臺灣博碩士論文加值系統

聚苯胺(Polyaniline ,Pan)的結構複雜，一般而言，在Emeraldine 結構且酸性的範圍內較為穩定，由於其具有不錯的穩定性及可處理性，將具有陽離子交換特性的全氟磺酸鈉(Nafion)與無毒性的聚苯胺粉末態(Pan powder)混合均勻，以取代傳統的電聚合製備Pan方式，再加入可添加大量修飾劑、電極表面可以更新、取得容易價格便宜的碳粉(Graphite powder) 製備成Pan/Nafion複合式碳粉電極，經實驗發現此系統對NH4+離子偵測具有很好的靈敏度。在Pan/Nafion複合式碳粉電極前端接上固定化尿素水解酵素(Urease)之controlled porous glass 反應器（Reactor），使尿素(Urea)經酵素反應器中的酵素催化之後，產生NH4+離子，在電極上產生離子交換反應，以還原電流的形式輸出，此電極對尿素的反應有很好的再現性及靈敏度，線性範圍由5uM~7.5mM，偵測極限為5uM (S/N=3)，並應用於人體血液中尿素含量的分析。將麩胺酸去氫酶 (Glutamate dehydrogenase，GLDH)與輔酶(NAD+)混入碳粉電極內部，製備成GLDH/NAD+/Pan/Nafion electrode。此電極利用GLDH配合NAD+將麩酸鹽（L-glutamate，glu）催化產生NH4+離子，再由Pan/Nafion 碳粉電極偵測，可得到一個還原訊號。glu的線性範圍由500uM-5mM (r=0.9821)，偵測極限為500uM ，此電極再現性不錯(CV=4.25%，n=40)。

A Polyaniline/Nafion（Pan/Nafion）carbon paste electrode was prepared by mixing the polyaniline emeraldine salt powder with nafion and graphite to construct the urea & ammonia sensor. the urease was immobilized on the controlled porous glass（CPG）as the enzyme reactor and amounted before the Pan/Nafion electrode in flow injection analysis (FIA) system to analyze the urea in blood samples .The ratio of nafion, polyaniline and graphite for the construction of the electrode and the optimal conditions were studied. The detection limit of this sensor for urea was 5 �嵱 and the linearity from 5 �嵱 to 7.5 mM was obtained in FIA. The reproducibility and stability were acceptable (RSD, 6.3 %, n=43) and the response time ca 72s. The real samples from blood dialysis patients were analyzed and compared with the urea test kit method.
A nicotinamide adenine dinucleotide (NAD+) dependend glutamate dehydrogenase (GlDH) was studied for glutamic acid analysis. The GlDH ， NAD+， Pan， Nafion was mixed to preparaed the GlDH/NAD+/Pan/Nafion paste electrode. The ratio of GlDH and NAD+ for the Pan/Nafion electrode and the optimal conditions were studied. The detection limit of this sensor for glutamic acid was 500 �嵱 and the linearity from 500 �嵱 to 5 mM was obtained in FIA. The reproducibility was acceptable (RSD, 4.25 %, n=40).The real samples from food additive were studied.

目錄
摘要………………………………………………………………………I
目錄……………………………………………………………………II
表目錄…………………………………………………………………Ⅴ
圖目錄…………………………………………………………………Ⅵ

壹、緒論…………………………………………………………………1
一、前言…………………………………………………………………1
二、生物感測器…………………………………………………………3
三、酵素固定化…………………………………………………………7
四、碳粉電極之應用……………………………………………………9
五、化學修飾電極………………………………………………………10
六、聚苯胺塗覆電極……………………………………………………12
七、 全氟磺酸鈉 (Nafion) 的簡介…………………………………17
八、麩酸鹽（L-glutamate）的簡介…………………………………19
九、實驗目的……………………………………………………………23

貳、實驗部分……………………………………………………………25
一.藥品…………………………………………………………………25
二.儀器…………………………………………………………………26
三.方法…………………………………………………………………27
四.實驗步驟……………………………………………………………28

