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Polyurethane grafted calcium alginate hydrogel microspheres intended for bovine serum albumin imprinting were prepared and characterized. The grafting modification was performed prior to the assembling of protein template with hydrogel, which was advantageous to maintain protein conformation and construct more accessible imprinted sites. It was found that the imprinted hydrogel had higher affinity to template molecules after modification. Investigations of grafting ratio influence on specific rebinding and competitive properties indicated that grafted side chains were beneficial to the selective recognition of protein. [ABSTRACT FROM AUTHOR]