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超氧阴离子自由基和过氧亚硝酸根离子荧光测定法的研究

Study on Fluorescent Method for the Determination of Superoxide and Peroxynitrite Anion Radicals

作者: 专业:分析化学 导师:高吉刚 年度:2010 学位:硕士  院校: 山东农业大学

Keywords

Spectrofluorometric method, Peroxynitrite, Superoxide anion radical, Superoxide dismutase, Fluorescent probe

        自由基是人体生命活动中多种生化反应的中间代谢产物,其中超氧自由基(O2·-)和过氧亚硝酸根离子(ONOO-)是具有代表性的两种自由基,它们在各种致病过程中都是重要的介导因素,与癌症、机体炎症、组织过氧化、蛋白质交联变性、DNA损伤和细胞信号转导等都有着直接的关系。O2·-不仅自身具有毒性,而且可以经过一系列反应生成其它活性氧自由基,进一步对生物体产生损伤作用,且停留时间较长。超氧自由基是生物活性最高的氧中心自由基之一,它在生命过程和化学反应中作为电子受体,形成不同的活性氧自由基链中的第一个自由基,经过一系列反应生成其它的氧自由基。和其它的活性氧相比,超氧自由基不是很活泼,但其寿命很长,因此,超氧自由基危害较大,捕获超氧自由基进行研究具有重要的意义。ONOO-是NO·与O2·-相互反应的产物。ONOO·能导致DNA损伤、酶抑制、细胞死亡和细菌中毒并且能够引起一系列疾病。通过对自由基的检测研究,可以帮助我们更好的解释疾病产生的原因,采取有效的预防措施,减少机体的伤害程度。目前测定O2·-与ONOO-的方法主要有电子自旋共振法(ESR)或电子顺磁共振法(EPR)、高效液相色谱法(HPLC)、化学发光法(CL)、荧光法以及电化学方法等。然而,由于生物体内活性氧自由基的半衰期非常短,稳态浓度极低,所以真正适用于检测生物活体内O2·-与ONOO-的方法还是比较少的。这就迫切需要科研工作者开发对O2·-与ONOO-能够专一识别、快速响应的荧光探针用以动态观测研究某些器官中活性氧自由基的产生、代谢、相互转化及其动态损伤生物机体的过程。本论文利用荧光分析技术测定活性氧自由基的方法,此技术具有灵敏度高、选择性好、操作简便等优点。本文合成了三种新型荧光探针,利用探针对(O2·-)和(ONOO-)进行了检测,提出了新的检测方法。具体研究内容分为以下三个方面:(1)设计合成了一种新型的荧光探针香草醛缩苯胺,建立检测O2·-和SOD活性的荧光方法,研究测定方法的最佳条件,并用于测定大蒜﹑洋葱的SOD活性的测定,并与邻苯三酚自氧化法测定出的SOD活性进行了比较。(2)设计合成新型的荧光探针荧光素酰肼,用荧光光谱法检测ONOO-,研究测定方法的最佳条件,探讨了其它物质对体系的干扰影响及荧光探针的检测单一性,研究测定方法的灵敏度,精密度。(3)设计合成新型的荧光探针罗丹明6G酰肼,用荧光光谱法检测ONOO-,研究测定方法的最佳条件,探讨了其它物质对体系的干扰影响及荧光探针的检测单一性,研究测定方法的灵敏度,精密度。
    Numerous reactive oxygen species (ROS) are generated in the course of biology metabolism. Superoxide anion radical (O2·-) and peroxynitrite (ONOO-) are two typical ones. They have important relations with aging and diseases.O2·- is a toxic oxidizing and can generate other reactive oxygen species by a series of reactions. These reactive oxygen species have further damages to organisms and long resident time. Peroxynitrite, generated from the diffusion-controlled reaction between the nitrogen monoxide and superoxide radicals can cause lipid peroxidation. Peroxynitrite causes DNA damage, enzyme inhibition, apoptosis and bacterial toxicity and has been implicated in various diseases.About 95% of the total free radicals in the body are reactive oxygen species; therefore the research on the ROS has a special significance to human body. Researches of reactive oxygen species can help us explain the causes of diseases better, strengthen effective preventive measures and reduce the injury extent of the body.Because ROS is active, direct determination is difficult. At present, many methods for the indirect determination of superoxide anion radicals and Peroxynitrite have been developed. After having a comprehensive understanding of these methods, based on fluorescence analysis with the advantages of high sensitivity, selectivity and simple operation, the spectrofluorimetric method was used to detect reactive oxygen species. In the paper, three fluorescent probes were synthesized and their selectivity for O2·- and peroxynitrite were studied. In addition, the effects of scavenging of reactive oxygen species by some antioxidants were discussed. Three aspects of investigation have been carried out specifically:(1)A novel fluorescent probe, Vanilla aldehyde shrinks was synthesized. The spectrofluorimetric method was developed for the determination of O2·- and SOD activity. The optimum experimental conditions were discussed. Furthermore, SOD activity in onions was successfully determined. The results obtained by the suggested method were also compared with those obtained from the standard method.(2) A novel spectrofluorimetric method using fluorescent hydrazide as fluorescent probe was developed for the determination of peroxynitrite. The sensitivity and precision of the method were studied.(3) A novel fluorescent probe, rhodamine 6G hydrazide was synthesized. The spectrofluorimetric method was developed for the determination of peroxynitrite .The effects of interferences and the selectivity for peroxynitrite of the fluorescent probe was investigated. The sensitivity and precision of the method were studied.In the paper, fluorescent hydrazide was synthesized and the study on molecular mechanism of the interaction between peroxynitrite and fluorescent hydrazide by fluorescent spectroscopy was carried out firstly. These results are satisfied.
        

