臺灣博碩士論文加值系統

本實驗以混合聚3-己基噻吩Poly-(3-hexylthiophene),P3HT/十八烷胺octadecylamine，ODA 作為Langmuir 單分子層模板，藉由單分子層與葡萄糖氧化酵素(Glucose oxidase，GOx)的作用力，將水溶液中的GOx 吸附至氣/液界面，以製備P3HT/ODA/GOx 混合單分子層。由單分子層的表面壓-分子佔據面積等溫曲線(π-A isotherm)、表面壓-時間等溫曲線(π-t isotherm)，了解P3HT/ODA/GOx 混合單分子層在氣/液界面上的行為。以原子力顯微鏡觀察P3HT/ODA/GOx 混合LB 薄膜表面形態。並將混合單分子層沉積於白金基板，製備成生物感測器後進行電化學分析，探討單分子層形態對於感測效能的影響。調整P3HT/ODA 溶液的濃度，可以控制P3HT 於氣/液界面的聚集程度。當P3HT 聚集程度的減少，P3HT 分子能於氣/液界面展開，進而穩固吸附至界面的GOx 分子。感測結果也發現，隨著P3HT 聚集程度改善，酵素電極的感測效能會隨之提升，其靈敏度可提升至4.2μA/cm2．mM，電流變化斜率為0.106μA/cm2．s。結果說明利用ODA 改善P3HT 聚集行為的可行性，及其應用於生物感測器上的酵素固定材料及電子媒介層的發展性。

In order to prepare poly-(3-hexylthiophene) (P3HT)/octadecylamine (ODA)/ glucose oxidase (GOx) mixed monolayer,P3HT/ODA mixed monolayer was used as a template to adsorb GOx from solution to air/liquid interface by interactions between the template and GOx. The behaviors of the P3HT/ODA/GOx mixed monolayer at air/liquid interface were studied by the pressure-area (π-A) isotherm and the pressure-time (π-t) isotherm. The morphology of the P3HT/ODA/GOx mixed LB film was examined using an atomic force microscope (AFM). The monolayers were then transferred on Pt substrates to fabricate biosensors for sensing study on LB films characteristics. The P3HT aggregation at air/liquid interface can be controlled by regulating the concentration of the P3HT/ODA solution.P3HT chains extend at air/liquid interface while the degree of P3HT aggregation decrease and they fix the adsorbed GOx molecules.The sensing experiments demonstrate that performance of sensors is enhanced with improvement of the P3HT aggregation.The sensor exhibits the highest current sensitivity (4.2μA/cm2.Mm) and the lowest the slope of current change (0.106μA/cm2.s). It is possible to improve the P3HT aggregation by interactions between ODA and P3HT and to fabricate P3HT on sensors for immobilizing enzyme and transferring electron.

總目錄
中文摘要.........................................................................................................................I
ABSTRACT.........................................................................................................................II
致謝.........................................................................................................................III
總目錄.........................................................................................................................IV
表目錄.........................................................................................................................VI
圖目錄.........................................................................................................................VI
第 1 章.緒論.........................................................................................................................1
1-1 .前言............................................................................................................................1
1-2 .動機及目的................................................................................................................5
第 2 章.文獻回顧.................................................................................................................6
2-1 .糖尿病簡介................................................................................................................6
2-2 .生物感測器簡介......................................................................................................10
2-2-1 .生物感測器的起源與發展..............................................................................10
2-2-2 .生物感測器原理及結構..................................................................................11
2-2-3 .生物感測器特性與能力..................................................................................12
2-3 .葡萄糖生物感測器..................................................................................................15
2-3-1 .蛋白質..............................................................................................................15
2-3-2 .酵素(24-26)......................................................................................................21
