臺灣博碩士論文加值系統

本研究以化學自由基聚合法以聚吡咯包覆聚醯胺66靜電紡絲纖維製備電極，以大腸癌循環腫瘤細胞表面接枝專一性抗體(Anti-EpCAM conjugated Biotin)於電化學分析技術偵測大腸癌循環腫瘤細胞，並利用電聚合摻雜生物素技術，進行釋放後培養細胞之應用。結果指出，利用電化學分析法於大腸癌循環腫瘤細胞三株含有EpCAM表現細胞分別為HCT-116、HT-29及DLD1綜合偵測結果分析發現，空白聚吡咯包覆聚醯胺66靜電紡絲纖維其初始電流值為576.37μA，而各別偵測電流響應於5顆細胞平均偵測電流值降至509.47μA、10顆細胞降至459.58μA、15顆細胞降至406.24μA、20顆細胞降至297.55μA，隨著細胞數增加呈現線性正相關。而在釋放大腸癌循環腫瘤細胞結果，以電極表面摻雜生物素蛋白施加外加電場方式作為釋放循環腫瘤細胞的開關機制，以本實驗結果具備最低粗糙度5.67μm聚吡咯摻雜生物素蛋白包覆聚醯胺66靜電紡絲纖維過程可達到78%的釋放率效果。而後續在進行釋放後培養24小時生長觀察發現，細胞仍具備高度的活性於基材貼附、偽足伸長的生長過程。本研究成功製備聚吡咯包覆聚醯胺66三維奈米結構電極於大腸癌循環腫瘤細胞提供高靈敏度偵測及良好的釋放效率。

In this study, we electrospun Nylon 66 fibrous mats to deposit Polypyrrole on the fiber surfaces as electrode substrate. Anti-EpCAM was sequentially immobilized on the Polypyrrole-deposited Nylon 66 fibrous mats as assays to detect the circulating tumor cells (CTCs) from specimens with current change. The current of blank fibrous assay was 576.37μA. After five CTCs attachment, the current decreased to 509.47μA, indicated that the electrode of fibrous mats could detect the CTCs with high sensitivity. With increase of the amount of CTCs on the fibrous mats, the current of the fibrous mats decreased linearly. The results suggest that the fibrous mat could capture the CTCs from the specimen and detect the CTCs rapidly.
The captured CTCs are usually amplified with culture for gene therapy in future. The Polypyrrole-deposited Nylon 66 fibrous mats were doped biotin for release of cultured cells. We doped biotin in the Polypyrrole-deposited Nylon 66 fibrous mats. The results indicate that Polypyrrole-deposited Nylon 66 fibrous mats with lower roughness (5.67μm) could achieve higher releasing ratio (78%). Overall, Polypyrrole-deposited Nylon 66 fibrous mats not only detect the CTCs sensitively, but also well-performed in CTCs culture and release.

