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研究生: 林奕儒
I-Ju Lin
論文名稱: 鮑氏不動桿菌之紫膜生物光電感測晶片的開發
Development of purple membrane-based photoelectric chips for Acinetobacter baumannii detection
指導教授: 陳秀美
Hsiu-Mei Chen
口試委員: 吳雪霞
Hsueh-Hsia Wu
葉旻鑫
Min-Hsin Yeh
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: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 138
中文關鍵詞: 鮑氏不動桿菌紫膜生物傳感器生物光電晶片
外文關鍵詞: Acinetobacter baumannii, purple membrane, biosensor, photoelectric chips
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鮑氏不動桿菌 (Acinetobacter baumannii)為造成嚴重院內感染之病原體,其能引起肺炎、腦膜炎,甚至是高死亡率的菌血症,因此快速且靈敏的辨別出A. baumannii蔚為重要,唯有即時鑑定病原體才能增加施藥準確率,且避免用藥錯誤產生抗藥性並能有效降低死亡率。細菌視紫質 (bacteriorhodopsin, BR)存在於古生嗜鹽菌 (Halobacterium salinarum)的紫色細胞膜 (purple membrane, PM)中,其為受光激發會產生跨膜質子梯度差的光驅動質子泵,將此特性透過外接電路即可量測到光電流訊號。本論文即運用以PM為光電訊號轉換器之原理所製備的PM生物感測晶片,以針對A. baumannii具有高度專一性的核酸適體為辨識因子,開發A. baumannii aptamer-PM複合生物光電感測晶片,用以檢測A. baumannii。首先對晶片製程中核酸適體架橋的種類與長度、檢測緩衝液中Na+濃度進行探討,尋求對檢測效果最為顯著之製備條件。如此以最佳製程製備的A. baumannii aptamer-PM複合生物光電感測晶片可檢測A. baumannii最低濃度至1E-1 CFU/mL,接續測試其對台灣常見七種菌血症菌株之分辨能力,結果顯示本論文開發的A. baumannii aptamer-PM複合生物光電感測晶片對A. baumannii具有顯著區別能力 (p<0.01,具有顯著差異性)。最後再利用結合辨識因子的奈米金進行訊號增強的探討,發現如此製程對A. baumannii之檢測限能降低至濃度1E-2 CFU/mL。


Acinetobacter baumannii is a pathogen causing severe nosocomial infection, resulting in meningitis, pneumonia, or even lethal bacteremia. Therefore, the rapid and sensitive identification of A. baumannii is important for precision antibiotics to avoid the development of antibiotic resistance and to reduce the mortality rate. Bacteriorhodopsin (BR) exists in the purple membrane (PM) of Halobacterium salinarum and acts as a light-driven proton pump, generating a cross-membrane proton gradient which drives photocurrents through an external circuit. This thesis employs a previously developed sensor with PM as a photoelectrical signal transducer to further devise an aptamer-PM complex biosensor for A. baumannii detection. The biosensor contains specific aptamers against A. baumannii as the recognition element. For optimal biosensor development and detection, different aptamers were investigated, as well as the lengths of their conjugation linkers with PM and the Na+ concentration in the detection buffer. The best developed A. baumannii aptamer-PM complex biosensor sensitively detected A. baumannii down to 1E-1 CFU/mL and significantly discriminated A. baumannii from 6 other common bacteremia-causing strains found in Taiwan (p < 0.01). Finally, to further enhance the detection sensitivity, gold nanoparticles conjugated with the optimal recognition aptamer were used to label the microbes captured on the A. baumannii aptamer-PM complex sensing chip, resulting a detection limit of 1E-2 CFU/mL.

