表面電位對於蛋白質吸附行為之影響

Abstract

細胞外基質會影響許多細胞的行為，如細胞貼附、細胞伸展等等，而細胞外基質中的各種蛋白質，像是纖維連接蛋白、層黏連蛋白及膠原蛋白，更是在其中扮演了非常重要的角色。藉由研究這些蛋白質在不同性質的表面上的貼附行為對於了解細胞在不同環境中的反應有很大的幫助。 由本實驗室之前的研究可以知道，藉由調控金基材上二元自組裝單層膜中表面官能基的胺基與羧基比例，可以得到一系列不同的表面電位。本研究利用石英振盪微天平（QCM-D）及6-胺基-1-己基硫醇和6-羧基-1-己基硫醇的自組裝單層膜分子修飾的鍍金石英晶片來探討表面電位對於細胞外基質蛋白質和基材間交互作用的影響。此外，實驗中使用磷酸緩衝溶液來控制環境中的酸鹼值及離子強度，並藉由電動力分析儀及動態光散射分析儀分別量測以二元自組裝單層膜修飾的金基材及蛋白質的表面電位。在吸附過程中，同時偵測石英晶片的共振頻率變化及能量消散，並利用黏彈性模型可計算得到表面的吸附重量變化。另外，藉由能量消散對共振頻率變化作圖，其斜率可讓我們得到吸附蛋白質之機械性質資訊，而此機械性質資訊也可利用原子力顯微鏡掃描得到的蛋白質吸附層之表面形貌相互印證。 本研究發現，縱然在表面都是帶負電性的狀況下，低電荷密度的蛋白質依然可以吸附在高電荷密度的自組裝單層膜表面上。雖然在巨觀條件下靜電作用是排斥的作用力，但在微觀條件下由於高電荷密度的自組裝單層膜將蛋白質極化產生偶極誘導偶極力，所以促進此吸附行為。另一方面，在較低電位的自組裝單層膜修飾之表面，蛋白質被極化的現象較不明顯，在靜電作用主導之下會發現其吸附量會降低。除此之外，分子間作用力也會使蛋白質聚集成不同的結構，進而影響吸附後的表面形貌及其吸附速率。

Extracellular matrix (ECM) proteins such as fibronectin, laminin and collagen play an important role in many cellular behaviors including cell adhesion, cell spreading, etc. Understanding their adsorption behavior on surfaces of different nature is helpful for studying the cellular response to environments. It is known that by tailoring the chemical composition in binary amine and carboxylic acid terminated self-assembled monoalyers (SAMs) modified gold substrate, different surface potentials can be obtained. To examine how the surface potential affects the interaction between ECM proteins and the substrates, a quartz crystal microbalance with dissipation detection (QCM-D) using binary-SAMs-modified Au on quartz crystal is used. The ionic strength and pH are controlled by phosphate buffer solution at 37 °C and the zeta-potential of modified Au and protein is determined with electrokinetic analyzer and dynamic light scattering, respectively. During adsorption, the shift of resonate frequency (f) and the energy dissipation (D) are acquired simultaneously and the weight change is calculated using the viscoelastic model. The result reveals that the low charge-density protein can be adsorbed on highly charged SAM even both surfaces are negatively charged. This behavior is attributed to the highly charged SAM polarized the protein microscopically and the Debye interaction allows the adsorption although the macroscopic electrostatic interaction discourage the adsorption. For SAM-modified surface of moderate potential, proteins are not polarized and electrostatic interaction dominated hence less adsorption is observed. Besides, the intermolecular force that allows protein to self-assemble into macroscopic structure also lead to change in adsorption morphology and kinetics. This difference can also be identified from the D-f plot.