A nanoparticulate iridium oxide (IrOx) thin film has been developed as a redox-active matrix material for an advanced generation glucose biosensor, in which IrOx serves as the non-physiological mediator, replacing oxygen in the enzymatic re-oxidation of glucose oxidase (GOx). Ethanolic solutions of Nafion and an Ir sol were mixed with an aqueous GOx solution and then deposited on a Au support. The Ir nanoparticles were then oxidized electrochemically to IrOx and the resulting films (IrOx-GOx-Nafion) were tested for their glucose response in both oxygen- and argon-saturated solutions, with the oxygen content in both solutions monitored by a Pt electrode. The sensors that are regenerated largely by O-2 are characterized by a Michaelis-Menten K-m' value of similar to 30 mM or more and i(max) values of at least 20 mu A cm(-2). Under fully deareated conditions, the sensors lose only similar to 50% of their response to glucose, clearly indicating that a dual oxygen-regeneration and IrOx mediation mechanism is operative for the biosensor under these conditions. Under optimized conditions, involving a controlled GOx:Ir ratio, only the Ir oxide sites in the film serve to mediate GOx regeneration, giving K-m', (10-15 mM) and i(max) values that are independent of the O-2 content of the solution. (C) 2010 Elsevier Ltd. All rights reserved.