生物標記物質在生物檢測上是非常重要的。而隨著奈米科技（Nano-technology）的發展，許多奈米物質被應用於生物檢驗上，金奈米粒子（Gold-nanoparticles）即是最常被利用的一種。另外本研究亦開發了一種增強金奈米粒子之電化學訊號的簡單方法，就是利用還原劑將金離子沉積在金奈米粒子表面，使得金奈米粒子放大而進一步增加電化學偵測訊號。
實驗分別利用三明治型免疫分析法（Sandwich immunoassay）及藉由蛋白質間（Streptavidin-biotin）的鍵結能力將兔子免疫球蛋白（RIgG）及 DNA 序列片段固定在免疫分析盤（Microtiter plate）上，再利用已標記金奈米粒子（直徑分別為 5，10 nm）之二級抗體（Anti-RIgG labeled gold-nanoparticles）及 Streptavidin 將標記物質接上，接著把金奈米粒子放大後再將金粒子溶下來，以電化學方波剝除法（Square wave stripping voltammetry）來偵測溶液中的金離子而完成電化學偵測。實驗結果在抗體偵測部份 RIgG 濃度 1 ~ 500 pg/mL 範圍中得到一良好的線性範圍（R2 = 0.9975），而此系統偵測極限約為 0.25 pg/mL，且偵測訊號之再現性非常好，在空白溶液（Blank solution）及 RIgG 濃度為 20 pg/mL 的溶液下其相對標準偏差值（R.S.D.）分別為 2.8 %（n = 11）及 2.4 %（n = 9）。而在 DNA 的偵測部份，實驗是針對甲狀腺乳突癌（Papillary thyroid carcinomas）檢體中所含有的 DNA 變異（Mutant）片段來檢測，結果不僅能區分互補或其他非互補之 DNA 片段，對微量 DNA 片段亦良好的定量結果（線性範圍在 0.52 ~ 1300 aM 之間，R2 = 0.9982），在真實樣品的偵測方面本系統也能明顯區分出有無變異之 DNA 片段樣品，且此分析方法擁有極靈敏的檢測能力（偵測極限約為 0.52 aM，訊號再現性於空白溶液及 DNA 濃度為 130 aM 的溶液之 R.S.D. 值分別為 2.1 %，n = 11 及 2.4 %，n = 12）。
本研究另外開發出將其生化偵測系統移至微晶片（Microchip）中，結合奈米組合電極（GNEE，Gold nanoelectrode ensemble）及微管道系統來進行電化學即時偵測。實驗主要是利用不同物質因受電場影響而其在微管道中的遷移速率（Mobility）會不同，使得其不同物質會在微管道中混合反應，接著反應產物即可在管道下游被電化學系統所偵測。實驗先用尿素（Urea）及尿素酶（Urease）二種物質在微管道中混合測試，再用已標記金奈米粒子之蛋白質（Streptavidin-Au）、還原劑及金離子溶液同時在微管道中混合偵測。對於未混合前的待測物偵測結果（氨離子：R.S.D. = 2.5 %，n = 6、濃度線性範圍為 0.02 ~ 5.0 mM，R2 = 0.9778；金離子：R.S.D. = 1.9 %，n = 3、濃度線性範圍為 0.0375 ~ 3.75 g/mL，R2 = 0.9842）可證明此系統之偵測能力，且應用在混合後之生物樣品檢測結果也相當好（尿素：線性範圍為 0.1 ~ 50 mM，R2 = 0.9657；Streptavidin-Au：線性範圍為 0.2 ~ 100 ng/mL，R2 = 0.9507），顯示本實驗成功開發出奈米組合電極微管道晶片系統，未來可藉由金奈米粒子的標記對於不同檢測目標設計，應用性可大幅提高。

Determination of bio-material by using enzyme, fluorophore or metal-nanoparticles as markers is very important. Generally, gold-nanoparticles have been used frequently as marker for increasing the sensitivity in bio-chemical assay.
In this research, gold-nanoparticles were used as marker for immunoassay, DNA sequence assay, and protein analysis. However, the size of gold-nanoparticles affects directly the results of electrochemical detection. For improving the sensitivity of electrochemical method, enlargement of gold-nanoparticles was used in this study. By electroless deposition, Au will be deposited on the surface of gold-nanoparticles. The electrochemical response will thus be increased substantially.
