高性能微奈米流體樣品濃縮晶片之研製與其生醫應用

在此篇論文中，主旨在詳述此研究開發一種高效能微奈米樣品預濃縮晶片的過程，此晶片利用微奈米流體中的電動現象，具有將生物分子快速聚集，進而高度濃縮於微量體積下的功能，經由性能的強化，可進一步應用於多點免疫分析與多流道同時快速檢測，結合螢光與表面電漿共振偵測系統，進行高靈敏度的生物分子檢測。在本文中，首先著眼於研究理論的探索，濃縮的機制首在電滲透流於奈米流道中產生的離子選擇性，在外加電場下，由於奈米流道尺寸與匹配的緩衝溶液配合，造成電雙層的重疊效應，阻止帶負電的離子與蛋白質分子通過奈米流道，進而在微奈米流道交界處，產生負離子分子遠多於正離子的濃度極化現象，同電性相斥而形成離子空乏區，藉由增強電場加速空乏區的形成及穩固，接著改變微米流道兩端的電場差，進行蛋白質分子的高速聚集，快速達成高倍率濃縮效應。接續的研究方法章節，首先開發奈米尺寸流道於不同材質的製程，採用的是濕蝕刻技術與晶片接合封裝的方式來製作一維的奈米流道，初期分別完成玻璃與鈉玻璃兩種材質的製程，兩者主要差異為熱熔接合時的溫度差異，以及重力施壓的重量控制，後期為了將金膜薄層置於密封的微米流道中，改採用PDMS與鈉玻璃接合的常溫製程；再者，本研究利用電阻電路模型，模擬流場中離子流的數值與流道尺寸及流道中溶液導電度的關係，推導出一套設計微奈米濃縮晶片的規則，進而可根據應用性的需求，變化晶片中流道的尺寸與圖形，以滿足最小啟動濃縮機制之電流閾值。透過有效的設計與製作，此研究產出的奈米濃縮晶片的效能，可於五分鐘內將蛋白質分子濃縮十萬倍以上，並藉由良好的電場調控，可長時間定位濃縮寬帶於實驗區。配合電路模型的建立，微奈米流道的設計規則也藉變化尺寸的濃縮實驗加以驗證，以使整體電路計算後的電阻值，能與預計施予的電壓值相配合，同時也由實驗中，驗證電滲透流速與微米流道尺寸的關係，以期在設計上與啟動電流值相配合，達到最佳的濃縮效率。接續，本研究進一步增進了晶片的功能，首先在不改變晶片圖形的情況下，藉由調整電壓值，改變空乏區的長度，調整壓差使其在流道不同處濃縮，控制壓差使其在該處固定，進而開發出奈米濃縮晶片之 iii 樣品定位功能，且應用至免疫分析，可達成單一流道多點免疫分析之應用。為了將濃縮晶片應用在未知疾病抗原的免疫分析檢測，需要多條流道放置不同的抗體以做為檢測感測器，因此，本研究在不增加接地流道的設計下，同時在接地流道上下端各放置一條濃縮流道為雛形，以做為多流道設計之先期試驗；藉由電阻模型之模擬設計，能在適當的電壓控制下，完成雙流道濃縮及定位之實現。最後，本研究將此濃縮晶片應用於癌症抗原的免疫分析檢測，首先搭配螢光顯微鏡擷取抗原抗體接合前後的影像，藉由螢光強度的差異來分析接合後的結果；接著，利用高度濃縮效應，分別提高單位面積下的抗體固化數量與抗原數量，以縮短免疫流程時間及提高偵測的靈敏度；由於螢光免疫分析需將螢光物質預先嵌合於蛋白質上，以做為螢光偵測標記，因此，本研究另採用角度調制式表面電漿共振光學技術，作為免疫分析的偵測工具，同時，將其與濃縮晶片結合，組成無標記高靈敏度生物分子檢測平台，初期的研究已顯示在進行高倍濃縮下的抗原蛋白質寬帶會立即在表面電漿偵測平台上顯現大幅度的角度變化，故若將未濃縮前的偵測極限濃度大幅降低，應可提升系統數量級以上的的靈敏度。總結此研究的主要貢獻，首先在於提供一個完整的微奈米流體樣品濃縮晶片製程，此製程可根據研究需要，變化材質與製程方法，具有多方應用性。同時首提出一專門應用於設計奈米濃縮晶片的電阻電路模型，兼具濃縮啟動機制與濃縮效率的考量，也可使用於變化晶片圖形以供應用所需。同時，此研究亦開發晶片的多功能性，並進而運用於多點免疫分析與多樣品同步篩檢，最後將運用無標記光學技術，縮短檢體抽取後的體外生化流程，實現一微小化快速檢疫分析平台。

