Imaging localized horseradish peroxidase on a glass surface with scanning electrochemical/ chemiluminescence microscopy.

Scanning electrochemical/chemiluminescence microscopy (SECM/SCLM) was developed for simultaneous imaging of chemiluminescence and topography of immobilized horseradish peroxidase (HRP) on a glass substrate. When the SECM tip for electrochemical generation of H2O2 scanned over the immobilized spot in an aqueous luminol solution, the immobilized enzymes catalyzed the oxidation of luminol to emit chemiluminescence, which was detected by a photon counter. The photon-counting intensity was plotted against the position of the tip to give a two-dimensional image, indicating the activity of immobilized enzymes. Since the current was sensitive to the topography of substrate, the topography image was also obtained by mapping the oxygen reduction current.