Organic-inorganic Hybrid Hollow Spheres Applied In Biologic Affinity Separation

In this paper, the hollow silica spheres with micropores and macropores on theshell were prepared and the characteristic of spheres was discussed. Afterfunctionalized, the hollow silica spheres could be used as column materials of affinityseparation to purify trypsin after coupling with ovomucoid.We use template replication and sol-gel method together to prepare the hollowsilica spheres. In this paper, the polystyrene (PS) particle was used as rigid template,the surfactant was used as soft template, and the PS particle was coated with sol-gel,after calcining the double templates in the air, the hollow silica sphere withmicropores and macropores on the shell could be prepared. The form and size ofhollow spheres are related to the template. At 70℃, the uniform PS particles wereprepared with the ratio of reaction—100mL ethanol: 20mL styrene: 4.0g PVP: 0.1gAIBN. The gelation time is correlative with the ratio of reaction and the kind andquantity of catalyst. The thickness of shell is about 35-50nm confirmed from TEMand it is relation to the mass ratio of TEOS and PS. When the ratio is 2/3, the hollowsilica spheres have well shape and are dispersive. Many uniform mesopores on theshell (the adsorption average diameter 3.2nm accounted by BJH) and a small fractionof shell cracked can be observed from SEM and TEM. The BET surface area and thetotal pore volume is about 235m~2/g, 0.1533cm~3/g.In order to immobilize more protein on the shell of hollow silica sphere, thesphere was functionalized by the way of chemical modification. First, introduction ofamino groups as an initiator site on the silica surface was achieved by the treatment ofthe silica withγ-aminopropyltriethoxysilane. Polyamidoamine dendrimer waspropagated from silica surface by repeating two processes: (1) Michael addition ofmethyl acrylate to surface amino groups, and (2) amidation of the resulting esters withethylenediamine. The amino group was confirmed by FT-IR and the amino content ofthe resulting hollow silica sphere increased to 0.56mmol/g. The functional hollow silica spheres use 5％concentration of glutaraldehyde crosslinked with ovomucoid.The crosslinking ratio was 9.35％measured by ultraviolet spectrometer at 280nm.The hollow silica spheres still keep better shape after coupling with ovomucoid whichis observed from TEM. And form AFM we can see that the surface of spheres changeroughly. The small particles on the surface of spheres may be ovomucoid. Theeffluent solution was collected once per 10min and the specific activity of purifiedenzyme was measured by UV spectrometer. The specific activity of purified enzymeachieved 9456.29 (U/rag). It was 8.7 times the activity of original enzyme. And72.98％total activity of enzyme was recovered. The aim of affinity separating trypsinwas achieved basically.