臺灣博碩士論文加值系統

本研究提出一個結合微流體式紙分析平臺進行駕駛人濫用愷他命之定點快速唾液篩檢。此一新穎的毒品檢測平臺，優點為可同時且有效偵測唾液中藥物濃度、無須複雜的設備與額外能源、避免冗長的檢測過程，此外，此偵測系統證實能直接使用於未過濾唾液檢體，藉由搭配有效單株抗體，對待測檢體進行高特異性篩選，達到唾液中愷他命之快速定點篩檢目的。
此平臺已證實可發展為路邊定點篩檢之有效平臺，在資源侷限的環境下，具備高檢測靈敏度、高專一性以及高度重複性、並有效縮短檢測時間，達到快速篩選的目的，初步實驗結果顯示，使用微流體式紙分析平臺結合唾液檢測可達到愷他命偵測極限為 10 ng/mL，並能於 30 分鐘內進行單組十項樣品同時測試，或於 6 分鐘內完成單一樣品之檢測。
 

A novel oral fluid testing platform based on microfluidic paper‐based analytical devices (µPADs) is proposed for on-site high-throughput monitoring of driving under the influence of drug (DUID). A key advantage of the proposed technology is the ability to simultaneously and effectively measure low concentration of ketamine in oral fluid without the need of complex equipment, power sources and long processing time. The detection system will provide key elements including (a) adequate, consistent and high recovery oral fluid collection without dilution, (b) selection of antibodies for high-specific immunoassay screening of specific drug classes, (c) high sensitive and high throughput µPAD fabrication and characterization for capturing and concentrating abused drugs in oral fluid and (d) surface modification for high density of different types of antibodies binding on cellulose substrates, offering a robust and rapid solution for on-site drug screening. As a result, a ketamine assay with a detection limit of 10 ng/mL, and a single assay can be completed in 6 min.

目錄
誌謝-i
摘要 ii
Abstract iii
目錄 iv
圖目錄 viii
表目錄 xi
第一章前言 1
1.1 研究動機 1
1.1.1 愷他命的簡介 2
1.1.2 愷他命的藥理作用與代謝 3
1.1.3 濫用藥物檢測之生物樣品 5
1.1.4 使用唾液進行駕駛人之藥物濫用檢測之探討 8
1.1.5 我國及世界各國對於愷他命於尿液及唾液檢測之閾值 9
1.1.6 偵測愷他命及其代謝物方法之文獻探討 11
1.2 本文實驗原理與檢測平臺之回顧探討 13
1.2.1重點照護檢驗（Point-of-Care Test） 13
1.2.2 酵素免疫分析法 (Enzyme-linked immunosorbent assay, ELISA） 13
1.2.3 紙的檢測 17
1.2.4微流體式紙平臺回顧 19
1.2.5微流體式紙酵素免疫分析平臺與傳統酵素免疫分析平臺之優缺點比較 21
1.2.6微流體式紙平臺之製作 22
1.2.7 微流體式紙平臺之應用及未來發展 23
1.3 研究目標 23
第二章材料與實驗方法 25
2.1實驗藥劑與樣品 25
2.2儀器設備 25
2.3溶液及樣品配置 26
2.3.1 愷他命抗體稀釋 26
2.3.2 BSA 溶液配置 26
2.3.3 Ketamine HCl樣品配置稀釋 26
2.3.4 Ketamine-HRP溶液稀釋配置 26
2.3.5 PBS Buffer 配置 27
2.3.6 TMB 溶液配置 27
2.4 微流體試紙分析元件製備 27
2.5最佳化參數測試 27
2.5.1使用不同濃度 Ketamine 抗體的呈色結果 27
2.5.2不同濃度 Ketamine-HRP競爭效果測試 28
2.5.3分析於加熱版 (Hotplate) 上不同加熱溫度之影響 28
2.5.4測試不同清洗時間對於呈色影響 28
2.5.5 酵素受質反應時間 29
2.6 標準曲線製作 29
2.6.1 Ketamine HCl 稀釋於磷酸緩衝溶液中測試 29
2.6.2 Ketamine HCl稀釋於唾液中測試 30
2.6.3 Image J軟體個別分析紅、綠、藍 (RGB) 值 30
2.7真實樣品之檢測 31
2.8最佳化參數測試實驗流程圖 31
2.9真實樣品檢測驗流程圖 32
第三章結果與討論 33
3.1 微流體式紙分析元件流程及製備 33
3.1.1微流體式紙分析元件應用於口腔內液體之 Ketamine檢測之實驗流程 33
3.1.2微流體試紙分析元件製備 34
3.2最佳化參數測試 35
3.2.1使用不同濃度 Ketamine抗體的呈色結果 35
3.2.2不同濃度 Ketamine-HRP競爭效果測試 37
3.2.3分析於加熱版上不同加熱溫度之影響 39
3.2.4測試不同清洗時間對於呈色影響 41
3.2.5 酵素受質反應時間測試 43
3.2.6 最佳化參數 45
3.3 標準曲線測試及分析 45
3.3.1 Ketamine HCl稀釋於磷酸緩衝溶液中測試 45
3.3.2 Ketamine HCl稀釋於唾液中測試 47
3.3.3 Ketamine HCl稀釋於唾液中後進行過濾之測試 49
3.3.4標準曲線分析與比較 50
3.3.5 RGB 值分析 51
3.3.6單組與單一樣品測試時間 51
3.4 真實樣品檢測 52
3.4.1 真實樣品取得與檢測 52
第四章 結論 55
參考文獻 57

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