生物功能化纳米SiO_2微球的构建及对谷胱甘肽S-转移酶的捕获分离

Construction of Bio-functional Nanosized Silica Microspheres for Capture and Separation of Glutathione S-Transferase

采用巯基丙基三甲氧基硅烷(MPS)一步水解法制备了表面带有巯基(—SH)的纳米SiO2微球(nSiO2-SH),探讨了水/醇体积比、反应温度、MPS初始浓度及反应时间对nSiO2-SH微球形貌的影响,并分析了反应机理.制备的nSiO2-SH微球进一步与还原型谷胱甘肽(GSH)中的—SH反应生成双硫键(—S—S—),在微球表面键合上GSH分子,得到了生物功能化的纳米nSiO2-GSH微球.通过扫描电子显微(SEM)、透射电子显微镜(TEM)、傅里叶变换红外光谱(FTIR)和热重分析仪(TG)对样品的表面形貌、尺寸和组成等进行了表征.利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)检测样品对谷胱甘肽S-转移酶(GST)的分离效果,结果表明,nSiO2-GSH微球能从混合蛋白中特异性吸附GST,达到了分离GST的目的.

Nanosized silica microspheres with sulfydryl group（nSiO2-SH） were synthesized with（3-mercaptopropyl）trimethoxysilane（MPS） via one-step hydrolyzing process,and the effects of volume ratio of water to ethanol,reaction temperature,reaction time and the volume of MPS on the product morphologies were discussed.Additionally,the possible reaction mechanism was also proposed.The prepared nSiO2-SH microspheres reacted further with glutathione through disulfide bond（—S—S—） to produce nSiO2-GSH microspheres.Their morphology,size and ingredient were characterized by scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,Fourier transform infrared spectrometry（FTIR） and thermogravimetric（TG） analysis.The behavior of capture and separation of glutathione S-transferase（GST） was detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis（SDS-PAGE） method.The results show that GST can be specifically adsorbed on nSiO2-GSH microspheres in the mixed protein solution.Therefore,nSiO2-GSH microspheres have potential applications in the separation,purification and detection of the GST-tagged fusion proteins.