高聚物微流控芯片及固定化酶芯片的研究
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摘要
21世纪被称为“生物工程的时代”和“高信息化时代”,科学技术将获得更加迅猛的发展。分析化学将处于广泛的、深刻的、激烈的巨大变革时期，不断向微型化、自动化、仿生化、信息化等方向发展，微流控芯片技术将成为其重要的发展方向。微流控芯片的目的是通过化学分析设备的微型化与集成化，最大限度的把分析实验室的功能集成到几平方厘米的芯片上。在蛋白质组学研究正成为基础科学的研究的前沿时代，微流控芯片在蛋白质组学中的应用将具有更深刻的意义。微流控芯片技术只需要很微量的蛋白质样品即可在芯片上实现在线酶解、分离、富集、检测。本论文通过紫外光照射的方法使聚二甲基硅氧烷（PDMS）微流控芯片的微通道内壁产生自由基，从而将丙烯酸单体嫁接，再分别通过聚二烯丙基二甲基氯化铵（PDDA）和介孔材料将酶固定在芯片微通道中，实现了蛋白质在PDMS微流控芯片上的酶解。这两种方法固定的酶都具有很高的活性和稳定性。该结果建立了芯片在线酶解的新技术平台。
在第三章中研究了PDMS微流控芯片的制作问题。由PDMS基片直接封装的微流控芯片在实验过程中很容易发生渗漏现象。通过氧等离子体处理PDMS基片使其表面产生活性基团-OH，两基片永久性的粘合从而制得PDMS微流控芯片，但是氧等离子体发生器的价格昂贵，反应的参数也很难控制，而且产生的自由基团很不稳定。本文利用自制的简单模具发展了一种制作一体化PDMS微流控芯片的新技术，避免了芯片在粘合过程中出现的问题。
第四章中研究了两种在一体化微流控芯片通道中固定化酶的方法。这两种方法首先都是通过紫外光处理PDMS使其表面产生自由基团，从而
嫁接丙烯酸单体。第一种方法是通过PDDA将酶固定。PDDA是一种聚阳离子物质，在很宽的pH范围内都会电离生成季铵阳离子而带正电荷与丙烯酸单体层静电吸附，胰蛋白酶（trypsin）通过PDDA被固定在芯片的微通道中。第二种方法利用介孔材料的大量-OH将酶固定。蛋白质溶液以10 μL/s的速度流过PDMS微流控芯片已固定了trypsin的通道，收集酶解溶液用质谱、毛细管电泳方法检测。结果证明这两种方法固定的酶都具有很高的活性和稳定性。
本论文通过反射红外光谱分析检测到PDMS基片在经过丙烯酸处理后表面有很强的-COOH特征吸收峰，表征了丙烯酸单体嫁接到PDMS基片表面。通过扫描电镜测定、接触角测定、FITC标记等方法说明经过丙烯酸单体修饰后的基片可以分别通过聚阳离子物质PDDA和介孔材料将trypsin固定。
纯蛋白质溶液经过毛细管电泳分离检测只有一个峰，而通过芯片微通道后的溶液经过毛细管电泳分离检测，电泳谱图检测出许多小峰，这些小峰是蛋白质在芯片上酶解产生的小肽段。酶解溶液经过MALDI-TOF/TOF-MS检测，经 Mascot搜索，所得的序列覆盖率、匹配的肽段、蛋白得分都很高，与文献报道的溶液酶解方法得到的结果一致，也证实了本文报道的两种方法固定的酶都具有很高的活性。用PDDA方法固定有酶的PDMS微流控芯片在冰箱中放置12天后，蛋白质在芯片上酶解溶液的质谱检测结果与前一次测定结果没有差别，证明该方法固定的酶具有很高的稳定性。介孔材料由于具有较大的比表面积和孔体积，孔径均一且在纳米尺寸可调，形貌可以控制，表面容易官能团化等特点，显示出在生物大分子固定分离等方面将会具有很广泛的应用前景。本文通过检测各种介孔材料对酶的吸附量，选择了一种经过微波处理的介孔材料，并应用于PDMS微流控芯片固定化酶，蛋白质溶液在该芯片上酶解溶液经
过质谱检测，结果证实介孔材料在微流控芯片固定化酶方面具有广阔的应用前景。
本论文的研究成果有：
1、利用自制的简单模具提出了一种制作一体化PDMS微流控芯片的新技术；
2、研究出两种在PDMS微流控芯片上固定胰蛋白酶的新方法，第一种方法是通过聚阳离子物质PDDA将酶固定，第二种方法是利用介孔材料将酶固定；
3、实现了蛋白质在PDMS微流控芯片上的酶解，建立了芯片在线酶解的新技术平台。
期望进一步的研究能够实现蛋白质等生物样品在芯片上的在线预处理、分离、酶解、检测等，从而为蛋白质组学的研究提供一种快速、高效、高通量、高灵敏度的技术平台。
Following the rising demands for monitoring of large number of bioaffinity interactions or biomarkers at cell lever, the development of miniaturized , high throughput chemical systems has grown dramatically in recent years. In 1990s, Manz introduced the concept of micro total analysis systems(μTAS). The field of microfluidic chip has become a growing research area in the last decade for creating inexpensive, minute sample volumes, fast and parallel analytical tools applied to different fields such as DNA, protein digestion, separation and detection. In the first chapter, a summarized development in this field was reviewed.
