Preparation Of Nanoelectrode And Real-time Monitoring Of Intercellular Neurotransmitters

The nano-electrochemical field is the intersection between nanoscience and electrochemical technology,and the development of new technologies brings unprecedented opportunities to the real-time monitoring of cells.As a tool of nanoelectrochemical technology,carbon fiber nano-electrode(CFNE)has attracted more and more attention.However,the field faces many problems to be solved.First of all,the resolution of detection methods is low because of the undersize vesicles and the oversize electrode.One to one high-resolution detection can't be achieved,which causes inaccurate detection results.Secondly,it's hard to maintain the normal physiology condition of detected cell,which means not only cell activity maintaining,but also accurate testing results.For these problems,a series of studies were carried out in this paper.The research focus of this paper includes the preparation of carbon fiber nanoelectrode with dimension less than 100 nm,the study of the properties of the electrodes and the applications of single vesicle detection using nanoelectrode.It includes the following aspects.(1)Preparation of CFNE within 100 nm.In order to achieve accurate analysis of nano-sized objects in cell,it's necessary to make electrode diameter match with the vesicles in size.According to the characteristics that the size of vesicles is a few tens of nanometer,firstly,the study was focused to improve the size of nanoelectrode.The fabrication method of CFNE was further improved based on the flame etching method.By adjusting the etching flame and etching angle,the tip of nanoelectrode can be controlled within 100 nm or smaller.Carbon fiber was used as the electrode tip,which has fast electron transfer rate in electrochemical reaction.A greater detection coverage of target molecules and a more sensitive detection were achieved.Compared with the traditional gold electrode,it has a better capacity to decrease nonspecific adsorption,which makes a long time detection in the cell with a stable and accurate signal.Because of the small electrode tip size,the relative activity of cells can be kept during the cell monitoring process.The size and length electrode head is controllable,which can be adjusted according to the experiment requirement.The fabrication of the electrode also provides the conditions for future studies on the transport of intercellular substances.(2)Synthesis of vesicles similar to those inside cell,and detection through the ultra-fine carbon fiber nanoelectrode.A precise quantitative analysis of the detected substances was achieved.The dynamic process of vesicles release on the electrode surface was also studied through these statistics of vesicles kinetics.The detection efficiency can reach up to 90% by combining the control of the concentration of molecules wrapped in vesicle and the size-controlled nanoelectrode.A single vesicle detection technique with high temporal and spatial resolution has been constructed.In the aspect of spatial resolution,the single vesicle can be detected.The events that single vesicle or multiple vesicles simultaneously collide on electrode surface can be distinguished accurately.In the aspect of temporal resolution,the detection limit can be as low as 0.1 ms,which is far exceeding the required ms level for vesicle kinetics release for more than five times the theoretical value.This work shows a comprehensive study about the work mode of vesicles on the electrode surface,and provides a useful research platform for further exploration of the mechanism of cell exocytosis.