在支撐式脂質雙層膜中傳輸、過濾、以及無標定偵測細胞膜物質

Transportation, filtration, and label-free detection of membrane associated species in supported lipid bilayers

胡書愷(Shu-Kai Hu)

指導教授 ： 趙玲

國立臺灣大學/工學院/化學工程學研究所/博士(2018年)

https://doi.org/10.6342/NTU201800995

摘要

細胞膜蛋白質掌管了細胞之間的訊息溝通與各項生理反應，許多疾病是因其功能失常而產生。現今有超過六成的藥物標靶目標為膜蛋白，瞭解其結構與功能，以及其與候選藥物之間的作用，對於藥物發展及疾病治療非常重要。然而，由於膜蛋白的兩性分子特性，將其從脂質雙層膜中取出往往會造成膜蛋白結構的喪失，造成後續研究上的困難。在本論文中，我們在支撐式脂質雙層膜平台上發展各式分離純化與測試之技術，來讓膜蛋白可以在它們原本的脂雙層中被研究。
首先，我們運用微流道來發展測量膜物質在支撐式脂質雙層膜中流動性的平台。水流力先前已被發現可被用來造成各式膜中物質在支撐式脂質雙層膜中的移動，然而其中之輸送現象的機制還尚未明白。主要原因是有別於一般水溶性物質單純在水中的運動，膜中物質的運動同時受到水以及膜兩種流體之流動所影響。並且，脂質雙層膜本身的流動情形也同時會受水流所影響，其複雜性造成了解析上的困難。我們利用許多已知結構的膜中物質，來測試其被水流推動所產生之速度以瞭解並歸納出適當模型。此模型將膜中物質在系統中的力平衡式，表達成膜中物質與系統中各式介質的相對速度乘上相對應的阻抗，以連結膜中物質大小與其速度的關係，有助於未來在分離過程中瞭解不同大小之膜中物質會被分開的程度。另一方面，為了避免微流道中層流所導致的流場分布造成分散現象，我們流場圖案化技術將脂質膜和待測試膜物質置於流道中間之流場較均勻處，以增加量測流動性時的準確性。實驗與理論模型一致的結果表示我們的模型可有效描述此複雜的輸送現象。
接著，我們在支撐式脂質雙層膜中加入可光固化的脂質分子為材料，建造出奈米等級的障礙物以改良膜平台，發展出二維膜中的填充床和篩網。原子力顯微鏡結果顯示奈米結構障礙物的密度可以被紫外光施加強度所調控。當膜物質流經填充床，膜物質的移動速度隨障礙物密度線性下降，使我們可有效的控制膜物質在膜中的流動性。另一方面，我們在可光固化膜中加入不可被光固化的脂質分子，並利用膜分相的現象創造出膜中篩網。藉由調整可光固化和不可光固化脂質分子之比例，篩網中的孔隙大小可以被控制，而將不同大小之膜物質進行過濾。
更進一步，我們將支撐式脂質雙層膜與石墨烯電晶體結合，發展出無標定檢測的平台以解決現今免疫染色法因為額外加入螢光分子而影響了生物分子彼此之辨識能力，而導致錯誤的判斷。我們發現以二氧化矽為基板的石墨烯電晶體在水溶液中不穩定的表現是因為石墨烯與二氧化矽基板中會形成約一奈米的水層。此水層扮演著隔絕層的效果，可以將二氧化矽帶給石墨烯的電洞參雜效應、電子散射、遲滯現象給大幅減弱，使我們能獲得高度穩定性且快速反應之電晶體特性，以利於生物感應器之應用。我們也發展了利用閘極電壓加速水層生成的方法。
此外，之前的研究對於式否能將支撐式脂質雙層膜形成在石墨烯/二氧化矽基板上仍舊有一些爭論，主要因為石墨烯擁有將周圍螢光熄滅的能力，導致常見的螢光判定支撐式脂質膜是否生成的方式無法被使用。為解決此問題，我們發展出改良式螢光漂白後恢復技術來驗證脂質雙層膜之存在。我們發現介於石墨烯與二氧化矽基板間的水層存在會妨礙支撐式脂質雙層膜形成。在搭建出脂質雙層膜於石墨烯表面上後，我們成功利用石墨烯電晶體量化地偵測到帶電物質吸附到膜上。由於偵測目標物質與石墨烯之間被脂質雙層膜分開了約五奈米的距離，使用比一般生理條件離子濃度還要低的緩衝液為環境以降低水溶液中的離子屏蔽效應是必要的。
在本論文所發展之新方法均是為了使膜蛋白能被保護在它們原始的脂雙層環境中，來進行分離純化以及量測其配體作用，以達到未來藥物篩選之目標。我們展示出水流能被利用為驅動力，在支撐式脂質雙層膜平台中對不同大小之膜物質進行分離，並利用改良過的平台以達成不同的分離需求。我們也發展出脂質膜-石墨烯電晶體來對膜物質相關配體作用做無標定檢測。未來我們將這些方法與含有感興趣膜蛋白的真實細胞膜做結合，進行藥物篩選開發等應用。

