臺灣博碩士論文加值系統

由於支撐式脂雙層膜系統平面的結構非常適合用在許多表面分析工具上，因此已經被廣泛地使用在膜蛋白與脂質膜特性的研究之中。然而，介於脂雙層膜與基材之間存在著相當敏感的平衡交互作用，可是仍有許多地方卻還未被了解。在這本論文中，我們使用extended-Derjaguin-Landau-Verwey-Overbeek (DLVO)理論以及其他相關的理論來估計脂雙層膜在不同條件下的能量分布，預測形成我們有興趣的脂雙層膜型態的實驗條件，以此呈現我們的實驗結果與理論模型的預測是具有一致性的。
在本論文第一段的研究的目標主要是在具有奈米柵欄結構的基材上形成自由態(Free-standing)的脂雙層膜。可是相較於自由態的情形，貼附態(Contour-following)的脂雙層膜卻是大家比較容易觀測的到的，說明了形成自由態脂雙層膜的條件是較為嚴苛的，且需要更多的訊息來了解適當的形成條件。因此這裡的目標是透過估計系統在自由態或貼附態情形條件下的總能量，並以此決定各個實驗條件下哪種狀態的脂雙層膜是較為穩定的。對脂雙層膜與基材互動的情形來看，當脂雙層膜與基材貼附的越靠近能夠得到較多的親和力能量上的回饋，然後當脂雙層膜需要藉由彎曲才能與基材構形狀貼附的話，反而會需要付出能量使其彎曲，所以是否能量能夠得到回饋或是需要付出能量決定了脂雙層膜的型態。我們使用DLVO理論和Helfrich型變理論連結總能量大小與各個實驗條件之間的關係，透過實驗上調控溶液的離子強度、使用不同種類的脂質、和三種不同形狀的基材來驗證理論預測的正確性。由於實驗上較為困難直接觀察奈米尺度下脂雙層膜在基材上的型態，我們發展出藉由觀察螢光漂白(Fluorescence recovery after photobleaching，FRAP)後的形狀改變的方法，來觀測脂雙層膜在奈米柵欄結構的基材上的型態為何。我們的實驗結果與理論預測具有相當好的一致性，表示我們發展的模型能夠預測形成自由態脂雙層膜所需的條件。
在第二段的研究中，主要是針對如何提高支撐式脂雙層膜在黃金基材表面上流動性。由於許多像是表面電漿共振儀(Surface Plasmon Resonance)等的重要分析工具需要黃金表面作為其應用技術的平台，可是由於脂雙層膜與黃金基材具有相當強的親和力，導致脂雙層膜在黃金表面的流動性大幅下降，進而限制支撐式脂雙層膜在表面電漿共振技術上的應用。為此我們使用DLVO理論與Lifshitz理論計算脂雙層膜與黃金的親和力大小，發現脂雙層膜與黃金基材間強親和力無法使脂雙層膜以特定的水層距離隔開於基材表面來維持其流動性。我們在形成於黃金基材表面上的第一層脂雙層膜上再形成流動性較好的第二層脂雙層膜以避免第二層脂雙層膜與黃金基材表面的直接接觸。我們也預測出第二層脂雙層膜能穩定的存在於系統中。實驗上，為了能夠讓第二層脂雙層膜能夠較容易的形成於在黃金表面，我們在黃金基材表面創造奈米柵欄結構，以幫助脂雙層膜囊胞破裂來形成脂雙層膜。透過FRAP對脂雙層膜流動性的分析，以及比較單層脂雙層膜螢光亮度，我們觀測出具有高流動性的第二層脂雙層膜出現在於系統中並覆蓋60% 的基材表面。此外，COMSOL之模擬結果也和以上的觀察結果相符合。這些現象均指出第二層脂雙層膜能夠自發性地形成於具有奈米柵欄結構的黃金基材表面，並且同時保持脂雙層膜的流動性，使得支撐式脂雙層膜在表面分析上將有更廣泛地應用與可能性。

Supported lipid bilayers (SLBs) have been widely used to study protein-lipid membrane interactions because their planar geometry is suitable for many surface analytical tools. However, there exists a delicate interaction balance between the support and the SLB, and the interaction is still not fully understood. In this thesis, we used the extended Derjaguin-Landau-Verwey-Overbeek (DLVO) theory and other relevant theories to estimate the energies of various membrane states and predict the conditions required for the interested membrane states to occur. We also showed that our experimental results are consistent with the model predictions.
In the first part of this study, we intended to create free-standing SLBs on nano-structured supports for biosensor applications. However, membranes following the support surface contour are more frequently observed than are free-standing membranes on structured supports, indicating that the parameter range suitable for the formation of free-standing SLBs might be narrow and more information is necessary to understand the required conditions. The objective was to estimate the system energies of free-standing and contour-following membrane states, and determine which state is the more energetically favorable under various conditions. For a lipid membrane preferring to stay close to the support, an energy reward occurs when they are in close proximity; however, increasing the contact area on a structured surface can result in an energy penalty because of the bending of the lipid bilayer. Whether the energy reward or the energy penalty dominates could determine the membrane state. We used the extended Derjaguin-Landau-Verwey-Overbeek (DLVO) theory and the Helfrich bending theory to relate the energy sizes to experimentally controllable parameters. We experimentally examined whether the membrane state followed the model prediction when we used various buffer ionic strengths, various lipid types, and nano-grating supports with three different geometries. Because it is difficult to observe the experimental membrane state directly at the nanoscale, we developed a method to use the fluorescence recovery shape change after photobleaching to distinguish experimental membrane states at the micron-scale. Our experimental results closely matched the theoretical predictions, suggesting that the developed model can be used to predict the suitable conditions for the formation of free-standing bilayers on nano-structured solid supports.
In the second part of this study, we intended to enhance the fluidity of the lipid membrane on the gold surface. The gold surface is required for many powerful surface analytical tools such as surface plasmon resonance (SPR). However, the SLB on the gold surface has found to have poor fluidity probably because of the strong membrane-support affinity, which impedes the biosensing applications of the SLB platform. By using DLVO theory and Lifshitz theory to calculate the membrane-gold total interaction energy, membrane-gold affinity is too strong to keep a certain lubricant water layer between them. We found out that it is possible to form a second lipid bilayer on the first lipid bilayer contacting the gold surface. Compared with the first bilayer having a strong membrane-gold affinity and lack of a lubricant water layer for membrane fluidity, the calculation shows that the second bilayer has a water layer between itself and the first bilayer and could have a significant fluidity. However, we found that it is experimentally difficult to form a second bilayer by the conventional vesicle deposition method because of the slow kinetics of the vesicle rupture. We created a nano-grating structure on the gold surface to enhance the lipid vesicles to rupture. We examined the membrane fluidity by FRAP and the formation of a second bilayer by comparing the fluorescence intensity with the intensities of some standard samples with known membrane states. The fluorescence intensity result showed that the second bilayer was spontaneously formed on the Au-coated nano-grating support and covered around 60 % surface area. The COMSOL simulation result also supported the formation of the second bilayer and the surface coverage ratio. These results all showed that the nano-gating geometry could facilitate the formation of the mobile second lipid bilayer on the gold surface, which has a great potential to be incorporated with some surface analytical tools to study interested biomolecular interactions.

