本文選題：超微電極　切入點(diǎn)：電化學(xué)　出處：《華中科技大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：H202是生物體系中存在的一種重要小分子,在許多生理過程中發(fā)揮重要作用。對于植物,不利的環(huán)境脅迫包括干旱、洪澇、紫外照射、極端溫度等會導(dǎo)致包含H202作用的植物防御機(jī)制的激活。因此檢測H202在植物的應(yīng)激反應(yīng)過程中的產(chǎn)生和消亡對更好地了解植物的應(yīng)激機(jī)制,對于篩選和培育抗逆性作物具有重要意義。在細(xì)胞水平,正常細(xì)胞中H202含量的升高可以誘發(fā)癌變,H202在腫瘤細(xì)胞中大量積累,并在腫瘤細(xì)胞的生存、增殖和凋亡的調(diào)控過程中發(fā)揮作用。同時(shí)腫瘤細(xì)胞對H202含量的變化非常敏感,H202含量的過度增加或減小都可能誘導(dǎo)腫瘤細(xì)胞凋亡。因而,探究抗癌藥物作用下腫瘤細(xì)胞產(chǎn)生H202的濃度隨時(shí)間連續(xù)變化狀況,對研究腫瘤細(xì)胞的凋亡機(jī)制,從而預(yù)防和治療腫瘤是極有意義的。 化學(xué)發(fā)光法、熒光法和分光光度法等檢測生物體內(nèi)的過氧化氫都需要示蹤物和不穩(wěn)定的化學(xué)探針,這些方法很難做到氧爆發(fā)的動(dòng)態(tài)在體檢測。另外,這些方法都需要將植物樣品分離或磨碎,并且過程比較復(fù)雜和耗時(shí)。而且,分離組織本身對植物就是一種脅迫,有可能引起植物的包括相關(guān)抗性基因表達(dá)和連續(xù)的生理學(xué)過程等的防御反應(yīng)。此外,考慮到非生物脅迫下的H202水平變化的動(dòng)態(tài)監(jiān)測,連續(xù)在體檢測更加合適,但是這種測量方式很難通過光學(xué)探針實(shí)現(xiàn)。 因?yàn)榫哂袠O小的體積、良好的生物相容性和較高的靈敏度,微電極和超微電極已經(jīng)被廣泛用于生物在體的電活性小分子檢測。本研究采用電化學(xué)方法,利用納米材料構(gòu)建微電極和超微電極,進(jìn)行植物在體和癌細(xì)胞外的H202的檢測,主要研究內(nèi)容和結(jié)果如下： (1)利用對H202具有較好氧化還原催化能力的血紅蛋白(Hb)修飾碳纖維超微電極,并在單壁碳納米管(SWCNTs)的幫助下不僅保持較高的催化能力而且提高了電子轉(zhuǎn)移速率,降低工作電壓到-O.1V。這樣,在不需要添加氧化還原媒介和額外抗干擾層的情況下,避免了其他電活性物質(zhì)的干擾,構(gòu)建了對H202具有較高靈敏度和選擇性的微型生物傳感器。并成功在體檢測了蘆薈在受到鹽脅迫后連續(xù)19.5小時(shí)內(nèi)的氧爆發(fā)過程。 (2)對H202具有強(qiáng)催化能力的辣根過氧化物酶(HRP)在室溫離子液的幫助下修飾在覆蓋了一層SWCNTs的碳纖維超微電極表面。結(jié)果表明,該修飾電極不僅保持了HRP的氧化還原催化活性,并且對H202具有很高的親和力和靈敏度,檢出限為0.13μM。在沒有外加選擇性滲透膜的情況下對生物環(huán)境中的常見電活性小分子具有很好的抗干擾作用。該傳感器分別成功檢測到HeLa細(xì)胞在刺激劑抗壞血酸,抗癌藥物10-羥基喜樹堿的作用下的H202釋放過程。 (3)為了更加全面了解脅迫條件下植物體內(nèi)小分子的濃度變化狀況,構(gòu)建一種電化學(xué)傳感電極束,與傳統(tǒng)光學(xué)探針方法相比,可在不影響植物生長或不分離植物任何部位的情況下,實(shí)現(xiàn)植物體內(nèi)H202、NO和pH的同時(shí)、實(shí)時(shí)檢測。利用旱脅迫下的油菜作為模式生物,該陣列檢測到了旱脅迫下,NO在旱脅迫開始后11小時(shí)和22小時(shí)處的兩個(gè)較大的峰,分別對應(yīng)3.1μM和2.6μM的NO濃度；H202的含量從旱脅迫開始后20.8小時(shí)開始增加,一直到整個(gè)檢測結(jié)束,其含量在45小時(shí)內(nèi)增加了2.5μM；pH被觀察到在前18小時(shí)相對穩(wěn)定,之后持續(xù)增加。在整個(gè)檢測過程中,油菜莖中的pH增加最大值大約為1.08。由于陣列直徑小于lmm,可以在植物發(fā)育的不同階段實(shí)施實(shí)時(shí)監(jiān)測,并且電極的制作簡單、造價(jià)低、響應(yīng)速度快,該技術(shù)也有望用于幫助理解脅迫信號中的NO、H2O2和pH的相互機(jī)制。 (4)在鉑微柱電極上修飾SWCNTs和Hb,并利用該電極成功檢測到抗旱型油菜和野生型油菜在同樣的旱脅迫條件下的氧爆發(fā)的差異。在野生型油菜中,檢測到在旱脅迫7.8小時(shí)開始出現(xiàn)氧爆發(fā),并在10.3小時(shí)和26.5小時(shí)分別產(chǎn)生兩個(gè)比較大的峰。和野生型相比,抗旱型油菜在旱脅迫開始后共40個(gè)小時(shí)中只觀察到在15.8小時(shí)處的一個(gè)氧爆發(fā)的峰�？购敌陀筒嗽诤得{迫下的H202積累量比較小,這可能是因?yàn)槠渑cH202清除有關(guān)的酶的活性更高,這也進(jìn)一步解釋了抗旱型油菜在旱脅迫下的較高的存活率。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：Q-33;O657.1

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