近年來螢光顯影技術進步，在生物領域上提供有效的工具。除了螢光顯影具有良好的選擇性及靈敏度外，最大的優勢在於具有極佳的空間解析度與時間解析度(Spatial and temporal resolution)。近年來螢光顯影甚至運用到手術上，作為外科醫生在手術上的輔助的工具，稱之為螢光導引手術，突顯實際應用價值。而在螢光顯影中，螢光探針扮演重要的角色，其中，Pdots有大的Absorption cross section與高的量子產率(Quantum yield)、極佳的光穩定性、低的細胞毒性、適當的奈米顆粒(小於30 nm)，以及容易生物偶聯等優點，適合應用在生物領域上，並且已有一系列不同放光波長的Pdots，儘管如此，仍缺少一個良好具有近紅外光螢光的Pdots。近紅外光有較深的穿透度，較少的背景干擾與對生物體傷害較小，然而目前近紅外光的Pdots會由於分子與分子間的堆疊(π–π stacking)造成嚴重的螢光消光(Quenching)，減少實際應用價值，本篇論文中主要致力於設計並合成具有近紅外光性質，並且能減少因堆疊造成螢光消光的高分子材料。
　　Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,1,3}-thiadiazole)](PFBT)為常用的高分子，具有許多優點，然而放光只在540 nm，我們將高分子結構中Benzo-{2,1,3}-thiadiazole(BT)部分的硫(Sulfur)，置換成硒(Selenium)，成為Benzo-{2,1,3}-selenadiazole(BS)，希望藉此產生較小的能階而造成螢光紅移，卻又不對整體結構產生太大的改變，造成嚴重的堆疊現象而螢光消光。實驗結果證明，比起PFBT Pdots，PFBS Pdots放光有紅移的現象(~40 nm)，並且當Fluorene與BS比例為70:30時(PFBS30)，有良好的量子產率(~44 %)，最後應用在生物領域上，PFBS30修飾Streptavidin後標記在細胞上，藉由流式細胞儀與共軛焦顯微鏡可以證實，經PFBS30-streptavidin Pdots成功標記細胞且具有專一性吸附。
　　然而PFBS Pdots放光並未到達近紅外光區域，因此以BS為基底修飾上Thiophene成為4,7-Di(thiophen-2-yl)benzo[c][1,2,5]selenadiazole (DBS)，希望藉由增加結構的共軛系統結構來將放光推至近紅外區。不過DBS高分子因嚴重的堆疊現象而螢光消光，於是在DBS的Thiophene上修飾不同種類的長碳鏈以避免堆疊，結果發現，修飾上己基(Hexyl group)的DBS(C6)可以提升Flourene與DBS比例至90:10(PFDBS(C6)10)，同時具有不錯的量子效率(~15 %)與近紅外放光光譜(710 nm)。另外在高分子結構上以共價鍵，修飾羧酸官能基，有利於標記細胞。最終應用在生物領域上，PF-COOH3-DBS(C6)10修飾Streptavidin後標記在細胞上，藉由流式細胞儀與共軛焦顯微鏡可以證實，PF-COOH3-DBS(C6)10-streptavidin Pdots成功標記細胞且具有專一性吸附。

Semiconducting polymer dots (Pdots) have recently emerged as a new class of extraordinarily bright fluorescent probes with promising applications in biological imaging and sensing. Herein we synthesized a novel series of highly emissive orange-fluorescent copolymers, poly[9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,19-3}-selenadiazole)] (PFBS) and tuned the ratio of fluorene to benzoselenadiazole (BS) from 98 : 2 to 50 : 50 to investigate the influence of BS molar ratio on the emission properties of the resulting Pdots. An optimal quantum yield of 44% could be obtained for PFBS Pdots at a fluorene to BS ratio of 70 : 30. These PFBS Pdots also exhibited great photostability and superior single-particle brightness. We next conjugated biomolecules onto the surface of these PFBS Pdots and demonstrated their ability for specific cellular labeling without any noticeable nonspecific binding. We are now working on the synthesis of near-infrared Pdots based on this BS unit and anticipate this series of ultrabright Pdots will be very useful in a variety of in vitro and in vivo bioimaging applications.
In recent years, semiconducting polymer dots (Pdots) have emerged as a new class of extraordinarily bright fluorescent probes with burgeoning applications in biological imaging and sensing. With the increasing demand for near-infrared (NIR)-emitting probes for in vivo biological measurements, the direct synthesis of semiconducting polymers that can form Pdots with ultrahigh fluorescence brightness are extremely lacking due to the severe aggregation-caused quenching of the NIR chromophores in Pdots. Here we describe the design and synthesis of dithienylbenzoselenadiazole (DBS)-based NIR-fluorescing Pdots with ultrahigh brightness and excellent photostability. More importantly, the fluorescence quantum yields of these Pdots could be effectively increased by the introduction of long alkyl chains into the thiophene rings of DBS to significantly inhibit the aggregation-caused emission quenching. Additionally, these new series of DBS-based Pdots can be excited by a commonly used 488 nm laser and show a fluorescence quantum yield as high as 36% with a Stokes shift larger than 200 nm. Single-particle analysis indicates that the per-particle brightness of the Pdots is at least 2 times higher than that of the commercial quantum dot (Qdot705) identical laser excitation and acquisition conditions. We also functionalized the Pdots with carboxylic acid groups and then conjugated biomolecules onto the surface of Pdots to demonstrate their capability for specific cellular labeling without any noticeable nonspecific binding. Our results suggest that these DBS-based NIR-fluorescing Pdots will be very useful in a variety of bioimaging and analytical applications.

論文審定書 i
謝誌 ii
中文摘要 iv
Abstract vi
圖目錄 xii
表目錄 xvii
化學結構縮寫表 xviii
第一部分 1
第一章　前言 1
第二章　實驗 11
2-1　實驗藥品 11
2-2　實驗儀器 13
2-3　合成部分 17
2-4　Pdots 製備方法 41
2-5　Bioconjugation 45
2-6　細胞標記 46
第三章　結果討論 49
3-1　設計與反應探討 49
3-2　Pdots製備與顆粒大小 54
3-3　光學性質探討 56
PFBS、PFBT-DBS與PFBT-DBS(C6)各別的吸收光譜和放光光譜 64
3-4　計算莫耳吸收係數 69
3-5　單一顆粒亮度(Single particle brightness)與光穩定性(Photostability) 71
3-6　專一標記細胞 74
第四章　結論 79
第五章　參考資料 79
第二部分 82
第一章　前言 82
第二章　實驗 83
2-1　實驗藥品 83
2-2　實驗儀器 84
2-3　合成部分 86
三　結果與討論 91
3-1　合成探討 91
第四章　結論 93
第五章　參考資料 93
第三部分 94
第一章　前言 94
第二章　實驗 94
2-1　實驗藥品 94
2-2　實驗儀器 95
2-3　合成部分 96
第三章　結果與討論 97
3-1　合成探討 97
第四章　結論 99
第五章　參考資料 99

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