發展並整合微型化分析系統在現代分析化學中是極為重要且有助益的，因其具有可降低成本、易於自動化及高分析效率等優點。微型化分析系統目前已被廣泛應用在生化分析、藥物篩選及化學反應等領域上。然而，將系統微型化通常會伴隨一些問題並且會面臨到實用性的挑戰。例如，在晶片型電泳中，電壓進樣法由於具有便利及易於操作的特點，使得它成為最為廣用的進樣法。但以電壓法進樣時，卻經常伴隨著進樣誤差（injection bias）及對中性分析物進樣效率差的缺點。晶片型電泳的另一個問題是以光學法偵測分析物時，由於晶片可容許的進樣量低及蝕刻管道的光路短，使得偵測靈敏度通常無法達到實際要求。本研究的目的便是要在晶片型電泳上發展一套連續自動化取樣系統及線上濃縮法來解決上文所提之進樣及靈敏度的問題。
在晶片型電泳中，取樣是相當重要的步驟，因為此步驟將直接影響分析結果。本研究的第一個主題便是要以連續壓力取樣法（flow-through sampling）為基礎，發展出一套可在單一晶片上連續分析不同樣品的自動取樣系統。在此系統中，我們將原本針對高效能液相層析系統所設計的自動取樣器串聯到電泳晶片上，並且將整個實驗流程，包括取樣(sampling)、進樣(injection)、分析、訊號擷取及清洗等步驟完全自動化。在實驗中，不同濃度的鹽基桃紅精(Rhodamine B)經由自動取樣器取樣後，以水壓法(hydrodynamic flow)將之傳送至晶片中，然後依序被進樣至微管道中分析，而從自動取樣器取得的每一個樣品(0.2-1.0µL)則可供多次的進樣(1-10nL)及電泳分析。此外，系統中的連接管柱所造成的變異(variance)雖然不會影響電泳分析結果，卻會造成管柱外樣品的擴散及稀釋，因此我們亦針對整個系統的變異做理論推導。本研究結果顯示，相較於傳統的手動電壓進樣法，本系統之自動化取樣、清洗連接管柱及微管道除可免除人為操作外，所需樣品量及處理時間均大為減少。
傳統晶片型電泳由於不同晶片間的差異、重新置換樣品、微管道操作條件的重新建立及電壓進樣誤差等原因，在定量分析上一直無法有很好的表現。本研究的第二個主題便是要整合第一部份所發展的自動化連續壓力取樣系統的優點與廢液移除裝置，以改善傳統電泳型晶片在定量分析效能上的不足並且延長系統操作時間。研究結果顯示以異硫氰酸熒光素標記之雌激素(FITC-labeled estrogen)或鹽基桃紅精為分析物，此整合裝置在超過三小時(360次分析)的操作時間下，仍然具有相當好的再現性（R.S.D＜6.6%）。另外，針對一系列濃度的磷酸化胜肽(phosphopeptide)建立校正曲線後發現，在不使用內標準品的情況下，線性(R2)及再現性分別為0.9961及3.16%；而在使用內標準品(non-phosphopeptide)的情況下，線性及再現性則稍微改善為0.9986及2.27%。我們亦針對此系統之定量分析法在激酶磷酸化分析(kinase phosphorylation assay)上的應用做一簡短的討論。
由於可容納樣品體積少及蝕刻管道的光徑短，使得微晶片電泳的靈敏度在以光學偵測器為偵測手段時受到限制。在本研究的第三個主題，我們發展了一套整合型微流體裝置，50nL的DNA樣品被壓力注射到微管道內並被等速電泳法濃縮成很窄的帶寬，之後，此濃縮樣品被導入一均勻的膠電泳管道進行分離及偵測。此設計提供了可使用不同分離介質的可能性，在本研究中，我們使用了一種黏度大於前導離子緩衝液的分離介質來分離濃縮後的DNA標準品，結果顯示此DNA標準品可在1.5公分內被分離，並且由於黏度的關係還造成了更進一步的濃縮效應。相較於傳統膠電泳晶片，本研究所發展之等速膠電泳晶片在可忽略的分離度損失下，靈敏度提升了150倍而分離時間則減少了一半。此外，相較於其它預濃縮方法，等速電泳的一個獨特優點是其具有提升具大範圍濃度差異之分析物的偵測能力，我們導出了一套整合了Kohlrausch regulation function (KRF)及帶變寬效應(band broadening effect)的理論來解釋所觀察到的現象。我們亦將此特性應用在一個聚合酶連鎖反應(polymerase chain reaction,PCR)樣品上，此樣品含有放大效應相差甚大之兩段聚合酶連鎖反應產物。而結果顯示此等速膠電泳晶片可對放大效應較差的一段產物有較佳的濃縮效果，使得在同時偵測兩段放大效應相差甚多之產物的信心度提升。

Development and integration of miniaturized analytical system in modern analytical chemistry is of great importance and beneficial due to the reduced cost, ease of automation, and high analysis efficiency and so on. The miniaturized analytical system, which is widely known as microdevice, has been applied in many fields including bioanalysis, drug screening, and chemical reactions, etc. However, problems accomplished with the miniaturization arise and challenge the practicability of microdevices. In microchip electrophoresis, for example, sampling is typically realized by electrokinetic injection because of the simplification. Electrokinetic injection, however, usually accomplished with injection bias and low injection efficiency for neutral analytes. Another problem of microchip electrophoresis is the poor sensitivity due to the low sample loadability and shallow depth of microchannels for optical detection. The target of this study is to solve the sampling and sensitivity problems of microchip electrophoresis by using flow-through based automatic sampling method and on-line concentration technology, respectively.
