Spatial resolution of single-cell exocytosis by microwell-based individually addressable thin film...

We report the fabrication and characterization of microwell-based individually addressable microelectrode arrays (MEAs) and their application to spatially and temporally resolved detection of neurotransmitter release across a single pheochromocytoma (PC12) cell. The microwell-based MEAs consist of 16 4-μm-width square ultramicroelectrodes, 25 3-μm-width square ultramicroelectrodes, or 36 2-μm-width square ultramicroelectrodes, all inside a 40 × 40 μm square SU-8 microwell. MEAs were fabricated on glass substrates by photolithography, thin film deposition, and reactive ion etching. The ultramicroelectrodes in each MEA are tightly defined in a 30 × 30 μm square area, which is further encased inside the SU-8 microwell. With this method, we demonstrate that these microelectrodes are stable, reproducible, and demonstrate good electrochemical properties using cyclic voltammetry. Effective targeting and culture of a single cell is achieved by combining cell-sized microwell trapping and cell-picking micropipet techniques. The surface of the microelectrodes in the MEA was coated with collagen IV to promote cell adhesion and further single-cell culture, as good adhesion between the cell membrane and the electrode surface is critical for the quality of the measurements. Imaging the spatial distribution of exocytosis at the surface of a single PC12 cell has also been demonstrated with this system. Exocytotic signals have been successfully recorded from eight independent 2-μm-wide ultramicroelectrodes from a single PC12 cell showing that the subcellular heterogeneity in single-cell exocytosis can be precisely analyzed with these microwell-based MEAs.