臺灣博碩士論文加值系統

一直以來結核病被視為最嚴重的傳染性疾病之一，隨著科學與醫療技術的進步，其診斷方法也不斷地推陳出新，但卻無法發展出一套快速、方便且有效的診斷方法。本研究採用金奈米粒子比色法感測分析與微流體試紙分元件(mPADs)於結核病的診斷。使用未作修飾的金奈米粒子與單股檢測的寡核苷酸序列來達到快速診斷，並利用DNA的雜交作用(hybridization)使單股的寡核甘酸序列與結合分枝桿菌鍵結再一起，以此做為檢測結合分枝桿菌的感應探針，最後以加入食鹽溶液(NaCl)來誘導金奈米粒子聚集進而使膠體溶液產生顏色變化，藉此變色機制來進行結核病之診斷。此方法免除了複雜以及耗時的硫醇化或其他表面改質等方法來製作金奈米粒子生化感測探針的過程，此外並與微流體試紙分析元件做結合，提供了濃縮偵測樣品、提高靈敏度，並可同時記錄多個不同檢測結果的顏色變化，接著利用智慧型手機等裝置將檢測結果傳輸至雲端計算，以得到檢測結果資料。利用此方法可以排除使用精密、複雜的儀器來進行高靈敏度的數據分析，同時也具備了平行多工病理判讀的功用。目前研究結果顯示，此檢測平臺能輕易地檢測到從病患萃取出的結核病分枝桿菌目標序列濃度2.6 nM，並能在1小時內完成結核病的分析，吾人相信此平臺具備應用於資源匱乏之地區，並對結核病進行立即性診斷與即時監控的可能性。

Tuberculosis has always been considered one of the most serious infectious disease, with the advancement of science and medical technology, people continuously find new ways of diagnosing tuberculosis from old theories, but still unable to develop a real-time, convenient and effective diagnostics. In this study, a colorimetric sensing strategy employing gold nanoparticles and a paper assay platform has been developed for tuberculosis diagnosis. Unmodified gold nanoparticles and single-stranded detection oligonucleotides are used to achieve rapid diagnosis, through the theory of DNA hybridization, single-stranded detection oligonucleotides and mycobacterium tuberculosis (MTB) will combine together to become a MTB-detection probe, and the addition of high-concentration sodium chloride solution will induce gold nanoparticles aggregate and make the colloid color change which is regarded as the mechanism to proceed the diagnosis of tuberculosis. This method avoids using complicated and time-consuming thiolated or other surface-modified probe preparation process. Besides combining with paper assay platform can also eliminate the use of sophisticated equipment for data analysis, the color variance for multiple detection results was simultaneously collected and concentrated on cellulose paper with the data readout transmitted for cloud computing via a smartphone. The results show that the 2.6 nM tuberculosis mycobacterium target sequences extracted from patients can easily be detected, and the turnaround time after the human DNA is extracted from clinical samples is approximately 1 hour. We believe the proposed platform possesses the potential for tuberculosis diagnosis monitoring in resource constrained settings.

致謝 i
摘要 iii
Abstract iv
目錄 v
圖目錄 vii
表目錄 ix
第一章 前言 1
1.1研究動機 1
1.1.1 結核病的來源 1
1.1.2 結核病對人體的影響 2
1.2偵測方法回顧 3
1.2.1 結核病診斷技術發展 3
1.2.2 奈米材料之應用於生醫檢測 5
1.2.3 金奈米粒子之特性 10
1.2.4 金奈米粒子合成方法 11
1.2.5 層析紙(Chromatography paper)之發展與應用 15
1.2.6 層析紙的檢測 15
1.2.7 層析紙的應用 17
1.3本論文使用之原理與方法 22
1.3.1金奈米粒子之化學與光物理特性 22
1.3.2金奈米粒子於結核病診斷之應用 24
1.3.3本研究之目標 25
第二章 材料與實驗方法 26
2.1實驗藥劑與樣品 26
2.2儀器設備 27
2.3溶液配置 28
2.3.1緩衝溶液 28
2.3.2 Single-stranded DNA(ss-DNA) 溶液配置 28
2.3.3氯化鈉溶液 29
2.4金奈米粒子的製備 29
2.5從脊髓組織萃取出的結核病DNA 30
2.6用聚合?連鎖反應(PCR)製備IS6110序列的123鹼基對(bp)擴增因子至目標結核分枝桿菌 30
2.8層析紙分析元件圖形製作 31
2.7結核病於層析紙基材分析元件之比色法診斷 31
2.9真實樣品檢測 32
第三章 結果與討論 34
3.1金奈米粒子比色法結合微流體試紙分析元件於結核病診斷之實驗步驟 34
3.2不同加熱時間於結核病檢測之最佳化測試 35
3.3不同加熱溫度於結核病檢測之最佳化測試 39
3.4不同DNA濃度比例於結核病檢測之最佳化測試 43
3.5不同單股檢測DNA序列對結核病檢測之影響 47
3.6不同單股序列長度對結核病檢測之影響 52
第四章 結論 57
參考文獻 58

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