【作者】 贺冬秀；

【导师】 陈波；

【作者基本信息】 湖南师范大学 ， 分析化学， 2011， 博士

【摘要】 量子点(QDs)具有独特的物理化学性质和优良光谱性能,已经引起了生物医学、材料学与光学等领域科研工作者的浓厚兴趣和广泛关注,在生物医学、发光二极管与太阳能电池等领域显示了极其广阔的应用前景。本论文瞄准量子点这一重要的研究方向,在对量子点研究现状进行简要综述的基础上,以量子点的制备、表征以及量子点在癌相关物质检测与药物研究中的应用为主线,主要开展了以下几个方面的工作：1.采用巯基乙酸(TGA)、L-半胱氨酸(L-cys)和巯基乙胺(CA)三种稳定剂在水相中直接合成了三种水溶性的CdTe QDs,通过简单的光活化操作将其量子产率提高了一倍。采用紫外可见吸收光谱和荧光发射光谱对这三种量子点的光学性能进行了表征,结果表明：选用同一稳定剂合成QDs,其紫外吸收峰和荧光发射峰随反应时间的延长均有明显红移；当选用不同的稳定剂合成QDs,相同的反应时间,可以得到不同粒径的量子点。即通过控制反应时间或采用不同稳定剂都很容易获得发射波长在520-580 nm之间的不同粒径的量子点。并对这三类量子点的细胞毒性进行了评价。为本论文量子点的后续应用研究奠定基础。2.将花生凝集素(PNA)与水溶性CdTe QDs通过共价连接形成QDs-PNA偶联产物。该偶联产物经过凝胶柱的分离纯化后,分别采用荧光、紫外、红外光谱、SDS-PAGE电泳和胸腺细胞凝集试验对其进行了表征,表明该偶联产物具有良好的光谱性能和生物活性,可作为实际应用的功能化量子点荧光探针。基于丁抗原与花生凝集素选择性结合的特性,利用该探针对肠癌中高表达的T抗原进行检测,且与传统的荧光有机染料标记的免疫荧光分析进行了比较。实验结果表明：该功能化的荧光探针能够有效地识别肠癌的相关T抗原,从而为T抗原的检测以及肠癌的临床诊断与预后判断提供了一种新方法。3.将自制的性能优良的两种不同颜色的水溶性CdTe量子点分别与花生凝集素、刀豆凝集素偶联。将偶联产物经过凝胶柱的分离纯化,并分别用荧光、紫外、红外光谱、SDS-PAGE电泳和细胞凝集试验对其表征,获得两种具有优良光谱性能和良好生物活性的凝集素化量子点荧光探针。基于凝集素能与不同糖类专一性结合的特性,将这两种荧光探针成功应用于肺癌和肝癌细胞膜表面表达的糖复合物标记检测,从而为癌细胞膜表面的不同糖复合物标记表征提供一条新的思路。4.基于尼群地平明显猝灭TGA-QDs和L-cys-QDs量子点荧光,利用这两种水溶性TGA-QDs和L-cys-QDs量子点作为荧光探针,考察了缓冲体系、缓冲液浓度、缓冲液的pH值、反应时间、量子点粒径大小、量子点的浓度等多种因素的影响,发展了两种灵敏度高、选择性强的荧光猝灭法,对药片中尼群地平进行了准确测定。同时探讨了尼群地平导致TGA-QDs荧光猝灭的机制。在优化的实验条件下,两种方法的线性范围分别为1.09-65.4μg/mL和0.38-77μg/mL,检测下限分别为0.11μg/mL和0.28μg/mL。与传统的尼群地平光度方法相比,这两种方法操作简便、快速、灵敏度高,测定结果与中国药典中的标准方法比较,结果满意。5.通过考察中草药中七种酚酸、十种黄酮、五种蒽醌和十三种生物碱类有效成分对水溶性CdTe TGA-QDs的荧光强度影响。研究结果表明：在同等条件下,酚酸、黄酮和蒽醌类化合物酸性越强,其对量子点荧光猝灭程度越大；而对于生物碱而言,生物碱对CdTe TGA-QDs荧光猝灭程度与其碱性强弱亦相关,碱性较强的季铵盐类生物碱对CdTe TGA-QDs荧光有猝灭作用,非季铵盐类生物碱对CdTe量子点荧光猝灭较弱或不猝灭。这一结论在南香橘皮提取液中得到初步验证。6.基于芦荟大黄素、大黄素、大黄酸、大黄酚和大黄素甲醚五种蒽醌化合物能不同程度猝灭CdTe量子点荧光,本章继续研究了五种蒽醌化合物的浓度对水溶性CdTe TGA-QDs的荧光猝灭程度的影响。结果表明在一定的浓度范围内,五种蒽醌化合物的浓度与量子点相对荧光强度呈良好相性关系。同时运用荧光光谱法对大黄素和水溶性量子点相互作用进行了探讨。结果显示大黄素吸附在量子点上形成复合物从而导致量子点荧光猝灭,大黄素导致量子点荧光猝灭方式为静态猝灭。基于量子点荧光猝灭法难以对五种蒽醌类化合物同时定量分析,一种简便、灵敏的高效液相色谱-荧光检测新方法被发展和确证,用于同时分析虎杖、何首乌、大黄及大黄复方制剂中的五种蒽醌化合物。更多还原

