發展同時具有表面電漿共振以及電漿波導共振之微結構晶片以研究葡萄糖之跨細胞膜傳輸現象

Abstract

研究膜上通道蛋白質進行跨膜運輸的相關現象對於了解生物機制及應用扮演著重要的角色，而現今在研究通道蛋白運作時(尤其在離子的遷移)主要使用的工具為膜片箝制(Patch Clamp)技術，此技術雖然已經發展得相當成熟、精確，然而卻需要經過高強度訓練和經驗豐富的操作人員來操作，以及需要相對高昂的設備成本的門檻。我們希望能在無需使用螢光或放射物等標記的前提下，建構出一個能提供通道蛋白運輸物質總量之動態資訊的技術平台。 我們在本實驗中提供兩種平台: 具蝕刻結構之表面電漿共振平台以及電漿波導/表面電漿共振混合模態平台。兩種平台均具有微米等級的二氧化矽厚膜蝕刻陣列結構，以提供脂質膜將未被蝕刻的區塊作為支撐基點，而在此結構上形成跨孔洞型態之脂質膜，以區隔孔洞內部空間以及蝕刻結構外部的空間。具蝕刻結構之表面電漿共振平台為在將石英玻璃基材蝕刻後，於微米等級的孔洞底部沉積薄膜金層，以表面電漿共振模態(Surface Plasmon Resonance)來偵測孔洞內部空間之物質濃度累積變化。至於電漿波導/表面電漿共振混合模態平台，則是可容納兩種電磁波共振模態同時存在，即表面電漿共振模態及電漿波導共振模態(Plasmon-waveguide Resonance)。在此蝕刻的結構中，孔洞底部鍍有裸露的薄膜金以作為表面電漿共振的介質，用以偵測孔洞內部空間之物質累積變化；未被蝕刻的區塊一方面作為脂質膜的支撐基點，一方面由其特定厚度之二氧化矽和底部金膜形成電漿波導共振模態的存在介質，用以偵測脂質膜上的吸附現象以及膜外溶液之改變。 本次實驗我們將海拉細胞(Hela cell)的細胞膜鋪於這兩種特殊結構的平台，來示範葡萄糖通過葡萄糖運輸蛋白質(Glut 1 and Glut2)的運輸行為，以及相對應之藥物 (細胞鬆弛劑及根皮素) 可如何調控其運輸行為。我們期待可將此平台推廣至可動態監控不同種類之通道膜蛋白運送物質的機制，以及配體(Ligand)和藥物對於通道蛋白質的結合現象。

Studying species transport across lipid membranes by membrane transport proteins is important for various biological applications. Although patch-clamp technique is well developed for recording the ion transport across lipid membranes, the technique requires well trained personals for the challenging and delicate operation. In this study, we created two types of platforms: grating structured SPR chip and PWR/SPR combined chip. Both types of chips have sub-micron sized grating pore array structures to allow lipid membranes to span over the pores and separate the space inside the pore from the outside environment. In the grating structured SPR chip, the gold film was only coated at the bottom of the pore and only the change of refractive index in the pore region close to the gold surface can be sensed by the surface plasmon resonance. The change of refractive index can be correlated to the target species concentration change and therefore the chip can be used to detect how much target species is transported into the pore region across the lipid membrane. The structure of the PWR/SPR combined chip is similar to the one of the grating structured SPR chip, and the only difference is that the gold film was not only at the bottom of the grating pore but also below the grating top region composed of the silicon dioxide layer. The geometry of the PWR/SPR combined chip allowed us to use the surface plasmon resonance (SPR) to detect the refractive index change in the pore region, which is correlated to the target species concentration inside the pore, and the plasmon-waveguide resonance (PWR) to simultaneously monitor the change of refractive index at the top silica surface, which is correlated to the binding events occurring on the lipid membrane surface. Here, we used giant plasma membrane-derived vesicles (GPMV) from Hela cell to span across the sub-micron sized pore structure to demonstrate how these two platforms can be used to study the glucose transport through the corresponding transporters (Glut 1, Glut2). In the future, we plan to use these platforms to monitor how various inhibitors or ligands could influence the transport dynamics of interested membrane transport proteins.