纳米电极的制备及细胞间神经递质的实时监测
发布时间：2018-09-12 05:03
【摘要】：纳米电化学领域是纳米科学技术与电化学技术的交叉领域,新技术的发展为细胞的实时监测提供了前所未有的机遇。碳纤维纳米电极(CFNE)作为一种纳米电化学技术的工具也越来越引起人们重视。但是该领域也面临着亟待解决的问题,首先就是囊泡尺寸太小,而电极尺寸过大,检测方法的分辨率低,无法实现一对一的高分辨率检测,从而导致检测结果不准确。其次是无法维持检测对象的正常状态,既要保证细胞的生理活性,又不影响检测效果。针对这些问题,我们进行了两方面的研究,本论文的研究工作主要包括100 nm尺寸的碳纤维纳米电极的制备、电极各方面性质的研究以及利用纳米电极进行单囊泡检测方面的应用。主要包括以下几个方面:(1)制备100 nm的CFNE,在细胞内部要实现对纳米尺寸研究对象的精确分析,电极直径和囊泡相匹配是必要条件。针对囊泡尺寸在几十纳米左右这一特点,本工作中首先改进了纳米电极的尺寸,基于火焰刻蚀的方法,将CFNE的制作方法进一步完善,通过调节刻蚀火焰强度以及刻蚀角度,得到的纳米电极尖端可以达到100 nm。电极用碳纤维作为尖端材料,在电化学反应中电子传输速率快,可以实现对靶标分子更灵敏的检测;与传统的金电极相比,有比较好的抗非特异性吸附的能力,能够在细胞内部持续进行长时间检测而不对信号造成较大影响;电极尖端尺寸小,细胞监测过程中不会导致细胞死亡,可以保证其相对活性;电极尖端的尺寸以及长度可控性强,能够根据实验需要可控的进行调整。电极制作方法的改善也为以后进行细胞间物质的传递与运输的探究提供了条件。(2)本研究通过合成与细胞内部相似的囊泡,再通过超精细CFNE对其进行检测,可以实现对检测物质的准确定量分析,同时对其囊泡动力学进行统计得到囊泡在电极表面释放的动态过程。调控囊泡中包裹分子的浓度,配合可控性的纳米电极,检测效率可以达到90%以上。我们构建了一种具有超高时空分辨率的单囊泡检测技术,在空间分辨率上面,可以实现对单个囊泡的检测,准确区分单个囊泡或者多个囊泡同时在电极表面发生碰撞的情况;在时间分辨率上面,检测可以低至0.1 ms,而囊泡胞吐释放是在ms级别,超过了理论值5倍以上。这项工作对囊泡在电极表面的作用方式进行了更为深入的研究,可为进一步探究细胞胞吐释放机理提供更为有保障的研究平台。
[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.
【学位授予单位】：中国科学院研究生院(上海应用物理研究所)
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.1;Q25
[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.
【学位授予单位】：中国科学院研究生院(上海应用物理研究所)
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.1;Q25
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