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標題: Manipulating pH in Microchannel for Affinity Chromatographic Elution
微管道中操縱 pH 值以進行親和性色層分析之溶析
作者: Huang, Szu-Chi
關鍵字: pH
pH 值
Affinity chromatographic
出版社: 食品暨應用生物科技學系所
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摘要: Affinity chromatography is a very popular technique for modern biochemists. Usually, researchers switch the different elution buffer to alter system pH for manipulating the affinity between ligands and receptors. However, the mechanical microvalve is a technical issue especially when adopting affinity chromatography into microchip. During isoelectric focusing electrophoresis (IEF), a pH gradient was automatic generated via free ampholytes mobilization along the axis from anode to cathode after designated electric potential applied. In this study, we demonstrated an integrated microfluidic platform, which facilitates affinity chromatographic elution by electrically developed pH gradient in a 129 mm length microchannel. The parameters for generating pH gradient in the microchannel were discussed in this study. The micro-column was packed at designed position by seizing the chromatographic matrix through different microstructural height. The result revealed that target protein IgG was separated from reference protein albumin by flushing microfluidic column with 0.1 M CaCl2 (pH 7.00). Furthermore, the electric field strength of 0.8 V/cm was adopted for 5 minutes to shift the pH at designated column position to adjust the affinity between target protein and chromatographic sepharose without changing eluting solution. The fluorescent and electrophoretic results confirm this integrated micro-platform successfully separated target IgG from reference albumin and subsequently recovered purified IgG by electric manipulating local pH in the microchannel. The electrical elution process without replacing internal solution demonstrated in this study shows the potential on the application of lab-on-a-chip in future.
親和性色層分析是生物化學相關研究中常用以純化分離蛋白質的方法。在進行親和性色層分析時,需要改變配體以及目標物之間之親和性,來達到分離或純化的目的。常用作法是更換注入之流洗緩衝液,改變配體及目標物之間的結合強度,進而達到溶析目標物之目的。但應用於微流體系統時則因微閥控制及液體混合不易,增加系統設計及操作的複雜度。本研究利用電解質液體中的離子會在通電後形成梯度的原理,整合建構一個可通電形成 pH 梯度總長度為 129 mm 之液體微流路結構及純化蛋白質用途之親和性色層分析管柱的微流體晶片系統平台,並討論用以產生 pH 梯度之通電參數。研究中利用微通道結構的高低差限制將親和性樹脂充填封裝於晶片的指定位置,並討論設計管柱位置對於pH梯度形成與親和性色層分析的溶出操作效果。螢光影像結果顯示,以 0.1 M CaCl2 (pH 7.00) 流洗液沖提注入晶片上親和性色層分析管柱之螢光標誌 IgG 以及對照用之 albumin 樣品,可成功的分離兩種蛋白質。隨後在不改變流洗液狀況下,以電場強度 0.8 V/cm 下通電 5 分鐘,可改變微通道中指定位置管柱的 pH 值,影響目標蛋白質與固定的層析樹脂間親和性以回收純化的 IgG。此平台實現了單一流路中不切換變更溶液種類而操控親和力結合的目標,可供未來實驗室晶片設計之參考。
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