摘要 I
Abstract III
致謝 V
圖目錄 XI
表目錄 XVI
式目錄 XVII
第1章 緒論 1
研究動機與目的 1
研究背景 5
第2章 文獻回顧及實驗理論 7
2-1-1 循環腫瘤細胞介紹 7
2-1-2 循環腫瘤細胞生物特性標記 10
2-1-2-1 生物上皮黏附因子 10
2-1-2-2 目前捕捉及標定循環腫瘤細胞應用技術介紹 13
2-1-3 循環腫瘤細胞捕捉後培養於個人化治療之應用 15
2-1-3-1 循環腫瘤細胞釋放 17
2-1-3-2 循環腫瘤細胞培養 21
2-1-3-3 循環腫瘤細胞電誘導釋放理論 23
2-1-3-3-1 循環伏安分析法 25
2-1-3-3-2 計時電位聚合法 28
2-1-2-3-3 電化學阻抗分析法 28
2-1-2-4 靜電紡絲纖維結構於電雙層性質的貢獻 29
2-1-2-5 循環腫瘤細胞電訊號偵測技術 30
2-2 偵測/培養型電極奈米纖維基板製備 32
2-2-1奈米纖維製備技術 33
2-2-2靜電紡絲發展 36
2-2-3靜電紡絲原理 37
2-3靜電紡絲纖維基板於生物技術應用 38
2-3-1聚醯胺66 40
2-4導電高分子 42
2-4-1聚吡咯 45
2-4-2電化學聚合法 46
2-4-3導電高分子摻雜系統 47
2-4-4化學氧化聚合法 52
2-5靜電紡絲介面優勢於偵測、捕捉循環腫瘤細胞 53
2-6材料性質於抗生物沾黏之應用 57
2-7生物分子與材料介面固定法 59
2-7-1生物分子固定法 59
2-7-2共價鍵固定法(EDC-NHS reaction) 60
2-7-3生物親和法(Streptavidin-biotein system) 61
第3章 分析儀器原理 64
3-1 高解析度場發射電子顯微鏡 (Scanning Electron Microscope, SEM) 64
3-2 穿透式電子顯微鏡 (Field-emission transmission electron microscope，FE-TEM) 65
3-3 共軛焦顯微鏡 (Confocal microscopy, CLSM) 70
3-4 恆電位分析儀 (Potential Stat) 71
3-5接觸角量測儀(Contact Angle Meter) 72
3-6 光電子能譜儀 (X-ray Photoelectron Spectroscope, XPS) 74
3-7 螢光光譜儀 75
3-8 三維光學輪廓儀 76
第4章 實驗設計與方法 78
4-1實驗設計目的與流程架構 78
4-2實驗藥品 80
4-3實驗儀器 83
4-4實驗步驟 85
4-4-1靜電紡絲工程 85
4-4-2靜電紡絲溶液製備 86
4-4-3聚吡咯包覆聚醯胺66功能化關係 86
4-4-4基本電極奈米纖維表面製備流程 87
4-4-4-1製備生物素摻雜聚吡咯於捕捉與釋放循環腫瘤細胞之功能化介面 87
4-4-4-2製備表面專一性抗體於偵測循環腫瘤細胞之功能化介面 88
4-4-5 電化學反應系統設計於循環腫瘤細胞分析 88
4-4-5-1電解質條件 89
4-5電極表面專一性於循環腫瘤細胞 89
4-6奈米纖維功能化性質抗沾黏之應用 89
4-7生物檢體標定與染色 90
4-7-1血液檢體處理White blood cell、Buffy coat 90
4-7-2細胞固定與染色 91
4-7-3細胞於EpCAM蛋白標定與染色 91
4-7-3-1 HCT-116、HT-29、DLD1 91
4-7-3-2 Hela cell 92
4-8螢光標準曲線分析 92
