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用于细胞内活性氧自由基检测的远可见、近红外荧光探针的设计合成及应用研究

用于细胞内活性氧自由基检测的远可见、近红外荧光探针的设计合成及应用研究

论文编辑
论文导师 唐波,论文学位 博士,论文专业 植物学
论文单位 山东师范大学,点击次数 62,论文页数 106页File Size2658K
2007-05-20论文网 http://www.lw23.com/lunwen_434341647/
superoxide anion radical; peroxide hydrogen; near-IR fluorescent probe; cell imaging; simultaneous measure
生物体内活性氧自由基水平直接与生物的生理、病理相关,高选择性识别与定量自由基是研究自由基参与生命活动机制的前提。本文以荧光素、萘荧光素为荧光发色团,基于非氧化机理,设计合成了系列远可见、近红外荧光分子探针,实现了细胞内超氧阴离子自由基及过氧化氢的检测及可视化成像;将荧光素系列探针与萘荧光素系列探针优化组合,筛选出互不干扰的两种探针用于超氧阴离子自由基及过氧化氢的同时检测,**实现同一体系中两种自由基的同时分别定量。 本论文主要内容如下: 1、基于超氧阴离子自由基的亲核性,大家设计合成了一种用于识别超氧阴离子自由基的新型荧光探针—双(二苯基次膦酰基)荧光素(FP-1),并通过元素分析,红外,核磁共振及质谱对探针结构进行表征,建立了测定O2-·的普通荧光法。探针试剂本身没有荧光,与O2-·反应后荧光显著增强,激发和发射波长分别位于490 nm和530 nm。该探针能高选择性地捕获O2-·,而同倍共存的其它活性氧(ROS)及一些生物样品中常存在的还原性化合物不干扰。该方法在两段O2-·浓度范围内,1×10-10~1×10-8 mol l-1和1×10-6~8×10-6 mol l-1,与荧光增强呈现良好的线性关系,*低检测限为4.6 pM,具有很高的灵敏度。利用激光共聚焦成像技术对探针孵育的正常巨噬细胞、PMA刺激的巨噬细胞及Tiron试剂处理的巨噬细胞荧光成像,照片显示探针对微摩尔级的O2-·产生响应。实验表明该荧光探针对于O2-·的识别与检测具有选择性好、灵敏度高、水溶性好及生物兼容性等优点,且可实现细胞内O2-·的可视化示踪。研究结果将为植物体内超氧阴离子自由基的检测及生理、病理研究提供实用可靠的探针及实验方法。 2、设计合成了一种新型红色荧光探针—双(二苯基次膦酰基)萘荧光素(PNF-1),用于检测细胞内超氧阴离子自由基。该探针是基于O2-·的强亲核性促使分子中次膦酸酯迅速水解生成荧光性化合物-萘荧光素来检测超氧的,其荧光发射谱位于662 nm处,能有效避免细胞自发荧光的干扰,提高了检测的选择性和灵敏度。实验方法的*佳条件及线性范围被获得,检测限为0.1 nM,具有很高的灵敏度。共聚焦细胞成像显示探针可以对细胞内纳摩尔级的O2-·产生响应(RAW 264.7)。结果显示该荧光探针对于O2-·的识别与检测具有选择性好、灵敏度高、膜渗透性好等优点,可实现O2-·的可视化示踪,对于活性氧的研究具有重要意义。 3、设计合成了一种有效用于水相中检测超氧阴离子自由基的新型红色荧光探针—双(二苯基次膦酰基)-5(6)-羧基萘荧光素,用元素分析、红外、核磁共振等手段对探针结构进行了表征,建立了测定水相中O2-·的普通荧光法。探针试剂本身无荧光,经O2-·亲核性水解次膦酯产生强荧光产物-5(6)-羧基萘荧光素,激发和发射波长分别位于588nm和667nm。该探针高选择性捕获O2-·,同倍共存的其它活性氧(ROS)不干扰。该方法线性范围1.0×10-7~1.0×10-5mol l-1,检出限为0.25 nM,具有良好的灵敏度 4、基于H2O2能特异性催化水解磺酸酯,大家**性地设计合成了两种新型近红外荧光探针-双(对甲苯磺酰基)萘荧光素(NFDS-1)和双(全氟辛烷磺酰基)萘荧光素(NFDS-2),用于细胞内过氧化氢的检测。探针试剂本身没有荧光,与过氧化氢反应后产生荧光产物萘荧光素,激发和发射波长分别为602/692 nm,位于近红外区,有效避免了细胞自发荧光的背景干扰。实验表明,脂溶性探针NFDS-1具有良好的膜穿透性,可选择性捕获细胞内的过氧化氢,并且可以对细胞内纳摩尔级过氧化氢含量的变化产生响应。尽管NFDS-1的水溶性欠佳,但在二甲基亚砜促溶下,仍然可以有效进行水相中过氧化氢的检测。方法的线性范围为6.0×10-9~4.0×10-6 mol l-1,检测限为81.5 pM。研究结果将为植物体内过氧化氢的检测及信号转导研究提供实用可靠的探针及实验方法。 5、本文设计合成了新型绿色荧光探针-双(对甲苯磺酸酯基)荧光素(FS-1)和双(对甲苯磺酸酯基)-二氯荧光素(FS-2),用于检测细胞内过氧化氢。检测原理是基于H2O2能特异性催化水解磺酸酯,给出荧光产物荧光素。实验表明,探针FS-1和FS-2在模拟生物体系中检测过氧化氢,具有良好的选择性和灵敏度,且线性范围宽。其它种类活性氧和一些生物体内存在的还原性化合物,包括O2.-,.OH,NO,ONOO-,对苯二酚,谷胱甘肽和酯酶等基本不干扰。细胞成像实验表明,探针FS-1和FS-2用于PMA刺激或外加不同浓度H2O2孵育的小鼠腹膜巨噬细胞均呈现明亮的绿色荧光,且能对细胞内微摩尔级H2O2浓度变化产生响应。证明两探针均具有良好的膜渗透性、高的选择性及良好的灵敏度。 6、利用双(对甲苯磺酸酯基)萘荧光素及双(二苯基次膦酰基)荧光素联合测定肺纤维化鼠的胸腔灌洗液的上清液及巨噬细胞内过氧化氢和超氧阴离子自由基的量,以及肺组织细胞中该两种自由基的同时定量,定性给出了肺纤维化程度与自由基量的关系。
