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研究生:陳晏琦
研究生(外文):Yen-Chi Chen
論文名稱:奈米銀/二氧化鈦/幾丁聚醣複合材料之抗菌效能與應用之研究
論文名稱(外文):Synthesis and characterization of nano-silver / titanium dioxide / chitosan composites and their antimicrobial applications
指導教授:余國賓余國賓引用關係
指導教授(外文):Kuo-Pin Yu
學位類別:博士
校院名稱:國立陽明大學
系所名稱:環境與職業衛生研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2017
畢業學年度:106
語文別:中文
論文頁數:119
中文關鍵詞:奈米銀抗菌劑二氧化鈦熱還原臨界濃度抗真菌活性建築材料幾丁聚醣光化學還原存活率
外文關鍵詞:Silver nanoparticles (AgNPs)Antimicrobial agentTitanium dioxideThermal reductionCritical concentrationAntifungal activityBuilding materialsChitosanPhotochemical reductionSurvival rate
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近年來,因微生物易引起人體健康不良的效應,抗菌的相關材料已引起廣泛的討論。其中,奈米銀(Silver nanoparticles, AgNPs)被視為理想的新穎抗菌產品,因銀已被證實具良好的抗菌效能,且當特徵尺寸為奈米等級時,可獲得更大的表面積,增加與微生物接觸機率,造成微生物直接或間接地損傷或死亡。本研究目的為開發低毒性且穩定的AgNPs,評估AgNPs對於不同微生物的抗菌效果、探討影響因素與實際應用於建材防黴之可行性。另外,為增加AgNPs的生物相容性,本研究亦著手於生物奈米複合材料的開發。研究內容分為以下三部分:
第一部份: 利用熱還原提升TiO2負載奈米銀之抗菌效果及其特性分析
利用兩種不同類型的二氧化鈦(Titanium dioxide, TiO2)粉末(P25、Merck TiO2)及兩種熱處理溫度(120、200℃)合成奈米銀(AgNPs/ TiO2)。透過穿透式電子顯微鏡(TEM)分析確認AgNPs具良好的分散性,其粒徑分佈為3.2 - 5.7 nm。X射線光電子能譜儀 (XPS)及X光繞射(XRD)的結果指出,當熱處理達到200℃時,約67% - 85%的銀離子(Ag+)可被還原成金屬銀(Ag0)。利用抑菌圈(Zone of inhibition)測試評估AgNPs對於三種真菌(A.niger, P. spinulosum和S. chartarum)及兩種細菌(E. coli和S. epidermidis)的抗菌活性。結果發現,Ag0對於細菌及真菌的抗菌活性約為Ag+的1.5 - 3.3倍。當TiO2表面的AgNPs分散程度於 0.2-0.7 μg-Ag/m2時,可表現出最佳的殺菌效果,對於細菌及真菌的最小抑菌濃度 (Critical concentration, Cc) 分別為13.48和25.4 μg/ mL。
第二部份: 利用熱還原奈米銀提升建材防黴性能
本部份將上述合成的AgNPs/P25 TiO2應用於三種常見的室內建材上(石膏板、水泥板及軟木夾板),觀察在95% 相對溼度(relative humidity, RH) 環境下,對於三種真菌 (A.niger, P. spinulosum和S. chartarum)的長效抗菌能力。AgNPs/P25 TiO2的合成變項為兩種不同AgNPs濃度 (2和5wt%),及兩種不同的熱製備溫度(120、200℃),並於建材上負載四種不濃的銀含量(0.025、0.05及0.5μg/cm2和抑制瓊脂培養基上的各真菌生長所需之最小抑菌濃度(Cc))。實驗結果發現,AgNPs/P25 TiO2-200℃顯示出最佳的抗菌效果,當銀負載量為0.5μg/cm2時,所有黴菌在觀察時間內(28天)可完全被抑制生長,且不影響建材的美觀;當銀負載量為0.05μg/cm2時,石膏板及水泥板表現出較高的抗菌性,並可抑制70%以上的黴菌生長。另外,逐步迴歸分析(stepwise linear regression)的結果指出,影響黴菌生長於建材的主要因子為AgNPs的熱製備溫度(β Coefficient= -0.739 – -0.51),且熱製備溫度亦與還原金屬銀(Ag0)濃度達高度正相關(相關係數= 0.781)。本研究結果表示AgNPs/P25 TiO2具實際應用於抗菌建材發展之潛力。
第三部份: 幾丁聚醣奈米銀複合材料的製備與其抗菌成效
本研究以生物聚合物(幾丁聚醣)合成奈米銀複合材料(AgNPs@CS-TiO2),用以增進AgNPs的生物相容性及抗菌效果。經SEM觀察指出,AgNPs@CS-TiO2以直徑約1.7 mm的幾丁聚醣-二氧化鈦微球為主體;利用光化學沉積法將硝酸銀還原並附著於上的AgNPs粒徑約為6.69-8.84 nm (TEM image)。XPS及可見光紫外光分光光譜儀(UV-VIS)的結果更進一步指出,AgNPs@CS-TiO2表面的AgNP主要為還原態金屬(Ag0)。另外,透過傅里葉轉換紅外光譜(FTIR)分析得知,AgNPs除沉積於TiO2表面外,亦可與chitosan上的amide II官能基配位。最後,分別利用最小抑菌濃度(MIC)、最低殺菌濃度(MBC)及殺菌試驗評估AgNPs@CS-TiO2對於細菌(S. epidermidis、E. coli)與真菌孢子(A. niger spores)的抗菌效能。結果發現,AgNPs@CS-TiO2複合材料展現出比單一成分(幾丁聚醣、TiO2)更高的抗細菌效果,並可在3.3小時內可殺死90%的真菌孢子。
