基于微流控芯片的免疫传感器和蛋白质组学研究
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摘要
微流控芯片已经在生物化学、医学检验、药物合成筛选、环境监测等领域得到了广泛的应用。微流控芯片结合了生物技术和微电子加工技术等,将实验室中多种仪器的功能集成到芯片上进行处理,具有微型化、检测效率高、操作简单、检测成本低、易于集成化等优点。目前普遍使用的微流控芯片的制作材料是高分子聚合材料,但是其仍存在非特异性吸附等问题。研究结果表明,对高分子材料表面进行修饰改性可以改善它的性质,使聚合物芯片更有利于实际生物样品分析。本论文以高分子聚合物芯片为分析平台,通过有效的修饰方法对微流控芯片通道表面进行修饰和改性,成功实现了芯片通道内生物分子的固定,为生物分析提供了新的研究方法。
论文的第一章综述了微流控芯片加工方法、制作材料、检测技术和实际应用等方面的研究进展,并强调了对微流控芯片通道表面进行改性和修饰的必要性和重要性；同时,详细介绍了免疫分析、生物传感器和蛋白质组学的发展状况、主要研究内容和应用领域；最后总结了本论文研究的目的和意义。
本论文研究工作分为三章,以聚甲基丙烯酸甲酯(PMMA)和聚对苯二甲酸乙二醇酯(PET)微流控芯片作为研究对象,分别采用不同的表面修饰方法,包括聚乙烯亚胺衍生法、纳米金胶层层组装法、氧化铝溶胶-凝胶法,将S100B蛋白抗体、核酸适配体和胰蛋白酶有效地固定在微流控芯片通道表面,发展了以微流控芯片为技术平台的酶联免疫分析、核酸适配体特异识别蛋白定量分析以及酶反应器用于蛋白鉴定的新方法。具体内容如下：
一、基于核酸适配体的微流控芯片用于生物传感器的分析研究
本工作将核酸适配体(aptamer)固定在纳米金颗粒修饰的PMMA微流控芯片通道的内表面,利用筛选出来的核酸适配体能够高度特异性地识别人凝血酶蛋白的特点,进而将凝血酶蛋白捕获在芯片表面,同时凝血酶蛋白再与标记有生物素的二级核酸适配体相结合,最后利用生物素-亲和素特异性相互作用的性质,将碱性磷酸酶固定,碱性磷酸酶可以催化底物产生灵敏的电化学信号,从而达到检测人凝血酶蛋白的目的。本方法同时结合了微流控芯片反应速度快、耗样量少、灵敏度高等优点,最后测得的人凝血酶蛋白的检出限可以达到1pM,线性范围宽,从1pM-100pM,将该方法用于实际人血清样品中凝血酶蛋白的检测,结果令人满意。
二、微流控芯片用于脑损伤标志物S100B蛋白的高灵敏度分析检测
在本工作中,我们发展了一种基于微流控芯片的电化学免疫传感器用于低浓度的脑损伤标志物S100B的分析检测。利用聚乙烯亚胺(PEI)对聚甲基丙烯酸甲酯(PMMA)芯片通道表面进行氨基化修饰,用于S100B蛋白抗体的固定。PEI是一种具有丰富氨基官能团的高分子聚合物,它能够覆盖在PMMA表面引入氨基官能团,因此被广泛用于蛋白质和DNA的固定。S100B蛋白抗体固定后,通过抗原-抗体特异性反应,将S100B蛋白和碱性磷酸酶标记的二抗依次结合在一起,形成了微流控芯片通道内的具有三明治夹心结构的酶联免疫反应体系。在芯片微通道的末端,利用三电极电化学检测体系记录下用碱性磷酸酶催化4-氨基苯基磷酸盐(PAPP)时产生的还原电流信号,从而检测S100B蛋白的浓度,检出限低至0.1pgmL-1,线性范围为0.1pg mL-1-100pg mL-1.整个温育反应过程和检测过程被集成在微芯片装置上,具有高灵敏度、特异性和分析时间短等优点。因此,该方法为临床免疫检验提供了一种新的多功能的检测平台。
三、氧化铝溶胶-凝胶修饰微流控芯片酶反应器用于低丰度蛋白的酶解鉴定
本工作通过材料表面酯键的水解作用,产生-OH、-COOH等活性基团,进一步通过它们和溶胶-凝胶缩合反应,在聚对苯二甲酸乙二醇酯(PET)芯片通道内部构建了溶胶-凝胶网络结构,实现了胰蛋白酶的包埋固定,制备了A12O3溶胶-凝胶修饰的PET芯片酶反应器,用于低丰度蛋白的检测。A12O3溶胶-凝胶固定在PET微流控芯片的通道表面,提供了一个具有亲水性和生物相容性的界面,用于胰蛋白酶的包埋固定。由于将大量的胰蛋白酶限定在了狭小的微通道中,痕量的标准蛋白在几秒内可以在芯片酶反应器上完成酶解步骤。我们从鼠肝细胞中提取的复杂样品来评估A12O3溶胶-凝胶微酶反应器酶解效能以及应用的可行性,得到了较好的结果。这种蛋白的快速酶解鉴定方法的好处在于方法简单,易于操作,有望在蛋白质组学中得到进一步应用,实现快速、高效、在线的复杂蛋白样品的酶解鉴定。
Microfluidic chips have been widely recognized as a powerful technology that will play an important role in future biological analysis to meet the large-scale and high-throughput requirements. Their miniaturized architectures provide a number of distinct advantages, such as high sample processing rates, low manufacturing costs, advanced system integration, and reduced volumes of samples and analytes.
In chapter 1, recent research progress on microfluidic chips, including fabrication techniques, materials, detection system and analytical applications, is described. In the end of this part, the purpose and significance of our study on microfluidics-based bioanalytical application are emphasized.
