Patch clamping on plane glass-fabrication of hourglass aperture and high-yield ion channel recording
Patch clamping on plane glass-fabrication of hourglass aperture and high-yield ion channel recording
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DOI：
10.1039/b901025d
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发表时间：
2009-01-01
期刊：
影响因子：
6.1
通讯作者：
Wo, Andrew M.
中科院分区：
文献类型：
--
作者：
Chen, Chang-Yu;Tu, Ting-Yuan;Wo, Andrew M.
平面膜片钳彻底改变了离子通道测量，消除了传统微移液管方法的费力操作，实现了高通量。然而，由于微加工工艺，gigasseal地层的低产量和/或相对较高的成本是两个主要缺点。本文提出了在玻璃基板上贴片夹紧的一种经济的解决方案，而不牺牲千兆位的收率。采用两段式CO2激光钻孔方法，在150 μ m硼硅覆盖玻璃上生成了一个指定直径的沙漏状、漏斗状、表面光滑且无碎屑的孔径。以1 ~ 3 μ m的孔径作为膜片钳芯片，对人胚胎肾、中国仓鼠卵巢和Jurkat T淋巴瘤细胞进行封闭阻力测试，封闭成功率分别为62.5%、43.6%和66.7%。结果还证实了对内源性表达离子通道的全细胞和单通道记录，以证实不同贴片结构的能力。
Planar patch-clamp has revolutionized ion-channel measurement by eliminating laborious manipulation from the traditional micropipette approach and enabling high throughput. However, low yield in gigaseal formation and/or relatively high cost due to microfabricated processes are two main drawbacks. This paper presents patch clamping on glass substrate-an economical solution without sacrificing gigaseal yield rate. Two-stage CO2 laser drilling methodology was used to generate an hourglass, funnel-like aperture of a specified diameter with smooth and debris-free surfaces on 150 mu m borosilicate cover glass. For 1-3 mu m apertures as patch-clamp chips, seal resistance was tested on human embryonic kidney, Chinese hamster ovary, and Jurkat T lymphoma cells with a gigaseal success rate of 62.5%, 43.6% and 66.7% respectively. Results also demonstrated both whole-cell and single channel recording on endogenously expressed ion channels to confirm the capability of different patch configurations.