Due to the outbreak of the infectious diseases caused by different pathogenic microbes and the development of antibiotic resistance, many researches are searching for new antimicrobial agents. Silver nanoparticles (AgNPs) are considered to be a novel antimicrobial agent, due to their high surface area to volume ratio and the unique chemical and physical properties, moreover, the excellent antimicrobial properties of silver to microbes is well known. This study aimed to develop stable AgNPs with low toxicity to evaluate the antimicrobial effect of AgNPs on different microorganisms and explore the influencing factors and practical applications of AgNPs in building materials. To increase the biocompatibility of AgNPs, this study also embarked on the development of bionanocomposites. The study is divided into the following three parts:
Part 1: Enhanced antimicrobial efficacy of thermal-reduced silver nanoparticles supported by titanium dioxide
The antimicrobial efficacy of silver nanoparticles (AgNPs) is influenced by many factors, including the particle size, AgNP oxidation state and support materials. In this study, AgNPs are synthesized and supported by two types of TiO2 powders (P25 and Merck TiO2) using two heat-treatment temperatures (120 and 200°C). The formation of well-dispersed AgNPs with diameters ranging from 3.2 to 5.7 nm was confirmed using transmission electron microscopy. X-ray photoelectron spectroscopy and X-ray diffraction indicated that the majority of the AgNPs were reduced from Ag+ to Ag0 at 200°C. The AgNP antimicrobial activity was determined by the zone of inhibition against three fungi, A. niger, P. spinulosum and S. chartarum, and two bacteria, E. coli (Gram-negative) and S. epidermidis (Gram-positive). The antimicrobial activity of metallic AgNPs was more pronounced than that of the silver nitrate complex and some antimicrobial drugs. The AgNPs exhibited optimal antimicrobial efficacy when the AgNP dispersion on the surface of TiO2 was in the region between 0.2 and 0.7 μg-Ag/m2. The minimum (critical) AgNP concentrations needed to inhibit the growth of bacteria (E. coli) and fungi (A. niger) were 13.48 and 25.4 μg/mL, respectively. The results indicate that AgNPs/TiO2 nanocomposites are a promising disinfectant against both bacteria and fungi.
