微晶片分析技術是檢測雲端服務的應用課題之一，優點在於極低樣品及試劑需求量，因此結合分離、偵測兩項功能的微晶片設計變成個人可攜式移動平台，樣品分析速度也較以往提升數十倍。由於微晶片分析的感測區域較以往分析技術縮小數十至數百倍，因此需要更高靈敏度的分析工具，本論文主旨在於開發高靈敏度的微晶片電泳電化學偵測系統，本研究提出低深寬比微流道結構來提高電極反應面積，藉由降低流道深度來換取微流道寬度的方式增加實際電極有效的反應面積，因微流道的實際截面積沒有太多的增加，所以實際電泳所需的電泳流並不會大幅提升。在最佳化後，使用寬度400 μm深度7.5 μm的微流道，多巴胺、鄰苯二酚與尿酸偵測極限(k=3)約在20nM左右，相較於使用寬度 50 μm深度15 μm 的微流道所得到的多巴胺與鄰苯二酚的靈敏度相較起來，其靈敏度提高4.7(高濃度6.6倍)與11.83倍，顯示低深寬比微流道結構確實可增加分析靈敏度。
    而使用低深寬比微流道搭配白金去耦合電極進行實驗時，發現進行電泳其間去耦合電極會產生自由基，此自由基對於低氧化電壓的干擾物可快速進行氧化反應，對高氧化電位的物種則反應性較差，因此可增加低深寬比微流道結構的選擇性，並以乙醯氨酚作為實際應用。使用分離流道長度2 cm搭配白金去耦合電極長度2.5 mm時，可將10 μM的多巴胺與4-胺基苯酚的電流訊號從38.21 nA降至0.242 nA，解析度由1.35提昇至2.11，乙醯胺酚的線性為0.5 μM-300 μM，且與尿酸達到完全分離的狀態，整體實驗可在50秒內對尿液，血清以及感冒成藥中的乙醯氨酚完成電泳檢驗。
    第二部分是開發檢測新鮮度的指標-組織胺及多胺的偵測方法。近年來食安問題愈來愈受大眾的重視，食材新鮮度未來可預測將是重要檢測指標之一，其中生物胺類分子如組織胺及多胺，由於分子安定性極高，目前較常用來作為新鮮度以及安全度的檢測指標，因此開發生物胺的快速檢測技術具有一定的重要性及商業應用性。
    發展組織胺偵測原理是藉由銅與組織胺之間的配位能力，使存在於銅金屬電極表面的氧化層溶解，在電壓輔助下，藉由銅層再生產生的氧化電流來量測分析物濃度，因此可在無酵素輔助下分析組織胺。經最佳化後，組織胺線性範圍為1-750 μM，最後搭配液相層析儀增加組織胺與其他物質的選擇性，組織胺在液相層析儀的分析線性為10-2500 μM，已符合實際分析所需的濃度範圍，最後以秋刀魚做為實際應用實例，檢測該食材在解凍時新鮮度的變化。
    而在多胺的量測機制上，多正電荷的多胺可以降低在低導電度的緩衝溶液中帶負電荷物質在電極表面進行電子轉移的阻抗，不僅降低負電分子過電壓且增加其電流值，藉由增加的電流值來測定多胺的濃度，最佳化後，腐胺與屍胺的線性範圍可達200 μM，亞精胺與精胺的線性範圍為可達25 μM。利用此方法的優點是不需在電極上修飾任何酵素即可進行偵測，且偵測電位低可大幅度降低常見的易氧化物質的干擾，實驗最後搭配液相層析儀增加多胺在生物胺上的選擇性。

Microchip analysis is one of the interesting topics in the current research due to its potential application in the field of internet of thing (IOT). The goal of this thesis focuses on developing a high sensitivity glass based microfluidic electrophoresis chip coupled with the electrochemical detection module. In order to reduce the cost of the whole detection system, in this research, the whole electrodes and their connecting circuits were fabricated by filling a filling conducted carbon ink into laser ablatedcavities on a glass substrate. The microchannels were also fabricated by casting PDMS prepolymer against a master and generating its replica, however, a screen printed technique (SPT) combing a wet etch process was also adapted to substitute the traditional MEMS process in the fabrication a glass mold. Thus, the width of the microchip can be defined in the process of SPT and the depth of the channel can be controlled based on the duration of the etching time.
