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博碩士論文 etd-0704115-105308 詳細資訊
Title page for etd-0704115-105308
論文名稱
Title
使用高亮度螢光奈米顆粒結合不同分析平台,並應用於:(1)檢測生物體內鉛離子;(2)使潛伏指紋可視化
Applications of Highly Fluorescent Semiconducting Polymer Dots:(1) In Vitro Detection of Lead Ions;(2) Visualization of Latent Fingerprints
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
116
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2015-07-07
繳交日期
Date of Submission
2015-08-05
關鍵字
Keywords
半導體高分子奈米顆粒、寧海德林、孔洞材質、胺基酸、聚乙二炔、螢光共振能量轉移、鉛離子、非孔洞材質、螢光訊號比值
polydiacetylenes, poly(vinyl alcohol), Semiconducting Polymer Dots, sandwich complex, blue shift, Porous, Ninhydrin, Förster resonance energy transfer, hydrophobic interactions, Ruhemann's purple
統計
Statistics
本論文已被瀏覽 5721 次,被下載 35
The thesis/dissertation has been browsed 5721 times, has been downloaded 35 times.
中文摘要
本篇研究利用螢光高分子奈米顆粒(Semiconducting Polymer Dots, Pdots)的良好的光學特性、高能量傳遞與低生物毒性等優點來設計分析探針。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,在此我們利用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分析主體,並應用於不同的分析平台上,應用分別為金屬離子的偵測以及潛伏指紋的可視化。

(一) 開發比色法與螢光雙偵測系統的半導體高分子奈米探針,並可快速定性與
定量生物體中的鉛離子:
在日常生活中,鉛金屬往往無所不在。而鉛金屬要進入人體非常容易,其途徑有分呼吸與食入方式,當血液中的鉛蓄積到一定程度後,會使人記憶力、智力下降,影響生育能力,出現精神障礙、失眠等問題。鉛中毒對兒童的危害更大,容易引起難以復原兒童智力發育障礙。
  因此在此我們開發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。我們將聚乙二炔(Polydiacetylene, PDA)包覆在半導體高分子奈米顆粒(Semiconducting Polymer Dots, Pdot)外,再利用PDA的羧酸與另外修飾上的15-crown-5 ether官能基對鉛離子具有選擇性形成三明治體,使PDA共振結構變形進一步變色,而達到快速定性功效;另一方面,當PDA共振結構破壞時,Pdot中包覆的近紅外光695 nm染劑就會因為缺少PDA包覆,使染劑流至Pdot外,而原本的螢光共振能量轉移(Förster resonance energy transfer, FRET)機制因此中斷,螢光將從波長715 nm移動至650 nm左右,此時藉由螢光訊號比值的改變,進一步達到定量功能。

(二) 結合半導體高分子奈米顆粒與寧海德林,使孔洞及非孔洞材質中的潛伏指紋具有明顯呈色與高亮度螢光:
  在社會中犯罪行為層出不窮,使警察尋找在犯罪現場找到任何有關犯人的證據,除了人證物證等,還有指紋採證。因為指紋獨一無二,因此可藉指紋採證,來達到捕捉犯人。但在現今指紋採證中,多使用單純粉末法及一些螢光微弱化學試劑,因此在一些背景干擾下,採證不易;同時這些試劑通常在僅能用於單一物質:介面孔洞材質或非孔洞材質(Porous or Non-Porous),因此指紋採證上通常有諸多不便的地方。
  我們利用螢光高分子奈米顆粒(Pdots)當作指紋的螢光發光主體,同時再以寧海德林試劑對指紋胺基酸有良好的呈色效果,使指紋同時擁有顯色與螢光效果。利用Pdots本身疏水性與指紋中分泌的油質類產生交互作用而吸附一起,同時產生螢光藍移現象,進一步讓指紋呈現高對比螢光顯影,此外,將開環後Poly(styrene-alt-maleic anhydride, PSMA)與寧海德林(Ninhydrin)試劑進行酯化鍵結,再包覆在Pdots表面上,讓指紋上的氨基酸可與寧海德林反應,生成明顯紫色指紋,此時Pdots可對於不同物質介面進行指紋顯色,如對於有孔洞材質時,可使用寧海德寧達到指紋顯色;相對於無孔洞材質時,則可以利用Pdots疏水性進一步使指紋呈現出來,最終達到可同時在許多不同物質介面上進行指紋的顯色與螢光效果。
Abstract
1. Dual Colorimetric and Fluorescent Sensor Based On Semiconducting Polymer Dots for Ratiometric Detection of Lead Ions in Living Cells
Recently, semiconducting polymer dots (Pdots) have become a novel type of ultrabright fluorescent probes which hold great promise in biological imaging and analytical detection. Here we developed a visual sensor based on Pdots for Pb2+ detection. We first embedded near-infrared (NIR) dyes into the matrix of poly[(9,9-dioctylfluorene)- co-2,1,3-benzothiadiazole-co-4,7-di(thiophen-2-yl)-2,1,3-benzothiadiazole] (PFBT-DBT) polymer and then capped the Pdots with polydiacetylenes (PDAs), in which parts of the PDAs were prefunctionalized with 15-crown-5 moieties to form Pdots. The high selectivity of these Pdots for lead ions is attributed to the formation of 2:1 15-crown- 5-Pb2+-carboxylate sandwich complex on the Pdot surface. After Pb2+ chelation, the conjugation system of the PDA was perturbed and strained, causing a chromatic change of the PDA from blue to red. At the same time, the encapsulated NIR dyes were liable to leach out that resulted in an emission variation of the Pdots. Accordingly, lead ions can be recognized by either color change or emission variation of the Pdots. We also loaded these nanoprobes into live HeLa cells through endocytosis, and then monitored changes in Pb2+ levels within cells, demonstrating their utility for use in cellular and bioimaging applications. In addition, we fabricated easyto-prepare test strips impregnated with Pdot-poly(vinyl alcohol) films to identify Pb2+ in real samples, which proved their applicability for in situ on-site detection. Our results suggest that this Pdot-based visual sensor shows promising potential for advanced environmental and biological applications.

