何國川Ho, Kuo-Chuan臺灣大學:化學工程學研究所李達人Li, Ta-JenTa-JenLi2010-06-302018-06-282010-06-302018-06-282009U0001-2007200911290700http://ntur.lib.ntu.edu.tw//handle/246246/186915本研究利用溶膠-凝膠法(Sol-gel method)製備肌酸酐(Creatinine, Cre)的分子模版。在實驗上以氯化鋁(Aluminum chloride, AlCl3)為功能性單體與肌酸酐作用，四乙氧基矽烷(Tetraethoxysilane, TEOS)為交鏈劑，鹽酸為催化劑，水為溶劑配製成溶膠，然後在60 °C的環境下縮合一天得到塊狀的凝膠。之後利用水將肌酸酐萃取出來得到肌酸酐分子模版。非分子模版的製備過程除了不加入肌酸酐之外其餘的步驟都與分子模版相同。分子模版的效能測試中，本研究發現以Cre:AlCl3:TEOS=1:3:10(莫耳數比)所製備出來的分子模版在50, 100, 150, 200 μM的肌酸酐濃度之下的模版效率分別為1.93, 1.93, 1.83, 2.17。在干擾物測試上，本研究選擇的四種干擾物分別為肌酸(Creatine, Cn)，N-羥基琥珀酼亞胺(N-hydroxysuccinimide, NHS)，L-酪氨酸(L-tyrosine, L-tyr)以及乙醯酚胺(Acetaminophen, APAP)。分子模版對於四種干擾物的選擇性分別為7.98(Cre/Cn)、39.98(Cre/NHS)、13.87(Cre/L-tyr)、6.51(Cre/APAP)。非分子模版亦對於肌酸酐有選擇性，它們分別為4.00(Cre/Cn)、9.20(Cre/NHS)、16.72(Cre/L-tyr)、6.34(Cre/APAP)。研究對於矽醇基(Silanol group)、路易斯酸位置(Lewis acid site)以及氯化鋁對於肌酸酐吸附造成的影響做了一些探討。在氯化鋁與矽醇基方面，本研究發現有添加與沒添加氯化鋁的分子模版在不同濃度下的吸附量有明顯的差異，在20, 40, 60, 80, 100 μM下相差的量分別為0.84, 1.18, 1.85, 2.49, 2.98 mg/g，至於造成差異的原因本研究首先推論是氯化鋁的添加使得粒子變為較小顆，而有較多的矽醇基裸露在外面，此推論在微卡計測試、傅立葉轉換紅外線光譜以及掃描式電子顯微鏡的檢測中獲得證實。本研究也將TEOS的量提高一倍，然後比較分子模版與非分子模版的吸附量，結果發現吸附量均較之前來得低，但是模版效率卻提高(在50, 100, 150, 200 μM下的模版效率分別為2.52, 2.12, 2.30, 2.41)，推論吸附量較低的原因為，TEOS的量增加一倍將使得粒子變為較大顆，裸露在外面的矽醇基變得較少，而模版效率提高的原因是，TEOS的量提高使得矽醇基在模印位置的比例增加。研究在製備分子模版的過程中加入氯化鋁，希望鋁能夠與矽基材結合形成路易斯酸位置，但是在萃取過程中發現只有約20%的鋁與矽基材結合，而且是否形成路易斯酸位置並沒有直接證據。有鑑於此，本研究在製程中加入覆蓋(Capping)的步驟，將粒子表面的矽醇基與六甲基二矽氮烷(Hexamethyldisilazane, HMDS)反應成以三甲基矽(Trimethylsilyl)為終端的基團，然後偵測覆蓋後的粒子對於肌酸酐的吸附量，以了解路易斯酸位置對於肌酸酐吸附的貢獻。結果發現經過覆蓋後的粒子完全不會吸附肌酸酐，本研究提出可能的原因為，路易斯酸位置在粒子的內層，而粒子經過覆蓋的步驟後變得非常的疏水，因此高度親水性的肌酸酐不易靠近而擴散到此位置。A creatinine (Cre) imprinted polymer was fabricated by the sol-gel method. In this experiment, aluminum chloride (AlCl3) was chosen as the functional monomer, tetraethoxysilane (TEOS) as the cross-linker, hydrochloric acid (HCl) as the catalyst, and deionized water (DIW) as the solvent to prepare a sol. And then, the sol was put at 60 °C for 1 day for gelation to get a gel monolith. After that, Cre was extracted by DIW to get a Cre imprinted polymer. Non-imprinted polymer (NIP) was prepared in the same manner except for adding Cre during preparation. n the performance tests, the imprinting efficiencies of Cre imprinted polymer, fabricated with the molar ratio of Cre:AlCl3:TEOS=1:3:10, were 1.93, 1.93, 1.83, and 2.17 at the concentration of 50, 100, 150, and 200 μM, respectively. In the interferences tests, four compounds, including creatine (Cn), N-hydroxysuccinimide (NHS), L-tyrosine (L-tyr), and acetaminophen (APAP), were chosen as interferences. The selectivities of Cre imprinted polymer were 7.98(Cre/Cn), 39.98(Cre/NHS), 13.87(Cre/L-tyr), and 6.51(Cre/APAP), respectively. NIP was also found selective to Cre, and the selectivities were 4.00(Cre/Cn), 9.20(Cre/NHS), 16.72(Cre/L-tyr), and 6.34(Cre/APAP), respectively. his research also did some investigations on the effects of silanol groups, Lewis acid sites, and AlCl3 toward the adsorption of Cre. In the aspect of silanol groups and AlCl3, it was observed that the imprinted polymer, with adding AlCl3 during fabrication, adsorbed much more Cre than that without adding AlCl3 did. The differences of adsorption amount were 0.84, 1.18, 1.85, 2.49 and 2.98 mg/g at the concentration of 20, 40, 60, 80 and 100 μM, respectively. As for the reasons for making the differences, we first postulated that adding AlCl3 made the particles smaller, more silanol groups being on the surface. This postulation was substantiated by using microcalorimeter to measure the interactions, Fourier transform infrared spectrometer (FTIR) to observe the absorbance of silanol groups, and scanning electron microscope (SEM) to observe the morphology of surface. In addition, the amount of TEOS was doubled to see the effects toward the adsorption amount and the imprinting efficiency. It was shown that the adsorption amounts were lower than that of previous, but the imprinting efficiencies were upgraded a little. (The imprinting efficiencies were 2.52, 2.12, 2.30, and 2.41 at the concentration of 50, 100, 150, and 200 μM, respectively.) Adsorption amounts being lower was considered to be caused by higher cross-linking degree, thus leaving less silanol groups on the surface, while imprinting efficiencies being higher was considered to be caused by higher proportion of silanol groups in the total imprinted sites. uring preparation, AlCl3 was added for the purpose of forming Lewis acid sites in the silica matrix. However, it was shown that only 20% of Al incorporated with silica matrix, and whether the Lewis acid sites was formed was not very clear. In view of this, the silanol groups were capped by reacting with hexamethyldisilazane (HMDS) to give tirmethylsilyl (TMS) terminating groups. The adsorption amounts of capped particles were tested to see the contribution of Lewis acid sites to Cre adsorption singly. However, it was observed that the capped particles didn’t adsorb Cre. Explanations to this phenomenon were given in this research. Lewis acid sites maybe formed in the inner layer of the particles, which became very hydrophobic after capping, so it was not easy for a highly hydrophilic compound, Cre, to diffuse into.