曙红B、CdTe@谷胱甘肽量子点与HSA的相互作用及检测没食子酸的酸度敏感探针研究
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摘要
人血清白蛋白(HSA)是人体内重要的运输蛋白质,能与许多内源及外源物质相结合,起到存储和转运的作用。研究HSA与小分子配体的相互作用过程及相互作用机理对临床、药理学和毒理学的研究具有重要意义。量子点因其独特的性质(尤其是光学性质)越来越受到科学工作者们的青睐,已在荧光探针、生物标记和细胞成像等方面表现出强大的应用前景。基于以上研究背景,在前人研究工作的基础上,本论文运用分子对接及各种光谱技术分别研究了染料曙红B、谷胱甘肽(GSH)修饰的CdTe量子点与HSA间的相互作用；合成了检测没食子酸的酸度敏感荧光探针-CdTe@巯基乙酸量子点,并建立了检测没食子酸的新方法。论文分为以下几个部分：
第一章：介绍了人血清白蛋白(HSA)和量子点的结构、功能、性质(尤其是其光谱特性)等；总结了用于研究小分子与HSA相互作用的各种光谱法；简要介绍了量子点在痕量物质检测方面的应用。
第二章：在模拟的生理条件下利用多种光谱技术和分子对接方法考察了曙红B与HSA的相互作用。用分子对接技术模拟了曙红B与HSA的结合位置和结合方式,计算了相互作用的Gibbs自由能；通过荧光猝灭数据确定了相互作用的结合常数、结合位点数及相关热力学参数,并对相互作用的机理进行了考察；用红外、紫外、同步荧光、圆二色性光谱证明了曙红B对HSA二级结构的影响；计算了曙红B与HSA的氨基酸残基之间的距离。
第三章：在水相中合成了谷胱甘肽(GSH)修饰的CdTe量子点,并用荧光光谱(FL),紫外-可见吸收光谱(UV-vis)对合成的量子点进行了表征,结果表明合成的量子点粒径较为均一,表面缺陷较少。用荧光光谱法对该量子点与HSA之间的相互作用进行了研究,通过实验测定的结合常数、基本的热力学参数确定了量子点与HSA的键合模式及作用机理。同步荧光光谱表明量子点可引起HSA二级结构的变化。
第四章：在水相中合成了尺寸分布较为均一的巯基乙酸(TGA)修饰的CdTe量子点,考察了体系pH值对该量子点荧光强度的影响。实验结果表明其荧光强度在pH4.0-6.5的范围内,随着体系pH值的升高而增强,据此原理建立了以自行合成的CdTe@TGA量子点为酸度敏感荧光探针检测药物没食子酸的新方法。在最佳条件下,该方法的线性范围为4.00-32.00μg/mL,检测限为0.44μg/mL,线性相关系数为0.9967。该方法已用于人工合成样品中没食子酸含量的检测,结果令人满意。
Human Serum Albumin (HSA) is the important carrier protein in human body, which can bind different categories of endogenous and exogenous small molecules, and play a role in storage and transit. Investigating the binding mechanism between small molecules and HSA is significative in diagnostics, pharmaceutical and toxicology. Because semiconductor nanocrystals (also known as quantum dots) have many unique properties (especially the optical properties), they are favored by more and more researchers. In fluorescence probe, biological biomarkers and cell image, quantum dots have shown great potential applications. Based on the abovementioned research background, we have used molecular docking approach and multi-spectroscopic technology to study the binding characteristics of HSA with Eosin B and homemade CdTe@GSH quantum dot. Furthermore, we used homemade CdTe@TGA quantum dots as pH-sensitive fluorescent probe for detection of Gallic acid.
This thesis is divided into four chapters:
Chapter1:The structures, functions and properties of QDs and HSA were introduced. Various spectroscopic methods were reviewed, which are often used to study the binding characteristics between small molecules and HSA. Meanwhile, the methods used to detect trace materials by QDs and the latest applications were introduced.
Chapter2:Under simulated physiologic conditions, the interaction of Eosin B with HSA was investigated by the combination of fluorescence, UV, FT-IR, CD spectroscopic and molecular binding technologies. Through molecular docking method, the binding site and binding mode were discussed. Fluorescence quenching data revealed the binding constants and the number of binding sites. The thermodynamic parameters and binding mechanism were also discussed. The results of synchronous fluorescence, UV, FT-IR and CD spectra indicated that secondary structure of HSA was changed in the presence of Eosin B. In addition, the binding distance was estimated according to the Forster non-radiative resonance energy transfer theory.
Chapter3:CdTe@GSH QDs were synthesized in aqueous solution. The obtained QDs were characterized by UV-visible absorption spectrum and fluorescence spectrum, which indicates that as-prepared QDs are nearly monodisperse and homogeneous. The interaction between the as-prepared QDs and HSA was studied by fluorescence technique. The results were discussed on the binding parameters, the quenching mechanism, the nature of forces involved in the interaction, and the effect of QDs on the conformation of HSA.
