全内反射荧光法研究蛋白质与壳聚糖的相互作用
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摘要
壳聚糖作为重要的天然聚合物,以良好的生物相容性、生物可降解性,对细胞组织不产生毒性影响,在生物医学等方面得到了广泛的研究应用。壳聚糖对蛋白质有良好的亲和能力,研究蛋白质与壳聚糖在界面上的相互作用、理解蛋白质与壳聚糖分子在界面接触时的吸附过程变化对蛋白药物的开发和生物化学研究具有非常重要的意义。全内反射荧光法是研究界面蛋白质吸附的一种有效的手段。它利用指数衰减的倏逝波选择性激发介质表面的荧光团分子,产生全内反射荧光,具有高度的界面(表面)特异性,从而可有效排除本体干扰,获取界面信息,而且又具有非破坏性,能够实时、原位检测蛋白质吸附过程。本文致力于改进实验室已有的固/液界面全内反射荧光分析的部件,考察壳聚糖超薄膜的固定方案,继而研究三种模型蛋白在壳聚糖超薄膜上的吸附情况。此外,还对罗丹明6G(R6G)在石英/水界面上的吸附进行研究,进一步认识咕吨染料在界面的情况。
论文分为四章:
第一章综述了全内反射荧光法在生物体系中的研究应用,以及壳聚糖与蛋白质相互作用的研究概况。简要介绍全内反射荧光法的原理和应用,对目前全内反射荧光法在生物体系,特别是固/液界面蛋白质吸附的研究作了详尽的概述,同时还介绍了壳聚糖的性质、应用以及它与蛋白质相互作用研究的状况,最后提出整篇论文的设想。
第二章在现有可行的实验条件下,以石英片为载体,改进实验室已有全内反射荧光部件,使其易于进行表面修饰,并且使样品的检测体积减少至200μL。考察了旋涂法与化学键合法固定壳聚糖薄膜的情况,同时应用全内反射恒波长同步荧光分析方法,监测壳聚糖表面固定情况。最后确定了以硅烷化试剂自组装在石英载体上引入活性基团进行表面接枝壳聚糖的化学键合法,并对接枝方法进行了优化,获得了表面均一的壳聚糖超薄膜。
第三章用荧光素异硫氰酸酯(FITC)标记常见的模型球型蛋白(牛血清白蛋白、溶菌酶、牛血纤维蛋白原),应用全内反射荧光法分析它们分别与壳聚糖超薄膜相互作用的情况,以及对牛血纤维蛋白原和牛血清白蛋白的二元竞争吸附体系进行研究。
首先成功用FITC标记各种模型蛋白质,标记率在0.6-3.8之间,观测其吸收、荧光波长均有所红移,荧光各向异性值有6-10倍的增长。
牛血清白蛋白(BSA)在壳聚糖上随着本体浓度增加,出现两个吸附增长区。随浓度增加,吸附在壳聚糖表面上的BSA可能从平躺取向排布向竖直取向变化;在平衡浓度大于400μg/mL以上可能出现多层吸附。pH对BSA在壳聚糖上的吸附作用也有显著的影响,随着pH增加,BSA在pH 6.5附近出现了极值,说明主要以静电相互作用驱动了BSA吸附。离子强度的增加,屏蔽了蛋白质与壳聚糖表面、蛋白质-蛋白质分子之间的静电斥力,使得BSA的吸附量增加;而当盐浓度大于0.1 mol/L之后,由于壳聚糖分子链的刚性变小,容易成团,与蛋白质接触面积减小,导致吸附量下降。
此外,还考察了另外两种模型蛋白吸附随本体溶液浓度变化的影响,以及对模型蛋白的吸附过程进行动力学方程拟合。牛血纤维蛋白原的界面荧光随着蛋白质本体浓度的增加而增加;在低浓度下,双常数速率方程能够得到较好拟合性,而在高浓度下,准二级动力学模型能够得到较好的拟合性。而溶菌酶在考察浓度范围内,界面信号随浓度增长变化曲线与Langmuir吸附等温线形状近似。溶菌酶对壳聚糖的表面亲和能力比BSA更好,这可能由于壳聚糖与溶菌酶的底物结构相似,溶菌酶易吸附于壳聚糖膜上。溶菌酶的吸附动力学过程可用准二级吸附动力学模型描述,随着吸附初始浓度的增加,吸附速率常数k也总体有一个数量级的增长。
比较壳聚糖膜上BSA与牛血纤维蛋白原在生理条件下竞争吸附的情况,在壳聚糖界面上,牛血纤维蛋白原的相对竞争吸附能力要大于BSA。这表明壳聚糖也能够正常驱动牛血纤维蛋白原的吸附,激发血小板和凝血因子而发生凝血。
第四章利用全内反射恒波长同步荧光法直接测量在石英/水界面上的罗丹明6G(R6G)。通过对其本体、界面荧光光谱的分析,观察到R6G分子在亲水石英/水界面上出现5 nm红移,红移的原因主要是染料分子旋转运动受到限制,石英表面的刚性限制了分子的自由度,同时,石英/水界面更低的极性也对红移产生影响。同时还考察了R6G在石英/水界面上的吸附情况与本体溶液的浓度、pH以及离子强度的关系。
As an important natural polymer, chitosan has been applied extensively in biomedicine for its biocompatibility, biodegradation, as well as non-toxicity to cellular tissues. There is good affinity between chitosan and protein. For the development of protein drugs and the research of biochemistry, it is significant to probe the interaction between chitosan and protein, and to observe changes of adsorption when both of them contact at interface. Total internal reflection fluorescence spectroscopy (TIRF) is an effective technique to investigate protein adsorption at interface. The total internal reflection fluorescence generates from interfacial fluorophore, which is selectively excited by evanescent wave. Therefore, there