基于微电极的生物体系内过氧化氢等活性分子的传感检测技术

发布时间：2018-03-06 04:12

本文选题：超微电极　切入点：电化学　出处：《华中科技大学》2015年博士论文　论文类型：学位论文

【摘要】：H202是生物体系中存在的一种重要小分子,在许多生理过程中发挥重要作用。对于植物,不利的环境胁迫包括干旱、洪涝、紫外照射、极端温度等会导致包含H202作用的植物防御机制的激活。因此检测H202在植物的应激反应过程中的产生和消亡对更好地了解植物的应激机制,对于筛选和培育抗逆性作物具有重要意义。在细胞水平,正常细胞中H202含量的升高可以诱发癌变,H202在肿瘤细胞中大量积累,并在肿瘤细胞的生存、增殖和凋亡的调控过程中发挥作用。同时肿瘤细胞对H202含量的变化非常敏感,H202含量的过度增加或减小都可能诱导肿瘤细胞凋亡。因而,探究抗癌药物作用下肿瘤细胞产生H202的浓度随时间连续变化状况,对研究肿瘤细胞的凋亡机制,从而预防和治疗肿瘤是极有意义的。化学发光法、荧光法和分光光度法等检测生物体内的过氧化氢都需要示踪物和不稳定的化学探针,这些方法很难做到氧爆发的动态在体检测。另外,这些方法都需要将植物样品分离或磨碎,并且过程比较复杂和耗时。而且,分离组织本身对植物就是一种胁迫,有可能引起植物的包括相关抗性基因表达和连续的生理学过程等的防御反应。此外,考虑到非生物胁迫下的H202水平变化的动态监测,连续在体检测更加合适,但是这种测量方式很难通过光学探针实现。因为具有极小的体积、良好的生物相容性和较高的灵敏度,微电极和超微电极已经被广泛用于生物在体的电活性小分子检测。本研究采用电化学方法,利用纳米材料构建微电极和超微电极,进行植物在体和癌细胞外的H202的检测,主要研究内容和结果如下： (1)利用对H202具有较好氧化还原催化能力的血红蛋白(Hb)修饰碳纤维超微电极,并在单壁碳纳米管(SWCNTs)的帮助下不仅保持较高的催化能力而且提高了电子转移速率,降低工作电压到-O.1V。这样,在不需要添加氧化还原媒介和额外抗干扰层的情况下,避免了其他电活性物质的干扰,构建了对H202具有较高灵敏度和选择性的微型生物传感器。并成功在体检测了芦荟在受到盐胁迫后连续19.5小时内的氧爆发过程。 (2)对H202具有强催化能力的辣根过氧化物酶(HRP)在室温离子液的帮助下修饰在覆盖了一层SWCNTs的碳纤维超微电极表面。结果表明,该修饰电极不仅保持了HRP的氧化还原催化活性,并且对H202具有很高的亲和力和灵敏度,检出限为0.13μM。在没有外加选择性渗透膜的情况下对生物环境中的常见电活性小分子具有很好的抗干扰作用。该传感器分别成功检测到HeLa细胞在刺激剂抗坏血酸,抗癌药物10-羟基喜树碱的作用下的H202释放过程。 (3)为了更加全面了解胁迫条件下植物体内小分子的浓度变化状况,构建一种电化学传感电极束,与传统光学探针方法相比,可在不影响植物生长或不分离植物任何部位的情况下,实现植物体内H202、NO和pH的同时、实时检测。利用旱胁迫下的油菜作为模式生物,该阵列检测到了旱胁迫下,NO在旱胁迫开始后11小时和22小时处的两个较大的峰,分别对应3.1μM和2.6μM的NO浓度；H202的含量从旱胁迫开始后20.8小时开始增加,一直到整个检测结束,其含量在45小时内增加了2.5μM；pH被观察到在前18小时相对稳定,之后持续增加。在整个检测过程中,油菜茎中的pH增加最大值大约为1.08。由于阵列直径小于lmm,可以在植物发育的不同阶段实施实时监测,并且电极的制作简单、造价低、响应速度快,该技术也有望用于帮助理解胁迫信号中的NO、H2O2和pH的相互机制。 (4)在铂微柱电极上修饰SWCNTs和Hb,并利用该电极成功检测到抗旱型油菜和野生型油菜在同样的旱胁迫条件下的氧爆发的差异。在野生型油菜中,检测到在旱胁迫7.8小时开始出现氧爆发,并在10.3小时和26.5小时分别产生两个比较大的峰。和野生型相比,抗旱型油菜在旱胁迫开始后共40个小时中只观察到在15.8小时处的一个氧爆发的峰。抗旱型油菜在旱胁迫下的H202积累量比较小,这可能是因为其与H202清除有关的酶的活性更高,这也进一步解释了抗旱型油菜在旱胁迫下的较高的存活率。
[Abstract]:H202 is a kind of important small molecules exist in biological systems, play an important role in many physiological processes. In plants, adverse environmental stresses including drought, flood, ultraviolet radiation, extreme temperature will lead to the activation of plant defense mechanisms including H202 function. So the detection of H202 in response to stress the process of plants the rise and fall of a better understanding of the mechanism of plant stress, has important significance for the selection and cultivation of crop resistance. At the cellular level, increase the content of H202 in normal cells can induce carcinogenesis, H202 in tumor cells of large accumulation, and in the survival of cancer cells, play a role in regulation of proliferation and apoptosis. At the same time is very sensitive to changes of tumor cells on H202 content, H202 content of the excessive increase or decrease may induce the apoptosis of tumor cells. Therefore, exploring the tumor cells to anticancer drugs produced by the action of H2 The continuous change of 02 concentration with time is of great significance for the study of the mechanism of apoptosis of tumor cells and the prevention and treatment of tumors.
Chemiluminescence detection, biological fluorescence and spectrophotometry in hydrogen peroxide to tracer and unstable chemical probes, these methods are difficult to achieve dynamic oxygen burst in vivo detection. In addition, these methods need to be separated or ground plant samples, and the process is complicated and time-consuming. Moreover, separation the organization itself is a kind of plant stress, may cause the expression of related genes including physiological and continuous learning process of plant defense responses. In addition, taking into account the dynamic monitoring of H202 levels under abiotic stress, for in vivo detection is more appropriate, but this measurement is difficult to achieve through the optical probe.