參.結果與討論…………………………………………………………31
一、Urease reactor/Pan/Nafion 電極………………………………31
1.Pan/Nafion電極製作的探討………………………………………31
2.最佳實驗條件………………………………………………………32
3.校正曲線及偵測極限的探討………………………………………35
4.電極的再現性………………………………………………………36
5.干擾物的影響………………………………………………………36
6.真實樣品之測定……………………………………………………36

二、GLDH/NAD+/Pan/Nafion 電極……………………………………37
1.GLDH與Pan/Nafion複合式電極的組成…………………………37
2.最佳實驗條件………………………………………………………38
3.校正曲線及偵測極限的探討………………………………………40
4.電極的再現性………………………………………………………41
5.干擾物的影響………………………………………………………41
6. GLDH/NAD+/Pan/Nafion碳粉電極穩定度…………………………42

肆.結論與未來展望……………………………………………………43

伍.參考文獻……………………………………………………………58














表目錄
表1、生物辨識元件的分類…………………………………………………4
表2、生物感測器的類型……………………………………………………5
表3、信號轉換元件的分類…………………………………………………6
表4、各類酵素電極的應用…………………………………………………11
表5、Pan/Nafion複合式修飾電極之文獻比較……………………………18
表6、應用Glutamate oxidase(GlOx)偵測glutamate之文獻比較……21
表7、應用GLDH偵測Glutamate之文獻比……………………………….22
表8、Urease/Pan/Nafion electrode實驗最佳化條件…………………35
表9、Urease/Pan/Nafion複合式修飾電極之干擾物對偵測urea的影響……………………………………………………………………56
表10、Urease/Pan/Nafion複合式修飾電極之真實樣品測定…………56
表11、GLDH/Pan/Nafion複合式修飾電極之實驗最佳化條件…………40
表12、GLDH/Pan/Nafion複合式修飾電極之干擾物的影響……………57


圖目錄
圖1.生物感測器之結構圖……………………………………………………3
圖2.Polyaniline的五種氧化還原態………………………………………14
圖3.Nafion的結構圖………………………………………………………17
圖4.Pan/Nafion複合式電極結合Urease reactor偵測urea的反應
示意圖…………………………………………………………………24
圖5.GLDH/NAD+/Pan/Nafion複合式修飾電極偵測L-glutamate之反應示意
圖……………………………………………………………………24
圖6.實驗儀器裝置圖………………………………………………………30
圖7.Nafion含量的探討………………………………………………….45
圖8.碳粉含量之最佳化……………………………………………………45
圖9.Urease/Pan/Nafion複合式修飾電極之波峰圖……………………46
圖10.Urease/Pan/Nafion 複合式修飾電極之最佳工作電位…………46
圖11.Urease/Pan/Nafion複合式修飾電極之最佳緩衝溶液pH值……47
圖12.Urease/Pan/Nafion複合式修飾電極之最佳流速選擇……………47
圖13.Urease/Pan/Nafion複合式修飾電極之反應器中尿素酵素濃度
最佳化………………………………………………………………48
圖14.樣品注入量對Urease/Pan/Nafion複合式修飾電極反應之影響.48
圖15.Urease/Pan/Nafion.複合式修飾電極之NH4+ 與urea的校正曲線以及其線性範圍………………………………………………………49
圖16.Urease/Pan/Nafion複合式修飾電極之再現性……………………50
圖17.GLDH/NAD+/Pan/Nafion複合式修飾電極之GLDH濃度最佳化……50
圖18.GLDH/NAD+/Pan/Nafion複合式修飾電極之NAD+濃度最佳化的條件51
圖19.GLDH/NAD+/Pan/Nafion複合式修飾電極之工作電位的選擇………51
圖20.GLDH/NAD+/Pan/Nafion複合式修飾電極之最佳緩衝溶液pH值…52
圖21.GLDH/NAD+/Pan/Nafion複合式修飾電極之最佳流速………………52
圖22.GLDH/NAD+/Pan/Nafion複合式修飾電極之NH4+的校正曲線………53
圖23.GLDH/NAD+/Pan/Nafion複合式修飾電極之L-glutamate的
校正曲線……………………………………………………………54
圖24.GLDH/NAD+/Pan/Nafion複合式修飾電極之再現性…………………55
圖25.GLDH/NAD+/Pan/Nafion複合式修飾電極之穩定性…………………55

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