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博碩士論文 etd-0616115-115319 詳細資訊
Title page for etd-0616115-115319
論文名稱
Title
蛋白質合成螢光金奈米簇做為硫醇及生物分子感測器
Protein-directed synthesis of luminescent gold nanoclusters for sensing bio-molecules
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
145
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
2014-07-10
繳交日期
Date of Submission
2015-07-30
關鍵字
Keywords
過氧化氫、生物感測器、硫醇分子、螢光、酵素活性分析、金奈米簇、pH值、尿素
Gold nanoclusters, biosensor, fluorescence, thiol, enzyme activity assay, hydrogen peroxide, pH value, urea, thimerosal
統計
Statistics
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中文摘要
本篇研究為使用以蛋白質作為模板之螢光金奈米簇作為分析平台,針對特定種類之生物小分子進行偵測,利用金奈米簇具有之獨特光學性質對特定分析物進行感測,藉由對照其螢光訊號之差異值對分析物進行定量。後續可以配合各類需求採用不同合成策略來製備金奈米簇,藉此達到快速、簡易、高靈敏性及選擇性之分析目的。
(一) 以第六型溶菌酶修飾金奈米簇作為螢光探針偵測硫醇分子-探討其螢光消光機制並用於偵測疫苗中的硫柳汞
本研究中,為使用第六型溶菌酶Lyz VI作為蛋白質模板修飾之金奈米簇Lyz VI@Au8作為螢光感測器,由於Lyz VI@Au8在晶體結構上的特性,故易受到硫醇分子的影響而導致螢光消光,而硫醇分子對金奈米簇的蝕刻反應(Core-Etching) 先前亦未有完整的探討,因此在對此機制進行詳細的研究。首先比較在不同酸鹼性環境下,硫醇分子對金奈米簇Lyz VI@Au8造成的螢光消光程度的影響,之後再進一步討論帶有不同官能基之硫醇分子對金奈米簇Lyz VI@Au8螢光造成的消光影響。由實驗結果得知,在鹼性條件下,使用帶有羧酸基且碳鏈長度較短之硫醇物種對於金奈米簇Lyz VI@Au8造成的螢光消光現象最為明顯,在此推論造成金奈米簇Lyz VI@Au8螢光消光的主因為當其表面有多個硫醇分子時,此時硫醇分子會先和金奈米簇Lyz VI@Au8形成Au-S鍵結,由於硫醇分子會將其所帶有的電荷傳導 (Electron Injection) 給金奈米簇Lyz VI@Au8,因此會致使金奈米簇Lyz VI@Au8表面電荷密度過高,故導致其碎裂來釋放此靜電張力,故會造成金奈米簇Lyz VI@Au8螢光消光的現象。而在後續可將硫醇分子造成的螢光消光機制應用於偵測硫柳汞 (Thiomersal) 。硫柳汞為疫苗中常用的抗腐劑,由於其會在水溶液中會部份解離形成硫柳酸及乙基汞,而硫柳酸之化學結構為具有硫醇基及羧酸基的分子,故會造成金奈米簇Lyz VI@Au8的螢光消光,而其降解後之產物乙基汞及2,2'-二硫代二苯甲酸則不會對金奈米簇Lyz VI@Au8造成影響,因此可對硫柳汞進行定量,定量結果其線性範圍為4 ~ 100 μM,偵測極限為1 μM,並且成功應用於偵測疫苗樣品中硫柳汞的含量。此外可藉由加入硫醇基物種來對金奈米簇Lyz VI@Au8進行蝕刻作用,將其模板蛋白質釋出,故可使用含有硫醇官能基之分子,如:榖胱甘肽來調控金奈米簇Lyz VI@Au8之模板蛋白質活性,日後於藥物調控以及生醫領域的研究方面上,本研究也具備潛在的應用性存在。
(二) 利用螢光物質標記牛血清蛋白質合成之金奈米簇使用螢光比例法作為活性氧化物質及酸鹼探針
在本次研究中,使用標記異硫氰酸螢光素FITC (Fluorescein Isothiocyanate) 之牛血清蛋白質作為模板,之後以一步的合成方法來合成金奈米簇,因此可以得到具有525 nm及670 nm兩道不同放射波長的螢光探針,其中525及670 nm分別為FITC和金奈米簇所放射的波長。由於金奈米簇受到活性氧化物質氧化時會使其氧化價數產生改變,故會導致金奈米簇螢光消光,但FITC則不受影響,因此可以利用兩者螢光強度比值IF525 nm/IF670 nm應用活性氧化物質的偵測,在此選用過氧化氫作為反應物進行測試,其偵測極限為12 μM。後續將此偵測機制應用於葡萄糖以及乙醯膽鹼酯酶活性含量的檢測,其偵測極限各別為32 μM及0.4 unit/L,同時也可以對乙醯膽鹼酯酶之抑制劑-含磷農藥Paraoxon進行偵測,所測的酵素半抑制濃度IC50值為1.41 nM。此外由於在不同的酸鹼環境中,FITC的分子結構會產生改變,導致其螢光強度有所變化,而牛血清蛋白質合成之金奈米簇BSA@AuNCs的螢光強度則受到環境酸鹼性干擾較小,因此也可對環境之酸鹼性進行偵測,其線性範圍為pH 6.0 ~ pH 8.0,同時可以區分pH範圍為0.1的差異。後續將此偵測機制應用於尿素的偵測上,且可成功應用於人體血清樣品中尿素的定量,同時和傳統醫事檢驗方法對照,這兩種偵測方法所得到的實驗數值為接近的。
Abstract
The main idea of this thesis described the strategies of utilizing protein-directed synthesis gold nanoclusters as optical platform to sense specific bio-molecules. By the unique photoluminescence properties of gold nanoclusters, the gold nanoclusters served as a fluorescence probe for thiol and bioactive small molecules. Furthermore, the gold nanoclusters-based sensor had not only established a facile, simplicity, sensitivity, and selectivity approach to determination of target analyte, but also provided a new class of promising nanomaterials to design the assay for other bio-molecules and related species, which may play critical roles in the physiological and biological field.
