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系統識別號 U0026-2301201421350000
論文名稱(中文) 創新鍍金奈米孔洞陣列誘發表面增顯拉曼散射以增強檢測微量特定生物標記
論文名稱(英文) Innovative Au-coated Nano-cavities Array Induced SERS effect Reinforced for Trace Detection of Specific Biomarker
校院名稱 成功大學
系所名稱(中) 材料科學及工程學系
系所名稱(英) Department of Materials Science and Engineering
學年度 102
學期 1
出版年 103
研究生(中文) 姚智凱
研究生(英文) Chih-Kai Yao
學號 n58981040
學位類別 博士
語文別 中文
論文頁數 103頁
口試委員 指導教授-廖峻德
口試委員-溫添進
口試委員-林弘萍
口試委員-張世慧
口試委員-楊裕雄
口試委員-呂昱瑋
中文關鍵字 表面增顯拉曼散射  空間強化鍍金奈米孔洞  拉曼位移  免標定定性  染色標定定量  生物標記  核酸雜合反應 
英文關鍵字 surface-enhanced Raman scattering  spatially reinforced Au-coated nanocavity  Raman shift  non-labeling qualification  dye-labeling quantification  biomarker  hybridization 
學科別分類
中文摘要 在奈米級結構中引發電場強化進而誘發表面電漿拉曼散射效應需要綜合考慮增顯機制與製程技術等因素。而由於此技術能用於生醫檢測應用基板之開發,故在近年來被廣泛討論。本研究利用奈米壓痕製程技術,於金表面上製作週期性之奈米壓痕孔洞結構陣列利用縮減壓痕間距改變孔洞結構深寬比變化開發一具空間強化效能的SERS活性基板,提升表面電漿子侷限化之效果,能提升增顯因子至9.0×107。
針對蛋白質類生物標記分子的分析中可證實,本研究所設計奈米壓痕孔洞結構陣列檢測HBV-cAg與HCV-cAg時,在10-8 M低濃度下可有效分辨兩者之圖譜差異性,證實此基板具有免標定的圖譜定性分析效能。此外,導入簡易染色法對於極低濃度之蛋白分子之染色定量分析,證實於極低濃度(10-7~10-10 M)下區間內,其染劑分子之拉曼增顯強度與蛋白分子濃度之對數值呈現線性關係。
針對核酸類生物標記分子的分析中可證實,SR-nAu基板能有效的定義出不同亞型的禽流感病毒特殊基因片段以及其雜合前後之狀態的差異性。此外,藉由核酸引子改質的基板能夠成功的藉由核酸特有的雜合反應由互補與非互補的核酸序列中分離出標的核酸,由拉曼圖譜中738 cm-1處的特性峰消失以及最強特性峰位置由1575 cm-1位移至1554 cm-1處可明確辨識雜合反應是否發生。故可證明本研究設計之SERS活性基板能有效的捕捉並檢測出檢體中是否具有特定核酸序列。
最後,將上述所設計之SERS活性基板嵌入微流道結構內,整合成一簡易檢測平台,並以此驗證所設計之微流道檢測平台能成功辨識微量特定生物標記分子。其對蛋白質體與核酸序列等生物標記分子之檢測極限能成功拓展至約奈米等級的莫耳濃度等級。基於基礎與應用性之研究,其結果顯示:SR-nAu基板是非常有前景且具有很大的潛力應用於檢測工具,特別適用於極少量的生物標記之快速篩檢領域。
英文摘要 To reinforce electromagnetic coupling of light in nano-structure has been a major challenge in biomedical diagnosis because of the need to consider various enhancement mechanisms of and fabrication challenges. In this study, a novel “Spatially reinforced Au nano-cavities” (SR-nAu) were designed with a reduced tip-to-tip displacement to localize the surface plasmons by properly controlling the aspect ratio of cavity structure, its optimal EF of Surface-enhanced Raman scattering (SERS) for R6G can be increased to 9.0×107.
In the study of recognizing biomarker as protein, the SR-nAu substrates are competent to distinguish the difference between HBV-cAg and HCV-cAg within a limit of 10-8 M and be employed for quantitative distinction of antigen concentration simultaneously. A linear relationship between Raman intensities of CBBG with various antigen concentrations can be found over a concentration range of 10-7 to 10-10 M.
