使用物理障礙發展不受空氣-水界面破壞之磷脂雙層膜平台

Abstract

支撐式脂雙層膜平台具有可以保存生物分子的結構，以及允許生物分子能夠在平台上自由移動的特性，因此被認為可與各式表面分析技術結合，來發展成絕佳的生物檢測平台。然而，傳統的支撐式磷脂雙層膜在暴露於空氣-水界面後會受到破壞且失去其結構的完整性，限制了其廣泛的應用。在本論文中，有別於以往文獻所用之化學修飾方法，我們創建了獨特的物理障礙，以發展不受空氣-水界面破壞的磷脂雙層膜平台。這些物理障礙能夠攔截水層於障礙間的脂雙層膜上以避免空氣-水的界面張力直接作用於雙層膜而造成破壞。第一部份的研究中，我們使用磷脂酶A2在膜上水解反應生成的障礙來做為系統中的物理障礙。由於這些物理障礙具有抗洗滌劑與強力貼附在基材表面的性質，允許我們在利用洗滌劑清洗反應完的磷脂酶和脂質後，能夠將欲組成的脂雙層膜形成於障礙之間。在螢光顯微鏡下，我們直接觀察到空氣-水界面通過後，水層被截於障礙間並保護脂質膜受破壞的情形。螢光漂白回復測試結果也驗證出，受物理障礙保護的雙層膜在通過空氣-水界面後又回到水溶液環境下，仍保有原本不受影響的流動性。在第二部分的研究中，我們使用光顯影製程來形成光阻物理障礙，以避免脂雙層膜在生物檢測之試劑交換時受到空氣氣泡的破壞。藉由改變物理障礙的間距和微流道系統中的操作流速，我們進一步發現當空氣-水界面移動速度增加時，需要更短的物理障礙間距才能達到良好的保護雙層膜效果。螢光漂白回復測試方法也同樣顯示出，受光阻物理障礙保護的雙層膜在回到水溶液環境下仍保有與通過空氣-水界面之前相當的流動性。另外，鏈霉抗生物素蛋白與生物素脂質分子間鍵結能力測試，也顯示出被物理障礙保護的雙層膜上的受體能和配體反應的能力亦不受空氣-水界面而影響。本研究顯示我們能成功使用物理障礙的方法以避免脂雙層膜被空氣-水界面破壞，進而保存其完整性與流動性。此法突破了先前相關技術均會降低雙層膜流動性及造成空間障礙的問題，更進一步提升本技術在生物感測平台上的應用潛力。

Supported lipid bilayers (SLBs) have been thought as desirable platforms for various biosensing applications. The bilayer structure allows the embedded membrane species to maintain their native orientation, and the two dimensional fluidity is crucial for numerous biomolecular interactions to occur. However, the lipid bilayers easily delaminate and lose their natural structure after being exposed to air-water interface. Here, for the first time, we demonstrated using physical confinement instead of chemical modifications to create air-stable membranes. The physical confinement could trap some water above the lipid bilayers to prevent the air-water interface from directly contacting and peeling the lipid bilayers. In the first part of this thesis, the physical confinement was generated by the obstacle network induced by a peripheral enzyme, phospholipase A2 (PLA2). The enzyme and reacted lipids can be washed away from the obstacle network which is detergent-resistance and strongly bonded to the solid support. With the property, the obstacle framework on the solid support was reusable and lipid bilayers with the desired composition can be refilled and formed in the region confined by the obstacle framework. Fluorescence recovery after photobleaching (FRAP) results showed that the diffusivities of the SLBs before drying and after rehydration are comparable, indicating the air-stability of the physically confined membrane. In addition, we observed that the obstacles could trap a thin layer of water after the air-water interface passed through the SLB. In the second part of this thesis, we used patterned photoresist obstacle grating to protect SLBs from destroying by an air bubble. We varied the patterned obstacle distances and found that the grating geometry criterion was more restricted when the air-water interface moving speed was faster. The FRAP measurement from the unaffected confined SLBs showed that the fluidity remained unchanged after an air-bubble treatment. In addition, the interaction assay result from the streptavidin and biotinylated lipid in the confined SLBs suggested that receptors on the SLBs remained the interaction ability after air-bubble treatment. These results showed that the SLB platform physically confined by the obstacle grating structure can preserve not only the membrane fluidity but also the accessibility to the outside environment. Integrating with a device for reagent transport and exchange, this platform has a great potential to be applied with surface analytical tools to create more robust in-vitro cell membrane related bioassays in the future.