臺灣博碩士論文加值系統

丙酮（acetone）是糖尿病患反應血糖控制的重要指標，利用氨基酸序列為仿生物材料的辨識元件，並以壓電晶體（piezoelectric crystal）作為換能器元件，製成丙酮呼氣檢測的生物電子鼻。本研究以Cytochrome P4502E1蛋白為模版用以尋找可能與丙酮結合的配位基，本研究採用分子模擬方法模擬蛋白催化部位氨基酸片段與受質（丙酮）的結合狀態，經能量計算後找出與目標氣體結合狀態最穩定的氨基酸序列；並探討氨基酸序列材料的固定化方法，在晶片表面固定形成一層穩定的生物薄膜，作適當的溫度處理，提昇整體氣體感測器的靈敏度。進行呼氣測試時，氣體感測器以模組的方式組成，模組配置為兩支對丙酮指標氣體具特異性探針（探針編號E1, E2）、兩支對胺類指標氣體具特異性探針（探針編號N1, N2）和兩支對酸類指標氣體具特異性探針（探針編號A1, A2）；並將模組對樣品氣體所測得的靈敏度，進行分析及統計。依據疾病指標氣體的濃度，顯現指標氣體的靈敏度高於其它干擾氣體，以此偵測指標氣體。固定化方法研究發現，在晶片表面固定一層11-Mercapto- undecanoic acid（MDA），形成一層排列整齊而穩定的自排性單層膜（Self-assembled monolayer, SAM），再將適量濃度的目標氨基酸序列固定於晶片表面，可以提升探針靈敏度2-15倍。經熱處理50-70℃之探針對感測低感度氣體丙酮、氯仿的靈敏度上升7-15%。臨床呼氣檢測發現，本模組對糖尿病酮酸症（Diabetic Ketoacidosis, DKA）患者呼出內含丙酮的氣體具顯著辨識效果，並可區分健康人與糖尿病患者呼出的丙酮濃度。本模組可繼續開發應用於多種疾病指標氣體的呼氣檢測器。

Acetone is an important exhale gas indicator for diabetic patients. The purpose of this study is to develop a gas phase biosensor which consists of multi-array piezoelectric (PZ) quartz crystal devices with peptides as recognized materials. Cytochrome P4502E1 was used as a template searching the plausible binding site with acetone molecule. To find a proper length and sequence of peptides for detecting target acetone compound, molecular simulation was applied to determine the best catalytic and substrate binding site by comparing the binding energy at the lowest level. Then, used the peptide as recognized materials for biosensor fabrication. These peptides formed a layer of biofilm on the surface of PZ gold electrodes by different immobilized methods that had been evaluated. To raise the sensitivity of the sensor chips, heat treatment was applied in this study. In the exhale analysis, the gas sensor module comprised three pairs of characteristic chips, the first pair chips dedicated to detect acetone(probe code:E1, E2), the second pair chips dedicated to detect amine gases(probe code:N1, N2), and the third pair chips dedicated to detect acid gases(probe code:A1, A2). The sensitivity of gas sensor module chips were also measured. According to the pathological concentration of the breath acetone of diabetic patients, the result showed the sensitivity of acetone chips to target compound acetone was much higher than other none-target compounds. It also showed that the sensitivity of acetone chips could be improved 2-15 folds by pre-immobilizing a self-assembled monolayer(SAM) on PZ electrode surface before recognized peptide molecules be immobilized. The sensitivity of chips to acetone and chloroform has been improved 7-15% after heat pre-treatment. The best condition for heat treatment was 50-70℃. The result of clinical test showed that this sensor module could clearly discriminate the exhale gas from Diabetic Ketoacidosis (DKA) patients and healthy person. It also could differentiate the diabetic patients and healthy person. Finally, this technical plateform might be applied to develop a multi-diseases detection system by continuing intensive research in the future.

