Development of sub-micron patterned carbon electrodes for immunoassays.

Sub-micron sized domains of a carbon surface are derivatized with antibodies using biotin/avidin technology. These sites are spatially-segregated from, and directly adjacent to, electron transfer sites on the same electrode surface. The distance between these electron transfer sites and enzyme-loaded domains are kept to a minimum (e.g. less than a micron) to maintain the high sensitivity required for the measurement of enzyme-linked cofactors in an enzyme-linked immunoassay (ELISA). This is accomplished through the use of photolithographic attachment of photobiotin using an interference pattern from a UV laser generated at the electrode surface. This allows the construction of microscopic arrays of active ELISA sites on a carbon substrate while leaving other sites underivatized to facilitate electron transfer reactions of redox mediators; thus maximizing sensitivity and detection of the enzyme mediator. The carbon electrode surface is characterized with respect to its chemical structure and electron transfer properties following each step of the antibody immobilization process. The characterization of specific modifications of micron regions of the carbon surface requires analytical methodology that has both high spatial resolution and sensitivity. We have used fluorescence microscopy with a cooled CCD imaging system to visualize the spatial distribution of enzyme immobilization sites (indicated by fluorescence from Texas-Red labeled antibody) across the carbon surface. The viability of the enzyme attached to the surface in this manner was demonstrated by imaging the distribution of an insoluble, fluorescent product.