This article describes the preparation of tosyl-activated nonmagnetic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [P(HEMA-GMA)] microspheres by dispersion polymerization and tosyl-activated magnetic poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) [P(HEMA-EDMA)] microspheres by multistep swelling polymerization method and precipitation of iron oxide inside the pores. These new approaches show that monodisperse microspheres, 2.3 mu m, respectively 4.1 mu m, in diameter can be produced in high yields avoiding aggregation and with the advantage of being free of aromatic moieties. To demonstrate their potential for diagnostic applications, both types of microparticles have been coated with capture and detection antibodies (DAs), respectively. Immunoassay protocols have then been developed for the dosage of ferritin using an automated affinity platform combining microchannel chips and electrochemical detection. The assay performance using the above magnetic microspheres has been compared with that obtained with commercial tosyl-activated beads. Finally, the possibility to combine functionalized magnetic and nonmagnetic microspheres has been evaluated in view of amplifying the number of enzymatic labels in the immuno-complex. At a ferritin concentration of 119.6 ng/mL, a signal-to-noise ratio of 150.5 is obtained using 0.2 mg/mL of anti-ferritin-coated P(HEMA-GMA)-DA microspheres against a value of 158.8 using free DA in solution. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 532542, 2013