【作者】 丁玲；

【导师】 周培疆；

【作者基本信息】 武汉大学 ， 环境科学， 2011， 博士

【摘要】 量子点(quantum dots, QDs)是一种由Ⅱ-Ⅵ族或Ⅲ-Ⅴ族元素组成的半导体纳米颗粒,具有优越的光学性能,被广泛的应用于生命科学和医学等领域。目前,研究较多的是含重金属Cd的量子点,但量子点中Cd的存在,必然会对生物体及环境产生不利的影响,因而,其生物学毒性效应成为新的研究热点。量子点进入生物有机体内,表面会结合蛋白,同时会影响蛋白质的结构和功能等,可以通过研究量子点和蛋白质之间的作用来了解量子点的生物效应。此外,光照条件是影响量子点结构稳定性的重要因素,并影响其细胞毒性效应,但对于量子点所引起的光毒性的研究很少,光照条件下量子点与蛋白质的作用及其微观机制研究也尚未见报导。因此,制备一种具有低毒、高量子产率、好的稳定性和生物相容性等优点的量子点,同时,研究光作用对量子点的影响,并通过光作用前后的量子点与蛋白质的相互作用来了解其生物学毒性效应具有重要的意义。本论文从量子点的毒性、荧光效率、稳定性等目前在应用中存在的实际问题出发,通过选用不同的稳定剂、表面修饰剂和不同的合成方法来研究ZnSe量子点的制备。对制备的量子点进行不同类型光照,以研究光作用对量子点结构和性能的影响,探讨其光作用机理。以牛血清白蛋白(BSA)作为模型蛋白,从分子水平上研究量子点与血清蛋白的相互作用,具体研究内容及结论如下：(1)先后以巯基乙酸(TGA)和谷胱甘肽(GSH)为稳定剂,分别用水热法和微波法制备ZnSe QDs,优化合成条件,对其结构进行表征；并通过BSA对微波优化合成的ZnSe/GSH QDs进行交联修饰。结果表明,采用GSH水热合成的ZnSe QDs荧光性能和稳定性优于TGA合成的ZnSe QDs;微波合成的ZnSe/GSH QDs优于水热合成的量子点。在前驱体溶液pHH10.5、95℃下加热回流60min、反应物摩尔比GSH:Zn:Se=1.6:1:0.1优化条件下,微波反应得到的ZnSe/GSH QDs,荧光量子产率可达到42%左右,荧光发射峰半峰宽为25nm,粒径约为2-3nm,其表面具有羟基与羧基两种官能团。BSA交联修饰的ZnSe/GSH QDs荧光强度增大,形成了QDs-BSA复合荧光探针体系。(2)用紫外可见吸收光谱、荧光光谱、圆二色谱和等温滴定微量热研究量子点和BSA之间的相互作用,同时计算它们之间结合的热力学常数、结合位点数、结合分子间的距离。结果表明,ZnSe QDs对BSA荧光强度的减小是形成一种不发荧光的复合物的静态猝灭过程。三种QDs与BSA的结合常数K从大到小顺序依次为ZnSe/TGA QDs、CdSe/L-cysteine QDs、ZnSe/GSH QDs,其主要原因就是表面基团的不同。ZnSe/GSHQDs和BSA的相互作用是一种焓熵共同驱动过程,静电作用和氢键是它们之间主要的作用力,而离子强度和pH值也会影响其相互作用。(3)将ZnSe QDs分别进行避光、紫外和白炽灯光照作用,用紫外吸收光谱、荧光发射光谱、等离子体发射光谱和高分辨率透射电镜、X射线衍射、红外光谱考察了光作用对ZnSe QDs结构和性能的影响。结果表明,光照作用下ZnSe QDs的物理尺寸和元素含量发生了变化,但ZnSe QDs的晶形结构未见明显变化。和避光ZnSe/GSH QDs相比,紫外光照ZnSe/GSH QDs的球形度、尺寸分布和分散性均较好,但也有少量团聚现象。紫外光照的ZnSe/GSH QDs中S元素的含量相对较多。光作用对QDs中O-H键的伸缩振动特征峰影响较大,对其它键的影响很小,可能光照导致ZnSe/GSH QDs表面的-SH基团和-OH基团产生了化学键合作用。综合比较,紫外光对ZnSe/GSH QDs物理和化学性能的影响要大于白炽灯光和避光条件。(4)用荧光光谱法、圆二色谱法和红外光谱法等技术研究不同类型光作用后ZnSe/GSHQDs和BSA的相互作用,同时计算了结合的热力学常数、结合位点数。结果表明,三种光作用QDs对BSA的荧光猝灭机理均为静态猝灭,结合常数K大小顺序依次为紫外光QDs、白炽光QDs、避光QDs。紫外光作用的ZnSe/GSH和ZnSe/TGAQDs与BSA结合的热力学参数ΔH<0,ΔS<0,表明其主要作用力为氢键和范德华力；避光和白炽灯光QDs与BSA结合的△H<O和△S>0,其结合反应以静电作用力为主,疏水作用和配位作用为辅。光作用改变了QDs的表面配体结构,间接作用于QDs对BSA构象的影响程度,紫外光照QDs对BSA构象影响较大。QDs对BSA构象的影响,主要是BSA的酰胺带及氨基酸残基发生变化,BSA的主链结构仍然以α螺旋为主。(5)用计算机分子对接模拟法研究了三种不同表面包被的QDs与BSA相互作用的空间结合方式和结合位点和结合强度。结果表明,三种表面包被的QDs均可与BSA分子表而处结合,有多个结合位点,但作用力类型和强度不同。巯基乙酸包被的QDs结合的作用力类型主要是极性作用力,包括氢键和范德华力,结合强度较弱。L-半胱氨酸包被的QDs主要通过氢键和疏水力结合,结合强度较强。谷胱甘肽包被的QDs大部分通过氢键相结合,结合强度很强。在这几种表面包被的QDs中,谷胱甘肽包被的QDs与BSA作用的强度最大,L-半胱氨酸包被的QDs与BSA的作用次之,巯基乙酸包被的QDs与BSA作用最小。分子模拟结果与光谱分析结果基本一致。更多还原

【Abstract】 Quantum dots(QDs) is a kind of semiconductor nanoparticles consisting of Ⅱ～Ⅵ or Ⅲ～Ⅴ family elements. Quantum Dots, with excellent optical properties, are widely used in the life sciences and medicine. At present, QDs containing heavy metals Cd, were studied by most of the researches. However, the QDs in the presence of Cd will have a certain of negative impact on organisms and the environment. So its biological toxic effects become a new researching foucus. The surface of QDs entering into the bio-organic body can bind protein, which can affect protein structure and function. We can understand the biological toxic effects of QDs through the interaction study between QDs and protein. In addition, the lighting conditions are the important factors that affect the structures stability of quantum dot and their cytotoxic effect. But the