Towards the development of multianalyte electrochemical immunoassays three individually addressable microelectrode array (MEA) type working electrodes and a reference electrode were integrated into a 4 muL volume, planar electrochemical cell. To model the simultaneous determination of multiple antigens in the cell with enzyme linked immunosorbent assays (ELISAs) glucose oxidase (GOx), alkaline phosphatase (ALP), and beta-galactosidase (beta-GAL) were immobilized site specifically onto the individual MEA surfaces and the biocatalitic activity of these surface confined enzymes were evaluated by measuring the products of the enzyme catalyzed reactions directly on the gold MEA surfaces by chronoamperometry or by imaging the enzyme patterned microelectrode array surfaces by Scanning Electrochemical Microscopy (SECM). ALP and beta-GAL were selected as model enzymes because they are the most commonly used enzymes labels in ELISAs. In these measurements glucose, ascorbic acid phosphate (AAP), and p-aminophenyl-beta-D-galactopyranoside (PAPG) served as enzyme substrates, respectively. The electrochemical surface area of the gold MEAs did not change during the multistep immobilization process. All enzyme modified MEAs presented selective and proportional responses to their substrates and the response characteristics of the enzyme modified sensors were identical in separate and simultaneous calibration protocols, i.e., there was no cross-contamination between the closely placed MEAs. The SECM images of the enzyme patterned MEA surfaces suggest that nonspecific adsorption is negligible on the insulating polyimide surface of the MEA separating the individual microelectrode sites.
Liu, Y., R. E. Gyurcsányi, G. Jágerszki, J. D. DeNuzzio, E. Lindner