过氧化氢定量释放及其与金属蛋白相互作用的SECM-SPR原位研究

发布时间：2018-01-14 23:23

本文关键词：过氧化氢定量释放及其与金属蛋白相互作用的SECM-SPR原位研究　出处：《中南大学》2011年硕士论文　论文类型：学位论文

【摘要】：过氧化氢与羟基自由基作为活性氧中的重要分子,广泛参与细胞增殖、分化和凋亡等多种细胞调节过程,其在生物体内引发的过氧化反应是细胞不可逆损伤的分子基础,与肿瘤、心血管疾病、自身免疫病、衰老、癌症等疾病的发生密切相关,因此采用实时原位的技术方法从结构和动力学角度研究H202或OH·引发生物分子过氧化机制是揭示和预防机体氧化应激伤害的关键。但是,由于活性氧物种固有的高反应活性和低稳定性(平均寿命只有微秒甚至纳秒级),如何在现有的技术条件下有效地控制H202和OH·浓度仍然是一个难题。本论文中,我们针对这一现存问题,建立了基于H2O2/OH·可控释放和高效捕获的扫描电化学显微镜—表面等离子体激元共振联用技术(SECM-SPR),实时原位研究了H2O2/OH·定量诱导的微区内金属蛋白的氧化过程,结果显示： (1)采用SECM-SPR可实现对H202的可控释放和现场原位检测。通过电化学还原02实现了H202在SECM针尖上的定量释放,利用SECM针尖与SPR基底之间亚微米级的短程间距有效抑制H202的扩散损耗和副反应损耗,实现了在SPR基底上的高效捕获。相比于非在线的荧光检测结果,采用SECM-SPR联用技术可实现基底对H202的100%捕获,原位释放并捕获到的H202产率高于非在线检测的结果,且检测误差较小。 (2)采用SECM-SPR实现了对OH-诱导铜金属蛋白氧化反应的实时原位灵敏监测。通过控制探针电位定量释放H202,扩散至SPR基底,与组装在基底的铜金属硫蛋白(Cu12+-MT)反应生成OH·,SPR现场监测OH·引发MT的原位不可逆氧化损伤。采用荧光法证实了反应过程中产生了OH·。H2O2释放量可与Cu12+-MT的氧化量呈良好线性关系。
[Abstract]:Hydrogen peroxide and hydroxyl radical, as important molecules in reactive oxygen species, are widely involved in the regulation of cell proliferation, differentiation and apoptosis. The peroxidation induced by it is the molecular basis of irreversible cell damage, which is closely related to the occurrence of cancer, cardiovascular disease, autoimmune disease, aging, cancer and other diseases. Therefore, it is the key to reveal and prevent oxidative stress injury from the point of view of structure and kinetics by using real-time in situ technique to study the mechanism of biological molecule peroxidation induced by H 202 or OH 路. Because of the inherent high reactive activity and low stability of the reactive oxygen species (the average life span is only microsecond or even nanosecond). How to effectively control the concentration of H202 and OH 路under the existing technical conditions is still a difficult problem. In this paper, we aim at this existing problem. A SECM-SPRR technique based on controllable release and high performance capture of H _ 2O _ 2 / OH was established by using SECM-SPRs with a scanning electrochemical microscope (SEM) and surface plasmon resonance (SPR). The oxidation process of metalloproteins in microdomain induced by H _ 2O _ 2 / OH 路was studied in situ in real time. The results showed that: 1) the controllable release and in-situ detection of H202 can be realized by SECM-SPR, and the quantitative release of H202 on the tip of SECM can be realized by electrochemical reduction 02. The short range distance between SECM tip and SPR substrate can effectively suppress the diffusion loss and side reaction loss of H202. Compared with the off-line fluorescence detection results, the 100% capture of H202 on the substrate can be realized by using SECM-SPR combined with SECM-SPR technology. The yield of H 202 released in situ and captured is higher than that of non-line detection, and the detection error is small. The real-time in situ sensitive monitoring of OH- induced oxidation of copper metalloproteins was realized by SECM-SPR. The H202 was released quantitatively by controlling the potential of the probe and then diffused to the SPR substrate. It reacts with copper metallothionein (Cu _ (12) -MTT) assembled on the substrate to form OH 路. In situ irreversible oxidative damage induced by OH 路H _ 2O _ 2 was monitored by SPR. It was proved by fluorescence method that the amount of OH 路路H _ 2O _ 2 released during the reaction was comparable to that of Cu12. The oxidation amount of-MT shows a good linear relationship.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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