Single-Entity Protein Electrochemistry of Diffusion-Limited Enzymes

Single-entity electrochemistry has recently emerged as a promising method for label-free exploration of the catalytic functions of individual enzymes. However, skepticism within the scientific community regarding the applicability of the method for single enzyme measurements has arisen due to issues in the experimental data presented in the literature and limited theoretical modeling of such data. Here, we address these concerns through a thorough experimental investigation of two diffusion-limited enzymes, catalase and superoxide dismutase, employing a combination of protein film voltammetry and single-entity protein electrochemistry measurements. We then introduce a novel theoretical model for simulating the current responses, generated by the reduction of the product of the enzymatic reaction of single enzyme molecules at the electrode. This model is based on a combination of finite element simulations using COMSOL Multiphysics and random walk simulations. It incorporates the diffusion-limited enzymatic kinetics of the investigated enzymes and introduces a geometry that mimics the substrate diffusion channel of the enzyme. Our work demonstrates that the experimentally detected current signals align with the simulated current signals, affirming that they can be attributed to the catalytic activity of single enzymes detected via the product of the enzymatic reaction.