二氧化鋯奈米粒子塗佈管柱之製備及其毛細管電層析分離含鐵與磷酸化蛋白質之應用

Abstract

本研究將二氧化鋯以溶膠凝膠方式塗佈於熔融矽毛細管內壁作為毛細管電層析靜相，首先以氯氧化鋯zirconium (IV) oxychloride溶於乙醇與水形成澄清透明的溶膠凝膠溶液，導入毛細管內，與管壁的矽氧基進行縮合反應。將所製備完成的管柱，以磷酸緩衝溶液在不同的pH值進行電滲流測試，由於磷酸會與二氧化鋯錯合，發現即使在低於二氧化鋯的pI值 (pH 5-6) 時，管柱電滲流仍為正向而非理論上的逆向。本研究將其應用於分離含鐵的血紅蛋白 (Hb) 及肌紅帶白 (Mb) ，經由磷酸緩衝溶液的pH值與濃度等改變，皆獲得流析順序為Mb > Hb，符合文獻中Hb對二氧化鋯奈米粒子的binding constant較Mb大。並於20 mM、pH 9硼酸緩衝溶液中發現血紅蛋白存在α、β subunit兩個波峰，加入20 % (v/v) MeOH作為有機修飾劑增加疏水性，可提升分離效率。本研究進一步將其應用於分離磷酸化蛋白質，如卵白蛋白 (OVA) 、α-酪蛋白 (α-casein) 及牛血清蛋白 (BSA)， 與含鐵蛋白質，如伴蛋白 (ConA)及運鐵蛋白 (Tf) 之分析。研究發現磷酸化蛋白質α-casein、BSA隨著磷酸緩衝溶液pH值的改變，其滯留時間改變較ConA及Tf明顯，且緩衝溶液濃度增加，動相會與分析物競爭，使得層析峰的分離較為快速及波峰變窄。此研究證明二氧化鋯奈米表面的特殊錯合行為會影響毛細管電層析的分離機制，尤其與含有磷酸官能基的分析物進行配位基交換，可達到更有效的分離。本研究進一步發現此管柱具有高度優異性分離異構物，在40 mM、pH 9之硼酸緩衝溶液條件下，可得到14種以上OVA醣蛋白異構物，並以此條件進行雞蛋、鴨蛋等真實樣品中醣蛋白之分析，發現雞蛋、鴨蛋的OVA構形有明顯差異，但其流析順序均為ConA > OVA，由標準添加法得知紅色雞蛋的ConA濃度為0.72 mM。

A ZrO2 nanoparticles(NPs) coated column was prepared through the sol-gel reaction of zirconium(IV) oxychloride with silanol groups of the fused-silica capillary. The condensation reaction was carried out at 350 oC for 8 h. Electroosmotic flow (EOF) measurements and SEM image were used for the characterization of the ZrO2 on the inner wall of capillary. Below the pI value (pH 5-6), cathodic EOF was elucidated that the phosphate buffer adsorb tightly on the zirconia surface resulting in a negative charge surface. In this work, iron-binding-, phosphorylated- and glyco-protein were selected as the model compounds. For the optimization, the effects of pH, concentration and buffer type as well as the organic modifier on the separation were studied. The results of iron-binding proteins show that the retention time is Mb > Hb. It is corresponding to the binding constants of ZrO2 NPs. α-, and β-subunit of Hb could be separated in borate buffer (20 mM, pH 9.0), MeOH (20 %, v/v). A greater affinity of α-casein and BSA toward the stationary phase was indicated by the comparison with that of ConA and Tf as pH increase. Interestingly, 14 peaks of glycoisoforms of OVA were observed under borate buffer (40 mM, pH 9.0). The established method was also applied to the determination of ConA and OVA in egg-white of chicken and duck egg. The concentration of ConA in chicken egg-white is 0.72 mM.