【作者】 何定庚；

【导师】 何晓晓； 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2009， 硕士

【摘要】 二氧化硅纳米材料作为无机纳米材料的重要成员之一,由于具有制备简单、性质稳定、表面易修饰、良好的生物相容性等优良的物理和化学性质,已在化学、工程和生物医学等学科领域得到了广泛的应用。在进一步拓展二氧化硅纳米材料应用的同时,开展形貌可控的二氧化硅纳米材料的制备研究对于二氧化硅纳米材料制备和应用研究都具有非常的重要意义。本论文开展了不同形貌的二氧化硅纳米材料制备研究,此外,将本研究小组发展的基于二氧化硅荧光纳米颗粒和SYBR Green I的双色流式细胞术进一步应用于大肠杆菌O157:H7的检测研究。主要包括以下两个方面的工作:一、多聚赖氨酸为模板的形貌可控性二氧化硅纳米材料制备以多聚赖氨酸为内核材料,通过正硅酸乙酯在油包水反相微乳液中水解后制备不同形貌二氧化硅纳米材料的研究,探讨了多聚赖氨酸构象变化与二氧化硅纳米材料形貌变化的关系。结果表明通过采用多聚赖氨酸同一种模板材料,可以分别制备球形结构、棒状核壳结构和管形结构三种不同形貌的二氧化硅纳米材料。在此基础上,以Hela细胞为代表,初步考查了制备的不同形貌的二氧化硅纳米材料的细胞吞噬情况,初步研究结果表明不同形貌的二氧化硅纳米材料和Hela细胞孵育24h后,均能进入细胞,且随机地分布在细胞质中。二、二氧化硅荧光纳米颗粒和SYBR Green I双色流式细胞术(FSiNP@SG-FCM)用于大肠杆菌O157:H7的检测研究将本研究小组发展的基于二氧化硅荧光纳米颗粒和SYBR Green I的双色流式细胞术应用于大肠杆菌O157:H7的检测研究,通过大肠杆菌O157:H7抗体修饰的二氧化硅荧光纳米颗粒对大肠杆菌O157:H7的识别与结合以及SYBR Green I对大肠杆菌O157:H7的核酸标记染色,完成对大肠杆菌O157:H7的双色标记,再通过流式细胞术的免分离检测方法对大肠杆菌O157:H7进行快速、灵敏检测。研究结果表明,该方法对PB缓冲液体系中大肠杆菌O157:H7的检测下限为2.8×10~3cfu/ml,比基于FITC标记的传统流式细胞术具有低一个数量级的检测下限。更多还原

【Abstract】 As one of the important members of inorganic nanomaterials, silica nanomaterials have been widely applied in chemical, engineering and biomedical fields, with their extraordinary properties including simple synthesis, high stability, easy modification and good biocompatibility. Studies on shape-controlled preparation of silica nanomaterials are very necessary and important for the development of silica nanomaterials. In this paper, preparation of silica nanomaterials with various morphology has been studied, and the previously developed, fluorescent nanoparticles and SYBR Green I based two-color flow cytometry has also been further applied to detect E. coli. O157: H7.1. Shape-controlled preparation of silica nanomaterials by using poly-L-lysine as the template.We selected poly-L-lysine as the core material and prepared silica nanomaterials with various morphologies through TEOS hydrolysis in the reverse microemulsion. The influence of poly-L-lysine solutions with different pH and concentrations on the preparation of silica nanomaterials with various morphologies has been systematically examined. And by using circular dichroism to analyse the secondary structures of poly-L-lysine under different conditions, the relationship between the structure changes of poly-L-lysine and the different morphologies of silica nanomaterials has been further explored. The results showed that silica nanomaterials could be prepared into spherical, rod-like and the tube-shaped structures, respectively, by using the same template material poly-L-lysine. In addition, with Hela cells as the representative cell line, we initially examined whether the prepared silica nanomaterials with different morphologies could be taken up by cells. And the preliminary results showed that, after a 24h-incubation with Hela cells, all the three silica nanomaterials with different morphologies could get into the cells, and randomly distributed in the cytoplasm.2. Fluorescent nanoparticles and SYBR Green I based two-color flow cytometry (FSiNP@SG-FCM) for detection of E. coli. O157:H7A method using an improved two-color flow cytometric analysis by a combination of fluorescent silica nanoparticles and SYBR Green I has been developed for the rapid detection of E. coli O157:H7. Firstly, antibody of E. coli. O157:H7 was conjugated with RuBpy-doped silica nanoparticles. Then, E. coli. O157:H7 was specially labeled with antibody-conjugated RuBpy-doped silica nanoparticles, and subsequently stained with a nucleic acid dye SYBR Green I, followed by multiparameter determination with flow cytometry. This assay allowed for detection of as low as 2.8×10~3 cfu/ml E. coli. O157:H7 in buffer, which was decuple lower than the detection limit of the FITC-based conventional flow cytometry. This proposed FSiNP@SG-FCM method will be promising for rapid detection of E. coli. O157:H7 in water and food.更多还原