In immunoassay and DNA sequence assay, traditional 96-wells microtiter plate was used for immobilizing antibody or oligonucleotide, and the gold-nanoparticles were marked subsequently base on the immunoreaction or protein reaction of streptavidin and biotin. After gold-nanoparticles were enlarged, they were dissolved and transferred to an electrochemical cell for square wave stripping voltammetry（SWSV）analysis. Under optimal experimental condition, dynamic range of 1 ~ 500 pg/mL and 0.52 ~ 1300 aM were found respectively for RIgG and Target DNA analysis, and a good linear relationship（R2 = 0.9975 and 0.9982）. The relative standard deviation（R.S.D.） of blank were 2.8 % and 2.4 %（n = 11）for immunoassay and DNA assay, respectively. And the variance was 2.4 %（n = 9）and 2.4 %（n = 12）for immunoassay and DNA assay, respectively. The detection limit（based on S/N = 3）of RIgG and DNA were 0.25 pg/mL and 0.52 aM, respectively. They are very competitive compared with similar results reported in the literature.
Additional, a gold nanoelectrode ensemble（GNEE）coupled microchip system was developed for bio-electrochemical analysis. Due to the difference in mobility of urea and urease were mixed and allowed the enzymatic reaction to proceed in microchannel. The enzymatic product NH4+ was determined by the coupled GNEE at the outlet of the channel. Another experiment of streptavidin conjugated gold-nanoparticles（streptavidin-Au）, reductant and gold-ion（Au3+）solution was be applied here, too. The product, NH4+ or Au3+ was passed through downstream of microchannel and detected by GNEE of electrochemical system. Satisfactory linear relationship（R2 = 0.9778 and 0.9657）were found from 0.1 mM to 50 mM for NH4+ and urea in the range of 0.02 mM to 5.0 mM, respectively. The other satisfactory linear relationship（R2 = 0.9842 and 0.9507） were found between 3.75 mg/mL and 3.75 g/mL for Au3+ and streptavidin-Au in the range of 0.2 ng/mL to 100 ng/mL, respectively. Variances of 2.5 %（n = 6）was found for analysis of with the microchip system.

論文提要....................................................................................................................................i
英文摘要（Abstract）.............................................................................................................iii
誌謝...........................................................................................................................................v
目錄..........................................................................................................................................vi
附圖目錄...................................................................................................................................x
圖目錄......................................................................................................................................xi
表目錄....................................................................................................................................xiii
第壹章 、緒論..........................................................................................................................1
一、前言...................................................................................................................................1
二、免疫分析簡介...................................................................................................................1
1. 免疫分析的基本理論...........................................................................................................1
2. 酵素免疫分析.......................................................................................................................2
3. 免疫球蛋白簡介...................................................................................................................4
三、DNA 簡介..........................................................................................................................7
1. DNA 的基本理論..................................................................................................................7
2. DNA 的結合與其構造........................................................................................................10
四、酵素簡介.........................................................................................................................13
五、晶片電泳簡介.................................................................................................................14
1. 毛細管電泳.........................................................................................................................14
2. 微機電晶片.........................................................................................................................17
六、無電電鍍方法簡介.........................................................................................................18
1. 無電電鍍.............................................................................................................................18
2. 奈米組合電極的特性.........................................................................................................22
七、金奈米粒子放大方式簡介.............................................................................................24
八、實驗目的.........................................................................................................................26
第貳章 、免疫分析................................................................................................................27
一、前言.................................................................................................................................27
二、實驗.................................................................................................................................28
1. 藥品與溶液配製.................................................................................................................28
（1）藥品...............................................................................................................................28
（2）溶液之配製...................................................................................................................30
2. 儀器設備.............................................................................................................................32
3. 電化學系統.........................................................................................................................34
4. 實驗操作流程及電化學偵測.............................................................................................34
（1）實驗操作步驟...............................................................................................................34
（2）電化學偵測...................................................................................................................37
三、結果與討論.....................................................................................................................38
1. 金奈米粒子放大結果.........................................................................................................38
（1）以電子顯微鏡觀測金奈米粒子之放大效果...............................................................38
（2）以電化學方式觀測金奈米粒子之放大效果...............................................................40
2. 實驗各項參數之探討.........................................................................................................42
（1）電化學方波剝除伏安法之各項參數探討...................................................................42
（2）電化學方波剝除伏安法之電沉積時間.......................................................................43
（3）金離子溶液及還原劑之比例.......................................................................................45
（4）金奈米粒子之放大時間...............................................................................................47
（5）GaRIgG-Au 培養濃度的影響.......................................................................................49
3. 實驗結果.............................................................................................................................51
（1）電化學方波剝除伏安法之訊號再現性.......................................................................51
（2）RIgG 的定量偵測.........................................................................................................52
（3）真實樣品中之干擾物影響..........................................................................................56
