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.