臺灣博碩士論文加值系統

中文摘要
胰島素是一種多胜肽荷爾蒙，由胰臟製造與分泌並參與醣類代謝，進而提供細胞能量以維持生理功能，當身體出現異常，胰島素分泌不足或是胰島素作用不良時，血糖無法維持平衡，而出現糖尿病。膠體奈米金粒子是在免疫層析檢測試片中最常用來標幟抗體的一種，因為其具有穩定性,且肉眼易觀察之優點。此外，金探針標幟抗體並不需要反應時間、清洗步驟及與酶反應等費時的程序來產生信號。本研究目的為開發檢測人類胰島素之競爭型與三明治型奈米金免疫層析試片，以提供初步檢測胰島素以及胰臟健康情況。實驗結果顯示，此自製式競爭型免疫層析試片能於緩衝溶液與小鼠血清中進行檢測，且不會與其它蛋白質有交叉反應，可檢測範圍為5-100 µg人類胰島素，檢測時間約15-20分鐘。本研究所製備的可拋棄式免疫層析試片具有下列優點：1. 高度專一性與靈敏度；2. 樣品不需前處理；3. 所需樣品量少；4. 操作方便；5. 檢測快速；6. 低成本；7. 沒有交叉反應；8. 不需要任何儀器之使用；9. 試片可長期保存。本研究所開發之試片預期可應用於健康檢查之糖尿病篩檢及胰臟健康狀態之評估。

Abstract
Insulin is a peptide hormone that produced and secreted by the pancreas to involve in regulating glucose metabolism and provide cellular energy to maintain the physiological function. When the body is abnormal, the insulin secretion becomes dysfunctional so that the insulin secretion will be unable to maintain blood sugar balance resulted in diabetes. The colloidal gold nanoparticles are the most commonly used detector reagent in these strips for visualization of signals because of their stability and easy visual observation. Moreover, gold probes do not require time-consuming procedures such as incubations, washing steps and enzymatic reactions to generate a signal.
The main purpose of this study is to develop the competitive and sandwich format which based on nano-gold immunochromatographic strip for detection of insulin. We had successfully developed competitive nano-gold immunochromatographic strip system, the strip can provide diabetes screening and the pancreas health diagnosis.
According to the experimental results, the self-assembled immunochromatographic strip can determine human insulin in PBS and mice serum. This results also showed no cross-reaction with other protein, detect the range of 5-100 μg of human insulin and detection time was about 15-20 minutes. The advantages of self-assembled immunochromatographic strip was highly specific and sensitive, no pretreatment, low sample requirement, easy to operate, low cost, rapid determination, no cross-reaction, long-term preservation, no machine needed etc. In this study, the development of the immunochromatographic strip expected to be used for homecare and point-of-care of diabetes, also assess the health status of the pancreas.

目錄
中文摘要 I
英文摘要 II
致 謝 IV
目錄 V
圖表目錄 VII
附錄目錄 VIII
第一章 緒論 1
1-1、研究動機 1
1-2、糖尿病流行病學 2
1-3、糖尿病簡介 2
1-4、人類胰島素 ( insulin ) 6
1-5、人類胰島素 ( insulin ) 作用機轉 7
1-6、免疫分析 ( Immunoassay ) 原理 8
1-7、免疫分析法 11
1-8、免疫層析試片 ( Immunochromatographic strip ) 14
1-8-1、免疫層析試片之組成 14
1-8-2、免疫層析試片檢測之原理 17
1-9、傳統免疫層析檢測試片使用之方法 18
1-9-1、乳膠 19
1-9-2、膠體金 19
1-10、工作大綱 22
第二章、材料與方法 22
2-1、材料 22
2-1-1、試劑 22
2-1-2、儀器設備 23
2-2、免疫層析試片製作 24
2-2-1、膠體金粒子合成 24
2-2-2、膠體金抗體製備過程 24
2-2-3、試片製作 25
2-2-4、試片檢測 25
2-3、免疫層析試片填塞( blocking )效果測試 26
2-4、交叉反應測試 26
2-5、保存期限 26
第三章、結果 27
3-1、人類胰島素競爭型奈米金免疫層析試片檢測 27
3-2、人類胰島素三明治型奈米金免疫層析試片檢測 27
3-3、免疫層析試片填塞效果 28
3-4、交叉反應測試 28
3-5、保存期限 28
第四章、討論 29
4-1、免疫層析試片之製備與檢測 29
4-2、保存期限 30
4-3、膠體金粒子合成 30
4-4、競爭型免疫層析試片 30
4-5、三明治型免疫層析試片 31
第五章、結論 32
參考文獻 33
圖表目錄
圖 1、競爭型試片陽性結果 37
圖 2、競爭型試片陰性結果 38
圖 3、利用競爭型試片檢測緩衝溶液中不同濃度之人類胰島素 39
圖 4、利用競爭型試片檢測小鼠血清中不同濃度之人類胰島素 40
表 1、CLIA方法與本研究試片(STRIP)檢測不同濃度胰島素結果之比較.41
圖 5、利用三明治型試片檢測緩衝溶液中不同濃度之人類胰島素 42
圖 6、競爭型試片填塞配方測試 43
圖 7、三明治型試片填塞配方測試 44
圖 8、以競爭型試片進行交叉反應測試 45
圖 9、競爭型試片於室溫下之保存期限測試 46
圖 10、競爭型試片於冷藏中之保存期限測試 ( D0-D20 ) 47
圖 11、競爭型試片於冷藏中之保存期限測試 ( D30-D60 ) 48
圖 12、競爭型試片於冷藏中之保存期限測試 ( D90 ) 49
附錄目錄
附錄 1、各種免疫分析方法之比較 50
附錄 2、99年國人十大主要死因統計(6) 51
附錄 3、細胞內胰島素之合成 52
附錄 4、人類胰島素胺基酸序列 53
附錄 5、胰島素訊息傳遞路徑(31) 54
附錄 6、免疫分析法原理 55
附錄 7、人類免疫球蛋白之基本單元結構 56
附錄 8、抗原與抗體之間的作用力(11) 57
附錄 9、免疫層析試片之組成(22) 58
附錄 10、三明治型免疫層析檢測流程圖(22) 59
附錄 11、競爭型免疫層析檢測流程圖(22) 60
附錄 12、膠體金粒徑大小與其外觀顏色之特性比較 61
附錄 13、本實驗之主要工作流程 62

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