2. The Detection of Latent Fingerprints on Porous/Non-Porous Surfaces Using Combination Semiconductor Polymer Dots and Ninhydrin for Colorimetric and Highly Fluorescence
In the 21st century, this is vitally important in crime scene investigation for tracking the links between the scene,victims and the perpetrators.The impressions left by the friction ridge skin on the palmar surfaces of the hands, most often referred to as fingerprints, not only demonstrate contact but also are sufficiently unique to enable personal identification. So there are many fingerprint reagents on detection of laterent fingerprints. But most of them can only be used on a single material surfaces (Porous or Non-Porous).Here, we developed the new fingerprint fluorescent reagent based on Semiconducting Polymer Dots (Pdots) for detection of laterent fingerprint. This is can be used on porous surface besides non-porous. Meanwhile, the Pdots can reveal the fingerprint with high contrast fluorescence result from blue shift. The hydrophobic interactions between the Pdots and fingerprint residue play an important role in the blue shift. We combine the Pdots and Ninhydrin in order to reveal fingerprint with color. Ninhydrin is most commonly used to detect amino acid in fingermarks. When reacting with amino acid, a deep blue or purple color known as Ruhemann's purple is produced. Ultimately fingerprints simultaneously are revealed on porous/nonporous surfaces with color and fluorescence.
目次 Table of Contents
論文審定書………………………………………….………………..……………....….i
誌謝………………………………………….…………………..……………..…….….ii
中文摘要………………………………………….…………………..……………..….iii
英文摘要……………………………………………………………..………………….v
目錄………………………………………………………………….…….…..……….vii
圖目錄……………………………………………………………….….….……..…..…x
表目錄……………………………………………………………….………....…..…..xv
縮寫表……………………………………………………………….………....……....xv

第一章、 緒論………………………………………………………………....….…….1
一、前言………………………………………………………………………...….........1
二、歷史發展………………………………………………………………….…..…..…3
三、製備方式………………………………………………………………...…….……14
四、螢光共振能量轉移(FERT)………………………………………………...………15
五、Pdots之應用.................................................................................................19
5-1. Ion sensor ………………………………….…………..…………………....…….19
5-2. Biosensor …………………………………..…………..………………...….…….20
5-3. Bioimaging ……..…………………………..…………………………….….…….21
六、研究動機…………………………………………………………………....…..…..22

第二章、 開發比色法與螢光雙偵測系統的半導體高分子奈米探針 ,並可快速定
性與定量生物體中的鉛離子..................................................................................23
一、前言………………………………….………………………………...……....…….23
二、實驗部分.......................................................................................................26
2-1.實驗藥品………………………………………………………..………………...….26
2-2.儀器裝置…………………………………………………………………………......29
2-3.樣品配製方法……………………………………………………………………......31
2-4.實驗步驟…………………………………………………………………………......34
三、實驗設計及原理……………………………………………………...…………......40
四、實驗結果與討論.............................................................................................43
4-1.半導體高分子奈米顆粒以及NIR染劑的選擇…………….……..………....….......43
4-2.PDA-crown-enclosed NIR695-doped Pdots的物理及光學特性探討……....…..44
4-3.探討NIR染劑的含量………………………………………………………...…........47
4-4. 奈米探針選擇性…………………………………………………...………...…......48
4-5.螢光檢測Pb2+離子濃度………………………………………………..….….….....50
4-6.以PDA吸收光譜、螢光生命期及分離染劑證明反應機制正確性……....……......53
4-7.0.3% NIR695-PFBT-DBT半導體高分子奈米顆粒於生物體上的應用…….........57
4-8.真實樣品的測定………………………………..…….……………………………....62
4-9.試紙製備與應用…………………………………….………………..……………....63
五、結論………………………………………………………………………...…….......65

第三章、結合半導體高分子奈米顆粒與寧海德林,使孔洞及非孔洞材質中的潛伏指紋具有明顯呈色與高亮度螢光..........................................................................................................................66
一、 前言……...…………………………………………….…………………….……....66
二、 實驗部分........................................................................................................68
2-1.實驗藥品………………………………………………….....……….……….……….68
2-2.儀器裝置……………………………………………………….....…………….……..71
2-3.樣品配製方法……………………………………………………….....……….….….73
2-4.實驗步驟………………………….…………………………………….....…….….…74
三、 實驗設計及原理…………………………………………………………......……....79
四、實驗結果與討論...............................................................................................82
4-1. Pdots疏水性交互作用……………………………………………………......……..82
4-2. Polymer Dots與光學特性………………………………………………….…...…..84
4-3.以FRET提升螢光量子產率………………..……………………………...…….......86
4-4.以Pdots進行指紋顯影……………….……..…....…………………………...……..88
4-5. PFBT-TTFQ Pdots指紋特徵點辨識………..……...…………………….....…….90
4-6. 未來工作 : Pdots-Ninhydrin 指紋顯影…………….....……………………...…….92
五、結論………………………...……………….……………..... ..….…………………93

第四章、參考文獻……………………………………….…….….....…………...……...94

第五章、附錄.……………………………………….…….….………....…...…………..98
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