细胞器靶向探针合成及荧光成像研究

发布时间：2018-03-05 00:36

本文选题：荧光探针　切入点：次氯酸根　出处：《哈尔滨工业大学》2017年博士论文　论文类型：学位论文

【摘要】：基于小分子荧光探针的荧光成像技术广泛地应用在生物学、医学、诊断学等学科领域。但该技术在很大程度上受制于小分子荧光探针的性质,如发光效率、光稳定性、选择性、灵敏性、生物兼容性等。近年来,科学家尝试着通过各种方法改善小分子荧光探针的化学和光学性能,如调整探针的吸收/发射波长到近红外区域(650-900 nm)、设计特异性识别位点提高探针的选择性、灵敏性,引入亲水、亲脂性基团增加探针的水溶性和脂溶性,并将大量荧光探针用于生物相关性分子、离子、酶等的检测中。其中,由于次氯酸/次氯酸根及铜离子在维持细胞正常生理功能中扮演着重要的角色而引起了广泛关注。但到目前为止,受探针自身性质的制约和复杂生物环境的影响,人们仍然无法完全了解ClO~-或铜离子在细胞,尤其是在细胞器层次所涉及的生理、病理过程中的作用。因此,设计具有细胞器靶向检测功能的小分子荧光探针用于监测ClO~-或铜离子在细胞中的行为具有很大的意义。针对ClO~-或铜离子荧光探针设计及检测中存在的一些关键问题,本论文开展了长波长荧光染料的设计合成、模型探针的设计合成、细胞器靶向定位检测探针的设计合成以及荧光成像检测等工作,具体研究内容如下:制备出含Rhodol与吩噻嗪结构单元的杂化近红外荧光染料PtzRhlMe,并在其结构上进行修饰得到具有线粒体靶向定位功能的近红外染料PtzRhlMe-Mito。利用分子中内置的反应位点,探针PtzRhlMe和PtzRhlMe-Mito能在PBS-乙醇体系中实现了对ClO~-的比率荧光检测,并具有高选择性、高灵敏性和快速响应等优点。探针能用于活细胞外源ClO~-的比率成像检测,同时也通过荧光成像检测了应激细胞Qg源的ClO~-。尤其是,PtzRhlMe-Mito能成功标记细胞线粒体并通过比率成像监测其中ClO~-浓度的变化,成功构建了线粒体靶向检测功能的ClO~-荧光探针模型。基于此,利用SNAr H反应继续合成含罗丹明与吩噻嗪结构单元的杂化近红外荧光染料Ptz RhEt,并对其结构修饰得到具有线粒体靶向定位功能的近红外探针PtzRhEt-Mito。基于类似识别机理,探针PtzRh Et和PtzRhEt-Mito能在PBS-乙腈(乙醇)溶液中实现对ClO~-的快速、高选择性、高灵敏性比率荧光检测。其中,探针PtzRhEt能用于外源和内源ClO~-的比率成像检测,探针Ptz RhEt-Mito能成功标记细胞线粒体并通过比率成像检测ClO~-。研究不但为了解ClO~-在细胞内的生理、病理功能提供了有用的工具,还为长波荧光染料的合成提供了新的思路。通过缩合反应合成了含萘香豆素与罗丹明结构单元的杂化长波长荧光染料NCR,并对其结构修饰得到铜离子模型探针NCR-Cu。利用铜离子催化的罗丹明螺环内酰胺水解开环反应,探针NCR-Cu在HEPES-乙腈溶液中实现了对铜离子的选择性“turn-on”荧光响应并可用于活细胞内铜离子的荧光成像检测,构建了铜离子模型探针。在此模型探针基础上,通过SNAr H反应合成了含吩恶嗪与罗丹明结构单元的杂化近红外铜离子荧光探针PhxRh-Golgi。探针PhxRh-Golgi在HEPES-乙腈溶液中实现了对铜离子的选择性“turn-on”荧光响应。细胞成像实验表明,探针PhxRh-Golgi可实现活细胞内铜离子的荧光成像检测。共定位细胞成像实验表明,探针可靶向检测细胞高尔基体内的铜离子,证实细胞内过量的铜离子在高尔基体内的存在。通过引入吸电子基进行对Fluoran染料修饰,成功调控了其分子内PET机制有关的荧光特性。在此基础上,制备出荧光探针ABAF-Cu和AF-ClO。其中,探针ABAF-Cu在HEPES-乙腈溶液中实现了对铜离子的选择性“turn-on”荧光响应,并通过荧光成像成功检测了活细胞内铜离子。AF-ClO在PBS-乙醇溶液中实现了对ClO~-的选择性“turn-on”荧光响应,并实现了对活细胞内ClO~-的荧光成像检测。研究开辟了Fluoran染料在荧光探针领域的应用。
[Abstract]:Medical imaging technology of small molecule fluorescent probes are widely used in biology, diagnosis based on the nature of science and other fields. But the technology is largely subject to the small molecule fluorescent probe, such as luminous efficiency, light stability, selectivity, sensitivity, biological compatibility and so on. In recent years, scientists try to improve chemical and the optical properties of small molecule fluorescent probe through a variety of methods, such as adjusting the probe absorption / emission wavelength to the near infrared region (650-900 nm), the design of specific recognition sites to improve probe selectivity, sensitivity, hydrophilic, lipophilic groups increase probe water-soluble and fat soluble, and a large number of fluorescent probes for biological relevance molecular detection, ion, enzyme and other. Among them, the hypochlorous acid / hypochlorite and copper ion in maintaining normal physiological function of cell plays an important role and has aroused wide concern. But so far, affected by the nature of their own probe constraints and complex biological environment, people still do not fully understand ClO~- or copper ions in the cell, especially the physiological involved in organelle level, pathological process. Therefore, small molecule fluorescent probe with organelle targeting detection function for to monitor the behavior of ClO~- or copper ions in the cell is of great significance. Aiming at some key problems of ClO~- or copper ion fluorescent probe design and test, this paper carried out the design and synthesis of long wavelength fluorescent dyes, synthetic probe design model, design and synthesis of target organelles to detect probe and fluorescence imaging the detection work, the specific contents are as follows: to prepare hybrid containing Rhodol