論文提要

H2O2因用途廣泛，且為許多氧化酶作用後的產物，是一個非常重要的分析物，其偵測方法以電化學與luminol化學冷光法為主，甚少利用tris(2,2’-bipyridyl)ruthenium(II)（Ru(bpy)32+）電化學冷光系統。本論文第參章利用H2O2抑制tris(2,2’-bipyridyl)ruthenium(II)/ Tripropylamine（TPA）電化學冷光的特性，在流動注入系統中加入固定有葡萄糖氧化酶的矽凝膠管柱，使注入的葡萄糖與氧化酶作用，即時反應生成H2O2；生成之H2O2繼而進入反應器中，測得對Ru(bpy)32+ /TPA電化學冷光的抑制訊號，以對葡萄糖進行間接定量。對H2O2與葡萄糖的偵測極限分別為0.10、1.0μM。另藉由參考管柱、酵素管柱之雙管柱設計，簡便地扣除維他命C與尿酸的干擾訊號，使本系統成功應用於血清與飲料等真實樣品中的葡萄糖含量分析。
本論文中以冷凍碎裂方式製作含酵素之矽凝膠顆粒，除了溶膠-凝膠法所擁有的優點外，尚有製作簡易、時間縮短，製得之矽凝膠顆粒易於長期保存，並且不需經乾燥，避免了凝膠於乾燥過程中持續釋出的甲醇、及凝膠結構與內部孔洞縮小對酵素構成壓力等優點。因此法製得的酵素管柱對相同濃度分析物連續注入偵測具有很好的再現性，於本論文第肆章與第伍章中以同樣方式製作含Ru(bpy)32+ 的矽凝膠顆粒，並於第伍章中探討Ru(bpy)32+ 固定於水合凝膠與乾燥凝膠中電化學冷光反應性的差異。
Ru(bpy)32+ 試劑本身非常穩定，其電化學冷光反應是可逆的，因此許多人致力於將Ru(bpy)32+ 固定在電極表面，常見的方法有在電極上形成Langmuir-Blodgett film、self-assembled films、或利用聚合物（如Nafion film、silica gel..）在電極表面形成薄層，將Ru(bpy)32+ 固定於薄層中；這些方法需要仔細且多步驟的電極製作過程。本論文第肆章與第伍章中分別設計了管柱式與平面式電化學冷光反應器，將含Ru(bpy)32+ 矽凝膠顆粒填充於工作電極周圍的空間，藉電極與Ru(bpy)32+ 矽凝膠顆粒緊密接觸進行電化學冷光反應，所注入之待測液可分別直接與工作電極及凝膠顆粒中的Ru(bpy)32+ 作用，提高反應的靈敏度。
本論文設計的管柱式與平面式電化學冷光反應器內容積分別約為50、80μL，溶液連續流經填充Ru(bpy)32+ 矽凝膠顆粒之電化學冷光反應器24小時以上、其中施加固定電位10小時以上，凝膠顆粒中Ru(bpy)32+ 仍維持良好的反應性；尤其以Ru(bpy)32+ 乾燥凝膠填充平面式電化學冷光反應器、對相同濃度NADH連續注入105次所得RSD為2.8 %，證明本論文固定Ru(bpy)32+ 的方法適用於流動注入系統。以流動注入系統配合管柱式電化學冷光反應器對TPA測定線性範圍為0.020μM – 5.0μM，實際可測得之偵測極限為0.020μM，低於一般論文的方法。平面式電化學冷光反應器應用於NADH、NADPH測定的線性範圍分別為0.50μM – 5.0 mM、1.0μM – 3.0 mM；結合去氫酶測定葡萄糖與葡萄糖-6-磷酸的線性範圍分別為5.0μM – 0.50 mM、10.0μM – 1.0 mM。
第伍章中配合測定使用之去氫酶可單獨固定於矽凝膠顆粒中、填充成為反應管柱；亦可共價鍵結於Ru(bpy)32+ 矽凝膠顆粒表面，於電化學冷光反應器中同時進行酵素反應與放光反應。前者的優點為酵素與試劑分開固定，若其中一者不宜使用時可單獨替換；後者優點為電化學冷光反應器中酵素反應之後的產物即時與Ru(bpy)32+ 矽凝膠顆粒進行電化學冷光反應，減少因流動擴散的稀釋效應。
Ru(bpy)32+ 固定於乾燥凝膠中進行電化學冷光反應，反應訊號與矽凝膠顆粒結構本身均較穩定，可長時間應用於分析測定。但Ru(bpy)32+ 固定於水合凝膠中的電化學冷光反應訊號約為乾燥凝膠中的1.5 – 2 倍，因此如何提升Ru(bpy)32+ 於乾燥凝膠中的反應性是值得進一步研究的方向。

Based on the linear relationship between concentration of H2O2 and the decrease of electrochemiluminescence (ECL) intensity in a Ru(bpy)32+/TPA system, procedures for the indirect determination of glucose with a flow injection analysis were developed. By passing solutions of glucose through a FIA system containing a glucose oxidase (GOx) immobilized sol-gel column and an ECL system of Ru(bpy)32+ and TPA, glucose can be determined optimally with a detection limit of 1.0 μM in a linear dynamic range of 1.0 – 200.0 μM. A repetitive injection of glucose (100 μM) and human serum solutions gave satisfactory reproducibility with relative standard deviations of 1.3 (N=31) and 3.9 % (N=42) respectively. Interference due to the presence of ascorbic acid, uric acid or other reducible agents in solution can be corrected by passing sample solutions through another sol-gel column that contained no GOx. From the agreement between the contents of glucose in human serum and soft drink analyzed by the developed method and those obtained by the spectroscopy method based glucose assay kit and satisfactory recovery of glucose from interferent containing solutions, the feasibility of the developed method for real sample analysis was confirmed.