2-3-3 .葡萄糖感測器之應答機制..............................................................................23
2-4 . Langmuir-Blodgett 簡介.........................................................................................28
2-4-1 . Langmuir-Blodgett 單分子層.........................................................................28
2-4-2 . Langmuir-Blodgett 薄膜製備.........................................................................36
2-4-3 .Langmuir-Blodgett 技術沉積生化感測薄膜..................................................40
2-5 .導電高分子..............................................................................................................41
2-5-1 .位置規則性聚3-烷基噻吩.............................................................................42
2-5-2 .聚3-己基噻吩單分子層行為.........................................................................45
第 3 章.實驗.......................................................................................................................48
3-1 .實驗藥品..................................................................................................................48
3-2 .實驗儀器簡介..........................................................................................................49
3-2-1 .Langmuir-Blodgett 沉積裝置.........................................................................49
3-2-2 .表面壓測量原理..............................................................................................50
3-2-3 .原子力顯微鏡(Atomic Force Microscope，AFM) ........................................52
3-2-4 .圓二色光譜儀(Circular Dichroism Spectroploarinter ， CD)......................54
3-2-5 .傅立葉轉換紅外線光譜儀(Fourier Transform Infrared Spectrometry ，FTIR) ..........................................................................................................................61
3-2-6 .電化學分析方法簡介......................................................................................64
3-3 .實驗步驟..................................................................................................................69
3-3-1 .導電高分子/界面活性劑混合單分子層等溫線測量.....................................70
3-3-2 .酵素溶液配製..................................................................................................70
3-3-3 .導電性高分子/界面活性劑/葡萄糖氧化酵素langmuir-Blodgett 混合薄膜等溫線測量....................................................................................................................70
3-3-4 .導電性高分子/界面活性劑/葡萄糖氧化酵素各類混合LB 薄膜製備方法71
3-3-5 .使用圓二色光譜儀進行薄膜上固定化酵素之構形測定..............................71
3-3-6 .使用傅立葉轉換紅外線光譜儀比較電極上固定酵素量..............................72
3-3-7 .緩衝溶液配置..................................................................................................72
3-3-8 .葡萄糖溶液配製..............................................................................................73
3-3-9 .酵素修飾電極的電化學分析方法..................................................................73
第 4 章.結果與討論...........................................................................................................75
4-1 .聚3-己基噻吩/十八碳胺混合單分子層於氣/液界面的行為探討.......................75
4-1-1 .聚3-己基噻吩單分子層於氣/液界面的行為探討........................................75
4-1-2 .添加十八碳胺對於聚3-己基噻吩單分子層影響之探討.............................80
4-1-3 .不同濃度對於P3HT/ODA 混合單分子層影響之探討................................83
4-2 .葡萄糖氧化酵素在氣/液界面的吸附行為探討.....................................................88
4-2-1 .不同Langmuir 單分子模板對葡萄糖氧化酵素吸附行為的影響................88
4-2-2 .不同聚集程度單分子層做為模板對GOx 吸附行為的影響........................90
4-3 .Langmuir-blodgett 沉積技術製備電流式葡萄糖感測器.......................................94
4-3-1 .改變P3HT/ODA 比例對感測能力影響之探討............................................95
4-3-2 .分子膜聚集行為對感測能力之影響..............................................................99
4-3-3 .改變壓縮條件對感測能力之影響................................................................104
4-3-4 .改變副相濃度對感測能力之影響................................................................109
第 5 章.結論與建議.........................................................................................................115
