We report the fabrication and characterization of carbon microelectrode arrays (MEAs) and their application to spatially and temporally resolve neurotransmitter release from single pheochromocytoma (PC12) cells. The carbon MEAs are composed of individually addressable 2.5-mu m-radius microdisks embedded in glass. The fabrication involves pulling a multibarrel glass capillary containing a single carbon fiber in each barrel into a sharp tip, followed by beveling the electrode tip to form an array (10-20 mu m) of carbon microdisks. This simple fabrication procedure eliminates the need for complicated wiring of the independent electrodes, thus allowing preparation of highdensity individually addressable microelectrodes. The carbon MEAs have been characterized using scanning electron microscopy, steady-state and fast-scan voltammetry, and numerical simulations. Amperometric results show that subcellular heterogeneity in single-cell exocytosis can be electrochemically detected with MEAs. These ultrasmall electrochemical probes are suitable for detecting fast chemical events in tight, spaces, as well as for developing multifurictional electrochemical microsensors.

This chapter contains sections titled: Electrochemistry Inside and Outside Single Nerve Cells Amperometry Experiments with Adrenal Chromaffin Cells Experiments at PC12 Cells Experiments at Mast Cells Experiments in Pancreatic β-Cells Artificial Liposomal Cell Models of Exocytosis Fast-Scan Cyclic Voltammetry Patch Amperometry Single-Cell Measurements in Microvials Single-Cell Discoveries Made Using Electrochemistry Conclusions Acknowledgments References

A liposome model of exocytosis has been used to examine the nanometer environment of the space between the electrode and the membrane. First, this has been used to test models of coulometric efficiency for different size vesicles with 5- and 33-mum electrodes. The resulting model has a best fit that suggests that the liquid space is similar to300 nm across the gap. Given this dimension, the volume of the electrode-membrane space is not large enough to accommodate the volume of larger vesicles in cells such as the mast cells of the beige mouse. Second, the model suggests that flow of solution from the exocytosis event is more important than diffusion. Flow from the finite vesicle volume past the electrode leads to less charge passed. Third, and finally, this system can be used to model transport in the synapse and so it is possible to examine the idea that transmitter flows in addition to diffusing from the synapse. This model should be useful in understanding and quantifying the escape of transmitter from synapses in vivo.