目 錄

頁次
授權書 -------------------------------------------------------------------------I
致 謝 --------------------------------------------------------------------------II
中文摘要 ----------------------------------------------------------------------III
英文摘要 ----------------------------------------------------------------------IV
目 錄 --------------------------------------------------------------------------VI
圖 目 錄 --------------------------------------------------------------------- IX
表 目 錄 ----------------------------------------------------------------------XII
第一章 緒論 ----------------------------------------------------------------1
1.1 研究緣由 --------------------------------------------------------------1
1.2 研究目的 --------------------------------------------------------------4
第二章 研究背景與文獻回顧 -------------------------------------------5
2.1 生物感測器 -----------------------------------------------------------5
2.1.1 生物感測器的發展 ----------------------------------------------6
2.1.2 生物感測器的分類 ----------------------------------------------10
2.1.3 生物感測器的市場潛力 ----------------------------------------12
2.1.4 壓電石英晶體 ----------------------------------------------------13
2.1.4.1 壓電效應 ------------------------------------------------------13
2.1.4.2 石英晶體微天平 ---------------------------------------------15
2.1.4.3 壓電石英晶體測定原理 ------------------------------------18
2.2 呼氣診斷（Breath Diagnosis） -----------------------------------19
2.2.1呼氣分析儀器 -----------------------------------------------------23
2.2.2呼氣診斷的臨床應用與展望 -----------------------------------26
2.3 糖尿病（Diabetes Millitus） --------------------------------------27
2.3.1 糖尿病簡介 -------------------------------------------------------27
2.3.2 糖尿病的檢驗 ----------------------------------------------------32
2.3.3 糖尿病患者的呼氣檢測器 -------------------------------------34
2.4 細胞色素（Cytochrome）P450 ------------------------------------35
2.4.1 Cytochrome P4502E1 ---------------------------------------------37
2.4.2 Cytochrome P4502E1的酵素催化部位 -----------------------38
2.4.3 將P450應用於感測器 ------------------------------------------41
2.5 固定化方法 -----------------------------------------------------------44
2.6生物資訊在探針序列設計的應用 ---------------------------------46
2.6.1 同源模擬（Homology）的基本原理及作法 ----------------46
2.6.2 嵌合模擬(Docking)的基本原理及作法 ----------------------48
第三章 實驗方法與材料 --------------------------------------------------49
3.1 實驗儀器與晶片 -----------------------------------------------------50
3.2 計頻軟體 --------------------------------------------------------------53
3.3 目標氨基酸片段的篩選 --------------------------------------------57
3.3.1 分子模擬(Molecular Simulation) ------------------------------58
3.3.1.1同源模擬（Homology） -------------------------------------59
3.3.1.2嵌合模擬（Docking） ---------------------------------------61
3.4 氨基酸序列合成 -----------------------------------------------------63
3.4.1氨基酸序列合成的儀器與材料 ----------------------------------65
3.5探針製備 ---------------------------------------------------------------66
3.5.1 晶片表面清潔 ----------------------------------------------------67
3.5.2 固定化方法 -------------------------------------------------------68
3.6 最適淬火條件處理 --------------------------------------------------70
3.7 氣體測試 --------------------------------------------------------------71
3.7.1 單一氣體測試 ----------------------------------------------------72
3.7.2組成感測模組 -----------------------------------------------------74
3.7.3 模擬檢體測試 ----------------------------------------------------75
3.8實驗數據處理 --------------------------------------------------------76
3.9 臨床實測 --------------------------------------------------------------78
3.10 統計分析 ------------------------------------------------------------79
第四章 結果與討論 --------------------------------------------------------80
4.1 目標氨基酸片段的篩選 --------------------------------------------80
4.1.1 模擬結果與探針測試的比較 ----------------------------------92
4.1.2 分子模擬結果討論 ----------------------------------------------94
4.2 晶片表面處理 --------------------------------------------------------94
4.2.1 單層處理與多層處理的比較 ----------------------------------95
4.2.2 單層處理與空白晶片的比較 ----------------------------------99
4.2.3 表面處理結果討論 ----------------------------------------------100
4.3 探針熱處理 -----------------------------------------------------------101
4.3.1 探針熱處理的比較 ----------------------------------------------102
4.3.2 探針熱處理的討論 ----------------------------------------------102
4.4 組成感測模組 --------------------------------------------------------104
4.4.1 模組探針的特異性 ----------------------------------------------104
4.4.2 丙酮靈敏度與濃度的關係 -------------------------------------110
4.4.3 組成感測模組的討論 -------------------------------------------111
4.5 模擬檢體測試 --------------------------------------------------------111
4.5.1 模擬檢體測試結果 ----------------------------------------------112
4.5.2 模擬檢體測試的討論 -------------------------------------------115
4.6 臨床測試 --------------------------------------------------------------115
4.6.1 採樣 ----------------------------------------------------------------115
4.6.2 呼氣測試結果及分析 -------------------------------------------116
4.6.3 階層聚類分析與主成份分析 ----------------------------------126
4.6.4分析結果討論 -----------------------------------------------------129
第五章 結論與建議 --------------------------------------------------------130
第六章 參考文獻 -----------------------------------------------------------132
圖 目 錄

