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標題: | 發展可用於生物感測應用之支撐式細胞膜平台 Development of Supported Cell Plasma Membrane Platforms for Biosensing Applications |
作者: | Po-Chieh Chiang 蔣伯頡 |
指導教授: | 趙玲 |
關鍵字: | 奈米柵欄結構,支撐式細胞膜,細胞膜-石墨烯電極,電生理應用,生物感測應用, nano-grating structured support,supported plasma membrane,biomembrane-Graphene,electrophysiological applications,biosening applications, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | 發展出能具有天然跨膜蛋白的支撐式細胞膜平台,對於研究膜蛋白會很有幫助。本論文的目的在於製造各式適合脂質膜形成之奈米結構基材,發展可將細胞基質膜由細胞取下並破裂形成支撐式細胞膜平台之方法,以及將支撐式細胞膜和石墨烯電晶體結合以偵測細胞膜中離子通道的電性質。
在本論文的第一部分,我們利用奈米壓印的技術,成功地製備出含有奈米柵欄結構的熔融石英基材,以獲得具有橫跨結構並且大面積的連續式支撐式脂質膜。然而,由於要直接用顯微鏡觀察在奈米尺度下之膜型態是困難的,因此我們開發了使用光漂白後的螢光恢復(FRAP)為基礎之技術來判斷實驗中得到的奈米級膜型態。另外,在金表面上形成具有流動性的脂雙層膜,可以使用在生物感測方面進行脂質-膜和相關生物分子的交互作用。然而,透過一般脂質囊泡熔合的方法,在金表面形成具有流動性的支撐式脂質膜仍是很有挑戰性的。因此我們建構一個具有奈米柵欄結構之金表面,其結構可促進脂質囊泡破裂形成含有流動性的支撐式脂質膜。我們從光漂白後螢光恢復的結果,可以觀察到第一層直接形成在金表面上的脂雙層膜並不具流動性,然而形成在金表面邊角之第二層脂雙層膜具有高流動性,這結果為我們提供一個可以透過調控結構上邊角密度來製造出在金表面上具流動性脂雙層膜的可能性。 在論文第二部分,我們專注在發展一個可以研究細胞膜蛋白的仿生物細胞膜環境的平台。到目前為止,要進行並研究細胞膜蛋白在原生脂質膜環境下仍然具有挑戰性。若能夠直接將從細胞取下,並將含有真實細胞膜蛋白的巨型細胞膜囊泡(GPMV),鋪在基材形成支撐式細胞膜,將可使細胞上的膜蛋白在相仿原生脂質膜環境中,來被各式表面分析技術來進行研究。然而,巨型細胞膜囊泡因為有具高含量的蛋白及高含量的膽固醇,並不是很容易破裂形成支撐式細胞膜。在本論文中,我們驗證了透過空氣水介面造成壓縮的處理,可以有效地造成巨型細胞膜囊泡破裂形成支撐式細胞膜,。而透過多次空氣水介面的處理,可以明顯的增加基材上鋪平細胞膜的覆蓋率。而未被細胞膜佔據的區域,我們則加入人工脂質囊泡填補,以形成連續的支撐式細胞膜。我們同時證明從海拉細胞(HeLa cell)取下的細胞膜平台,不僅含有原生的脂質,並且含有具有流動性的水通道蛋白(AQP3),此方法可便利地產生出具有連續性的細胞膜平台,使這些膜蛋白能夠在平台上被移動以及進行進一步生物感測上的應用。 在論文第三部分中,我們進一步將支撐式細胞膜與石墨烯電極做結合以進一步在平台上直接偵測膜蛋白的活性。在建構生物性-石墨烯電極平台時,首先要將巨型細胞膜囊泡有效地在石墨烯基材的表面打破並鋪成區塊狀細胞膜。我們發現利用改良式的鹼性(RCA)清潔步驟來清潔石墨烯表面,可以明顯地提升細胞膜在平台上的覆蓋率,而且經過改良式清潔步驟處理過的石墨烯基材,仍然保有其完整之電性質。能夠監測並記錄膜傳輸蛋白及離子通道的功能活性,可以為病理學和藥理學研究提供有幫助的資訊。目前通常是透過電生理量測技術來鑑定膜上傳輸蛋白和離子通道的活性。然而,傳統電生理量測系統操作不易,並且效率尚低。在此,我們利用發展出之細胞膜-石墨烯電極平台去記錄離子通道的電流,以達成電生理的檢測。此論文中,我們選用在藥物開發上極具重要性的鈉鉀離子泵(Na+/K+ ATPase)來作為我們研究的目標,並成功地顯示出我們可用發展出的平台來檢測鈉鉀離子泵的活性如何受其抑制劑影響。未來我們希望能利用此具便利性的細胞膜-石墨烯電極平台進一步探索藥物篩選的可能性,並簡化傳統電生理系統的量測。 Developing a supported plasma membrane platform to investigate transmembrane proteins has become important for protein structure identification. Providing a sufficient space between a membrane and a platform support can prevent exposed extracellular domains of an integral membrane protein from interacting directly with the solid support. In addition, it is important to incorporate transmembrane proteins into free-standing lipid bilayers. In the first part of this thesis, we created nano-grating structures on fused silica substrate by nano-imprinting technique in order to providing a free-standing large-area continuous planar SLB platform for various applications. In addition to the nanofabrication of the substrate, we developed a method to use the fluorescence recovery after photobleaching (FRAP) technique to distinguish experimental membrane states. The method allowed us to overcome the difficulty to observe the nanoscale membrane states from a fluorescence microscope. In addition, forming fluid supported lipid bilayers on a gold surface can also enable various lipid-membrane-associated biomolecular interactions for biosensing detection. However, forming fluid SLBs on a gold surface through lipid vesicle deposition continues to pose a challenge. we constructed nano-grating structures on a gold surface to facilitate lipid vesicle rupture for forming a mobile layer of SLBs. Observations based on fluorescence recovery after photobleaching showed that SLBs on the