Formation and imaging of microscopic enzymatically active spots on an alkanethiolate-covered gold electrode by scanning electrochemical microscopy

Microscopic, enzymatically active spots on self-assembled monolayers (SAMs) of alkanethiolates on gold were obtained by a combination of localized desorption induced using the scanning electrochemical microscope (SECM) followed by chemical derivatization. Starting from a SAM of dodecanethiolate on gold, localized desorption of alkanethiolates creates microscopic areas of an uncovered gold surface surrounded by a dense Au alkanethiolate layer. The renewed gold surface chemisorbs an amino-derivatized disulfide (cystaminium dihydrochloride) in a second step. Periodate-oxidized glucose oxidase was attached covalently to the terminal amino functions to create a stable, catalytically active pattern of the enzyme on the alkanethiolate SAM. The enzymatic activity was mapped using the imaging capabilities of SECM, The generator-collector mode (amperometric H2O2 detection) was advantageously used, as the feedback mode leads to interferences due to concurrence between mediator regeneration by the enzymatic reaction and by the heterogeneous electron transfer at the gold regions from which the blocking dodecanethiolate layer had been desorbed. Rising backgrounds due to H2O2 accumulation in the bulk solution can be prevented by adding minute amounts of the enzyme catalase to the working solution. By catalyzing the H2O2 decomposition, the lifetime of H2O2 is adjusted to prevent its accumulation in the bulk phase yet to allow its diffusion across the gap between the enzyme-modified region and the collecting electrode. Perspectives for creating miniaturized multienzyme structures, which will become accessible by repeating the desorption and covalent enzyme immobilization steps using different enzymes in each cycle, are highlighted.