臺灣博碩士論文加值系統

發展具有生物惰性與生物活性調控功能表面的智慧材料，是現今主要生醫材料研究課題。而在本研究主要以溫感性的高分子(N-isopropylacrylamide)與雙離子性高分子(zwitterionicsulfobetaine methacrylate) 以低壓電漿聚合法接枝於醋酸纖維膜中(cellulose acetate)，以成為可調控之生物感應膜。並製備出混合式與雙層結構的改質膜，以探討微生物吸附之可逆效果。表面化學組成與性質鑑定以傅立葉紅外線光譜儀(Fourier Transform Infrared Sepectromter)、表面化學分析儀(X-ray photoelectron spectroscopy)、接觸角量測儀(contact angle) 、與掃描式電子顯微鏡(Scanning Electron Microscopy)觀察改質膜材之定性與定量分析。改質膜於生物環境中展現出高的溫度感應性、膨潤度與親水能力以達到調控大腸桿菌(Escherichia coli)的貼附行為。而對於非離子性的NIPAAm含量較高的接枝膜，由於在高溫中展現出疏水特性，以致含有較高的細菌貼附。反之SBMA含量高的接枝膜，藉由表面的親水特性，展現出抗微生物沾黏之特性。而本研究以雙層結構探討微生物的可逆吸脫附效果，發現可有效控制微生物脫附速率。這種藉由環境感應而調控表面特性的改質膜，對於智慧材料的生物應用上，有很大貢獻與發展。

A great challenge in the smart biomaterials is to design a dual-functional bioinert/bioactive surface with reversible bioadhesive property. This work describes a novel tunable bioadhesive membrane of cellulose acetate (CA) grafted with thermoresponsive N-isopropylacrylamide (NIPAAm) and zwitterionic sulfobetaine methacrylate (SBMA). Two grafting structures of PNIPAAm-SBMA copolymer layer, mix- and bi-layer grafting, were controlled using low pressure plasma-induced graft polymerization and varing NIPAAm and SBMA molar ratios. The chemical composition and microstructure of the various surface-modified CA membranes were characterized by Fourier transform infrared spectroscopy (FT-IR), contact angle, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) measurements. The prepared membranes exhibited controllable temperature-dependent swelling behaviors and surface hydrophilicity which highly regulates the attachment of Gram-negative bacteria (E. coli). At a physiological temperature, the high content of the non-ionic polyNIPAAm in grafting copolymeric layer exhibits a high bacterial attachment due to the interfacial exposure of polyNIPAAm-rich hydrophobic domains. A relatively high content of polySBMA in grafting copolymeric layer exhibits reduced amounts of bacterial attachment due to the interfacial hydration of polySBMA-rich hydrophilic segments. This work shows that the bioadhesive properties of CA membranes with bi-layer copolymer grafting can be effectively controlled to be potentially useful for triggered bacteria detachment. The tunable-bioadhesive behavior of temperature-sensitive
PNIPAAm-SBMA grafted CA membranes makes this smart membrane appropriate for biomedical applications.

目錄
中文摘要 ................................................................................................................ I
英文摘要 .............................................................................................................. II
致謝 ..................................................................................................................... IV
目錄 ...................................................................................................................... V
圖目錄 .............................................................................................................. VIII
表目錄 ............................................................................................................... XII
第一章 緒論 ......................................................................................................... 1
研究系統一、接枝含非離子與雙離子混合式結構之醋酸纖維智能膜於可逆性微生物貼附控制之研究 ........................................................................... 1
研究系統二、接枝含非離子與雙離子雙層式結構之醋酸纖維智能膜於可逆性微生物貼附控制之研究 ........................................................................... 1
第二章 文獻回顧 ................................................................................................. 2
2.1生醫材料之簡介 ......................................................................................... 2
2.1.1生醫材料之發展 ................................................................................... 2
2.1.2 生醫材料之分類 .................................................................................. 3
2.1.3 抗沾黏性生醫材料之發展與應用 ...................................................... 4
2.2 智能材料 ................................................................................................. 8

2.2.1智能材料之介紹 ................................................................................... 8
2.2.2 智能材料之分類 ................................................................................ 10
2.2.3智能膜材之發展與應用 ..................................................................... 18
2.3 微生物貼附 ............................................................................................... 19
2.3.1微生物貼附之簡介 ............................................................................. 19
2.3.2微生物表面之結構介紹 ..................................................................... 21
2.3.3 微生物表面作用力 ............................................................................ 23
2.3.4抗微生物沾黏之材料發展與應用 ..................................................... 25
2.4 材料改質法 ............................................................................................... 25
2.4.1 電漿改質法 ........................................................................................ 25
2.4.2臭氧改質與熱聚合法 ......................................................................... 30
2.4.3原子自由基轉移聚合法 ..................................................................... 31
第三章 實驗藥品、設備與方法 ....................................................................... 33
3.1 實驗藥品 ................................................................................................... 33
3.2實驗設備 ................................................................................................... 35
3.3 實驗方法 ................................................................................................... 37
3.3.1 微生物之選擇 .................................................................................... 37
3.3.2菌種培養 ............................................................................................. 37
3.3.3醋酸纖維膜浸泡單體溶液之製備 ..................................................... 38
3.3.4薄膜表面改質 ..................................................................................... 39

