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研究生： 黃真瑜
Huang, Chung-Yu
論文名稱： 應用微接觸技術製備薄膜型肌酸酐分子模版
Using a Microcontact Technology to Prepare Thin-Film Creatinine Imprinted Polymer
指導教授： 周澤川
Chou, Tse-Chuan
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2006
畢業學年度： 94
語文別： 中文
論文頁數： 147
中文關鍵詞： 解離常數 、肌酸酐 、微接觸技術 、分子模版 、微熱卡計
外文關鍵詞： Freundlich isotherm, Creatinine, calorimeter, Biosensor, 2-(trifluoromethyl)-acrylic acid
相關次數： 點閱：161  下載：1 
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    • 在臨床的腎臟疾病診斷時，人體血清或尿液中的肌酸酐濃度的檢測，可以協助醫護人員判斷病人的腎功能是否正常。此研究希冀藉由製備薄膜型肌酸酐分子模版，來測試人體尿液樣本中的肌酸酐濃度，再進一步組裝成可應用在檢測人體體液的生物感測器，由於此薄膜型肌酸酐分子模版為生物感測器中最重要的元件，其各種參數會深深的影響其辨識及檢測的效果，故本研究將尋找最佳化的方法製備吸附性與辨識性效能最大之肌酸酐分子模版，其中將探討的變因，包括功能性或交聯劑單體的選擇及比率、肌酸酐分子模版的效率評估模式等。結果顯示，以微熱卡計觀察目標分子與單體的吸放熱情形，配合所製備之薄膜型肌酸酐分子模版，可得到相同的趨勢，亦即與目標分子具有放熱情形之單體在製備分子模版後，在相同的吸附條件下，具有較佳之吸附效果，例如交聯劑EGDMA比TEGDMA及PEG400DMA對肌酸酐的吸附量較低，而功能性單體則以TFMAA比其他單體之MAA，AA，4VP，NVP以及Styrene對肌酸酐的吸附力最強。第二部分是探討不同的變因對分子模版吸附量的影響，單體組成為TFMAA與EGDMA以1:1莫耳組成時，起始劑為單體的10 mole%，所形成之分子模版在進行4小時之吸附後，其最高吸附量為4.65±0.08 g/cm2。此外，吸附溫度高於或低於室溫，其吸附量也會有明顯的提升。吸附常數或解離常數可經由使用不同濃度的肌酸酐溶液對分子模版吸附效能，其Kd(解離常數)為10mg/dL及N值(模版上辨識性孔洞之總濃度)為5.23mg/dL。最後，本研究進行類似物，如肌酸、尿酸及尿素等，分子之競爭吸附與真實樣品，人體中之尿液之吸附，發現所製備之分子模版對肌酸酐分子具有極佳之辨識情形。

    The creatinine concentrations in serum or urine are very important determinants to help doctors to diagnose patient’s kidney function. In this study, we prepared thin film creatinine imprinted polymer to examine the creatinine concentration in human urine, and further to fabricate the biosensor for body fluid testing. As a result, the thin-film creatinine-imprinted polymer is a key element which influences the recognizing ability and sensitivity. Synthesis of MIPs, therefore, is needed to meet the requirement. Many factors including species and ratios of functional monomers or crosslinkers are as important as procedures of MIP synthesis and evaluatation. The results indicate that exothermal or endothermal reactions monitored by the isothermal titration calorimeter between target and monomer molecules could be correlated with the rebinding amount of creatinine to the creatinine MIPs. For example, MIPs composed of EGDMA have lower creatinine adsorption than TEGDMA or PEG400DMA, and TFMAA shows the highest creatinine adsorption among MAA, AA, 4VP, NVP and Styrene. Different MIPs synthesis procedures are compared for optimization, MIPs compose of TFMAA and EGDMA in the mole ratio 1:1, the initiator 10 mole% of monomer and four hours MIPs rebinding in 10mg/mL creatinine solution. The highest rebinding amount could reach 4.65±0.08 g/cm2. In addition, rebinding temperature at higher or lower than the room temperature, the rebinding amount is obviously promoted. The equilibrium adsorption constant or dissociation constant can be calculated by immsering MIPs in creatinine solutions with different concentrations for MIPs assessments. The dissociation constant (Kd) and number (N) of MIPs with composition (EGDMA/MAA=1:1) are 10mg/dL and 5.23mg/dL, respectively. Finally, several creatinine analogues such as the creatine, uric acid or urea is employed for competition rebinding on MIPs, and human urine (real sample) is also tested and MIPs show good recognition results.

