GENERATION OF BIOTIN AVIDIN/ENZYME NANOSTRUCTURES WITH MASKLESS PHOTOLITHOGRAPHY/

Micrometer-sized domains of a carbon surface are modified to allow der ivatization to attach redox enzymes with biotin/avidin technology. The se sites are spatially segregated from and directly adjacent to electr on transfer sites on the same electrode surface. The distance between these electron transfer sites and enzyme-loaded domains must be kept t o a minimum (e.g., less than 5 mu m) to maintain the fast response tim e and high sensitivity required for the measurement of neurotransmitte r dynamics. This is accomplished through the use of photolithographic attachment of photobiotin using an interference pattern from a UV lase r generated at the electrode surface. This will allow the construction of microscopic arrays of active enzyme sites on a carbon fiber substr ate while leaving other sites underivatized to facilitate electron tra nsfer reactions of redox mediators, thus maximizing enzyme activity an d detection of the enzyme mediator. The ultimate sensitivity of these sensors will be realized only through careful characterization of the carbon electrode surface with respect to its chemical structure and el ectron transfer properties following each step of the enzyme immobiliz ation process. The characterization of specific modifications of micro meter regions of the carbon surface requires analytical methodology th at has both high spatial resolution and sensitivity. We have used fluo rescence microscopy with a cooled CCD imaging system to visualize the spatial distribution of enzyme immobilization sites (indicated by fluo rescence from Texas Red-labeled avidin) across the carbon surface. The viability of the enzyme attached to the surface in this manner was de monstrated by imaging the distribution of an insoluble, fluorescent pr oduct, An atomic force microscope was used to obtain high-resolution i mages that probe the heterogeneity of the enzyme sites.