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研究生:方嘉嘉
研究生(外文):Chia-Chia Fang
論文名稱:高分子螢光奈米顆粒結合免疫色層分析試紙應用於癌症標記蛋白檢測
論文名稱(外文):Development of semiconducting polymer dots-based Immunochromatography test strip for cancer biomarker diagnosis
指導教授:詹揚翔
指導教授(外文):Yang-Hsiang Chan,
學位類別:碩士
校院名稱:國立中山大學
系所名稱:化學系研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:102
中文關鍵詞:螢光訊號比值螢光共振能量轉移癌症標記物免疫色層分析試紙半導體高分子奈米顆粒
外文關鍵詞:Förster resonance energy transferRatiometricCancer biomarkerImmunochromatography test stripSemiconducting polymer dots
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本篇研究利用螢光高分子奈米顆粒 (Semiconducting Polymer Dots, Pdots) 具備良好的光學特性、高能量傳遞與低生物毒性等等不同優點而來設計生物分析探針。除此之外,還結合FRET (Förster resonance energy transfer) 能量傳遞,發展出更多不同層面的應用,因此,我們利用了螢光高靈敏度與高亮度特性,將半導體高分子奈米顆粒作為主要檢測材料,希望搭配不同方法用以檢測不同癌症標記物。

(一) 高分子螢光奈米顆粒結合免疫色層分析試紙應用於癌症標記蛋白檢測
在現代生活中,癌症好發率逐年往上升,但大多檢測相對耗費較長時間,與特地地點及專業人員在場,才能進行檢測,因此希望可以針對對癌症抗原進行快速篩檢的目標物,並搭配免疫色層分析試紙來進行檢測,希望可以有效的降低檢測的時間,並可以進行快速與有效的定性與定量,以減少時間與資源的消耗。
我們在此題目中,因此我們開發出一種能夠快速達到定性與定量的半導體高分子奈米顆粒探針,我們將具不同螢光顏色的半導體高分子奈米顆粒(Semiconducting Polymer Dots, Pdot),修飾上對不同目標抗原具有專一性的抗體,再利用試紙上畫有測試線與控制線,當存在目標物時藉由液體的毛細現象的流動到達測試線與控制線,並在測試線上形成三明治體結構,而達到快速檢測效果,並具有定性的結果;另一方面,在不同濃度的目標物存在時,藉由測試線與控制線上所鍵結的亮度比值判斷,來進一步做到定量效果。

關鍵字: 半導體高分子奈米顆粒、免疫色層分析試紙、癌症標記物、螢光訊號比值

(二) 、利用FRET發展雙顯色高分子螢光奈米顆粒於免疫色層分析試紙
現今市售化試紙用以檢測癌症標記抗原的產品越趨多元,所使用的材料與檢測方法變化也越多,如果可以將單一顏色吸收或是放光改為直接判別顏色差異,在兩個方法比較之下,判別方式也可以較為直觀與快速。
而因此在免疫色層分析試紙上,通常是藉由測試線與控制線上顯色能夠來有效判別檢測結果,但當需要進一步的有效判別,還是必須透過兩條線上的亮度差異來進行比值的結果判讀,不過大多數材料多屬單一顏色的吸收深淺或放光強度差異來判斷,而我們期望可以利用半導體共軛高分子此材料,於可見光區可以具有多種不同放光波長,藉由吸收與放光具有重疊Pdots彼此之間具有的螢光共振能量轉移 (Förster resonance energy transfer, FRET) 效率,希望可以在不需要額外數據處理的階段,進一步的避免了儀器操作或是軟體輸出等等,來對於測試線與控制線上亮度比例的微小差距進行定量,改為使用顏色來立即判別,即可有效區別出該檢測檢體的濃度高低差異,做出該檢測目標物結果的判斷。

關鍵字: 半導體高分子奈米顆粒、免疫色層分析試紙、螢光共振能量轉移
In this study, fluorescent polymer nanoparticle (Pdots) with good optical properties, high energy transfer, low toxicity and different advantages were used as fluorescent probe combined with test paper to detect cancer biomarker. In the first part of my study, the test paper was drawn two lines on it, control line and test line which can selectively attach to the Pdots bioconjugated with antibody and the test paper could be easily told that either one line bright (negative) or two lines bright (positive). In the second part of study, we improved the experiment method by combining Pdots with FRET mechanism. It could be applied on more challenging cancer biomarker detection and lower the expense of the detection procedure. Therefore, based on our study’s outcome pdots combined with test paper had great potential on point-on-care cancer diagnosis.