1.結論..........................................................................................................................115
2.建議..........................................................................................................................117
第 6 章.參考文獻.............................................................................................................118
表目錄
表格2-1.血糖控制指標................................................................................................................. 9
表格2-2.Ⅰ型糖尿病與Ⅱ型糖尿病之特徵................................................................................. 9
表格4-1.界面濃度為33.51 Å 2/UNIT，溶液濃度與溶液灑佈量變化表.................................... 85
表格4-2.不同模板吸附GOX 單分子層構形組成表................................................................. 93
表格4-3.不同混合比例P3HT/ODA/GOX LB 膜電極的感測特性。....................................... 98
表格4-4.固定界面濃度為33.51 Å 2/UNIT，不同溶液濃度之P3HT/ODA/GOX LB 膜電極的感測特性。............................................................................................................................................ 103
表格4-5.不同溶液濃度於壓膜40MN/M 沉積之P3HT/ODA/GOX LB 膜電極的感測特性。............................................................................................................................................................ 108
表格4-6.不同溶液濃度於副相濃度20PPM 沉積之P3HT/ODA/GOX LB 膜電極的感測特性。............................................................................................................................................................ 113
圖目錄
圖 1-1.金屬，半導體與絕緣體的導電度分佈........................................................................... 4
圖2-1.圖示第一型糖尿病與第二型糖尿病的差異.............................................................. 8
圖2-2.生物感測器的基本構造示意圖....................................................................................... 14
圖2-3.(A)胺基酸的基本構造及(B)胜肽鍵的形成。................................................................. 16
圖2-4.蛋白質的一級結構。....................................................................................................... 16
圖2-5.(A)胜肽平面及(B)胜肽平面間的限制轉動。................................................................. 17
圖2-6.蛋白質的二級結構，(A)Α-HELIX 及(B)Β-SHEET。.......................................................... 18
圖2-7.蛋白質的四種結構。....................................................................................................... 20
圖2-8.酵素反應之模型鑄型學說(INGOT THEORY).............................................................. 22
圖2-9.電化學法偵測葡萄糖的原理示意圖。........................................................................... 25
圖2-10.生物分子固定法示意圖。............................................................................................. 27
圖2-11 .LANGMUIR 單分子層之形成。...................................................................................... 29
圖2-12.氣/液界面上單分子層表面壓-每分子佔據面積等溫線。........................................... 32
圖2-13.LANGMUIR-BLODGETT 膜之沉積，(A)親水性固體基板移出佈滿單分子層之液面，(B)疏水性固體基板浸入佈滿單分子層之液面。................................................................................... 36
圖2-14.三種LANGMUIR-BLODGETT 膜的型式。....................................................................... 39
圖2-15.烷基噻吩單體三種耦合方式，以及四種可能的耦合序列................................... 44
圖2-16.圖示說明(A)位置規則性聚3-烷基噻吩(B)非位置規則性聚3-烷基噻吩........... 44
圖2-17.三種製備POLYMER LB 膜方法示意圖(A)A : MONOMER 在氣/液界面形成單分子層，轉移至基板上行程LB 膜後，再行聚合反應；(B)B：POLYMER 在氣/液界面形成單分子層，再轉移至固體基板上形成LB 膜；(C)C：MONOMER 形成單分子層後，在氣/液界面行聚合反應後，再轉移至固體基板上形成LB 膜。................................................................................................. 47
圖3-1.KSV MINITROUGH 沉積裝置............................................................................................ 49
圖3-2.威氏平板(WILHELMY PLATE)法示意圖........................................................................... 51
圖3-3.TAPPING MODE AFM 示意圖。........................................................................................ 53
圖3-4.光線由電場與磁場所構成，可經由平面極化器濾出線性偏極光。........................... 56
圖3-5.顯示左旋及右旋圓偏極化光的結合(A)兩個波長向量相同的振幅，是平面偏極化光的結果(B)兩個波長向量不同的振幅，不再是圓而顯示為橢圓。...................................................... 56