3.3.5控溫型膜表面親疏水性量測 ............................................................. 41
3.3.6材料表面鑑定與分析 ......................................................................... 41
3.3.7含水量(degree of swelling)測試 ......................................................... 42
3.3.8細菌貼附之測試 ................................................................................. 42
第四章 醋酸纖維膜混合式接枝NIPAAM與SBMA高分子之智能膜材含水層轉移與菌種可逆性之研究 ......................................................................... 44
4.1研究目的 ................................................................................................... 44
4.2 改質條件與定性分析 ............................................................................... 46
4.3 微生物可逆貼附測試 ............................................................................... 58
第五章 醋酸纖維膜(CELLULOSE ACETATE)異相改質SBMA與NIPAAM之雙層高分子智能膜含水層轉移與菌種可逆性之研究 ............... 63
5.1研究目的 ................................................................................................... 63
5.2雙層異相結構之定性分析 ....................................................................... 63
5.3雙層改質之微生物可逆貼附測試 ........................................................... 69
第六章 結論 ....................................................................................................... 76
參考文獻 ............................................................................................................. 77

圖目錄
第二章文獻回顧
圖2- 1 接枝PMAA膜材，對於環境PH值得改變，表面AFM型態 ......... 11
圖2- 2 PLGA(poly L-glutamic acid)的等電點大約在5.5左右，因此環境PH大於等電點時，電性即發生轉變 .............................................................. 12
圖2- 3 基材接枝PLGA後，藉由等電點對環境的變化，改變本身電性，使的膜材間的孔洞發生填補效果之示意圖 .............................................. 12
圖2- 4 spiropyran-containing copolymer (SPMA/MMA)藉由紫外光照射下，型態的轉變示意圖 ...................................................................................... 13
圖2- 5 NIPAAm結構圖 ..................................................................................... 14
圖2- 6 PNIPAAm-AZ感溫型高分子接枝膜，對於色胺酸(tryptophan)的流通效果示意圖 .................................................................................................. 14
圖2- 7含溫度響應高分子膜對細菌之感應控制圖 ........................................ 15
圖2- 8高分子SBMA與NIPAAm以1:1混合(S50-N50)溫度敏感性示意圖 ...................................................................................................................... 16
圖2- 9摻混SBMA與NIPAAm比例之水膠與TCPS對於細菌貼附測試 (a) S. epideridis表面貼附測試(b) Escherichia coli表面貼附測試 ........... 17
圖2- 10摻混比例水膠對於人類纖維瘤細胞(HT-1080)脫附測試 ................. 18
圖2- 11微生物貼附於表面的主要影響原因分類表 ....................................... 19
圖2- 12微生物貼附於表面的先後順序 ........................................................... 20

圖2- 13細菌表層及內部之構造圖 ................................................................... 23
圖2- 14 不同菌種於不同PH值下的表面電荷 ............................................... 24
圖2- 15電漿反應示意圖 ................................................................................... 28
第三章 實驗藥品、設備與方法
圖3- 1 E-coli 之生長曲線 ................................................................................. 38
圖3- 2低壓電漿設備外觀圖 ............................................................................. 40
第四章 醋酸纖維膜(cellulose acetate)混合式接枝NIPAAm與SBMA高分子之智能膜材含水層轉移與菌種可逆性之研究
圖4- 1接枝於纖維膜表面之結構主要分為(A)混合式高分子接枝與(B)雙層 異相接枝 ...................................................................................................... 44
圖4- 2 (a) 摻混型與(b)異相雙層結構藉由降溫過程，各高分子鏈段之LCST與UCST相互轉變，以致含水層相互擾動與遷移之示意圖 ................. 45
圖4- 3不同濃度之(a)SBMA(5%wt、10%wt、20%wt)與(b)NIPAAm(5%wt、
10%wt)藉由氬氣低壓電漿(150W、30sccm)分批時間改質於醋酸纖維膜中(5S、15S、30S、60S)之接枝度圖 ...................................................... 48
圖4- 4 SBMA與NIPAAm依不同混合比例，接枝於醋酸纖維膜材上，以秤重量測各別條件之接枝度圖 .................................................................. 49
圖4- 5 混合式高分子接枝於膜材表面之油相變溫接觸角量測圖 ................ 49
圖4- 6 以ATR-FTIR對於不同摻混比例、改質30秒之(a)全波段、(b)S=O、(c)N-H、(d)C-O-C特徵峰譜圖 .................................................................. 51

