Detection of CO₂ Locally Generated by Formate Dehydrogenase Using Carbonate Ion-Selective Micropipette Electrodes

Many technologies involve immobilizing catalysts such as enzymes on surfaces, and the catalytic activities or functional efficiencies of these surface-bound catalysts can vary depending on orientations, localized binding sites, active sites, and intrinsic molecular nature. Accurate and rapid quantification of reaction products from surface-immobilized catalysts is crucial for understanding the selectivity, mechanisms, and reaction dynamics of catalytic systems and for revealing heterogeneous catalytic activities and reaction sites for applications such as biosensors and energy conversion/generation systems. Here, we demonstrate the feasibility of localized enzymatic activity measurements using microscale carbon dioxide (CO₂)-sensitive ion-selective electrode (ISE) pipettes (0.5-2.5 μm tip radius) as a probe, with in situ potentiometric scanning electrochemical microscopy (SECM). We develop carbonate (CO₃^2-) ionophore-incorporated ISEs exhibiting a Nernstian response (26.7 mV/decade) with a detection limit of 1.72 μM and explore surface-immobilized formate dehydrogenase (FDH) activity by detecting CO₂ generated by the enzymatic reaction via potentiometric measurements. SECM is used for real-time spatial/temporal investigation of FDH immobilized onto the surface at a micrometer-scale resolution. Moreover, unlike voltammetric techniques based on faradaic reactions, the potentiometric measurements using ISEs allow highly sensitive and selective detection of CO₃^2-, rendering efficient quantification of CO₂ without interference from solution composition changes arising from faradaic processes. The total amount of CO₂ generated at an FDH-immobilized Au ultramicroelectrode is quantified as a function of coenzyme, i.e., NAD⁺, and substrate, i.e., formate, concentrations both in constant tip-sample distance mode and variable depth mode. Finally, we demonstrate the use of the ISE to quantify CO₂ levels in blood serum.