頁次
圖2.1典型的（生物）化學感測器裝置 -----------------------------5
圖2.2葡萄糖酵素電極的作用原理 ----------------------------------8
圖2.3機械性與電性的交互作用 -------------------------------------14
圖2.4壓電效應示意圖 -------------------------------------------------14
圖2.5石英晶體及壓電石英晶片 -------------------------------------15
圖2.6石英晶體的軸系 -------------------------------------------------16
圖2.7石英晶體各種切割方向及角度 -------------------------------17
圖2.8石英晶體各種切割角度震盪頻率與溫度的關係 ----------17
圖2.9石英晶片的波動情形 -------------------------------------------18
圖2.10有機揮發物（VOCs）在體內流動的式意圖 ---------------20
圖2.11脂肪分解路徑 ----------------------------------------------------30
圖2.12酮體的交互關係 -------------------------------------------------31
圖2.13酮體的形成、釋放與利用 -------------------------------------31
圖2.14人類細胞色素P450的基因家族 ------------------------------36
圖2.15P450IIE1的誘導物及受質 -------------------------------------38
圖2.16CytochromeP4502E1的酵素催化位置 -----------------------40
圖2.17電子在膜上P450s的傳遞路徑 --------------------------------41
圖2.18由酵素所製成的氧化電極生物感測器 ----------------------42
圖2.19由酵素所製成的離子選擇性場效電晶體生物感測器 ----43
圖2.20由酵素所製成表面漿膜共振（SPR）生物感測器 --------43
圖2.21自我排列單層膜示意圖 ----------------------------------------45
圖3.1壓電石英氣體分析儀裝置 -------------------------------------50
圖3.2氣體測試反應槽圖 ----------------------------------------------51
圖3.3壓電石英晶體感測器示意圖 ----------------------------------51
圖3.4壓電石英晶片的尺寸及照片 ----------------------------------52
圖3.5SMELL軟體的主畫面 ------------------------------------------53
圖3.6參數設定圖 -------------------------------------------------------54
圖3.7頻率範圍設定圖 -------------------------------------------------54
圖3.8頻率穩定的測定畫面 -------------------------------------------55
圖3.9探針吸附與脫附的頻率變化圖 -------------------------------56
圖3.10計算吸附前後的頻率差值圖 ----------------------------------56
圖3.11SWISS-MODEL的First Approach mode網頁 -------------60
圖3.12t-Boc及F-moc保護基的結構式 -----------------------------63
圖3.13氨基酸序列合成的反應流程 ----------------------------------64
圖3.14EDC/NHS reaction scheme --------------------------------------69
圖3.15固定化方法二及固定化方法三圖示 -------------------------70
圖3.16探針熱處理方法 -------------------------------------------------71
圖4.1Cytochrome P4502E1的三級結構模擬 ----------------------81
圖4.2編號A序列與丙酮的親和模擬 -------------------------------83
圖4.3編號B序列與丙酮的親和模擬 -------------------------------84
圖4.4編號C序列與丙酮的親和模擬 -------------------------------85
圖4.5編號D序列與丙酮的親和模擬 -------------------------------86
圖4.6編號E序列與丙酮的親和模擬 --------------------------------87