light toxicity caused by QDs have been little studied. The interaction of quantum dots under light conditions and protein and their microscopic mechanism of interaction have not been reported. Therefore, the preparation of a kind quantum dots with low toxicity, high quantum yield, good stability and biocompatibility advantages, and the study of the role of light impacting on the quantum dots, and the role of light through quantum dots before and after the interaction with proteins to understand the its toxic effects, have all important significance.This paper starts from the toxicity, the fluorescence efficiency and stability of QDs in the current practical problem of its application. Different stabilizers, surface modifiers and different synthesis methods are adopted to study the preparation of ZnSe QDs. QDs are irradiated by different types of light to study the effects of role of light on the structure and properties of QDs, and to explore its mechanism of action of light. Bovine serum albumin was selected as a model protein to study the interaction between QDs and protein at the molecular level. The contents and conclusions of the research are as follows:(1) Thioglycolic acid (TGA) and glutathione (GSH) were used as stabilizers, and ZnSe QDs were prepared by hydrothermal and microwave methods, respectively. The synthesis condition were optimized. The structure of QDs were characterized. ZnSe/GSH QDs that prepared by microwave method were modified by BSA cross-linking. Results showed that, the fluorescence and stability properties of ZnSe/GSH QDs is much better than ZnSe-TGA QDs’s in the hydrothermal synthesis. The microwave method is superior to hydrothermal method for ZnSe/GSH QDs’s synthesis. The optimal microwave synthesis of ZnSe/GSH QDs conditions were that the moral ratio of GSH:Zn:Se was1.6:1:0.1, pH10.5, the heating time60min, and the heating temperature95℃. In optimal condition, we obtained the ZnSe/GSH QDs, with a half peak width of emission peak25nm, diameter about2-3nm, and hydroxyl and carboxyl two kinds of functional groups in their surface. The fluorescence intensity of ZnSe/GSH QDs modified by BSA cross-linking increased. QDs-BSA complex fluorescent probe system was formed.(2) The interaction of QDs and BSA was investigated by UV-visible absorption spectrum, fluorescence spectrum, circular dichroism and isothermal titration calorimetry, and the thermodynamic constant, binding sites, and the distance between molecules of QDs and BSA interaction were calculated. The results showed that QDs cause distinct quenching on the intrinsic fluorescence of BSA, the quenching mechanism is static quenching mode with the formation of non-fluorescent complexes. The magnitude order of binding constants for three QDs is ZnSe/TGA QDs, CdSe/L-cysteine QDs, ZnSe/GSH QDs. The main reason is that the QDs have different surface groups. ZnSe/GSH QDs and BSA interaction is a common enthalpy-entropy-driven process. The main interaction forces are electrostatic and hydrogen bonding.The ionic strength and pH also affected the interaction between them.