The safe immobilisation of intact biomolecules plays a crucial role in the construction of enzyme electrodes. Among others the approach based on sol-gel technology seems to be promising. The protein encapsulation into a sol-gel silicate matrix enables to preserve its activity and preventing its leaching to the surrounding solution [1]. The capability to reduce dioxygen directly to water without reactive intermediates formation is one of the most important properties of laccase [2]. In recent years the successful application of electrodes modified by laccase for electrocatalytic reduction of dioxygen was reported. This system can be potentially applied in biofuel cells. Sol-gel process was successfully employed for immobilisation of laccase on the electrode surface. The efficient electron exchange between electrode and encapsulated laccase is achieved. This is because the porous hydrophilic silicate film allows fast redox mediator diffusion between immobilised enzyme and electrode. Among other factors the distribution of laccase immobilised in silicate film affects the efficiency of electrochemical devices employing this enzyme as catalyst. Therefore there is a current interest to estimate this parameter. Here we propose to employ for this purpose scanning electrochemical microscopy (SECM) [3]. This method allows to monitor distribution and activity of enzyme immobilised on flat surface [3]. In this work we present results of the investigation of laccase distribution within sol-gel processed silicate film by SECM. As film support glass plate was used, because of its flat surface, strong affinity to silicate film and lack of electric conductivity. The last property allows to eliminate positive feedback in SECM generation-collection mode experiment. This method was used in combination with confocal laser scanning microscopy. The ability of the enzyme to reduce dioxygen and to generate the oxidised form of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS^2-) was employed for imaging the studied film. The produced anion radical was detected on ultramicroelectrode positioned above laccase encapsulated in the film.

[1] S. Braun, S. Rappoport, R. Zusman, D. Avnir, M. Ottolenghi, Mater. Lett. 10 (1990) 1.
[2] E.I. Solomon, U.M. Sundaram, T.E. Machonkin, Chem. Rev. 96 (1996) 2563.
[3] G. Wittstock, M. Burchardt, S. Pust, Y. Shen, C. Zhao, Angew. Chem. Int. Ed. 46 (2007) 1584.

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