Fabricating and imaging carbon-fiber immobilized enzyme ultramicroelectrodes with scanning electrochemical microscopy

The scanning electrochemical microscope (SECM) is used to image the activit y of enzymes immobilized on the surfaces of disk-shaped carbon-fiber electr odes. SECM was used to map the concentration of enzymatically produced hydr oquinone or hydrogen peroxide at the surface of a 33-mum diameter disk-shap ed carbon-fiber electrode modified by an immobilized glucose-oxidase layer. Sub-monolayer coverage of the enzyme at the electrode surface could be det ected with micrometer resolution. The SECM was also employed as a surface m odification tool to produce microscopic regions of enzyme activity by using a variety of methods. One method is a gold-masking process in which micros copic gold patterns act as mask for producing patterns of chemical modifica tion. The gold masks allow operation in both a positive or negative process for patterning enzyme activity. A second method uses the direct mode of th e SECM to produce covalently attached amine groups on the carbon surface. T he amine groups are anchors for attachment of glucose oxidase by use of a b iotin/avidin process. The effect of non-uniform enzyme activity was investi gated by using the SECM tip to temporarily damage an immobilized enzyme sur face. SECM imaging can observe the spatial extent and time-course of the en zyme recovery process.