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研究生:吳致廣
研究生(外文):Jhih-Guang Wu
論文名稱:利用石英晶體微天平探索人體C反應蛋白與具磷酸膽鹼官能基表面之交互作用
論文名稱(外文):Critical Study of the Recognition between C-Reactive Protein and Surface-Immobilized Phosphorylcholine by Quartz Crystal Microbalance with Dissipation
指導教授:羅世強
口試日期:2017-07-14
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:材料科學與工程學研究所
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:47
中文關鍵詞:人體C反應蛋白(CRP)磷酸膽鹼官能基(PC)34-乙烯二氧基噻吩(EDOT)石英晶體微量天平-耗散偵測系統(QCM-D)霍夫梅斯特序列(Hofmeister series)
外文關鍵詞:C-reactive protein (CRP)phosphorylcholine (PC)34-ethylenedioxythiophene (EDOT)quartz crystal microbalance with dissipation (QCM-D)Hofmeister series
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心血管疾病是全球的主要死亡原因之一,為了在發病前有更充裕的時間面對心血管疾病,我們可以妥善運用和心血管疾病有關的生物指標,評估人體罹患心血管疾病的風險,並提早投入醫療資源,防患於未然。人體C反應蛋白(C-reactive protein, CRP)就是一種用於評估心血管疾病的生物指標。根據研究,在有鈣離子的環境下,CRP和磷酸膽鹼官能基(phosphorylcholine, PC)會選擇性吸附,此外,PC官能基是極具親水性的兩性離子,可以抵抗非特定性吸附蛋白質的沾粘,以維持生物檢測器的功能。因此,我們將PC官能基和3,4-乙烯二氧基噻吩導電高分子(3,4-ethylenedioxythiophene, EDOT)結合,並用電化學聚合的方式,聚合成具PC官能基的導電高分子薄膜,再藉由調控電聚合溶液的高分子單體比例,來調整高分子薄膜表面的PC官能基密度,而這些帶有不同官能基密度的表面,將會對CRP有不同的偵測極限和範圍。CRP和PC官能基的特定性吸附,將由石英晶體微量天平-耗散偵測系統(quartz crystal microbalance with dissipation, QCM-D)監控,藉由石英震盪頻率的改變,來估量CRP吸附在具PC官能基表面之質量變化。經由QCM的偵測,由單體組成是100% PC官能基的EDOT單體溶液電聚合而成的表面有最好的抗沾黏性質,並可以吸附最多的CRP,偵測範圍落在0.25~5 mg/L,正好符合「高靈敏人體C反應蛋白試驗」(high-sensitive CRP test, hs-CRP teat)的範圍(1~3 mg/L)。霍夫梅斯特序列(Hofmeister series)是探討蛋白質在水中的狀態和水溶液中陰離子的交互關係;根據霍夫梅斯特序列,在我們的系統中導入不同種類和濃度的陰離子,透過改變水溶液中的陰離子種類和陰離子濃度,再藉由從QCM觀察到的蛋白質吸附量,來討論不同的陰離子對高分子表面性質和蛋白質吸附的關聯性。
Cardiovascular disease is the main cause of death in the recent years. Being able to prevent the sudden death caused by the disease, we could get help by using the biomarker to detect the cardiovascular disease and level the risk of cardiovascular disease in advance before the health turning worse. The detection of the human C-reactive protein (CRP) is a potential method to know the risk of cardiovascular disease in advance. It has been found that CRP has strong affinity to phosphorylcholine (PC) in the solutions contained calcium ion. Moreover, phosphorylcholine is one kind of zwitterions which usually provides non-fouling surfaces and prevents the non-specific binding of proteins. To demonstrate this concept, a phosphorylcholine-functionalized 3,4-ethylenedioxythiophene (EDOT-PC) was used to create conducting PEDOT films with PC functional groups on the surfaces. The density of PC groups can be fine-tuned by changing the composition of monomer solutions for the electropolymerization. By changing the surface density of PC groups, the conducting polymer surface can be applied for different linear ranges and detection limits for the CRP detection. The specific interaction of CRP with PC groups was determined by quartz crystal microbalance with dissipation (QCM-D). By monitoring the change of quartz frequency, the change of mass during protein binding can be estimated. The detection range is 0.25~5 mg/L which fit the range of “high sensitive CRP test” (hs-CRP test). Hofmeister series discuss the relation between the conformation of the protein and the kind of the anion. According to the Hofmeister series, the different salt ions can change the water structure and the protein property in water. Therefore, we introduced the different salts into our system to discuss how these anions affected the surface property and protein binding.
