Patch clamping on plane glass—fabrication of hourglass aperture, high-yield ion channel recording, and microfluidics integration
Date Issued
2009
Date
2009
Author(s)
Chen, Chang-Yu
Abstract
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 process are two main drawbacks. This work 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 by a simple process of reflow of melted glass on 150 μm borosilicate cover glass, providing smooth, debris-free, and freshly activated aperture surface. Two-phase flow simulation illuminates details of the reflow process via Navier-Stokes equation coupled with level set method, and the results reveal that the computation captures the dominant physics of the glass reflow process. In addition, a systematic investigation of the laser drilling procedures was comprehensively studied to ascertain proper parameters for fabricating desired apertures.For 1-3 μm apertures as patch-clamp chips, gigaseal formation and ion channel recording were demonstrated in various cell types. PC-12 cells were proven, for the first time, capable of gigaseal formation. Statistical seal resistance was then tested on human embryonic kidney (HEK-293T), Chinese hamster ovary (CHO-K1), and Jurkat T lymphoma cells with success rate of gigaseal of 62.5%, 43.6% and 66.7% respectively. Results demonstrate both whole-cell recording on endogenously expressed ion channels of HEK-293T cells, and single channel recording on Jurkat cells by cell-attached patch to confirm the capability of different patch configurations. Planar patch-clamp chips were further integrated with microfluidic concentration generator to provide rapid solution exchange. A series of linear concentrations of fluid mixture could be rapidly generated by adjusting relative flow rate of each inlet and deliver to the location of patched cell. Endogenous volume-regulated chloride channels in HEK-293T cells were successfully examined in various osmolarities generated by the linear concentration generator. Furthermore, a logarithmic microfluidic concentration generator was designed to provide either spatial or temporal concentration by the same design rule. This approach is conceivably beneficial in dose-response assays for high throughput screening of drugs. The planar patch-clamp chips described herein provide an alternative fabrication process with low cost and high-yield of gigaseal formation. It was proven practical in different patch configurations for ion channel recording, and successfully integrated with microfluidics for rapid solution exchange. It is likely to be further leveraged to perform single cell study, or patch-clamp array configuration for the purpose of compound identification and/or drugs evaluation.
Subjects
ion channel
patch-clamp chip
hourglass
laser drilling
glass reflow
microfluidic
concentration generator
Type
thesis
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