Microelectrode control of surface-bound enzymatic activity

Microelectrodes have been used to control the microscopically local reaction environment of surface-bound alcohol dehydrogenase (ADH). A surface comprised of agarose beads coated with immobilized ADH was prepared on a microscope slide and exhibited maximum activity toward the oxidation of ethanol, in the presence of nicotinamide adenine dinucleotide (NAD(+)), at a pH of 9.0. Microelectrode control of activity was achieved by submerging the slide in a solution of pH 6.0, well below the optimum value, and generating hydroxide at the microelectrode tip through the reduction of oxygen or water. An alkaline "sphere of influence" was set up around the microelectrode tip that, when positioned in close proximity to the enzyme surface, created a favorable reaction environment. The increased enzymatic activity was monitored by observing fluorescence of the reduced cofactor, NADH, using a fluorescence microscope equipped with an imaging camera. The fluorescent sphere diameter was characterized as a function of time, potential, and solution buffer strength. Optimum spatial resolution for enzymatic control was 7-12 μm.