4-9顯微鏡試片製作 93
4-9-1掃描式電子顯微鏡試片 93
4-9-2螢光顯微鏡試片 93
4-9-3穿透式電子顯微鏡試片 94
4-9-4綠光干涉儀試片 94
4-10奈米纖維於細胞捕捉與電釋放訊號分析 94
4-11奈米纖維電偵測訊號分析捕捉循環腫瘤細胞 95
第5章 實驗結果與討論 96
5-1聚醯胺66奈米纖維基板於聚吡咯導電高分子包覆分析 96
5-1-1不同包覆過程時間於基板之纖維尺度及表面結構 96
5-1-2不同包覆過程時間於基板之導電性質 98
5-1-3聚吡咯包覆Nylon66證明之元素分析 99
5-1-3-1聚吡咯包覆Nylon66 IR 光譜圖 99
5-1-3-2聚吡咯包覆Nylon66 XPS能譜圖 100
5-1-4聚吡咯包覆後電誘導於粗糙度變化響應 102
5-2基板應用於捕捉後釋放大腸癌循環腫瘤細胞分析 103
5-2-1電極表面摻雜生物素之表面改質技術 104
5-2-1-1電極表面改質摻雜生物素 IR光譜分析 105
5-2-1-2功能化電極表面及通電釋放生物素 XPS圖譜分析 106
5-2-1-3電極表面改質摻雜生物素 循環伏安圖分析 109
5-2-2電極表面摻雜過程粗糙度變化分析 110
5-2-3電極表面於釋放大腸癌循環腫瘤細胞之電訊號分析 111
5-2-4電誘導於釋放循環腫瘤細胞與電極表面粗糙度關係 113
5-2-5電誘導釋放循環腫瘤細胞於PNF奈米纖維培養螢光圖 115
5-2-5-1電誘導釋放CTCs於PNF奈米纖維培養SEM圖 117
5-3 PNF電極應用於電化學定量與捕捉循環腫瘤細胞分析 118
5-3-1電極表面於偵測大腸癌循環腫瘤細胞專一性改質分析 119
5-3-2電化學分析技術於偵測大腸癌循環腫瘤細胞 120
5-3-2-1 循環伏安法於偵測循環腫瘤細胞 120
5-3-2-1-1 偵測系統之循環伏安法於HCT-116電訊號分析 120
5-3-2-1-2 偵測系統之循環伏安法於HT-29電訊號分析 121
5-3-2-1-3 偵測系統之循環伏安法於DLD1電訊號分析 122
5-3-2-1-4偵測結果於HCT-116、HT-29、DLD1 123
5-3-3電化學阻抗分析技術於偵測循環腫瘤細胞 124
5-3-3-1定量電化學阻抗分析於0-1000顆HCT-116 124
5-3-3-2定量電化學阻抗分析於0-1000顆HT-29 125
5-3-3-3定量電化學阻抗分析於0-1000顆DLD1 126
5-3-3-4電化學阻抗偵測技術於循環腫瘤細胞結果 127
5-3-4 電化學分析技術於定量大腸癌循環腫瘤細胞 127
5-3-4-1循環伏安法分析於定量HCT-116、HT-29、DLD1 127
5-3-4-2電化學阻抗分析於定量HCT-116、HT-29、DLD1 131
5-3-5電流綜合分析於HCT-116、HT-29、DLD1 134
第6章 結論 136
第7章 參考文獻 137





















圖目錄
圖 1 1: 2017美國十大癌症死亡統計人數 2
圖 1 2:2017年美國女性(右)、男性(左)罹癌名列第三名統計圖 2
圖 1 3:美國針對各癌症相對五年存活率統計 3
圖 1 4:個體基因型、基因表現對於藥效治療差異示意圖 4
圖 2 1:原位癌進入血液循環系統示意圖 8
圖 2 2:循環腫瘤細胞轉化及入侵其他細胞與周圍組織過程 8
圖 2 3:量測腫瘤大小與抽血檢驗血液中的CTC數目相比較。CTC數目(紅)與腫瘤大小(藍)，(A)肺癌，(B)肺癌，(C)大腸癌，(D)胰臟癌 9