誌謝....................................................................................................................................I文摘要...........................................................................................................................II文摘要.........................................................................................................................IV錄................................................................................................................................VII目錄.............................................................................................................................XI目錄.......................................................................................................................XVIII號說明.......................................................................................................................XX寫說明......................................................................................................................XXI素委員會號碼及名稱對照表...............................................................................XXVI一章 緒論.....................................................................................................................1 1-1 前言..............................................................................................................1 1-2 肌酸與肌酸肝的發現史..............................................................................3 1-2-1 肌酸的發現史...................................................................................3 1-2-2 肌酸酐的發現史...............................................................................4 1-3 肌酸與肌酸酐的代謝..................................................................................5 1-3-1 肌酸在人體內的合成.......................................................................5 1-3-2 肌酸在人體內的利用.......................................................................7 1-3-3 肌酸酐在人體內的代謝...................................................................9 1-4 肌酸肝的臨床意義....................................................................................12 1-4-1 腎臟的基本介紹.............................................................................12 1-4-2 腎功能的檢測指標.........................................................................14 1-5 肌酸酐的檢測方法....................................................................................18 1-5-1 Jaffé氏反應.....................................................................................18 1-5-2 勞埃德試劑.....................................................................................19 1-5-3 光動力學法.....................................................................................19 1-5-4 酵素法.............................................................................................20 1-5-5 肌酸酐分子模版.............................................................................26 1-5-5-1 肌酸酐分子模版粉體高效能液相層析感測.................26 1-5-5-2 肌酸酐分子模版粉體螢光式感測................................ 34 1-5-5-3 肌酸酐分子模版感測器................................................ 36 1-6 研究動機....................................................................................................44二章 原理...................................................................................................................45 2-1 分子模版技術的介紹................................................................................45 2-1-1 分子模版的起源與發展.................................................................45 2-1-2 分子模版的原理.............................................................................48 2-1-2-1 結合.................................................................................49 2-1-2-2 聚合.................................................................................52 2-1-2-3 萃取.................................................................................53 2-1-3 製備分子模版的要素.....................................................................54 2-1-3-1 目標分子.........................................................................54 2-1-3-2 功能性單體.....................................................................55 2-1-3-3 交鏈劑.............................................................................56 2-1-3-4 起始劑.............................................................................58 2-1-3-5 溶劑.................................................................................59 2-1-4 分子模版效能測試.........................................................................60 2-1-4-1 批次再吸附.....................................................................60 