Microspots of GOD-HRP bienzyme for scanning chemiluminescence microscopy with higher resolution

Scanning chemiluminescence microscopy (SCLM) was applied to visualize bienzyme reaction at a microspot (50 mum in radius) composed of glucose oxidase (GOD) and horseradish peroxidase (HRP). In an aqueous solution Containing glucose, a glass capillary tip was scanned over the bienzyme spot with luminol injected continuously from the tip to induce local chemiluminescence. The GOD catalyzes the oxidation of glucose by oxygen to form H2O2 which oxidizes luminol in the presence of HRP to generate chemiluminescence. Since the enzymatic luminescence reaction proceeds only within a bienzyme spot, even an array of spots was clearly imaged by SCLM without any significant interference between neighbor spots.