圖 2 4:腫瘤細胞與血液中細胞濃度關係 9
圖 2 5:全血樣本經磁性磁珠修飾抗體分離CTCs過程 13
圖 2 6:微流道發展技術示意圖 14
圖 2 7:藉由圖騰化(左)及微流道(右)之特異性捕獲標靶細胞示意圖 14
圖 2 8:個人化醫療目的 15
圖 2 9:量身打造針對最適藥物對於個人化醫療 16
圖 2 10:光感型功能化磁珠鍵結抗體捕捉循環腫瘤細胞後磁性分離後光釋放循環腫瘤細胞 18
圖 2 11:特定醣類高分子經由功能化捕捉循環腫瘤細胞後以裂解酶將其醣類高分子斷鍵釋放循環腫瘤細胞 18
圖 2 12:材料基板生物降解型釋放循環腫瘤細胞 19
圖 2 13:功能化磁珠鍵結抗體捕捉循環腫瘤細胞後磁性分離後利用 19
圖 2 14 :溫感控制常以設計高分子低臨界溶解溫度(LCST)做捕獲與釋放循環腫瘤細胞 20
圖 2 15:以摻雜系統捕捉及電釋放循環腫瘤細胞 21
圖 2 16:藉由通電作為開關機制釋放專一性捕獲的循環腫瘤細胞作後續培養示意 21
圖 2 17:神經細胞生長在非方向性(A)/方向性(B)靜電紡絲基材上貼附伸出偽足及循環腫瘤細胞生長在具有生物相容性材料上(C)及一般玻片(D)上伸出偽足差異特性的情形 22
圖 2 18:藉由從臨床檢體捕獲後釋放至特定培養載台的少量循環腫瘤細胞培養過程 23
圖 2 19:由生物素Biotin陰離子摻雜入聚吡咯Ploypyrrole導電高分子於循環腫瘤細胞捕獲與釋放 24
圖 2 20:專一性電位釋放(圖左)於電位對於細胞活性影響(圖中)專一性捕獲抗體接枝於電性改變下變性響應(圖右) 24
圖 2 21:循環伏安圖實際曲線 25
圖 2 22:電化學阻抗頻譜原理 29
圖 2 23:循環腫瘤細胞專一性電極製備之循環伏安/電流訊號變化 30
圖 2 24:細胞培養後藉由摻雜系統機制電釋放循環伏安法 31
圖 2 25:電流訊號定量於捕獲電極表面循環腫瘤細胞 31
圖 2 26:電化學阻抗分析Impedance spectroscopy(EIS) 32
圖 2 27:融溶式靜電紡絲技術 34
圖 2 28:氧化鋁之化學沉積纖維圖騰化技術 34
圖 2 29:壓印翻模柱狀纖維結構技術 34
圖 2 30:延展法纖維結構製備技術 35
圖 2 31:溼式紡絲技術 35
圖 2 32:靜電紡絲製程技術 35
圖 2 33:靜電紡絲工程應用領域 37
圖 2 34：靜電紡絲形成示意圖 38
圖 2 35:靜電紡絲纖維應用於組織工程 39
圖 2 36:靜電紡絲工程應用於中空結構藥物的填埋於電釋放技術 39
圖 2 37：電雙層形成高縱橫比的尼龍奈米纖維模擬圖 41
圖 2 38：(A)高縱橫比的奈米纖維及(B)電雙層結構圖 42
圖 2 39:導電高分子電子傳導過程 43
圖 2 40:藉由摻雜效應改變導電高分子本質能隙示意圖 44
圖 2 41:三極式電化學聚合法反應裝置 47
圖 2 42:本質型導電高分子經由摻雜形成p-type/ n-type型過程 48
圖 2 43:摻雜離子反應於電子與電洞形成過程 49
圖 2 44:不同摻雜濃度之生物素摻雜於聚吡咯表面比較捕捉循環腫瘤細胞數量關係 50
圖 2 45:聚乳酸奈米纖維以化學聚合法以聚吡咯做表層包覆 52
圖 2 46:以DNA互補關係於電化學分析系統下偵測電訊號示意圖 53
圖 2 47:藉由電極陣列形式作為開關於電訊號分析系統下應用 54
圖 2 48:電化學阻抗分析技術於生物樣本定量示意圖 55
圖 2 49:循環伏安法分析技術於電極製備改質證明(a)、電子轉移效率測試(b)及電流變化於生物樣本定量示意圖 55