2-1-4-2 層析.................................................................................62 2-1-5 分子模版的應用.............................................................................63 2-2 溶膠-凝膠法的介紹...................................................................................66 2-2-1 溶膠-凝膠法製備二氧化矽的反應...............................................66 2-2-1-1 水解.................................................................................66 2-2-1-2 縮合.................................................................................67 2-2-2 pH值對於溶膠-凝膠法的影響.......................................................69 2-2-3 共溶劑對於溶膠-凝膠法的影響...................................................71 2-2-4 含水量對於溶膠-凝膠法的影響...................................................71 2-2-5 溶膠-凝膠法在分子模版製備上的應用.......................................72 2-3 微卡計的介紹............................................................................................81三章 實驗設備與方法...............................................................................................85 3-1 實驗藥品....................................................................................................85 3-2 實驗儀器....................................................................................................87 3-3 製備方法....................................................................................................88 3-3-1 利用溶膠-凝膠法製備肌酸酐分子模版.......................................88 3-3-2 利用溶膠-凝膠法製備非分子模版 .............................................89 3-3-3 加入覆蓋步驟於肌酸酐分子模版的製備 ...................................91 3-4 分析方法....................................................................................................93 3-4-1 萃取液的分析.................................................................................93 3-4-1-1 肌酸酐校正曲線的製作.................................................93 3-4-1-2 利用UV/Vis吸收光譜估算肌酸酐的萃取量................93 3-4-1-3 感應藕荷電漿發射光譜定量萃取液中的鋁.................93 3-4-2 分子模版與非分子模版的吸附測試.............................................94 3-4-2-1 干擾物校正曲線的製作.................................................94 3-4-2-2 吸附時間的決定.............................................................94 3-4-2-3 分子模版及非分子模版對於肌酸酐吸附量的評估.....95 3-4-2-4 分子模版及非分子模版對於干擾物吸附量的評估.....95 3-4-3 等溫微卡劑滴定.............................................................................96 3-4-4 傅立葉轉換紅外線光譜儀分析.....................................................98 3-4-5 掃描式電子顯微鏡分析.................................................................98四章 結果與討論 .....................................................................................................99 4-1 製備方法與配方的決定..........................................................................101 4-2 肌酸酐分子的萃取..................................................................................104 4-3 萃取液UV/Vis吸收波形偏移原因的討論.............................................109 4-4 檢量線的製作..........................................................................................115 4-5 吸附時間的決定......................................................................................121 4-6 模版效率測試..........................................................................................123 4-7 干擾測試..................................................................................................126 4-8 不同氯化鋁量對於肌酸酐吸附量的影響..............................................130 4-9 應用微卡計評估作用力..........................................................................135 4-10 覆蓋表面矽醇基並觀察吸附量............................................................138 4-11 提高交鏈劑比例對於吸附量以及模版效率的影響............................142 4-12 利用掃描式電子顯微鏡觀察粒子的表面型態....................................144 4-13 利用傅立葉轉換紅外線光譜儀觀測粒子的官能基............................147 4-14 綜合討論................................................................................................150五章 結論與建議.....................................................................................................155 5-1 結論..........................................................................................................155 5-2 建議..........................................................................................................158六章 參考文獻.........................................................................................................161錄A 酵素委員會號碼的介紹..................................................................................175錄B 自述..................................................................................................................1764868000 bytesapplication/pdfen-US氯化鋁覆蓋肌酸酐肌酸酐吸附量肌酸酐分子模版六甲基二矽氮烷路易斯酸位置微卡計測試矽醇基Aluminum chloride (AlCl3)CappingCreatinine (Cre)Cre adsorption amountCre imprinted polymerHexamethyldisilazane (HMDS)Lewis acid sitesMicro-calorimeter testsSilanol groupsthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/186915/1/ntu-98-R96524031-1.pdf