Small Electrochemical Sensors and Single Cells : Zeptomoles, Milliseconds and the Nature of Reality in Exocytosis
소속 :
연사 : Prof. Andrew Ewing(Chalmers University of Technology)
일시 : 2015-11-26 16:30 ~
장소 : 25-1동 국제회의실
Electrochemical methods provide a powerful approach to investigate neurotransmitter release and storage from and in single cells and single nanometer vesicles.
Electrochemical cytometry is a new method that can be used to separate nanometer vesicles, lyse them on an electrode surface, and amperometrically detect the active contents of each vesicle in a high throughput manner. We began with a hybrid capillary-microfluidic device surrounding the electrode to rapidly determine levels of aminergic transmitters in vesicles. More recently, we have developed a new method of electrochemical cytometry we call vesicle impact cytometry to measure the total content of single neurotransmitter vesicles. The electrochemical response to single adrenal chromaffin vesicles filled with hormone transmitters as they impact a 33-um diameter disk-shaped carbon electrode will be shown. The vesicles appear to adsorb onto the electrode surface and sequentially spread out over the electrode surface trapping their contents against the electrode. These contents are then oxidized and a peak results for each vesicle that bursts. A large number of current transients can be observed if the concentration of vesicles is high relative to the area of the electrode. We have also been able to accomplish this type of cytometry in the cytoplasm of living PC12 and adrenal cells. Comparison of the contents of these biological vesicles to the release of catecholamine from single cells supports the concept that only a fraction of transmitter is released during exocytosis.
We have also used amperometry to examine post-spike feet that would be expected if the fusion pore were closing again with open and closed exocytosis and to examine exocytotic release at varicosities in the fly larvae. We conclude that normal exocytosis is open and closed and only a fraction of the transmitter is released for each opening!
Electrochemical cytometry is a new method that can be used to separate nanometer vesicles, lyse them on an electrode surface, and amperometrically detect the active contents of each vesicle in a high throughput manner. We began with a hybrid capillary-microfluidic device surrounding the electrode to rapidly determine levels of aminergic transmitters in vesicles. More recently, we have developed a new method of electrochemical cytometry we call vesicle impact cytometry to measure the total content of single neurotransmitter vesicles. The electrochemical response to single adrenal chromaffin vesicles filled with hormone transmitters as they impact a 33-um diameter disk-shaped carbon electrode will be shown. The vesicles appear to adsorb onto the electrode surface and sequentially spread out over the electrode surface trapping their contents against the electrode. These contents are then oxidized and a peak results for each vesicle that bursts. A large number of current transients can be observed if the concentration of vesicles is high relative to the area of the electrode. We have also been able to accomplish this type of cytometry in the cytoplasm of living PC12 and adrenal cells. Comparison of the contents of these biological vesicles to the release of catecholamine from single cells supports the concept that only a fraction of transmitter is released during exocytosis.
We have also used amperometry to examine post-spike feet that would be expected if the fusion pore were closing again with open and closed exocytosis and to examine exocytotic release at varicosities in the fly larvae. We conclude that normal exocytosis is open and closed and only a fraction of the transmitter is released for each opening!