In my research, by using surface modification approaches, such as PEI, AuNPs nanoparticles assembly, and A12O3 sol-gel. By using these methods, the antibodies, aptamer or protease can be effectively immobilized/adsorbed within the microchannels to devise microfluidic immunoassay or enzymatic reactors combined with electrochemistry method or mass spectrometry; the microchips are capable of performing the analysis of proteins in real samples. The detailed points of this thesis are expanded as follows:
1) In this work, we designed an aptamer-based microfluidic chip biosensor and applied it to the detection of low-level human thrombin. Gold nanoparticles are coating on the surface of the modified PMMA microchannel, and sulfhydryl-labeled first-rate aptamer is fixed on AuNPs. Through the specific binding of aptamer and thrombin, the thrombin protein and biotin-labeled second-rate aptamer are connected, and ALP-labeled avidin is linked to biotin. DPV is used to detect the electronic signals from the substrate solution of PAPP and a low detection limit of 1pM has obtained. The biosensor showed high sensitivity, selectivity and broad linear response. The results indicate that the microfluidic chip combined with apatmer-protein interaction has great potential in clinical diagnosis detection.
2) A simple and sensitive microchip-based method has been proposed to determine low level of S100B, biomarker of neurological disease, coupled with electrochemical detection system. Firstly, poly(ethyleneimine) (PEI) was applied to modify the poly(methyl methacrylate) (PMMA) microchannels. PEI contained abundant NH2 groups which can covalently immobilize S100B monoclonal antibody in the next step. Afterward, the antigen S100B and polyclonal rabbit anti-S100 were sequentially immobilized through antigen-antibody specific interaction. Finally, the anti-rabbit IgG alkaline phosphatase conjugate (ALP-conjugate) was bound to the polyclonal rabbit anti-S100 in microchannnel. Using three-electrode electrochemical detection system, the microchip-based immunosensor had the detection limit of S100B down to 0.1 pg mL-1, and achieved a detectable linear concentration range of 0.1 pg mL-1～100pg mL-1 by differential pulse voltammetry (DPV). The on-chip immunosensor can not only provide rapid and sensitive detection for target proteins but also be capable of resisting to non-specific adsorption of proteins. The result of the experiment shows that the proposed approach is highly sensitive and specific, which is feasible and has the potential application in clinical analysis and diagnosis.
