視紫紅質用於ITO表面修飾及與共軛高分子間的能量轉移研究

Abstract

從嗜鹽古細菌的細胞膜上發現的細菌視紫紅質HmBRI之突變種HmBRI-D94N是一種由光驅動的氫離子幫浦。HmBRI-D94N內部的全反式retinal在吸收光能之後改變構形，進而使HmBRI-D94N依序放出和吸收一個氫離子。這個光敏感蛋白質可以在光照下瞬間酸化周遭環境，造成一個短時間的pH值變化。這在生醫領域裡可以做為感光裝置元件，藥物傳遞材料和偵測元件等應用。本實驗分成兩部分，第一部分為利用具有螢光性質的共軛高分子去改變HmBRI-D94N氫離子幫浦功能所需使用的驅動波長。HmBRI-D94N是吸收波長為550 nm附近的綠光而受驅動，結合會放出綠色螢光的共軛高分子，理論上可以在其他波長的光照下驅動HmBRI-D94N，延伸HmBRI-D94N的應用範圍。本實驗使用兩種高分子，poly(p-phenyleneethynylene)的離子性高分子PPESO3，以及poly[2,7-(9,9-dihexylfluorene)-alt-4,7-benzothiadiazole)] (PFBT)的奈米粒子。這兩種共軛高分子都具有藍光的吸收波段以及綠光的螢光波段，並且都有著吸收紅外光的雙光子吸收的性質。以高穿透跟低傷害的紅外光激發高分子，藉由螢光共振能量轉移(FRET)間接激發HmBRI-D94N為此實驗的目標。第二部分為氧化銦錫(ITO)導電玻璃的表面改質，目的是讓HmBR-D94N I能夠依附在導電玻璃之上。首先使用三乙酸基胺(NTA)跟鎳離子，跟HmBRI-D94N上的組氨酸標籤(histidine tag)形成錯合物。這方法雖然能成功固定HmBRI-D94N在ITO上，但不夠穩定，容易在水中解離。因此在形成錯合物之後，再用1-乙基-3-(3-二甲基氨基丙基)碳醯二亞胺 (3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine，簡稱EDC)跟N-羥基琥珀醯亞胺(N-Hydroxysuccinimide，簡稱NHS)使HmBRI-D94N跟NTA之間形成更穩定的醯胺鍵結。結合HmBRI-D94N的氫離子釋放能力跟ITO因為環境酸鹼值改變電動勢的性質，可以作成偵測蛋白質交互反應的元件。

A new kind of bacteriorhodopsin HmBRI was found in Haloarcula marismortui. HmBRI-D94N is a light driven proton pump, which is functioned by a series of conformational changes of the retinal inside the protein in the presence of light illumination. It can pump out a proton and change the pH value of environment in a very short time. HmBRI-D94N can be engineered for application for biotechnology, including molecular memory devices, light-triggered drug delivery, and a protein sensor. The first part of this study focuses on changing the wavelength of stimulating light to drive HmBRI-D94N, by introducing water-soluble conjugated polymer. The proton-pump functionality of HmBRI-D94N is triggered by absorbing primarily green light. Theoretically speaking, a polymer which can convert other wavelengths into green fluorescence should be capable of activating HmBRI-D94N by Förster resonance energy transfer (FRET). PPESO3 (conjugated polyelectrolyte) and poly(fluorine-alt-benzothiadiazole) (PFBT, water-soluble nanoparticles) are examined for this purpose. The results indicate that the FRET can take place between PFBT and HmBRI-D94N. The second part is the surface modification of indium tin oxide (ITO) glass by HmBRI-D94N, aiming at stably fastening HmBRI-D94N onto the ITO glass. A combination of nitrilotriacetic acid and nickel ion was employed to absorb HmBRI-D94N by establishing the coordination between polyhistidine-tag of HmBRI-D94N and nickel. This modification can be achieved; however, the coordination is not stable enough. HmBRI-D94N can be easily removed from the ITO surface by water and in the presence of imidazole‎. (3-(Ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine (EDC) and N-hydroxysuccinimide (NHS) were then utilized to build covalent bond between HmBRI-D94N and NTA. The corresponding device performs good stability against water and even imidazole, validating that HmBRI-D94N can be stably fastened on the ITO glass by our approaches.