Chapter4:CdTe@TGA QDs were synthesized in aqueous solution. The relationship between its fluorescence intensity and pH was investigated. The experimental results showed that in the pH range4.0-6.5, the QDs was pH-sensitive. And a new method was developed to detect Gallic acid by employing CdTe@TGA QDs as pH-sensitive fluorescence probe. Under the optimal conditions, the linear range of this method was4.00-32.00μg/mL. The detection limit was0.44μg/mL and the correlation coefficient was0.9967. The method was applied to detect Gallic acid in synthetic samples and the obtained results were satisfactory.
第一章：介绍了人血清白蛋白(HSA)和量子点的结构、功能、性质(尤其是其光谱特性)等；总结了用于研究小分子与HSA相互作用的各种光谱法；简要介绍了量子点在痕量物质检测方面的应用。
第二章：在模拟的生理条件下利用多种光谱技术和分子对接方法考察了曙红B与HSA的相互作用。用分子对接技术模拟了曙红B与HSA的结合位置和结合方式,计算了相互作用的Gibbs自由能；通过荧光猝灭数据确定了相互作用的结合常数、结合位点数及相关热力学参数,并对相互作用的机理进行了考察；用红外、紫外、同步荧光、圆二色性光谱证明了曙红B对HSA二级结构的影响；计算了曙红B与HSA的氨基酸残基之间的距离。
第三章：在水相中合成了谷胱甘肽(GSH)修饰的CdTe量子点,并用荧光光谱(FL),紫外-可见吸收光谱(UV-vis)对合成的量子点进行了表征,结果表明合成的量子点粒径较为均一,表面缺陷较少。用荧光光谱法对该量子点与HSA之间的相互作用进行了研究,通过实验测定的结合常数、基本的热力学参数确定了量子点与HSA的键合模式及作用机理。同步荧光光谱表明量子点可引起HSA二级结构的变化。
第四章：在水相中合成了尺寸分布较为均一的巯基乙酸(TGA)修饰的CdTe量子点,考察了体系pH值对该量子点荧光强度的影响。实验结果表明其荧光强度在pH4.0-6.5的范围内,随着体系pH值的升高而增强,据此原理建立了以自行合成的CdTe@TGA量子点为酸度敏感荧光探针检测药物没食子酸的新方法。在最佳条件下,该方法的线性范围为4.00-32.00μg/mL,检测限为0.44μg/mL,线性相关系数为0.9967。该方法已用于人工合成样品中没食子酸含量的检测,结果令人满意。
Human Serum Albumin (HSA) is the important carrier protein in human body, which can bind different categories of endogenous and exogenous small molecules, and play a role in storage and transit. Investigating the binding mechanism between small molecules and HSA is significative in diagnostics, pharmaceutical and toxicology. Because semiconductor nanocrystals (also known as quantum dots) have many unique properties (especially the optical properties), they are favored by more and more researchers. In fluorescence probe, biological biomarkers and cell image, quantum dots have shown great potential applications. Based on the abovementioned research background, we have used molecular docking approach and multi-spectroscopic technology to study the binding characteristics of HSA with Eosin B and homemade CdTe@GSH quantum dot. Furthermore, we used homemade CdTe@TGA quantum dots as pH-sensitive fluorescent probe for detection of Gallic acid.
This thesis is divided into four chapters:
Chapter1:The structures, functions and properties of QDs and HSA were introduced. Various spectroscopic methods were reviewed, which are often used to study the binding characteristics between small molecules and HSA. Meanwhile, the methods used to detect trace materials by QDs and the latest applications were introduced.
Chapter2:Under simulated physiologic conditions, the interaction of Eosin B with HSA was investigated by the combination of fluorescence, UV, FT-IR, CD spectroscopic and molecular binding technologies. Through molecular docking method, the binding site and binding mode were discussed. Fluorescence quenching data revealed the binding constants and the number of binding sites. The thermodynamic parameters and binding mechanism were also discussed. The results of synchronous fluorescence, UV, FT-IR and CD spectra indicated that secondary structure of HSA was changed in the presence of Eosin B. In addition, the binding distance was estimated according to the Forster non-radiative resonance energy transfer theory.
Chapter3:CdTe@GSH QDs were synthesized in aqueous solution. The obtained QDs were characterized by UV-visible absorption spectrum and fluorescence spectrum, which indicates that as-prepared QDs are nearly monodisperse and homogeneous. The interaction between the as-prepared QDs and HSA was studied by fluorescence technique. The results were discussed on the binding parameters, the quenching mechanism, the nature of forces involved in the interaction, and the effect of QDs on the conformation of HSA.
Chapter4:CdTe@TGA QDs were synthesized in aqueous solution. The relationship between its fluorescence intensity and pH was investigated. The experimental results showed that in the pH range4.0-6.5, the QDs was pH-sensitive. And a new method was developed to detect Gallic acid by employing CdTe@TGA QDs as pH-sensitive fluorescence probe. Under the optimal conditions, the linear range of this method was4.00-32.00μg/mL. The detection limit was0.44μg/mL and the correlation coefficient was0.9967. The method was applied to detect Gallic acid in synthetic samples and the obtained results were satisfactory.
引文
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