Because of its small size, high sensitivity and compatibility of good biological microelectrode and microelectrode has been widely used in biological detection of electroactive molecules of the body. This study used electrochemical methods, construct microelectrode and microelectrode using nano materials, detection of H202 plants in the body and the outside of cancer cells, the main research the contents and results are as follows:
(1) use has good catalytic redox ability of hemoglobin H202 (Hb) modified carbon fiber microelectrode, and single-walled carbon nanotubes (SWCNTs) with the help of not only maintain high catalytic ability but also improve the electron transfer rate, reduce the working voltage to the -O.1V., do not need to add in the redox medium and extra anti interference layer, to avoid interference with other electroactive substances, constructed a micro biosensor with high sensitivity and selectivity to H202. And in vivo measurement of the outbreak of aloe under salt stress after 19.5 hours of continuous oxygen in the process.
(2) horseradish peroxidase has strong catalytic ability of H202 (HRP) modified carbon fiber covered with a layer of SWCNTs ultra microelectrode surface at room temperature ionic liquid with the help of the modified electrode. The results show that the HRP not only keeps the redox catalytic activity, and the H202 has a very high affinity and sensitivity the detection limit was 0.13 M., which has a good anti interference effect in the absence of external conditions on the selective permeable membrane biological environment in common electroactive molecules. The sensor was successfully detected in HeLa cell stimulating agent ascorbic acid, anticancer drug 10- hydroxycamptothecin under the action of the H202 release process.
(3) to a more comprehensive understanding of the concentration changes of plants under stress conditions of small molecules, constructing an electrochemical sensing electrode beam, compared with the traditional optical probe method, can not affect the plant growth or any parts of the plant are not isolated cases, implementation of plant H202, real-time detection of NO and pH at the same time. Under drought stress. The use of rape as a model organism, the array was detected under drought stress, the larger of the two peak of NO in drought stress after 11 hours and 22 hours at the concentration of NO, corresponding to the 3.1 M and 2.6 M; the content of H202 from 20.8 hours after the beginning of drought stress began to increase until the end, detection, its content increased 2.5 M in 45 hours; pH was observed to be relatively stable in the first 18 hours after increasing. During the whole testing process, the rape stem pH increase in maximum value is about 1.08. because the array diameter is less than l Mm can implement real-time monitoring at different stages of plant development, and the fabrication of electrodes is simple, low cost and fast response. This technology is also expected to help understand the mutual mechanism of NO, H2O2 and pH in stress signals.
(4) SWCNTs and Hb on Pt micro column electrode, and the electrode successfully detected differences in drought resistant rape and rape in the same wild type under drought stress. The outbreak of the oxygen in the wild type rapeseed, detected in drought stress at 7.8 hours hypoxia outbreak, and in 10.3 hours and 26.5 hours respectively produce two large peaks. Compared with the wild type, drought resistant rape in drought stress after the start of a total of 40 hours were only observed in the outbreak in an oxygen 15.8 hours at peak. The drought resistant Rapeseed under drought stress H202 accumulation is relatively small, this may be due to the with the H202 scavenging activity of enzymes related to higher, which further explains the survival rate of rape in high drought resistance under drought stress the.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q-33;O657.1

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