研究生: |
黎閔哲 Min-Che Li |
---|---|
論文名稱: |
即時檢測抗體及核酸適體於紫膜生物光電晶片上之固定化穩定性及開發大腸桿菌檢測晶片 Real-time monitoring the stability of antibodies and aptamers immobilized on purple menbrane-based photoelectric biochips and development Escherichia coli detection |
指導教授: |
陳秀美
Hsiu-Mei Chen |
口試委員: |
林景堉
Ching-Yu Lin 蔡伸隆 Shen-Long Tsai |
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: | 2018 |
畢業學年度: | 106 |
語文別: | 中文 |
論文頁數: | 137頁 |
中文關鍵詞: | 抗體 、核酸適體 、紫膜 、光電晶片 、大腸桿菌 |
外文關鍵詞: | antibodies, aptamers, purple menbrane, phoelectric biochips, Escherichia coli |
相關次數: | 點閱:215 下載:0 |
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太古嗜鹽菌Halobacterium salinarum紫膜 (purple membrane, PM) 內具有獨特光敏感蛋白,被稱為細菌視紫質 (bacteriorhodopsin, BR)。由於BR受光後會使PM膜兩側產生質子梯度差而誘導光電流產生,且此光電流與入射光強度成正向關係,因此再利用菌體本身可散射光特性,本實驗室先前已分別開發以抗體及核酸適體為辨識分子的PM膜微生物感測器。本研究首先以連續注流式即時檢測系統延續探討抗體及aptamer被固定化於PM膜上及微生物被吸附於辨識分子上之穩定性,發現兩種辨識分子在10 μL/min低流速時均具有最佳穩定性;反之150 μL/min高流速時皆會被脫附於晶片表面,其次,分別被抗體及核酸適體辨識分子捕捉於PM膜晶片上之Escherichia coli K-12及Lactobacillus acidophilus兩種微生物,在100 μL/min 較高流速下也會完全脫離固定於PM晶片上之辨識分子,因此可藉由調高流速而使辨識分子-PM晶片再生利用。此外,在10 μL/min 低流速流場中辨識分子-PM晶片可連續檢測不同濃度之微生物體,研究中發現抗體-PM晶片測菌時所需平衡時間為30分鐘;核酸適體-PM晶片則只要5分鐘,都比原先靜態吸附檢測菌時需要反應兩小時更為迅速。再者,本研究也發現空白PM晶片在流場下所能承受之最大流速為2 mL/min,因此可再次藉由粉體及菌體之光遮蔽效應直接以空白PM晶片進行檢測。於250 μL /min高流速下,發現空白PM晶片能檢測到體積平均粒徑為45 μm Al2O3粉末之最低濃度為0.2 ppb;E. coli K-12能被檢測到的最低濃度為102 CFU/mL。最後,我們以E. coli核酸適體取代現有固定化在PM膜上之抗體辨識分子,先以模擬軟體探討其二級結構並選出最適化固定化與檢測條件,再分別對E. coli、L. acidophilus及Bacillus subtilis進行檢測,結果發現能檢測到E. coli之最低濃度為1 CFU/10 mL;然而對於106 CFU/mL L. acidophilus及Bacillus,此E. coli感測晶片仍分別有45 %及29 %因非特異性吸附所引起的光電流下降,因此未來需調整檢測條件,以提升其專一性。
Halobacterium salinarum purple membranes (PM) contain a unique light-sensitive protein called bacteriorhodopsin (BR). When BR is illuminated, a proton gradient is formed across PM, which drives photocurrent production. The generated photocurrent is linearly correlated with the illumination intensity; thus both antibody-PM and aptamer-PM composite sensors have previously been developed in this laboratory to detect microorganisms based on the fact that bacteria scatter light. In this study, a real-time flow-injection analysis system was employed to investigate the stability of the immobilized antibodies and aptamers as well as the captured bacteria on PM-coated chips in a shear flow. Both recognition molecules exhibited the best stability at 10 μL/min, while their dissociation from the chip surface was observed at 150 μL/min. In addition, Escherichia coli K-12 and Lactobacillus acidophilus cells captured by the antibody-PM and aptamer-PM sensor chips, respectively, were completely detached from their respective immobilized recognition molecules at 100 μL/min, suggesting the feasibility of regenerating either sensor chip simply by raising flow rates. Moreover, microorganisms at different concentrations were readily detected at 10 μL/min with only 5-min and 30-min equilibrium time observed upon each cell injection for the aptamer-PM and antibody-PM chips, respectively, which were significantly shorter than what was observed (2 h) in a static detection. The maximal flow rate for the bare PM chip to sustain its full photoelectric activity was 2 mL/min, so we subsequently employed the bare PM chip to directly detect microparticles and bacteria based on their light scattering effects. At 250 μL /min, the bare PM chips directly detected Al2O3 powders with a volume-based particle size of 45 μm with a detection limit of 0.2 ppb, and a limit of 102 CFU/mL for E. coli K-12 was obtained. Finally, an E. coli aptamer-PM sensor chip was prepared by first simulating the secondary structure of the E. coli aptamer for its optimal immobilization and detection conditions. The chip readily detected E. coli K-12 with a limit of 1 CFU/10 mL. However, the chip also exhibited 45 % and 29 % photocurrent reductions on the detection of 106 CFU/mL L. acidophilus and Bacillus subtilis, respectively, indicating nonspecific adsorption of both cells. More investigations of the detection condition will be conducted to improve the chip selectivity in the future.
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