are several characteristics for evanescent wave inducing fluorescence, such as surface specificity, elimination of bulk interference and non-destruction. It has been successful to investigate protein adsorption in situ, real-time by this technique. This dissertation is concerned on the improvement of instrumental device of TIRF for probing solid/liquid interface, the option of suitable method for immobilizing chitosan, and the analysis of interaction between model proteins and ultrathin chitosan film which was immobilized on silica substrate. In addition, the adsorption behavior of rhodamine 6G (R6G) at silica/water interface was also concerned. The dissertation is composed of following parts:
In the first chapter, the application of total internal reflection fluorescence technique in the research of biological systems and the advance on the study of interaction between chitosan and protein were reviewed. The principle and application of TIRF were introduced briefly. The research on biological systems, especially on the study of protein adsorption at solid/water interface, was described in detail. Then the property and application of chitosan were introduced, as well as the hot topics on the study of chitosan-protein interaction. The plan of dissertation was put forward at the last part of this chapter.
In the second chapter, a new TIRF cell was developed based on the present experimental conditions. Microfluid cell made up of silica slices, of which detection volume was minimized to 200μL, was more easily for modification and cleaning. The immobility of chitosan films was monitored by total internal reflection synchronous fluorescence (TIRSF). Two methods (spin coating and chemical bonding) were employed to immobilize chitosan onto the silica substrate. We finally applied chemical bonding method to fabricate chitosan film, which utilized self-assembly membrane to introduce active group for surface grafting. The protocols of grafting were optimized as well. As a result, the uniform ultrathin chitosan film was obtained.
In the third chapter, the interactions between model FITC labeled proteins (BSA, Fibrinogen, Lysozyme) and ultrathin chitosan film were investigated by TIRF, respectively. The competitive adsorption between BSA and fibrinogen was studied as well.
Firstly, it was successful to label model proteins with FITC, whose labeling ratios were between 0.6 to 3.8. Red shifts were observed both in absorption and fluorescence spectra. There was 6-10 times increase in the value of fluorescent anisotropy of labeled proteins.