	The next purpose of this thesis is increasing the sensitivity of the microchip electrophoretic system. Since the effective area of working electrode is limited by the width of the channel, a low-aspect ratio microchannel is proposed to increase the width of the channel and the effective reaction area of the working electrode. However, the depth of channel is reduced to minimize the electrophoretic current of whole electrophoretic system. After the optimization, a microchannel with 400 μm in width and 7.5 μm in depth was adopted. The detection limit of this system was estimated as 15.5, 13.55 and 26.5 nM (k=3) for dopamine, catechol, and uric acid, respectively. Compared to a traditional channel (50 μm in width, 15 μm in depth), the detection limit of this low-aspect ratio is improved by 6.6 to 11.83 folds, which proves the low-aspect ratio channel can increase the analytical sensitivity of microchip.
    In the second project, the performance of the decoupler electrode on the microchip was investigated. It is found that the maximum electric field of a carbon ink based decoupler electrode is restricted to below 175 V/cm due to the formation of the hydrogen gas in the higher electric field.The strategy of this study is modifying some oxygen sensitive materials in the decoupler electrode to enhance the electron transfer rate of oxygen. After investigation, the thionine, gold, platinum and palladium modified carbon electrode can increase the maximum electric field to 300, 346, 400, and 440 V/cm, respectively.
    Subsequently, a novel method to improve the selectivity of a microchip electrophoresis without alternating any separation condition was proposed. It is found that the modified decoupler electrode can produce free radicals during electrophoresis. This phenomenon makes the decoupler electrode possesses high selectivity to pre-oxidize the easily oxidized substances, but shows less influence to the compounds with high oxidative potential. Therefore, for a complicate sample, this system can eliminate the influence of antioxidants and increase the resolution of any electrochemical based microchip. By using this phenomenon, a novel method for rapid determination of acetaminophen was reported on a microchip with an effective separation length of 2 cm couple with the platinum decouple electrode. In the optimal condition, the signal of interferences such as dopamine and 4-aminophenol can be eliminated over than 99% and the resolution of acetaminophen with these interferences shows a dramatic increment from 1.35 to 2.11. Thus, this system can rapidly determine the acetaminophen within 50 seconds with the linearity from 0.5 to 300 μM. 
		The second part focuses on developing a reliable method to evaluate the freshness level of food. In this study, histamine and polyamine are used as the index to monitor the freshness of fish, and the histamine can be directly monitored on a bared copper electrode due to the strong chelate capability between the cupric (II) ion and histamine. After investigation, the operating potential and buffer were optimized as 200 mV and 100 mM phosphate buffer, pH 10.0, respectively. A suitable dynamic range from 1 to 750 μM for histamine determination with a sensitivity of 15 nA/μM (R= 0.999) was achieved on a typical FIA system. Although the linearity of this scheme after integrating with HPLC shows a significant decrement from 10 to 2500 μM (0.5-125 nmol per injection), it is still an adequate range for freshness quality definition of seafood. Finally, the feasibility of this scheme in real sample application was demonstrated by evaluating the histamine level in a fresh and defrost saury fish.
    In polyamine measurement mechanism, the role of polyamine can reduce the charge transfer resistance of negatively charged molecules such as ferricyanide that due to the multiple positive charges in the lower conductivity buffer solution. It not only reduces the overvoltage of the ferricyanide, but also increase the magnitude of the current. After optimization, the optimized condition of the buffer condition, operating potential and flow rate were optimized as pH 8, 50 mM borate buffer containing 1 mM ferricyanide, 0 mV, and 0.5 mL/min, respectively. The dynamic rage at the FIA system were estimated as 1-200 μM, 1-200 μM, 0.1-25 μM and 0.075-25 μM for putrescine cadaverine, spermidine and spermine, respectively. The advantage of this method for detecting polyamine were enzyme free detecting, and reduced the influence of antioxidants because of the low operating potential. Finally, the FIA system was integrated with HPLC in order to increase the selectivity of the polyamine from other biogenic amines.