超氧阴离子自由基和过氧亚硝酸根离子荧光测定法的研究

符号说明4-5
目录5-8
中文摘要8-10
ABSTRACT10-11
1 引言12-29
    1.1 自由基的性质13-16
        1.1.1 超氧阴离子自由基的性质13
        1.1.2 过氧亚硝酸根离子的结构与性质13-16
    1.2 自由基的产生16-18
        1.2.1 超氧阴离子自由基的产生16-17
        1.2.2 过氧亚硝酸根离子的产生17-18
    1.3 活性自由基的检测方法18-28
        1.3.1 超氧阴离子自由基的检测方法18-23
        1.3.2 过氧亚硝酸根离子的检测方法23-28
    1.4 论文研究内容及意义28-29
2 材料与方法29-35
    2.1 仪器与试剂29-30
    2.2 实验方法30-35
        2.2.1 香草醛缩苯胺为探针测定 0_2·~-和 SOD 活性30-32
            2.2.1.1 香草醛缩苯胺荧光试剂的合成与表征30
            2.2.1.2 0_2·~-的测定30
            2.2.1.3 0_2·~-清除率的测定30
            2.2.1.4 样品 SOD 提取液的制备及 SOD 活性的测定30-31
            2.2.1.5 邻苯三酚自氧化法测定 SOD 活性31-32
        2.2.2 荧光素酰肼为探针测定 ONOO的荧光光谱研究32-33
            2.2.2.1 荧光素酰肼荧光试剂的合成与表征32
            2.2.2.2 ONOO~-溶液的制备32-33
            2.2.2.3 ONOO~-的测定33
        2.2.3 罗丹明6G 酰肼为探针测定 ONOO~-的荧光光谱研究33-35
            2.2.3.1 罗丹明6G 酰肼荧光试剂的合成与表征33
            2.2.3.2 ONOO~-溶液的制备33-34
            2.2.3.3 ONOO~-的测定34-35
3 结果与讨论35-55
    3.1 香草醛缩苯胺为探针测定 0_2·~-和 SOD 活性荧光分析研究35-42
        3.1.1 测定原理35-36
        3.1.2 荧光探针与 0_2·~-反应前后的激发光谱与发射光谱36
        3.1.3 酸度和缓冲用量的影响36-37
        3.1.4 反应温度的影响37-38
        3.1.5 反应时间的影响38
        3.1.6 邻苯三酚浓度的影响38-39
        3.1.7 荧光探针浓度的影响39-40
        3.1.8 试剂加入顺序的影响40
        3.1.9 方法的重现性及干扰试验40
        3.1.10 0_2·~-清除率实验40-41
        3.1.11 生物样品中 SOD 活性的测定41-42
    3.2 荧光素酰肼为探针测定 ONOO~-的荧光法研究42-48
        3.2.1 荧光探针与 ONOO~-反应前后的激发光谱与发射光谱42-43
        3.2.2 酸度的影响43-44
        3.2.3 反应温度的影响44
        3.2.4 反应时间的影响44-45
        3.2.5 荧光探针浓度的影响45
        3.2.6 ONOO~ˉ浓度的影响45-46
        3.2.7 试剂加入顺序的影响46
        3.2.8 反应机理46-47
        3.2.9 线性范围、精密度47
        3.2.10 干扰物质的影响47-48
        3.2.11 生物样品的测定48
    3.3 罗丹明6G 酰肼为探针测定 ONOO~-的荧光法研究48-55
        3.3.1 荧光探针与 ONOO~-反应前后的激发光谱与发射光谱48-49
        3.3.2 酸度的影响49
        3.3.3 反应温度的影响49-50
        3.3.4 反应时间的影响50-51
        3.3.5 荧光探针浓度的影响51
        3.3.6 试剂加入顺序的影响51-52
        3.3.7 ONOO~-浓度的影响52
        3.3.8 反应机理52-53
        3.3.9 线性范围、精密度53
        3.3.10 干扰物质的影响53
        3.3.11 生物样品的测定53-55
4 结论55-56
    4.1 香草醛缩苯胺为探针测定 0_2·~-和 SOD 活性荧光分析研究55
    4.2 荧光素酰肼为探针测定 ONOO~-荧光分析研究55
    4.3 罗丹明6G 酰肼为探针测定 ONOO~-荧光分析研究55-56
参考文献56-65
致谢65-66
攻读学位期间发表的学术论文66
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