Secondly, with increase of concentration of FITC-BSA in bulk solution, two growth regions of interfacial intensity of FITC-BSA were present on chitosan film. It is suggested that FITC-BSA will adsorb on chitosan film with the orientations changing from horizontal to vertical, and as multilayer structure at high concentration (more than 400μg/mL).The adsorption of FITC-BSA depended with pH variations. With pH increasing, there was an extreme value of interfacial intensity approximately at pH 6.5. It is illustrated that the electrostatic attraction was the main driving force to adsorb BSA. Increasing of ionic strength screened the repulsive forces between protein-chitosan, protein-protein at pH 7.4. As a result, the amount of adsorbates was increased. However, higher concentration of supporting electrolyte induced the decrease in BSA adsorption for chitosan chains tended to curling to be conglobation, which resulted in fewer sites to contact with protein.
Thirdly, we investigated the concentration effects and adsorption kinetics of other two model proteins. For fibrinogen, interfacial intensity was increased with the initial concentration. At low concentration, Double Constant Equation was fitted greatly to the adsorption kinetics curves. At high concentration, Pseudo-second order model appeared as the best fitter to adsorption kinetics curves. For lysozyme, interfacial intensity changing with concentration of FITC-Lyz was in the same shape as Langmuir adsorption isotherm. The affinity of lysozyme to chitosan was much better than that of BSA for the structure of chitosan was similar to substrate of enzyme. Pseudo-second order model could describe adsorption processes of lysozyme on chitosan film exactly. With increase of initial concentration of lysozyme, there was one-order magnitude increase in adsorption rate constant (k).
And then, the study of competitive adsorption of BSA and fibrinogen on chitosan film at physiological condition was carried out. It was revealed that the relative competitive adsorption capacity of fibrinogen was much larger than that of BSA. The result confirmed that chitosan, as other common haemostatic, would adsorb blood protein and activate platelet and coagulation factor for clotting.
In the fourth chapter, total internal reflection synchronous fluorescence (TIRSF) was developed as a direct and successful tool to investigate a xanthene dye (R6G) at the silica/water interface. From analysis of fluorescence spectra both in bulk and interface, 5 nm red shift of fluorescence spectra at hydrophilic silica/water interface were observed. The bathochromic shift was mainly due to the limitation of rotational movements of the dye. The rigidity of the silica surface restricted the freedom of the molecules. The lower polarity of microenvironment at the silica/water interface than that in aqueous environment should also make a contribution to the phenomenon. Meanwhile, the effects of bulk concentration, pH, and ionic strength on the adsorption of R6G were studied as well.
论文分为四章:
第一章综述了全内反射荧光法在生物体系中的研究应用,以及壳聚糖与蛋白质相互作用的研究概况。简要介绍全内反射荧光法的原理和应用,对目前全内反射荧光法在生物体系,特别是固/液界面蛋白质吸附的研究作了详尽的概述,同时还介绍了壳聚糖的性质、应用以及它与蛋白质相互作用研究的状况,最后提出整篇论文的设想。
