Electron transfer kinetics at a biotin/avidin patterned glassy carbon electrode

Photolithographic techniques using a laser interference pattern were used t o attach photobiotin to micron-sized stripes on the surface of a carbon ele ctrode. Fluorophore-tagged avidin was attached to this spatially-patterned biotin with essentially no loss in spatial resolution. The kinetics of the glassy carbon surface were examined to see if electron transfer sites could indeed be segregated from the attachment sites of photobiotin-immobilized avidin. The ECL of luminol and SECM were used to verify the segregation bet ween underivatized sites (which exhibit normal electron transfer kinetics) and extensively derivatized biotin/avidin surfaces (which presumably exhibi t slow electron transfer kinetics). Both techniques were found to be capabl e of differentiating the protein-covered surface fi om bare carbon with suf ficient resolution to tell whether a significant portion of the carbon surf ace is still active and available to detect the product of an enzyme genera ted analyte. These results indicate that extensive biotin/avidin derivatiza tion of the surface does decrease the electron transfer rate of a carbon el ectrode, and that the photolithographic approach was able to modify specifi c sections of the electrode surface, while leaving other regions untouched and available for facile electron transfer. This leads to a more general pr otocol for the construction of enzyme-based biosensors which utilize diffus able mediators. (C) 1998 Elsevier Science S.A. All rights reserved.