Enzymatic tools serve important functions in all areas of human life. Research aimed at comparing the catalytic rates of functionally overlapping enzymes of interest (EOIs) is hampered by expensive and time-consuming growth and purification procedures. Assaying the performance of already purified EOIs requires further laborious optimization. These assays are typically colorimetric and employ a downstream signal producing enzyme such as NADH-coupled dehydrogenases, which complicate assay optimization with their own unique chemical and pH requirements. Electrocatalysts such as 2,2,6,6-tetramethylpiperidine 1-Oxyl (TEMPO) have much broader pH ranges and substrate scopes (TEMPO oxidizing aldehydes and non-tertiary alcohols) than signal enzymes and output their catalytic rates directly as current density. The field of electrochemistry is also making strides in enzyme-electrode immobilization, with several works now reporting methods to anchor enzymes onto electrode surfaces directly from cell lysate, avoiding lengthy column-purification procedures.

This work proposes a hybrid electrolytic cell in which the catalytic rate of immobilized enzyme is quantified as current density produced by TEMPO-mediated oxidation of its products. The rate of production of carbon paper electrodes has been expedited and their functional area has been standardized through novel cutting and waxing procedures, demonstrating surface area coefficient of variation values up to an order of magnitude lower and production rates an order of magnitude higher than those of traditionally produced electrodes. Variation in electrode-loading of pyrene-anchored TEMPO has also been minimized, demonstrating current density variation identical to that of electrode functional area. Current work is investigating enzyme immobilization architectures employing electrode-immobilized DNA and Zinc-finger anchored enzyme to maximize enzyme loading, as well as enzyme-electrocatalyst reactivity to minimize background signal. In summary, we aim to develop an electrochemical assay of enzyme activity able to quantify the catalytic rates of a wide range of aldehyde and non-tertiary alcohol producing EOIs while minimizing the lengthy enzyme purification and assay optimization procedures of traditional colorimetric assays.