Abstract Microfluidic System for Planar Patch-Clamp Electrode Arrays Xiaohui Li Yale University 2006 The patch clamp has been widely accepted as a standard technique for fundamental studies of ion channel proteins, and discovery of drugs that affect these proteins. Traditional patch clamp has a very low throughput which has been proven to be a bottleneck for the drug discovery process. Planar patch-clamp electrode array, which is scaleable and easy to use, provides a potential way to solve this problem. We present a microfluidic system integrated with disposable cell-interface partitions for simultaneous patch clamp recordings. A disposable partition is made by bonding an air-blown PDMS partition, which has a 2 µm air-blown aperture, to a small glass washer. Then it is reversibly sealed to the fluidic system having fluid exchange channels with isolation valves and Ag/AgCl electrodes. Fluid channels are molded from PDMS using microlithographically defined molds. At the cross-over point, channels in different layers formed a valve. Ag/AgCl electrodes are fabricated with standard microfabrication techniques. The suitability of PDMS valves and microfabricated Ag/AgCl electrodes for patch clamp measurement are examined in this report. Gigaseal patch recordings from RBL-1 cells are obtained with a 24% success rate. Our system allows simultaneous recordings from valve-isolated electrodes. Microfluidic System for Planar Patch-Clamp Electrode Arrays A Dissertation Presented to the Faculty of the Graduate School of Yale University in Candidacy for the Degree of Doctor of Philosophy by Xiaohui Li Dissertation Director: Mark A. Reed Dec 2006 ii Copyright © 2007 by Xiaohui Li All rights reserved. iii Acknowledgements This thesis was written based on five-year collaborating work. During the study period, I obtained tremendous help and support from people with various backgrounds. Hereby, I would like to acknowledge it and extend my gratitude to them. Without their tireless and patient help, I would not expect to accomplish the thesis successfully. I am deeply indebted to my supervisors: professor Mark A. Reed and professor Fred J. Sigworth. Their constructive advising provided me great help through my research period and also in drafting this thesis. I have been under their supervision for 5 years. I owe them immense gratefulness for teaching me research skills as well as an attitude to both research and life. I greatly appreciate that I have the opportunity to work with them and learn from them. I also thank the rest of my previous and current advisory committee members: professor Katepalli R. Sreenivasan, professor Marshall Long, professor Alessandro Gomez , professor Juan de la Mora , professor Tso-Ping Ma and professor James Duncan. They have provided very helpful guidance to make my PhD research progress move forward smoothly. My colleagues in the two research groups under professor Mark A. Reed and professor Fred J. Sigworth have supported me a lot in my research work. I am especially grateful to Dr. Kathryn G. Klemic who directly supervised me on the research and is always ready to help. I also thank Dr. Youshang Yang and Ms. Yangyang Yan for their helpful advice on cell culture and patch clamp techniques. I thank Dr. James F. Klemic, iv David Routenberg, Eric Stern, Aric Sanders, Ryan Munden, Stan Guthrie, Dr. Wenyong Wang, Dr. Ilona Kretzschmar, Dr. Glenn Martin, Dr. Menno de Jong, Dr. Takhee Lee, Dr. Guosheng Cheng and Dr. Nilay Pradhan for their cooperative support and for the wonderful suggestions they have ever given during the cleanroom and general lab work. I appreciate Dr. Liguo Wang, Dr. Shumin Bian, Dr. Qiuxing Jiang, Dr. David Chester and Puey Ounjai for their help in the biological laboratory. I thank my friends who have helped me in my academic research and studies. Dr. Zhengting Jiang helped me in using the scanning electron microscope at the Department of Geology and Geophysics. Dr. Rustom Bhiladvala is my first friend in US and introduced me to the technology of microfabrication. Many friends helped me out unselfishly: Linlin Wang, Chris Liu, Zhongping Bao, Biao Li, Yifan Chen, Beelee Chua, Huiming Bu, Dechao Guo, Yanxiang Liu, Weiwei Deng, Yu Xiang, Jian Xu, Leidong Mao and etc. It is my great pleasure to come to know them during the journey of my life. I also have a church home in New Haven. I have met many brothers and sisters in the Calvary Baptist Church. Their spiritual and physical support has given me enormous encouragement during the last three years especially whenever I came across difficulty. I am especially grateful to Huiyuan Chen, Weihua Niu, Tiehong Wang, and pastor Roc Wang. Without their vast enthusiasm, splendid planning, and unreserved efforts, our wedding would have never been like