胡文聰臺灣大學：應用力學研究所莊信弘Chuang, Hsin-HungHsin-HungChuang2007-11-292018-06-292007-11-292018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/62450膜片嵌制（patch clamp）技術在標準電生理分析中是一種發展相當完備、且成為研究離子通道及轉運蛋白相當有力的工具。儘管膜片嵌制技術擁有較一般分析方法中更優渥的資訊含量，但是此技術在操作上卻是需要技術相當純熟的人工操控，才可將玻璃吸管精準地移動到所要記錄的細胞上，過程中所耗費的時間與精力造成實驗產量必然是相當少量的。 本論文將以一多層壓克力層為元件之紀錄腔體，再加上以PDMS為基礎的紀錄晶片來整合膜片嵌制技術所有的必要條件於一個整體系統中，有所謂晶片嵌制系統（Patch-on-A-Chip system）的電生理紀錄方法。以PDMS為材料的膜片嵌制晶片主要是仿照玻璃微管前的尖端外型，經過表面處裡後的PDMS膜片嵌制晶片變得更有吸引力，對細胞膜表面也更容易且迅速地形成Giga 歐姆吸附。此晶片嵌制系統不僅改良了傳統膜片嵌制技術中所不可避免的缺點外，此系統裝置更提供多孔電生理紀錄以提高資料點產量。 我們建構了一個可以在Xenopus oocytes細胞上表現inactivation-removed Shaker potassium channels 的非同步記錄多孔訊號的紀錄晶片，此紀錄晶片是以PDMS為材料製作而成的彈性薄膜，在精準的對位中貼附在已打洞的玻璃蓋玻片上而形成的記錄晶片。我們的實驗結果更指出，若將整個記錄系統整合時將可達到 ”高通量電生理篩選”（High-throughput Electrophysiological Screening）之目的。The patch clamp technique, in the standard electrophysiological assays, is a well-developed and powerful tool for the study of ion channels and transporters. Despite claims that the patch clamp technique has superior characteristics of high information content, the technique is suffered by the laborious and slow manual manipulations of the glass pipette onto the cell by highly skilled technicians with consequential low throughput. This thesis presents a multiple acrylic layers device（Recording Chamber） that integrates all the necessary conditions in the patch clamp technique on a Poly（dimethylsiloxane）（PDMS）-based Patch-on-A-Chip（Recording Chip）system. PDMS is an attractive material which to mimic the tip of the glass micropipette that can rapidly forms gigaohm seals to a cell membrane after surface treatment. The recording system is not only to avoid the inherent shortcomings found in the conventional patch clamping but also provides Multiple-throughput apertures for electrophysiological recording to raise the throughput of data points. We demonstrate a recording chip, which is fabricated by an elastomeric membrane using PDMS material conformal contact onto an apertured glass cover slip after a precise alignment, allows patch clamp recording on Xenopus oocytes expressing inactivation-removed Shaker potassium channels at different apertures of the recording chip asynchronously. Our results have indicated the potential of high-throughput electrophysiological screening with an integration of recording system.ABSTRACT 1 中文摘要 2 CHAPTER 1：INTRODUCTION 5 1.1 Introduction 5 1.1.1 High-Throughput Screening in Context 6 1.1.2 The Definition of High-Throughput Screening 8 1.2 Current Approaches to High-Throughput Screening 8 1.2.1 Downsizing and Automation of High-Throughput Screening 9 1.2.2 Microfluidics and High-Throughput Screening 10 1.2.3 A brand-new assay of High-Throughput Screening-Patch-on-a-Chip 11 1.3 RESEARCH OVERVIEW 13 CHAPTER 2：THE MARVELOUS CONNECTION BETWEEN PATCH-CLAMP TECHNIQUES AND BIOMEMS TECHNOLOGIES 16 2.1 INTRODUCTION TO CONVENTIONAL PATCH-CLAMP TECHNIQUES 16 2.1.1 Introduction to Ion Channels and Ionic Current Flow 16 2.1.2 Ion Channels in Drug Discovery 18 2.1.3 Patch Clamping as the Gold Standard for Ion-Channel Assays 20 2.1.4 Introduction to Voltage Clamp 21 2.1.5 Why Patch-on-a-Chip？ 23 2.2 INTRODUCTION TO CHIP-BASED PATCH-CLAMPING （PATCH-ON-A-CHIP） 23 2.2.1 Historical background of the development of Patch-on-a-Chip 24 2.2.2 The main application in Drug Discovery Technology 26 2.2.2.1 AVIVA Biosciences Corp. (San Diego, CA, U.S.A.) / Axon Instruments, Inc. (Union City, CA, U.S.A.) 26 2.2.2.2 Cytocentrics CCS (Reutlingen, Germany) 27 2.2.2.3 Flyion GmbH (Tubingen, Germany) 29 2.2.2.4 Ionworks HT – Molecular Devices corp.（Sunnyvale, CA, USA） 30 2.2.2.5 Sophion Biosciences (Ballerup, Demark). 32 2.2.2.7 Yale University (New Haven, CT, U.S.A.) / Axon Instruments (Union City, CA, U.S.A.). 33 2.3 CONCLUSION 33 CHAPTER 3：MATERIALS, METHODS, AND EXPERIMENTAL SETUP 34 3.1 PREPARATION OF XENOPUS OOCYTE 34 3.1.1 Maintenance of Xenopus laevis 34 3.1.2 Romoval of an Ovarian Lobe 35 3.1.3 Removal of Follicular Cell Layer 37 3.1.4 Injection of Oocytes 38 3.2 Microfabrication of High-Throughput Screening Chip 39 3.2.1 Fabrication of elastomeric membrane 39 3.2.1.1 Soft lithography 39 3.2.1.2 The excellent properties of PDMS material 41 3.2.1.3 Microfabrication of Recording Chip 42 3.3 RECORDING CHAMBER DESIGNING 44 3.4 ELECTRICAL MEASUREMENT 48 CHAPTER 4：RESULT AND DISCUSSION 49 4.1 Fabrication of Recording Chip 49 4.1.1 Fabrication of elastomeric membrane 49 4.1.2 Aperture Alignment 50 4.1.3 The application of Recording Chamber 50 4.2 Electrophysiological result 51 4.2.1 Voltage Clamp Capability 51 4.2.2 The Advantages of our Chip-based Patch-clamping 56 4.2.3 The disadvantages of our Chip-based Patch-clamping 56 CHAPTER 5：CONCLUSION AND FUTURE WORK 57 5.1 Conclusion 57 5.2 Future work 57 Reference 582360599 bytesapplication/pdfen-US多通量電生理篩選軟微影膜片箝制Soft LithographyPatch ClampMultiple-Throughput Electrophysiological Screeningthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/62450/1/ntu-93-R91543066-1.pdf