The formation of nanofibrillar polyaniline-polyvinyl sulfonate (Pani-PVS) composite by electropolymerization of aniline in the presence of ferrocenium hexafluorophophate (FcPF(6)) and its application in mediated-enzyme biosensor using the horseradish peroxidase/hydrogen peroxide (HRP/H2O2) enzyme-substrate system is reported. The electropolymerization was carried out at glassy carbon electrodes (GCE) and screen printed carbon electrodes (SPCE) in a strongly acidic medium (HCl). Scanning electron microscopy (SEM) images showed that 100 nm diameter nanofibrils were formed on the SPCE in contrast to the 800-1000 nm cauliflower-shaped clusters which were formed in the absence of FcPF6. A model biosensor (GCE//Pani-PVS/BSA/HRP/Glu), consisting of horseradish peroxidase (HRP) immobilized by drop coating atop the GCE//Pani-PVS in the presence of bovine serum albumin (BSA) and glutaraldehyde (glu) in the enzyme layer casting solution, exhibited voltammetric responses characteristic of a mediated-enzyme system. The biosensor response to H2O2 was very fast (5 s) and it exhibited a detection limit of 30 mu M (3 sigma) and a linearity of up to 2 mM (R-2 = 0.998). The relatively high apparent Michaelis-Menten constant value (K-M(app) = 1.7 mM) of the sensor indicated that the immobilized enzyme was in a biocompatible microenvironment. The freshly prepared biosensor was successfully applied in the determination of the H2O2 content of a commercial tooth whitening gel with a very good recovery rate (97%). (C) 2009 Elsevier Ltd. All rights reserved.