Individually Addressable Thin-Film Ultramicroelectrode Array for Spatial Measurements of Single Vesicle Release

Abstract

Thin-film platinum ultramicroelectrode arrays (MEAs) with subcellular microelectrodes were developed for the spatial measurement of neurotransmitter release across single cells or clusters of single cells. MEAs consisting of 16, 25, and 36 square ultramicroelectrodes with respective widths of 4, 3, and 2 μm were fabricated on glass substrates by photolithography, thin-film deposition, and reactive ion etching. The electrodes in each MEA are tightly defined in a 30 μm × 30 μm square, which is potentially useful to measure exocytosis across a single cell or clusters of single cells. These MEAs have been characterized with scanning electron microscopy and cyclic voltammetry and show excellent stability and reproducibility. Culturing PC12 cells on top of the MEAs has been achieved by modifying the array with a poly(dimethylsiloxane) chamber and coating a thin layer of collagen IV on top of the electrode surface. The electrochemical response to dopamine has been characterized after coating the surface with the cell-adhering molecules and then with cells attached. Amperometric detection demonstrates that individual exocytotic events can be recorded at these arrays with spatial resolution for dynamic electrochemical measurements near 2 μm. In contrast to previous single-cell experiments, the effect of dopaminergic drugs on imaging single vesicle exocytotic release from PC12 cell clusters is presented at cell clusters incubated with the dopamine precursor and Parkinson's therapy agent, L-3,4-dihydroxyphenylalanine, and at cell clusters incubated with the vesicular monoamine transport inhibitor, reserpine. The results of electrochemical imaging demonstrate that the drug effect on PC12 cell clusters is consistent with previous single-cell experiments.