致病原檢測晶片元件之設計與製造： 吸收力驅動微流式細胞儀與面對面漸張式交流電滲流微濃縮器

李彥昌

指導教授 ： 謝文馨

國立中正大學/工學院/機械工程學系/博士(2016年)

摘要

本研究之目的為微流體元件之設計與製造，包含二種微流體元件： 一、吸收力驅動微流式細胞儀(AMCC)與 二、面對面漸張式交流電滲流微濃縮器。第一個部分，AMCC運用高吸水性材料之吸收力以驅動微流式細胞檢測晶片中之溶液，使該晶片具有不需外在動力即能運作及易微型化之優勢。探討了 AMCC內部流體流動特性、微結構尺寸對 AMCC流速與流體聚焦寬度的影響、雷射誘導螢光檢測系統參數最佳化、AMCC與流式細胞儀(BD, FACSCalibur)兩者螢光測試結果之比較以及 AMCC螢光偵測極限。由實驗結果顯示，在 AMCC中，高吸水性材料可造成相當穩定之微流道流速及流體聚焦現象，並可經由不同的微流道尺寸設計以控制 AMCC內流體流速與流體聚焦寬度(平均流速約由 1.6 mm/s到18.5 mm/s、流體聚焦寬度約由 4 μm到 23 μm)。本研究並成功的將 AMCC與雷射誘導螢光檢測系統結合，進行螢光校正顆粒之螢光檢測，且結果與現行流式細胞儀檢測結果符合，證實了以高吸水性材料做為微流體晶片流體驅動源的構想。第二個部分，面對面漸張式交流電滲流微濃縮器由一含頂部電極(氧化銦錫, indium tin oxide, ITO)之上板與一含底部電極(含三細長電極與一三角形電極)之下板所構成，上下板電極間利用蓋玻片作間隙形成流體反應室，利用三細長電極配合三角形電極與其連接處形成之電極漸張區域，將周圍流體反應室的待測物引導至底部電極中心，在底部電極中心附近產生流場停滯點，形成一濃縮區，並利用共軛焦顯微鏡分析垂直電極方向的濃度分佈，以對此濃縮器之濃縮區大小與分布更佳的了解，且此微濃縮器具備可重覆濃縮的功能。相較於其他文獻，在相似的施加電場下，本研究之濃縮器濃縮螢光乳膠顆粒(直徑 200 nm) 所得之螢光增強因子 3.9~9.1倍於其他文獻，濃縮雙股 DNA (T4 GT7 DNA)生物溶液所得之螢光增強因子 1.4~5.7倍於其他文獻。

關鍵字

微流式細胞儀 ； 超吸水性材料 ； 雷射誘導螢光 ； 水力聚焦 ； 螢光偵測 ； 軟微影技術 ； 交流電滲流 ； 微濃縮器 ； 非對稱電極對 ； 螢光增強因子 ； 濃度增強因子

並列摘要

The purpose of this research is the design and manufacture of microfluidic components, including two kinds of microfluidic components: (1) absorbing the force driving the micro flow cytometer (AMCC) and (2) AC electro-osmotic (AC-EO) microconcentrator using a face-to-face, asymmetric electrode pair with expanded sections in the bottom electrode. This work developed an absorbent-force-driven microflow cytometer chip (AMCC), in which solutions were driven by the absorbent force of superabsorbent materials to allow chip operation without external power and easy miniaturization. The polydimethylsiloxane (PDMS) cover of the microflow cytometer chip containing microchannels and reservoirs was fabricated by soft lithography and then bonded to a glass substrate. Then, superabsorbent material was put into contact with the microchannel’s end to drive the test solution from the reservoir to the superabsorbent material through the microchannel, forming a complete AMCC. The flow characteristics inside the AMCC, the impact of the microstructure size on the flow velocity and hydrodynamic focusing width of AMCC, and the optimized laser-induced fluorescence (LIF) detection system parameters were investigated in this work. Results showed that superabsorbent materials allowed stable microchannel flow and hydrodynamic focusing and that the flow rate and hydrodynamic focusing width of the AMCC could be controlled by varying the microchannel dimensions. The AMCC was integrated with the LIF detection system to detect the fluorescence of calibration particles, and the fluorescence results were consistent with those from a large-scale flow cytometer (BD, FACSCalibur), confirming the successful use of superabsorbent material as the fluid-driving source in an AMCC. An AC-EO microconcentrator using a face-to-face, asymmetric electrode pair with expanded sections in the bottom electrode is proposed in this study. The electrode pair of the AC-EO microconcentrator is composed of a larger top electrode (30 mm x 60 mm) and a bottom electrode (containing three slim electrodes and a triangular electrode). In the expanded section at the connection of a slim electrode and the triangular electrode, an electroosmosis flow transports test samples far away from the triangular electrode to the stagnation zone inside the triangular electrode through the slim electrode for concentration. On the three sides of the triangular electrode, vortices bring test samples surrounding the triangular electrode to the stagnation zone. By these two electroosmosis flow fields, the microconcentrator can concentrate test samples near and far from the triangular electrode to its central area, achieving a highly efficient sample concentration. The measured concentration distribution in the vertical electrode direction by confocal microscopy indicates that the concentration process occurs above the electrode surface. The capability of the proposed AC-EO concentrator in the repeated concentration and release of test samples is verified by a reversible switch test. The performance of the proposed AC-EO concentrator in concentrating latex particles and T4 GT7 DNA is better than those reported in the literature under similar average electric field strength. The fluorescence enhancement factor is 3.9 to 9.1 times better when concentrating latex particles and the concentration enhance factor, 1.4 to 5.7 times better when concentrating T4 GT7 DNA.

並列關鍵字

Microchip ； Microflow cytometer ； Superabsorbent material ； PDMS ； LIF ； Hydrodynamic focusing ； Fluorescence detection ； Soft lithography ； AC electroosmotic flow ； Microconcentrator ； asymmetric electrode pair ； Fluorescence enhancement factor ； Concentration enhancement factor

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