臺灣博碩士論文加值系統

聚苯胺經常被用來作為感測器的導電材料之用，但是其在中性和鹼性溶液中會因為電化學活性及導電性喪失而顯得不穩定。本研究即討論三種含硫酸根聚陰離子當作聚苯胺的對離子，以提供酸性的微環境來穩定聚苯胺，使其可以成為電流式氨離子的檢測電極。另一方面，我們利用逐層組裝方式製作多層膜，並利用架橋反應使多層膜表面穩定化，不至於在水溶液中溶出。最後我們利用多層膜包覆和固定酵素使聚苯胺修飾電極可以進一步成為尿素電極。
研究中利用鍍金的氧化鋁板，浸入硫酸基聚電解質與苯胺摻混液中並進行電聚合而形成電極，聚合調控利用循環伏安法，並控制電壓在-0.2 ~ 0.8 V之內，以電位循環數來控制聚苯胺厚度。我們先製作Au/PAni-Poly(sodium 4-styrene-sulfonate)(PSS)及Au/PAni-Poly(vinyl sul-fate , Potassium salt)(PVS)的改質電極，並與Nafion摻雜的電極做比較。發現Nafion雖因為具有高質子傳導率而被普遍用以製備質子感測膜材，但是其高價位及溶解性限制其應用發展。而PVS和PSS則為水溶性，較易與聚苯胺形成複合物。修飾電極表面以光電子能譜分析儀、掃瞄式電子顯微鏡和電變色分析鑑定。ESCA分析顯示在168 eV有硫酸基團的能峰，表示當聚苯胺形成時，水溶性的聚電解質可以沈積吸附在電極上。SEM 觀察到PAni/polyelectrolyte composites表面上顆粒孔洞之間會佈滿奈米粒子，這情形可以增加有效的電極面積。而此種複合物表面亦顯示在施加-0.8到0.8 V的電位時，電極顏色會有由綠（還原態）轉到藍（氧化態）的可逆變化。
在銨離子偵測上，分別以循環伏安法(Cyclic Voltammetry；CV)、計時安培法(Chronoamperometry；CA)進行不同濃度NH4+的檢測，此修飾電極可以展現聚苯胺的對銨離子偵測的電活性，而且對PSS-PAni修飾電極而言在0 ~ 400 mM的偵測範圍，還原電流對銨離子濃度有良好的線性關係，靈敏度為6.17±0.099 μA(mM)-1cm-2。至於pH變化（在6.9-7.6之間）對電流反應可以忽略。為了增加電極穩定性，利用重氮樹脂(Diazo-resin)塗佈在電極最外層，再照光使電極表面形成網狀結構，可使電極靈敏度在測定4次後仍能維持90%。
在尿素偵測方面，以利用帶正電的Poly(allylamine hydrochloride) (PAH)包覆天然帶電的酵素製備逐層組裝的電極結構。水接觸角測定在PAH和酵素（或是PSS）之間形成鋸齒狀的接觸角度分佈，這也證實了逐層組裝多層膜的形成。接著利用此技術製備PSS-PAni/PSS/(PAH/Urease)5的電極，測試後發現在60 ~ 130 mg/dL的尿素濃度下與電流反應有不錯的線性關係，靈敏度為1.203±0.028 μA dL(mg)-1cm-2。但是考慮到穩定化電極效果，利用DAR來固定化尿素酵素時，可能因為照光固化DAR導致酵素失活，而使電極對尿素無感測性。因此我們改利用含環氧末端基聚乙二醇（PEGDE）來固定化酵素，製備PSS-PAni/PSS/(PAH/PEG/Urease)n電極，當接上一層酵素後，初步偵測在尿素濃度在0.5 ~ 9 mg/dL之間與反應電流有良好的線性區間，靈敏度為0.197±0.061 μA dL(mg)-1cm-2。組裝兩層固定化酵素後，可將偵測區間推至0.5 ~ 30 mg/dL的尿素濃度範圍，靈敏度為0.125±0.022 μA dL(mg)-1cm-2。顯示調整酵素層數可以控制偵測區間。

Polyaniline (PAni) has been used frequently for the construction of bio-sensors. However, a prime limitation is its instability at basic or neutral pH because of the loss of its electrochemical activity and conductivity. In this study, three available sulfonated polyanions serving as the counterion and providing an acidic microenvironment to stabilize PAni, are used to fabricate an amperometric sensor for ammonium ion deteection. Additionally, the electrodes were coated in multilayered polyelectrolytes using layer-by-layer techniques. And they were stabilized by crosslinking reactions on the out-most surfaces to prevent dissolution in aqueous solution. Furthermore, the multilayered electrodes were rendered to encapsure and immobilize urease for serving as urea sensors.
