【作者】 张鹏飞；

【导师】 王柯敏； 羊小海；

【作者基本信息】 湖南大学 ， 分析化学， 2009， 硕士

【摘要】 量子点(Quantum dots, QDs)是一种新型半导体荧光纳米探针,与传统有机荧光染料相比,具有许多优良的光谱特性,在分子诊断、靶向治疗、生物医学成像与生物传感等方面得到了广泛应用,并极大地推动了生命分析化学、生物医学工程等研究领域的飞速发展。本论文在实验室已有的量子点合成技术平台上,以量子点与细胞相互作用为主线,主要开展了以下几个方面的工作:1.磷脂修饰量子点的制备针对脂溶性量子点在生物分析研究中面临的相转移及其功能化修饰问题,分别采用普通磷脂(DPPC)与聚乙二醇功能化磷脂(PEG-DPPE)修饰量子点,在对其进行紫外可见吸收光谱、荧光发射光谱、荧光显微成像、透射电子显微镜成像等系列表征后,考察了两种修饰方法所得量子点光谱性质、分散性、光稳定性的差异。结果表明:普通磷脂修饰量子点得到多个量子点的团聚体,与之相比,采用聚乙二醇功能化磷脂修饰的量子点单分散性较好、光稳定性强,可用于后续实验。2.量子点与细胞非特异性相互作用研究非特异性吸附是纳米颗粒应用于细胞成像研究面对的首要问题之一。在第一部分工作基础上,为了更好的将修饰后的量子点应用于细胞成像研究中,分别采用带有氨基、羧基、甲基的聚乙二醇功能化磷脂修饰得到不同功能表面基团的量子点,结合激光共聚焦成像技术,对不同功能基团表面的量子点与细胞非特异性作用进行研究。结果表明:量子点表面电荷对量子点与细胞的非特异性作用影响显著,氨基功能化后带正电荷的量子点最容易与细胞发生非特异性作用,羧基功能化后带负电荷的量子点次之,甲基功能化后基本不带电荷的量子点与细胞非特异性作用最小。量子点与细胞的作用是一个依赖于量子点的浓度、表面电荷、培育时间与温度,并受培养基中血清影响的耗能过程。因此,可以有针对性地对量子点的表面进行修饰或改性并将其应用于细胞成像研究。3.血管生成素-量子点复合物特异识别细胞及其内化研究血管生成素(Angiogenin)是一种与肿瘤发生、发展密切相关的蛋白质。在前面两部分工作的基础上,借助核酸适体,将血管生成素固定在量子点上,并进一步考察了量子点-血管生成素复合物对目标细胞的选择性识别与内化情况。由于细胞表面血管生成素受体的作用,血管生成素-量子点复合物能选择性地结合到人脐带静脉内皮细胞(HUVEC)和人宫颈癌细胞(Hela)上;荧光共定位实验和Z-轴扫描结果进一步证实,复合物能进入靶细胞,最终主要定位在溶酶体中。结合以上实验结果,构建了一种特异性识别表面表达有血管生成素受体细胞的纳米探针,有望在肿瘤血管生成及肿瘤细胞早期诊断与靶向治疗等基础研究方面发挥重要作用。更多还原

【Abstract】 Compared with conventional organic fluorescent dyes, quantum dots show unique size-dependent optical properties, which make them appealing as a new class of fluorescent probes, and have gained increasing applications in the fields of molecular diagnosis, target therapy,biomedical imaging and biosensor, etc, which promote the evolution of bio-analytical chemistry and biomedical engineering. The following several works have mainly performed by taking the interaction of quantum dots with cells as the line of this dissertation.1. Encapsulation of Quantum Dots in Phospholipid MicellesPhase transfer is an essential and nontrivial step for QDs that render solubility only in nonpolar organic solvents to be useful as biological reporters. In this study hydrophobic QDs were encapsulated with phospholipid and PEG-phospholipid. The abtained micelles were characterized by UV-Visible spectroscopy, fluorescence spectroscopy, fluorescence microscopy imaging and Transmission Electron Microscope, respectively. The results showed that QDs encapsulated in poly(ethylene glycol)-phospholipid micelles with one QD per micelle was more feasible than that encapsulated in phospholipid micelles with many QDs per micelle. The as-prepared water-compatible QDs all hold high photostability and narrow size-distribution.2. Nonspecifical Binding Study of Functional QD Micelles to CellsA major problem for the application of nanoparticles in cellular imaging is that nanoparticles tend to bind nonspecifically to cellular membranes. Based on the first part work, functional QD micelles with chemically reactive groups (e.g., NH2- , COOH-, and CH3-) were prepared by encapsulation of QDs into functional PEG-phospholipid micelles, and their nonspecifical binding to COS-7 cells were investigated by using confocal laser scanning microscopy. The results indicated that nonspecifical binding of QD micelles depended on particle concentration and incubation time. The surface charge of QD micelles and the serum in cell culture medium also affected the nonspecifical binding. In particular, functional QD micelles with highly charged surface groups, such as carboxylic acids and amines, have been shown to more strong nonspecific binding to cells than that with little charged methyl groups. These results offered a foundation for further better biological application of quantum dots in cell biological field by pertinent modification of their surface to minimize nonspecific cellular binding of QDs.3. The Selectively Binding and Cellular Internalization Study of QD-angiogenin ConjugatesAngiogenin is closely correlated with the occurrence and development of cancers. Based on above work, QD-angiogenin conjugates, prepared using an aptamer that bridges the inorganic fluorophores and angiogenin, were employed to recognize target cells. The cellular internalization study of the conjugates was followed. With the function of angiogenin receptor, QD-angiogenin conjugates could be selectively bound to human umbilical vein endothelial cells and human cervical carcinoma cells. Z-axis scanning studies demonstrated that the QD-angiogenin conjugates were internalized to intracellular organelles of target cells. Subcellular localization and fluorescent colocalization studies indicated that the conjugates mainly located in organelle lysosome after entering the cells. The QD-angiogenin conjugates could be potentially utilized as a novel cellular recognition system in the field of tumor angiogenesis, tumor early diagnosis and target therapy.更多还原