In this study, hydrogel microparticles containing enzyme-linked magnetic nanoparticles (MNPs) were prepared. Peroxidase (POD), a model enzyme, was covalently immobilized on the surface of MNPs using 3-aminopropyltriethoxysilane (APTES), and the resultant POD-linked MNPs were entrapped within various shapes of hydrogel microparticles using photopatterning. Pre-immobilizing POD on the MNP surface made it possible to use hydrogels prepared from high molecular weight PEG without enzyme leaching, which enhanced the reaction rate of entrapped enzymes by reducing resistance to mass transport. Quantitative assays showed a linear correspondence between fluorescence intensity and H(2)O(2) concentrations below 15 mM. (C) 2009 Elsevier Ltd. All rights reserved.