臺灣博碩士論文加值系統

(1) 甲基安非他命的檢測
毒品是指非醫療目的，卻過度使用，產生迷幻、中樞神經興奮的作用，對身體有極大危害，且經由毒品所衍生出來的各種犯罪時有所聞，因此取締毒品有其必要性。本研究希望偵測的毒品為甲基安非他命(methamphetamine, 簡稱 MA)，是台灣近幾年來最流行的毒品之一，成癮後容易產生精神分裂或憂鬱症，甚至造成身體多種器官損傷。
目前衛生署所採用的檢驗方式包含初步檢驗（使用酵素免疫分析）與確認檢驗（使用氣相層析質譜儀），本研究最終目的希望將fiber-optic particle plasmon resonance (FOPPR) 系統作為初步檢驗，並以LC-MS (liquid chromatography-mass spectrometry) 作為確認檢驗，整合成一套線上分析方法，有效的定性與定量甲基安非命。由於FOPPR對於直接偵測小分子較不靈敏，故先將MA與蛋白質結合，衍生化成MA-protein conjugate，先用FOPPR進行感測後，再使用脫附溶液如glycine buffer等，將MA-protein conjugate從光纖上脫附下來，進入LC-MS進行結構鑑定。

(2) 較小蛋白質在FOPPR系統與ESI-MS的偵測極限比較
由於FOPPR系統對於大分子量的蛋白質具有較好的靈敏度，LC-MS則是在偵測小分子量的分子具有較佳的靈敏度，當該蛋白質與探針分子結合常數較小時，與FOPPR二者之間為互補關係。因此對於較小分子量的蛋白質，需要藉由實驗比較二者檢量線之間的偵測極限 (limit of detection, LOD) 與線性範圍，以選擇適當的定量工具。
我們選擇了二種不同分子量的蛋白質進行比較，實驗結果而言，分子量較大的OVA (44.3 kDa) 在FOPPR與LC-MS偵測極限較為接近；分子量較小的myoglobin (17 kDa) LC-MS的偵測極限比FOPPR低。

(1) Determination of illicit drugs methamphetamine (MA)
Use of illicit drugs often causes addictions, resulting in severe harms to health. Illicit drugs have also been responsible for a recent surge of diverse crimes and a series of social problems in Taiwan. In this study, we aim to integrate FOPPR biosensor with LC-MS to develop a detection system for illicit drug determination as an alternative method to standard protocols which are not able to carry out fast screening test of large amount of suspected samples and confirmation test of much smaller amount of identified samples with one instrumentation system.
We firstly target on detecting methamphetamine (MA, M.W. 149 Da) because of a recent increase in the numbers of addicts to this stimulant drug. Since FOPPR has no ideal sensitivity to determine small molecule such as MA, therefore MA was conjugated with bovine serum albumin (BSA) as an adduct to improve sensitivity. We started with detecting MA-BSA conjugate using FOPPR biosensor and then desorpted the conjugate from fiber and flushed the stripped conjugate to inject into LC-MS for structure confirmation.

(2) Comparison detection limit of small protein between FOPPR and LC-MS
FOPPR system is known to provide superior sensitivity in detection of large protein. On the contrary, LC-MS system is a more suitable tool to detect small molecules. Therefore LC-MS and FOPPR were complementary tools to detect molecules of various molecule weights. However, when the binding constant between probe molecule and the target protein is small, the sensitivity and linear range differences between using FOPPR and using LC-MS are in need of investigations to choose the more suitable tool for quantitations.
We studied two proteins ovalbumin (OVA) and myoglobin of which the molecular weights are 44.3 kDa and 17 kDa, respectively, and found that, LC-MS and FOPPR have similar detection limits to determine OVA and LC-MS has superior detection limit than FOPPR to determine myoglobin.

目錄 iii
圖目錄 vii
表目錄 xi
第一章 緒論 1
1-1 研究動機 1
1-1-1 濫用藥物甲基安非他命的檢測 1
1-1-2 較小蛋白質在FOPPR系統與LC-MS偵測極限比較 3
1-2 文獻回顧 4
1-3 免標記光纖式奈米生物感測器 9
1-3-1 光纖簡介與漸逝波 9
1-3-2 粒子電漿共振 12
1-3-3 自我組裝單層 13
1-3-4 免標記光纖式奈米生物感測器 15
1-4 液相層析電噴灑質譜儀 (LC-ESI-MS) 17
1-4-1 電噴灑游離源 18
1-4-2 四極柱質量分析器 20
第二章 實驗方法 23
2-1 實驗藥品與溶液配製 23
2-2 實驗儀器與工具 27
2-3感測光纖製備 29
2-4 圓球形金奈米粒子合成與固定化金奈米粒子於光纖表面 30
2-4-1有機相圓球形金奈米粒子合成步驟與固定化 30
2-4-2水相圓球形金奈米粒子合成步驟與固定化 31
2-5 修飾混合自我組裝單層與探針分子於金奈米粒子表面 33
2-6 免標記光纖式奈米生物感測器 35
2-6-1 免標記光纖式奈米生物感測器架構 35
2-6-2 感測晶片封裝 36
2-6-3 粒子電漿共振感測器靈敏度 37
2-6-4 利用免標記光纖式奈米生物感測器偵測生化樣品 38
2-7 合成甲基安非他命牛血清蛋白共軛物 38
2-8 間接型酵素免疫分析法 (Indirect-ELISA) 檢測甲基安非他命結合牛血清蛋白共軛物 (MA-BSA) 39
2-9液相層析電噴灑游離質譜儀 41
2-9-1 流動相 (mobile phase) 41
2-9-2 偵測參數設定 42
第三章 結果與討論 44
3-1 金奈米粒子SEM圖 44
3-2濫用藥物甲基安非他命檢測 45
3-2-1 FOPPR感測器靈敏度 45
3-2-2 甲基安非他命抗體與甲基安非他命牛血清蛋白共軛物鍵結能力與非特異性吸附測試 47
3-2-3 利用FOPPR檢測甲基安非他命牛血清蛋白共軛物 51
3-2-4 利用質譜儀檢測甲基安非他命牛血清蛋白共軛物 56
3-3較小蛋白質在FOPPR系統與LC-MS偵測極限比較 64
3-3-1 FOPPR偵測卵白蛋白 64
3-3-2 LC-MS偵測卵白蛋白 66
3-3-3 FOPPR偵測肌紅蛋白 69
3-3-4 LC-MS偵測肌紅蛋白 70
3-3-5 FOPPR與LC-MS偵測極限比較 74
第四章 結論 75
4-1 濫用藥物甲基安非他命的檢測 75
4-2 較小蛋白質在FOPPR系統與LC-MS偵測極限比較 75
第五章 參考文獻 76

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