【摘要】：納米電化學(xué)領(lǐng)域是納米科學(xué)技術(shù)與電化學(xué)技術(shù)的交叉領(lǐng)域,新技術(shù)的發(fā)展為細(xì)胞的實(shí)時(shí)監(jiān)測提供了前所未有的機(jī)遇。碳纖維納米電極(CFNE)作為一種納米電化學(xué)技術(shù)的工具也越來越引起人們重視。但是該領(lǐng)域也面臨著亟待解決的問題,首先就是囊泡尺寸太小,而電極尺寸過大,檢測方法的分辨率低,無法實(shí)現(xiàn)一對一的高分辨率檢測,從而導(dǎo)致檢測結(jié)果不準(zhǔn)確。其次是無法維持檢測對象的正常狀態(tài),既要保證細(xì)胞的生理活性,又不影響檢測效果。針對這些問題,我們進(jìn)行了兩方面的研究,本論文的研究工作主要包括100 nm尺寸的碳纖維納米電極的制備、電極各方面性質(zhì)的研究以及利用納米電極進(jìn)行單囊泡檢測方面的應(yīng)用。主要包括以下幾個(gè)方面:(1)制備100 nm的CFNE,在細(xì)胞內(nèi)部要實(shí)現(xiàn)對納米尺寸研究對象的精確分析,電極直徑和囊泡相匹配是必要條件。針對囊泡尺寸在幾十納米左右這一特點(diǎn),本工作中首先改進(jìn)了納米電極的尺寸,基于火焰刻蝕的方法,將CFNE的制作方法進(jìn)一步完善,通過調(diào)節(jié)刻蝕火焰強(qiáng)度以及刻蝕角度,得到的納米電極尖端可以達(dá)到100 nm。電極用碳纖維作為尖端材料,在電化學(xué)反應(yīng)中電子傳輸速率快,可以實(shí)現(xiàn)對靶標(biāo)分子更靈敏的檢測;與傳統(tǒng)的金電極相比,有比較好的抗非特異性吸附的能力,能夠在細(xì)胞內(nèi)部持續(xù)進(jìn)行長時(shí)間檢測而不對信號造成較大影響;電極尖端尺寸小,細(xì)胞監(jiān)測過程中不會導(dǎo)致細(xì)胞死亡,可以保證其相對活性;電極尖端的尺寸以及長度可控性強(qiáng),能夠根據(jù)實(shí)驗(yàn)需要可控的進(jìn)行調(diào)整。電極制作方法的改善也為以后進(jìn)行細(xì)胞間物質(zhì)的傳遞與運(yùn)輸?shù)奶骄刻峁┝藯l件。(2)本研究通過合成與細(xì)胞內(nèi)部相似的囊泡,再通過超精細(xì)CFNE對其進(jìn)行檢測,可以實(shí)現(xiàn)對檢測物質(zhì)的準(zhǔn)確定量分析,同時(shí)對其囊泡動力學(xué)進(jìn)行統(tǒng)計(jì)得到囊泡在電極表面釋放的動態(tài)過程。調(diào)控囊泡中包裹分子的濃度,配合可控性的納米電極,檢測效率可以達(dá)到90%以上。我們構(gòu)建了一種具有超高時(shí)空分辨率的單囊泡檢測技術(shù),在空間分辨率上面,可以實(shí)現(xiàn)對單個(gè)囊泡的檢測,準(zhǔn)確區(qū)分單個(gè)囊泡或者多個(gè)囊泡同時(shí)在電極表面發(fā)生碰撞的情況;在時(shí)間分辨率上面,檢測可以低至0.1 ms,而囊泡胞吐釋放是在ms級別,超過了理論值5倍以上。這項(xiàng)工作對囊泡在電極表面的作用方式進(jìn)行了更為深入的研究,可為進(jìn)一步探究細(xì)胞胞吐釋放機(jī)理提供更為有保障的研究平臺。
[Abstract]:The field of nano-electrochemistry is the intersection of nanotechnology and electrochemical technology. The development of new technology provides an unprecedented opportunity for the real-time monitoring of cells. Carbon fiber nanoelectrode (CFNE) has attracted more and more attention as a tool of nanoelectrochemical technology. However, this field also faces some urgent problems. Firstly, the size of vesicle is too small, the electrode size is too large, the resolution of the detection method is low, and the high resolution detection method can not realize one-to-one, which leads to inaccurate detection results. Secondly, it is impossible to maintain the normal state of the detection object, not only to ensure the physiological activity of the cell, but also not to affect the detection effect. In view of these problems, we have carried out two aspects of research. The research work in this thesis mainly includes the preparation of carbon fiber nanoelectrodes with the size of 100 nm. The study of the properties of the electrode and the application of nano-electrode in the detection of single vesicle. The main contents are as follows: (1) the preparation of 100 nm CFNE, is necessary for the accurate analysis of nanoscale size. The matching of electrode diameter and vesicle is a necessary condition. In this work, the size of nano-electrode is improved firstly. Based on the method of flame etching, the fabrication method of CFNE is further improved, and the etching flame intensity and etching angle are adjusted. The nanoelectrode tip can be up to 100 nm.. The electrode uses carbon fiber as the tip material, and the electron transport rate in the electrochemical reaction is fast, so the target molecule can be detected more sensitively. Compared with the traditional gold electrode, the electrode has a better ability to resist non-specific adsorption. The electrode tip size is small and the cell monitoring process will not lead to cell death, which can ensure its relative activity. The size and length of the electrode tip are controllable and can be adjusted according to the need of the experiment. The improvement of electrode preparation method also provides conditions for the further study of intercellular material transfer and transport. (2) in this study, the vesicles similar to those in cells were synthesized, and then detected by hyperfine CFNE. The dynamic process of vesicle release on the electrode surface can be obtained by statistical analysis of the vesicle kinetics. The concentration of encapsulated molecules in the vesicles was regulated, and the detection efficiency was over 90%. We constructed a single vesicle detection technique with super-high spatial and temporal resolution. On the spatial resolution, we can detect the single vesicle and distinguish the collision of single vesicle or multiple vesicles at the same time on the electrode surface. In time resolution, the detection can be as low as 0. 1 ms, and the release of vesicle exocytosis is at the ms level, which is more than 5 times the theoretical value. This work provides a more secure platform for further research on the mechanism of cell exocytosis.
【學(xué)位授予單位】：中國科學(xué)院研究生院(上海應(yīng)用物理研究所)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O657.1;Q25

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本文編號：2237990