Sampling is a critical step in microchip electrophoresis because it affects the analytical results in a straightforward way in the very early stage of analysis. The first study (Chapter 4) is to develop an automatic sampling system based on flow-through sampling for sequential analysis of different samples in a single microchip. An autosampler, which was originally designed for high performance liquid chromatography (HPLC), was on-line connected to the microchip and the whole process including sample loading and injection, analysis and data acquisition as well as washing were all automated. Rhodamine B at different concentrations was first loaded into a hydrodynamic flow stream by the autosampler, delivered to the microchip, and then sequentially injected into the electrophoretic microchannels for analysis and detection. Using this sampling system, each loaded volume (0.2-1.0µL) can be injected for dozens of electrophoretic analyses (1-10nL for each injection). In addition, the variances caused by the external connections, which did not affect the electrophoretic analysis but would cause band broadening of the loaded sample in the hydrodynamic flow stream, were theoretically deduced. Such a design allows sequential analysis of a series of samples while the running buffer is continuously pumped into the connection capillary as well as microchannels for washing without human intervention. Using this sampling system, the required sample amount and handling time can be greatly reduced compared to the manual electrokinetic injection method.
Quantitative analysis is problematic for microchip electrophoresis for several reasons including chip-to-chip variation, discontinuous sample re-loading, channel reconditioning, and electrokinetic injection bias. In the second study (Chapter 5), the flow-through based automatic microchip electrophoresis system developed in the first study was integrated with the waste-removing function to demonstrate a more quantifiable and more reproducible performance than manual electrokinetic injection method for quantitative analysis and prolong the analysis time. Using the flow-through microchip with waste-removing function, FITC-labeled estrogen or Rhodamine B could be continuously analyzed without significant changes (R.S.D. < 6.6%) in signal intensity for over 3 h (360 analyses), which is sufficient for a complete set of quantitative analysis. With the use of a phosphorylated kinase substrate as the model, a calibration curve for quantitative analysis of phosphopeptides were constructed and results indicate that both R2 value of the linearity and R.S.D. values of the peak intensity were around 0.9961 and 3.16%, respectively, without the use of an internal standard. These values were slightly improved to be around 0.9986 and 2.27%, respectively, with the use of a non-phosphopeptide counterpart as the internal standard. The potential of this flow-through system for the development of a kinase phosphorylation assay based on the quantitative method was also briefly discussed.
Due to the low sample loadability and shallow depth of microchannels, the sensitivity of microchip electrophoresis is limited, especially when optical detector is used for detection. In the third study (Chapter 6), we demonstrated an integrated microfluidic device in which 50nL of DNA sample is hydrodynamically injected and concentrated by isotachophoresis (ITP) into narrow bands and then subsequently introduced into a homogeneous gel electrophoresis (GE) channel for separation and detection. This design renders flexibilities in choosing different buffers for separation regardless of the stacking buffers used. In this study, a sieving gel buffer with a higher viscosity than the leading buffer was utilized to separate DNA markers in a short distance (1.5 cm) as well as to cause a further stacking as the sample zone enters the separation channel. Compared to conventional microchip gel electrophoresis (MGE), the sensitivity of ITP-GE microchip was increased by 150-fold and the separation time was reduced by 2-fold without noticeable loss in resolution for the fragments of X 174 digest. In addition, compared to other pre-concentration methods, one unique advantage of ITP stacking is the enhancement of detection capability for samples with a wide dynamic range. This observation is explained based on a model derived from Kohlrausch regulating function (KRF) coupled with the band broadening effect occurred during the separation by gel electrophoresis. We further demonstrated that such characteristics is useful for detecting two products amplified from a multiplex polymerase chain reaction (PCR) in which one product is poorly amplified compared to the other and the much more enhanced S/N ratio for the weak signal than for the strong signal has greatly increased the confidence for detecting both products.

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