臺灣博碩士論文加值系統

Halobacterium salinarum 古生嗜鹽菌紫色細胞膜(purple membrane, PM)內含有一單方向光驅動質子泵：細菌視紫質(bacteriorhodopsin, BR)，其受脈衝式及連續式雷射激發後分別會產生極快B1B2及微分D1D2光電流響應。本研究利用LabVIEW自動化量測系統，針對BR受光激發後所產生之兩種光電流響應進行訊號快速擷取。研究分為兩部份：第一部份以B1B2光電流訊號之量測來分析PM膜固定化於ITO導電基材上之定向性，結果顯示在PM固定化架橋中添加氧化石墨烯，並對晶片再施以剪應流清洗，可使PM膜排列接近同向且趨近單層，而使得B1光電流訊號大幅增加，同時B2訊號強度減少。接著以微陣列方式掃描PM晶片，進一步證實上述論點。第二部份以D1D2微分光電流分析為量測參數，並利用受檢物可遮蔽入射光而導致PM晶片光電流訊號下降之特性，製作陣列式PM免疫光電感測晶片。首先在PM晶片上分別固定化avidin與NeutrAvidin，接著與高濃度E. coli 反應後，晶片光電流強度分別下降57 %及23 %；接著分別以avidin與NeutrAvidin先固定E. coli抗體於PM晶片上，之後再進行相同菌濃度檢測，晶片光電流訊號則分別下降40 %與50 %，顯示NeutrAvidin對E. coli有較低的非特異性吸附，並以之固定化抗體的晶片對E. coli有較佳檢測靈敏度。同樣以LabVIEW系統掃描陣列式PM免疫光電晶片，可同步且快速地檢測數種菌株。

The purple membrane (PM) of Halobacterium salinarum contains an unidirectional light-driven proton pump, bacteriorhodopsin (BR), which generates a fast (B1B2 ) and a slow (D1D2) photocurrent response upon excitation by a pulse and CW laser, respectively. This research aimed to collect and analyze those two photocurrent responses through a home-built LabVIEW-controlled automatic data acquisition system. This thesis contains two parts. First, the immobilization orientation of b-PM patches on ITO electrodes was investigated by analyzing the B1B2 photocurrent responses. A PM monolayer with nearly uniform orientation could be achieved by a fabrication process with addition of graphene oxide in the linker for PM immobilization and with a followed-up washing over the PM-fabricated chip using a shear flow. This resulted in significant enhance of B1 signals as well as reduction of B2 signals. Microarray-scanning of the as-prepared PM chips also yielded the same supportive observation. Secondly, a PM-based immuno- photoelectric array chip was developed with the D1D2 differentiation photocurrent as the measuring parameter as well as with the fact that PM photocurrents decline when the incident light is blocked by analytes. The detection of Escherichia coli using avidin-bound PM chips with and without prior immobilization of E. coli antibodies resulted in 40% and 57% photocurrent reductions, respectively. On the other hand, 50% and 23% photocurrent reductions were observed when NeutrAvidin-bound PM chips were used to detect E. coli with and without prior immobilization of E. coli antibodies, respectively. Therefore, NeutrAvidin had a lower nonspecific adsorption toward E. coli than avidin, and the prepared antibody-bound PM chips with NeutrAvidin had better detection sensitivity. Finally, simultaneous and fast detection of different organisms was demonstrated by using a LabVIEW-controlled array scanning system.

中文摘要 I
Abstract II
目錄 III
表目錄 V
圖目錄 VI
第一章 緒論 1
第二章 文獻回顧 2
2-1 細菌視紫質 (Bacteriorhodopsin，BR) 2
2-1-1 Halobacterium salinarum 2
2-1-2 Bacteriorhodopsin (BR) 3
2-1-3 BR光循環 4
2-1-4 BR的光電響應 6
2-2 PM光電晶片製備方法 12
2-3 LabVIEW介紹 14
2-3-1人機介面視窗 (Front panel) 15
2-3-2程式方塊視窗 (Block diagram) 16
2-3-3 圖示及連接器 (Icon and connector pane) 17
2-3-4資料擷取的原理 18
2-3-4-1類比訊號 18
2-3-4-2數位訊號 19
2-3-5示波器訊號取樣概念 20
2-3-5-1 頻寬 (Bandwidth) 21
2-3-5-2 取樣速率 (Sampling rate) 23
2-3-5-3 解析度 (Resolution) 25
第三章 實驗 27
3-1實驗目的 27
3-2 實驗設備 29
3-3 實驗流程 31
3-4 實驗量測 32
3-4-1 微分光電流訊號量測（D1、D2） 32
3-4-2 陣列式掃描脈衝光電流訊號量測（B1、B2） 33
第四章 結果與討論 34
4-1 LabVIEW自動化系統建立 34
4-1-1 示波卡的選擇 34
4-1-2 電控四軸移動平台與控制器 38
4-2 添加氧化石墨烯 (GO)對於以生物親和吸附製備PM晶片的影響 39
4-2-1 脈衝雷射自動化B1B2訊號偵測系統 41
4-2-2 添加GO對B1B2光電流響應之影響 46
4-2-2-1 ITO基材在不同pH值電解液的光電流背景訊號 46
4-2-2-2 ITO基材在不同濃度電解液的光電流背景訊號 48
4-2-2-3逐層塗覆晶片的B1B2光電流響應 51
4-2-2-4 微流清洗對PM晶片B1B2光電流響應之影響 53
4-2-3 微陣列掃描探討GO添加對晶片B1B2光電流響應分佈之影響 57
4-3 製備PM生物感測晶片 74
4-3-1 連續式雷射自動化PM光電流量測系統 75
4-3-2陣列式PM生物感測晶片建立 77
4-3-2-1 以固定不同抗體之陣列式PM晶片探討對菌檢測的特異性 79
4-3-2-2 溶液震盪速率於PM晶片光電流響應的影響 81
4-3-2-3 以NeutrAvidin取代avidin固定化經biotin修飾之抗體 82
4-3-2-4 NeutrAvidin固定化濃度的最適化 85
4-3-3以NeutrAvidin為架橋之抗體-PM晶片對大腸桿菌直接檢測之檢量線 91
第五章 結論 93
第六章 參考文獻 94

