Development of sub-micron patterned carbon electrodes for immunoassays

Sub-micron sized domains of a carbon surface are derivatized with antibodie s using biotin/avidin technology. These sites are spatially-segregated from , and directly adjacent to, electron transfer sites on the same electrode s urface. The distance between these electron transfer sites and enzyme-loade d domains are kept to a minimum (e.g. less than a micron) to maintain the h igh 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 patter n from a UV laser generated at the electrode surface. This allows the const ruction of microscopic arrays of active ELISA sites on a carbon substrate w hile leaving other sites underivatized to facilitate electron transfer reac tions 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 specif ic modifications of micron regions of the carbon surface requires analytica l methodology that has both high spatial resolution and sensitivity. We hav e used fluorescence microscopy with a cooled CCD imaging system to visualiz e the spatial distribution of enzyme immobilization sites (indicated by flu orescence from Texas-Red labeled antibody) across the carbon surface. The v iability of the enzyme attached to the surface in this manner was demonstra ted by imaging the distribution of an insoluble, fluorescent product. (C) 1 999 Elsevier Science B.V. All rights reserved.