Part 2: Novel Mold-Resistant Building Materials Impregnated with Thermal-Reduced Nano-Silver
In this study, we evaluated the long-term antifungal effectiveness of 3 types of interior building materials (gypsum board (GB), cement board (CB) and softwood plywood (S-PW)) impregnated with the thermal-reduced silver nanoparticles supported by titanium dioxide (AgNPs/TiO2) under 95% relative humidity for four weeks. AgNPs/TiO2 were synthesized under two thermal reduction temperatures (120 and 200°C) with two different AgNP weight percentages (2 and 5 wt%). Four different silver loading levels (0.025 μg/cm2, 0.05 μg/cm2, 0.5 μg/cm2 and the critical concentration (Cc) to inhibit fungal growth on agar plates), and three fungal species (A. niger, P. spinulosum and S. chartarum) were used in the experiments. The 200°C thermal-reduced AgNPs/TiO2 demonstrated an excellent antifungal efficiency: mold growth was almost completely inhibited at a very low silver loading level of 0.5 μg/cm2 over a period of 28 days. Additionally, AgNPs/TiO2 exhibited higher antifungal activity in GB and CB than on S-PW (on which mold growth was reduced by more than 70%) at a silver loading level of 0.05 μg/cm2. A stepwise regression model for the major effect factors indicated that the thermal reduction temperature of AgNPs was the key factor inhibiting fungal growth on building materials (standardized β= -0.739 – -0.51).

Part 3: Synthesis of chitosan/TiO2/silver nanocomposites and their antimicrobial activities
Chitosan, which exhibits high biocompatibility, has attracted attention for developing nontoxic fabrications of antibacterial materials. This study demonstrates an important bactericidal application using chitosan as the matrix to load TiO2 and silver nanoparticles (AgNPs @ CS-TiO2) to promote the antimicrobial efficiency and improve their biocompatibility. SEM showed that the AgNPs @ CS-TiO2 catalyst was consisted of chitosan–titania microspheres with diameters of approximately 1.7 mm. AgNPs were reduced with diameters of 6.69–8.84 nm through photochemical deposition. The results of XPS and UV-vis spectroscopy further indicated that the AgNPs growth on the surface of CS-TiO2 were mainly formed by metallic Ag0. In addition, Fourier transform infrared spectroscopy confirmed that the AgNPs can form a chelate with CS-TiO2 through metal coordination of the Ag+ and the amide II groups of chitosan. Finally, minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and inactivation assay were used to evaluate the antibacterial activity of AgNPs @ CS-TiO2 against bacterial and fungal spores. The AgNPs @ CS-TiO2 composite exhibits stronger antibacterial effects than each single-component materials and is able to kill 90% of fungal spores within 3.3 hours.
摘 要 I
Abstract III
目 錄 VI
表目錄 IX
圖目錄 X
附件目錄 XII
名詞解釋 XIII
第一章 緒論 1
1. 研究背景與目的 1
1.1. 研究背景 1
1.2. 研究目的 2
1.3. 研究架構 3
2. 文獻回顧 4
2.1. 微生物的健康效應 4
2.2. 銀/奈米銀的抗菌機制 7
2.3. 奈米銀尺寸及形狀對於抗菌效能影響 10
2.4. 微生物對金屬的抗性機制 11
