用MicroBCA方法定量检测硅烷化表面固定的纤维连接蛋白

摘要

使用microBCA方法（micro-bicinchoninicacid protein assay）,定量研究了人纤维连接蛋白（fi-bronectin,Fn）在硅烷化石英玻璃表面实现共价固定的情况。首先对石英玻璃进行化学清洗,使表面的羟基得以充分暴露。再利用硅烷偶联剂（APTE）将氨基基团引入,并通过EDC/NHS催化体系把人纤维连接蛋白以酰胺键的形式共价固定于该表面。最后用Mi-croBCA方法对该表面固定化的生物分子（Fn）进行量化表征。应用水接触角、X射线光电子能谱（X-rayphotoelectron spectroscopy,XPS）、原子力显微镜（atomic force microscope,AFM）等方法对各步处理效果进行跟踪分析。结果表明Fn在硅烷化石英玻璃表面实现了固定,一定范围内,硅烷化表面的Fn固定量随所使用Fn孵化液浓度的增加而增大。为生物材料表面改性与修饰研究中生物大分子的量化表征提供了一种新方法。%In this paper,after being sonic cleaned by acetone,ethanol and distilled water,the quartz glass was chemically activated for obtaining hydroxyl functional groups on its surface.Silanization was then performed by incubating the activated quartz glass in 3% solution of（3-aminopropyl）-triethoxysilane（APTE）（from Sigma-Aldrich） in anhydrous alcohol for 12h at room temperature.Fibronectin（Fn） was dissolved in physiology saline water for achieving different concentrations.To immobilize Fn molecule onto the silanized quartz glass surface,the samples were respectively incubated into 5,25,50μg/mL Fn solution containing NHS/MES/EDC system for 4h at 37℃.The water contact angle measurement,X-ray photoelectron spectroscopy（XPS） and atomic force microscope（AFM） were used to investigate the characteristics of the surface after each modification procedure.Especially,the immobilized fibronectin was quantificated by the micro-bicinchoninic acid（MicroBCA） protein assay（from Pierce） method.The results indicated that,the fibronectin was successfully immobilized on the silanized quartz glass surface.The density of the immobilized Fn molecule on surface was positively correlated with the input concentration of the incubating Fn solution in a certain range.The work supplied a new assay to quantify the immobilized biomolecule on biomaterials surface.