可配合原子力顯微鏡量測之非對稱脂質雙層膜成膜平台的建構

Abstract

脂質雙層膜在生物體中普遍存在,其存在對細胞功能來說至關重要。自然界的脂質雙層膜大部份以非對稱形式存在,例如人體中紅血球的細胞質膜。此類脂質雙層膜更加類似原生細胞 膜,使細胞上成分,如膜蛋白,在其上作用時更能貼近原始生物體。 本論文所提出之人工脂質雙層膜成膜系統是以水膠與液珠表面自發形成的脂質單層膜接 觸形成雙層膜。水相液珠在油相環境內成膜避免液珠的揮發,為加強所形成的脂質雙層膜之機 械強度,以水膠作為支撐介質使雙層膜的穩定時間延長。以此系統先行建構對稱脂質雙層膜, 利用倒立螢光顯微鏡進行光學觀察,膜片箝制放大器量測雙層膜電訊號,探討此系統成膜成功 率及膜穩定性和影響因素。實驗結果此系統成膜成功率達 80%,且利用顯微鏡成功記錄脂質雙層膜的形成與持續時間,最長膜壽命記錄為 15 分。利用氯化銀電極控制液珠接觸水膠形成脂 質雙層膜,膜片箝制放大器量測成膜過程,脂質雙層膜形成至膜破裂的電容值變化,得膜壽命 約 1 分鐘,單位面積電容值為 0.32 μF/cm2。實驗發現水膠的平坦度以及表層水厚度會對成膜成功率造成影響,而由光學及電訊號膜壽命差異可知電極插入液珠與否也會影響脂質雙層膜的穩定。 未來將改變液珠上單層膜分子種類,形成非對稱脂質雙層膜。也將置入細胞膜蛋白,量測 是否有離子通道電訊號形成,同時也結合 AFM 原子力顯微鏡,達到更多樣化、多元化的細胞 膜蛋白特性量測,以達到穩定性高且泛用度廣之人工脂質雙層膜平台。

Lipid bilayer membrane is very common in organism, and it is a very important component of a biological cell. Most of lipid bilayer membranes are asymmetric, such as the membrane of erythrocyte. The properties of artificial asymmetric bilayer are more like the original cell membrane. When protein or some cell components work on it, the behavior of the component will more close to biological organism in nature. This lipid bilayer formation system is based upon connecting aqueous volumes immersed in a hydrophobic phase containing lipids. Briefly, a lipid monolayer self-assembles at the water-oil boundary of the aqueous components, and when two components are brought into contact, a bilayer spontaneously forms between them. This formation system and method have several advantages. Full-oil-phase environment prevent aqueous droplet evaporation in oil chamber. To increase the lifetime of bilayer, hydrogel-supporting design makes bilayer strong. In experiments, the symmetric bilayer has been formed to discuss the factors that might afford stability and lifetime of bilayer. In bilayer formation experiment with optical image, the optical image was viewed by using inverted fluorescence microscopy, and formation success rate is 83%. The formation and lifetime of bilayer recorded in video clip through CCD. The longest lifetime of bilayer is 15 minutes. The electrophysiological recording measures by using patch clamp. The electrophysiological signal was detected through all formation timeline, and the longest lifetime of bilayer is about 1 minute. Bilayer capacitance per area is 0.32 μF/cm2. The lipid bilayer formation system will soon to be use to form asymmetric bilayer. Further more, membrane also will insert into the device to record ion channel signals, this can make sure the feasibility of the system. The final goal is that using atomic force microscopy to measure the dynamic situation when proteins are working, quantify the values of protein’s displacement