(3) ZnSe QDs were irradiated by dark, ultraviolet light and incandescent light, respectively. The affect of light on the structure and property of QDs were investigated by UV-visible absorption spectrum, fluorescence spectrum, ICP-AES, HRTEM, XRD and FTIR. Results showed that the physical size and element contents of ZnSe QDs under different light were changed, but the crystal structure of ZnSe QDs was not changed obviously. Compared with dark ZnSe/GSH QDs, UV irradiation ZnSe/GSH QDs possessed good sphericity, size distribution and dispersion, but there were also a small amount of agglomeration. The content of S elements in UV irradiation ZnSe/GSH QDs was higher relatively. The role of light impact on the O-H stretching vibration peaks greatly, however, little impact on other bonds. This is because that the light makes the ZnSe/GSH QDs’s surface-SH group and-OH groups interact by chemical bond. Comprehensive comparison, UV light impact on physical and chemical properties of ZnSe/GSH QDs is greater than incandescent and dark conditions. (4) The interaction of ZnSe/GSH QDs illuminated and BSA was investigated by fluorescence spectroscopy, circular dichroism and FTIR, and the thermodynamic constant and binding sites were calculated. The results showed that three kinds of ZnSe/GSH QDs illuminated quenched on the intrinsic fluorescence of BSA, the quenching mechanism is static quenching. The magnitude order is UV light QDs, incandescent light QDs, Dark QDs. The values of△H and△S are negative (△H<0,△S<0). These results indicate that hydrogen bonds and van der Waals interactions play a major role in the binding reaction between UV light QDs and BSA. The△H<0and△S>0suggested that electrostatic interactions play a major role in the binding reaction between UV light QDs and BSA.but the hydrophobic and coordination interactions are also important. Light changed the QDs surface ligand structure, which effected indirectly on the conformation of BSA. UV QDs conformational change of BSA greater impact. The amino acids microenvironment of BSA was sensitive to the binding of QDs to BSA, while the secondary structure of BSA was still predominant a-helix.(5) The spatial binding mode of QDs to BSA was modeled by Autodock and Sybyl8.1software. Result indicated that QDs capped by TGA, QDs capped by L-cysteine and QDs capped by GSH all can bind with surface of BSA. There were many binding sites, but their interaction force type and strength were different. The type of interaction force of QDs capped by TGA and was mainly polar interaction that include van der Waals force and hydrogen bond. The bonding strength was weak. QDs capped by L-cysteine combind with hydrophobic bond and hydrogen bond, and bonding strength was strong. QDs capped by GSH are combind strongly by hydrogen bond, and bonding strength was very strong. Among the surface modifiers, the interaction force of BSA and QDs modified by GSH is the most strongest, the interaction force of BSA and QDs modified by L-cysteine was the second, interaction force of BSA and QDs modified by TGA was the smallest. Molecular modeling results were in accordance with the spectral analysis’s partly.更多还原