The levels of various reactive oxygen species (ROS) in organism have been associated with biologic physiology and pathology. Recognition and quantifying of free radicals are a premise to study ROS biological functions. In this dissertation, we designed and synthesized a series of fluorescein- and naphthofluorescein-fluorescent probes for detecting and imaging superoxide anion radical (O2-·) and peroxide hydrogen (H2O2) in living cells or aqueous solutions, based on a nonredox mechanism; and performed the simultaneous optical measurement of superoxide anion radical and peroxide hydrogen within living cells from Wistar rats of bleomycin-induced lung fibrosis. The main results of this dissertation were shown as follows: 1. Based on the nucleophilicity of O2-·, a novel green fluorescent probe, bis(diphenylphosphinacyl) fluorescein (PF-1), was synthesized and characterized with elemental analysis, IR, 1H NMR, 31P NMR, and chemical ionization mass spectrometry (CIMS). The synthesis method is simple and original. Upon treatment with O2-?, PF-1, a closed, colorless, and non-fluorescent lactone, was transformed into an open, colored, and fluorescent product (λex/em = 490/530 nm). The fluorimetric experiments of the probe showed that the sensitive fluorogenic reagent features a high selectivity for O2-? over other intracellular ROS and biological compounds, wide response ranges (1×10-10 - 1×10-8 mol l-1 or 1×10-6 - 8×10-6 mol l-1) and low detection limit (4.6 pM) owing to its nucleophilic mechanism. Using fluorescence microscope, cell-derived O2-? were located in live cells. The specific response of PF-1 to O2-? was confirmed by adding a nonenzymatic superoxide scavenger. These experimental results show that PF-1 is an excellent fluorescent probe, which possesses good selectivity, high sensitivity, good water solubility, and prompt reactivity. 2. A near-IR fluorescent probe, bis(diphenylphosphinacyl) naphthofluorescein (PNF-1), was synthesized and characterized with elemental analysis, IR, 1H NMR, 31P NMR, and chemical ionization mass spectrometry (CIMS). The synthesis method is simple and original. The design strategy for the probe is based on the nucleophilic mechanism of O2-·to mediate deprotection of the probe to naphthofluorescein (λex/em = 602/662 nm), while the emission spectrum of naphthofluorescein is just in a spectral region of low background fluorescence interference in biological systems. Upon reaction with O2-·, the probe exhibits a strong fluorescence response and high selectivity for O2-·only, rather than the other reactive oxygen species and some biological compounds. PNF-1 linear calibration curve was obtained and the detection limit was 0.1 nM. The phosphinate-based probe, a new type of fluorescent probe, is cell permeable and can detect nanomolar changes in O2-·concentrations in living cells, using confocal microscopy. 