二氧化鋯非水解型溶膠凝膠塗佈管柱的製備及其於毛細管電層析分離蛋白質的應用

Abstract

本研究以非水解型溶膠凝膠法將二氧化鋯修飾於熔融矽毛細管內壁作為靜相。依序將前驅物zirconium isopropoxide propanol complex與zirconium chloride 的氯仿溶液填入毛細管，以梯度式升溫法從室溫升溫 300 oC 高溫反應塗佈一層於管壁並以一個小時反應時間，使烷氧基與管壁的矽氧基進行反應。將如此製備的二氧化鋯管柱，以磷酸緩衝溶液在不同的 pH 值下進行電滲流測試，由於二氧化鋯在約 pH < 6時表面電位會帶正電，在此時電滲流理應呈現逆向，但由於磷酸離子會錯合在二氧化鋯管壁上，形成一層遮蔽層使管壁表面帶負電。因此在pH 的偏低時，只觀察到二氧化鋯管柱的電滲流較空管小的趨勢，而在 pH = 3 的電滲流才呈現逆相。將此二氧化鋯管柱應用在ovalbumin (OVA)、bovine serum albumin (BSA)、conablumin (ConA) 及apo-transferrin (apoTf) 的分離。由於二氧化鋯的水解穩定度較二氧化矽高，可以在極酸或是極鹼的環境下進行偵測。由研究發現，最佳化條件為磷酸緩衝溶液 (10 mM , pH 8)，並加入 5% MeOH (v/v) 當有機修飾劑，所獲得的流析順序為 ConA > apo-Tf > OVA > BSA，而且可以進一步分離出OVA 的五種醣蛋白異構物與 BSA 的兩種構形異構物。此研究之分離機制，除了電泳效果之外，應是基於二氧化鋯奈米表面的特殊化學，在磷酸緩衝溶液中，所形成的鋯－磷酸錯合物會與分析物進行配位基交換，進而達到較為優異的分離效果。

A ZrO2 nanoparticle-coated open-tubular column was prepared through the non-hydrolysis sol-gel reaction of zirconium isopropoxide propanol complex and zirconium chloride with silanol groups of the fused-silica capillary. The condensation reaction was carried out by gradient temp. raising to 300oC to form a monolayer. EOF measurements and SEM image were used to monitor the completion of reactions. The property of ZrO2 on the inner wall of capillary was significantly affected by the running buffer. Cathodic EOF at pH between 4~6 was indicated. It can be elucidated that the negatively charge phosphate ion in the buffer absorb strongly on the surface of ZrO2 resulting in the formation of phosphate ions-capped ZrO2 NPs complex. We selected four proteins, such as conalbumin (ConA), apo-transferrin (apoTf), ovalbumin (OVA), and bovine serum albumin (BSA) as model compounds, whose molecular weights and pI values are alike to each other. They could be separated with phosphate buffer (10 mM, pH 8.0) with 5 % MeOH and an applied voltage of 20 kV. In addition, five peaks of glycoisoforms of OVA and two forms of BSA were observed under these conditions. The column was also used to separate egg-white proteins. In addition to separating OVA and ConA, it can resolve three microheterogeneities of OVA and two forms (iron-free and iron-saturated) of ConA. In comparison with the retention behavior of the analytes on the bare fused-silica column, the new column of high resolving power seems to be predominantly derived from the ligand exchange of the analytes with the phosphate adsorbed onto the ZrO2 .