3) An on-chip microreactor for highly efficient proteolysis has been demonstrated using BSA, myoglobin and cytochrome c as model substrates. Alumina sol-gel exhibits a simple surface modification protocol in PET microchannels for enzyme immobilization. The standard proteins were confidently identified with a low femtomole per analysis at a concentration of 0.5ngμL-1 with the digestion time less than few seconds. Our findings also suggest that this on-chip microreactor can be used for the detection of proteins from real biological samples. This versatile system is a good example of proteolytic reactions occurring in the alumina gel-derived microchannels, where the network not only provides a support for encapsulation of enzymes, but also acts as microreactor to facilitate the protein digestion. Such on-chip enzymatic reactor coupled to MALDI-TOF-MS/MS or 2D-LC-ESI-MS/MS systems could be applied to profile the complex protein extracts in proteomic research.
论文的第一章综述了微流控芯片加工方法、制作材料、检测技术和实际应用等方面的研究进展,并强调了对微流控芯片通道表面进行改性和修饰的必要性和重要性；同时,详细介绍了免疫分析、生物传感器和蛋白质组学的发展状况、主要研究内容和应用领域；最后总结了本论文研究的目的和意义。
本论文研究工作分为三章,以聚甲基丙烯酸甲酯(PMMA)和聚对苯二甲酸乙二醇酯(PET)微流控芯片作为研究对象,分别采用不同的表面修饰方法,包括聚乙烯亚胺衍生法、纳米金胶层层组装法、氧化铝溶胶-凝胶法,将S100B蛋白抗体、核酸适配体和胰蛋白酶有效地固定在微流控芯片通道表面,发展了以微流控芯片为技术平台的酶联免疫分析、核酸适配体特异识别蛋白定量分析以及酶反应器用于蛋白鉴定的新方法。具体内容如下：
一、基于核酸适配体的微流控芯片用于生物传感器的分析研究
本工作将核酸适配体(aptamer)固定在纳米金颗粒修饰的PMMA微流控芯片通道的内表面,利用筛选出来的核酸适配体能够高度特异性地识别人凝血酶蛋白的特点,进而将凝血酶蛋白捕获在芯片表面,同时凝血酶蛋白再与标记有生物素的二级核酸适配体相结合,最后利用生物素-亲和素特异性相互作用的性质,将碱性磷酸酶固定,碱性磷酸酶可以催化底物产生灵敏的电化学信号,从而达到检测人凝血酶蛋白的目的。本方法同时结合了微流控芯片反应速度快、耗样量少、灵敏度高等优点,最后测得的人凝血酶蛋白的检出限可以达到1pM,线性范围宽,从1pM-100pM,将该方法用于实际人血清样品中凝血酶蛋白的检测,结果令人满意。
二、微流控芯片用于脑损伤标志物S100B蛋白的高灵敏度分析检测
在本工作中,我们发展了一种基于微流控芯片的电化学免疫传感器用于低浓度的脑损伤标志物S100B的分析检测。利用聚乙烯亚胺(PEI)对聚甲基丙烯酸甲酯(PMMA)芯片通道表面进行氨基化修饰,用于S100B蛋白抗体的固定。PEI是一种具有丰富氨基官能团的高分子聚合物,它能够覆盖在PMMA表面引入氨基官能团,因此被广泛用于蛋白质和DNA的固定。S100B蛋白抗体固定后,通过抗原-抗体特异性反应,将S100B蛋白和碱性磷酸酶标记的二抗依次结合在一起,形成了微流控芯片通道内的具有三明治夹心结构的酶联免疫反应体系。在芯片微通道的末端,利用三电极电化学检测体系记录下用碱性磷酸酶催化4-氨基苯基磷酸盐(PAPP)时产生的还原电流信号,从而检测S100B蛋白的浓度,检出限低至0.1pgmL-1,线性范围为0.1pg mL-1-100pg mL-1.整个温育反应过程和检测过程被集成在微芯片装置上,具有高灵敏度、特异性和分析时间短等优点。因此,该方法为临床免疫检验提供了一种新的多功能的检测平台。
三、氧化铝溶胶-凝胶修饰微流控芯片酶反应器用于低丰度蛋白的酶解鉴定
本工作通过材料表面酯键的水解作用,产生-OH、-COOH等活性基团,进一步通过它们和溶胶-凝胶缩合反应,在聚对苯二甲酸乙二醇酯(PET)芯片通道内部构建了溶胶-凝胶网络结构,实现了胰蛋白酶的包埋固定,制备了A12O3溶胶-凝胶修饰的PET芯片酶反应器,用于低丰度蛋白的检测。A12O3溶胶-凝胶固定在PET微流控芯片的通道表面,提供了一个具有亲水性和生物相容性的界面,用于胰蛋白酶的包埋固定。由于将大量的胰蛋白酶限定在了狭小的微通道中,痕量的标准蛋白在几秒内可以在芯片酶反应器上完成酶解步骤。我们从鼠肝细胞中提取的复杂样品来评估A12O3溶胶-凝胶微酶反应器酶解效能以及应用的可行性,得到了较好的结果。这种蛋白的快速酶解鉴定方法的好处在于方法简单,易于操作,有望在蛋白质组学中得到进一步应用,实现快速、高效、在线的复杂蛋白样品的酶解鉴定。
Microfluidic chips have been widely recognized as a powerful technology that will play an important role in future biological analysis to meet the large-scale and high-throughput requirements. Their miniaturized architectures provide a number of distinct advantages, such as high sample processing rates, low manufacturing costs, advanced system integration, and reduced volumes of samples and analytes.
