色谱系统的发展和现状进行综述。对其系统所使用的进样方式和色谱柱材料进行总结。同时,也对高分辨分离系统在多相微流控中的应用进展进行了介绍。
第二章发展了一种基于微流控芯片的无阀液相色谱系统。该系统由加工了色谱分离柱和平衡柱的微流控芯片、注射泵和激光诱导荧光检测器组成。仅需通过切换注射泵的开关状态即可实现无阀的纳升级门式进样。在样品和分离通道中同时加工等长的整体柱,分别作为色谱分离柱和压力平衡柱。.平衡柱通过平衡充样通道和分离通道中的背压简化了门式进样过程。在门式进样模式下,可以实现进样量的范围为0.4-2.4nL。我们将该系统初步应用于荧光标记胺类分子的分离,表明了该系统在液相色谱分离中的可行性。该系统具有结构简单、操作方便、进样体积易于改变和柱容量大的优点。
第三章发展了一种基于微流控液滴技术的液相色谱-质谱分析系统,为纳升级液滴中复杂组分的分析提供了有效的方法。发展了一种可灵活可控地进行液滴精确寻址的毛细管液相色谱和液滴阵列芯片的接口,实现了包括纳升级液滴内进行酶抑制反应、4nL的样品引入、毛细管液相色谱的快速分离、电喷雾质谱的非标记检测在内的多步操作。该系统初步应用于液滴中细胞色素P450(CYP1A2)的抑制剂的筛选和IC50值的测定。每次液滴测定的样品消耗量为100nL。与常规384孔板系统相比,样品消耗量降低了10-100倍。
Microfluidics is the science and technology that manipulate small (nL～fL scale) amounts of fluids, using channels with dimensions of tens to hundreds of micrometer. Its objective is to integrate all of the units in chemical and biological analysis, including sample preparation, reaction, separation, detection, on a microfluidic chip to build the micro total analysis system. High-resolution microanalytical technique plays a very important role in chemical and biological analysis. Currently, there is a growing demand for the development of liquid chromatography systems for microfluidic systems.
In Chapter1, the applications of microfluidic chip-based liquid chromatography systems, as well as sample injection techniques and chromatography columns are reviewed. The recent progress in high-resolution separation approaches for droplet-based microfluidic systems is also reviewed.
In Chapter2, a microfluidic chip-based liquid chromatography system with valveless gated sample injection method and monolithic columns was developed. The valveless LC system consisted of a microchip with a separation column and a balance column, two syringe pumps and a laser induced fluorescence detector. Two monolithic columns with the same length were fabricated in the sample and separation channel served as pressure-balance column and LC separation column, respectively. Nanoliter-scale sample injection was achieved by simply switching the ON and OFF states of the syringe pumps under gated injection mode, without the need of any mechanical valve. The balance column facilitated the gated injection process by balancing the back pressure in the sample channel with that in the separation channel. Under the gated injection mode, the sample injection volumes could be varied in the range of0.4-2.4nL. The present system was applied to the fast separation of two fluorescence-labeled amines.The advantages of the present system include simple system structure, ease of operation, convenience for varying injection volume, and high sample loading capacity.
In Chapter3, the combination of droplet-based microfluidics with liquid chromatography-mass spectrometry was achieved, for providing a fast separation and high-information-content detection method for analysis of nanoliter-scale droplets with complex compositions. A novel interface method for capillary LC and droplet array chip with significant flexibility in accurate addressing and sampling of interesting droplets on demand was developed. With this method, multistep operations including parallel enzyme inhibition reactions in nanoliter droplets,4-nL sample injection, fast separation with capillary LC, and label-free detection with ESI-MS were successfully realized. The present system was further applied to the screening for inhibitors of cytochrome P450(CYPI A2) and the measurement of IC50value of the inhibitor. The sample consumption for each droplet assay was100nL, which is reduced10to100times compared with conventional384multi-well plate systems usually used in high-throughput drug screening.
