毛细管液相色谱热膨胀泵及蛋白质微分离新方法研究
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摘要
微型化是现代科学技术发展的一个趋势,在分析化学领域尤其如此。为降低样品消耗量和分析成本,提高分析灵敏度和分析速度,减少环境污染,发展微型化分析仪器,微分离分析已成为现代色谱研究的重要方向,研究热点集中于毛细管液相色谱、毛细管电泳及微流控芯片三方面,与之相配套的分析仪器及分离分析技术与方法都得到了迅猛发展。其中,微流量输液泵是微分离分析系统的关键部件,液体的微流量驱动与控制技术是实现微分离分析的前提和基础,而蛋白质的微分离分析研究有助于人们从更深层次认识生命活动的规律。目前,微流量高压输液泵是制约毛细管液相色谱发展和液相色谱微型化方面的瓶颈之一,而微量、低浓度、复杂蛋白质的分离分析也是研究者致力于解决的问题。本论文针对微流量输液泵和蛋白微分离分析研究中的热点难点问题,研制出能够连续恒流输出并用于实际操作的毛细管液相色谱热膨胀泵,发展了低浓度蛋白的固相荧光衍生检测和微量复杂蛋白双相柱分离分析新方法。全文共分五章,主要内容如下：
第一章,文献综述。本章总结了微流量输液泵及相应的液体微流量驱动与控制技术,概述了液相色谱微流泵的特殊要求和发展现状,介绍了蛋白质微分离分析常用的分离技术和检测技术,最后,阐述了本论文的选题意义。
第二章,采用液体热膨胀原理的微流量高压输液泵的基础研究。热膨胀泵无动态密封,无单向阀等活动部件,不需高精度的机械加工,成本只有同类设备的10%甚至更低,更避免了传统机械泵所固有的机械脉动、动态密封渗漏及磨损问题,无需分流可实现微流量高压输出,经济环保,同时便于实现微型化。本章系统梳理了以耐压腔体内液体体积受热膨胀为根本驱动力的微流泵的输液过程,详细分析了体积热膨胀、液体压缩及输液过程中泵腔内液体质量损失对流量输出的影响,讨论了压力对体积热膨胀系数的影响,针对实际应用引入泵初始化温度等一系列修正,发展完善了热膨胀泵输液理论及通过控制温度来精确控制微流量输出的方程,使理论脉络更清晰,方程更实用。随后,我们根据上述理论和方程,选择水为工作对象,计算确定了适合毛细管高效液相色谱流量要求的泵腔体积,使用高强度金属作为泵体材料,采用电热源直接加热法和PID温度控制系统对泵体实施高精度温度控制,加工制造出模型机,对其基础性能进行了考察并优化了热膨胀泵的加热方式和泵体设计。根据实验结果,利用液体热膨胀的原理,对于一定体积的受热液体,通过程序升温控制可以实现10-9～10-9L/min内微流量的精确输出,其输出压力可达到10 MPa以上,并有望应用于UPLC系统。我们还对微型化热膨胀泵-纳流泵进行了初步研究。
第三章,首次研制出具备连续恒流输液能力的毛细管液相色谱热膨胀泵并成功应用于nano-HPLC。该泵由四元耐压加热泵体、微流泵程控系统、切换阀、压力传感器等连接构成。我们采用上一章发展完善的热膨胀输液理论和流量输出控制方程,设计了双十通阀切换热膨胀泵系统,以“多泵联用-阀切换轮替”的方式,实现了10-5-10-9L/min范围内微流量的连续恒流输出；发明了“双压力平衡程控阀切换”技术,进一步保证了热膨胀泵运行过程中系统压力和流量输出的稳定性；在仪器化梯度生成方法的探索上也取得了有益的进展,可以实现包括台阶式梯度、线性梯度等梯度流型的微流量输出。该热膨胀微流泵系统应用于nano-HPLC显示出良好的输液性能,500 nL/min时输液稳定性RSD=4%,色谱峰保留值RSD小于2%。随后,我们进一步验证了建立以热膨胀微流泵为驱动的nano-HPLC与激光诱导荧光(LIF)检测器联用的国产分离检测系统的可行性,拓展了热膨胀微流泵的应用范围。
第四章,发展了用于低浓度蛋白样品荧光衍生的毛细管固相微反应器新方法,研究并优化了蛋白固相荧光标记的反应条件,能实现10-8M和pmol量级蛋白的衍生和检测,并将该方法成功应用于实际样品的分析。本章采用溶胶凝胶法及匀浆填充法相结合的方法,在毛细管中制备了用于蛋白荧光标记的固相载体填充床,不需精密微加工技术,采用商业色谱填料,制备方法简单易行；选择荧光素异硫氰酸酯(FITC)作为荧光衍生试剂,发展了蛋白的固相荧光衍生方法,将固相萃取与固相衍生结合起来,可以方便的处理小体积样品和低浓度样品,取得了优于传统溶液衍生的效果；发展了基于毛细管体积排阻色谱的微升级荧光衍生蛋白纯化方法,有效降低荧光小分子的干扰。该方法为拓展蛋白荧光检测在微分离分析中的应用提供了基础。