In chapter 1, recent research progress on microfluidic chips, including fabrication techniques, materials, detection system and analytical applications, is described. In the end of this part, the purpose and significance of our study on microfluidics-based bioanalytical application are emphasized.
In my research, by using surface modification approaches, such as PEI, AuNPs nanoparticles assembly, and A12O3 sol-gel. By using these methods, the antibodies, aptamer or protease can be effectively immobilized/adsorbed within the microchannels to devise microfluidic immunoassay or enzymatic reactors combined with electrochemistry method or mass spectrometry; the microchips are capable of performing the analysis of proteins in real samples. The detailed points of this thesis are expanded as follows:
1) In this work, we designed an aptamer-based microfluidic chip biosensor and applied it to the detection of low-level human thrombin. Gold nanoparticles are coating on the surface of the modified PMMA microchannel, and sulfhydryl-labeled first-rate aptamer is fixed on AuNPs. Through the specific binding of aptamer and thrombin, the thrombin protein and biotin-labeled second-rate aptamer are connected, and ALP-labeled avidin is linked to biotin. DPV is used to detect the electronic signals from the substrate solution of PAPP and a low detection limit of 1pM has obtained. The biosensor showed high sensitivity, selectivity and broad linear response. The results indicate that the microfluidic chip combined with apatmer-protein interaction has great potential in clinical diagnosis detection.
2) A simple and sensitive microchip-based method has been proposed to determine low level of S100B, biomarker of neurological disease, coupled with electrochemical detection system. Firstly, poly(ethyleneimine) (PEI) was applied to modify the poly(methyl methacrylate) (PMMA) microchannels. PEI contained abundant NH2 groups which can covalently immobilize S100B monoclonal antibody in the next step. Afterward, the antigen S100B and polyclonal rabbit anti-S100 were sequentially immobilized through antigen-antibody specific interaction. Finally, the anti-rabbit IgG alkaline phosphatase conjugate (ALP-conjugate) was bound to the polyclonal rabbit anti-S100 in microchannnel. Using three-electrode electrochemical detection system, the microchip-based immunosensor had the detection limit of S100B down to 0.1 pg mL-1, and achieved a detectable linear concentration range of 0.1 pg mL-1～100pg mL-1 by differential pulse voltammetry (DPV). The on-chip immunosensor can not only provide rapid and sensitive detection for target proteins but also be capable of resisting to non-specific adsorption of proteins. The result of the experiment shows that the proposed approach is highly sensitive and specific, which is feasible and has the potential application in clinical analysis and diagnosis.
3) An on-chip microreactor for highly efficient proteolysis has been demonstrated using BSA, myoglobin and cytochrome c as model substrates. Alumina sol-gel exhibits a simple surface modification protocol in PET microchannels for enzyme immobilization. The standard proteins were confidently identified with a low femtomole per analysis at a concentration of 0.5ngμL-1 with the digestion time less than few seconds. Our findings also suggest that this on-chip microreactor can be used for the detection of proteins from real biological samples. This versatile system is a good example of proteolytic reactions occurring in the alumina gel-derived microchannels, where the network not only provides a support for encapsulation of enzymes, but also acts as microreactor to facilitate the protein digestion. Such on-chip enzymatic reactor coupled to MALDI-TOF-MS/MS or 2D-LC-ESI-MS/MS systems could be applied to profile the complex protein extracts in proteomic research.
引文
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