    目錄 中文摘要……………………………………………………… Ⅰ 英文摘要……………………………………………………… Ⅲ 致謝…………………………………………………… Ｖ 目錄…………………………………………………… ⅤI 圖目錄………………………………………………… ⅩI 表目錄………………………………………………… ⅩⅤ 專有名詞對照表……………………………………… ⅩVI 第一章 緒論………………………………………… 1 1-1 肌酸酐……………………………………………………… 1 1-1-1 肌酸酐之簡介…………………………… 1 1-1-2 肌酸酐之臨床意義……………………… 2 1-2 肌酸酐與腎臟疾病的關係…………………… 5 1-3 肌酸酐的歷史發展…………………………… 6 1-4 肌酸酐之檢測分析…………………………… 6 1-5 實驗動機與目的……………………………… 11 第二章 原理………………………………………… 15 2-1 分子模版技術………………………………… 15 2-1-1 分子模版高分子原理…………………… 16 2-1-2 分子模版之材料特性選擇……………… 19 2-1-2-1 目標物及功能性單體……………… 20 2-1-2-2 交聯劑之選定……………………… 22 2-1-2-3 起始劑……………………………… 23 2-1-3 聚合反應………………………………… 23 2-1-4分子模版之型態………………………… 26 2-2 HPLC之原理………………………………… 29 2-3 微熱卡計之簡介…………………………… 30 2-4分子模版等溫吸附之系統原理……………… 32 2-4-1 分子模版動力式之推導……………… 41 第三章 實驗步驟………………………………… 43 3-1 藥品與儀…………………………………… 43 3-1-1 藥品…………………………………… 43 3-1-2 儀器…………………………………… 45 3-2 實驗流程與步驟…………………………… 47 3-2-1 玻璃清洗與改質……………………… 47 3-2-1-1 蓋玻片之清洗與改質…………… 47 3-2-1-2 載玻片之清洗與改質…………… 50 3-2-2 微接觸分子模版薄膜 (micro-contact polymer film)之壓印與聚合……………………… 51 3-2-3 目標分子之移除………………………… 54 3-2-4 吸附分析之偵測方法…………………… 55 3-2-4-1 HPLC之濃度測定…………………… 55 3-2-4-1-1 目標分子之吸附……………… 56 3-2-4-1-2 肌酸酐模版之競爭吸附……… 57 3-2-4-2 微熱卡計之吸放熱等溫滴定……… 58 3-2-4-2-1微熱卡計之操作與前處理……… 58 3-2-4-2-2欲滴定樣品及注射針之製備…… 60 3-2-5 微接觸分子模版薄膜之特性分析…… 61 3-3 研究架構……………………………………… 62 第四章 結果與討論………………………………… 63 4-1 應用微接觸法製備肌酸酐之分子模版……… 63 4-1-1肌酸酐分子模版之交聯劑的選擇………… 64 4-1-1-1交聯劑對已吸附肌酸酐玻璃之滴定吸放熱… 64 4-1-1-2 交聯劑與目標分子之吸附情形…… 72 4-1-2 選擇與肌酸酐分子作用之單體……………… 74 4-1-2-1 單體對已吸附肌酸酐玻璃之滴定吸放熱 ……………………………………………… 74 4-1-2-2 單體官能基與目標分子之吸附情形 81 4-2 以AA系列為功能性單體探討影響分子模版選擇性吸附之因素……………………………………………… 85 4-2-1分子模版在聚合時影響之各種因素……… 85 4-2-1-1 製備分子模版的方法對吸附效能的影響… 85 4-2-1-2 功能性單體與交聯劑之選擇……… 89 4-2-1-3 單體和交聯劑比例組成之選擇……… 92 4-2-1-4 起始劑的用量對分子模版吸附能力之影 響……………………………………… 94 4-2-2 分子模版清洗後吸附影響之各種因素……… 97 4-2-2-1 分子模版吸附溫度對吸附之影響…… 97 4-2-2-2 分子模版吸附時間對吸附效能之影響…… 100 4-2-2-3 吸附濃度的高低對AA系列吸附之影響 102 4-2-2-3-1 使用AA當功能性單體製備分子模版吸附不同濃度的肌酸酐溶液對吸附效能之影響……… 102 4-2-2-3-2 使用MAA當功能性單體製備分子模版吸附不同濃度的肌酸酐溶液對吸附效能之影響…… 107 4-2-2-3-3 使用TFMAA當功能性單體製備分子模版吸附不同濃度的肌酸酐溶液對吸附效能之影響… 111 4-2-3 使用TFMAA為功能性單體製備肌酸酐分子模版在肌酸酐、肌酸、尿酸與尿素溶液中之選擇性…………117 4-2-4 使用TFMAA為功能性單體製備肌酸酐分子模版對人體尿液的吸附……………………………………… 125 4-2-5 使用TFMAA當功能性單體製備微接觸分子模版薄膜用AFM分析薄膜表面…………………………………128 4-3 以Styrene與TFMAA為功能性單體製備肌酸酐分子模版測試分子模版的吸附量………………………………130 4-3-1 使用Styrene與TFMAA為功能性單體製備肌酸酐分子模版在肌酸酐、肌酸、尿酸與尿素溶液中之選擇性…………………………………………………………133 第五章 結論…………………………………………… 135 第六章 綜合討論與建議……………………………… 140 參考文獻 ………………………………………………143 自述 ………………………………………………147 圖目錄 Fig. 1-1 The formation of human urine…………………………… 5 Fig. 1-2 Schematic depiction of the IPCCA sensor concept……… 8 Fig. 1-3 Preparation of capacitive chemosensors for creatinine......... 9 Fig. 1-4 Imprinted effect of the creatinine imprinted (poly (4-Vpy-co-DVB)) in different creatinine solutions………………… 11 Fig. 1-5 The sensor was operated easily myself…………………..... 14 Fig. 1-6 The sensor can be used to examine other substances……… 14 Fig. 2-1 Schematic showing the formation of a MIP……………….. 16 Fig. 2-2 Pattern of cake core………………………………………... 28 Fig. 2-3 The apparatus of isothermal titration calorimeter…………. 31 Fig. 2-4(a)(b) Binding isotherm for polymer 1…………………….. 