圖 2 50:摻雜系統改質介面(A)(B)(D)於表面接枝專一性分子及先以介面改質技術(C)作後生物分子鍵結示意圖 56
圖 2 51:電極表面改質後於接枝專一性響應分子抗體-抗原示意圖 57
圖 2 52:非專一性分子貼附於基材表面抗非專一性沾附示意圖 58
圖 2 53:電極纖維表面BSA阻斷效應於專一性改質電訊號偵測CTCs 58
圖 2 54：三種固定化技術於物理性、共價鍵及生物親合性法 59
圖 2 55：EDC與NHS共價鍵結固定反應 60
圖 2 56：固定脂於Poly(acrylonitrile-co-maleic acid)奈米纖維表面 61
圖 2 57:鏈親和素SA增加抗體接枝密度示意圖 62
圖 2 58:摻雜系統於纖維表面電釋放循環腫瘤細胞示意圖 62
圖 2 59：偵測循環腫瘤細胞上將生物素化的Anti-EpCAM藉由生物親和性反應鍵結奈米纖維上鏈親和素SA提升抗體接枝密度 63
圖 3 1：掃描式電子顯微鏡構造示意圖 65
圖 3 2:穿透式電子顯微鏡結構示意圖 67
圖 3 3:穿透式電子顯微鏡影像及繞射原理示意圖 68
圖 3 4:亮場與暗場成像示意圖 69
圖 3 5：共軛焦雷射掃描顯微鏡技術示意圖 71
圖 3 6:電化學分析系統電極配置圖 72
圖 3 7: 液滴表面張力示意圖 73
圖 3 8：光電子產生示意圖 74
圖 3 9：Mirau干涉原理與干涉條紋 76
圖 3 10：干涉儀架構示意圖 77
圖 4 1:電極介面底材製備及應用流程圖 79
圖 4 2:電誘導奈米纖維釋放循環腫瘤細胞實驗流程圖 79
圖 4 3:電化學定量技術與循環腫瘤細胞捕捉流程圖 80
圖 5 1: Nylon66奈米纖維於不同時間包覆聚吡咯表面形貌(A)Nylon66、 (B) 聚吡咯包覆Nylon66於7小時、(C) 聚吡咯包覆Nylon66於14小時、(D) 聚吡咯包覆Nylon66於24小時 97
圖 5 2:Nylon66奈米纖維包覆聚吡咯穿透式電子顯微鏡成像形貌 98
圖 5 3:聚吡咯以化學聚合法包覆Nylon66(PNF)基板製備過程 99
圖 5 4:C1s, N1s 譜於聚吡咯包覆Nylon66前後變化能譜圖 101
圖 5 5:S2p, Cl2p3 譜於聚吡咯包覆Nylon66前後變化能譜圖 101
圖 5 6:S2p, Cl2p3 譜於聚吡咯包覆Nylon66前後相對強度能譜圖 102
圖 5 7:PNF通於氧化/還原電位下接觸角的響應(A)通電前(B)通電後PNF 103
圖 5 8:PNF通於氧化/還原電位下接觸角的重複性響應 103
圖 5 9:標準PNF基板表面鍍金技術ESS與電聚合生物素ES1-ES10表面改於電子顯微鏡成像圖 104
圖 5 10:黑線與紅線IR圖譜表示PNF奈米纖維成功製備過程後於電極鍍金技術電聚合成功摻雜生物素於電極表面藍線IR圖譜 105
圖 5 11:藉由1-10分鐘摻雜時間XPS於C、N、O、S譜分析 106
圖 5 12:1-10分鐘摻雜過程於電極表面硫含量分析 107
圖 5 13: Au4f圖譜得知PNF表面鍍金技術前後特徵訊號能譜 108
圖 5 14:電極表面改質生物素C1s,N1s釋放前與釋放後相對強度能譜 108
圖 5 15:電極表面改質生物素S2p釋放前與釋放後相對強度能譜圖 109
圖 5 16:A圖PBS緩衝溶液、B吡咯單體氧化還原電位、C圖生物素加入影響吡咯氧化還原電位掃描10次循環伏安圖譜、D圖疊圖於A、B、C之單次循環伏安譜 110