第五章,发展建立了弱阴离子交换-反相(WAX-RPLC)双相柱纳流液相色谱两维分离系统,结合高灵敏度的激光诱导荧光(LIF)检测方法,用于微量蛋白复杂体系的微分离分析。我们将两种不同的固定相——WAX和RPLC填料,分段装填在一根毛细管柱内,既充分利用两种分离模式的正交性有效提高了分离体系峰容量,又减少了两维连接中的死体积和分析系统的复杂性,避免了微量样品在多维毛细管液相色谱阀切换中的损失和复杂的接口制作工艺。本章描述了该两维系统的构建方法,以蛋白质酶解肽段混合物和微量人胚肾细胞提取蛋白为样品,建立了双相柱两维分离的工作流程,对体系的性能优化和实际分离能力进行了初步考察,具有制作简单,操作方便,灵敏度高、峰容量大的优点,为微量蛋白复杂体系的微分离分析提供了一条可行的技术路线。
综上所述,本论文围绕微流量输液泵和蛋白微分离分析研究中的热点难点问题,研制了毛细管液相色谱热膨胀泵,发展完善了热膨胀输液理论,实现了热膨胀泵高压下微流量连续恒流输出的技术突破并成功应用于nano-HPLC,有助于解决液相色谱微流泵高压输液和微型化方面的难题,也有利于我国分析仪器事业实现跨越式发展,无论在科学研究还是技术应用领域都具有重要意义；同时发展的低浓度蛋白样品固相荧光衍生方法和弱阴离子交换-反相色谱双相柱纳流高效液相色谱分离-激光诱导荧光检测方法,拓展了蛋白微分离分析手段,为实现液相色谱单细胞分析提供了重要基础。
Miniaturization is a trend of modern science and technology, especially in analytical chemistry. Separation and analysis in micro-scale has been an important task for the development of modern chromatography. In the miniaturized system, the sample consumption and analysis costs can be greatly reduced, high detection sensitivity and fast analysis speed can be obtained, environmental pollution can be minimized, and miniaturized analytical instruments would be possible. In recent years, capillary HPLC, capillary electrophoresis and micro-fluidic chip have attracted extensive research interest and developed rapidly in instruments and methods. It has been recognized that micro-flow pump is a key component of the miniaturized system, micro-fluidic driving and controlling is its basic technology, and protein separation and analysis in micro-scale would lead to a better understanding of life. A pump that can produce high pressure and constant flow at micro-flow rate range is always desired for capillary HPLC and miniaturization of HPLC. The separation and analysis of trace, low concentration and complex protein is concerned by experts. In this study, we focused on developing a novel thermal expansion pump that can produce continuous flow for capillary HPLC, inventing a solid-support florescent derivatization method for protein in low concentration and establishing a new 2D-nano-HPLC separation method employing a biphasic column for trace protein analysis. The research work in this thesis is divided into five chapters.