3. Bis(diphenylphosphinacyl)-5(6)-carboxylnaphthofluorescein was synthesized and characterized with elemental analysis, IR and 1H NMR. The design strategy for the probe is based on the nucleophilic mechanism of O2-·to mediate deprotection of the probe to naphthofluorescein (λex/em = 588/667nm). Upon reaction with O2-·, the probe, a closed, colorless, and non-fluorescent lactone, was transformed into an open, colored, and fluorescent product. The fluorimetric experiments of the probe showed that the sensitive fluorogenic reagent features a strong fluorescence response and high selectivity for O2-·in a aqueous solution. 4. In this chapter, we presented the synthesis, fluorescence properties, and biological applications of naphthofluorescein disulfonates (NFDS-1 and NFDS-2), as near-IR fluorescent imaging probes to detect intracellular H2O2. The probe NFDS-1 features high selectivity for H2O2 over competing with other intracellular ROS and some biological compounds, a wide dynamic response range (6.0×10-9-4.0×10-6 mol l-1) and low detection limit (81.5 pM) owing to its nonredox mechanism, and far-visible excitation and near-IR fluorescence emission profiles to minimize cell and tissue damage while avoiding native fluorescence from native cellular species. Furthermore, we have demonstrated the value of this probe by measuring living cell-derived H2O2 and the nanomolar concentration of exogenous H2O2 within living macrophages. We found that NFDS-1 is an excellent fluorescence reagent in detecting intracellular H2O2 and can respond to nanomolar change in H2O2 concentrations within living cells. 5. We designed and synthesized fluorescein disulfonates (FS-1 and FS-2) as fluorescence imaging probes for intracellular H2O2, which were characterized with elemental analysis, IR, 1HNMR. FS-1 and FS-2 are the closed, colorless, and non-fluorescent lactones. Upon treatment with H2O2, hydrolytic deprotection of the probes generated subsequently open, colored, and fluorescent products. The fluorescein-based reagents feature excellent selectivity toward H2O2 over competing with cellular ROS (O2.-, .OH, NO and ONOO-) and some biological compounds (HQ, GSH, Vc and esterase), large dynamic response ranges owing to their dual colorimetric/fluorometric detection mechanism, and long-wavelength visible excitation and emission profiles to minimize cell and tissue damage. The sulfonate-based probes are cell-permeable and can detect mircomolar changes in O2-·concentrations in living cells, using confocal microscopy. 6. The concentrations of H2O2 and O2-·in the clear solutions, the macrophages from the pleural lavage fluid and the lung cells of Wistar rats of bleomycin-induced lung fibrosis were simultaneously detected by applying the two probes, naphthofluorescein disulfonates (NFDS-1) and bis(diphenylphosphinacyl) fluorescein (PF-1). Compared with the clear solutions from normal lung and fibrosis lung, the qualitative relation between the grades of lung fibrosis and the levels of H2O2 and O2-·was expounded.
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