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研究生:李郁佳
研究生(外文):Li, Yu-Jia
論文名稱:開發功能性奈米材料於碘離子與凝血酶檢測和抑菌敷料應用
論文名稱(外文):Development of Functional Nanomaterials for Detection of Iodide Ions and Thrombin and Antibacterial Dressing Application
指導教授:黃志清黃志清引用關係
指導教授(外文):Huang, Chih-Ching
口試委員:黃志清何彥鵬黃郁棻吳彰哲林翰佳許邦弘
口試委員(外文):Huang, Chih-ChingHo, Yen-PengHuang, Yu-FenWu, Chang-JerLin, Han-JiaHsu, Pang-Hung
口試日期:2016-01-11
學位類別:博士
校院名稱:國立臺灣海洋大學
系所名稱:生命科學暨生物科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:104
語文別:中文
論文頁數:94
中文關鍵詞:金奈米粒子纖維薄膜雷射脫附游離法碘離子凝血酶碳量子點抑菌劑
外文關鍵詞:Gold nanoparticlesCellulose membranesLaser-induced desorption/ionization mass spectrometryIodide ionsThrombinCarbon quantum dotsAntibacterial agents
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  • 下載下載:24
  • 收藏至我的研究室書目清單書目收藏:0
本論文第一部份研究為利用雷射脫附游離質譜法(laser desorption/ionization mass spectrometry, LDI-MS)於混和纖維薄膜(mixed cellulose ester membrane, MCEM)吸附金奈米粒子(gold nanoparticles, Au NPs)以偵測碘離子。由於碘離子會沉積於金奈米粒子表面並形成金碘鍵結,即可藉由脈衝雷射擊打偵測金碘離子團簇訊號來定量溶液中碘離子。搭配上混和纖維薄膜,可使金奈米粒子均勻分散於薄膜表面及減少質譜背景雜訊干擾,使訊號強度標準偏差小於10%,且能在高鹽類環境分析真實樣品(如:海水及尿液),此方法可簡單、快速並具有高靈敏度(偵測極限為5 × 10−10 M)和選擇性的於生物樣品中檢測碘離子。第二部分研究主要為凝血酶檢測,凝血酶生成(thrombin generation, TG)為凝血系統中重要的一環,可藉以診斷凝血有關的各種健康問題。本實驗利用LDI-MS於混和纖維薄膜吸附纖維蛋白原(fibrinogen)–金奈米粒子偵測凝血酶。凝血酶會與金奈米粒子表面之纖維蛋白原反應,形成不溶性纖維蛋白(fibrin)並抑制LDI下金團簇離子的形成,使其質譜訊號下降,我們藉此偵測凝血酶濃度,此方法具有高選擇性與高靈敏度,於血漿中偵測極限可達2.5 pM。另外利用此方法亦可應用於凝血酶抑制劑的篩選。
於第三部分抑菌劑開發研究中,我們利用兩步合成法合成亞精胺(spermidine,Spd)修飾的碳量子點(spermidine–carbon quantum dots,Spd–CQDs)應用於抑制細菌生長。首先使用檸檬酸銨(ammonium citrate)煅燒180 ℃製成螢光碳量子點(CQDs),接著將碳量子點粉末與亞精胺混合加熱,以不同溫度熱裂解亞精胺於碳量子點上。亞精胺修飾之碳量子點其表面多胺基使碳量子點帶有正電,與細菌接觸時(如大腸桿菌)會造成細菌穿孔死亡,藉此可抑制細菌的生長。Spd–CQDs不僅對大腸桿菌(E. coli)、綠膿桿菌(P. aeruginosa)、枯草芽孢桿菌(B. subtilis)與金黃色葡萄球菌(S. aureus)有抑制效果,也可抑制多抗藥性金黃色葡萄球菌(MRSA)。在體外細胞毒性和溶血分析中皆顯示Spd–CQDs具優越的生物相容性及材料安全性。我們亦進行動物抑菌實驗,以SD大鼠作為實驗模型,於其背部兩側開創並使其傷口感染MRSA,以Spd–CQDs作為敷料的條件下明顯觀察到傷口癒合快速、上皮恢復較好且有效的形成膠原蛋白。此研究結果表示Spd–CQDs在做為抗菌劑及臨床皮膚開創感染敷料上極具應用潛力。

The first part of this thesis, we report an simple method for the detection of iodide (I–) ions by using gold–iodide hybrid cluster ions on gold nanoparticles (Au NPs)–modified mixed cellulose ester membrane (Au NPs–MCEM) by pulsed laser desorption/ionization mass spectrometry (LDI-MS). When I− ions were deposited and concentrated on the surfaces of Au NPs (32 nm) via strong Au+I− interaction on the MECM, the Au NPs-MCEM was observed to function as an efficient surface-assisted LDI substrate with very low background noise. When pulsed laser radiation (355 nm) was applied, I− binding to Au NPs ions induced the enhancement of the desorption and ionization efficiency of gold-iodide hybrid cluster ions from the Au NPs surfaces. The reproducibility of the probe for both shot-to-shot and sample-to-sample (both less than 10%) ion production was also improved by homogeneous nature of the substrate surface. Thus, it allows the accurate and precise quantification of I− ions in high-salinity real samples (i.e., edible salt samples and urine) at the nanomolar range. This novel LDI-MS approach provides a simple route for the high-speed analysis of I− ions with high sensitivity and selectivity in real biological samples.