【Abstract】 The quantum dots (QDs) exhibit unique physical and chemical properties and excellent luminescent properties, which have a very widespread potential applications in biomedicine, LED, solar batter etc. The quantum dots have attracted the extensive interest and wide concern of the researchers in the fields of biomedicine, materials and optics. Aimed at the important research direction of the quantum dots, the present research condition of quantum dots was summarized, and then the following several works have mainly performed by taking quantum dots preparation, characterization and quantum dots applications in detection of the substances associated with cancer and medicine study as the line of this dissertation.1. Three water-soluble CdTe quantum dots were directly synthesized in water with three different thiol molecules:thioglycolic acid, L-cysteine and cysteamine. The quantum yield of the newly made CdTe quantum dots was doubled through simply light-activated operation. The optical properties of the three quantum dots were characterized by using ultraviolet vision (UV-Vis)spectra and fluorescence emission spectra. The results show with prolongation of the reaction time at the same stabilizer ultraviolet absorption peak and fluorescent emission peak both present obvious red shifts. With the different modifiers at the same reaction time, the quantum dots with different diameters can be gained. That is to say that the quantum dots whose emission wavelength is between 520-580 nm are readily available by controlling the reaction time or using different modifiers. And the cytotoxicities of these three quantum dots have been evaluated, which provides a powerful foundation for further applied research of quantum dots.2. The CdTe QDs-PNA coupling products were prepared through modifying with peanut (Arachis hypogaea) agglutinin (PNA) on the surfaces of the water-soluble CdTe QDs. The coupling products were characterized by using fluorescence, ultraviolet, infrared spectroscopy, SDS-PAGE electrophoresis and thymus cell agglutination test after gel column purification. The coupling products can be used as the QDs fluorescence probe with high optical properties and good bioactivity. Based on the binding properties that PNA strongly recognizes the T antigen, the fluorescent probe was used to detect T antigen highly expressed in the patients with colorectal carcinoma and compared with conventional fluoroimmunoassay labeled by organic dye-labeled fluorescent probe. The experiment results demonstrate that the QDs-PNA fluorescence probes can effectively recognize T antigen and exhibit good sensitivity and exceptional photostability, which would provide a new method in detecting T-antigen and offer a novel way for the diagnosis and prognosis of colorectal cancer. 3. Two CdTe QDs conjugated to agglutinin were prepared through modifying with peanut agglutinin (PNA) and concanavalin agglutinin on the surfaces of the two colors of water-soluble CdTe QDs, respectively. The two coupling products were characterized by using fluorescence, ultraviolet, infrared spectroscopy, SDS-PAGE gel electrophoresis and cell agglutination test after gel column purification. Based on the property that lectin specific binding with different corresponding carbohydrate, these two fluorescence probes were successfully used to label and detect the carbohydrate complexes expressed on the cell surface of lung and liver cancer, which can provide a new method that characterizes different carbohydrate complexes on cell membrane surface.4. With the prepared water-soluble TGA-QDs and L-cys-QDs in chapter two used as fluorescent probes, two novel approaches were developed for sensitive and selective detection of nitrendipine in tablet based on the fluorescence quenching of aqueous CdTe quantum dots by nitrendipine. Meanwhile, different effect factors on the detection were investigated, including buffer concentration, buffer pH, reaction time, quantum dots size and quantum dots concentration. The mechanism of fluorescence quenching of TGA-QDs caused by nitrendipine was further studied. Under the optimized conditions, the linear ranges of the two methods were 1.09-65.4μg/mL and 0.38-77μg/mL, and the detection limits were 0.11μ.g/mL and 0.28μg/mL, respectively. Compared with traditional spectrophotometry for nitrendipine detection, the two methods were simple, fast, and highly sensitive. The results were in good agreement with that of the national standard method in Chinese Pharmacopoeia.5. Seven phenolic acids, ten flavonoids, five anthraquinones and thirteen alkaloids which have an effect on the fluorescence intensity of aqueous CdTe TGA-QDs were investigated in Chinese herbal medicine. The findings indicate that under the same conditions the degree of fluorescence quenching of water-soluble CdTe TGA-QDs increase with increasing acidity of the phenolic acids, flavonoids and anthraquinones; quaternary ammonium alkaloids have a strong influence on the fluorescence intensity of aqueous CdTe TGA-QDs while non-quaternary alkaloids have weak or no influence. The conclusion has been preliminary verification in the extract of the citrus peel of the Nanxiang.6. The effect of the five anthraquinones concentration on the fluorescence intensity of aqueous CdTe TGA-QDs was investigated based on the conclusion that aloe-emodin, rhein, emodin, chrysophanol and physcion can quench the fluorescence of CdTe TGA-QDs. The results show that it has good linearity between the concentration of anthraquinones and relative fluorescence intensity of quantum dots at a certain concentration range. The interaction of emodin and water-soluble quantum dots has been explored by using fluorescence spectra. The results showed that complex substance formed by emodin adsorbed on the QDs leads to the fluorescence quenching of CdTe quantum dots and the way of fluorescence quenching is static quenching. Because the quantum dots fluorescence quenching methods was difficult for the simultaneous analysis of five anthraquinones, a simple and sensitive HPLC with fluorescence detection was developed and validated to a simultaneous analysis of five anthraquinones in P.cuspidatum, Polygonum multiflori, Rhubarb and compound preparation.更多还原