Copolymer of vinylferrocene, acrylamide and 2-(diethylamino)ethyl methacrylate was synthesized by free-radical polymerization. Proton nuclear magnetic resonance and inductively coupled plasma-optical emission spectroscopy confirmed successful incorporation of ferrocene moieties in the copolymer. The effect of monomer feed ratio on copolymer molecular weight was studied and it was found that the increase in 2-(diethylamino)ethyl methacrylate fraction in the monomer feed facilitated incorporation of vinylferrocene and led to decrease in copolymer molecular weight. The synthetic process was scaled up in a pilot plant and an ultrafiltration method was developed to reduce residual levels of each monomer below 0.1% w/w. The copolymer displayed reversible redox electrochemical characteristics of the ferrocene moieties and mediation of glucose oxidation with enzyme flavin adenine dinucleotide-glucose dehydrogenase in phosphate buffered saline. A diffusion coefficient of 3.36 x 10(-7) cm(2)/s for the dissolved polymeric mediator was estimated from Randles-Sevcik analysis. The copolymer and the enzyme were mixed with water-based carbon ink and then screen-printed to form working electrodes. These biosensors established equilibrium current responses in less than 3 min following step changes of glucose concentration within the clinically relevant range of 8-735 mg/dL when exposed to a continuous flow of blood at 15 mu L/min. The calibration plot of three biosensors aligned closely to a quadratic regression line (R square 0.9909) suggesting reasonable sensor measurement reproducibility and low levels of ferrocene leaching out of the polymeric mediator during the 7.5 h test period. A Michaelis constant for the sensor was estimated at 655 mg/dL. (C) 2010 Elsevier Ltd. All rights reserved.