In this study, the electrodes were fabricated by immersing gold-coated alumina plates into the mixture of sulfonated polyelectrolytes and aniline solution, and then electropolymerized under cyclic voltammetric (CV) control in a potential range of -0.2 V to 0.8V. The polyaniline thickness was determined by the number of scan cycles. Two modified electrode sys-tems were developed: Au/PAni-Poly(sodium 4-styrene-sulfonate) (PSS), and Au/PAni-Poly(vinyl sulfate, Potassium salt) (PVS), compared with the Nafion-coated electrode system. Nafion used to be a common ion-sensitive membrane due to its high proton conductivity. However, its high cost and limited solubility has constrained its uses. PVS and PSS are water-soluble polymers, easily incorporating with PAni to form the composites. Surface analysis by electron spectroscopy for chemical analysis (ESCA) and scan-ning electron microscope (SEM), and the electrochromic property for the PAni composites provided the convenient tools to characterize the electrode fabrication. ESCA analysis illustrated that the sulfur peak at 168 eV was de-rived from the sulfonate groups, verifying that the water-soluble polyelec-trolytes can be deposited and adsorbed on the electrode as polyaniline form-ing. Also the SEM photographs of PAni/polyelectrolyte composites showed that the pores between particles were filled with some nanoparticles, leading to an increase of effective electrode area. Reversible color changes were ob-served between green (reduced state) and blue (oxidized state) with the ap-plication of the potentials between -0.8 and 0.8 V to the PAni/polyelectrolyte composites.
On the aspect of sensing ammonium ions, CV and chronoamperometry (CA) methods were applied for detecting various concentration of ammo-nium solution. The modified electrodes exhibited electroactivity of PAni in ammonium ion detection and also showed the linear dependence of reduc-tion current on the ammonium ion concentration in a range of 0-400 mM, with the sensitivity of 6.17±0.099 μA(mM)-1cm-2. The pH effect was found insignificant to the response (ranging from pH 6.9 to 7.6). For increasing the stability of the electrodes, the diazo-resin (DAR) was introduced to the coat on the outmost layer and then cured by UV irradiation, forming the covalent network between the layers of polyelectrolytes, and giving a 90 % of re-mained sensitivity after detecting 4 times.
On the application of urea detection, the LbL structures of electrodes were prepared by encapsulating naturally charged enzyme using cationic Poly(allylamine hydrochloride) (PAH). Water Contact angle measurement showed a zigzag profile of contact angle between PAH and the enzyme (or PSS), verifying the formation of LbL multilayer. The electrode system of PSS-PAni/PSS/(PAH/Urease)5 was prepared and found that linear relation-ship between the current response and the urea concentration ranging from 60-130 mg/Dl with the sensitivity of 1.203±0.028 μA dL(mg)-1cm-2. As con-sidering the electrode stabilization, DAR was used on the outmost electrode surface, however, UV curing process possibly denatured the immobilized urease. Thus, the multilayer systems were change to introduce epoxyl chain-ended poly(ethylene glycol) (PEGDE) to immobilize the urease layers, forming the PSS-PAni/PSS/(PAH/PEG/Urease)n modified electrodes. It was found that the electrode having one bilayer (n=1) displayed the linear de-pendence of current response on the urea concentration ranging from 0.5-9 mg/dL with the sensitivity of 0.197±0.061 μA dL(mg)-1cm-2; and that having two bilayer (n=2) displayed the linear dependence ranging from 0.5-30 mg/dL of urea concecntration with 0.125±0.022 μA dL(mg)-1cm-2 sensitivity, showing the detectable range was dependent and controllable on the number of enzyme layers.

