本論文研究目的為利用中空金屬奈米粒子開發新型生醫感測器，並應用於過氧化氫之檢測。一準確且快速檢測過氧化氫的系統在眾多領域當中皆相當重要，且應用極其廣泛，包括臨床診斷、生物分析及食品安全。而過氧化氫亦是許多酵素反應中的副產物，舉例來說，透過葡萄糖氧化酶的催化，葡萄糖可被氧化形成葡萄糖酸並產生過氧化氫。至今，已開發許多檢測過氧化氫的方法，如分光光度檢測法、電化學法、螢光光度計等。然而，這些技術皆需具備複雜、精密且昂貴的儀器，無法進行就地檢測甚至回饋於開發中國家或落後地區。因此，針對過氧化氫的檢測需求及其應用，開發便宜、快速、攜帶方便且容易使用的檢測系統成為一新穎的研究目標。
在本研究中，透過合成不同中空貴金屬奈米粒子並使其自組裝於高分子基板上，並開發兩種方法以檢測過氧化氫。第一部分使用中空銀鈀奈米粒子，運用鈀獨特的催化特性，將其製備為中空結構以增加具活性之表面積，透過自組裝在聚對苯二甲酸乙二酯 (Polyethylene terephthalate, PET) 基板上排成單層奈米粒子陣列，以此催化過氧化氫與2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) 之氧化還原反應，藉由觀察ABTS 之顏色變化以檢測過氧化氫，並探討於不同反應條件及環境中，中空銀鈀奈米粒子的催化活性。驗證中空銀鈀奈米粒子於酸性下具仿生過氧化酶的特性，反應可於30 分鐘內達飽和，且能在室溫下進行反應。接著以動力學分析比較中空銀鈀奈米粒子與天然酵素辣根過氧化酶 (Horseradish peroxidase, HRP) 之催化活性，發現本研究中所製備的中空銀鈀奈米粒子催化活性與HRP 相近，且線性範圍相當寬廣，落於5 mM~500 mM 之間。最後將中空銀鈀奈米粒子自組裝於PET 基板上，並成功於基板上進行過氧化氫的檢測，線性範圍為2 mM~10 mM，亦可以肉眼觀察到明顯的顏色變化，進而透過比色法輕易的判別過氧化氫濃度。
第二 部分中，製備吸收波段位於500 nm 附近之中空銀金奈米粒子，活用過氧化氫的強氧化特性，使得含銀量偏高的中空銀金奈米粒子，與過氧化氫產生氧化還原反應，並以紫外-可見光光譜儀與穿透式電子顯微鏡驗證奈米粒子與不同含量過氧化氫反應後，結構及光譜的改變。銀氧化形成銀離子並增添中空銀金奈米粒子之結構孔洞性，而結構的改變促使奈米粒子在吸收波段產生紅移並改變其顏色，因此得以觀測不同顏色來監測過氧化氫濃度 ，接著探討在各環境下中空銀金奈米粒子與過氧化氫的氧化還原能力。成功驗證中空銀金奈米粒子於過氧化氫檢測的可行性後，將奈米粒子自組裝於PET 基板，製備PET 檢測試片，並測試此基板上的氧化還原反應可於室溫且中性環境下進行，而反應於30 分鐘達飽和，接著以三原色分析 (分別為紅色―Red、綠色―Green、藍色―Blue，RGB) 建立過氧化氫檢量線，線性範圍為 5 µM~50 µM 之間，而偵測極限為4.3 µM。接著再將中空銀金奈米粒檢測試片與特定酵素結合進行生物標的物的檢測 (如葡萄糖、尿酸及膽固醇等)，而本研究中，已成功進行膽固醇的檢測，並以RGB 分析製備膽固醇檢量線，線性範圍為 10 µM~50 µM 之間，且偵測極限可達6.0 µM。於此檢測平台進行生物標的物分析時，因顯著的顏色變化，而可以比色法輕易的判別生物標的物濃度 (如本實驗中所進行的膽固醇檢測)。未來期望將此生醫感測器推廣至更多生物標的物的濃度監測，甚至於實際樣品 (如尿液或血液) 或病人的血清。