關鍵字

膜蛋白 ； 石墨烯 ； 藥物篩選 ； 細胞膜 ； 光固化 ； 脂質 ； 無標定檢測

並列摘要

Cell membrane proteins are vital in cellular communication, function, and health. In fact, the intention of more than 60% of drug targets is to interact with membrane proteins to alter cellular biochemical pathways. Understanding their functions, structures, and interactions with drug candidates are important in drug developments. However, cell membrane proteins are amphiphilic and have structures adapted to the cell membrane lipid bilayer environment, leading to the difficulty in maintaining their structures and functions after they are extracted from cell membranes for further characterization. In this study, we developed new methods to separate and characterize membrane-embedded species in supported lipid bilayers (SLBs), so that they can be studied in their native lipid bilayer environment.
First, we developed an effectively controlled SLB platform for examining membrane species mobility under in-lipid-membrane forced convection in microfluidic channels. Although hydrodynamic flows have been applied to transport membrane species in SLBs for separation purposes based on the concept of chromatography, the transport mechanism is still unclear. The major reason is that the membrane species are convected by not only hydrodynamic flow but also the surrounding fluid lipid membrane which is also influenced by hydrodynamic force simultaneously, resulting in the difficulty to analyze the transport mechanism. Therefore, we developed a model in the form of the applied driving force and the resistances that the membrane species encountered to predict the mobility. On the other hand, we used a microfluidic device for controlling the flow to arrange the lipid membrane and the tested membrane species in the desirable locations in order to obtain a SLB platform which can provide clear mobility responses of the species without disturbance from the species dispersion effect. The consistency between the experimental results and the model predictions suggests that our model based on lateral drag and sliding frictions between the species and the lipid leaflets can be used to describe the mobility of membrane species.
Second, we used a type of crosslinkable diacetylene phospholipids, DiynePC, to create “2-D barriers” in order to construct two conventional separation processes, chromatography and filtration, in SLBs. Our atomic force microscopy result shows that the nano-scaled structure density of the “2-D packed-bed” can be tuned by the UV dose applied to the DiynePC membrane. When the model membrane biomolecules were forced to transport through the packed-bed region, their concentration front velocities were found to linearly decrease with the UV dose, indicating the successful creation of packed obstacles in SLBs. On the other hand, we mixed uncrosslinkable lipids with DiynePC and used the phase segregation to constructed 2-D filters. The cutoff size of the filter can be manipulated by the lipid molar ratio to sieve different hydrophilic size membrane associated species.
Third, we combined graphene field effect transistors (GFETs) with the SLB platform in order to label-free detect ligand binding at the lipid membrane, since the current immunodetection methods may alter the biomolecular interactions. GFETs provide rapid responses with high sensitivity. We discovered the instability of the electrical performance of silica-based GFETs in aqueous environment because of the water intercalation between graphene and SiO2 substrate. The water layer can act as an isolated film which can significantly reduce the influence from SiO2, including the hysteresis phenomena. In addition, we developed the method to accelerate the formation of water layer for highly stable and fast response GFETs.
Furthermore, whether the SLB can form on graphene/SiO2 was still controversial because the fluorescence quenching effect of graphene. To eliminate the confusion, we developed a modified fluorescence recovery after photo-bleaching (FRAP) method to verify the existence of SLBs on graphene area in our system, and we found the water intercalation would hamper the formation of SLBs on graphene/SiO2 substrate. We successfully used GFETs to detect the molecule adsorption onto the SLBs formed above graphene channels. We successfully used GFETs to quantitatively detect the molecule adsorption onto the SLBs formed above graphene channels. Low ionic strength was required to reduce the screening effect for larger responses since the analytes and graphene were separated by SLBs.
The works in this thesis are intended to developed new methods to separate membrane proteins and detect the ligand binding to the membrane proteins for potential drug screening purpose. We used model membrane species to demonstrate that we can separate membrane associated species based on their hydrophilic sizes and hydrophobic sizes, incorporate conventional separation processes into lipid bilayer platforms to tune the separation efficiency, and detect the ligand binding at the lipid bilayer platforms with a label-free GFET technique. In the future, we will further combine these methods with cell membrane patches with membrane proteins from interested cells to achieve drug screening applications.

並列關鍵字

supported lipid bilayer ； membrane protein ； purification ； separation ； crosslinkable lipid ； graphene ； field effect transistor ； label-free ； biosensor

參考文獻

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