I. Understanding of Thiol-Induced Etching of Luminescent Gold Nanoclusters:Control of Lysozyme Activity and Sensing of Thimerosal
This study reported that Lysozyme Type VI (Lys VI)-stabilized Au8 clusters serve as a model to probe how distinct types of alkanethiol ligands affect the core etching of AuNCs. By monitoring the fluorescence of Au8 clusters, we determined that thiogly-
colic acid (TGA) -induced core etching of Au8 clusters was substantially faster at pH 9.0 than it was at pH 3.0. This can be attributed to more efficient electron injection from TGA to Au8 clusters at pH 9.0, facilitating the core etching of Au8 clusters. Because long-chain mercaptoalkanoic acids attached to Au8 clusters are considerably disordered and exhibit a high density of gauche defects, the ability of mercaptoalkan-
oic acid to etch Au8 clusters increased when the alkyl chain length was decreased.
The thiol analogs exhibited the following trend in the core etching of Au8 clusters at pH 9.0:TGA > 2-Mercaptoethanol > 1-Octanethiol. These results indicate that the carboxyl group of ligand is a key element for the core etching of Au8 clusters. We also disclosed that Au8 clusters can protect Lys VI activity against denaturation and act as a fluorescent probe to detect thimerosal in vaccines.

II. Fluorescein-5-isothiocyanate-conjugated protein-directed synthesis of gold nanoclusters for fluorescent ratiometric sensing of an enzyme–substrate system
This study describes the synthesis of a dual emission probe for the fluorescent ratio-
metric sensing of hydrogenperoxide (H2O2) , enzyme activity, and environmental pH change. Green-emitting fluorescein-5-isothiocyanate (FITC) was conjugated to the amino groups of bovine serum albumin (BSA) .This FITC-conjugated BSA acted as a template for the synthesis of red-emitting gold nanoclusters (AuNCs) under alkaline conditions.Under single wavelength excitation, FITC/BSA-stabilized AuNCs (FITC/
BSA-AuNCs) emitted fluorescence at 525 and 670 nm, which are sensitive to changes in solution pH and H2O2 concentration, respectively. The effective fluorescence quen-
ching of AuNCs by H2O2 enabled FITC/BSA-AuNCs to ratiometrically detect the H2O2 product-related enzyme system and its inhibition, including glucose oxidase-
catalyzed oxidation of glucose, acetylcholinesterase/cholineoxidase-mediated hydro-
lysis and oxidation of acetylcholine, and paraoxon-induced inhibition of acetylcholin-
esterase activity. When pH-insensitive AuNCs were used as an internal standard, FITC/BSA-AuNCs offered a sensitive and reversible ratiometric sensing of a 0.1-pH unit change in the pH range 5.0–8.5. The pH-induced change in FITC fluorescence enabled FITC/BSA-AuNCs to detect an ammonia product-related enzyme system. This was exemplified with the determination of urea in plasma by urease-mediated hydrolysis of urea.
