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研究生:林郁婷
研究生(外文):Yu-Ting Lin
論文名稱:利用結合表面電漿共振與電漿波導共振之晶片研究膜蛋白傳輸現象
論文名稱(外文):Monitoring Transport Behaviors of Cell Membrane Transporters by a Surface Plasmon and Plasmon-Waveguide Resonance Combined Chip
指導教授:趙玲趙玲引用關係
指導教授(外文):Ling Chao
口試日期:2017-07-20
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學工程學研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:79
中文關鍵詞:表面電漿共振電漿波導共振微米級孔洞陣列通道蛋白質傳輸動態
外文關鍵詞:surface plasmon resonanceplasmon-waveguide resonancesub-micron grating structuremembrane transport proteinstransport kinetics
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能夠瞭解物質藉由膜上通道蛋白跨膜運輸的現象,對於生物醫藥開發等相關領域來說相當的重要。現今表面電漿共振現象常被應用於免標定的生物感測平台,主要利用光學折射率變化來偵測晶片表面生物分子吸附脫離的情況。為了延伸其應用,我們設計了一個結合表面電漿共振(SPR)與電漿波導共振 (PWR)的平台,並利用模擬驗證出此結構能表現出兩個用於偵測孔洞內部與外部區域濃度的特徵共振角。在本研究中,我們在奈米級厚度的金薄膜上鍍二氧化矽薄膜後,藉由蝕刻製造出微米等級的孔洞陣列,再鋪上由化學發泡法從海拉細胞(Hela cell)取出的含有蛋白質之細胞膜至平台上。其中,細胞脂質膜具有阻隔洞內與洞外物質擴散的功用,也能使通道蛋白質被保持在其原始環境中。蝕刻的孔洞則提供了類似細胞內部的空間,以讓傳輸的物質在洞內進行累積,藉由金薄膜配合雷射所產生的表面電漿共振現象(SPR)和在二氧化矽上方產生的電漿波導共振,可用來同時偵測孔洞內部與外部的物質濃度變化所造成的折射率改變。由於只有特定物質能通過對應之通道蛋白進行傳輸,因此,洞內之濃度變化能夠反映通道蛋白的傳輸行為。這些實驗證明此平台應可用於研究不同藥物對通道蛋白所造成之抑制或促進功效,以及可即時偵測通道蛋白之傳輸動態,在醫藥檢測及研發藥物等廣泛用途具有相當的潛力。
Surface plasmon resonance (SPR) is a powerful label-free and contact-free technique for chemical and biological sensing experiments. However, the traditional use of SPR instruments is for molecular interactions on the surface of metallic film. In order to extend the application of SPR, we integrated the concept of plasmon-waveguide resonance (PWR) and proposed the idea of PWR/SPR combined chip in order to measure the transport behaviors of cell membrane transport proteins. The PWR/SPR combined chip is composed of a silica layer with sub-micron sized pores on a thin gold film. The geometry allows us to use SPR to detect the refractive index change in the pore region, which is correlated to the target species concentration inside the pore, and PWR to simultaneously monitor the change of refractive index at the top silica surface. We deposited the giant plasma membrane vesicles (GPMV) derived from cells onto the PWR/SPR combined chip to construct the lipid membrane with the membrane proteins suspending over the sub-micron sized pores. Consequently, the detection area can be divided into two regions including the space inside the pores and the region above the grating structure. By using COMSOL simulation, we confirmed that this system allows us to simultaneously measure the change of refractive indices in the two regions across the lipid membrane. We experimentally demonstrated how the platform can be used to study how various inhibitors or ligands can influence the glucose transport through the corresponding membrane transport proteins (Glut1, Glut2) in Hela cell plasma membranes.
Acknowlegement i
摘要 iv
Abstract v
Table of Content vi
Figure Captions ix
Table Captions xiv
Chapter 1 Introduction 1
1.1 Cell Membrane Species 3
1.2 Surface Plasmon Resonance (SPR) 4
1.2.1 Angle Scanning Mode 7
1.2.2 Real Time Mode 8
1.3 Plasmon Waveguide Resonance (PWR) 9
1.4 Giant Plasma Membrane Vesicles (GPMV) 10
1.5 Cell Membrane Transport 12
1.6 Glucose Transporter Family 12
1.6.1 Glucose Transporter Family 12
1.6.2 Glucose Transporter 1 (GLUT1) and Glucose Transporter 2 (GLUT2) 13
1.7 Glucose Transport Kinetics 14
Chapter 2 Materials and Method 18
2.1 Materials 18
2.2 Apparatus 19
2.3 Materials of Homemade SPR System 20
2.4 COMSOL Simulation 20
2.5 Fabrication of a PWR / SPR Combined Chip 21
2.6 Standard Test of Chips 23
2.7 Cell Culture and Labeling 24
2.8 GPMV Preparation and Deposition Method 24
2.9 Fluorescence Microscopy and Fluorescence Recovery After Photo-bleaching (FRAP) 25
2.10 Preparation of Solutions 25
2.11 Giant Plasma Membrane Vesicles (GPMVs) Deposition on the Chip 26
2.12 Fabrication of Flow Chamber 26
2.13 Glucose Transport Experiments 27
2.13.1 Find Position with GPMV Membranes Over the Pores 27
2.13.2 Glucose Transport Experiment with Solution Injection Process 27
2.14 Using MATLAB Code to Determine the Resonance Angle 29
Chapter 3 Results and Discussions 30
3.1 PWR/SPR Combined Chip 30
3.1.1 Simulation Results of Different Grating Size 30
3.1.2 Simulation Results of Different Etching Depth 32
3.1.3 The Independent Feature of Two Resonance Peaks 33
3.2 The Examination of Parameters through Different Group of Chips 36
3.3 Optical Microscopy 37
3.4 Sensitivity Test 40
3.4.1 Optimizing Substance Deposition Pparameters 41
3.4.2 Optimizing Annealing Parameters 42
3.4.3 Optimizing Exposure Energy and Development Time 44
3.4.4 Optimizing Etching Time 47
3.5 Fluorescence Microscope Imaging and Fluorescence Recovery after Photobleaching (FRAP) 49
3.6 Angle Scanning Mode 51
3.6.1 Minimizing the Laser Spot Size to Avoid Multiple SPR Resonance Peaks 51
3.6.2 Comparison of Positions with and without Membrane 55
3.6.3 The Variations between Different Detecting Positions and Chip Samples 62
3.7 Real Time Mode 64
3.7.1 Simple Diffusion 64
3.7.2 Transport Kinetics 65
3.8 Filtering and Smoothing the Detected Signal by MATLAB 66
3.9 Proposed Mechanism of Glucose Transport across the GPMV Membrane 67
3.10 Nonlinear Fitting Method 70
Chapter 4 Conclusion 75
Chapter 5 References 77
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