臺灣博碩士論文加值系統

紫膜（purple membrane，PM）為Halobacterium salinarum細胞膜的一部份，其膜上的細菌視紫質（bacteriorhodopsin，BR）蛋白具有光電響應能力。本研究主要以AFM探討分別以活化avidin或與鍵結有biotin的氧化石墨烯（b-GO）之混合物為架橋時，在雲母片上固定化修飾有biotin之PM（b-PM）的差異。結果發現活化avidin架橋層原本的粗糙度為0.48 nm，在添加0.02 mg/mL b-GO形成複合架橋層後下降為0.35 nm；同時，架橋層的黏滯力也由未添加前的5.93 nN下降至4.81 nN。觀察b-PM層，b-GO添加使b-PM的塗覆由多層降為單層，且BR三聚體的間距由7.9 nm縮小至5.4 nm。進一步探討以相同製程將b-PM塗覆於ITO之晶片的微分光電流響應，發現添加b-GO後光電流密度由649 nA/cm2下降至417 nA/cm2；推測是因為ITO基材表面過於粗糙。此外，微量添加b-GO可提高脈衝雷射光電流響應的B1/B2比值，表示PM的固定化方向一致性提高。另一方面，藉著調整被papain水解之PM的共價鍵結固定化pH值，可製備不同定向的PM晶片。AFM分析發現，在中性pH鍵結時，PM膜內具坑洞，然而在酸性pH鍵結時，膜片內部有碎痕，顯示這兩種膜的固定化方向相反；同時這兩種PM膜內部BR三聚體的間距分別是5.1和5.5 nm。探討共價鍵結固定化PM晶片的微分光電流響應，在中性塗覆時被水解與完整PM膜的響應分別為221 nA/cm2與185 nA/cm2；將這兩種PM膜在酸性pH固定化，所得的響應則分別下降為39 nA/cm2和11 nA/cm2，顯示固定化pH與PM水解會對晶片光電流響應有影響。

Purple membrane (PM) is a cellular component of Halobacterial salinarum containing bacteriorhodpsin (BR), which is able to generate photoelectric responses. This study mainly used AFM to examine biotinylated PM (b-PM) affinity-adsorbed on mica with either activated avidin or the mixture of activated avidin and biotinylated grapheme oxide (b-GO) as a linker. The surface roughness and adhesion force of the linker layer both decreased from 0.48 nm and 5.93 nN to 0.35 nm and 4.81 nN, respectively, when the activated-avidin linker was added with 0.02 mg/mL b-GO. Moreover, b-GO addition reduced stacking of immobilized b-PM patches and decreased the spacing between BR trimers from 7.9 to 5.4 nm. Furthermore, the CW differential photocurrent densities generated by b-PM immobilized on ITO were reduced from 649 to 417 nA/cm2 upon b-GO addition, which was attributed to high roughness of ITO. Moreover, the addition with 0.005 mg/mL b-GO enhanced the B1/B2 component ratio of PM fast photocurrents responding to pulse-laser illumination, implying an improvement of the orientation uniformity of immobilized b-PM. Papain-digested PM patches covalently immobilized at different orientations were attained by adjusting reaction pHs. Digested PM immobilized at neutral pH had a pitted interior, while the one attached at acidic pH appeared to be cracked, suggesting these two PMs were in opposite orientations. In addition, their spacing distances between BR trimers were 5.1 to 5.5 nm, respectively. The differential photocurrent densities of digested and intact PMs covalently attached at neutral pH were 221 and 185 nA/cm2, respectively. However, the values both decreased to 39 and 11 nA/cm2, respectively, indicating the effect of immobilization pH and papain digestion on PM photocurrents.

中文摘要 Ⅰ
英文摘要 Ⅱ
致謝 Ⅲ
目錄 Ⅳ
表目錄 Ⅵ
圖目錄 Ⅶ

第一章 緒論 1

第二章 文獻回顧 2
2-1 Bacteriorhodopsin(BR) 2
2-1-1 H. salinarum 2
2-1-2 BR光循環 3
2-1-3 BR之光電流訊號 5
2-1-4 蛋白酶papain降解PM 6
2-2 氧化石墨烯 7
2-2-1 石墨烯與氧化石墨烯 7
2-2-2 GO於生物感測器上的應用 9
2-3 原子力顯微鏡(AFM) 11
2-3-1 AFM簡介 11
2-3-2 AFM原理 12
2-3-3 AFM探針設計與校正 14
2-3-4 力曲線分析(analysis of force curve) 18
2-3-4-1 力曲線分類 19
2-3-4-2 接近與撤回曲線 22
2-3-4-3 接觸區域(contact regime)分析 23
2-3-4-4 凡德瓦力分析 26
2-3-4-5 黏滯力(adhesion force)分析 29
2-3-5 AFM之掃描方式 34
2-3-6 AFM液相分析 40
2-3-7 利用AFM觀察PM膜的CP與EC側 47

第三章 實驗 50

3-1 實驗目的 50
3-2 實驗藥品 52
3-3 實驗設備 53
3-4 實驗流程 55

第四章 結果與討論 56

4-1 b-GO添加對於以生物親和固定化法製備之PM晶片的影響 56
4-1-1 以AFM分析GO與b-GO 56
4-1-2 以AFM分析b-GO與活化avidin的混合物 65
4-1-3 以AFM分析b-GO與活化avidin混合物的塗覆層 65
4-1-4 以AFM分析PM塗覆層 70
4-1-5 以微分光電流響應探討b-GO添加之影響 78
4-1-6 以脈衝光電流響應探討b-GO添加之影響 79
4-2共價鍵結法置備定向性PM晶片 85
4-2-1 以AFM分析共價鍵結法固定化之PM 86
4-2-2 以微分光電流探討以共價鍵結法固定PM之影響 92

第五章 結論 95

第六章 參考文獻 96

附錄 102

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