In Chapter 1, micro-flow pumps and technologies of micro-fluidic driving were summarized, requirements and current technologies of micro-flow pumps for capillary HPLC were included, separation methods and corresponding detection techniques commonly used for protein separation and analysis in micro-scale were introduced. The research background was demonstrated.
In Chapter 2, the basic research of thermal expansion pump (TEP, for short) was done. The TEP has almost no moving part and no mechanical consumptive hardware to change frequently. It is running in silence. Its overall costs of manufacture and routine running are very low. It is also easy to fabricate and assembly without complicated technologies. TEP utilizes the volume expansion of liquid in a heating chamber for fluid delivery, which is mainly depends on temperature elevation. The mechanism of TEP was investigated and described more clearly. Three main factors to influence the accuracy of fluidic output, including volume thermal expansion, liquid compression, and liquid mass loss in the heating chamber during output, were analyzed in detail and a(T, P)was discussed for special. Theoretical equation for controlling fluidic output of this pump by accurate temperature control had been established and pump initial temperature was taken into account for practical application. According to the theory, water was selected as the liquid medium used for TEP and chamber volume of TEP was estimated to match capillary HPLC. Copper and stainless steel were chosen as the pump material. The heating control system was set up based on PID algorithm and relevant software was completed. The prototype was fabricated and optimized in heating control and pump structure. The novel pump is capable of generating a stable and continuous flow at high pressure (above 10MPa) from nano-liters to micro-liters per minute without splitting and demonstrated by a series of experiments. It is expected to apply in the UPLC system. We also explored its miniature potential.
In Chapter 3, we developed, for the first time, a TEPs system that is capable of generating continuous flow for capillary HPLC, and applied it in nano-HPLC system coupled with laser induced fluorescence detector (LIF). The TEPs system employing two groups of thermal expansion pumps (TEPs) working by turns were fabricated, and a controlling strategy for the pump system to maintain a continuously delivery without pressure fluctuation even at switching point was also developed. Both isocratic and gradients of binary solvent delivery by the TEPs were performed. Reproducibility and standard deviation at different flow rates were determined. A novel nano-HPLC system employing the TEPs system was set up for the first time. A result of RSD=4% for flow and RSD=2% for retention times at 500 nL/min was achieved. We have successfully established a nano-HPLC-LIF system, consisting of the TEPs system as the main innovation.
In Chapter 4, a method based on solid-support reaction was initially developed to realize fluorescent derivatization of picomoles of protein at concentrations as low as 10-8 M, and the method has been successfully applied to real samples. A simple, low-cost homemade capillary C18 cartrideges was fabricated as the solid-support reactor with Sol-gel and slurry packing methods. FITC was selected as fluorescent reagent and effects of reaction conditions on solid-support has been evaluated and optimized. Fluorescent derivatiozation of protein in 10-8 M with FITC on solid-support were realized for the first time. Compared with solution derivatization, lower detection limit of protein can be obtained by this method. And the use of the capillary solid-support reactor allows easy handling of protein with small volume. We also developed a method based on capillary-size exclusion chromatography (cSEC) for fluorescent labeled protein purification withμL volume, effectively reducing the interference of fluorescent intruders in analysis.
In Chapter 5, a novel nano-HPLC system for two-dimensional separation was first reported, which coupling weak anion exchange chromatography (WAX) and reverse phase chromatography (RPLC) by employing a biphasic column. Combining with high sensitive laser-induced fluorescence (LIF) detection method, the system was applied for trace complex protein analysis. We packed WAX resin and RP C18 particles in a fused silica capillary column (100μm i.d.) in sequence. There are many advantage by using this biphasic column:1. the flow rate was decreased, thus the sensitivity of system was increased; 2. WAX separated by charge and RP separated by hydrophobicity, the two dimensions of chromatography were orthogonal, which improved peak capacity of system effectively; 3. the unique biphasic column effectively eliminated the valves and dead volume commonly associated with complex chromatography. In our initial research, we described the structure and separation process of this system, optimized and demonstrated its detection sensitive and separation capability roughly for trace complex peptides and proteins analysis.