35 Fig. 2-5 (a)Five-point binding isotherm of polymer 1. (b)Scatchard plot of polymer 1 from five-point binding isotherm and calculated Scatchard curve using the function: B/F ＝ 16.252F0.4999-1………. 36 Fig. 2-6 Experimental isotherms of indole and 2-methylindole adsorption………………………………………………………….. 39 Fig. 2-7 Scatchard plots of the experimental data for indole and 2-methylindole asorption………………………………………….. 39 Fig. 2-8 Freundlich plots for indole and 2-methylindole adsorption.. 40 Fig. 2-9 Langmuir plots for indole and 2-methylindole adsorption 40 Fig. 3-1 The process to clean cover glasses……………………….. 49 Fig. 3.2 The preparation scheme of MIPs…………………………. 52 Fig. 3-3 Micro-contact Creatinine-imprinted polymer film based on functional monomer……………………………………………….. 53 Fig. 3-4 Micro-contact non-imprinted polymer film based on functional monomer……………………………………………….. 53 Fig. 3-5 Micro-contact imprinted polymer film put in absorbed creatinine solution vessel………………………………………….. 55 Fig. 3-6 The chart of sealing ampoule…………………………….. 58 Fig. 3-7 The singal of static calibration of tritration cell…………... 59 Fig. 4-1 4-1 UV calibration of creatinine by HPLC at 238nm in buffer solution………………………………………………………. 68 Fig. 4-2 4-2 UV calibration of creatine by HPLC at 214nm in buffer solution……………………………………………………………… 68 Fig. 4-3 UV calibration of uric acid by HPLC at 280nm in buffer solution……………............................................................................ 69 Fig. 4-4 UV calibration of urea by HPLC at 206nm in buffer solution……………………………………………………………… 69 Fig. 4-5 (a) The isothermal titration of crosslinkers (EGDMA, squares; TEGDMA, circles; PEG400DMA, triangles) to creatinine-absorbed glass slide (filled) and (b) blank glass slide (empty)……………………………………………………………… 70 Fig. 4-6 Use a series of cross-linkers based on ethylene glycol dimethylacrylate to titrate covered Creatinine glass (PEG400DMA, triangles; TEGDMA, circles; EGDMA, squares)…………………... 71 Fig. 4-7 Rebinding of Creatinine to the NIP made with various Crosslinkers; EGDMA, TEGDMA and PEG400DMA…………….. 73 Fig. 4-8 Use acidic functional monomer to titrate covered Creatinine glass (TFMAA, triangles; MAA, circles; AA, squares)… 79 Fig. 4-9 Use functional monomer to titrate covered Creatinine glass (4VP, squares; Styrene, triangles; NVP, circles)……………………. 80 Fig. 4-10 Rebinding of Creatinine to the NIP made with various acidic functional monomer; MAA and TFMAA……………………. 83 Fig. 4-11 Rebinding of Creatinine to the NIP made with various function monomer; 4VP, NVP and Styrene………………………… 84 Fig. 4-12 Rebinding of Creatinine to the different prepared Molecular Imprinted Polymers methods in MIP and NIP………….. 88 Fig. 4-13 Rebinding of Creatinine to the MIP and Non-MIP made with various functional monomers and various crosslinkers……….. 91 Fig. 4-14 Rebinding of Creatinine to the MIP and NIP made with different ratio in TFMAA and EGDMA……………………………. 93 Fig. 4-15 Rebinding of Creatinine to the MIP and NIP made with various initiator amount…………………………………………….. 