可配合原子力顯微鏡量測之雙層脂質膜成膜平台之開發

Abstract

在生物細胞當中，位於細胞膜上的膜蛋白對於細胞整體的健康扮演著重要的角色，由於膜蛋白的動態特性目前尚不清楚，因此一直是科學界重要的研究題目之一。本研究以達成開發配合原子力顯微鏡進行膜蛋白的動態量測的脂質雙層膜成膜平台為目標，提出了兩個成膜裝置設計，分別為液珠垂直式脂質雙層膜成膜平台以及液珠傾斜式脂質雙層膜成膜平台。在液珠垂直式成膜方法中雖然以光學量測觀察到脂質雙層形成的影像，但是由於電生理量測尚未成功且此方法的脂質雙層膜形成成功率大約20%，因此希望可以在垂直於水平面的脂質膜被形成後進行旋轉以配合原子力顯微鏡的量測而提出液珠傾斜式脂質雙層膜成膜平台。液珠傾斜接觸式成膜可以由液珠中液體的不同分為水性液珠接觸式成膜以及磁流體液珠接觸式成膜。水性液珠接觸式成膜方法中，水平成膜的脂質雙層膜維持時間最長達到3個小時且成功率大約為80%，且藉由量測在水性液珠當中混合溶血素後鑲嵌於脂質雙層膜所產生的離子通道電流確認雙層脂質膜的存在，此外還利用傾斜平台搭配電生理量測觀察到此系統可以在0∘至45∘的傾角範圍穩定形成脂質雙層膜。磁流體液珠接觸式成膜方法中，其中一顆水性液珠混合了磁流體且液珠中磁流體與水性溶液體積比為1：300，此系統成功以磁鐵非接觸式移動液珠以形成脂質雙層膜，並且也由插入的金屬電極得到形成脂質膜時的電容充放電訊號。

In this research, two designs of lipid bilayer formation method have been demonstrated for the goal of application of dynamically scanning membrane protein by atomic force microscopy. First, the hydrogel-droplet vertical bilayer formation system is utilized to form lipid bilayer. Although a bilayer is successfully formed by this system, there is no reasonable electrophysiology signal being measured and the lipid bilayer formation success rate is only 20%. It is not good enough for the application of atomic force microscopy dynamically scanning process. Besides, the oblique droplet interface bilayer formation system is also provided to lipid bilayer formation and the atomic force microscopy dynamically scanning. For the two aqueous droplets system, this method demonstrates lipid bilayer formation, membrane protein insertion and oblique lipid bilayer formation. In this system, success rate of the lipid bilayer formation is about 80% and the longest bilayer life time is about three hours. There is a step-liked current signal caused by the membrane protein insertion which provides an evidence of lipid bilayer existence. An oblique lipid bilayer formation experiment indicates that a droplet interface bilayer can be formed at 0∘to 45∘related to horizontal. In addition, this system provides a new method of droplet transporting. An aqueous droplet is mixed with magnetic fluid in a particular ratio, 1:300, so that the droplet can be transported by magnet rapidly before lipid bilayer formation. In this bilayer formation system, a charge/discharge signal of lipid bilayer capacitance is also been measured by inserting two electrodes into the droplets.