Feedback mode SECM study of laccase and bilirubin oxidase immobilised in a sol-gel processed silicate film.

Thin silicate films with immobilised enzymes catalysing dioxygen reduction, i.e. laccase and bilirubin oxidase (BOD), were deposited on glass and poly(methyl 2-methylpropenoate) (Plexiglas) surfaces in a sol-gel process by sol drop evaporation. Scanning electrochemical microscopy (SECM) images and approach curves were recorded using hexacyanoferrate(iii) as mediator in the feedback mode. Confocal laser scanning microscopy (CLSM) images in the reflection mode showed larger film thickness close to the edge of the film and laccase aggregates within the film. SECM images obtained using different dioxygen concentrations showed that the film edge and laccase aggregates exhibit higher enzymatic activity towards dioxygen reduction. SECM current-distance curves enabled the determination of kinetic information at the particular regions of the samples after numerical fitting of model parameters. The heterogeneous first order rate constant at the film border was estimated to be ca. 19 times higher than the value obtained when approaching to the centre of the film. The reason of higher laccase surface concentration at the film edge is carefully discussed. For comparison of laccase and BOD activities, silicate spots of 50 microm diameter were deposited on a single Plexiglas sample and examined using SECM. BOD exhibits much higher activity especially at neutral pH.