中文摘要……………………………………………………………...............Ⅱ
英文摘要……………………………………………………………………...Ⅳ
誌謝…………………………………………………………………………...Ⅶ
目錄
第一章 緒論…………………………………………...………………………1
1-1腎臟疾病臨床生化檢測………………………………….………1
1-2 研究背景…………………………………………………………3
第二章 原理與文獻回顧……………………………………………………...6
2.1生物感測器………………………………………………………..6
2.2尿素檢測分析……………………………………………………..9
2.3聚苯胺（polyaniline；PAni）……………………………………12
2.4 Nafion的簡介……………………………………………………15
2.5 NH4+的檢測……………………………………………………...16
2.6 Multilayer自我組裝多層奈米薄膜修飾電極…………………..17
2.7照光穩定多層膜結構--重氮樹酯的使用……………………….18
第三章 實驗方法…………………………………………………………….20
3.1藥品及材料………………………………………………………21
3.2實驗儀器及原理…………………………………………………23
3.2.1真空濺鍍（Sputter）…………………………………………..23
3.2.2掃描式電子顯微鏡（Scanning Electron Microscope, SEM）….23
3.2.3.傅立葉轉換紅外線光譜儀（FTIR）………………………….23
3.2.4.元素分析儀Electron Spectroscopy for Chemical Analysis (ESCA)………..………………………………………………24
3.2.5.靜態接觸角(Contact angle)……………………………………24
3.2.6.循環伏安法(Cyclic Voltammetry)……………………………..24
3.2.7.計時安培法Chronoamperometry(CA)………………………..25
3.2.8.pH meter……………………………………………………….25
3.3電極基板製備……………………………………………………26
3.3.1基材的清洗…………………………………………………….26
3.3.2鍍膜（sputtering）…………………………………………….26
3.4苯胺純化…………………………………………………………27
3.5重氮樹酯合成……………………………………………………27
3.6修飾電極設計……………………………………………………27
3.6.1 Au/PAni電極…………………………………………………..28
3.6.2 Au/Nafion-PAni電極…………………………………………..28
3.6.3 Au/PSS-PAni電極……………………………………………..28
3.7 Au/PAni-PSS修飾電極進行多層膜製程……………………….29
3.8尿素酵素固定層的製備…………………………………………30
第四章.結果與討論…………………………………………………………..31
4.1電極表面的狀態變化……………………………………………32
4.1.1電極表面的電變色性………………………………………….32
4.1.2 表面構形……..……………………………………………….33
4.1.3 多層膜電極表面親疏水性質的變化………………………...35
4.1.4 表面元素分析鑑定…………………………...………………37
4.1.5電極表面分析：RAIR………………………………………...40
4.2修飾電極特性……………………………………………………41
4.2.1Au/PAni感測電極……………………………...………………41
4.2.2 Au/Nafion/PAni感測電極……………………………………..46
4.2.2.1 1wt% Nafion-PAni 電極………………………………...…..46
4.2.2.2 3wt% Nafion-PAni電極……………………………………..50
4.2.3 Au/PVS-PAni 感測電極...………………...…………………..54
4.2.4 Au/PSS-PAni 感測電極...…….…………...…………………..58
4.2.5 Au/PSS-PAni/(PSS/PAH)n感測電極…………………………..64
4.2.6 Au/PSS-PAni/(PSS/PAH)n/PSS/DAR感測電極 ...…………...67
4.2.7 NH4+電極的比較………………………………………………69
4.3 NH4+感測之干擾性測試…………………………………..…….70
4.3.1 pH值的影響…………………………………………………...70
4.3.2 多層膜的的感測阻力………………………………………...71
4.4重氮樹酯之影響…………………………………………………72
4.4.1 重氮樹酯合成及NMR分析………………………………….72
4.4.2 DAR保護電極效果…………………………………………...73
4.5酵素電極特性……………………………………………………74
4.5.1 Au/PSS-PAni+free Enzyme+Urea感測電極…………………. 74
4.5.2 Au/PSS-PAni/Urease/DAR感測電極……………….…………77
4.5.3 Au/PSS-PAni/PSS(PAH/Urease)5感測電極……….…………..79
4.5.4 Au/PSS-PAni/PSS/(PAH/PEG+Urease) 感測電極..…………..82
4.5.4 Au/PSS-PAni/PSS/(PAH/PEG/Urease)n感測電極……………84
4.6 結論……………………………………………………………..89
4.6.1在NH4+ sensor的方面………………………………………....89
4.6.2在Urease sensor的方面………………………………………..90
參考文獻………….…………………………………………………………..91
作者自述……………………………………………………………………...95

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陳明儀,李泰興,黃啟輝,楊先仁.,2004.遠東學報第21卷第3期,517頁