论文标题:量子点荧光探针的制备及其在化学生物分析中的应用研究
Preparation of Luminescent Quantum Dots and Their Applications in Chemical and Biological Analysis
论文作者
论文导师 王柯敏;谭蔚泓,论文学位 博士,论文专业 分析化学
论文单位 湖南大学,点击次数 380,论文页数 133页File Size8327K
2006-12-01论文网 http://www.lw23.com/lunwen_1735887/
Nanocrystals; Quantum dots; Ion detection; Protein detection; Cellular imaging; Nanorods
量子点是近年发展起来的一种新型荧光探针,与传统的有机荧光染料相比,具有许多优良的光谱性能,在生物化学、细胞生物学、分子生物学等研究领域显示了极其广阔的应用前景,已经引起了人们越来越广泛的重视。本论文瞄准这一重要的研究方向,在对当前迅速发展的量子点进行简要综述的基础上,以量子点的制备、量子点的性能表征以及量子点在化学生物分析中的应用为主线,主要开展了以下几个方面的工作: 一、直接制备了水溶性CdTe量子点,开展了基于水溶性量子点的Cu~(2+)离子测定研究。 以巯基乙酸为稳定剂,采用水相合成法成功制备了表面带有羧基的水溶性CdTe量子点,并对其进行了荧光发射光谱、紫外可见吸收光谱、荧光成像、原子力显微镜(AFM)成像、透射电子显微镜(TEM)成像等系列表征。实验结果表明,所制备的量子点具有良好的光学性能,其激发光谱宽且连续,发射光谱窄而对称,为本论文后续的拓展与应用研究提供了保证。在此基础之上,以该水溶性量子点作为荧光探针,基于荧光猝灭效应对Cu~(2+)离子进行了测定,并阐释了Cu~(2+)离子引起量子点荧光猝灭的机理,考察了缓冲体系、反应时间等多种因素的影响。在优化的实验条件下,该方法的检测下限为0.29μg/L,线性范围为2~200μg/L。与传统的Cu~(2+)离子荧光检测方法相比,该方法有效克服了有机荧光染料光稳定性差等缺陷,具有简单直接、条件温和等优点,发展了一种基于水溶性CdTe量子点的Cu~(2+)离子测定技术。 二、基于水溶性CdTe量子点,建立了一种用于人免疫球蛋白测定的新方法。利用上述水溶性CdTe量子点优良的光学性能,结合胶体金消光系数大、吸收光谱可调等特性,基于量子点与胶体金之间的荧光共振能量转移作用,发展了一种用于人免疫球蛋白测定的新方法。量子点的发射波长可以通过改变尺寸进行调控,从而保证了供体发射波长与受体吸收波长的良好重叠,促进了荧光共振能量转移的产生。该方法无需昂贵的实验仪器,设计灵活,操作简便,特异性好,安全可靠,线性范围为0.5~10μg/mL,检测下限为0.21μg/mL,为人免疫球蛋白的测定提供了一种高效、便捷的检测手段。 三、发展了一种基于水溶性CdTe量子点的新型荧光标记方法,在细胞水平上对多药耐药蛋白P-gp的表达进行了表征。 瞄准细胞水平上的纳米表征这一重要研究领域,结合免疫分析技术,以舌癌细胞多药耐药蛋白P-gp为检测底物,通过在水相法直接制备的CdTe量子点表面同时修饰生物素(Biotin)和聚乙二醇(Polyethyleneglycol, PEG),成功的在细胞水平上对多药耐药蛋白P-gp的表达进行了表征。研究结果表明,药物诱导之前,舌癌细胞的P-gp表达量较低,经过平阳霉素处理以后,其表达量明显升高。与传统采用的荧光标记技术相比,该方法显示出了良好的抗光漂白性能,可作为一种准确高效的识别手段,应用到细胞及亚细胞水平上的纳米表征这一前沿性研究领域。 四、制备了光稳定性更加优良的硅壳型CdTe量子点,并将其应用于肝实质细胞的识别。 基于油包水(W/O)反相微乳液体系,以上述水溶性CdTe量子点作为内核材料,采用硅烷化试剂在油包水形成的微囊中同步水解的方法,在温和的实验条件下,将多个量子点包被到同一硅壳中,从而制备了硅壳型量子点荧光纳米颗粒。研究结果表明,所得颗粒具有更加优良的光稳定性,连续照射2000 s以后,荧光强度仅下降了3.54%。此外,该颗粒大小均一,分散性好,并根据实验需要同步带上了氨基、磷酸基等化学基团,进而把对哺乳动物肝实质细胞有特异性识别作用的乳糖酸修饰到颗粒表面,实现了对肝实质细胞的识别。 五、制备了一种发蓝色荧光的半导体纳米晶体,促进了短波长发光纳米材料的相关研究进展。 对量子点进行硅壳包被能够进一步提高其光稳定性,但美中不足的是,不管是对单个量子点进行包壳,还是采用微乳液的方法将多个量子点包被到同一硅壳中,都要经过量子点的制备、硅烷化处理等多个相互独立的环节。为了简化操作步骤,本论文提出了以3-(巯基丙基)三甲氧基硅烷(MPS)为稳定剂,基于自组装的原理,直接制备硅壳型CdTe量子点的实验设想。然而,实验中发现,采用该路线并没有得到硅壳型CdTe量子点,却生成了一种性能优良的蓝色发光半导体纳米晶体,为蓝色发光材料的制备开辟了一条新的途径。因此,瞄准短波长发光纳米材料这一前沿研究领域,对所得产物进行了荧光光谱、荧光成像、透射电子显微镜(TEM)、高倍透射电子显微镜(HRTEM)、选区电子衍射花样等系列表征。研究结果表明,所制备的纳米晶体呈比较规则的棒状,分散性好,具有优良的光谱性能,且光稳定性好,最大发射波长在410 nm左右,表现出较强的蓝色荧光,是一种新型的蓝色发光半导体纳米材料。 六、以价格低廉的工业用导热油为溶剂,成功合成了性能优良的脂溶性CdSe量子点。 针对脂溶性量子点制备过程中所用溶剂成本较高这一缺陷,本论文采用高温胶体化学合成方法,以价格低廉的工业用导热油Dowtherm RP为溶剂,直接制备了脂溶性CdSe量子点。荧光光谱、吸收光谱、透射电子显微镜(TEM)成像、高倍透射电子显微镜(HRTEM)成像等测定结果表明,所合成的量子点大小均一,单分散性好,具有极其优良的光学性能,吸收光谱宽且连续,荧光发射光谱窄而对称,没有红色拖尾现象,半峰宽(Full Width at Half Maximum, FWHM)均在26~30 nm之间,且光稳定性强,连续照射3600 s以后,其荧光强度几乎没有变化。该方法为脂溶性CdSe量子点的低成本合成提供了可能,并有望应用于其他类型脂溶性量子点的制备研究。
Compared with conventional organic fluorescent dyes, quantum dots have a lot of unique optical properties, which make them appealing as a new class of fluorescent probes. In recent years, quantum dots have gained increasing attention and played important roles in the fields of biochemistry, cell biology and molecular biology, etc. Aiming at this important research direction, the development of quantum dots was summarized, and then the following several works have mainly performed by taking quantum dots preparation, quantum dots characterization and quantum dots applications in chemical and biological analysis as the line of this dissertation. 