Abstract

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 (CO32-) 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 voltametric techniques based on faradaic reactions, the potentiometric measurements using ISEs allow highly sensitive and selective detection of CO32-, 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.

Department(s)

Chemistry

Keywords and Phrases

carbonate ion-selective electrodes; CO detection 2; formate dehydrogenase; NADH regeneration; potentiometric measurements; scanning electrochemical microscopy

International Standard Serial Number (ISSN)

1936-086X; 1936-0851

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2025 American Chemical Society, All rights reserved.

Publication Date

08 Apr 2025

PubMed ID

40130603

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