本文選題：pH值 + 熒光探針�。� 參考：《西南大學》2016年碩士論文

【摘要】：背景:細胞內(nèi)pH值在細胞生物學過程中起著至關(guān)重要的作用,正常生理條件下,細胞外液的H+濃度約為40 nmol/L(pH=7.40),正常變動幅度約為0.1 0.2個pH單位,異常的pH值會干擾細胞功能,導(dǎo)致細胞生長和分裂發(fā)生突變,影響離子轉(zhuǎn)運、內(nèi)吞和肌肉收縮等生命過程,嚴重的可誘發(fā)癌癥和阿爾茨海默綜合癥。因此,研究細胞內(nèi)pH值的變化具有重要的理論意義和實踐意義。與玻璃電極法、核磁共振法、吸收光譜法等其他檢測pH值的方法相比,熒光法由于選擇性好、靈敏度高、操作簡便、實時檢測而且不損傷細胞等優(yōu)勢,已被廣泛用于環(huán)境和生物體內(nèi)pH、離子及細胞活性分子(如活性氧和CO2)的檢測。目前為止,文獻報道的檢測細胞內(nèi)pH值的傳感器主要包括:測量細胞質(zhì)pH值的近中性熒光探針(pH6.8 7.4)和檢測酸性細胞器(如溶酶體)的弱酸性熒光探針(pH 4.0 6.0),而鮮有文獻報道強酸性pH熒光探針(pH4.0)。雖然強酸性環(huán)境不利于大多數(shù)生物生存,但某些微生物如幽門螺桿菌和噬酸菌卻偏愛這樣的生存環(huán)境。因此,開發(fā)檢測強酸性環(huán)境的熒光傳感器是十分必要的。目的:本論文擬合成結(jié)構(gòu)新穎的羅丹明衍生物,篩選出受金屬離子干擾更小,量子產(chǎn)率更高,響應(yīng)更迅速,膜滲透性好,生物毒性較低的pH熒光探針,以便檢測生物體內(nèi)強酸性pH值。方法:鑒于羅丹明類染料具有良好開關(guān)效應(yīng)的螺環(huán)結(jié)構(gòu)和量子產(chǎn)率高、長波吸收、短波發(fā)射、水溶性好、生物毒性低等特點,本課題以羅丹明B為母體,先后通過成環(huán)、親核進攻、N-烷基化反應(yīng)合成了兩個結(jié)構(gòu)新穎的羅丹明衍生物。繼而測試了目標化合物在不同pH值條件下的紫外吸收光譜和熒光發(fā)射光譜,并將其應(yīng)用于大腸桿菌生物成像。結(jié)果:該論文成功合成了目標分子4、5,兩目標分子的紫外吸收強度和熒光強度均隨酸性pH的減小而增強,且在強酸性環(huán)境時變化得尤為明顯。其中,化合物4在pH=1.75 2.62范圍內(nèi)的熒光強度與pH值呈線性關(guān)系;化合物5在pH=1.70 4.10范圍內(nèi)其熒光強度與pH值呈線性關(guān)系。將目標分子導(dǎo)入大腸桿菌后,化合物4在pH=1.75,2.30顯示出強烈的紅色熒光,在pH=4.83處無熒光,且其熒光強度隨pH值增大而減弱;化合物5在pH=1.70,2.37,3.85均呈現(xiàn)出紅色熒光,且熒光強度逐漸減小。結(jié)論:終產(chǎn)物4、5能迅速響應(yīng)強酸性環(huán)境,且其熒光強度隨pH值的變化呈現(xiàn)出良好的線性、時間穩(wěn)定性、可逆性和不受金屬離子干擾的特性,且目標化合物4、5具有較低的生物毒性,能迅速穿透細胞膜檢測大腸桿菌胞內(nèi)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.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：O657.3

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本文編號：1831950