圖4.7編號F序列與丙酮的親和模擬 --------------------------------88
圖4.8編號G序列與丙酮的親和模擬 -------------------------------89
圖4.9編號H序列與丙酮的親和模擬 -------------------------------90
圖4.10編號B-1序列與丙酮的親和模擬 -----------------------------91
圖4.11探針對丙酮的實測值 -------------------------------------------93
圖4.12固定化方法一及方法二的比較 -------------------------------97
圖4.13固定化方法一及方法三的比較 -------------------------------98
圖4.14固定化方法二及方法三的比較 -------------------------------98
圖4.15探針熱處理的比較 ----------------------------------------------103
圖4.16E探針的單一氣體測試 -----------------------------------------105
圖4.17N1探針的單一氣體測試 ---------------------------------------107
圖4.18N2探針的單一氣體測試 ---------------------------------------107
圖4.19A1探針的單一氣體測試 ---------------------------------------109
圖4.20A2探針的單一氣體測試 ---------------------------------------109
圖4.21丙酮吸附與濃度的關係圖 -------------------------------------110
圖4.22健康人呼氣的模組感測圖譜 ----------------------------------112
圖4.23健康人的呼氣圖譜與模擬糖尿病酮酸症病人呼氣指標氣體丙酮圖譜之比較 ----------------------------------------------113
圖4.24健康人的呼氣圖譜與模擬肝硬化病人呼氣指標氣體氨圖譜之比較 ----------------------------------------------------------113
圖4.25健康人的呼氣圖譜與模擬肝硬化病人呼氣指標氣體乙酸圖譜之比較 -------------------------------------------------------114
圖4.26健康人的呼氣圖譜與模擬肝硬化病人呼氣指標氣體丙酸圖譜之比較 -------------------------------------------------------114
圖4.27健康人的呼氣模組感測圖譜 ----------------------------------120
圖4.28健康人的呼氣模組感測模式圖譜 ----------------------------120
圖4.29糖尿病一型患者的呼氣模組感測圖譜 ----------------------121
圖4.30糖尿病一型患者的呼氣模組感測模式圖譜 ----------------121
圖4.31糖尿病一型二型之間患者的呼氣模組感測圖譜 ----------122
圖4.32糖尿病一型二型之間患者的呼氣模組感測模式圖譜 ----122
圖4.33糖尿病二型患者的呼氣模組感測圖譜 ----------------------123
圖4.34糖尿病二型患者的呼氣模組感測模式圖譜 ----------------123
圖4.35糖尿病酮酸症患者的呼氣模組感測圖譜 -------------------124
圖4.36糖尿病酮酸症患者的呼氣模組感測模式圖譜 -------------124
圖4.37健康人與糖尿患者感測模式圖譜的比較 -------------------125
圖4.38臨床檢體樣品的主成分分析 ----------------------------------127
圖4.39臨床檢體樣品的聚類分析---------------------------------------128
表 目 錄

頁次
表2.1生物感測器的分類 --------------------------------------------11
表2.2可作為呼氣檢測診斷疾病的重要生物指標氣體 --------22
表2.3不同呼氣檢測分析方法的比較 -----------------------------25
表2.4丙酮與血糖濃度的關係 --------------------------------------35
表3.1疾病與呼出指標氣體的濃度 --------------------------------73
表4.1實測探針說明 --------------------------------------------------93
表4.2晶片的分層製備 -----------------------------------------------97
表4.3第一層（SAM1）與空白晶片的比較 ---------------------99
表4.4糖尿病人呼氣樣本採集結果 --------------------------------117

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