（4）和文獻中相似分析方法比較......................................................................................58
四、結論................................................................................................................................60
第參章 、DNA 分析..............................................................................................................61
一、前言................................................................................................................................61
二、實驗................................................................................................................................62
1. 藥品與溶液配製................................................................................................................62
（1）藥品..............................................................................................................................62
（2）溶液之配製..................................................................................................................63
2. 儀器設備............................................................................................................................65
3. 電化學系統........................................................................................................................66
4. 實驗操作流程及電化學偵測............................................................................................66
（1）實驗操作步驟..............................................................................................................66
（2）電化學偵測..................................................................................................................69
三、結果與討論....................................................................................................................70
1. 金奈米粒子放大結果........................................................................................................70
（1）以電子顯微鏡觀測金奈米粒子之放大效果..............................................................70
（2）以電化學方式觀測金奈米粒子之放大效果..............................................................73
2. 實驗各項參數之探討........................................................................................................75
（1）Coating DNA 培養濃度之影響....................................................................................75
（2）遮敝試劑（BSA-Biotin）之濃度探討.........................................................................77
（3）遮敝試劑（BSA-Biotin）之培養時間.........................................................................78
（4）DNA 雜交（Hybridization）時間探討........................................................................80
（5）Streptavidin-Au 之濃度探討.........................................................................................82
（6）Streptavidin-Au 之培養時間.........................................................................................83
3. 實驗結果.............................................................................................................................85
（1）電化學方波剝除伏安法之訊號再現性.......................................................................85
（2）Target DNA 的定量偵測...............................................................................................87
（3）真實樣品之偵測...........................................................................................................90
（4）和文獻中相似分析方法比較.......................................................................................93
四、結論.................................................................................................................................95
第肆章 、微管道晶片系統之生物分析................................................................................96
一、前言.................................................................................................................................96
二、實驗.................................................................................................................................98
1. 藥品與溶液配製.................................................................................................................98
（1）藥品...............................................................................................................................98
（2）溶液之配製...................................................................................................................99
1 金奈米組合電極製備所需之溶液......................................................................................99
2 微管道晶片製作所需之溶液............................................................................................101
3 微管道系統所需之溶液....................................................................................................101
2. 儀器設備...........................................................................................................................103
三、結果與討論...................................................................................................................107
1. 金奈米組合電極...............................................................................................................107
（1）金奈米組合電極製備.................................................................................................107
（2）金奈米組合電極特性.................................................................................................108
2. 微管道晶片製作...............................................................................................................111
（1）光罩製作.....................................................................................................................111
（2）石英玻璃母模製作.....................................................................................................113
（3）微管道晶片熱壓程序.................................................................................................115
（4）壓克力底板電極製作.................................................................................................116
（5）壓克力上之金奈米組合電極.....................................................................................120
（6）壓克力化學接合過程.................................................................................................121
（7）完成微管道晶片製作步驟.........................................................................................122
（8）微管道晶片系統暨電化學偵測系統.........................................................................125
3. 在微管道晶片系統中做尿素偵測...................................................................................126
（1）金奈米組合電極對 NH4+ 之反應性.........................................................................126
（2）微管道晶片系統的偵測步驟.....................................................................................128
（3）微管道晶片系統對 NH4+ 之反應性.........................................................................130
（4）分離電壓對分析物的影響.........................................................................................132
（5）NH4+ 在微管道晶片系統中的定量偵測...................................................................134
（6）微管道晶片系統中酵素催化的影響及樣品訊號再現性.........................................136
（7）尿素在微管道晶片系統中的定量偵測.....................................................................137
4. 在微管道晶片中做蛋白質偵測.......................................................................................140
（1）對金離子溶液反應之還原劑選擇.............................................................................140
（2）微管道晶片系統的偵測步驟.....................................................................................140
（3）微管道晶片系統對金離子（Au3+）之反應性........................................................142
（4）分離電壓對分析物的影響.........................................................................................143
（5）Au3+ 在微管道晶片系統中的定量偵測...................................................................145
（6）微管道晶片系統中的再現性測試.............................................................................146
（7）Streptavidin-Au 在微管道晶片系統中的定量偵測...................................................148
四、結論與未來展望...........................................................................................................150
第伍章 、總結......................................................................................................................152
第陸章 、參考文獻..............................................................................................................154

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