利用結合表面電漿共振與電漿波導共振之晶片研究膜蛋白傳輸現象

Abstract

能夠瞭解物質藉由膜上通道蛋白跨膜運輸的現象，對於生物醫藥開發等相關領域來說相當的重要。現今表面電漿共振現象常被應用於免標定的生物感測平台，主要利用光學折射率變化來偵測晶片表面生物分子吸附脫離的情況。為了延伸其應用，我們設計了一個結合表面電漿共振(SPR)與電漿波導共振 (PWR)的平台，並利用模擬驗證出此結構能表現出兩個用於偵測孔洞內部與外部區域濃度的特徵共振角。在本研究中，我們在奈米級厚度的金薄膜上鍍二氧化矽薄膜後，藉由蝕刻製造出微米等級的孔洞陣列，再鋪上由化學發泡法從海拉細胞(Hela cell)取出的含有蛋白質之細胞膜至平台上。其中，細胞脂質膜具有阻隔洞內與洞外物質擴散的功用，也能使通道蛋白質被保持在其原始環境中。蝕刻的孔洞則提供了類似細胞內部的空間，以讓傳輸的物質在洞內進行累積，藉由金薄膜配合雷射所產生的表面電漿共振現象(SPR)和在二氧化矽上方產生的電漿波導共振，可用來同時偵測孔洞內部與外部的物質濃度變化所造成的折射率改變。由於只有特定物質能通過對應之通道蛋白進行傳輸，因此，洞內之濃度變化能夠反映通道蛋白的傳輸行為。這些實驗證明此平台應可用於研究不同藥物對通道蛋白所造成之抑制或促進功效，以及可即時偵測通道蛋白之傳輸動態，在醫藥檢測及研發藥物等廣泛用途具有相當的潛力。

Surface plasmon resonance (SPR) is a powerful label-free and contact-free technique for chemical and biological sensing experiments. However, the traditional use of SPR instruments is for molecular interactions on the surface of metallic film. In order to extend the application of SPR, we integrated the concept of plasmon-waveguide resonance (PWR) and proposed the idea of PWR/SPR combined chip in order to measure the transport behaviors of cell membrane transport proteins. The PWR/SPR combined chip is composed of a silica layer with sub-micron sized pores on a thin gold film. The geometry allows us to use SPR to detect the refractive index change in the pore region, which is correlated to the target species concentration inside the pore, and PWR to simultaneously monitor the change of refractive index at the top silica surface. We deposited the giant plasma membrane vesicles (GPMV) derived from cells onto the PWR/SPR combined chip to construct the lipid membrane with the membrane proteins suspending over the sub-micron sized pores. Consequently, the detection area can be divided into two regions including the space inside the pores and the region above the grating structure. By using COMSOL simulation, we confirmed that this system allows us to simultaneously measure the change of refractive indices in the two regions across the lipid membrane. We experimentally demonstrated how the platform can be used to study how various inhibitors or ligands can influence the glucose transport through the corresponding membrane transport proteins (Glut1, Glut2) in Hela cell plasma membranes.