One of the major purposes of this study was to develop new immobilization approaches and flow cell designs for the fabrication of regenerable ECL-based sensors with improved sensitivity, convenience and long-term stability. Silica particulates were used as immobilization support in ECL sensors for TPA and NAD(P)H and in biosensors for glucose and glucose-6-phosphate（G6P）. The first ECL flow cell was fabricated from a glass tube, and a platinum wire was used as working electrode held at +1.3 V. The volume of the flow cell was about 50 μL. An Ag/AgCl electrode and a piece of Pt wire were used as the reference and counter electrode respectively and placed downstream of the working electrode. Ru(bpy)32+ immobilized silica particulates with 1/3 silica sol content showed the best performance for TPA determination, and the sensitivity of TPA determination was dependent upon the amount of Ru(bpy)32+ immobilized in silica particulates. The lowest level of analyte detected for TPA was 0.02μM, and linear range was from 0.02μM to 5μM.
Up to a certain concentration level, it was found that Ru(bpy)32+ was tightly held in silica particulates and did not leach out into aqueous solutions, even with continuous flow for up to ten hours. Ru(bpy)32+ immobilized silica particulates were characterized of well activity and high stability; that stored at 0℃ exhibited its original activity for up to one year.
The second ECL flow cell was fabricated from a piece of epoxy block supported Pt electrode (1 × 2 cm) as counter electrode, a piece glass window and a polyethylene spacer with 78 μL cell volume, two 2.0-cm length of 0.6-mm diameter platinum wires were used as working electrodes held at +1.1 V, and an Ag/AgCl electrode as reference electrode. All three electrodes were incorporated within the main body of the cell.
One of the biosensor design packed Ru(bpy)32+ incorporated silica particulates in the ECL flow cell, and a glucose dehydrogenase (GDH) immobilized silica sol-gel column is placed between the sample injection valve and the flow cell. The ECL response to samples containing glucose and cofactor (NADP) results from the Ru(bpy)33+ ECL reaction with NADPH produced by glucose dehydrogenase. This ECL biosensor was shown applicable for both NAD+- and NADP+- dependent enzymes, where NADH detection ranged from 0.50μM – 5.0 mM NADH and NADPH detection ranged from 1.0μM - 3.0 mM NADPH. Glucose can be determined in a linear dynamic range of 5.0 - 500 μM.
Another biosensor design immobilized glucose-6-phosphate dehydrogenase（G6PDH）onto the Ru(bpy)32+ -doped silica particulates through silica chemistry and then packed these particulates into the ECL flow cell. By passing samples containing G6P and cofactor (NAD) through the ECL flow cell, G6P can be determined in a linear dynamic range of 10.0 μM-1.0 mM.
The regenerable ECL biosensor was characterized of good reproducibility and well stability for flow injection analysis. A repetitive injection of NADH (100 μM) and G6P（500μM）gave satisfactory reproducibility with relative standard deviations of 2.8 %（N=105）and 2.8 % (N=40) respectively.