圖 5 17: PNF奈米纖維基板經由電極製備技術於1、3、5、7、10電聚合摻雜生物素過程材料表面粗糙度變化 111
圖 5 18:接枝專一性抗體於捕捉釋放循環腫瘤細胞電流密度變化 112
圖 5 19:接枝專一性抗體於捕捉釋放循環腫瘤細胞電化學阻抗頻譜變化 112
圖 5 20:電位-0.8伏特專一性釋放循環腫瘤細胞(A)陽性反應釋放HCT-116 (B)陰性反應電誘導於White blood cells 白血球 (C)陰性反應電誘導於Hela cells 子宮頸癌細胞 114
圖 5 21:不同摻雜時間及Biotin含量比較於釋放循環腫瘤細胞效率與選擇性 115
圖 5 22:利用PNF奈米纖維培養釋放後循環腫瘤細胞HCT-116 116
圖 5 23:三維培養HCT-116循環腫瘤細胞DAPI染色圖 116
圖 5 24:於釋放培養PNF基材表面1至12天細胞成長過程 116
圖 5 25:循環腫瘤細胞HCT-116貼附行為於PNF培養示意圖 117
圖 5 26:三維培養HCT-116循環腫瘤細胞電子顯微鏡成像圖 118
圖 5 27:表面專一性改質螢光圖譜證明於圖A未改質前、圖B接枝Streptavidin-FITC、圖C鍵結Biotin-Cy5、圖D螢光疊圖於圖B、C 119
圖 5 28:偵測系統之循環伏安法於HCT-116循環腫瘤細胞偵測電訊號分析 121
圖 5 29:偵測系統之循環伏安法於HT-29循環腫瘤細胞偵測電訊號分析圖 122
圖 5 30:偵測系統之循環伏安法於HT-29循環腫瘤細胞偵測電訊號分析圖 123
圖 5 31:定量電化學阻抗分析於0-1000顆HCT-116循環腫瘤細胞奈式曲線圖 124
圖 5 32:定量電化學阻抗分析於0-1000顆HT-29循環腫瘤細胞奈式曲線圖 125
圖 5 33:定量電化學阻抗分析於0-1000顆DLD1循環腫瘤細胞奈式曲線圖 126
圖 5 34:循環伏安法於HCT-116定量分析圖 128
圖 5 35:循環伏安法於HT-29定量分析圖 128
圖 5 36:循環伏安法於DLD1定量分析圖 129
圖 5 37:電流變化量分析於HCT-116定量檢量線 129
圖 5 38:電流變化量分析於HT-29定量檢量線 130
圖 5 39:電流變化量分析於DLD1定量檢量線 130
圖 5 40:電化學阻抗分析定量於HCT-116奈式曲線圖 131
圖 5 41:電化學阻抗分析定量於HT-29奈式曲線圖 132
圖 5 42:電化學阻抗分析定量於DLD1奈式曲線圖 132
圖 5 43:阻抗變化量分析於HCT-116定量檢量線 133
圖 5 44:阻抗變化量分析於HT-29定量檢量線 133
圖 5 45:阻抗變化量分析於DLD1定量檢量線 134
圖 5 46:綜合電流定量及偵測分析於HCT-116、HT-29、DLD1 135
圖 5 47:電極表面改質專一性蛋白Anti-EpCAM於大腸癌循環腫瘤細胞捕獲率 135


表目錄
表格 2 1：利用MOC-31與9-2抗體來檢測人類癌症中的EpEX與EpICD 12
表格 2 2: 吡咯單體於不同摻雜劑之最適化條件參數設定 51
表格 4 1: 聚乳酸於聚吡咯表層包覆反應濃度條件 86
表格 5 1: 導電奈米纖維基板(PNF)於不同包覆時間導電率變化 98
表格 5 2: 聚吡咯包覆Nylon66 纖維及PVD鍍金電極製備技術元素分析含量表 100


式目錄
式 2 1 25
式 2 2 26

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