新型有机荧光探针的设计合成和生物成像研究

发布时间：2018-04-04 21:54

本文选题：荧光　切入点：探针　出处：《吉林大学》2017年博士论文

【摘要】：近年来,荧光探针作为一种优秀的化学检测试剂,由于其具有高的灵敏度和选择性、操作简单、响应迅速等优点,不仅适合于传统实验,还可用于生物体内研究。尤其是在活体生物成像研究方面,荧光探针几乎不对细胞造成损伤,而且还可以实现活体的可视化研究,能够同时显示出较高的空间和时间分辨能力。因此,设计和合成新颖的荧光探针,利用它们识别特定的分析物,具有重要的意义。在本论文中,我们设计合成了基于不同原理的、可用于检测Cu~(2+)、Hg~(2+)、Cd~(2+)和ClO~-的三种新型荧光探针,主要研究内容如下:(1)设计合成了一种对于Cu~(2+)具有识别能力的新型罗丹明衍生物(RhS)。该荧光探针可用于测定饮用水和活体细胞中Cu~(2+)的浓度,并且具有可逆性。其原理为Cu~(2+)与RhS发生配位,RhS的螺酰胺环发生开环反应,导致其荧光发光强度增加,而其他金属离子无此性能,因此该探针对于Cu~(2+)具有较高的选择性。通过Job's点曲线和质谱(MS)确定了RhS和Cu~(2+)配位的化学计量比为1:1,该实验结果同时得到了密度泛函(DFT)理论计算的支持。该探针对于测定Cu~(2+)的线性范围和检出限分别为0.50~20.00和0.11μmol L-1。饮用水样品的分析结果显示该探针检测Cu~(2+)的回收率为100.4~101.2%,此外,探针还显示了其在活体He La细胞中用于监测Cu~(2+)的潜力。(2)基于荧光共振能量转移(FRET)机理,设计合成了一种可用于测定中性水样品中Hg~(2+)和监测活细胞内Hg~(2+)水平的荧光探针DRh。通过UV-Vis和荧光光谱实验,考察了探针DRh对于金属阳离子的选择性,结果表明探针DRh能够对Hg~(2+)进行高选择性的识别。通过Job's点曲线和质谱(MS)确定了DRh和Hg~(2+)配位的化学计量比为1:1,并结合密度泛函(DFT)和含时密度泛函(TD-DFT)理论计算研究了配位和发光机理。该探针对于测定Hg~(2+)的线性范围和检出限分别为0~30.00和0.008μmol L-1。中性水样品的分析结果显示检测Hg~(2+)的回收率为104.5~107.9%。此外,该探针还应用于活体HeLa细胞中Hg~(2+)的成像研究。(3)设计合成了一种水溶性基于荧光素的增强型荧光探针TF。该探针可用于荧光选择性的检测生理pH条件下的ClO~-和Cd~(2+),荧光增强幅度分别为64倍和4倍,增强原理分别为氧化反应和配位反应。探针对于ClO~-和Cd~(2+)的检出限分别为0.068μmol L-1和2.2μmol L-1。采用密度泛函(DFT)和含时密度泛函(TD-DFT)理论计算方法对ClO~-、Cd~(2+)与TF的结合方式与反应机理进行了研究。此外,该探针还显示出了在活体细胞中用于监测ClO~-和Cd~(2+)的潜力。
[Abstract]:In recent years, as a kind of excellent chemical detection reagent, fluorescent probe is not only suitable for traditional experiments, but also can be used in vivo because of its high sensitivity and selectivity, simple operation and rapid response.Especially in the study of in vivo biological imaging, fluorescent probes can hardly damage the cells, but also can realize the visual research in vivo, and can display high spatial and time resolution at the same time.Therefore, it is of great significance to design and synthesize novel fluorescent probes and use them to identify specific analytes.In this thesis, we have designed and synthesized three novel fluorescent probes based on different principles, which can be used for the detection of Cu~(2 and ClO-.The main research contents are as follows: (1) A novel Rhodamine derivative with recognition ability for Cu~(2 was designed and synthesized.The fluorescent probe can be used to determine the concentration of Cu~(2 in drinking water and living cells.The principle is that Cu~(2) reacts with the spiroamide ring of RhS, which leads to the increase of fluorescence intensity, but other metal ions do not, so the probe has a high selectivity for Cu~(2).The stoichiometric ratio of RhS and Cu~(2 is determined to be 1: 1 by Job's point curve and MS. The experimental results are supported by density functional theory.The linear range and detection limit of the probe for the determination of Cu~(2 were 0.50 渭 mol L ~ (-1) and 0.11 渭 mol L ~ (-1), respectively.The analysis of drinking water samples showed that the recovery rate of the probe for detecting Cu~(2 was 100.4 / 101.2. In addition, the probe also showed its potential for monitoring Cu~(2 in He-La cells in vivo, based on the fluorescence resonance energy transfer (FRET) mechanism.A fluorescent probe, DRH, which can be used to determine the level of Hg~(2 in neutral water samples and to monitor Hg~(2 in living cells, has been designed and synthesized.The selectivity of probe DRh to metal cations was investigated by UV-Vis and fluorescence spectra. The results showed that the probe DRh could recognize Hg~(2 with high selectivity.The stoichiometric ratio of DRh and Hg~(2 was determined to be 1: 1 by Job's point curve and MS). The coordination and luminescence mechanism were studied by means of density functional theory (DFT) and time-dependent density functional theory (TD-DFTT).The linear range and detection limit of the probe for the determination of Hg~(2 were 0 ~ 30.00 渭 mol L ~ (-1) and 0.008 渭 mol L ~ (-1), respectively.The analytical results of neutral water samples showed that the recovery of Hg~(2 was 104.5% 107.9%.In addition, the probe was applied to the imaging study of Hg~(2 in living HeLa cells. A water-soluble enhanced fluorescent probe based on fluorescein was designed and synthesized.The probe can be used for the selective fluorescence detection of ClO- and Cd~(2 at physiological pH. The fluorescence enhancement amplitude is 64 times and 4 times, respectively. The enhancement principle is oxidation reaction and coordination reaction, respectively.The detection limits for ClO- and Cd~(2 were 0.068 渭 mol L-1 and 2.2 渭 mol L-1, respectively.The binding mode and the reaction mechanism of ClOOO- (-) and TD-DFT2) with TF were studied by means of density functional theory (DFT) and time-dependent density functional theory (TD-DFTT).In addition, the probe shows the potential for monitoring ClO- and Cd~(2 in living cells.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3

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