Controlled on-chip stimulation of quantal catecholamine release from chromaffin cells using photolysis of caged Ca2+ on transparent indium-tin-oxide microchip electrodes.
Photorelease of caged Ca(2+) is a uniquely powerful tool to study the dynamics of Ca(2+)-triggered exocytosis from individual cells. Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca(2+), 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 (approximately 20 microm by 20 microm). We loaded bovine chromaffin cells with acetoxymethyl (AM) ester derivatives of the Ca(2+) cage NP-EGTA and Ca(2+) indicator dye fura-4F, then transferred these cells onto the working ITO electrodes for amperometric recordings. Upon flash photorelease of caged Ca(2+), a uniform rise of [Ca(2+)](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 [Ca(2+)](i) and a "priming" effect of sub-stimulatory [Ca(2+)](i) on the response of cells to subsequent [Ca(2+)](i) elevation, similar to previous reports using different techniques. We conclude that UV photolysis of caged Ca(2+) is a suitable stimulation technique for higher-throughput studies of Ca(2+)-dependent exocytosis on transparent electrochemical microelectrode arrays.
Chen
Xiaohui
X
Department of Biological Engineering, University of Missouri, Columbia, MO 65211, USA.
Gao
Yuanfang
Y
Hossain
Maruf
M
Gangopadhyay
Shubhra
S
Gillis
Kevin D
KD
D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002395:Catecholamines / Q000378:metabolism*; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology; D004563:Electrochemistry; D005089:Exocytosis; D056656:Lab-On-A-Chip Devices; D046208:Microchip Analytical Procedures / Q000379:methods*; D008839:Microelectrodes; D008954:Models, Biological; D055095:Optics and Photonics; D010782:Photolysis; D017971:Tin Compounds / Q000737:chemistry*
D002395:Catecholamines; D002413:Cations, Divalent; D017971:Tin Compounds; C109984:indium tin oxide; D004072:Digitonin; D002118:Calcium