基于喹啉为母体的双光子荧光化学探针的研究及其在细胞中的应用

发布时间：2018-04-19 03:06

本文选题：Cd~(2+) + Cys/Hcy　；参考：《安徽大学》2014年硕士论文

【摘要】：荧光化学探针是检测生物体内金属离子浓度以及小分子的重要工具,其高选择性、高灵敏度、合成简单、价格便宜以及很好的生物应用的性质,使其逐步成为主要检测工具。通过荧光化学探针与生物体内的活性物质参与化学反应的结果,可以帮助我们更好地从微观角度去了解生命活动。近些年来,荧光检测技术的应用主要在于临床诊断,生物成像,环境保护等方面。然而,目前荧光化学探针大部分为单光子荧光化学探针,单光子荧光化学探针不仅易使生物本体产生自发荧光干扰,而且短的激发波长还会对细胞产生光致毒和光漂白。与传统的单光子荧光化学探针相比,双光子荧光探针作为一个科学家感兴趣的研究课题,开始逐步替代单光子荧光探针。双光子荧光化学探针具有渗透到更深的组织能力和提供更高的空间分辨率以及局域激发在近红外光谱区(700-1000nm),长波激发可以降低光漂白以及减少对生物样品的光损伤,从而得到高对比度的三维(3D)图像。特别是,双光子共聚焦成像在细胞中能延长活细胞和组织的观察时间的特有优点,这使得生物科学家们更好地观察和研究活细胞和组织中的生命过程。在众多的荧光报告基团中,喹啉衍生物由于其良好的光谱特性、水溶性、易于修饰以及低毒性的特点,越来越多地应用于生物实验中。本文利用Sonogashira偶联等有机化学反应,以喹啉为母体,成功地制备了一系列能够选择性识别Cd2+口Cys/Hcy的双光子荧光化学探针,并通过核磁共振谱以及质谱等手段对目标探针分子进行了结构表征。一,本论文设计并合成了一例基于喹啉为母体,并在细胞溶酶体中定位的新型双光子荧光探针Lyso-QS1。当探针分子与Cd2+络合后,基于ICT,荧光谱图红移了60nm;基于PET机理,荧光逐渐增强。由于其对细胞毒性低,在双光子共聚焦成像,发现探针很容易地在活细胞的溶酶体中长时间观察Cd2+在细胞溶酶体中的成像,这也是我们第一个报道发现的。探针Lyso-QS1与Lysotracker Green商用品在7402细胞中进行了对比研究,研究表明了二者均在溶酶体定位,重叠率能达到90%以上。二.设计合成了一种新型比率计双光子荧光探针MQ高选择地来检测Cys/Hcy,基于ICTPPET机理可以作为Cys和Hcy的新型反应型探针。荧光探针在加入Cys/Hcy时,在710nm拥有大双光子吸收截面值为265GM,同时伴随着发射波长发生95nm的蓝移。与之前报道的双光子荧光探针检测Cys/Hcy相比,MQ与Cys/Hcy反应速度较快。反应机理主要通过1HNMR, Maldi-TOF MS和理论计算(DFT)来分析。细胞毒性测试表明了MQ对细胞毒性低,生物成像表明了在双光子激发下,MQ是一个能够穿透细胞膜来检测细胞内的Cys。
[Abstract]:Fluorescent chemical probe is an important tool for detecting the concentration of metal ions and small molecules in organism. It has the advantages of high selectivity, high sensitivity, simple synthesis, low cost and good properties of biological application, which makes it gradually become the main detection tool.The results of chemical reactions with fluorescent chemical probes and active substances in organisms can help us to understand the life activities from the microscopic point of view.In recent years, the application of fluorescence detection technology mainly lies in clinical diagnosis, biological imaging, environmental protection and so on.However, at present, most of the fluorescent probes are single photon fluorescence probes. Single photon fluorescence probes not only cause autofluorescence interference in biological bodies, but also produce phototoxicity and photobleaching on cells with short excitation wavelength.Compared with traditional single photon fluorescence probes, two photon fluorescence probes, as a subject of interest to scientists, are gradually replacing single photon fluorescence probes.Two-photon fluorescence probes have the ability to penetrate deeper tissues and provide higher spatial resolution and local excitation in the near infrared region of 700-1000 nm ~ (-1). Long wave excitation can reduce photobleaching and reduce photodamage to biological samples.The high contrast 3D image is obtained.In particular, two-photon confocal imaging has the unique advantage of prolonging the observation time of living cells and tissues in cells, which makes biological scientists better observe and study the life processes in living cells and tissues.Quinoline derivatives have been widely used in biological experiments due to their good spectral properties, water solubility, easy modification and low toxicity among many fluorescent reporting groups.In this paper, a series of two-photon fluorescence probes which can selectively recognize Cd2 mouth Cys/Hcy have been successfully prepared by using organic chemical reactions such as Sonogashira coupling and quinoline as parent.The structure of the target probe was characterized by NMR and MS.In this thesis, a novel two-photon fluorescence probe Lyso-QS1 was designed and synthesized based on quinoline as parent and located in lysosome.When the probe molecule is complexed with Cd2, the fluorescence spectrum shifts to 60 nm based on ICT.Based on the mechanism of PET, the fluorescence intensifies gradually.Because of its low cytotoxicity, the probe was found to be easy to observe Cd2 imaging in lysosomes of living cells for a long time in two-photon confocal imaging, which is the first reported finding.Lyso-QS1 and Lysotracker Green were compared in 7402 cells. The results showed that both of them were located in lysosome, and the overlap rate was over 90%.II.A novel ratio meter two-photon fluorescence probe MQ is designed and synthesized to detect cysts in high selectivity. Based on the ICTPPET mechanism, it can be used as a new reactive probe for Cys and Hcy.With the addition of Cys/Hcy, the fluorescence probe has a large two-photon absorption cross section of 265 GM in 710nm, and the blue shift of 95nm occurs with the emission wavelength.Compared with the two-photon fluorescence probe reported previously, the reaction rate between Cys/Hcy and Cys/Hcy is faster.The reaction mechanism was analyzed by 1H N M R, Maldi-TOF MS and theoretical calculation.Cytotoxicity tests showed that MQ was less cytotoxic, and biometric imaging showed that MQ was a cell penetrating cell membrane capable of detecting intracellular Cyss under two-photon excitation.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914;O657.3

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