Immobilization of enzyme on nitrocellulose film for pH-EGFET type biosensors

口試委員審定書 授權書 ii 中文摘要 iv 英文摘要 v 誌謝 vi 第一章 緒論 1.1 研究背景 1 1.2 研究目的與動機 4 1.3 論文架構 6 第二章 理論分析與探討 2.1 生物感測器之種類與應用 8 2.2 酵素固定化技術 11 2.3 延伸式閘極感測場效電晶體之原理 13 2.4 相轉換法（Phase inversion process） 15 2.5 成膜理論 18 2.6 尿素之生理作用機制 22 2.7 尿素循環 25 第三章 材料方法 3.1 實驗材料 26 3.2 實驗流程 27 3.3 感測器之製作 28 3.4 量測系統 29 3.5 硝化纖維素之表面活化 30 3.6 尿素感測器之酵素固定化作用 31 第四章 結果與討論 4.1 硝化纖維素溶液之phase inversion process 32 4.2 硝化纖維素的濃度與旋轉塗佈轉速會影響pH感測度 34 4.3 尿素感測器之反應電壓 35 第五章 結論 36 參考文獻 37 圖表目錄 圖1-1、生物感測器之原理 44 圖1-2、Scheme of the second measurement setup with the microreactor packed with lipase immobilised onto the NC sheets and lipase coupled to glass beads coated with keratin. 45 圖1-3、論文架構圖 46 圖2-1、電化學生物感測器之種類 47 圖2-2、硫酸溶液的氧化還原反應 49 圖2-3、pH Optical sensor 50 圖2-4、QCMs(quartz-crystal microbalances ) 51 圖2-5、MOSFET、ISFET、ENFET之構造 52 圖2-6、離子感測場效電晶體(ISFET) 53 圖2-7、酵素固定化方式(a)吸附(b)微粒包覆(c)包埋(d)交聯(e)共價鍵結 54 圖2-8、延伸式閘極感測場效電晶體（EGFET）之結構圖 55 圖2-9、非對稱離子選擇元件之等效電路 56圖2-10、Schematic representation of a ternary phase diagram of polymer/solvent/nonsolvent 57 圖2-11、Schematic repressentation of casting film/coagulant interface 58 圖2-12、尿素循環 59 圖3-1、實驗流程圖 60 圖3-2、SnO2/ITO玻璃元件基本架構 61 圖3-3、測量系統 62 圖3-4、硝化纖維素溶液配製流程 63 圖3-5、尿素感測器之酵素固定化 64 圖4-1、不同比例的去離子水於硝化纖維素溶液中其元件之感測度 65 圖4-2、添加不同比例去離子水之硝化纖維素溶液之SEM 66 圖4-3、不同濃度的硝化纖維素修飾於延伸式閘極感測場效電晶體表面之感測度 67 圖4-4、不同轉速塗佈修飾硝化纖維膜之元件其pH感測度 68 圖4-5、尿素反應曲線圖 69 圖4-6、尿素濃度64 mg/dl之反應電壓 70 圖4-7、尿素感測器校正曲線圖 71 圖4-8、尿素感測器穩定度測試 72 參考文獻 Abeyrathne PD, Lam JS. 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Sci., 87, 267. 黃冠勛。探討添加劑對巨型孔洞形成之影響。2002。中原大學化學工程系碩士學位論文。 陳佳琪。可拋棄式尿素感測器與前置放大器之研究。2003。中原大學電子工程系碩士學位論文。 王彥盛。利用酵素固定化方式製備尿素感測器之研究。2004。國立雲林科技大學電子與資訊工程研究所碩士論文。 謝振榮。2006年8月。物理雙月刊，廿十八卷四期，704-710。[[abstract]]本篇研究中，我們首先提出利用旋轉塗佈技術將硝化纖維膜固定於延伸式閘極感測場效電晶體，並將酵素固定於硝化纖維膜上用以探討蛋白質之固定效率。旋轉塗佈所使用的硝化纖維素溶液之濃度將會影響延伸式閘極感測場效電晶體之感測度，因此為了找出表面最佳化的條件且又能維持其感測度與蛋白質固定能力，針對硝化纖維溶液的配製包含固定化基材濃度、溶劑與非溶劑比例與旋轉塗佈的轉速做探討，其結果顯示配製硝化纖維溶液最主要的參數為9%硝化纖維、3%去離子水與3000 rpm之轉速，將此硝化纖維溶液旋轉塗佈於延伸式閘極感測場效電晶體上其感測度仍可維持在53±1.2 mV/pH，此外為了了解延伸式閘極感測場效電晶體經硝化纖維膜修飾後其固定蛋白質能力，我們利用尿素酶固定於經硝化纖維膜修飾的延伸式閘極感測場效電晶體，形成尿素感測器，此尿素感測器於尿素濃度為64 mg/dl之尿素溶液中其反應電壓達136mV，硝化纖維膜表面的多孔性形成一種低擴散障蔽，使此尿素感測器於反應時間約20秒時即達最大反應電壓。尿素感測器穩定度之測試，元件在尿素溶液中反覆量測，量測27次後此酵素感測器仍然可保持其良好的工作性能。與包埋固定法相互對照，硝化纖維膜之固定蛋白質能力相似，但其形成較低的擴散障蔽。 In this paper, we propose an EGFET biosensor which the enzyme was immobilized on the nitrocellulose film. A porous nitrocellulose layer formed by phase inversion process was deposited on SnO2/ITO gate directly by spin-coated technology. To reach a surface chemistry condition can corresponding EGFET has appropriate pH-sensitivity and protein immobilization capability, the optimized nitrocellulose film manufacturing conditions were discussed through factors of inverse phase process, spin speed of the spin coater concentration of nitrocellulose. To understand the protein immobilization ability of nitrocellulose modified EGFET, we immobilized the urease on nitrocellulose film which used as an urea biosensor. The device shows a response of 136mV in a urea concentration of 64mg/dl. The porous surface of nitrocellulose shows low diffusion barrier, which has a fast response time of 20 sec. After a stability test, we found the sensor could maintain its excellent performance for detection of urea samples at least repeat measured of 27 times during 60 days. It is concluded that the developed nitrocellulose modified EGFET is perspective for its biosensor application for the determination enzyme or antigen-antibody reaction that the proposed method to prepare a selective biological membrane