臺灣博碩士論文加值系統

本研究運用一新穎的膜材-具生物活性中孔洞氣凝膠於側向流免疫層析技術上，來偵測抗體間之專一性。實驗選用goat anti-mouse IgG和mouse IgG兩株抗體，作為實驗的待測物與測試線上之捕捉物質。在實驗過程中先將具有生物活性之氣凝膠鋪在試條上，再與mouse IgG進行反應，使抗體上之氨基接於氣凝膠之環氧官能基上，接著再進行側向流試驗，最後以帶有膠體金標定之goat anti-mouse IgG讀取之信號。
本論文結果發現，使用氣凝膠AG60-GL10在37℃下反應4小時為最佳反應條件，偵測結果比硝化纖維膜佳。在相同待測物質濃度及捕捉抗體量之下；觀察得到的結果顯示，氣凝膠訊號值高於傳統快篩膜材硝化纖維膜的訊號值約一倍之多。在偵測靈敏度方面，具生物性的氣凝膠也有較佳的表現。具生物性的氣凝膠於1μg/ml的濃度之下還能以肉眼辨識訊號，但硝化纖維膜肉眼可觀察的偵測極限卻達5μg/ml。

A novel material, bioactive mesoporouse silica, was applied in this study to the lateral-flow immunochromatographic technology for detecting a model biosample. The pair of mouse IgG and goat anti-mouse IgG was chosen as the model analyte and its corresponding recognition antibody, respectively. In the experimental procedure, the epoxy-modified bioactive mesoporouse silica was first spread on a plastic backing card in membrane layer, and then the goat anti-mouse IgG antibody was deposited to interact with the silica material as the test line. The alternative approach was mixing the recognition antibody with the silica material prior to spreading on the backing card. In both approaches, the epoxy group was able to interact with the amino functional group of the antibody to form an active biosensor for its target antigen. The detection result was designed to be read out from a visual signal, generated by nano-gold conjugated antibody.

The results indicated that the optimal condition was mixing the recognition antibody with the silica material at 37 oC for 4 hours, followed a frozen drying. In this condition, the detection limit of the model biosample by unaided eyes was 1μg/ml, which worked better than the conventional material, nitrocellulose. The influence of different dilution solvents for the preparation of nano-gold conjugated antibody was also investigated. The problems and changelings encountered in this study were also discussed as references for future works.

總目錄
中文摘要 I
ABSTRACT II
總目錄 III
圖目錄 VI
表目錄 VIII
第一章 緒論 1
1-1生物感測器 1
1-1.1生物感測器架構及原理 1
1-1.2生物感測器種類 4
1-1.3感測顯色物質 5
1-2奈米材料特性 6
1-3免疫層析試紙分析法 7
1-3.1側向流免疫層析法 7
1-3.2相關文獻回顧 10
1-3.3硝化纖維膜 14
1-3.4具生物活性中孔洞氣凝膠膜材 14
1-4研究動機 15
1-5實驗目的 16
第二章 理論背景 18
2-1快速檢測組件 18
2-2抗體標定金之原理 20
2-3抗原-抗體間之免疫偵測機制 21
2-4蛋白質分子與硝化纖維膜結合機制 23
2-5蛋白質分子與氣凝膠結合機制 24
第三章 材料、儀器與實驗方法 26
3-1材料 26
3-1.1標定膠體金抗體所需材料 26
3-1.2製作偵測試紙所需材料 26
3-1.3其他實驗藥品及生物試劑 27
3-2 實驗儀器及分析軟體 29
3-2.1 Linomat5半自動點樣儀(TLC) 29
3-2.2冷凍乾燥機(Freeze Drying) 29
3-2.3生物晶片掃描機 29
3-2.4掃描式電子顯微鏡(SEM) 30
3-2.5其他儀器及分析軟體 30
3-3實驗溶液製備程序 31
3-4實驗步驟 33
3-4.1膠體金抗體之製備 33
3-4.2將抗體探針固定於硝化纖維膜上 34
3-4.3將抗體探針固定於氣凝膠膜材上 37
3-4.4偵測試片元件之組裝 41
3-4.5偵測試片元件之規格 42
3-4.6於膜材上進行快速檢測 43
3-4.7訊號掃描與數據讀取 44
第四章 結果與討論 48
4-1緩衝溶液對膠體金抗體之影響 48
4-1.1以PBS buffer為緩衝溶液之探討 48
4-2測試片於SEM下之掃描結果 55
4-3具生物活性中孔洞二氧化矽之鑑定 59
4-4快速偵測測試 61
4-4.1 硝化纖維膜試片測試 61
4-4.2具生物活性中孔洞二氧化矽試片測試 64
4-4.3比較材料偵測試片之結果 70
第五章 結論 74
第六章 參考文獻 77
第七章 附錄 84

圖目錄
圖1- 1生物感測器之基本組成元件 3
圖1- 2側向流測試法運用於point-of-care和field-use上的優點 8
圖1- 3免疫偵測：競爭型 9
圖1- 4免疫偵測:三明治型 10
圖1- 5使用兩個接金複合物來提高訊號強度 12
圖1- 6透過還原劑的電子轉移在已標定的奈米金表面鍵結上銀離子 13

圖2- 1快速免疫檢測試劑元件組成 19
圖2- 2膠體金與抗體結合之作用力 20
圖2- 3抗體抗原間結合作用力 22
圖2- 4免疫偵測：兩相對應抗體的捕捉 22
圖2- 5蛋白質吸附機制 23
圖2- 6具生物活性的奈米孔洞二氧化矽薄膜 24
圖3- 1點樣前薄膜於紙張的固定方式(欲噴塗長度10公分為例) 34
圖3- 2 TLC點樣儀及其細部說明 35
圖3- 3具有氣凝膠薄層的Plastic film點樣前之步驟 38
圖3- 4快速偵測元件之組裝 41
圖3- 5快速檢測測試流程圖 43
圖3- 6 Photoshop 7.0軟體作介面 46
圖3- 7GenePixPro6.0操作介面 47
圖4- 1以不同的pH值來製備膠體金抗體 49
圖4- 2以pH值為9的 PBS buffer 稀釋抗體製備不同濃度的膠體金抗體 51
圖4- 3 於4℃下靜置12小時後的膠體金抗體 52
圖4- 4比較膠體金在不同緩衝溶液的聚集程度 54
圖4- 5 膜材於SEM下的表面結構 56
圖4- 6膜材於SEM下的側視圖 58
圖4- 7具生物活性膜材之FTIR鍵定 60
圖4- 8以Donkey anti-goat IgG為捕捉物質之訊號結果 62
圖4- 9以Mouse IgG為捕捉物質之訊號結果 63
圖4- 10於膠體金抗體不同濃度之顯色效果 65
圖4- 11捕捉抗體與具生物活性的氣凝膠於不同反應時間下的結果 67
圖4- 12不同稀釋溶劑 69
圖4- 13膠體金抗體於不同濃度下結果 71
圖4- 14改變捕捉物質的量之比較結果 73


表目錄
表4- 1具生物活性中孔洞二氧化矽之比表面積、孔體積與平均孔徑 59

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