1. Development of semiconducting polymer dots-based Immunochromatography test strip for cancer biomarker diagnosis
Recently, the incidence of cancer increased year by year, but the procedure cost longer time to detect, need professional staff and specific location. We developed a rapid method to detect cancer biomarker based on immunochromatographic test strips, which can reduce the detection time effectively, and detect accurately in order to decrease the detection time and resources.
We embedded the specific antibody into the different fluorescent colors matrix of 5-(5-(9,9-dioctyl-9H-fluoren-2-yl)-4-hexylthiophen-2-yl)-6,7-difluoro-2,3-bis(3-(hexyloxy)phenyl)-8-(4-hexylthiophen-2-yl)quinoxaline (TC6FQ), poly[(Z)-3-(4-(9,9-dioctyl-9H-fluoren-2-yl)phenyl)-2-phenylacrylonitrile] (PFCN), and 9,9-dioctyl-9H-fluorene (PFO), which selectively bound to the antigen and the sandwich structure was formed on the test line. The result determined by the brightness of two lines, and we used the brightness of the test line over the control line to determine the ratiometric.
Keyword:Semiconducting polymer dots、Immunochromatography test strip、Cancer biomarker、Ratiometric

2. Development of semiconducting polymer dots-based Immunochromatography test strip by FRET
There are many commercially test strips to detect cancer biomarker, and the more materials and methods are developed. If only the single absorption or fluorescence existence, it may hard to determine directly.
We used semiconductor conjugated polymer dots in the immunochromatographic test paper, the ratiometric of the test line over the control line always was the results. We purposed a new method to determine results conveniently, so we fabricated easy-to-prepare test strips by Förster resonance energy transfer (FRET) between contrasting color Pdots, had a variety of different emission wavelength in visible region. In this new idea, it doesn’t need additional instrumentation or software to process data and distinguish the results effectively at the different concentrations.
Keyword:Semiconducting polymer dots、Immunochromatography test strip、Förster resonance energy transfer
論文審定 i
摘要 ii
Abstract iv
目錄 vii
圖表目錄 x
縮寫表 xiv

第一章、 緒論 1
一、前言 1
二、歷史 3
第二章、Point-of-care (POC) 10
一、前言: 10
二、歷史: 11
三、應用 19
3-1. Electrochemical 19
3-2. Microfluid 21
3-3. 免疫色層分析試紙 22
四、免疫色層分析試紙 23
五、原理 25
5-1. 免疫分析: 25
5-2. 抗原 (Antigen): 25
5-3. 抗體 (Antibody): 25
5-4. 檢測方式: 27
5-5. 檢測材料: 29
六、研究動機: 31
第三章、Development of semiconducting polymer dots-based Immunochromatography test strip for cancer biomarker diagnosis 32
一、前言: 32
二、實驗藥品與器材: 34
2-1. 藥品: 34
2-2. 儀器: 37
2-3. 樣品配製方法 39
2-4. 實驗步驟 41
三、實驗設計原理 44
4-1. 半導體高分子奈米顆粒以及抗原的選擇 47
4-2. 半導體高分子奈米顆粒光學及物理特性探討 49
4-3. 抗原選擇性測試 53
4-4. 時間條件優化 55
4-5. 癌症抗原濃度定量 56
4-6. 真實樣品: 60
四、 結論: 61
第四章、Development of semiconducting polymer dots-based Immunochromatography test strip by FRET 62
一、前言: 62
二、實驗藥品與器材: 63
2-1. 藥品: 63
2-2. 儀器: 66
2-3. 樣品配製方法 68
2-4. 實驗步驟 69
三、實驗設計與原理: 71
四、實驗結果與討論: 75
4-1. 半導體高分子奈米顆粒: 75
4-2. FRET條件測試: 76
4-3. 實驗設計原理改善: 79
4-4. 條件改善: 81
4-5. 未來方向: 82
參考文獻 83
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