Controlled on-chip stimulation of quantal catecholamine release from chromaffin cells using photolysis of caged Ca2+ on transparent indium-tin-oxide microchip electrodes
Controlled on-chip stimulation of quantal catecholamine release from chromaffin cells using photolysis of caged Ca2+ on transparent indium-tin-oxide microchip electrodes
复制标题
DOI：
10.1039/b715308m
复制
发表时间：
2008-01-01
期刊：
影响因子：
6.1
通讯作者：
Gillis, Kevin D.
中科院分区：
文献类型：
--
作者：
Chen, Xiaohui;Gao, Yuanfang;Gillis, Kevin D.
笼中钙离子的光释放是研究单个细胞内钙离子引发胞吐的动力学的唯一有效工具。利用光刻和其他微制造技术，我们已经开发出透明的微芯片器件，能够光释放笼中的钙离子，并电化学检测单个细胞或细胞阵列中的量子儿茶酚胺分泌，作为开发高通量实验设备的一步。在玻璃盖上溅射沉积一层厚度为100 nm的透明氧化铟锡(ITO)薄膜，然后将其制成24个晶胞大小的工作电极(类似于20微米乘以20微米)。我们用钙离子笼NP-EGTA和钙离子指示剂Fura-4F的乙酰氧甲基(AM)酯衍生物负载牛嗜铬细胞，然后将这些细胞转移到工作的ITO电极上进行安培记录。当笼状钙离子在闪光下释放时，靶细胞内[Ca~(2+)](I)的均匀上升导致可氧化的儿茶酚胺的量子释放，通过ITO电极的安培法测量。我们观察到当[Ca~(2+)](I)迅速升高时，[Ca~(2+)](I)迅速升高时出现一串电流峰，以及亚刺激[Ca~(2+)](I)对细胞对随后[Ca~(2+)](I)升高的反应的‘启动’效应，这与以前使用不同技术的报道类似。我们的结论是，笼状钙离子的紫外光分解是一种适合于在透明电化学微电极阵列上高通量研究依赖于钙离子的胞吐作用的刺激技术。
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 (similar to 20 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+, 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.