2.5. 奈米銀的毒性 13
2.6. 建材黴菌與健康效應 15
2.7. 影響建材黴菌的生長因素 16
2.8. 裝修建材上的黴菌生長 17
2.9. 綠建材與建材防黴性能 19
2.10. 幾丁聚醣奈米複合材料 22
2.11. 幾丁聚醣的殺菌機制 23
2.12. 幾丁聚醣奈米銀的殺菌機制 24
參考文獻 25
第二章 利用熱還原提升TiO2負載奈米銀之抗菌效果及其特性分析 33
1. 研究背景與目的 33
2. 材料與方法 35
2.1. 材料與微生物培養 35
2.2. 奈米銀粉體(AgNPs/TiO2)製備 36
2.3. 奈米銀材料分析 37
2.4. 真、細菌菌液體製備 39
2.5. 抗菌試驗(Antimicrobial activity) 40
3. 結果與討論 41
3.1. 奈米銀材料特性 41
3.1.1. 奈米銀之型態與粒徑分佈 41
3.1.2. 奈米銀/二氧化鈦薄膜之表面形貌分析 43
3.1.3. 奈米銀/二氧化鈦薄膜之比表面積分析 44
3.1.4. 銀結構於不同溫度下之變化 45
3.1.5. 銀之化學組態與定量分析 46
3.2. 奈米銀抗菌性能 48
3.3. 奈米銀對於黴菌孢子型態的影響 53
4. 結論 55
參考文獻 56
附件 59
1. 利用ICP-OES測量AgNPs/TiO2在水中的銀釋放濃度 59
2. 臨界(最低)抑菌濃度計算 60
3. 不同熱製備溫度之AgNPs/TiO2抗菌成效(120 °C - 300 °C) 61
第三章 利用熱還原奈米銀提升建材防黴性能 63
1. 研究背景與目的 63
2. 材料與方法 65
2.1. 材料與微生物培養 65
2.2. 奈米銀粉體(AgNPs/TiO2)及菌液製備 66
2.3. 試驗建材 66
2.4. 建材保水率測試 66
2.5. 建材黴菌抗菌試驗 68
2.6. 資料處理與統計分析 70
3. 結果與討論 71
3.1. 建材保水率變化及黴菌生長 71
3.2. 奈米銀(AgNPs/TiO2)於建材上的抑菌 72
3.2.1. 石膏板(GB) 73
3.2.2. 軟木夾板(S-PW) 74
3.2.3. 水泥板(CB) 75
3.3. 迴歸分析 79
3.4. AgNP/TiO2的抗菌效能和特性 80
4. 結論 81
參考文獻 83
附件 85
1. 真菌孢子懸浮液製備流程及顯微鏡下之孢子型態 85
2. 已負載奈米銀建材浸漬無菌水之過程 86
3. 使用Image-J計算建材黴菌生長面積 87
4. 線性迴歸分析模式 88
5. 逐步迴歸分析模式(1)(2)(3) 89
第四章 幾丁聚醣奈米銀複合材料的製備與其抗菌成效 91
1. 研究背景與目的 91
2. 材料與方法 93
2.1. 材料與微生物培養 93
2.2. 幾丁聚醣-奈米銀複合材料製備(AgNPs@CS-TiO2) 95
2.3. 幾丁聚醣-奈米銀材料分析 96
2.4. 最低抑菌及殺菌濃度試驗 97
2.5. 殺菌試驗(Inactivation of A. niger by AgNPs@CS-TiO2) 98
3. 結果與討論 99
3.1. 幾丁聚醣-奈米銀複合材料特性 99
3.1.1. 幾丁聚醣-奈米銀微球表面形貌分析 99
3.1.2. 幾丁聚醣/奈米銀微球之型態與粒徑分佈 100
3.1.3. XPS analysis 101
3.1.4. FTIR spectra of AgNPs@CS-TiO2 102
3.1.5. UV-VIS spectroscopy of AgNPs/TiO2 and AgNPs@CS -TiO2 103
3.2. AgNPs@CS-TiO2抗菌性能 105
3.2.1. Antibacterial activity of AgNPs@CS-TiO2 against bacteria 105
3.2.2. The inactivation of Aspergillus niger spores 107
4. 結論 108
參考文獻 110
附件 113
1. AgNPs@CS-TiO2生物相容性 113
2. The zone of inhibition by AgNPs@CS-TiO2, CS and CS-TiO2 114
3. AgNPs/TiO2殺菌試驗流程 115
第五章 結論 117
未來研究項目 119
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part 2:
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15. Panchanathan, M.; Jayachandran, V.; Kalimuthu, S.; Kannan, S.; Se-Kwon, K., Biosynthesis, Antimicrobial and Cytotoxic Effect of Silver Nanoparticles Using a Novel Nocardiopsis sp. MBRC-1. BioMed Research International 2013,2013, 9.
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part 3:
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7. Ogar, A.; Tylko, G.; Turnau, K., Antifungal properties of silver nanoparticles against indoor mould growth. Sci Total Environ 2015,521-522, 305-314.
8. Huang, H. L.; Lin, C. C.; Hsu, K., Comparison of resistance improvement to fungal growth on green and conventional building materials by nano-metal impregnation. Building and Environment 2015,93, 119-127.
9. Chen, Y. C.; Shao, W. C.; Yu, K. P., A Study of Evaluating Fungal Growth and Influencing Factors on Interior Decoration Materials. JOURNAL OF ARCHITECTURE 2017,99, 37-54.
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part 4:
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