誌謝 i
摘要 ii
Abstract iv
Figure Captions xi
Table Captions xiii
1. Introduction 1
1.1. Supported Lipid Bilayers 6
1.2. Nano-imprinting Lithography 11
2. Theories 13
2.1. Extended-DLVO Theory to Describe Membrane-support Interaction 13
2.2. Lifshitz Theory to Describe van der Waals Interaction 15
2.3. Helfrich Elastic Bending Theory to Describe the Membrane Bending 18
3. Materials and Methods 20
3.1. Materials 20
3.2. Apparatus 21
3.3. Lipid Preparation 22
3.4. Sample Fabrication 22
3.4.1. Nano-grating Structured Fused Silica Fabrication 22
3.4.2. Au-coated Nano-grating Structured Fused Silica Fabrication 23
3.4.3. SEM Measurement of Nano-grating Structured Fused Silica 25
3.4.4. Polydimethylsiloxane Well Fabrication 25
3.4.5. Formation of Supported Lipid Bilayers and Substrate Cleaning Procedure 26
3.5. Fluorescence Photobleaching After Recovery 26
3.6. Controllable Occurrence of Free-standing Supported Lipid Bilayers on Nano-grating Fused Silica Supports 27
3.6.1. Obtaining Dx and Dy by 2-D FRAP Fitting by Matlab 27
3.6.2. Using COMSOL Software to Simulate Fluorescence Recovery Situations for Free-standing (FS) and Contour-following (CF) State Membranes 30
3.6.3. Details of Obtaining ESLB-solid Support 32
3.7. Spontaneous Formation of Multi-bilayers on Gold Supports Induced by Surface Structure 43
3.7.1. Membrane-Support and Membrane-Membrane Interaction Energy 43
3.7.2. Normalization of Fluorescence Intensity to Obtain the Amount of Lipid Bilayers Depositing on the Solid Support Surface 51
3.7.3. Obtaining Dx and Dy by 2-D FRAP fitting 53
4. Results 69
4.1. Controllable Occurrence of Free-standing Supported Lipid Bilayers on Nano-grating Fused Silica Supports 69
4.1.1. Total Energies of Various Membrane States on a Trenched Solid Support 69
4.1.2. Obtaining Theoretical Contour-following and Free-standing State Regions for the Prepared Supports 71
4.2. Spontaneous Formation of Multi-bilayers on Gold Supports Induced by Surface Structure 75
4.2.1. Theoretical Prediction of the Supported Multi-Bilayers Formation 75
4.2.2. The Mechanism to Form the 2nd Bilayer on the Gold Surface with Grating Structure 76
4.2.3. Effect of Au-Coating Thickness on Hamaker Constant 78
4.2.4. Using Fluorescence Intensity to Examine the Membrane Conformation 79
4.2.5. Significant SLB Fluidity on Au-coated Nano-grating Support but not on Au-coated Flat Support 81
5. Discussions 83
5.1. Controllable Occurrence of Free-standing Lipid Membranes on Nano-grating Structured Supports 83
5.1.1. Examining Experimental Membrane States by Using 2D Fluorescence Recovery After Photobleaching (FRAP) Measurement 83
5.1.2. Experimental Membrane States Influenced by Bending Constant, Ionic Strength, and the Nano-grating Structure 85
5.1.3. Comparison of the Experimental Membrane States and the Model Predictions 86
5.2. Spontaneous Formation of Multi-bilayers on Gold Supports Induced by Surface Structure 90
5.2.1. Using COMSOL Software to Simulate Fluorescence Recovery Situations for Lipid Membranes on the Au-Coated Nano-Grating Support 90
5.2.2. Explanations of why the 2nd bilayer is more easily to from on the gold surface than on the fused silica 91
6. Conclusion 96
6.1. Controllable Occurrence of Free-standing Lipid Membranes on Nano-grating Structured Supports 96
6.2. Spontaneous Formation of Multi-bilayer on Gold Supports Induced by Surface Structure 96
7. Appendix I: Fitting Results of Dx/Dy Ratio of Different Types Lipid and Ionic Strength Conditions 98
8. References 109

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