prepared grating supports had some fluidity, suggesting that a second layer of SLBs partially formed on top of the first layer in contact with the gold surface and extended along the grating structure. This provided us with an opportunity to pattern mobile lipid bilayers on gold surfaces by controlling the edge densities of the structures. In the second part of this thesis, we focused on creating a platform to study cell membrane proteins in a cell membrane-mimicking environment. Until now, it remained a challenge to process and study cell membrane proteins in their native lipid membrane environments. Being able to obtain cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes can allow membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Therefore, we demonstrated the possibility that using compression generated by air-water interface can efficiently rupture GPMVs to form supported membranes with native plasma membrane proteins. The GPMV patch coverage was significantly increased by applying multiple air-water interface treatments. The region of the support which was not covered by GPMV patches can be filled with artificially prepared lipid vesicles to form a continuous supported membrane. We demonstrated that not only lipids but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating a continuous supported membrane platform with mobile native transmembrane proteins can not only facilitate the study of membrane proteins by surface analytical tools, but also enable us to use native membrane proteins for biosensing applications. In the third part of this thesis, we integrated the GPMV membrane and graphene electrode as a biomembrane-Graphene system. In the process of building this biomembrane-Graphene platform, it was challenging to efficiently rupture GPMVs to form membrane patches on the graphene surface because the graphene surface can be easily contaminated. We followed a modified RCA (Radio Corporation of America) cleaning process to treat the graphene surface and successfully deposited a significant number of GPMVs on an intact graphene support. Being able to monitor and record the functional activities of membrane transporters and ion channels can provide useful information for pathologies and pharmacological studies. A reliable detection method is to identify their electrical activity by electrophysiological techniques. However, electrophysiological measurements are complicated and low throughput. Herein, we demonstrated this biomembrane-Graphene platform as a potential electrophysiological detection platform. In addition, we further explored the possibility of drug screening by using this biomembrane-Graphene platform. We chose a sodium-potassium ion pump (Na+/K+ ATPase) as our target as it is an important candidate for drug development. We demonstrated the activity of the ion pump can be detected in the absence and presence of a specific ion channel blocker on this biomembrane-Graphene platform. We envision that this relatively convenient biomembrane-Graphene system can be used for simplified electrophysiological detection, and can be widely used in biosensing and electrophysiological applications. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77817 |
DOI: | 10.6342/NTU201800826 |
全文授權: | 有償授權 |
電子全文公開日期: | 2023-06-08 |
顯示於系所單位: | 化學工程學系 |
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