新型罗丹明类pH荧光探针的设计及其生物应用

发布时间：2018-05-02 02:10

本文选题：pH值 + 荧光探针　；参考：《西南大学》2016年硕士论文

【摘要】：背景:细胞内pH值在细胞生物学过程中起着至关重要的作用,正常生理条件下,细胞外液的H+浓度约为40 nmol/L(pH=7.40),正常变动幅度约为0.1 0.2个pH单位,异常的pH值会干扰细胞功能,导致细胞生长和分裂发生突变,影响离子转运、内吞和肌肉收缩等生命过程,严重的可诱发癌症和阿尔茨海默综合症。因此,研究细胞内pH值的变化具有重要的理论意义和实践意义。与玻璃电极法、核磁共振法、吸收光谱法等其他检测pH值的方法相比,荧光法由于选择性好、灵敏度高、操作简便、实时检测而且不损伤细胞等优势,已被广泛用于环境和生物体内pH、离子及细胞活性分子(如活性氧和CO2)的检测。目前为止,文献报道的检测细胞内pH值的传感器主要包括:测量细胞质pH值的近中性荧光探针(pH6.8 7.4)和检测酸性细胞器(如溶酶体)的弱酸性荧光探针(pH 4.0 6.0),而鲜有文献报道强酸性pH荧光探针(pH4.0)。虽然强酸性环境不利于大多数生物生存,但某些微生物如幽门螺杆菌和噬酸菌却偏爱这样的生存环境。因此,开发检测强酸性环境的荧光传感器是十分必要的。目的:本论文拟合成结构新颖的罗丹明衍生物,筛选出受金属离子干扰更小,量子产率更高,响应更迅速,膜渗透性好,生物毒性较低的pH荧光探针,以便检测生物体内强酸性pH值。方法:鉴于罗丹明类染料具有良好开关效应的螺环结构和量子产率高、长波吸收、短波发射、水溶性好、生物毒性低等特点,本课题以罗丹明B为母体,先后通过成环、亲核进攻、N-烷基化反应合成了两个结构新颖的罗丹明衍生物。继而测试了目标化合物在不同pH值条件下的紫外吸收光谱和荧光发射光谱,并将其应用于大肠杆菌生物成像。结果:该论文成功合成了目标分子4、5,两目标分子的紫外吸收强度和荧光强度均随酸性pH的减小而增强,且在强酸性环境时变化得尤为明显。其中,化合物4在pH=1.75 2.62范围内的荧光强度与pH值呈线性关系;化合物5在pH=1.70 4.10范围内其荧光强度与pH值呈线性关系。将目标分子导入大肠杆菌后,化合物4在pH=1.75,2.30显示出强烈的红色荧光,在pH=4.83处无荧光,且其荧光强度随pH值增大而减弱;化合物5在pH=1.70,2.37,3.85均呈现出红色荧光,且荧光强度逐渐减小。结论:终产物4、5能迅速响应强酸性环境,且其荧光强度随pH值的变化呈现出良好的线性、时间稳定性、可逆性和不受金属离子干扰的特性,且目标化合物4、5具有较低的生物毒性,能迅速穿透细胞膜检测大肠杆菌胞内pH值。
[Abstract]:Background: intracellular pH plays an important role in the process of cell biology. Under normal physiological conditions, the H concentration of extracellular fluid is about 40 nmol / L ~ (-1) pH = 7.40%, and the normal range is about 0.1 ~ (0.2) pH unit. Abnormal pH value interferes with cell function. Leading to cell growth and division mutation, affecting ion transport, endocytosis and muscle contraction and other life processes, serious cancer and Alzheimer's syndrome can be induced. Therefore, it is of great theoretical and practical significance to study the change of intracellular pH. Compared with other methods, such as glass electrode method, nuclear magnetic resonance method, absorption spectrum method and so on, the fluorescence method has the advantages of good selectivity, high sensitivity, simple operation, real-time detection and no cell damage. It has been widely used in the detection of pH, ions and cellular active molecules (such as reactive oxygen species and CO _ 2) in the environment and organisms. So far, The reported biosensors for the detection of intracellular pH include: the near-neutral fluorescent probe pH 6.8 ~ 7.4 for measuring the pH value of the cytoplasm and the weak acid fluorescence probe for the detection of acidic organelles (such as lysosomes), pH 4.0 ~ 6.0, but rarely reported in the literature. Strong acid pH fluorescence probe (pH 4.0). Although strongly acidic environments are not conducive to the survival of most organisms, some microbes, such as Helicobacter pylori and phagocytic bacteria, prefer such environments. Therefore, it is necessary to develop a fluorescence sensor for the detection of strong acid environment. Objective: in this paper, Rhodamine derivatives with novel structure were synthesized, and pH fluorescence probes with less interference from metal ions, higher quantum yield, faster response, better membrane permeability and lower biological toxicity were screened out. In order to detect the pH value of strong acidity in organism. Methods: in view of the high snail structure and quantum yield of Rhodamine dyes with good switching effect, long wave absorption, short wave emission, good water solubility and low biological toxicity, Rhodamine B was used as the parent in this study. Two novel Rhodamine derivatives were synthesized by nucleophilic attack N-alkylation reaction. The UV absorption spectra and fluorescence emission spectra of the target compounds at different pH values were measured and applied to the biological imaging of Escherichia coli. Results: the target molecule 4H _ 5 was successfully synthesized in this paper. The UV absorption intensity and fluorescence intensity of the two target molecules increased with the decrease of acidic pH, especially in the strong acidic environment. The fluorescence intensity of compound 4 is linearly related to pH value in the range of pH=1.75 2.62, and the fluorescence intensity of compound 5 is linearly related to pH value in the range of pH=1.70 4.10. When the target molecule was introduced into Escherichia coli, compound 4 showed strong red fluorescence at pH ~ (1.75N) 2.30, no fluorescence at pH=4.83, and its fluorescence intensity decreased with the increase of pH value, and compound 5 showed red fluorescence at pH ~ (1.70) ~ 2.37 ~ 3.85, and the fluorescence intensity decreased gradually. Conclusion: the final product 4N5 can respond to the strong acidic environment rapidly, and its fluorescence intensity shows good linearity, time stability, reversibility and no interference with metal ions with the change of pH value. The target compound 4N 5 has low biotoxicity and can rapidly penetrate the cell membrane to detect the intracellular pH value of Escherichia coli.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3

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