In chapter 3, made of microfluidic chip was described. Poly(dimethylsiloxane) (PDMS) was selected as the chip material owing to its excellent characters including chemical inertness, low polarity, low electrical conductivity and elasticity. Oxygen plasma treatment is the traditional method to seal the PDMS films, but the surface treatment involves very complex mechanisms in response to the plasma type, material, atmosphere so that the operation conditions can’t be controlled easily, in addition the oxygen plasma generator is expensive. In this chapter a simple method to make intergraded PDMS microfluidic chip using a lab made mold was reported.
In chapter 4, two methods for immobilization of trypsin into the integrated PDMS chip microchannel were proposed. Acrylic acid(AA) was first grafted onto PDMS to yield hydrophilic surface by exposing to UV light, and then PDDA was electrostatically absorbed on the AA layer for
self-assembly of trypsin. The other method is that trypsin was immobilized by mesoporous material(FDU-1), then filled the mesoporous material into the microchannel, the mesoporous material was absorbed by AA. Protein solution was flowed in the microchannel with a rate of 10μL/s and collected, then detected by CE and MALDI-TOF/TOF-MS.
In this paper immobilization of enzyme in the microchannel was carried out via modifying the surface of PDMS microfluidic chip. The modified PDMS surface was characterized by the total attenuated reflection IR spectrometry, contact angle and image of scanning electron microscope.
Mesoporous materials have potential applications in bio-molecule separation and immobilization by controlling the size, dimension of the pore, modifying chemical groups in the pore and/or on the surface. In this paper, one kind of mesoporous material was selected for the application to the study of immobilization enzyme in microfluidic chip.
The selected protein solution was flowed through the microchannel then detected by MALDI-TOF/TOF-MS and CE. Only one peak was presented on the electrograms of CE in case of detecting the pure protein solution, while many peaks were occurred when the selected protein solution was detected. Many peaks of digested peptides were appeared on the MS spectrum. With Mascot search of the data, high sequence coverage and protein scores were obtained. The results prove the activity of the immobilized enzyme.
Microfluidic chip has presented a developing perspective in chemical monitoring. Chip based microsystems have achieved a great progress in the fields of Genomic, Proteomic, Clinical and Forensic analysis. Proteomic analysis has become more and more important with the end of gene sequence
detection. For the proteomic analysis, study of the sequence of protein is very important. A key of the techniques for the protein sequence detection is digested by enzyme. In this paper two novel methods have been provided for immobilization of enzyme in the microfluidic chip channel. Protein can be on line digested in the microchannel quickly at room temperature. This micro device can be combined with various kinds of detection approaches including CE separation, MALDI-TOF/TOF-MS, LIF. In summary, a new, sensitive, high throughput and useful technique has been established for the Proteomic study.
在第三章中研究了PDMS微流控芯片的制作问题。由PDMS基片直接封装的微流控芯片在实验过程中很容易发生渗漏现象。通过氧等离子体处理PDMS基片使其表面产生活性基团-OH，两基片永久性的粘合从而制得PDMS微流控芯片，但是氧等离子体发生器的价格昂贵，反应的参数也很难控制，而且产生的自由基团很不稳定。本文利用自制的简单模具发展了一种制作一体化PDMS微流控芯片的新技术，避免了芯片在粘合过程中出现的问题。
第四章中研究了两种在一体化微流控芯片通道中固定化酶的方法。这两种方法首先都是通过紫外光处理PDMS使其表面产生自由基团，从而
嫁接丙烯酸单体。第一种方法是通过PDDA将酶固定。PDDA是一种聚阳离子物质，在很宽的pH范围内都会电离生成季铵阳离子而带正电荷与丙烯酸单体层静电吸附，胰蛋白酶（trypsin）通过PDDA被固定在芯片的微通道中。第二种方法利用介孔材料的大量-OH将酶固定。蛋白质溶液以10 μL/s的速度流过PDMS微流控芯片已固定了trypsin的通道，收集酶解溶液用质谱、毛细管电泳方法检测。结果证明这两种方法固定的酶都具有很高的活性和稳定性。
本论文通过反射红外光谱分析检测到PDMS基片在经过丙烯酸处理后表面有很强的-COOH特征吸收峰，表征了丙烯酸单体嫁接到PDMS基片表面。通过扫描电镜测定、接触角测定、FITC标记等方法说明经过丙烯酸单体修饰后的基片可以分别通过聚阳离子物质PDDA和介孔材料将trypsin固定。
纯蛋白质溶液经过毛细管电泳分离检测只有一个峰，而通过芯片微通道后的溶液经过毛细管电泳分离检测，电泳谱图检测出许多小峰，这些小峰是蛋白质在芯片上酶解产生的小肽段。酶解溶液经过MALDI-TOF/TOF-MS检测，经 Mascot搜索，所得的序列覆盖率、匹配的肽段、蛋白得分都很高，与文献报道的溶液酶解方法得到的结果一致，也证实了本文报道的两种方法固定的酶都具有很高的活性。用PDDA方法固定有酶的PDMS微流控芯片在冰箱中放置12天后，蛋白质在芯片上酶解溶液的质谱检测结果与前一次测定结果没有差别，证明该方法固定的酶具有很高的稳定性。介孔材料由于具有较大的比表面积和孔体积，孔径均一且在纳米尺寸可调，形貌可以控制，表面容易官能团化等特点，显示出在生物大分子固定分离等方面将会具有很广泛的应用前景。本文通过检测各种介孔材料对酶的吸附量，选择了一种经过微波处理的介孔材料，并应用于PDMS微流控芯片固定化酶，蛋白质溶液在该芯片上酶解溶液经