第二章在现有可行的实验条件下,以石英片为载体,改进实验室已有全内反射荧光部件,使其易于进行表面修饰,并且使样品的检测体积减少至200μL。考察了旋涂法与化学键合法固定壳聚糖薄膜的情况,同时应用全内反射恒波长同步荧光分析方法,监测壳聚糖表面固定情况。最后确定了以硅烷化试剂自组装在石英载体上引入活性基团进行表面接枝壳聚糖的化学键合法,并对接枝方法进行了优化,获得了表面均一的壳聚糖超薄膜。
第三章用荧光素异硫氰酸酯(FITC)标记常见的模型球型蛋白(牛血清白蛋白、溶菌酶、牛血纤维蛋白原),应用全内反射荧光法分析它们分别与壳聚糖超薄膜相互作用的情况,以及对牛血纤维蛋白原和牛血清白蛋白的二元竞争吸附体系进行研究。
首先成功用FITC标记各种模型蛋白质,标记率在0.6-3.8之间,观测其吸收、荧光波长均有所红移,荧光各向异性值有6-10倍的增长。
牛血清白蛋白(BSA)在壳聚糖上随着本体浓度增加,出现两个吸附增长区。随浓度增加,吸附在壳聚糖表面上的BSA可能从平躺取向排布向竖直取向变化;在平衡浓度大于400μg/mL以上可能出现多层吸附。pH对BSA在壳聚糖上的吸附作用也有显著的影响,随着pH增加,BSA在pH 6.5附近出现了极值,说明主要以静电相互作用驱动了BSA吸附。离子强度的增加,屏蔽了蛋白质与壳聚糖表面、蛋白质-蛋白质分子之间的静电斥力,使得BSA的吸附量增加;而当盐浓度大于0.1 mol/L之后,由于壳聚糖分子链的刚性变小,容易成团,与蛋白质接触面积减小,导致吸附量下降。
此外,还考察了另外两种模型蛋白吸附随本体溶液浓度变化的影响,以及对模型蛋白的吸附过程进行动力学方程拟合。牛血纤维蛋白原的界面荧光随着蛋白质本体浓度的增加而增加;在低浓度下,双常数速率方程能够得到较好拟合性,而在高浓度下,准二级动力学模型能够得到较好的拟合性。而溶菌酶在考察浓度范围内,界面信号随浓度增长变化曲线与Langmuir吸附等温线形状近似。溶菌酶对壳聚糖的表面亲和能力比BSA更好,这可能由于壳聚糖与溶菌酶的底物结构相似,溶菌酶易吸附于壳聚糖膜上。溶菌酶的吸附动力学过程可用准二级吸附动力学模型描述,随着吸附初始浓度的增加,吸附速率常数k也总体有一个数量级的增长。
比较壳聚糖膜上BSA与牛血纤维蛋白原在生理条件下竞争吸附的情况,在壳聚糖界面上,牛血纤维蛋白原的相对竞争吸附能力要大于BSA。这表明壳聚糖也能够正常驱动牛血纤维蛋白原的吸附,激发血小板和凝血因子而发生凝血。
第四章利用全内反射恒波长同步荧光法直接测量在石英/水界面上的罗丹明6G(R6G)。通过对其本体、界面荧光光谱的分析,观察到R6G分子在亲水石英/水界面上出现5 nm红移,红移的原因主要是染料分子旋转运动受到限制,石英表面的刚性限制了分子的自由度,同时,石英/水界面更低的极性也对红移产生影响。同时还考察了R6G在石英/水界面上的吸附情况与本体溶液的浓度、pH以及离子强度的关系。
As an important natural polymer, chitosan has been applied extensively in biomedicine for its biocompatibility, biodegradation, as well as non-toxicity to cellular tissues. There is good affinity between chitosan and protein. For the development of protein drugs and the research of biochemistry, it is significant to probe the interaction between chitosan and protein, and to observe changes of adsorption when both of them contact at interface. Total internal reflection fluorescence spectroscopy (TIRF) is an effective technique to investigate protein adsorption at interface. The total internal reflection fluorescence generates from interfacial fluorophore, which is selectively excited by evanescent wave. Therefore, there are several characteristics for evanescent wave inducing fluorescence, such as surface specificity, elimination of bulk interference and non-destruction. It has been successful to investigate protein adsorption in situ, real-time by this technique. This dissertation is concerned on the improvement of instrumental device of TIRF for probing solid/liquid interface, the option of suitable method for immobilizing chitosan, and the analysis of interaction between model proteins and ultrathin chitosan film which was immobilized on silica substrate. In addition, the adsorption behavior of rhodamine 6G (R6G) at silica/water interface was also concerned. The dissertation is composed of following parts:
In the first chapter, the application of total internal reflection fluorescence technique in the research of biological systems and the advance on the study of interaction between chitosan and protein were reviewed. The principle and application of TIRF were introduced briefly. The research on biological systems, especially on the study of protein adsorption at solid/water interface, was described in detail. Then the property and application of chitosan were introduced, as well as the hot topics on the study of chitosan-protein interaction. The plan of dissertation was put forward at the last part of this chapter.
