Fabrication Of Size-controllable Untrasmall-disk Electrode And Its Application To Study Single Vesicle Release Kinetics And Fusion Pore Dynamics

Secretion plays an important role in the life span of the organisms. Micro-carbon fiber electrode (micro-CFE) amperometry is a good method to monitor cell secretion in real time, but conventional micro-electrode amperometry contains signal pollution owning to the diffusion of the transmitter from secretion position to active area of the electrode. To improve the spatio-temporal resolution of amperometry, we fabricated size-controllable micron or nano-disk carbon fiber electrode.An improved electrochemical etching procedure was employed to fabricate, for the first tiem, cylindrical fiber with controlled micron or nano-diameter. Afterwards, a facile insulation with polypropylene sheath was employed to completely insulate the whole body of the thinned fiber, and an ultrasmall-disk sensing area was finally produced by cutting of the insulated fibers. Scanning electron microscopy was employed to characterize the ultrasmall geometry size of the fabricated electrode and to show the tight adherence of the insulation sheath on the fiber. The cut ends of the electrodes were also shown to be smooth, clean and without obvious jagged layer. The fabricated micron or nano-disk carbon electrodes show ideal steady-state voltammetric behavior with satisfying reversibility.Then the etched micro-disk CFE was employed to monitor 5-HT secretion from the C. elegans NSM neurons, and results showed that, compared to the conventional micro-disk CFE, the etched small disk CFE possess higher sensitivity due to its improved signal-to-noise level, which enables minute amounts of 3000 oxidizable molecules to be detectable.Our further data also showed that UNC-31 plays an import role in synaptic vesicle (SV) docking, while UNC-13 plays an import role in the fusion step of SV in C. elegans. Meanwhile, the nano-disk CFE was employed to investigate fusion kinetics in PC12 cells. Results showed that it is avoid of diffusion broadening of the detected spikes, which is the inherent defect of the conventional micro- carbon fiber electrode technique.we also use a statistical methed to pick out the integrated events which reflect the realest information of fusion kinetics.The size-controllable micron or nano-disk carbon fiber electrode (CFE) was shown to be characterized by low noise, high sensitivity and high sptio-temporal resolution, and was demonstrated to be excellent for extra-cellular transmitter release detection at tiny structures and vesicle fusion kinetics analysis.