目次 Table of Contents
論文審定書 i
論文公開授權書 ii
謝誌 iii
中文摘要 v
英文摘要 vii
目錄 x
圖目錄 xiii
表目錄 xviii
第一章、以第六型溶菌酶修飾金奈米簇作為螢光探針偵測硫醇分子-探討其螢光消光機制並用於偵測疫苗中的硫柳汞 1
1.1 前言 1
1.2 實驗部分 3
1.2.1 實驗藥品 3
1.2.2 儀器設備 6
1.2.3 實驗溶液配置及樣品前處理步驟 8
1.3 實驗結果與討論 13
1.3.1 探討含有不同官能基以及含有不同碳鏈長度之相同官能基硫醇基物種對金奈米簇Lyz VI@Au8螢光消光程度探討 13
1.3.1.1 探討反應條件之酸鹼性對硫醇分子所造成金奈米簇Lyz VI@Au8螢光消光現象造成的影響 13
1.3.1.2 使用MALDI-TOF-MS、SEM、XPS以及ICP-MS驗證硫醇類分子對金奈米簇Lyz VI@Au8造成的蝕刻現象 17
1.3.1.3 探討使用不同碳鏈長度以及硫醇官能基數目對金奈米簇Lyz VI@Au8螢光消光現象造成的影響 22
1.3.1.4 比較使用其他類型蛋白質模板合成之金奈米簇和以第六型溶菌酶合成之金奈米簇之差異性 28
1.3.2 應用金奈米簇Lyz VI@Au8於偵測硫柳汞 33
1.3.2.1 偵測硫柳汞標準品測試及其最佳化條件探討 33
1.3.2.2 偵測硫柳汞之選擇性測試 39
1.3.2.3 偵測疫苗真實樣品中的硫柳汞含量 42
1.3.3 應用金奈米簇Lyz VI@Au8於控制其蛋白質模板之活性 44
1.4 結論 50
1.5 參考資料 51
第二章、利用螢光物質標記牛血清蛋白質合成之金奈米簇使用螢光比例法作為活性氧化物質及酸鹼探針 58
2.1 前言 58
2.2 實驗部分 61
2.2.1 實驗藥品 61
2.2.2 儀器設備 64
2.2.3 實驗溶液配置及樣品前處理步驟 67
2.3 實驗結果與討論 73
2.3.1 合成異硫氰酸螢光素標記牛血清蛋白質合成金奈米簇鑑定 73
2.3.1.1 螢光儀鑑定 73
2.3.1.2 膠體電泳鑑定 77
2.3.1.3 圓二色光譜儀鑑定 80
2.3.1.4 MALDI-TOF MS鑑定 82
2.3.1.5 螢光生命週期儀鑑定及吸收光譜儀鑑定 82
2.3.2 偵測過氧化氫之最適化實驗條件探討 85
2.3.3 偵測葡萄糖及選擇性測試 91
2.3.4 偵測乙醯膽鹼酯酶及其抑制劑測試 95
2.3.5 偵測pH值及酸鹼可調變化性測試 100
2.3.6 偵測尿素標準品測試 104
2.3.7 偵測尿素之選擇性測試 104
2.3.8 偵測血液真實樣品中的尿素含量 108
2.3.9 邏輯閘測試 110
2.3.9.1 AND/NAND邏輯閘設計概念 110
2.3.9.2 INHIBIT/REVERSE IMPLICATION邏輯閘設計概念 113
2.3.9.3 OR/NOR邏輯閘設計概念 116
2.4 結論 119
2.5 參考資料 120
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