張煥宗臺灣大學：化學研究所吳淑芬Wu, Shu-FenShu-FenWu2007-11-262018-07-102007-11-262018-07-102006http://ntur.lib.ntu.edu.tw//handle/246246/51784環境中之微生物影響人類健康甚鉅，其中大腸桿菌（Escherichia coli, E. coli）為監控水質最理想之指標微生物（indicator microorganism），因此建立有效及快速的大腸桿菌檢測方法，才能即時進行適當的防護措施。本研究係利用毛細管電泳暨雷射誘導螢光偵測於電滲流（electroosomotic flow, EOF）存在下，以含1.7% 聚環氧乙烯（poly(ethylene oxide), PEO）（Mw 4M g/mol）之Tris-borate緩衝溶液進行線上濃縮及分離大體積（約1.0 μL）之蛋白質樣品及細菌溶解產物（lysate）。為解決蛋白質吸附的問題，在樣品中加入0.01% PEO（Mw 300k g/mol），同時於樣品區帶前注入一小段（1.3 cm）0.2%之十二烷基硫酸鈉（sodium dodecyl sulfate, SDS）溶液塞，以提高濃縮與分離效率。將此技術應用於大腸桿菌溶解產物之分析，成功地從複雜基質中標定出其特徵蛋白質訊號峰。由於高濃縮效率提升偵測靈敏度，實際注入毛細管內的菌數只需約3 × 105個。此外，本方法樣品需求量低且不需繁複的前處理步驟，分析環境水樣中之大腸桿菌，可在一個工作天內得到結果，為大腸桿菌檢測提供一個快速、簡單及經濟的方法。The specific detection of Escherichia coli (E. coli) is essential for water quality control because its presence points directly to the presence of enteric disease-causing bacterial. We developed a rapid, easy and economical method for the analysis of bacterial lysates by capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF). Addition of 0.01% poly(ethylene oxide) (PEO) (Mw 300k g/mol) to the sample matrix and applied a 0.2% sodium dodecyl sulfate (SDS) plug (1.3 cm) before injecting large-volume (ca. 1.0 μL) sample zone can improve stacking and separation efficiencies. After injection of the large-volume samples, the proteins migrate against the electroosmotic flow (EOF) and enter 1.7% PEO (Mw 4M g/mol) zone; this process causes them to slow down and stack at the boundary between the PEO and sample zones. As a result, the characteristic peaks of E. coli lysate were identified by injecting as few as 3 × 105 cells. Owing to growth rapidly and predominance in fecal contaminated water, we could detect and identify culturable E. coli cells in 50 mL of pond water in campus within one working day.目錄 中文摘要 I 英文摘要 II 目錄 III 表目錄 VI 圖目錄 VII 第一章 毛細管電泳概論 1 1.1 前言 1 1.2 毛細管電泳發展史 1 1.3 毛細管電泳原理 4 1.3.1 電泳 4 1.3.2 電滲流（electroosmotic flow, EOF）與zeta電位 5 1.3.3 分離效率 7 1.4毛細管電泳之蛋白質分析 8 1.4.1 蛋白質分離 8 1.4.2 蛋白質偵測 11 1.5 線上樣品濃縮技術 13 1.5.1 場放大樣品堆積（field-amplified sample stacking, FASS） 14 1.5.2 pH調控堆積（pH-mediated stacking） 15 1.5.3 毛細管等速電泳（capillary isotachophoresis, CITP） 16 1.5.4 聚合物溶液線上濃縮與分離生物樣品 17 1.5.5 掃掠法（sweeping） 18 1.6 研究動機 19 1.7 參考文獻 21 1.8 本章圖表 31 第二章 大腸桿菌之毛細管電泳快速檢測技術 37 2.1 前言 37 2.1.1 細菌簡介 39 2.1.1.1 構造 39 2.1.1.2 生長 39 2.1.2 大腸菌類群（coliforms） 42 2.1.2.1 大腸桿菌（Escherichia coli, E. coli） 42 2.1.2.2 產氣腸桿菌（Enterobacter aerogenes, E. aerogenes） 43 2.1.2.3 腸道沙門氏菌（Salmonella enterica, S. enterica） 43 2.1.3以大腸桿菌作為指標微生物 44 2.1.4 大腸桿菌檢測方法 45 2.2 實驗方法 47 2.3 實驗部分 49 2.3.1 儀器裝置 49 2.3.2 毛細管電泳組裝 50 2.3.3 試藥 51 2.3.4 聚合物溶液配製 51 2.3.5 毛細管前處理 52 2.3.6 細菌樣品製備 52 2.3.6.1 菌株 52 2.3.6.2 菌株保存 53 2.3.6.3 細菌溶解產物（lysates）樣品之製備 53 2.3.6.4 實際環境樣品之細菌溶解產物製備 53 2.3.7 實驗流程 53 2.4 結果與討論 54 2.4.1 大體積蛋白質樣品之分析條件 54 2.4.2 