Formation of Supported Lipid Bilayer on Unconventional Supports by Tuning Lipid Bilayer Properties and Support Affinity
Date Issued
2014
Date
2014
Author(s)
Peng, Po-Yu
Abstract
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.
Subjects
支撐式脂雙層膜
奈米柵欄結構基材
柵欄結構懸掛式脂雙層膜
自由態
貼附態
extended- DLVO理論
Type
thesis
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