高等学校化学学报 ›› 2012, Vol. 33 ›› Issue (07): 1426-1431.doi: 10.3969/j.issn.0251-0790.2012.07.011

• 分析化学 • 上一篇    下一篇

共价固定亲和素的磁性纳米粒子用于快速测定蛋白A含量

孙术国1,2, 马美湖1, 覃芳1, 徐娅茹1, 余文珍1, 罗章1   

  1. 1. 华中农业大学食品科学技术学院国家蛋品加工技术研发分中心, 武汉 430070;
    2. 吉首大学化学化工学院, 吉首 416000
  • 收稿日期:2011-08-22 出版日期:2012-07-10 发布日期:2012-07-10
  • 通讯作者: 马美湖, 男, 博士, 教授, 博士生导师, 主要从事食品生物技术和蛋品科学研究. E-mail: mameihuhn@yahoo.com.cn E-mail:mameihuhn@yahoo.com.cn
  • 基金资助:

    国家现代农业产业技术体系建设专项资金(批准号: CARS-41-K23)资助.

Covalent Immobilization of Avidin onto Magnetic Nanoparticles and Application in Rapid Determination of Protein A

SUN Shu-Guo1,2, MA Mei-Hu1, QIN Fang1, XU Ya-Ru1, YU Wen-Zhen1, LUO Zhang1   

  1. 1. National R&D Center for Egg Processing, College of Food Science&Technology, Huazhong Agricultural University, Wuhan 430070, China;
    2. College of Chemistry and Chemical Engineering, Jishou University, Jishou 416000, China
  • Received:2011-08-22 Online:2012-07-10 Published:2012-07-10

摘要: 采用溶剂热法制备了Fe3O4磁性纳米粒子(MNPs), 以戊二醛为交联剂, 将亲和素共价固定于MNPs表面. 用透射电子显微镜(TEM)、 X射线衍射(XRD)、 紫外-可见吸收光谱(UV-Vis)、 傅里叶变换红外光谱(FTIR)和荧光光谱等手段对蛋白固定过程进行了监控和表征. 采用荧光光谱法评价了固定亲和素的磁性纳米粒子(Avi-MNPs)的活性, 并将Avi-MNPs应用于分光光度法测定蛋白A的含量. TEM结果表明, 功能化前后MNPs的粒度分布均匀, 粒径大小分别约为30和50 nm. XRD分析结果表明, MNPs与Fe3O4的特征衍射峰完全一致, 晶体纯度良好. UV-Vis, FTIR和荧光光谱结果表明, 亲和素已固定在MNPs表面. Avi-MNPs活性评价结果表明, 其结合生物素的活力为4.706 U/mg Avi-MNPs, 低于游离的亲和素活力(14.1 U/mg D-biotin). 该方法用于检测蛋白A含量比传统酶联免疫法省时、 省力, 且对检测仪器要求低.

关键词: 磁性纳米粒子, 亲和素, 共价固定, 活性评价

Abstract: Covalent immobilization of avidin onto Fe3O4 magnetic nanoparticales(MNPs) via a facile solvethermal synthetic method was carried out using glutaraldehyde as a crosslinking agent. Immobilization process was monitored and avidin immobilized magnetic nanoparticles(Avi-MNPs) were characterized by TEM, XRD, UV-Vis, FTIR and fluorescence spectroscopy. Evaluation of Avi-MNPs activity and its application for the determination of the concentration of protein A were also performed. TEM images show the average particle diameters of the non-functional MNPs and Avi-MNPs are approximately 30 and 50 nm, respectively, and all the nanoparticles have a uniform distribution. XRD analysis indicates that the characteristic diffraction peaks of MNPs are the same as Fe3O4 and MNPs are high purity. Firstly glutaraldehyde was linked to MNPs and then avidin was immobilized onto the surface of the functional MNPs, which were verified by UV-Vis, FTIR and fluorescence spectroscopy. The activity evaluation of Avi-MNPs shows that the activity is 4.706 U/mg of D-biotin bound/mg Avi-MNPs and lower than 14.1 U/mg D-biotin bound/mg free avidin. The Avi-MNPs were applied to the determination of the concentration of protein A, the results indicated that this method has an advantage of saving time and effort compared to the traditional enzyme-linked immunosorbent assay and can be carried out with a simple UV-Vis spectrophotometer.

Key words: Magnetic nanoparticales, Avidin, Covalent immobilization, Activity evaluation

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