In summary, this thesis focuses on the key problems of separation and analysis in micro-scale. We initially developed a novel thermal expansion pump for capillary HPLC, which is capable of generating continuous flow, and utilized it in fabrication of nano-HPLC-LIF system. This work will bring more breakthroughs both in scientific research and technical applications. We also developed a method of solid-support fluorescent derivatization for protein in low concentration, and established a nano-HPLC-LIF system employing WAX-RPLC biphasic column for trace complex protein analysis. Both of them provide an important foundation to realize the single-cell analysis by liquid chromatography in the future.
第一章,文献综述。本章总结了微流量输液泵及相应的液体微流量驱动与控制技术,概述了液相色谱微流泵的特殊要求和发展现状,介绍了蛋白质微分离分析常用的分离技术和检测技术,最后,阐述了本论文的选题意义。
第二章,采用液体热膨胀原理的微流量高压输液泵的基础研究。热膨胀泵无动态密封,无单向阀等活动部件,不需高精度的机械加工,成本只有同类设备的10%甚至更低,更避免了传统机械泵所固有的机械脉动、动态密封渗漏及磨损问题,无需分流可实现微流量高压输出,经济环保,同时便于实现微型化。本章系统梳理了以耐压腔体内液体体积受热膨胀为根本驱动力的微流泵的输液过程,详细分析了体积热膨胀、液体压缩及输液过程中泵腔内液体质量损失对流量输出的影响,讨论了压力对体积热膨胀系数的影响,针对实际应用引入泵初始化温度等一系列修正,发展完善了热膨胀泵输液理论及通过控制温度来精确控制微流量输出的方程,使理论脉络更清晰,方程更实用。随后,我们根据上述理论和方程,选择水为工作对象,计算确定了适合毛细管高效液相色谱流量要求的泵腔体积,使用高强度金属作为泵体材料,采用电热源直接加热法和PID温度控制系统对泵体实施高精度温度控制,加工制造出模型机,对其基础性能进行了考察并优化了热膨胀泵的加热方式和泵体设计。根据实验结果,利用液体热膨胀的原理,对于一定体积的受热液体,通过程序升温控制可以实现10-9～10-9L/min内微流量的精确输出,其输出压力可达到10 MPa以上,并有望应用于UPLC系统。我们还对微型化热膨胀泵-纳流泵进行了初步研究。
第三章,首次研制出具备连续恒流输液能力的毛细管液相色谱热膨胀泵并成功应用于nano-HPLC。该泵由四元耐压加热泵体、微流泵程控系统、切换阀、压力传感器等连接构成。我们采用上一章发展完善的热膨胀输液理论和流量输出控制方程,设计了双十通阀切换热膨胀泵系统,以“多泵联用-阀切换轮替”的方式,实现了10-5-10-9L/min范围内微流量的连续恒流输出；发明了“双压力平衡程控阀切换”技术,进一步保证了热膨胀泵运行过程中系统压力和流量输出的稳定性；在仪器化梯度生成方法的探索上也取得了有益的进展,可以实现包括台阶式梯度、线性梯度等梯度流型的微流量输出。该热膨胀微流泵系统应用于nano-HPLC显示出良好的输液性能,500 nL/min时输液稳定性RSD=4%,色谱峰保留值RSD小于2%。随后,我们进一步验证了建立以热膨胀微流泵为驱动的nano-HPLC与激光诱导荧光(LIF)检测器联用的国产分离检测系统的可行性,拓展了热膨胀微流泵的应用范围。
第四章,发展了用于低浓度蛋白样品荧光衍生的毛细管固相微反应器新方法,研究并优化了蛋白固相荧光标记的反应条件,能实现10-8M和pmol量级蛋白的衍生和检测,并将该方法成功应用于实际样品的分析。本章采用溶胶凝胶法及匀浆填充法相结合的方法,在毛细管中制备了用于蛋白荧光标记的固相载体填充床,不需精密微加工技术,采用商业色谱填料,制备方法简单易行；选择荧光素异硫氰酸酯(FITC)作为荧光衍生试剂,发展了蛋白的固相荧光衍生方法,将固相萃取与固相衍生结合起来,可以方便的处理小体积样品和低浓度样品,取得了优于传统溶液衍生的效果；发展了基于毛细管体积排阻色谱的微升级荧光衍生蛋白纯化方法,有效降低荧光小分子的干扰。该方法为拓展蛋白荧光检测在微分离分析中的应用提供了基础。
第五章,发展建立了弱阴离子交换-反相(WAX-RPLC)双相柱纳流液相色谱两维分离系统,结合高灵敏度的激光诱导荧光(LIF)检测方法,用于微量蛋白复杂体系的微分离分析。我们将两种不同的固定相——WAX和RPLC填料,分段装填在一根毛细管柱内,既充分利用两种分离模式的正交性有效提高了分离体系峰容量,又减少了两维连接中的死体积和分析系统的复杂性,避免了微量样品在多维毛细管液相色谱阀切换中的损失和复杂的接口制作工艺。本章描述了该两维系统的构建方法,以蛋白质酶解肽段混合物和微量人胚肾细胞提取蛋白为样品,建立了双相柱两维分离的工作流程,对体系的性能优化和实际分离能力进行了初步考察,具有制作简单,操作方便,灵敏度高、峰容量大的优点,为微量蛋白复杂体系的微分离分析提供了一条可行的技术路线。