In the second part, MCEM modified with Au NPs and fibrinogen (Fib) coupled with LDI-MS was used for monitoring of thrombin activity. Thrombin generation (TG) has an important part in the blood coagulation system, and monitoring TG is useful for diagnosing various health issues related to hypo-coagulability and hyper-coagulability. The LDI process produced Au cationic clusters ([Aun]+; n = 1–3) that we detected through MS. When thrombin reacted with fibrinogen on the Au NPs–MCEM, insoluble fibrin was formed, hindering the formation of Au cationic clusters and, thereby, decreasing the intensity of their signals in the mass spectrum. Accordingly, we incorporated fibrinogen onto the Au NPs–MCEMs to form Fib–Au NPs–MCEM probes to monitor TG with good selectivity (>1000-fold toward thrombin with respect to other proteins or enzymes) and sensitivity (limit of detection for thrombin of ca. 2.5 pM in human plasma samples). Our probe exhibited remarkable performance in monitoring the inhibition of thrombin activity by direct thrombin inhibitors. Analyses of real samples using our new membrane-based probe suggested that it will be highly useful in practical applications for the effective management of haemostatic complications.
In the third study, we demonstrated a two-steps method to synthesize spermidine–capped fluorescent carbon quantum dots (Spd–CQD) and it has great potential application as an antibacterial agent. First, the fluorescent carbon quantum dots (CQDs) were synthesized by pyrolysis of ammonium citrate in the solid state and then modified with spermidine by a simple dry heating treatment at different temperatures. We observed the Spd–CQDs (4.6 ± 0.8 nm) having highly positive surface charge and possess high antimicrobial activity. The Spd–CQDs exhibit great antimicrobial activity not only to non-multi-drug resistant E. coli, P. aeruginosa, B. subtilis, and S. aureus bacteria but also to the multi-drug resistant (MDR) bacteria, such as methicillin-resistant S. aureus (MRSA). In vitro cytotoxicity and hemolysis analyses have revealed superior biocompatibility of Spd–CQDs. Moreover, in vivo MRSA-infected wound healing studies in rats show faster healing, better epithelialization, and more efficient in the production of collagen fibers when using Spd–CQDs as a dressing material. This study suggests that the Spd–CQDs is a promising antimicrobial candidate for preclinical applications in treating wounds and skin infections.

