A novel biotinylated and enzyme-immobilized nanobio device was prepared with heterobifunctional composite latex particles. Hemispherical poly(glycidyl methacrylate-co-divinylbenzene)/polystyrene [P(GMA-DVB)/PSt] particles with epoxy and hydroxyl groups were prepared by soap-free seeded emulsion polymerization with P(GMA-DVB) seed particles. Hydroxyl groups were introduced to PSt chain terminals in the seeded stage by adding 2-mercaptoethanol as a chain-transfer agent. To obtain the desired hemispherical structure particles, we studied the effects of the preswelling process, the type and amount of solvents added in the seeded polymerization step, the weight ratio of the secondary monomer (styrene) to the seed particle (M/P), and the type of initiators. Under suitable conditions, heterobifunctional P(GMA-DVB)/PSt was obtained, which was confirmed by observing the binding of streptavidin-colloidal gold with transmission electron microscopy (TEM). To obtain biotinylated and enzyme-immobilized particles, 5-(N-succinimidyloxycarbonyl)pentyl D-biotinamide was first reacted with the hydroxyl group on the PSt domain of the particle. Pyruvate kinase (PK) was then directly immobilized to the biotinylated particles through a reaction with the epoxy group in the PGMA domain. The monolayer of PK on the latex particle surface was considered to be formed by covalent binding. The activity of immobilized PK was almost conserved, even after being stored at 4 degreesC for 48 days. (C) 2004 Wiley Periodicals, Inc.