大腸桿菌溶解產物之製備 58 2.4.3 大腸桿菌溶解產物之分析 59 2.4.4 不同菌種溶解產物之比較 61 2.4.5 實際水質樣品分析 64 2.5 結論 65 2.6 參考文獻 67 2.7 本章圖表 74 表目錄 Table 1-1 Representative CE-based concentration techniques for proteins or other biomolecules 31 Table 2-1 Comparisons of structure and physiological characteristic between Gram-positive and Gram-negative bacteria 74 Table 2-2 Generation times for some bacteria under optimal conditions of growth 75 Table 2-3 Quantitative investigations on the composition of human colon flora 76 Table 2-5 Bacterial strains 77 Table 2-5 Comparisons of current methods for the detection of Escherichia coli 78 圖目錄 Figure 1-1 Illustration of double layer and zeta potential. 32 Figure 1-2 Flow profiles of CE (A) and HPLC (B). 33 Figure 1-3 Schematic representation of field amplified sample stacking (FASS) mechanism. 34 Figure 1-4 Schematic representation of sample stacking and separation using polymer solution. 35 Figure 1-5 Schematic representation of sweeping mechanism. 36 Figure 2-1 Structure of a typical bacterium. 79 Figure 2-2 Cell wall structures of Gram-positive (left) and Gram-negative (right) bacteria. 79 Figure 2-3 Schematic representation of on-line concentration and separation of proteins in CE. 80 Figure 2-4 Schematic plot of the number of bound ligands per protein molecule (υ)as a function of the logarithm of the free SDS concentration (c). 81 Figure 2-5 Instrumental setup of capillary electrophoresis coupled laser induced fluorescence detection. 82 Figure 2-6 Flowchart of bacterial lysates preparation. 83 Figure 2-7 Structure of polymyxin B. 84 Figure 2-8 Effect of SDS plug on the separation of β-galactosidase isoforms 85 Figure 2-9 Effect of injection time on the separation of E. coli lysate. 87 Figure 2-10 Electropherograms of E. coli lysate obtained by injecting different cell number.. 88 Figure 2-11 Electropherograms of E. coli lysate which was obtained from 5 cells inoculated into 50 mL LB broth after different pre-culture time. 89 Figure 2-12 Electropherograms of E. aerogenes lysate obtained by injecting different cell number. 90 Figure 2-13 Electropherograms of S. enterica lysate obtained by injecting different cell number. 91 Figure 2-14 Electropherograms of the mixtures of bacterial lysates. 92 Figure 2-15 Linearity of E. coli characteristic peak area in the matrix of pond water with 10 μg/mL polymyxin B sulfate. 94 Figure 2-16 CE-LIF detection of E. coli in pond water (A); after 7.0 hr pre-culture (B); sample (B) with spiking of 4.5 × 105 E. coli (C). 952254356 bytesapplication/pdfen-US大腸桿菌指標微生物毛細管電泳暨雷射誘導螢光偵測線上濃縮細菌溶解產物Escherichia coliindicator microorganismcapillary electrophoresis coupled with laser-induced fluorescenceon-line concentrationbacterial lysatesthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/51784/1/ntu-95-R93223017-1.pdf