口試委員會審定書...............I
摘要..........II
Abstract.....III
目錄...........V
圖目錄.........VII
表目錄........VIII
前言與文獻回顧........................1
1.1 導電高分子介紹&導電高分子在生醫方面的運用..........3
1.2 心血管疾病起因與風險偵測..................4
1.3 人體C反應蛋白...............................5
1.4 磷酸膽鹼官能基製備抗沾黏表面(Non-fouling Surfaces) ........................5
1.5 霍夫梅斯特序列 (Hofmeister series) ......7
1.6 研究目標.....................8
第二章 實驗材料與方法.................................9
2.1 實驗藥品與儀器..............................10
2.1.1實驗用藥品................................10
2.1.2 儀器與量測................................11
2.2 EDOT-PC合成步驟............................12
2.3 電化學分析.........................................13
2.3.1 電化學聚合..................................13
2.3.2 電化學阻抗頻譜法分析.................14
2.4 高分子膜表面分析................................14
2.4.1 接觸角量測..............................14
2.4.2 場發射掃瞄式電子顯微鏡............14
2.4.3 原子力顯微鏡.......................15
2.5石英晶體微天平(QCM)簡介及操作....15
第三章 結果與討論........................18
3.1 高分子膜電化學性質...................18
3.1.1 電化學聚合..............................18
3.1.2 電化學阻抗頻譜法分析材料表面.20
3.2高分子膜表面性質.....................22
3.2.1 親疏水性....................22
3.2.2 表面形貌.............................24
3.3高分子膜和蛋白質的交互作用......27
3.3.1 鈣離子對CRP吸附之影響.....27
3.3.2 電化學聚合圈數對CRP吸附之影響..........29
3.3.3 PC官能基密度對蛋白質的吸附量之影響............31
3.3.4 各蛋白質吸附的耗散係數(dissipation)差異.....33
3.3.5 CRP偵測極限和範圍..............34
3.4 霍夫梅斯特序列(Hofmeister series) ....36
3.4.1 不同陰離子對高分子膜的影響......36
3.4.2 不同陰離子對蛋白質吸附的影響....37
3.5 人體血清溶液的偵測................39
第四章 結論....................................40
第五章 建議與未來工作.............................41
參考文獻.........42



圖目錄
圖一 反式聚乙炔示意圖......................1
圖二 心肌梗塞示意圖.................................3
圖三 CRP吸附示意圖...................................4
圖四 PC官能基...................................6
圖五 Hofmeister series簡表.........................7
圖六 EDOT-PC合成步驟............................12
圖七 三電極法示意圖.....................13
圖八 QCM外觀...................................17
圖九 利用循環伏安法電化學聚合poly(EDOT-OH) ...........19
圖十 利用循環伏安法電化學聚合poly(EDOT-PC) ............19
圖十一 電化學阻抗頻譜法對表面改質和CRP吸附的偵測............21
圖十二 不同表面對親疏水性和蛋白質吸附的影響.............22
圖十三 接觸角對EDOT-PC單體比例關係圖..23
圖十四 具有不同PC官能基密度表面的接觸角相片.......23
圖十五 不同PC官能基密度表面的AFM影像..........25
圖十六 不同PC官能基密度表面之SEM影像....25
圖十七 表面粗糙度對EDOT-PC單體比例關係圖.....26
圖十八 有無鈣離子對CRP吸附量的變化.....28
圖十九 鈣離子對CRP吸附的重要性.............28
圖二十 poly(EDOT-OH)電聚合圈數對CRP吸附之影響.........30
圖二十一 poly(EDOT-PC)電聚合圈數對CRP吸附之影響.........30
圖二十二 CRP和BSA吸附量對EDOT-PC單體比例關係圖..................................32
圖二十三 其他非特定性蛋白質的吸附量.....32
圖二十四 各種蛋白質吸附時,Frequency和Dissipation的變化.33
圖二十五 PC100在各種CRP濃度下的吸附曲線.........34
圖二十六 PC75在各種CRP濃度下的吸附曲線...........35
圖二十七 PC100和PC75的偵測範圍............35
圖二十八 PC100對各種陰離子和陰離子濃度之吸附量比較圖.....36
圖二十九 陰離子種類及陰離子濃度對BSA吸附之影響..........34
圖三十 陰離子種類及陰離子濃度對CRP吸附之影響........38

表目錄
表一 實驗用藥品...................9
表二 實驗用溶劑............................10
表三 實驗用蛋白質...........................10
表四 藥品簡寫...............................10
表五 實驗相關儀器..............................11
表六 hs-CRP test和PC100表面使用QCM偵測之數值比較.......39
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