过质谱检测，结果证实介孔材料在微流控芯片固定化酶方面具有广阔的应用前景。
本论文的研究成果有：
1、利用自制的简单模具提出了一种制作一体化PDMS微流控芯片的新技术；
2、研究出两种在PDMS微流控芯片上固定胰蛋白酶的新方法，第一种方法是通过聚阳离子物质PDDA将酶固定，第二种方法是利用介孔材料将酶固定；
3、实现了蛋白质在PDMS微流控芯片上的酶解，建立了芯片在线酶解的新技术平台。
期望进一步的研究能够实现蛋白质等生物样品在芯片上的在线预处理、分离、酶解、检测等，从而为蛋白质组学的研究提供一种快速、高效、高通量、高灵敏度的技术平台。
Following the rising demands for monitoring of large number of bioaffinity interactions or biomarkers at cell lever, the development of miniaturized , high throughput chemical systems has grown dramatically in recent years. In 1990s, Manz introduced the concept of micro total analysis systems(μTAS). The field of microfluidic chip has become a growing research area in the last decade for creating inexpensive, minute sample volumes, fast and parallel analytical tools applied to different fields such as DNA, protein digestion, separation and detection. In the first chapter, a summarized development in this field was reviewed.
In chapter 3, made of microfluidic chip was described. Poly(dimethylsiloxane) (PDMS) was selected as the chip material owing to its excellent characters including chemical inertness, low polarity, low electrical conductivity and elasticity. Oxygen plasma treatment is the traditional method to seal the PDMS films, but the surface treatment involves very complex mechanisms in response to the plasma type, material, atmosphere so that the operation conditions can’t be controlled easily, in addition the oxygen plasma generator is expensive. In this chapter a simple method to make intergraded PDMS microfluidic chip using a lab made mold was reported.
In chapter 4, two methods for immobilization of trypsin into the integrated PDMS chip microchannel were proposed. Acrylic acid(AA) was first grafted onto PDMS to yield hydrophilic surface by exposing to UV light, and then PDDA was electrostatically absorbed on the AA layer for
self-assembly of trypsin. The other method is that trypsin was immobilized by mesoporous material(FDU-1), then filled the mesoporous material into the microchannel, the mesoporous material was absorbed by AA. Protein solution was flowed in the microchannel with a rate of 10μL/s and collected, then detected by CE and MALDI-TOF/TOF-MS.
In this paper immobilization of enzyme in the microchannel was carried out via modifying the surface of PDMS microfluidic chip. The modified PDMS surface was characterized by the total attenuated reflection IR spectrometry, contact angle and image of scanning electron microscope.
Mesoporous materials have potential applications in bio-molecule separation and immobilization by controlling the size, dimension of the pore, modifying chemical groups in the pore and/or on the surface. In this paper, one kind of mesoporous material was selected for the application to the study of immobilization enzyme in microfluidic chip.
The selected protein solution was flowed through the microchannel then detected by MALDI-TOF/TOF-MS and CE. Only one peak was presented on the electrograms of CE in case of detecting the pure protein solution, while many peaks were occurred when the selected protein solution was detected. Many peaks of digested peptides were appeared on the MS spectrum. With Mascot search of the data, high sequence coverage and protein scores were obtained. The results prove the activity of the immobilized enzyme.
Microfluidic chip has presented a developing perspective in chemical monitoring. Chip based microsystems have achieved a great progress in the fields of Genomic, Proteomic, Clinical and Forensic analysis. Proteomic analysis has become more and more important with the end of gene sequence
detection. For the proteomic analysis, study of the sequence of protein is very important. A key of the techniques for the protein sequence detection is digested by enzyme. In this paper two novel methods have been provided for immobilization of enzyme in the microfluidic chip channel. Protein can be on line digested in the microchannel quickly at room temperature. This micro device can be combined with various kinds of detection approaches including CE separation, MALDI-TOF/TOF-MS, LIF. In summary, a new, sensitive, high throughput and useful technique has been established for the Proteomic study.
引文
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