研究生: |
吳欣穎 Xin-Ying Wu |
---|---|
論文名稱: |
細菌視紫質泛用型免疫光電晶片製備與原子力顯微鏡分析 Preparation and AFM Analysis of Bacteriorhodopsin Photoelectric Chips for Immunoassay |
指導教授: |
陳秀美
Hsiu-mei Chen |
口試委員: |
何明樺
Ming-hua Ho 王鐘毅 Chung-yih Wan |
學位類別: |
碩士 Master |
系所名稱: |
工程學院 - 化學工程系 Department of Chemical Engineering |
論文出版年: | 2015 |
畢業學年度: | 103 |
語文別: | 中文 |
論文頁數: | 107 |
中文關鍵詞: | 細菌視紫質 、紫膜 、原子力顯微鏡 、生物晶片 |
外文關鍵詞: | Bacteriorhodopsin, AFM, biosensor |
相關次數: | 點閱:290 下載:3 |
分享至: |
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細菌視紫質存在於Halobacterium salinarum古生嗜鹽菌紫膜(purple membrane, PM)中,為一種光驅動質子幫浦。本研究藉由PM光電流響應與入射光強度間的線性關係,發展出具有對微生物辨識與檢測能力的泛用性免疫檢測PM晶片;選用ITO導電玻璃為電極,PM為感測器核心的生物性光電訊號轉換器,並使用protein A定向化大腸桿菌抗體作為生物辨識元件,如此可定量偵測大腸桿菌的存在,檢測靈敏度可達 1 CFU/mL。研究中,並利用拉曼分析光譜及原子力顯微鏡鑑定並確認PM晶片之表面改質、Protein A 與抗體固定化及大腸桿菌吸附。此外,以AFM分析PM表面形貌,可區分被固定PM膜之定向性,在不同固定化基材下,以胞外側固定之PM膜可呈現三種不同形貌,分別為細小花紋、具粒狀皺褶的細小裂痕與筆直斷裂之裂痕形貌;而胞內側固定之PM膜在不同基材上的形貌皆無太大差異;pH值對膜表面形貌影響較小。
Bacteriorhodpsin (BR), a family of retinal-containing proteins, resides in the purple membrane (PM) of Halobacterium salinarum and functions as a light-driven proton pump. This study develops a PM-based immunesensor to recognize and detect microorganisms according to the linear relationship between the photocurrents generated by PM and the light-intensity illuminating on PM. ITO conductive glass was used as the electrode; PM was the signal transducer of the photoelectric biosensor; and anti-Escherichia coli antibodies orientationally immobilized by Protein A on PM chips were employed as the biological recognition element. The results show the photocurrents generated by the antibody-PM composite chip declined semi-log linearly with increasing of E. coli binding concentrations and a detection limit of 1 CFU/mL was achieved. The layer-by-layer fabrication of the antibody-PM composite chip as well as the binding of E. coli was confirmed by both Raman spectroscopy and AFM analysis. In addition, the orientations of PM patches immobilized on different substrates were investigated with AFM. Three different cracking topographies were observed for the PM patches attached with their extracellular side facing substrates: thin lightning-like cracks, thin cracks with tiny granules, and straight cracks. However, the topographies of the PM patches attached with their cytoplasmic side facing substrates were almost the same on different substrates. Finally, pH had a very little effect on the morphologies of both sides of immobilized PM.
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)
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" 2012a. 國立台灣科技大學化學工程研究所碩士論文 (2012a).
林承德 "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" 2012b. 國立台灣科技大學化學工程研究所碩士論文 (2012b).
翁永進 "原子力顯微鏡(AFM)介紹與應用" 開南商工 (2008)
黃柏竣 "還原劑、突變及量子點對細菌視紫質光電響應影響之探討" (2010)
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).
蔡孟訓 "自排性單層分子膜於Bacteriorhodopsin光電晶片製備之探討" 國立台灣科技大學化學工程研究所碩士論文 (2011).
蔡毓楨、薛富盛、呂福興、吳宗明"原子力顯微鏡實作訓練教材" 五南出版社 (2007).
AnaSpec SpA產品資訊http://www.anaspec.com/
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