臺灣博碩士論文加值系統

平面式片膜鉗制技術(planar patch-clamp technique)為一種革命性的離子通道量測方式，其除去於顯微鏡下操控傳統玻璃電極之繁瑣步驟，且可以符合快速篩檢的需求。然而，現行之片膜鉗制晶片部分之緊密電阻(seal resistance)具備十億歐姆阻抗(gigaseal)之比例低落，或是單一晶片製作費用昂貴歸咎於微製造技術之製程方式。鑒於此，本文提出一種經濟、簡易之製程方式，利用平面玻璃基材製作出片膜鉗制晶片，並具備達到優良緊密電阻之能力。以兩階段式二氧化碳雷射鑽孔製程方式，以簡易之玻璃回融機制，於150微米厚度之矽硼酸玻璃蓋玻片上製作出特殊沙漏構型、呈現漏斗狀之微孔洞；其微孔洞之表面具備平滑、無殘渣及新鮮高活性之特性。此玻璃回融機制藉由二相流之模擬計算，以納維-斯托克斯方程式(Navier-Stokes equation)搭配等位函數法(level set method)成功地描述玻璃回融之細節過程，證實本計算方式掌握此玻璃回融之主要物理機制。此外，本工作亦有系統地廣泛探討雷射鑽孔的參數對於微孔洞製程之影響，以期製作出符合需求之微孔洞。
製成之平面式片膜鉗制晶片具備1-3微米之微孔洞，在多種細胞測試下證實其可達到十億歐姆阻抗(gigaseal)之緊密電阻(seal resistance)以及量測細胞膜表面離子通道之能力。利用PC-12細胞首次證實此晶片具備達成gigaseal之能力；此外，三種細胞株達到gigaseal之統計，分別為human embryonic kidney (HEK-293T) 62.5%，Chinese hamster ovary (CHO-K1) 43.6%，以及Jurkat T lymphocyte 66.7%。實驗結果亦展現於HEK-293T細胞上量測到其本身之全細胞(whole-cell)離子通道電流，以及於Jurkat細胞上量測得單一離子通道(single-channel)之開闔電流，因此證實了本晶片可以不同形式之量測方式進行片膜鉗制技術。
平面式片膜鉗制晶片更進一步與微流體濃度產生器進行整合以提供快速之實驗流體交換。透過調整不同流體注入孔之相對流速，即可產生一系列具線性濃度之流體並傳送到待量測之細胞區域。透過此濃度產生器之整合，成功地在不同滲透壓之流體下進行離子通道量測，探討HEK-293T細胞本身之體積調節氯離子通道(volume-regulated chloride channel)。除此之外，本工作亦提出一種利用簡單原則所設計之對數型(logarithmic)微流體濃度產生器，可產生時間上或空間上之對數濃度流體，可用於高通量篩檢之藥物劑量反應分析。
本工作之平面式片膜鉗制晶片利用新穎之製造技術，使單一晶片價格降低，並具備產生高比例gigaseal之能力。本技術成功地在不同片膜鉗制方式下量測離子通道量電流，並與微流體系統整合以提供快速液體交換。此法可更進一步應用於其他單細胞分析，或擴展為片膜鉗制晶片陣列形式應用於化合物識別以及藥物評估等相關議題。

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.

序言與謝辭 i
中文摘要 ii
Abstract iv
目錄 vii
圖目錄 viii
1 Introduction 1
2 Material and methods 7
2.1 Planar patch-clamp chips 7
2.1.1 Glass substrate and laser setup 7
2.1.2 Principle of fabrication of the microaperture 7
2.1.3 Computational verification 10
2.1.4 Two-stage laser drilling methodology 12
2.2 Electrophysiology 14
2.2.1 Cell culture and preparation 14
2.2.2 Signals and solutions 15
2.2.3 Device operation 17
2.3 Microfluidic integration 19
2.3.1 Design of concentration generator 19
2.3.1.1 Linear concentration generator 19
2.3.1.2 Logarithmic concentration generator 20
2.3.2 Fabrication of microchannels 24
2.3.3 Concentration verification 24
3 Results and discussion 25
3.1 Physics of the formation of the hourglass aperture—Computational verification 25
3.2 Fabrication of microapertures—Two-stage laser drilling technique 27
3.3 Seal resistance characterization 36
3.4 Ion channel recording 45
3.5 Integration of planar patch-clamp chips and linear microfluidic concentration generator 52
3.6 Logarithmic concentration generator 56
4 Conclusions 61
Reference 63
Appendix A: Publication 68
Appendix B: Simulation 69
Appendix C: LabVIEW program 81

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