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

摘要

Thin silicate films with immobilised enzymes catalysing dioxygen reduction, i.e. laccase and bilirubinnoxidase (BOD), were deposited on glass and poly(methyl 2-methylpropenoate) (Plexiglasu0002) surfaces inna sol–gel process by sol drop evaporation. Scanning electrochemical microscopy (SECM) images andnapproach curves were recorded using hexacyanoferrate(III) as mediator in the feedback mode. Confocalnlaser scanning microscopy (CLSM) images in the reflection mode showed larger film thickness close tonthe edge of the film and laccase aggregates within the film. SECM images obtained using differentndioxygen concentrations showed that the film edge and laccase aggregates exhibit higher enzymaticnactivity towards dioxygen reduction. SECM current–distance curves enabled the determination ofnkinetic information at the particular regions of the samples after numerical fitting of model parameters.nThe heterogeneous first order rate constant at the film border was estimated to be ca. 19 times highernthan the value obtained when approaching to the centre of the film. The reason of higher laccase surfacenconcentration at the film edge is carefully discussed. For comparison of laccase and BOD activities,nsilicate spots of 50 mm diameter were deposited on a single Plexiglasu0002 sample and examined usingnSECM. BOD exhibits much higher activity especially at neutral pH.