目錄
第壹章 電化學冷光系統簡介...........................................……...............1
一、 有機分子之電化學冷光..........................……....................2
1. polyaromatic hydrocarbons (PAHs )之電化學冷光....2
2. 其它有機分子之電化學冷光……………..…..……...3
二、 陰極電化學冷光（Cathodic ECL）.…………..…………….4
1. 陰極電化學冷光機制…………………..……..……...4
2. luminol與tris(2,2’-bipyridyl)ruthenium(II) 之陰極電化學冷光…………………..……………….….……...5
3. 陰極電化學冷光於生化分析的應用………………...6
三、 luminol/H2O2 電化學冷光系統…………………...….…....6
1. luminol/H2O2 電化學冷光機制……………………...6
2. luminol/H2O2 電化學冷光於生化分析的應用...…....8
四、 tris(2,2’-bipyridyl)ruthenium(II)電化學冷光系統..……….9
1. tris(2,2’-bipyridyl)ruthenium(II) 與tripropylamine、oxalate電化學冷光機制……….…………..…….....10
2. tris(2,2’-bipyridyl)ruthenium(II) 與peroxidisulphate（S2O82－）電化學冷光機制.……………………….13
3. 將Ru(bpy)32+ 氧化為Ru(bpy)33+ 的三種方法：化學、光化學、電化學……………………………...……..14
4. tris(2,2’-bipyridyl)ruthenium(II) 電化學冷光系統於分析上的應用.……...………..…………..………….14
5. tris(2,2’-bipyridyl)ruthenium(II)/ tripropylamine電化學冷光於DNA-probe及免疫分析上的應用……....16
6. tris(2,2’-bipyridyl)ruthenium(II) 電化學冷光系統於HPLC、LC、CE上的應用…………………..……....20
7. 將tris(2,2’-bipyridyl)ruthenium(II) 固定於流動注入分析系統中發展reagentless sensor的文獻回顧......22
第貳章 溶膠-凝膠法簡介……………………..………………………....33
一、 溶膠-凝膠法原理………………………………….……....33
1. 矽烷氧化合物水解及縮合反應機制…………….....35
2. 以酸或鹼催化對水解、縮合反應及矽凝膠結構的影響…………..………………………………….……..37
3. 其它反應條件對水解、縮合反應及矽凝膠結構的影響………………………….……………….….……..38
4. xerogel 與aerogels………………………...…….....39
5. Tetramethoxysilane（TMOS）所形成矽凝膠之基本性質…………………………………………....…….....41
6. 凝膠結構表面的修飾…………………….…..….....42
二、 溶膠-凝膠法於固定生物分子的應用……….……...…....45
1. Ellerby 所發展適於固定生物分子的溶膠凝膠方法……………………………………………………..45
2. 蛋白質固定於凝膠中時之性質探討…….………....46
三、 tris(2,2’-bipyridyl)ruthenium(II) 固定於矽凝膠中之性質探討的文獻回顧……………..………………..……….....49
第參章 以過氧化氫對tris(2,2’-bipyridyl)ruthenium(II)/tripropylamine 電化學冷光的抑制效應建立間接測定葡萄糖之流動注入分析系統….…………………………………………………….…....55
一、 研究動機………………………………………..…….......55
二、 實驗部分.……………………………………..……..…....55
三、 結果與討論…………………………………………….....60
1. Ru(bpy)32+ /TPA電化學冷光系統測定H2O2 之分析條件探討.……………………….…….……….….....61
1-1. 工作電極電位的影響……….….………..…...61
1-2. TPA 濃度的影響……………..………..…......65
1-3. Ru(bpy)32+ 濃度的影響……….………...…....67
1-4. 載流溶液pH的影響…………..…………......69
1-5. 載流溶液流速的影響………..….…………....71
1-6. Ru(bpy)32+/TPA電化學冷光系統測定H2O2 之檢量線.………………………………………...72
2. Ru(bpy)32+ /TPA電化學冷光系統對葡萄糖之偵測.73
2-1. 以溶膠凝膠法將葡萄糖氧化酶固定於矽凝膠顆粒製備酵素反應器……………..……..…...73
