Optical stimulation of quantal exocytosis on transparent microchips
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Photorelease of caged Ca²⁺ is a uniquely powerful tool to study the dynamics of Ca²⁺-triggered exocytosis from individual cells. Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca²⁺ together with electrochemical detection of quantal catecholamine secretion from individual cells or cell arrays as a step towards developing high-throughput experimental devices. A 110 nm - thick transparent Indium-Tin-Oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (2̃0 [mu]m by 20 [mu]m). We loaded bovine chromaffin cells with acetoxymethyl (AM) ester derivatives of the Ca²⁺ cage NP-EGTA and Ca²⁺ indicator dye Fura-4F, then transferred these cells onto the working ITO electrodes for amperometric recordings. Upon flash photorelease of caged Ca²+С uniform rise of [Ca²⁺]i within the target cell leads to quantal release of oxidizable catecholamines measured amperometrically by the underlying ITO electrode. We observed a burst of amperometric spikes upon rapid elevation of [Ca²⁺]i and a "priming" effect of sub-stimulatory [Ca²⁺]i on the response of cells to subsequent [Ca²⁺]i elevation, similar to previous reports using different techniques. We conclude that UV photolysis of caged Ca²⁺ is a suitable stimulation technique for higher-throughput studies of Ca²⁺-dependent exocytosis on transparent electrochemical microelectrode arrays.
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Ph. D.