整合奈米流道預濃縮器與免疫分析球用於高靈敏抗原檢測

Integration of nanofluidic preconcentrator and immunobead's assay for high sensitivity biomarker detection

黃譯履(Yi-Lu Huang)

指導教授 ： 沈弘俊

國立臺灣大學/工學院/應用力學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10905

摘要

本研究結合預濃縮機制(preconcentration mechanism)以及布朗運動檢測技術(Brownian motion detection)成功開發一新型檢測系統，利用微影製程做出微米流道及微米氣動閥再以Nafion作為離子選擇性薄膜(ion-selective membrane)，將上述三者整合而成本實驗之檢測晶片，其中微米流道分為兩條，其一為濃縮流道(concentration channel)、其二為緩衝流道(buffer channel)而Nafion則橫跨兩流道。於濃縮流道中通入前列腺特異抗原(prostate specific antigen, PSA)及修飾於螢光小球(fluorescent microsphere)的前列腺特異抗體(anti-PSA)，利用氣動閥(pneumatic valve)捕捉樣本，且在去除電場以粒子追蹤測速儀(micro-particle tracking velocimetry, μ-PTV)針對螢光小球進行速度場分析，而螢光小球在抗原與抗體進行專一性鍵結時，因直徑變大會造成布朗運動速度下降，以此便可作為抗體抗原鍵結的依據。
預濃縮機制為施加一電位差於Nafion兩端，可產生濃度極化效應(ion concentration polarization, ICP)，意即一端為離子富集區(ion enrichment region)、另一端為離子空乏區(ion depletion region)，爾後在空乏區一側施加偏壓便可利用第二種電滲透流(electroosmosis of the second kind)使空乏區之高電壓側產生濃縮區塊(preconcentration plug)。
本研究同時利用可即時掌握預濃縮機制啟動時機之迴路電流監測預濃縮系統，由此便可固定預濃縮作用之時間長短。經由螢光小球及螢光劑驗證迴路電流、預濃縮機制及捕捉機制後，針對低濃度之前列腺特異抗原(0.2 ng/ml進行2分鐘之預濃縮作用，成功將濃度提升4000倍。本研究開發之免疫分析晶片成功量化低濃度之前列腺特異抗原，後續可直接應用於多種具檢測極限之生物檢體。

關鍵字

奈米流體預濃縮 ； 布朗運動 ； 前列腺特異抗原 ； 免疫螢光 ； Nafion ； 微粒子影像/追蹤測速儀

並列摘要

In this thesis, we have successfully developed a novel detection system by integrating preconcentration mechanism and Brownian motion detection. Fabricate a detection chip by coupling microchannels which including two channels: concentration channel and buffer channel, micro-pneumatic valve and Nafion as ion selection membrane which crossing two channels. We infuse prostate specific antigen (PSA) and fluorescent microsphere immobilized by anti-PSA into concentration channel, and capture the preconcentrated sample, then analyze the Brownian-velocity field by micro-particle tracking velocimetry (μ-PTV) after we remove the electric field. Observing the specific curve of Brownian velocity to quantify the preconcentration factor.
Preconcentration mechanism is applying a voltage drop in Nafion ends at first, can produce ion concentration polarization (ICP) which refers to one of the end becomes ion enrichment and the other becomes ion depletion. Then we apply a bias voltage to generate electroosmosis of the second kind to aggregate the preconcentration plug near the ion depletion region on the high voltage side.
We also apply dual-loop electric current to the detection system, which is able to observe the timing when concentration plug appears. Thus, we can define the time of preconcentration effect. After demonstrate the possibility of whole system, we preconcentrate low concentration of PSA (0.2 ng/ml) by 2 minutes, and raise the concentration over 4,000 fold successfully.
　　In summary, we have developed an immunoassays chip which is able to quantify PSA of low concentration. With the ability of our chip, quantify the concentration of various biopsies with the detection limit could be fulfilled.

並列關鍵字

Nanofluidic preconcentration ； Brownian motion ； Prostate specific antigen ； Immunofluorescence ； micro-Particle Tracking Velocimetry (μ-PTV)

參考文獻

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