Controlled On-chip Stimulation of Quantal Catecholamine Release from Chromaffin Cells Using Photolysis of Caged Ca2+ on Transparent Indium-Tin-Oxide Microchip Electrodes

摘要

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 100 nm - thick transparent Indium-Tin-Oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (∼20 μm by 20 μm). 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⁺, a 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.