The accurate and rapid determination of hydrogen peroxide (H2O2) is of great importance in many applications such as clinical diagnosis, bioanalysis, and food safety. Moreover, H2O2 is a side product of specific enzymatic reactions. For instance, glucose could be oxidized to produce gluconic acid and H2O2 in the presence of glucose oxidase (GOx). Up until now, there have already been many methods to detect H2O2, including spectrophotometry, electroanalysis, and fluorometry. However, these techniques require expensive or sophisticated instruments. Therefore, rapid and easy ways of H2O2 sensing are necessary.
In this thesis, we have developed two systems to detect H2O2 without any complicated equipment. Both of them are simple, low cost, and disposable. Most importantly, we could distinguish the color change by the naked eye. By these advantages, the sensor could be applied to develop point-of-care (POC) and clinical diagnosis platforms. The intrinsic catalytic ability of palladium in many specific reactions has been confirmed. Herein, our first system utilizes the enzymatic approach by using hollow silver/palladium nanostructures (Ag/Pd NSs) to replace commercial enzyme, Horseradish peroxidase (HRP), to catalyze the oxidization of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) by H2O2. By calculation of many kinetic constants from enzyme kinetics, we compared the catalytic activity of Ag/Pd NSs with HRP. Proved that the Ag/Pd NSs has high catalytic activity and similar results to HRP. We also did the catalytic reaction in different reaction conditions such as temperature, pH value and reaction time. The most suitable reaction conditions were under room temperature, acidic condition (pH=4.6) and 10 min in solution. Then, we have successfully immobilized a nanoparticle array onto the commercial polymer substrate, polyethylene terephthalate (PET), via a wet-chemical method. By mixing H2O2 with ABTS in an acetic buffer (pH=4.6) with the Ag/Pd NSs-coated PET sensing system, we could observe the increase in darkness of green color upon increasing H2O2 concentration by the naked eye. The high catalytic activity of Ag/Pd NSs might be attributed to the hollow structure (large surface area) and the intrinsic catalytic ability of palladium. The linear relationship was from 5 mM to 500 mM in solution-based test and 2 mM to 10 mM in PET-based detection system.
In another system, we immobilized hollow silver/gold nanostructures (Ag/Au NSs) onto PET substrates, similar to the first system. The detection process could be performed by directly dropping the H2O2 solution onto the Ag/Au NSs-coated PET sensing system, without any organic dyes. Because of the strong oxidizing ability of H2O2, the silver atoms in the Ag/Au NSs would be oxidized to silver ions and the Ag/Au NSs would transform to a more porous structure. Owing to the changes in structure and composition, the localized surface plasmon resonance (LSPR) peak of Ag/Au NSs would continuously shift to longer wavelength (red shift) and different colors would be obtained at various H2O2 concentrations, visible by the naked eye. We could successful detect the H2O2 under room temperature and neutral condition (pH 7.0) in 30 min. We obtained narrow linear range but low detection limit, 5 µM to 50 µM and 4.3 µM respectively. Finally, the Ag/Au NSs-based platforms could be utilized for clinical diagnosis, such as glucose, cholesterol or uric acid sensing. In this study, we have successfully combined the cholesterol oxidase to develop cholesterol sensing. The linear range was from 10 µM to 50 µM. The detection limit was low as 6.0 µM.
An overview of the two efficient sensing platforms, the second one shows lower detection limit but narrow linear range than first one. In the future, the applications of the sensing systems could extend to other fields that relate to H2O2.

目錄
致謝 I
中文摘要 II
Abstract IV
圖目錄 VI
表目錄 IX
第一章 序論 1
1.1 前言 1
1.2 論文架構 2
第二章 文獻回顧 3
2.1 過氧化氫與檢測 3
2.1.1 過氧化氫的應用 3
2.1.2酵素免疫分析法 (Enzyme-linked immunosorbent assay, ELISA) 4
2.1.3 無機材料檢測方法 5
2.2 金屬奈米粒子的簡介 7
2.2.1 實心金屬奈米粒子合成 7
2.2.2 中空金屬奈米粒子合成 9
2.2.3 金屬奈米粒子之侷域性表面電漿共振特性 (Localized surface plasmon resonance, LSPR) 12
2.2.4 金屬奈米粒子特殊催化性質與應用 14
2.2.5 金屬奈米粒子光學性質於生醫感測領域之應用 17
2.3 金屬奈米粒子與酵素的結合於疾病檢測之應用 18
2.3.1 葡萄糖 19
2.3.2 膽固醇 20
2.4 製備金屬奈米粒子陣列與方法 21
2.4.1金屬奈米粒子陣列於矽和玻璃基板 22
2.4.2 金屬奈米粒子陣列於紙基材 23
2.4.3 金屬奈米粒子陣列於高分子基板 24
第三章 結合高分子基板與金屬奈米粒子催化特性於過氧化氫檢測 26
3.1 研究目的 26
3.2 實驗 26
3.2.1實驗方法 27
3.3 研究結果與討論 31
3.2.1 中空銀鈀奈米粒子之形貌探討 31
3.2.2 中空銀鈀奈米粒子之光學性質 33
3.2.3 中空銀鈀奈米粒子之催化性質 34
3.2.4 中空銀鈀奈米粒子於不同環境之催化活性驗證 35
3.2.5 中空銀鈀奈米粒子之動力學分析 37
表3.1 催化活性比較。 39
3.2.6 中空銀鈀奈米粒子於過氧化氫之檢測 39
3.2.7 中空銀鈀奈米粒子檢測試片於過氧化氫之檢測 40
第四章 結合高分子基板與金屬奈米粒子氧化還原性質於過氧化氫檢測 43
4.1 研究目的 43
4.2 實驗 43
4.2.1 實驗方法 44
4.3 實驗結果與討論 47
4.3.1 中空銀金奈米粒子之形貌探討 48
4.3.2 中空銀金奈米粒子之光學性質探討 49
4.3.3 中空銀金奈米粒子與過氧化氫於水溶液中之氧化還原 50
4.3.4 中空銀金奈米粒子與過氧化氫於基板上之氧化還原 51
表4.1 不同LSPR 中空銀金奈米粒子與H2O2 反應前後之特徵吸收峰波谷位置差值。 52
4.3.5 中空銀金奈米粒子檢測試片於過氧化氫之檢測 56
4.3.6 中空銀金奈米粒子檢測平台於膽固醇之檢測 57
第五章 總結 59
參考文獻 60

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