圖4- 7 ESCA (a)硫譜(b)氮譜(c)碳譜分析量測各秒數處理之混合式膜材 .... 53
圖4- 8(a)SBMA依不同處理時間(5s、15s、30s、60s)表面SEM圖(b) NIPAAm依不同處理時間(5s、15s、30s、60s)表面SEM圖(c)固定處理時間(30s)不同摻混比例之表面SEM圖(d)未改質之醋酸纖維膜表面 ................... 57
圖4- 9混和接枝條件下，膜材於37℃與20℃之個別含水量測試 .............. 57
圖4- 10 CLSM之3小時細菌貼附圖，(a)3小時的貼附測試結束後，以共軛焦顯微鏡觀察貼附情況(b) 3小時的貼附測試結束後，將薄膜靜置於20℃培養液中1小時(c) 3小時的貼附測試結束後，將薄膜靜置於20℃培養液中2小時 .......................................................................................... 60
圖4- 11CLSM之24小時E-coli細菌貼附圖，(a)24小時的貼附測試結束後，以共軛焦顯微鏡觀察貼附情況(b) 24小時的貼附測試結束後，將薄膜靜置於20℃培養液中1小時(c) 24小時的貼附測試結束後，將薄膜靜置於20℃培養液中2小時 ......................................................................... 61
圖4- 12 E-coli貼附3小時之菌量相對百分比 ................................................ 62
圖4- 13 E-coli貼附24小時之混合式改質膜菌量相對百分比，由CLSM觀察出貼附量皆以死菌為主(>99%) .............................................................. 62
第五章 醋酸纖維膜(cellulose acetate)異相改質SBMA與NIPAAm之雙層高分子智能膜含水層轉移與菌種可逆性之研究
圖5- 1 製備不同SBMA接枝厚度之雙層膜，將SBMA以不同低壓電漿處理時間製備出不同厚度之底層(S100-15S、S100-30S、S100-60S)，再者
以NIPAAm固定30秒低壓電漿處理，即得雙層異相接枝結構(S100-15S_N100-30S、S100-30S_N100-30S、S100-60S_N100-30S) ....... 63
圖5- 2 單層SBMA改質膜之二碘甲烷油相接觸角 ....................................... 64
圖5- 3雙層改質膜之二碘甲烷油相變溫接觸角 ............................................ 65
圖5- 4雙層改質膜之ATR-FTIR圖譜(a)全譜圖(b)S=O特徵峰(c)N-H特徵峰(d)C-O-C特徵峰 ..................................................................................... 66
圖5-5雙層改質膜之SEM表面圖(a)未改質膜(b)N100-30S .......................... 67
圖5- 6雙層分改質膜之37℃與20℃控溫含水量變化與接枝度圖 .............. 69
圖5- 7雙層改質膜之3小時E-coli細菌貼附圖，(a)3小時的貼附測試結束後，以共軛焦顯微鏡觀察貼附情況(b) 3小時的貼附測試結束後，將薄膜靜置於20℃培養液中1小時(c) 3小時的貼附測試結束後，將薄膜靜置於20℃培養液中2小時 ......................................................................... 71
圖5- 8雙層改質膜之24小時E-coli貼附圖，(a)24小時的貼附測試結束後，以共軛焦顯微鏡觀察貼附情況(b) 24小時的貼 附測試結束後，將薄膜靜置於20℃培養液中1小時(c) 24小時的貼附測試結束後，將薄膜靜置於20℃培養液中2小時 ......................................................................... 72
圖5- 9E-coli貼附3小時之死菌與整體菌量相對百分比 ............................... 73
圖5- 10 E-coli貼附24小時之混合式改質膜菌量相對百分比，由CLSM觀察整體貼附皆以死菌為主要貼附(>99%).................................................. 74
圖5- 11(a)混合式結構(b)雙層結構之微生物可逆效果示意圖 ..................... 74

表目錄
第四章 醋酸纖維膜(cellulose acetate)混合式接枝NIPAAm與SBMA高分子之智能膜材含水層轉移與菌種可逆性之研究
表4- 1 ESCA硫氮譜之特徵峰面積值，硫譜面積主要可與SBMA含量成正相關；氮譜分峰為二級氮與三級氮(鍵結能各為399.18與401.9ev) .... 54
表4- 2由ESCA硫與四級氮面積百計算出接枝高分子之電性保有度 ........ 55
第五章 醋酸纖維膜(cellulose acetate)異相改質SBMA與NIPAAm之雙層高分子智能膜含水層轉移與菌種可逆性之研究
表5- 1雙層改質膜之ESCA分峰面積表 ........................................................ 67

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