what I had last October. I feel a deep sense of gratitude for my parents. They have always been supportive to me at their best in my life. The happy memory of my father constantly inspired me to v overcome obstacles and keep moving forward during my Ph.D. period and it will keep me on during the rest of my life. I would like to give my special thanks to my wife Baohui whose patient love enabled the completion of my thesis. This research has been supported and funded by NIH grant EB-002020 to F.J.S. I am very appreciative to Yale University for placing me in the world-top research environment with preeminent faculty and researchers and for providing all kinds of equipments to meet the experiment needs. vi Contents Chapter 1. Introduction...................................................................................1 1.1 Planar Patch-Clamp Electrode Array………………………………………….1 1.2 Outline of the Thesis…………………………………………………………..2 Chapter 2. Research Background……………………………………………5 2.1 Ion Channels…………………………………………………………………..5 2.2 Patch Clamp Technique……………………………………………………...10 2.2.1 Patch Clamp Configurations……………………………………….12 2.2.2 Whole-Cell Patch Clamp…………………………………………..15 2.2.3 Disadvantages of Traditional Patch Clamp………………………...15 2.3 Planar Patch Clamp…………………………………………………………..16 2.3.1 Cell Guidance in Planar Patch-Clamp……………………………..18 2.3.2 Planar Patch-Clamp Structure……………………………………...21 2.3.3 Materials for Planar Patch-Clamp………………………………….26 2.4 Microfluidics…………………………………………………………………30 2.5 Planar Ag/AgCl Electrodes…………………………………………………..34 vii Summary…………………………………………………………………………37 Chapter 3. Device Fabrication……………………………………………...39 3.1 A Disposable Planar PDMS Patch Partition…………………………………40 3.1.1 Partitions Molded with Microfabricated Silicon Master…………..40 3.1.2 Partitions Fabricated with the Air-Molding Technique……………44 3.2 PDMS Isolation Valves………………………………………………………47 3.2.1 Fabrication of PDMS Valves………………………………………47 3.2.2 Valve Isolation Resistance…………………………………………50 3.2.3 Valve Lifetime……………………………………………………..52 3.2.4 Simulation of the Valve Deformation……………………………...53 3.3 Fabrication of PDMS Microfluidics…………………………………………57 3.4 Planar Ag/AgCl Electrodes…………………………………………………..57 3.4.1 Fabrication of Ag/AgCl Electrodes………………………………..57 3.4.2 Lifetime of Planar Ag/AgCl Electrodes……………………………58 3.5 Assembly of the Microfluidic Device………………………………………..61 Summary…………………………………………………………………………63 Chapter 4. Results and Discussion…………………………………………65 viii 4.1 Cell Culture and Preparation…………………………………………………67 4.2 Recording Solutions………………………………………………………….67 4.3 Harvesting Cells……………………………………………………………...67 4.4 Recordings and Analysis……………………………………………………..67 4.5 Single Patch Electrode Measurement………………………………………..69 4.6 Compatibility with Commercial Planar Partitions…………………………...71 4.7 Simultaneous Measurement Isolated by Microfluidic Valves……………….71 4.8 Electrode Solution Exchange………………………………………………...73 4.9 Noise Comparison with Glass Pipette………………………………………..74 Summary…………………………………………………………………………77 Chapter 5. Conclusions and Future Direction…………………………..78 5.1 Summary of the Key Accomplishments……………………………………..78 5.1.1 Fabrication of Planar Partitions and the Microfluidic System……..78 5.1.2 Test of the Microfluidic System…………………………………...79 5.2 Suggestions for the Future Work…………………………………………….79 ix List of Figures 2.1 A cell membrane structure………………………………………………………...6 2.2 An ion channel protein…………………………………………………………….7 2.3 A gigaseal………………………………………………………………………...10 2.4 Cell-attached and whole-cell configurations…………………………………….11 2.5 Different configuration of conventional patch clamp……………………………14 2.6 A planar patch-clamp configuration……………………………………………..17 2.7 The CytoPatchTM chip……………………………………………………………19 2.8 Microfluidic chip for single cell patch-clamp measurement…………………….21 2.9 A smoothed DRIE etched aperture for planar patch clamp measurement……….23 2.10 A hollow SiO2 nozzle for planar patch-clamp measurement…………………...24 2.11 Side trapped patch-clamp array on a microfluidic platform……………………25 2.12 A Nanion glass chip…………………………………………………………….27 2.13 Planar PDMS patch-clamp recording system…………………………………..29 2.14 A planar silicon chip based patch-clamp system (QPatchTM)…………………..31 2.15 A pneumatically actuated valve………………………………………………...32 2.16 An elastomeric one-way diaphragm valve……………………………………...33 2.17 A very large scale microfluidic comparator chip……………………………….34 2.18 An exhaustible Ag/AgCl electrode……………………………………………..36 x 2.19 Structure of a thin-film Ag/AgCl electrode…………………………………….37 3.1 Schematic cross-section view of the microfluidic system……………………….39 3.2 Process of fabricating silicon master…………………………………………….41 3.3 SEM pictures of a microfabricated silicon master……………………………….42 3.4 Molding PDMS partition from the microfabricated silicon