In the second chapter, a new TIRF cell was developed based on the present experimental conditions. Microfluid cell made up of silica slices, of which detection volume was minimized to 200μL, was more easily for modification and cleaning. The immobility of chitosan films was monitored by total internal reflection synchronous fluorescence (TIRSF). Two methods (spin coating and chemical bonding) were employed to immobilize chitosan onto the silica substrate. We finally applied chemical bonding method to fabricate chitosan film, which utilized self-assembly membrane to introduce active group for surface grafting. The protocols of grafting were optimized as well. As a result, the uniform ultrathin chitosan film was obtained.
In the third chapter, the interactions between model FITC labeled proteins (BSA, Fibrinogen, Lysozyme) and ultrathin chitosan film were investigated by TIRF, respectively. The competitive adsorption between BSA and fibrinogen was studied as well.
Firstly, it was successful to label model proteins with FITC, whose labeling ratios were between 0.6 to 3.8. Red shifts were observed both in absorption and fluorescence spectra. There was 6-10 times increase in the value of fluorescent anisotropy of labeled proteins.
Secondly, with increase of concentration of FITC-BSA in bulk solution, two growth regions of interfacial intensity of FITC-BSA were present on chitosan film. It is suggested that FITC-BSA will adsorb on chitosan film with the orientations changing from horizontal to vertical, and as multilayer structure at high concentration (more than 400μg/mL).The adsorption of FITC-BSA depended with pH variations. With pH increasing, there was an extreme value of interfacial intensity approximately at pH 6.5. It is illustrated that the electrostatic attraction was the main driving force to adsorb BSA. Increasing of ionic strength screened the repulsive forces between protein-chitosan, protein-protein at pH 7.4. As a result, the amount of adsorbates was increased. However, higher concentration of supporting electrolyte induced the decrease in BSA adsorption for chitosan chains tended to curling to be conglobation, which resulted in fewer sites to contact with protein.
Thirdly, we investigated the concentration effects and adsorption kinetics of other two model proteins. For fibrinogen, interfacial intensity was increased with the initial concentration. At low concentration, Double Constant Equation was fitted greatly to the adsorption kinetics curves. At high concentration, Pseudo-second order model appeared as the best fitter to adsorption kinetics curves. For lysozyme, interfacial intensity changing with concentration of FITC-Lyz was in the same shape as Langmuir adsorption isotherm. The affinity of lysozyme to chitosan was much better than that of BSA for the structure of chitosan was similar to substrate of enzyme. Pseudo-second order model could describe adsorption processes of lysozyme on chitosan film exactly. With increase of initial concentration of lysozyme, there was one-order magnitude increase in adsorption rate constant (k).
And then, the study of competitive adsorption of BSA and fibrinogen on chitosan film at physiological condition was carried out. It was revealed that the relative competitive adsorption capacity of fibrinogen was much larger than that of BSA. The result confirmed that chitosan, as other common haemostatic, would adsorb blood protein and activate platelet and coagulation factor for clotting.
In the fourth chapter, total internal reflection synchronous fluorescence (TIRSF) was developed as a direct and successful tool to investigate a xanthene dye (R6G) at the silica/water interface. From analysis of fluorescence spectra both in bulk and interface, 5 nm red shift of fluorescence spectra at hydrophilic silica/water interface were observed. The bathochromic shift was mainly due to the limitation of rotational movements of the dye. The rigidity of the silica surface restricted the freedom of the molecules. The lower polarity of microenvironment at the silica/water interface than that in aqueous environment should also make a contribution to the phenomenon. Meanwhile, the effects of bulk concentration, pH, and ionic strength on the adsorption of R6G were studied as well.
引文
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