本研究以表面經過適當修飾改質之Halobacterium salinarium紫色細胞膜(purple membrane, PM)方向性地固定於ITO導電玻璃基板上，形成單層PM膜，並量測分析其光電訊號。首先，成功地針對嵌於PM內且具光驅動質子泵功能之細菌視紫質(bacteriorhodopsin, BR)之位於胞外側的Lys129殘基進行biotin鍵結之修飾或硫醇化。以對胺基具反應性的sulfo-NHS-LC-LC-biotin不論是對原生種(wild-type, WT)或突變種G241C PM進行修飾後，均可與帶有螢光分子之streptavidin結合，而觀察到紅色螢光訊號。但單獨就G241C PM而言，發現不論是以5 nm金奈米粒子或具硫醇基反應性的biotin衍生物皆無法與突變後之cysteine殘基之硫醇基順利反應，但添加高濃度的變性試劑於反應液中，則可稍微提高修飾率，可能此位置之硫醇基具有潛在的立體障蔽問題。不過，Lys129殘基已被硫醇化的WT PM則對於具硫醇基反應性的金奈米粒子與biotin衍生物分別有鍵結效果，意謂著Lys129處之屏蔽情形較Cys241處為低。最後，藉著光電響應分析，得知以沒用緩衝液調節酸鹼值的5%戊二醛溶液修飾帶胺基的ITO玻璃，所得晶片對streptavidin具有最佳鍵結效果，而且再固定化接有biotin分子之PM層後，可使受光激發後的光電流密度比未塗覆PM前之晶片高出4.5倍左右，證實BR光驅動質子泵功能之存在。此結果可作為未來製備可提高光電流密度之多層PM生物光電晶片研究之基石。

An unidirectional monolayer of Halobacterium salinarium purple membrane (PM), which had been differently chemically modified or mutagenized, was fabricated on conductive ITO glass through specific molecular interaction for photoelectric studies. First, the Lys129 residue of bacteriorhodopsin (BR), which is a photodriven proton pump protein embedded in PM, was successfully modified by either biotinylation or thiolation. The binding of fluorescently labeled streptavidin with either wild-type (WT) or G241C mutant PM, which had been each modified with amine-reactive sulfo-NHS-LC-LC-biotin, both yielded observable red fluorescent signals. Secondly, derivation of G241C PM was neither achieved by binding with 5 nm nanogold particles nor biotinylation with a thiol-active reagent, unless in the presence of high concentrations of denaturants for the latter case. Nevertheless, the same derivation treatments were both successfully achieved towards WT PM that had been thiolated on Lys129 of BR, suggesting that the steric hindrance on the mutated Cys241 residue is much more significant than that on Lys129. Finally, the photoelectric study revealed that 5 % glutaraldehyde prepared in deionized water without pH adjustment modified the amine–coated ITO glass best for the subsequent covalent binding of streptavidin. Further fabrication of the streptavidin-coated surface with a biotinylated PM led to a 5.5-folds increase in photoresponse, confirming the active function of the photodriven proton pump of BR. This study provides promising results for future preparation of unidirectionally multilayered PM photoelectric chips potentially with high photoresponse.

溫文興，"Bacteriorhodopsin突變蛋白之初步研究"，國立台灣科技大學化學工程研究所碩士論文(2004)

蘇志溫，"Bacteriorhodopsin生物晶片之光電響應"，國立台灣科技大學化學工程研究所碩士論文(2004)

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