目錄
中文摘要 i
Abstract iii
圖目錄 ix
第一章 1
1.1 脈衝雷射 1
1.2 碘離子的重要性 3
1.3 標準方法及奈米感測器檢測碘離子 3
1.4 凝血酶測定 5
1.5 奈米材料應用於抑菌 7
1.7 研究動機 9
第二章 利用金奈米粒子薄膜搭配雷射脫附游離法檢測尿液中碘離子 11
2.1 前言 11
2.2 實驗材料與方法 12
2.2.1 實驗藥品 12
2.2.2 儀器設備 13
2.2.3 金奈米粒子合成 14
2.2.4金奈米粒子薄膜的製備 15
2.2.5以雷射脫附游離法偵測碘離子 15
2.2.6分析真實水樣 15
2.3 實驗結果與討論 17
2.3.1 雷射脫附游離質譜儀偵測碘離子 17
2.3.2 碘離子感測器之優化參數 18
2.3.3 選擇性與靈敏度 19
2.3.4 真實樣品檢測 20
2.4 結論 21
第三章 22
利用金奈米粒子薄膜搭配雷射脫附游離法偵測凝血酶生成和篩選抗凝血劑 22
3.1 前言 22
3.2 實驗材料與方法 23
3.2.1 實驗藥品 23
3.2.2 儀器設備 24
3.2.3 金奈米粒子薄膜製備 25
3.2.4 Au NPs–MCEM作為基質搭配LDI-MS偵測凝血酶 25
3.2.5於血漿樣品中分析凝血酶和活化因子X 26
3.2.6. 抗凝血藥物篩選 27
3.3 結果與討論 28
3.3.1凝血酶誘導凝血纖維蛋白形成之分析 28
3.3.2金奈米粒子薄膜於雷射脫附游離質譜之參數 30
3.3.3 選擇性及實用性 32
3.3.4 抑制凝血酶實驗 33
第四章 36
合成亞精胺之碳量子點應用於抑菌探討 36
4.1 前言 36
4.2 實驗材料與方法 37
4.2.1 實驗藥品 37
3.2.2 儀器設備 38
3.2.3亞精胺–碳量子點的合成 39
3.2.4細菌樣品製備 40
3.2.5抑制細菌 40
3.2.6. 細胞毒性實驗 41
3.2.7. 動物實驗之傷口敷料 41
4.3 結果與討論 42
4.3.1碳量子點的鑑定與特性 42
3.3.2利用亞精胺–碳量子點抗菌活性 44
3.3.3 毒性測試與溶血實驗 45
3.3.4 傷口敷料癒合測試 46
3.4 結論 48
總結 48


圖目錄
圖1-1 金奈米粒子薄膜檢測碘離子之示意圖 50
圖1-2 金奈米粒子薄膜檢測碘離子之LDI-MS質譜圖 51
圖1-3 金奈米粒子檢測碘離子於不鏽鋼盤之LDI-MS質譜圖 52
圖1-4 金奈米粒子吸附於薄膜之比較 53
圖1-5 LDI-MS訊號再現性分析 54
圖1-6 比較金奈米粒子吸附不同種類薄膜分析圖 55
圖1-7 金奈米粒子薄膜偵測碘離子之雷射條件探討 56
圖1-8 金奈米粒子薄膜之選擇性 57
圖1-9 金奈米粒子薄膜檢測不同碘離子濃度之食鹽樣品 58
圖1-10 金奈米粒子薄膜分析人類尿液樣品 59
圖2-1 金奈米粒子薄膜檢測凝血酶之示意圖 60
圖2-2 金奈米粒子薄膜檢測凝血酶之SEM圖 61
圖2-3 金奈米分布於薄膜之SEM圖及金奈米粒子TEM圖 62
圖2-4 金奈米粒子薄膜檢測凝血酶之LDI-MS質譜圖 63
圖2-5 不同大小之金奈米粒子檢測凝血酶之比較 64
圖2-6 LDI-MS雷射擊打前後之金奈米粒子薄膜SEM圖 65
圖2-7 金奈米粒子薄膜於不同雷射強度檢測凝血酶之比較 66
圖2-8 不同雷射強度擊後之金奈米粒子薄膜SEM圖 67
圖2-9 與凝血酶反應之金奈米粒子薄膜不同雷射強度擊後之SEM圖 68
圖2-10 纖維蛋白原-金奈米粒子薄膜之選擇性 69
圖2-11 LDI-MS檢測不同濃度凝血酶於血漿樣品 70
圖2-12 纖維蛋白原-金奈米粒子薄膜對活性因子Xa之檢測 71
圖2-13 纖維蛋白原-金奈米粒子薄膜對凝血酶抑製劑之比較 72
圖3-1 亞精胺–碳量子點抑制大腸桿菌示意圖 73
圖3-2 亞精胺–碳量子點之TEM與吸收螢光光譜圖 74
圖3-3 碳奈米點之鑑定 75
圖3-4 多胺–碳量子點之MALDI圖 76
圖3-5 多胺–碳量子點之FT-IR圖 77
圖3-6 多胺–碳量子點之不同菌種抑制 78
圖3-7 多胺–碳量子點之碳量子點吸附大腸桿菌螢光顯微鏡 79
圖3-8 多胺–碳量子點抑菌之電子顯微鏡圖 80
圖3-9 多胺–碳量子點之溶血與細胞毒性 81
圖3-10 大鼠傷口測試 82
圖3-11 組織染色 83

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