基于pH响应涂层柱的毛细管电泳法在线富集和分离糖蛋白  

On-line enrichment and separation of glycoprotein by capillaryelectrophoresis based on pH response coating column

作　　者：张剑 徐彩凤 马彩莲 贺茂芳 张博[1] 刘春叶[1] ZHANG Jian;XU Caifeng;MA Cailian;HE Maofang;ZHANG Bo;LIU Chunye(School of Pharmacy,Xi’an Medical University,Xi’an 710021,China)

机构地区：[1]西安医学院药学院,陕西西安710021

出　　处：《色谱》2024年第9期903-908,共6页Chinese Journal of Chromatography

基　　金：陕西省科学技术厅社会发展项目(2023-YBSF-202);西安医学院大学生开放实验科研基金项目(2021DXS50,2021DXS68);西安医学院秦创原·科技成果产业化培育项目(21CYH01);西安医学院校内人才项目(2018XNRC03);西安医学院科研能力提升计划项目(2022NLTS063,2022NLTS069).

摘　　要：本研究通过静电自组装法制备了一种3-氨基苯硼酸(APBA)修饰的纳米金(AuNPs@APBA)涂层毛细管柱,该毛细管柱兼具抗非特异性吸附性和富集选择性,将其作为分离通道,建立了基于pH响应涂层柱的糖蛋白在线富集与分离方法。首先,采用柠檬酸钠还原法制备金纳米颗粒(AuNPs),再将APBA通过静电自组装修饰到AuNPs表面,制得AuNPs@APBA纳米材料;利用离子吸附作用将AuNPs@APBA吸附到毛细管内壁上,制得AuNPs@APBA涂层毛细管柱。在pH为8的条件下,糖蛋白能够与毛细管内表面的硼酸基团结合形成硼酸酯,从而被吸附;当pH为3时,硼酸酯发生解离并释放出糖蛋白,基于上述原理,该方法能够实现糖蛋白的选择性在线富集与分离。实验结果表明,与使用普通电泳法的裸柱相比,经AuNPs@APBA涂层毛细管柱富集后,卵清蛋白(OVA)的峰面积增大了2646倍,而牛血清白蛋白(BSA)的峰面积仅增大了847倍;并且,将鸡蛋清样品稀释1×10^(6)倍后,仍可利用该方法对糖蛋白进行检测。该方法样品用量少,操作简单,分离效率高,可用于实际样品中痕量糖蛋白的在线富集与分离。A capillary column coated with 3-aminophenylboronic acid(APBA)-functionalized gold nanoparticles(AuNPs@APBA)was prepared via electrostatic self-assembly.The coated column exhibited anti-nonspecific adsorption of glycoproteins,enabling selective online enrich-ment during capillary electrophoresis(CE).First,gold nanoparticles(AuNPs)were synthe-sized using the sodium citrate reduction method.Then,APBA was self-assembled electrostati-cally on the surface of the AuNPs to obtain [e-mail] nanomaterial was bonded to the inner wall of a capillary through ion adsorption to produce a AuNPs@APBA-coated capillary column.Glycoproteins were adsorbed via bond formation with boric acid groups under alkaline conditions(pH 8)to generate borate esters.Under acidic conditions(pH 3),the borate esters dissociated to release the glycoproteins,thereby achieving the selective online enrichment and separation of glycoproteins.The AuNPs and AuNPs@APBA were characterized using Fourier transform infrared spectroscopy,and their sizes and Zeta potentials were determined.In addition,the electroosmotic flow(EOF)of the AuNPs@APBA-coated capillary column was measured.The results showed that the surface of the AuNPs was successfully modified with APBA and that AuNPs@APBA was adsorbed on the inner wall of the capillary.The peak area of ovalbumin(OVA)on the AuNPs@APBA-coated column was 2646 times higher than that on a bare column via conventional electrophoresis.In contrast,the peak area of bovine serum albu-min(BSA)only increased by 847 times,indicating that the AuNPs@APBA coated column se-lectively enriched glycoproteins.Evaluation of the reproducibility and stability of this method revealed that the AuNPs@APBA coated capillary column could be used continually for 33-67 h.The relative standard deviations(RSDs)of the peak areas for intra-day(n=5)and inter-day(n=6)analyses were 22%and 30%,respectively.The developed method was successfully applied to enrich glycoproteins in a 1×10^(6)-fold diluted egg white sample.Glycoproteins were not detecte

关 键 词：毛细管电泳 在线富集 离子吸附 糖蛋白 苯硼酸 

分 类 号：O658[理学—分析化学]