Preparation and structural regulation of macroporous agarose microspheres for highly efficient adsorption of giant biomolecules

Macroporous agarose microspheres for bioseparation of giant biomolecules were prepared by a surfactant micellar swelling method, and the effects of preparation conditions on both particle size and its distribution and pore structure were systematically studied. Under the optimal condition with a surfactant amount of 20% (v/v), Triton X-100 to sodium dodecyl sulfate ratio (SDS) of 5:1 (w/w), an oil-absorbing-swelling time of 2.5 h, and a stirring speed of 250 rpm, macroporous agarose microspheres were successfully prepared, characterized by inverse size exclusion chromatography. Distribution coefficient (Kd) of dextran probes for agarose microspheres after being cross-linked was almost the same as that for agarose microspheres before being cross-linked. Inverse size exclusion chromatography results showed that macroporous agarose microspheres had a pore size distribution of more than 40 nm or even close to 60 nm, while that of conventional microspheres was no more than 40 nm. Macroporous agarose microspheres functionalized with diethylaminoethyl groups had a higher adsorption capacity and faster adsorption rate for hepatitis B surface antigen (HBsAg) compared with both commercial DEAE-agarose chromatographic media and conventional DEAE-agarose microspheres which were both based on agarose microspheres prepared using a traditional method. Confocal laser scanning microscopy results showed that macroporous agarose microspheres had a good binding ability to HBsAg, and this antigen completely entered the inside of the microspheres. Compared with conventional agarose chromatographic media, the macroporous agarose media combined macromolecular proteins more quickly and fully, having wider applications in the field of purification of giant biomolecules.