第一章首先对基于微流控芯片的液相色谱系统的发展和现状进行综述。对其系统所使用的进样方式和色谱柱材料进行总结。同时,也对高分辨分离系统在多相微流控中的应用进展进行了介绍。
第二章发展了一种基于微流控芯片的无阀液相色谱系统。该系统由加工了色谱分离柱和平衡柱的微流控芯片、注射泵和激光诱导荧光检测器组成。仅需通过切换注射泵的开关状态即可实现无阀的纳升级门式进样。在样品和分离通道中同时加工等长的整体柱,分别作为色谱分离柱和压力平衡柱。.平衡柱通过平衡充样通道和分离通道中的背压简化了门式进样过程。在门式进样模式下,可以实现进样量的范围为0.4-2.4nL。我们将该系统初步应用于荧光标记胺类分子的分离,表明了该系统在液相色谱分离中的可行性。该系统具有结构简单、操作方便、进样体积易于改变和柱容量大的优点。
第三章发展了一种基于微流控液滴技术的液相色谱-质谱分析系统,为纳升级液滴中复杂组分的分析提供了有效的方法。发展了一种可灵活可控地进行液滴精确寻址的毛细管液相色谱和液滴阵列芯片的接口,实现了包括纳升级液滴内进行酶抑制反应、4nL的样品引入、毛细管液相色谱的快速分离、电喷雾质谱的非标记检测在内的多步操作。该系统初步应用于液滴中细胞色素P450(CYP1A2)的抑制剂的筛选和IC50值的测定。每次液滴测定的样品消耗量为100nL。与常规384孔板系统相比,样品消耗量降低了10-100倍。
Microfluidics is the science and technology that manipulate small (nL～fL scale) amounts of fluids, using channels with dimensions of tens to hundreds of micrometer. Its objective is to integrate all of the units in chemical and biological analysis, including sample preparation, reaction, separation, detection, on a microfluidic chip to build the micro total analysis system. High-resolution microanalytical technique plays a very important role in chemical and biological analysis. Currently, there is a growing demand for the development of liquid chromatography systems for microfluidic systems.
In Chapter1, the applications of microfluidic chip-based liquid chromatography systems, as well as sample injection techniques and chromatography columns are reviewed. The recent progress in high-resolution separation approaches for droplet-based microfluidic systems is also reviewed.
In Chapter2, a microfluidic chip-based liquid chromatography system with valveless gated sample injection method and monolithic columns was developed. The valveless LC system consisted of a microchip with a separation column and a balance column, two syringe pumps and a laser induced fluorescence detector. Two monolithic columns with the same length were fabricated in the sample and separation channel served as pressure-balance column and LC separation column, respectively. Nanoliter-scale sample injection was achieved by simply switching the ON and OFF states of the syringe pumps under gated injection mode, without the need of any mechanical valve. The balance column facilitated the gated injection process by balancing the back pressure in the sample channel with that in the separation channel. Under the gated injection mode, the sample injection volumes could be varied in the range of0.4-2.4nL. The present system was applied to the fast separation of two fluorescence-labeled amines.The advantages of the present system include simple system structure, ease of operation, convenience for varying injection volume, and high sample loading capacity.
In Chapter3, the combination of droplet-based microfluidics with liquid chromatography-mass spectrometry was achieved, for providing a fast separation and high-information-content detection method for analysis of nanoliter-scale droplets with complex compositions. A novel interface method for capillary LC and droplet array chip with significant flexibility in accurate addressing and sampling of interesting droplets on demand was developed. With this method, multistep operations including parallel enzyme inhibition reactions in nanoliter droplets,4-nL sample injection, fast separation with capillary LC, and label-free detection with ESI-MS were successfully realized. The present system was further applied to the screening for inhibitors of cytochrome P450(CYPI A2) and the measurement of IC50value of the inhibitor. The sample consumption for each droplet assay was100nL, which is reduced10to100times compared with conventional384multi-well plate systems usually used in high-throughput drug screening.
引文
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