中文摘要 I Abstract II 致謝 III 目錄 IV 表目錄 VIII 圖目錄 XIII 第一章 緒論 1 第二章 文獻回顧 3 2-1 Acinetobacter baumannii 3 2-1-1 A. baumannii簡介 3 2-1-2 A. baumannii感染 4 2-1-3 菌血症 (bacteremia) 6 2-1-4 菌血症 (bacteremia)檢測方法 7 2-1-4-1 血液培養法 (blood cultures, BC) 7 2-1-4-2 PCR-電噴霧質譜法 (PCR-Electrospray Ionization Mass Spectrometry, PCR-ESI-MS) 8 2-1-4-3 16S 核醣體核糖核酸 (16S ribosomal RNA, rRNA)基因序列分析 9 2-1-5 A. baumannii檢測方法 10 2-1-5-1 環介導等溫擴增 (loop-mediated isothermal amplification, LAMP) 10 2-1-5-2 基質輔助雷射脫附游離飛行時間式質譜儀 (matrix-assisted laser desorption ionization-time of flight mass spectrometry, MALDI-TOF MS) 12 2-1-5-3 擴增片段長度多態性 (amplified fragment length polymorphism, AFLP) 13 2-2 細菌視紫質 (bacteriorhodopsin, BR) 15 2-2-1 Halobacterium salinarum (H. salinarum) 15 2-2-2 BR結構 17 2-2-3 BR光循環 18 2-2-4 BR光電響應 21 2-2-5 PM生物親和性之單層定向固定化 24 2-2-6 PM晶片之檢測與應用 26 2-2-7 AuNPs於PM晶片之應用 28 2-3 核酸適體 (aptamer) 29 2-3-1 核酸適體性質與篩選 29 2-3-2 以核酸適體應用於微生物檢測 31 2-4 奈米金粒子 (Gold nanoparticles, AuNPs) 32 2-4-1 AuNPs特性 32 2-4-2 DNA-AuNPs製備方法 33 2-4-3 AuNPs於生物傳感器之應用 35 第三章 實驗 36 3-1 實驗目的 36 3-2 實驗流程 37 3-3 量測 46 3-3-1 Cuvette系統之D1、D2微分光電流檢測 46 3-3-2 拉曼光譜分析 47 3-4 變異數分析 48 3-4-1 以Excel做單因子變數分析 49 3-4-2 以IBM SPSS Statistics ANOVA 49 第四章 結果與討論 50 4-1 A. baumannii aptamer-PM生物光電感測晶片製程探討 50 4-1-1 選擇標靶為A. baumannii之核酸適體 50 4-2 43 nt A. baumannii aptamer-PM生物光電感測晶片製程探討 52 4-2-1 以Oligoanalyzer 3.1模擬決定43 nt A. baumannii aptamer固定化於PM晶片之反應條件 52 4-2-1-1 固定反應溫度改變43 nt A. baumannii aptamer之固定化Na+濃度 52 4-2-1-2 固定Na+濃度改變43 nt A. baumannii aptamer之固定化反應溫度 56 4-2-2 以Sulfo-SIAB與NHS-PEGn-NHS為架橋製備43 nt A. baumannii aptamer-PM商務光電感測晶片 63 4-2-3 探討NHS-PEG2-NHS與43 nt A. baumannii aptamer之濃度關係 66 4-2-4 探討binding buffer中Na+對A. baumannii aptamer-PM生物光電晶片穩定度之影響 69 4-3 72 nt A. baumannii aptamer-PM生物光電晶片製程探討 73 4-3-1 以Oligoanalyzer 3.1模擬決定72 nt A. baumannii aptamer固定化於PM晶片之反應條件 73 4-3-1-1 固定反應溫度改變72 nt A. baumannii aptamer之固定化Na+濃度 73 4-3-1-2 固定Na+濃度改變72 nt A. baumannii aptamer之固定化反應溫度 79 4-3-2 探討立體障礙對72 nt A. baumannii aptamer之影響 86 4-4 比較43 nt與72 nt A. baumannii aptamer 88 4-5 對台灣常見七株菌血症菌株之檢測專一性 90 4-6 探討革蘭氏陰性菌對43 nt A. baumannii aptamer-PM生物光電感測晶片的影響 99 4-7 運用AuNPs降低43 nt A. baumannii aptamer-PM生物光電感測晶片檢測下限 103 4-7-1 測試AuNPs塗覆效果 103 4-7-2 探討AuNPs濃度與43 nt A. baumannii aptamer條數比例關係 105 4-7-3 以最佳化AuNPs濃度 (40 pM)與aptamer比例 (1:2000)檢測晶片再現性 108 4-7-4 對台灣常見七株菌血症菌株之檢測專一性 110 4-8 拉曼光譜分析 116 4-9 常人血液對43 nt A. baumannii aptamer-PM生物光電感測晶片檢測之影響 125 4-9-1 模擬常人血液對43 nt A. baumannii aptamer-PM生物光電感測晶片檢測的影響 126 4-9-2 模擬常人血液對使用AuNPs之43 nt A. baumannii aptamer-PM生物光電感測晶片檢測的影響 128 第五章 結論 130 第六章 參考文獻 132

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陳泓任. 檢測三磷酸酸腺苷、革蘭氏陽性菌和白血球之紫膜生物光電晶片之開發. 國立台灣科技大學, 2018.

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曾聖翔. 紫膜複合生物光電晶片應用於腦癌血清miRNA與免疫檢測之探討. 國立台灣科技大學, 2021.

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