1. Water-soluble CdTe quantum dots were synthesized directly and used for the detection of Cu2+. Water-soluble CdTe quantum dots were prepared in aqueous solution by using thioglycolic acid as the stabilizer and then characterized by fluorescence spectroscopy, UV-Visible spectroscopy, AFM and TEM, respectively. The results show that these quantum dots have good optical properties, such as narrow spectral line widths and continuous absorption profiles, which provide a powerful foundation for further applications. With this kind of water-soluble quantum dots as fluorescence probes, a novel approach was developed for sensitive and selective detection of copper ions based on fluorescence quenching. The mechanism of fluorescence quenching caused by copper ions was further studied. Meanwhile, different influence factors to the detection were investigated, including buffer concentration, quantum dots concentration, pH values and reaction time. Under the optimized conditions, the relative fluorescence intensity decreased linearly with the copper ions concentration in the range from 2 to 200μg·L-1 and the detection limit could reach 0.29μg·L-1. Compared with traditional fluorescence methods for copper ions detection, this quantum dots-based approach is more stable and effective and could be used as a new technique for the detection of copper ions. 2. A novel method was developed for the detection of human IgG based on water-soluble CdTe quantum dots. By using the above-prepared water-soluble CdTe quantum dots, a new method was established to detect human IgG based on the fluorescence resonance energy transfer between functional quantum dots and Au nanoparticles. Quantum dots have size-dependent emission spectrum, which can be overlapped easily by the absorbance spectrum of Au nanoparticles and thus facilitates the fluorescence resonance energy transfer. Based on this method, human IgG can be detected easily and specifically without expensive instrument. The relative fluorescence intensity increased linearly with human IgG concentration in the range from 0.5 to 10μg·mL-1 and the detection limit could reach 0.21μg·mL-1. Therefore, this method could be used as a novel technique in the detection of human IgG. 3. A novel fluorescence labeling method was developed to characterize the expression of P-gp at cellular level based on water-soluble CdTe quantum dots. Aiming at the advance research field related to cellular characterization by nanotechnology, the above-prepared water-soluble CdTe quantum dots were successfully conjugated with biotin and polyethylene glycol and further used for immunofluorescent labeling of multi-drug resistance related protein P-gp of toque cancer cells (Tca8113 and Tca8113/BLM). The results demonstrate that luminescence in Tca8113/BLM cells is brighter than that in Tca8113 cells, indicating that p-gp are over-expressed in BLM-induced Tca8113 cells (Tca8113/BLM). Compared with traditional fluorescent labeling methods, this approach has good stability against photobleaching, which would play an important role in the field of cellular and subcellular characterizations. 