2-2. Ru(bpy)32+ /TPA電化學冷光系統配合不同葡萄糖氧化酶含量之酵素反應器對葡萄糖的測定………………………………………………75
3. 維他命C與尿酸對Ru(bpy)32+ /TPA電化學冷光系統測定葡萄糖之干擾與解決方法………………….....77
3-1. 維他命C與尿酸對Ru(bpy)32+ /TPA電化學冷光系統的影響…………..……………..……...77
3-2. 添加尿酸的葡萄糖標準溶液測定之回收率...79
4. 血清與市售飲料中葡萄糖含量分析……….……....80
4-1. Ru(bpy)32+/TPA電化學冷光系統對血清與市售飲料中葡萄糖含量分析………..…..………...80
4-2. Ru(bpy)32+/TPA電化學冷光系統與Glucose assay kit（Sigma）對血清與飲料樣品中葡萄糖含量測定結果比較……………..…….……....82
4-3. Ru(bpy)32+/TPA電化學冷光系統應用於真實樣品分析之再現性………………...…………....82
5. 葡萄糖氧化酶矽凝膠填充管柱保存的穩定性.…...83
四、 結論……………………………………..……………….....84
第肆章 以SiO2 gel固定tris(2,2’-bipyridyl)ruthenium(II) 配合流動注入分析法設計非試劑消耗型電化學冷光偵測系統－I. TPA測定…………………………………………………….……..…....86
一、 研究動機.…………………………………….……...….....86
二、 實驗部分……………………………………….……….....86
三、 結果與討論……………………………..……….…..….....89
1. 製作矽凝膠時溶膠與緩衝溶液的比例對TPA測定的影響………………………………..…….……..........90
2. 矽凝膠中所含Ru(bpy)32+ 濃度對TPA測定的影響.96
3. Ru(bpy)32+ 固定於不同組成比例之矽凝膠中及所固定濃度對Ru(bpy)32+ /TPA ECL 反應性的影響..….98
4. 載流溶液流速對TPA測定的影響…………..…....101
5. 以不同頻率交替施加880 mV、1360 mV電位對TPA分析訊號的影響…………………………...……....102
6. 含Ru(bpy)32+ 矽凝膠填充反應器對TPA測定之訊號-時間圖與再現性…………………………………...104
7. 以含Ru(bpy)32+ 矽凝膠填充反應器應用於oxalate 偵測………………………………..………..….......108
四、 結論…………………………………..……………..….…110
第伍章 以SiO2 gel固定tris(2,2’-bipyridyl)ruthenium(II) 配合流動注入分析法設計非試劑消耗型電化學冷光偵測系統－II. NADH測定……………………………………………….……..…….…..115
一、 研究動機.……………………………..…………………..116
二、 實驗部分………………………………..….…...………...116
三、 結果與討論……………………………….….…………...119
1. Ru(bpy)32+ 水合凝膠顆粒填充之平面式電化學冷光反應器對NADH分析條件探討…….…………....120
1-1. 平面式電化學冷光反應器電極配置方式及施加電位對NADH電化學冷光偵測訊號的影響….…………………………..……….….....120
1-2. 矽凝膠製備時溶膠與緩衝液混合的體積比、凝膠中Ru(bpy)32+ 濃度對NADH分析訊號及背景放光的影響.………………………...…….122
2. Ru(bpy)32+ 水合凝膠顆粒填充之平面式電化學冷光反應器應用於NADH偵測.………..…..………...129
3. Ru(bpy)32+ 乾燥凝膠顆粒填充之平面式電化學冷光反應器應用於NADH偵測………………...….....133
4. Ru(bpy)32+ 乾燥凝膠顆粒填充之平面式電化學冷光反應器對NADH分析條件探討……………….....136
4-1. 載體溶液pH值對NADH電化學冷光偵測訊號的影響…………………………...…………..137
4-2. 反應器墊片厚度對NADH偵測檢量線的影響...……………………………….…………..139
5. Ru(bpy)32+ 乾燥凝膠填充之平面式電化學冷光反應器應用於NADPH偵測…………………………...143
6. Ru(bpy)32+ 乾燥凝膠填充之平面式電化學冷光反應器配合葡萄糖去氫酶及葡萄糖-6-磷酸去氫酶應用於葡萄糖及葡萄糖-6-磷酸偵測.………………....145
6-1. Ru(bpy)32+ 乾燥凝膠填充之平面式電化學冷光反應器配合固定於水合凝膠顆粒中之葡萄糖去氫酶應用於葡萄糖偵測……...………......145
6-2. 將葡萄糖-6-磷酸去氫酶結合於Ru(bpy)32+ 乾燥凝膠表面填充平面式電化學冷光反應器應用於葡萄糖-6-磷酸偵測……………....…....148
四、 結論………………………………………..……………...152
參考文獻……………………………………………..………...154

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