目  錄
目  錄	I
圖目錄	VIII
表目錄	XI
第一章緒論	1
前言	1
1-1電泳簡介	1
1-1-1電泳原理	1
1-1-2 電泳分離模式	3
1-1-3電滲流(Electroosmotic flow, EOF)	4
1-1-4分離效率及解析度	6
1-2微流道製作方法	7
1-2-1微影製作方法	8
1-2-2 乾蝕刻製作方法	10
1-2-3 高分子製作方法	11
1-2-3-1熱壓法	12
1-2-3-2射出成形法	13
1-2-3-3造模法	14
1-2-4 雷射燒除法	15
1-2-4-1 Nd-YAG雷射	15
1-2-4-2 準分子雷射(Excimer laser)	16
1-2-4-3 二氧化碳雷射(CO2 laser)	17
1-3 微晶片電泳的偵測方法	19
1-3-1 電化學偵測模式簡介	21
1-3-2 電化學偵測之工作電極製作	23
1-3-3 去耦合電極的介紹及製作	25
1-4 電化學量測法介紹	29
1-4-1 電化學直接量測法	30
1-4-2 電化學間接量測法	32
1-5 食材新鮮度指標	36
1-5-1 pH值	36
1-5-2 揮發性鹽基態氮 (Volatile basic nitrogen, VBN)	37
1-5-3 K值	39
1-5-4 組織胺	41
1-5-4-1 組織胺介紹	41
1-5-4-2 組織胺光化學偵測法	43
1-5-4-3 組織胺電化學偵測方法	47
1-5-5 多胺	49
1-5-5-2 多胺光化學偵測方法	52
1-5-5-3 多胺電化學偵測方法	53
1-6本實驗研究目的	57
第二章低深寬比微流道發開及其應用	60
2-1微全偵測系統簡介	60
2-1-1 目前微流道常見深寬比例	60
2-1-2目前寛流道製作方法	63
2-2本實驗目的	68
2-3 實驗部分	68
2-3-1 實驗藥品	69
2-3-2 儀器與設備	69
2-3-3流道製作流程	71
2-3-4電泳系統架設	72
2-4 結果與討論	74
2-4-1 流道形貌	74
2-4-2 低深寬比微流道的電泳行為探討	76
2-4-3蝕刻深度對電泳影響	79
2-4-4流道寬度對電滲流與訊號的影響	83
2-4-5去耦合電極的修飾	87
2-4-6分析特性	93
2-5結論	99
第三章 以電場誘導自由基生成效應發展微晶片電泳電化學干擾物消
除機制	100
3-1簡介	100
3-1-1 去除干擾物方法	101
3-1-2 乙醯胺酚介紹	104
3-2本實驗研究目的	107
3-3實驗部分	108
3-3-1藥品	108
3-3-2儀器與設備	109
3-3-3 流道製作流程	110
3-3-4去耦合電極修飾	110
3-3-5螢光實驗	111
3-3-6真實樣品製備	112
3-4結果與討論	113
3-4-1原理探討	119
3-4-2流道深度的影響	123
3-4-3去耦合電極長度的影響	128
3-4-4干擾物消除量測試	132
3-4-5乙醯胺酚分析特性	134
3-4-6真實樣品分析	136
3-5結論	138
第四章以鍍銅電極發展組織胺量測系統及新鮮度的應用	139
4-1簡介	139
4-1-1組織胺介紹	139
4-1-2 組織胺量測方法	140
4-2本實驗目的	144
4-3實驗部分	145
4-3-1藥品	145
4-3-2儀器	145
4-3-3電極製備與保存	146
4-3-4長時間電解與UV-Vis 量測	147
4-3-5 真實樣品製備與HPLC分析	147
4-4結果與討論	148
4-4-1分析機制的建立	148
4-4-2 最佳化條件	155
4-4-3 緩衝溶液條件最佳化	157
4-4-4分析特性	163
4-5 HPLC分析與真實樣品應用	169
4-6結論	173
第五章 發展電荷中和增加電子轉移速率的方法並應用於多胺分子的偵測	174
5-1簡介	174
5-2本實驗目的	178
5-3實驗部分	179
5-3-1藥品	179
5-3-2儀器	179
5-3-3 3,4-DHBA與ferricyanide中添加不同多胺濃度的阻抗分析量測	180
5-3-4 HPLC分離多胺實驗	181
5-4結果與討論	182
5-4-1 量測機制的建立	182
5-4-2 分析條件最佳化	192
5-4-2-1 溶液酸鹼值最佳化	192
5-4-2-2 緩衝溶液濃度最佳化探討	195
5-4-2-3偵測電位最佳化探討	197
5-4-2-4 Ferricyanide濃度最佳化探討	199
5-4-2-5流速最佳化	201
5-4-3分析特性	203
5-4-4 HPLC分析圖譜	207