關鍵字

微奈米樣品預濃縮晶片；濃度極化；離子空乏區；電阻電路模型；免疫分析；表面電漿共振

並列摘要

In this thesis, a micro/nanofluidic device with the capability of highly efficient trapping and concentrating biomolecules has been developed. With the advanced function of precisely multipoint immunosensing and multi-channel preconcentration, the nanofluidic preconcentrator has the potential for applying in highly sensitive detection of biomolecules. First, according to the electrokinetic theories of the nanofluidics, we build a novel electrical resistive network model to guide the design rules of various nanofluidic preconcentrators. In the prototype design for greatest concentration factor, two microchannels, one preconcentration microchannel and one ground microchannel are connected in the middle via 16 nanochannels. The preconcentrator is formed by a microchannels-patterned PDMS slab bonded with a nanochannels-patterned glass chip with the oxygen plasma treatment. The core fabrication techniques of the chip process are standard photolithography, nanochannel wet etching, microchannel molding, and PDMS-glass bonding alignment. After the design rule and the fabrication process were set up, the ion depletion region and electrokinetic trapping are generated to carry out the preconcentration with two sets of optimal voltage settings applied on the opposite ends of the nanofluidic chip. With the optimal voltage settings predicted by the model, the ionic current of the nanochannel in our preconcentrator is adjusted to be greater than the threshold value needed for the occurrence of the preconcentration, and a preconcentration factor greater than 105 is achieved in five minutes. Furthermore, we explore the length of the depletion region, improve a sample positioning capability of the preconcentrator by adjusting the applied voltage sets and manipulate the preconcentrated protein bands to multiple sites by a distance from several micrometers to several millimeters in the v preconcentration channel. At last, we made several changes in the geometry of the preconcentration channels to try out two concentrated protein in two separate microchannels. The multi-channel preconcentration capability is successful demonstrated by dividing the voltage sets on the ends of the microchannels. The multi-channel preconcentrator could be further added more preconcentration channels to apply in multiple biomarkers sensing simultaneously in the same chip. Finally, the preconcentrator with Au film was used to increase the limit of detection of immunoassay. By using fluorescence microscopy, the preconcentration effect is both demonstrated in the immobilization and immunoassay. The results indicates the preconcentration effect speeds up the antibody to immobilize onto the functionalized gold and enhances the signal of the antibody-antigen binding. Furthermore, we guided the sample positioning to complete the multipoint immunosensing. Otherwise, another observation method called surface plasmon resonance (SPR) system also has the preliminary results to combine the label-free detection with nanofluidic preconcentrator. In summary, a robust fabrication of micro/nanofluidic chips is accomplished for nanofluidic research, a resistive network model is developed and validated to optimize nanofluidic preconcentrators, a advanced precisely sample positioning function is applied to multipoint immunosensing, and a multi-channel preconcentrator has the potential for high throughput and highly sensitive sensing of low abundance analytes.

並列關鍵字

nanofluidic preconcentrator ； electrical resistive circuit model ； precise sample positioning ； multi-channel preconcentration ； multipoint immunosensing ； immunoassay ； SPR

參考文獻

1. B. Ziaie, A. Baldi, M. Lei, Y. Gu, and R. A. Siegel, “Hard and soft micromachining for BioMEMS: review of techniques and examples of applications in microfluidics and drug delivery,” Adv Drug Deliv Rev 56, 145– 172 (2004).

2. H. Daiguji, P. Yang, and A. Majumdar, “Ion transport in nanofluidic channels,” Nano lett 4, 137-142 (2004).

3. S. W. P. Turner, M. Cabodi and H. G. Craighead, “Confinement-induced entropic recoil of single DNA molecules in a nanofluidic structure,” Phys Rev Lett 88, 128103 (2002).

4. A. Manz, N. Graber, and H. M. Widmer, “Miniaturized total chemical analysis systems: a novel concept for chemical sensing,” Sens Actuators B 1, 244-248 (1990).

5. P. S. Dittrich, K. Tachikawa, and A. Manz, “Micro total analysis sysetems Latest advancements and trends,” Anal Chem 78, 3887-3907 (2006).

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高性能微奈米流體樣品濃縮晶片之研製與其生醫應用

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