4. CdTe quantum dots doped silica nanoparticles with excellent photostability were prepared and used to recognize liver cells. Based on the reverse microemulsion technique, luminescent quantum dots doped core-shell nanoparticles were prepared at moderate conditions by employing water-soluble CdTe quantum dots as the core and silica as the shell of the nanoparticles. In comparison with the pure water-soluble CdTe quantum dots, these nanoparticles showed superiority in photostability. This method is useful and simple for obtaining uniform quantum dots-doped nanoparticles without other rigorous experimental conditions and the nanoparticles have a good dispersibility. Moreover, these nanoparticles were modified with amine groups and phosphonate groups in one step, and further labeled with galactose receptor. With the function of galactose receptor, living liver cells were recognized by galactose receptor-modified quantum dots-doped nanoparticles successfully. 5. Semiconductor nanocrystal with blue emission was synthesized, which accelerates the development of short-wavelength-emitting nanomaterials. As is known to all, treatment of quantum dots by silica coating can further improve their photochemical stability. However, quantum dots preparation and silanization are separated in the reported methods. In order to simplify the operation, we designed another synthesizing route to prepare silica-coated CdTe quantum dot directly by using (3-mercaptopropyl)-trimeth-oxysilane as the stabilizer. Accidentally, a novel blue-light-emitting nanomaterials were obtained during this process. Aiming at the advance research field of short-wavelength-emitting nanomaterials, these products were further characterized by fluorescence spectroscopy, TEM, HRTEM and selected area electron diffraction (SAED) pattern, respectively. Nanorod structures can be seen distinctly from the TEM and HRTEM image. Furthermore, the results show that these nanorods have good luminescent characteristics with emission maximum at ~410 nm, exhibit good photostability and have a number of potential advantages, which could be used as a novel blue-emitting nanomaterials. 6. By using low-price heat-transfer fluid oil as solvent, hydrophobic CdSe quantum dots were synthesized successfully. According to the high temperature colloidal chemistry synthetic procedures, hydrophobic CdSe quantum dots were prepared successfully in heat-transfer fluid oil, which is very cheap and often used in industry. To further characterize these quantum dots, fluorescence spectroscopy, UV-Visible spectroscopy, TEM and HRTEM were performed, respectively. The results show that these hydrophobic CdSe quantum dots, with good dispersibility and uniformity, have excellent optical properties including continuous absorption profiles and narrow spectral line widths with the full width at half maximum (FWHM) at 26~30 nm. Moreover, no obvious change of fluorescence intensity of quantum dots was observed after it was excited for 3600 seconds by successive intense irradiation, which demonstrates that these quantum dots have excellent photostability. This approach is low-cost and safe for the synthesis of hydrophobic CdSe quantum dots and offered the possibility to produce other kind of hydrophobic quantum dots.
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