圖3-6.經由左旋和右旋圓偏極化光吸收度之差異，可測得圓二色光譜............................... 57
圖3-7.蛋白質在圓二色光譜儀上的二級結構吸收圖................................................... 60
圖3-8. 傅立葉轉換紅外線光譜儀儀器示意圖 ......................................................................... 63
圖3-9. ATR 原理示意圖 ............................................................................................................ 63
圖3-10.典型標準電位對時間關係圖.................................................................................. 65
圖3-11.典型可逆循環伏安圖.............................................................................................. 65
圖3-12.AMPEROMETRY 示意圖.................................................................................................. 67
圖3-13.CHRONOAMPEROMETRY 示意圖。(A)電位-時間之關係圖(B)時間-濃度之關係圖(C)電流-時間之關係圖................................................................................................................................. 68
圖3-14.實驗系統示意圖............................................................................................................. 69
圖3-15.沉積薄膜分析方法示意圖............................................................................................. 69
圖4-1.25℃下，固定界面濃度為33.51 Å 2/UNIT，氣/液界面上不同溶液濃度之P3HT 單分子層，副相為純水之表面壓-每分子佔據面積等溫曲線。(A)50PPM (B)100PPM (C)200PPM ............... 77
圖4-2.P3HT 結晶結構示意圖.................................................................................................... 78
圖4-3.25℃下，固定界面濃度為33.51 Å 2/UNIT，氣/液界面上不同溶液濃度之P3HT 單分子層，副相為純水，在表面壓為20MN/M 之AFM 影像分析圖。(A)50PPM (B)100PPM (C) 200PPM............................79
圖4-4.25℃下，固定界面濃度為33.51 Å 2/UNIT，氣/液界面上P3HT、P3HT/ODA 單分子層，副相為純水之表面壓-每分子佔據面積等溫曲線。(A)P3HT，(B)P3HT/ODA ............................... 81
圖4-5.25℃下，固定界面濃度為33.51 Å 2/UNIT，氣/液界面上(A)P3HT (B)P3HT/ODA 單分子層，副相為純水，在表面壓為20MN/M 之AFM 影像分析圖。 .................................................... 82
圖4-6.25℃下，固定界面濃度為33.51 Å 2/UNIT，氣/液界面上不同溶液濃度之P3HT/ODA 混合單分子層，副相為純水之表面壓-每分子佔據面積等溫曲線。P3HT 濃度:(A)100PPM (B)200PPM (C)500PPM (D) 1000PPM 與ODA 以莫耳比1:1 混合。.......................................................................... 85
圖4-7.25℃下，固定界面濃度為33.51 Å 2/UNIT，氣/液界面上不同溶液濃度之P3HT/ODA 混合單分子層，副相為純水，表面壓為20MN/M 之原子力顯微鏡表面分析圖。P3HT 濃度:(A)100PPM(B)200PPM (C) 500PPM (D) 1000PPM 與ODA 以莫耳比1:1 混合。.................................................. 87
圖4-8.25℃下，固定界面濃度為33.51 Å 2/UNIT 的ODA、P3HT/ODA 單分子層吸附GOX 分子(GOX CONC.=10PPM)的表面壓-時間吸附曲線。 .......................................................................... 89
圖4-9.25℃下，固定界面濃度為33.51 Å 2/UNIT，氣/液界面上不同溶液濃度之P3HT/ODA 混合單分子層吸附GOX 分子(GOX CONC.=10PPM)的表面壓-時間吸附曲線。 .................................. 92
圖4-10.不同混合比例P3HT/ODA/GOX LB 膜電極的響應電流密度-時間關係圖及響應電流密度-葡萄糖濃度校正曲線。............................................................................................................. 97
圖4-11.三種不同狀態下白金電極的循環伏安圖..................................................................... 98
圖4-12.固定界面濃度為33.51 Å 2/UNIT，不同溶液濃度之P3HT/ODA/GOX LB 膜電極的響應電流密度-時間關係圖及響應電流密度-葡萄糖濃度校正曲線。.................................................. 101
圖4-13 .固定界面濃度為33.51 Å 2/UNIT，不同溶液濃度之P3HT/ODA/GOX LB 膜電極的紅外線光譜圖。.................................................................................................................................... 102
圖4-14.不同溶液濃度於膜壓40MN/M 沉積之P3HT/ODA/GOX LB 膜電極的響應電流密度-時間關係圖及響應電流密度-葡萄糖濃度校正曲線。.................................................................... 106
圖4-15.不同溶液濃度於壓膜40MN/M沉積之P3HT/ODA/GOX LB 膜電極的紅外線光譜圖。............................................................................................................................................................ 107
圖4-16.不同溶液濃度於副相濃度20PPM 沉積之P3HT/ODA/GOX LB 膜電極的響應電流密度-時間關係圖及響應電流密度-葡萄糖濃度校正曲線。.............................................................. 111
圖4-17.不同副相濃度與不同沉積層數沉積之P3HT/ODA/GOX LB 膜電極的紅外線光譜比較圖。A)副相濃度:10PPM，沉積層數:5 層，B)副相濃度:10PPM，沉積層數:5 層。 ................... 112
圖4-18.無模板幫助吸附，不同副相濃度下GOX 自主吸附曲線。A)20PPM，B)10PPM。 .. 112
圖4-19. 不同 GOX 溶液濃度造成氣液界面分散差異示意圖。............................................ 114

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