and phenothiazine units near infrared fluorescent dye PtzRhlMe, and modified to obtain cable in its structure Particle target near infrared dye to PtzRhlMe-Mito. positioning function by using the built-in molecular reaction sites, PtzRhlMe and PtzRhlMe-Mito in the PBS- probe in ethanol system realizes the ratiometric fluorescence detection of ClO~-, and has high selectivity, high sensitivity and fast response etc. can be used to probe ratio imaging detection of viable cells with exogenous ClO~-, at the same time by fluorescence imaging to detect the cell stress source of Qg ClO~-. especially, PtzRhlMe-Mito can be successfully labeled cells and mitochondria by ratio imaging monitoring including ClO~- concentration changes, ClO~- fluorescent probe model successfully constructed the mitochondrial targeting detection function. Based on this, using the SNAr H reaction to synthesis of hybrid Luo Danming and phenothiazine structure unit near infrared fluorescent dye Ptz RhEt, and near infrared probe PtzRhEt-Mito. with mitochondrial targeting function of its structural modification A similar recognition mechanism based on PtzRh, Et and PtzRhEt-Mito probe in PBS- acetonitrile (ethanol) in the solution of ClO~- fast, high selectivity, high sensitivity ratio fluorescence test probe. Among them, PtzRhEt can be used for ratio imaging detection of exogenous and endogenous ClO~-, RhEt-Mito can successfully probe Ptz labeled cells and mitochondria by detection of ClO~-. ratio imaging not only to understand the physiology of ClO~- in cells, pathological function provides a useful tool, but also provides a new way for the synthesis of long wavelength fluorescent dye. Naphthalene containing coumarin and Luo Danming structural unit of the hybrid long wavelength fluorescent dye NCR was synthesized by the condensation reaction, and modifying the structure of copper ion probe model NCR-Cu. the use of copper ion catalyzed Luo Danming spiro lactam hydrolysis ring opening reaction, probe NCR-Cu realized the choice of "turn-on of copper ion in HEPES- acetonitrile solution "Fluorescence response and can be used for fluorescence imaging of living cells in the copper ion, copper ion probe model constructed. Probe on the basis of this model, the SNAr H was synthesized with hybrid phenoxazine and Luo Danming structural unit of the near infrared copper ion fluorescent probe PhxRh-Golgi. probe PhxRh-Golgi HEPES- in acetonitrile solution realizes selective response" copper ion on turn-on fluorescence. Cell imaging experiments demonstrated that the PhxRh-Golgi probe can realize the fluorescence imaging of living cells. The copper ion imaging experiment indicates that cell co localization of target, probe detection of copper ion Golgi cells in vivo, confirmed the presence of excess copper ion in cells in vivo. Golgi modified the Fluoran dye by introducing electron withdrawing, successful regulation of fluorescence of PET in the molecular mechanisms involved. On this basis, prepared by fluorescent probe ABAF-Cu and AF-C The lO. ABAF-Cu HEPES- probe in acetonitrile solution to achieve selective response to copper ion turn-on fluorescence, and successfully detected by fluorescence imaging in living cells of copper ion.AF-ClO in PBS- ethanol solution is realized in response to "ClO~- turn-on" selective fluorescence, and realizes the detection of fluorescence imaging in living cells ClO~- the research opens a new application of Fluoran dye fluorescent probes in the field.

【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q2-33;O657.3

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