Amperometric biosensors for hydrogen peroxide detection have been constructed using horseradish peroxidase (HRP) and two newly purified peroxidases extracted from tobacco (TOP) and sweet potato (SPP). The peroxidases were cross-linked to a redox polymer [poly(vinylimidazole) complexed with Os(4,4'dimethylbipyridine)(2)Cl] using poly(ethylene glycol) diglycidyl ether as the cross-linker. A comparative study with regard to their bioelectrochemical characteristics showed that, irrespective of peroxidase. the biosensors sensitivity was strongly influenced by hydrogel composition, curing procedure, film thickness and applied potential. The electrostatic interaction between the cationic redox polymer and the negatively charged peroxidases (TOP and SPP) enhanced the hydrogen peroxide signal. When operated in a Fl system, the optimized SPP biosensor (48% odor polymer, 23% cross-linker and 29% enzyme, w/w %) displayed the highest sensitivity for H2O2 (3.2 A M(-1)cm(-2)), a linear range up to 220 muM, a detection limit of 25 nM (calculated as 2S/N) and a response time of about 2 min. (C) 2000 Elsevier Science Ltd. All rights reserved.