Biofunctionalization of silica microspheres for protein separation

Mercapto-silica (SiO2–SH) microspheres were prepared via direct hydrolysis of 3-mercaptopropyltrimethoxysilane (MPS) in a basic aqueous solution. The content of surface thiol group (-SH) of SiO2–SH microspheres was measured by Ellman's reagent method and X-ray photoelectron spectroscopy (XPS) and the content of surface thiol group of SiO2–SH microspheres is strongly dependent on the reaction conditions. The thermal stability of SiO2–SH microspheres was evaluated by thermogravimetric (TG) analysis, which tended to reduce with the increase of content of surface thiol groups. SiO2–SH microspheres can be easily modified with reduced glutathione (GSH) to generate SiO2–GSH microspheres for the affinity separation of Glutathione S-transferase (GST). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to examine the validity of the separation procedure. The results showed that SiO2–GSH microspheres were efficient in GST affinity separation from mixed proteins. - Graphical abstract: The prepared SiO2–SH microsphere binding reduced glutathione (SiO2–GSH) as affinity precipitation support can capture selectively Glutathione S-transferase (GST) from mixed protein solution. Highlights: ► SiO2–SH microspheres were prepared in water using one-pot synthesis. ► The content of surface -SH was investigated by Ellman method and XPS spectra. ► The ratio of -SH to mass strongly depends on the reaction conditions. ► SiO2–SH microspheres were biofunctionalized by glutathione. ► SiO2–GSH can be used to capture selectively Glutathione S-transferase