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，2005
王世育，"細菌視紫質表面修飾及應用於單層膜光電晶片製備之研究"，國立台灣科技大學化學工程研究所碩士論文，2007
余安棣，"製備具高度方向性之Bacteriorhodopsin生物光電晶片"，國立台灣科技大學化學工程研究所碩士論文，2009
陳逸航，"定向性細菌視紫質晶片之光電與二倍頻響應探討"，國立台灣科技大學化學工程研究所碩士論文，2010
黃柏竣，"非特異性作用對親和吸附法製備細菌視紫質光電晶片之影響"，國立台灣科技大學化學工程研究所碩士論文，2012
林聖為，"利用自排性單分子膜與生物親和吸附增進PM晶片的製程"，國立台灣科技大學化學工程研究所碩士論文，2012
林承德，"氧化石墨烯濃度對以親和吸附作用製備細菌視紫質光電晶片之影響"，國立台灣科技大學化學工程研究所碩士論文，2013a
林其融，"AFM探討分別以結合氧化石墨烯之親和吸附與共價鍵結作用固定化之紫膜"，國立台灣科技大學化學工程研究所碩士論文，2013b
蕭子健、王智昱、儲昭偉，"虛擬儀控程式設計LabVIEW 8X"，高立圖書有限公司，2009
陳秀美、鄭凱如、胡孔政、蔡孟訓，"細菌視紫質光敏蛋白於生物感測的新型應用"，台灣化學工程學會化工會刊，2011，第58卷，第4期，82-92.
Balashov, S. P. Protonation reactions and their coupling in bacteriorhodopsin. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2000, 1460, 75-94.
Hampp, N. Bacteriorhodopsin as a photochromic retinal protein for optical memories. Chemical Reviews, 2000, 100, 1755-1776.
Hayashi, S.; Tajkhorshid, E.; Schulten, K. Molecular dynamics simulation of bacteriorhodopsin's photoisomerization using ab initio forces for the excited chromophore. Biophysical Journal, 2003, 85, 1440-1449.
Henderson, R.; Jubb, J. S.; Whytock, S. Specific labelling of the protein and lipid on the extracellular surface of purple membrane. Journal of Molecular Biology, 1978, 123, 259-74.
Hong, F. T.; Montal, M. Bacteriorhodopsin in model membranes. A new component of the displacement photocurrent in the microsecond time scale. Biophysical Journal, 1979, 25, 465-472.
Kawamura, I.; Ohmine, M.; Tanabe, J.; Tuzi, S.; Saitô, H.; Naito, A. Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR. Biochimica et Biophysica Acta (BBA) - Biomembranes, 2007, 1768, 3090-3097.
Lanyi, J. K. Proton transfers in the bacteriorhodopsin photocycle. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2006, 1757, 1012-1018.
National Instruments. LabVIEW User manual. 2003.
National Instruments. LabVIEW Top 10 things to consider when selecting a digitizer oscilloscope. 2014.
National Instruments. LabVIEW Introduction to data acquisition. 2014.
National Instruments. LabVIEW Tutorial: state machines. 2015.
National Instruments. LabVIEW Acquiring an analog signal: bandwidth, Nyquist Sampling Theorem, and aliasing. 2015.
Sharma, M. K.; Jattani, H.; Gilchrist, M. L. Bacteriorhodopsin conjugates as anchors for supported membranes. Bioconjugate Chemistry, 2004, 15, 942-947.
Su, T.; Zhong, S.; Zhang, Y.; Hu, K. s. Asymmetric distribution of biotin labeling on the purple membrane. Journal of Photochemistry and Photobiology B: Biology, 2008, 92, 123-127.
Wang, J. Vectorially oriented purple membrane: characterization by photocurrent measurement and polarized-Fourier transform infrared spectroscopy. Thin Solid Films, 2000, 379, 224-229.
Wang, J. P.; Yoo, S. K.; Song, L.; El-Sayed, M. A. Molecular mechanism of the differential photoelectric response of bacteriorhodopsin. Journal of Physical Chemistry B, 1997, 101, 3420-3423.