综上所述,本论文围绕微流量输液泵和蛋白微分离分析研究中的热点难点问题,研制了毛细管液相色谱热膨胀泵,发展完善了热膨胀输液理论,实现了热膨胀泵高压下微流量连续恒流输出的技术突破并成功应用于nano-HPLC,有助于解决液相色谱微流泵高压输液和微型化方面的难题,也有利于我国分析仪器事业实现跨越式发展,无论在科学研究还是技术应用领域都具有重要意义；同时发展的低浓度蛋白样品固相荧光衍生方法和弱阴离子交换-反相色谱双相柱纳流高效液相色谱分离-激光诱导荧光检测方法,拓展了蛋白微分离分析手段,为实现液相色谱单细胞分析提供了重要基础。
Miniaturization is a trend of modern science and technology, especially in analytical chemistry. Separation and analysis in micro-scale has been an important task for the development of modern chromatography. In the miniaturized system, the sample consumption and analysis costs can be greatly reduced, high detection sensitivity and fast analysis speed can be obtained, environmental pollution can be minimized, and miniaturized analytical instruments would be possible. In recent years, capillary HPLC, capillary electrophoresis and micro-fluidic chip have attracted extensive research interest and developed rapidly in instruments and methods. It has been recognized that micro-flow pump is a key component of the miniaturized system, micro-fluidic driving and controlling is its basic technology, and protein separation and analysis in micro-scale would lead to a better understanding of life. A pump that can produce high pressure and constant flow at micro-flow rate range is always desired for capillary HPLC and miniaturization of HPLC. The separation and analysis of trace, low concentration and complex protein is concerned by experts. In this study, we focused on developing a novel thermal expansion pump that can produce continuous flow for capillary HPLC, inventing a solid-support florescent derivatization method for protein in low concentration and establishing a new 2D-nano-HPLC separation method employing a biphasic column for trace protein analysis. The research work in this thesis is divided into five chapters.
In Chapter 1, micro-flow pumps and technologies of micro-fluidic driving were summarized, requirements and current technologies of micro-flow pumps for capillary HPLC were included, separation methods and corresponding detection techniques commonly used for protein separation and analysis in micro-scale were introduced. The research background was demonstrated.
