Enzyme Dynamics in Attoliter-Volume Electrochemical Zero-Mode Waveguides with On-Demand In-Situ Hydrogen Peroxide Delivery and Consumption

Physiological systems are not at equilibrium and undergo time-dependent fluctuations, making it challenging to relate in vitro studies to in vivo biomolecular behavior. To bridge this gap, enzyme dynamics can be studied in the presence of controlled perturbations that recapitulate the intracellular environment. Here, we report an approach to the study of reactive oxygen species (ROS) based on the in situ manipulation of hydrogen peroxide (H₂O₂) levels in functionalized nanopore-based electrochemical zero-mode waveguide (EZMW) arrays, with each nanopore presenting small numbers of immobilized horseradish peroxidase (HRP) enzyme molecules. H₂O₂ is generated or consumed within the attoliter volume of the EZMW nanopores by poising an embedded ring electrode to suitable potentials, and the resulting effect on apparent turnover of HRP under non-equilibrium conditions is monitored using the enzymatically accelerated conversion of the non-fluorescent probe Amplex Red to fluorescent resorufin. A Nafion membrane is placed on the top surface of the EZMW array, providing a cation permselective barrier to transport in, or out, of the EZMW nanopores, thereby improving the sensitivity of the experiment by sequestering enzymatically generated resorufin in the attoliter volume of the EZMW nanopores. By fabricating arrays presenting 441 individual reaction volumes in parallel, distinct changes in population dynamics in the presence of in situ H₂O₂ generation or consumption are characterized with respect to temporal evolution and magnitude of the H₂O₂ aliquot delivered. This approach presents a promising avenue for studying biomolecular reactions in spatiotemporally controlled chemical environments that can mimic the non-equilibrium conditions encountered in vivo.