The immobilization of catalase, with the formation of protein deposits with maintained activity and a homogeneous distribution of active sites, is crucial for neutralizing reactive oxygen species in biological and industrial applications. Thus, mapping the spatial distribution of activity towards hydrogen peroxide decomposition is essential for validating immobilization procedures and analyzing heterogeneous activity. For imaging the activity of immobilized catalase, we propose the use of mercury deposited on platinum (Hg@Pt) and mercury-gold amalgam microelectrodes as tips for scanning electrochemical microscopy (SECM). Hg@Pt or Hg-Au amalgam microelectrodes allow for the selective determination of local concentrations of dissolved oxygen in solutions containing hydrogen peroxide. These SECM tips can also locally deliver HO via a 2-electron oxygen reduction reaction (ORR). Asymmetric loop feedback mode (FB) and substrate generation/tip collection mode (SG/TC) were applied to image samples of catalase immobilized on glutaraldehyde-modified glass slides and spots of adsorbed enzyme on polystyrene Petri dishes delivered by means of a micropipette. Hg@Pt microelectrodes obtained by electrodeposition possess inlaid disk geometry at open circuit and become slightly recessed convex planes upon cathodic polarization. SG/TC SECM imaging results indicate that the decomposition rate of HO at micrometer-sized spots of adsorbed catalase is predominantly diffusion-limited. The proposed immobilization and activity imaging methods can be applied to samples with low surface concentrations of immobilized enzymes.
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