In the study of recognizing biomarker as DNA, specific target sequence of AIV, H7-T, H7-P, and its hybridization, within the nano-reservoir were mostly distinguishable. In addition, the probe-immobilized reservoir could be used to separate the match or mismatch DNA sequences with hybridization reaction. Raman shifts for the presence of 738 cm-1 and the change of major peak intensity from 1554 cm-1 (with hybridization) to 1575 cm-1 (without hybridization) could be clearly found as a recognizing basis.
Furthermore probe immobilized nano-reservoir was embedded into a micro-fluidic channel was competent to capture and distinguish the target biomarker. According to these results, the as-designed substrate are very promising and have high potential as a characterization tool for fast-screening detection of small quantity of target biomarkers
論文目次 摘要 I
Abstract II
誌謝 III
目錄 V
表目錄 VII
圖目錄 VIII
第一章導論 1
1.1 導論 1
1.2 研究動機 3
1.3 研究目的 5
第二章理論基礎與文獻回顧 7
2.1 振動光譜 7
2.2 拉曼光譜基本理論 9
2.2.1 拉曼散射原理 9
2.2.2 拉曼光譜之極化誘發理論 11
2.3 表面增顯拉曼散射光譜 13
2.3.1 表面增顯拉曼光譜之建立與發展 13
2.3.2 表面增顯拉曼光譜之電磁增顯機制 14
2.3.3 表面增顯拉曼光譜之化學增顯機制 17
2.4奈米結構對表面增顯拉曼散射之影響 20
2.4.1 週期性奈米結構對表面增顯拉曼散射之影響 21
2.4.2孔洞型奈米結構對於表面增顯拉曼散射效應之影響 24
2.4.3表面增顯拉曼散射應於生醫檢測應用 26
第三章材料與方法 31
3.1 實驗設計與流程 31
3.1.1實驗構想 31
3.1.2實驗設計 33
3.2 實驗材料與製備 35
3.2.1鍍金基板製作 35
3.2.2拉曼活性基板製作 36
3.2.3分子探針溶液製備 37
3.2.4生物標記-蛋白質選用 39
3.2.5 生物標記-核酸序列選用 39
3.2.6拉曼檢測與訊號處理 40
3.3 製程儀器 42
3.3.1電子束蒸鍍機 42
3.3.2奈米壓痕試驗機 42
3.4 分析儀器 45
3.4.1掃描式電子顯微鏡 45
3.4.2 顯微拉曼光譜儀 46
第四章以奈米壓痕製備SERS活性基板應用於分子探針之研究 48
4.1 不同參數之倒角錐型奈米孔洞陣列表面形貌分析 48
4.2 空間強化型奈米孔洞陣列之SERS效應評估 51
4.2.1 拉曼光譜量測之正規化與標準流程 51
4.2.2 SERS效果與增顯因子之評估 53
4.2.3 壓痕深度與間距變因對於SERS效果影響之探討 54
第五章 SERS活性基板應用於定性與定量分析肝炎抗原之效能評估 59
5.1標準化蛋白質樣品確效SERS活性基板之檢測極限 59
5.1.1 SERS活性基板用於標準蛋白質樣品之圖譜定性分析 59
5.1.2 SERS 活性基板用於標準蛋白質樣品之染色定量分析 62
5.2 SERS活性基板檢測不同亞型病毒型肝炎核心抗原 65
5.2.1 SERS活性基板用於病毒型肝炎核心抗原之圖譜定性分析 65
5.2.2 SERS 活性基板用於病毒型肝炎核心抗原之染色定量分析 66
第六章 SERS活性基板於鑑別特定核酸序列之檢測機制探討 69
6.1 評估SERS活性基板對於不同亞型禽流感核酸片段之辨識能力 69
6.1.1 評估SERS活性基板檢測濃度極限 69
6.1.2不同亞型禽流感核酸片段之表面增顯拉曼圖譜 72
6.2以表面增顯拉曼光譜探討特定核酸序列之各種雜合狀態 75
6.3 特定核酸序列改質於奈米結構表面之檢測應用 79
6.3.1 以電子能譜分析確定表面改質之成效 79
6.3.2改質後SERS活性基板對特定核酸序列之拉曼光譜分析 81
6.3.3改質後SERS活性基板辨識特定核酸序列之各種雜合狀態之探討 82
6.4 SERS活性基板與微流道之整合與應用 86
結論 90
未來發展 91
參考文獻 92
相關著作 100
自我介紹 103
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