Data for a series of fully integrated glucose oxidase, osmium redox polyelectrolyte layers deposited on thiolated gold electrodes by layer-by-layer self assembly was analysed using the relaxation and simplex method described in our earlier work (Flexer et al., 2008) [12]. The layer-by-layer assembly method allows fine control over the film thickness, enzyme loading, osmium and glucose concentrations with good reproducibility from electrode to electrode. In the analysis we combine the use of approximate analytical expressions with digital simulation to fit the data from an extensive set of experiments. The analysis shows a thickness dependence of the fraction of "wired enzyme molecules" and second order enzyme re-oxidation rate constant for thin films (below 300 nm) following changes in the multilayer film structure. For films thicker than 300 nm the kinetic data approach that of a redox hydrogel. (C) 2009 Elsevier B.V. All rights reserved.