96 Fig. 4-16 Rebinding of Creatinine to the MIP and NIP in different temperature…………………………………………………………. 99 Fig. 4-17 Rebinding of Creatinine to the MIP in different time……. 101 Fig. 4-18 Rebinding of different Creatinine concentration to the MIP and NIP………………………………………………………... 105 Fig. 4-19 Rebinding of different Creatinine concentration to the MIP and NIP………………………………………………………... 109 Fig. 4-20 Rebinding of different Creatinine concentration to the MIP and NIP………………………………………………………... 114 Fig. 4-21 Scatchard plot for Creatinine rebinding by imprinted polymer. [B vs. B/F]………………………………………………… 116 Fig. 4-22 Rebinding of different analogues to the Crn-MIP and NIP. 120 Fig. 4-23 HPLC analysis for Creatinine abosorbed results(a)sample, (b)MIP, (c)Non-MIP………………………………………………... 121 Fig. 4-24 HPLC analysis for Creatine abosorbed results (a)sample, (b)MIP, (c)Non-MIP………………………………………………... 122 Fig. 4-25 HPLC analysis for Uric acid abosorbed results (a)sample, (b)MIP, (c)Non-MIP………………………………………………... 123 Fig. 4-26 HPLC analysis for Urea abosorbed results (a)sample, (b)MIP, (c)Non-MIP………………………………………………... 124 Fig. 4-27 Rebinding of urine to the Crn-MIP and NIP……………... 126 Fig. 4-28 HPLC analysis for abosorbed results (a)original human body urine, (b)urine absorbed by MIPs, (c)urine absorbed by Non-MIPs…………………………………………………………… 127 Fig. 4-29 AFM images of the Creatinine-MIP surface……………... 129 Fig. 4-30 AFM images of the Creatinine-NIP surface……………… 129 Fig. 4-31 Rebinding of Creatinine to the MIP and NIP made with different ratio in TFMAA and Styrene……………………………… 132 Fig. 4-32 Rebinding of analogues to the MIP and NIP made with different ratio in TFMAA and Styrene……………………………… 134 表目錄 Table. 1-1 The stages of chronic kidney disease……………………. 2 Table. 1-2 Material was used to prepare sensor…………………….. 13 Table. 2-1 Comparison of natural biomolecules used in sensors (enzymes, receptors, antibodies) and MIPs………………………… 16 Table. 2-2 Examples of various templates used in molecular imprinting…………………………………………………………… 19 Table. 2-3 Template(Target) and analogues………………………… 21 Table. 2-4 Functional monomer……………………………………. 21 Table. 2-5 Crosslinkers……………………………………………... 23 Table. 4-1 Specificity of the binding of different concentration to AA-creatinine- imprinted polymer (MIP and NIP)…………………. 106 Table. 4-2 Specificity of the binding of different concentration to MAA-creatinine- imprinted polymer (MIP and NIP)………………. 110 Table. 4-3 Specificity of the binding of different concentration to TFMAA-creatinine- imprinted polymer (MIP and NIP)…………… 115 Table. 4-4 The equilibrium constant and binding site populations extracted from the Scatchard plot…………………………………... 115 Table. 4-5 HPLC analysis for Creatinine abosorbed results………... 121 Table. 4-6 HPLC analysis for Creatine abosorbed results………...... 122 Table. 4-7 HPLC analysis for Uric acid abosorbed results…………. 123 Table. 4-8 HPLC analysis for Urea abosorbed results……………… 124 Table. 4-9 HPLC analysis for Urine abosorbed results……………... 127 Table. 6-1 The effect of different factors for adsorption amount 142

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