In Chapter 2, the basic research of thermal expansion pump (TEP, for short) was done. The TEP has almost no moving part and no mechanical consumptive hardware to change frequently. It is running in silence. Its overall costs of manufacture and routine running are very low. It is also easy to fabricate and assembly without complicated technologies. TEP utilizes the volume expansion of liquid in a heating chamber for fluid delivery, which is mainly depends on temperature elevation. The mechanism of TEP was investigated and described more clearly. Three main factors to influence the accuracy of fluidic output, including volume thermal expansion, liquid compression, and liquid mass loss in the heating chamber during output, were analyzed in detail and a(T, P)was discussed for special. Theoretical equation for controlling fluidic output of this pump by accurate temperature control had been established and pump initial temperature was taken into account for practical application. According to the theory, water was selected as the liquid medium used for TEP and chamber volume of TEP was estimated to match capillary HPLC. Copper and stainless steel were chosen as the pump material. The heating control system was set up based on PID algorithm and relevant software was completed. The prototype was fabricated and optimized in heating control and pump structure. The novel pump is capable of generating a stable and continuous flow at high pressure (above 10MPa) from nano-liters to micro-liters per minute without splitting and demonstrated by a series of experiments. It is expected to apply in the UPLC system. We also explored its miniature potential.
In Chapter 3, we developed, for the first time, a TEPs system that is capable of generating continuous flow for capillary HPLC, and applied it in nano-HPLC system coupled with laser induced fluorescence detector (LIF). The TEPs system employing two groups of thermal expansion pumps (TEPs) working by turns were fabricated, and a controlling strategy for the pump system to maintain a continuously delivery without pressure fluctuation even at switching point was also developed. Both isocratic and gradients of binary solvent delivery by the TEPs were performed. Reproducibility and standard deviation at different flow rates were determined. A novel nano-HPLC system employing the TEPs system was set up for the first time. A result of RSD=4% for flow and RSD=2% for retention times at 500 nL/min was achieved. We have successfully established a nano-HPLC-LIF system, consisting of the TEPs system as the main innovation.
In Chapter 4, a method based on solid-support reaction was initially developed to realize fluorescent derivatization of picomoles of protein at concentrations as low as 10-8 M, and the method has been successfully applied to real samples. A simple, low-cost homemade capillary C18 cartrideges was fabricated as the solid-support reactor with Sol-gel and slurry packing methods. FITC was selected as fluorescent reagent and effects of reaction conditions on solid-support has been evaluated and optimized. Fluorescent derivatiozation of protein in 10-8 M with FITC on solid-support were realized for the first time. Compared with solution derivatization, lower detection limit of protein can be obtained by this method. And the use of the capillary solid-support reactor allows easy handling of protein with small volume. We also developed a method based on capillary-size exclusion chromatography (cSEC) for fluorescent labeled protein purification withμL volume, effectively reducing the interference of fluorescent intruders in analysis.
In Chapter 5, a novel nano-HPLC system for two-dimensional separation was first reported, which coupling weak anion exchange chromatography (WAX) and reverse phase chromatography (RPLC) by employing a biphasic column. Combining with high sensitive laser-induced fluorescence (LIF) detection method, the system was applied for trace complex protein analysis. We packed WAX resin and RP C18 particles in a fused silica capillary column (100μm i.d.) in sequence. There are many advantage by using this biphasic column:1. the flow rate was decreased, thus the sensitivity of system was increased; 2. WAX separated by charge and RP separated by hydrophobicity, the two dimensions of chromatography were orthogonal, which improved peak capacity of system effectively; 3. the unique biphasic column effectively eliminated the valves and dead volume commonly associated with complex chromatography. In our initial research, we described the structure and separation process of this system, optimized and demonstrated its detection sensitive and separation capability roughly for trace complex peptides and proteins analysis.
In summary, this thesis focuses on the key problems of separation and analysis in micro-scale. We initially developed a novel thermal expansion pump for capillary HPLC, which is capable of generating continuous flow, and utilized it in fabrication of nano-HPLC-LIF system. This work will bring more breakthroughs both in scientific research and technical applications. We also developed a method of solid-support fluorescent derivatization for protein in low concentration, and established a nano-HPLC-LIF system employing WAX-RPLC biphasic column for trace complex protein analysis. Both of them provide an important foundation to realize the single-cell analysis by liquid chromatography in the future.
引文
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