Abstract
We present a simple analytical method for studying the physical parameters governing redox-active enzyme kinetics using microscale electrodes. With the enzyme freely diffusing in solution, the interaction with its natural substrates produces a linearly increasing current corresponding to the reaction rate and the intrinsic thermodynamic properties of the enzyme. We show that external complications, such as artificial mediators or enzyme surface immobilization, can be avoided by using an unmediated, unmodified platinum microelectrode and that control over the dominating kinetic process can be readily achieved by changing the enzyme and (co)substrate concentrations. This is achieved using the glucose oxidase (GOx)/glucose system to compare with standard practice UV-Vis techniques, including the pH dependence of the enzyme activity. We illustrate how this straightforward chronoamperometric measurement is influenced by changes to reaction conditions commonly employed in enzyme investigations, including enzyme and oxygen concentrations as well as pH and the presence of chloride. Our method emphasizes that interpreting a simple increasing slope to analyze enzyme behavior requires ensuring adherence to initial rate assumptions, empirical observation of the current-concentration relationship, and insight from using the ping-pong framework. This enables a discussion of the bounds for evoking the commonly used Michaelis-Menten rate framework as well as the existing constraints of spectroscopy, contrasted with microscale voltammetry.