5-5結論	209
第六章 結論	210
參考資料	214
圖目錄
圖1- 1微影流程圖	11
圖1-2微晶片電泳電化學方法偵測模式	21
圖2- 1 一般深寬比與低深寬比微流道示意圖	68
圖2- 2微流道製作示意圖	71
圖2-3 玻璃全平面電極及低深寬比流道模組	73
圖2- 4 400 μm流道印製在玻璃與濕蝕刻後的光學圖	75
圖2- 5流道寬度400 μm 蝕刻10分鐘真實電泳圖譜	78
圖2- 6螢光顯微鏡下所觀測的樣品遷移情形	78
圖2- 7蝕刻時間對鄰苯二酚電流與滯留時間的影響以及鄰苯二酚與尿酸真實電泳圖譜	82
圖2-8不同流道寬度對鄰苯二酚的滯留時間與訊號的影響及鄰苯二酚與尿酸真實電泳圖譜	86
圖2- 9碳墨電極、白金、鈀對不同濃度過氧化氫的循環伏安圖	92
圖2- 10多巴胺、鄰苯二酚以及尿酸的校正曲線圖	95
圖2-11多巴胺、鄰苯二酚及尿酸真實電泳圖譜	96
圖3- 1乙醯胺酚代謝途徑	106
圖3- 2乙醯胺酚、尿酸、多巴胺、4-胺基苯酚在玻璃碳電極上的循環伏安圖譜	117
圖3- 3不同去耦合電極材質對多巴胺、4-胺基苯酚、乙醯胺酚及尿酸電泳圖譜的影響	118
圖3- 4多巴胺、4-胺基苯酚、乙醯胺酚及尿酸的電泳電流值	121
圖3- 5螢光放射圖譜	122
圖3-6流道深度對分析物訊號的影響	125
圖3- 7物質在薄層電極下進行電化學反應的濃度曲線分佈	126
圖3- 8不同長度去耦合電極對分析物電流的影響	130
圖3- 9分析物在不同長度去耦合電極的真實電泳圖譜	131
圖3- 10乙醯胺酚校正曲線	135
圖3- 11乙醯胺酚應用在真實樣品量測	137
圖4- 1組織胺結構	148
圖4-2添加不同濃度組織胺在銅電極上的循環伏安圖	151
圖4- 3銅電極量測組織胺可能機制1	152
圖4- 4銅電極量測組織胺可能機制2	152
圖4- 5硫酸銅及銅與組織胺紫外光-可見光光譜圖	154
圖4- 6銅線長時間電解紫外光-可見光光譜圖	154
圖4- 7電位最佳化探討	156
圖4- 8緩衝溶液pH值最佳化探討	158
圖4- 9緩衝溶液濃度最佳化探討	160
圖4- 10醋酸根濃度最佳化探討	162
圖4- 11組織胺校正曲線	167
圖4- 12連續量測20次10 μM組織胺的訊號	168
圖4- 13魚樣品於HPLC上分離實際圖譜	171
圖4- 14使用標準添加法量測魚樣品中的組織胺濃度實際圖譜	172
圖5-1多胺分子與衍生物反應示意圖	176
圖5- 2多胺結構圖	186
圖5- 3 鄰苯二酚、多巴胺及3,4-二羥基苯甲酸連續添加精胺循環伏安圖譜	189
圖5- 4正腎上腺素、腎上腺素、3,4-二羥基苯甲酸、3,4-二羥苯丙氨酸以及鐵氰化鉀連續添加精胺循環伏安圖	190
圖5- 5多胺分子對鐵氰化鉀及3,4-二羥基苯甲酸EIS圖譜的影響	191
圖5- 6緩衝溶液pH值最佳化探討	194
圖5- 7緩衝溶液濃度最佳化探討	196
圖5- 8偵測電位最佳化探討	198
圖5- 9 Ferricyanide濃度最佳化探討	200
圖5- 10流速最佳化探討	202
圖5- 11腐胺校正曲線	205
圖5- 12 多胺分離圖譜	208
表目錄
表1- 1不同水產品在新鮮度變化中的pH值	37
表1- 2 BAI值與食品狀態相關性	51
表2- 1常用於電泳分析微流道寬度與深度	62
表2- 2蝕刻10分鐘流道平均寬度及深度	76
表2- 3不同蝕刻時間對鄰苯二酚與尿酸滯留時間與電流訊號	81
表2- 4不同寛度對鄰苯二酚與尿酸滯留時間與電流訊號	85
表2- 5不同修飾物的背景電流值與電場承受度	91
表2- 6多巴胺、鄰苯二酚及尿酸分析特性	97
表2- 7比較實驗室以往偵測多巴胺與鄰苯二酚的分析特性	97
表2- 8與其他文獻比較多巴胺與鄰苯二酚的分析特性	98
表3- 1不同去耦合電極材質對於分析物電流影響	114
表3- 2過去實驗室發展微流道偵測的分析物	124
表3- 3不同時間下 與電極距離的關係	127
表3- 4去耦合電極對於不同濃度分析消除比例	133
表4- 1其他方法量測組織胺	165
表4- 2各干擾物干擾程度	166
表5- 1精胺對不同電荷反應物氧化峰電流的影響	187
表5- 2不同生物胺對於3,4-DHBA/Ferricyanide氧化/還原訊號的影響	187
表5- 3 胺官能基數量對電流訊號的影響	188
表5- 4多胺分析特性	205
表5- 5 干擾物干擾程度	206

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