研究成果:Using 1-D Spatially Resolved Silicon-Nanowire-Field-Effect-Transistor (SiNW-FET) Platform to Characterize Cell Membrane Proteins in Their Native Bilayer Environment.

 

建立分析膜蛋白的仿生膜檢測平台
Using 1-D Spatially Resolved Silicon-Nanowire-Field-Effect-Transistor (SiNW-FET) Platform to Characterize Cell Membrane Proteins in Their Native Bilayer Environment.

膜蛋白為目前主要藥物標靶和發展新穎治療方式的研究對象,能夠快速檢測出能與目標膜蛋白結合之配體可有助於新藥發展。現今用於檢測配體結合的主流方式為螢光免疫染色法,然而,螢光標記方法很有可能會改變生物分子交互作用,而造成不正確的檢測結果。本計劃利用場效電晶體技術,來發展可偵測膜蛋白和配體結合反應之無標記檢測法。我們選擇具有二維結構之石墨烯作為場效電晶體半導體材料,以方便和二維支撐式脂質雙層膜做結合。我們發現石墨烯表面高分子殘留物會干擾脂質膜形成,需用高溫淬火將之移除來使得脂質膜能容易形成。此外,我們也發現石墨烯上方脂質膜螢光幾乎會完全被石墨烯猝滅,無法以傳統螢光恢復法來驗證脂質膜的形成,因此本計畫開發一種新方法來測試脂質膜形成狀況,以找尋適合的成膜條件。我們更進一步發現緩衝液中的離子會逐漸吸附到石墨烯表面,而導致石墨烯的狄拉克點會在配體結合檢測期間隨時間顯著偏移。透過仔細控制微流體裝置中緩衝液添加量和流動持續時間,我們得以去除由離子吸附引起的效應。我們使用鏈黴親和素與具有生物素之脂質作為模型,來測試配體結合在本平台上產生的效應,結果顯示本平台的確能定量地檢測到配體與脂質膜上物質結合。將此計畫發展出的平台進一步與我們由大腸桿菌所得到之細胞膜囊泡做結合,將可使感興趣之膜蛋白在其天然脂質雙層環境中來進行無標記檢測。

Cell membranes are the gateway for the cells to send and receive signals. More than 60% of commercial drugs interact with membrane proteins to alter cellular biochemical pathways. Since membrane proteins are primary pharmaceutical targets, information about what ligands can bind to them is useful to facilitate drug discovery and development. Most of the current methods to identify the ligand binding to membrane proteins rely on immunodetection. However, immunodetection methods may alter biomolecular interactions. Here, we develop a label-free detection method based on field effect transistor (FET) technique, which can provide rapid and sensitive responses to the ligand binding. Supported lipid bilayers (SLBs) have been widely used as platforms for studying interactions between ligands and membrane proteins in vitro. To build a FET detection platform combined with SLBs, we chose graphene as the FET semiconductive material because the two-dimensionality is compatible with the planar bilayer platform and it has high sensitivity. We fabricated graphene field effect transistor (GFET) on silica substrates and investigated the formation of SLBs on the graphene/silica substrate. We discovered that the poly(methyl methacrylate) (PMMA) residues left on graphene surface can significantly influence the formation of SLBs on graphene and applying an extra annealing step to further clean the graphene surface is required for the robust SLB formation. In addition, because the fluorescent dyes in the SLB above graphene were almost completely quenched by graphene, which hinders us from using the conventional fluorescence recovery after photobleaching (FRAP) method to verify the formation of SLBs, we developed a modified FRAP method to examine whether SLBs successfully formed on the graphene/silica substrate. Furthermore, we discovered that ions in a regular buffer can gradually adsorb to the graphene surface and cause the Dirac point to shift significantly with time during our ligand binding detection. With careful control of the history of buffer addition and flow duration in a microfluidic device, we were able to eliminate the response caused by the ion adsorption and obtained the response only from the ligand binding. We used streptavidin-biotin-lipid as a model ligand-receptor to examine the GFET response to ligand binding on the SLB. Our results show that the SLB-GFET is capable to detect the streptavidin molecules binding onto the biotin-lipids embedded in the SLB, and the consistency between the electrical response and the fluorescent quantification result shows the quantitative detection capability of our GFET device. Incorporating this platform with the technique to obtain membrane vesicles from E. coli could allow us to study the interactions between potential drug candidates and interested membrane proteins in their native-like environment.