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.

Hydrophobic gating in biological transport proteins is regulated by stimulus-specific switching between filled and empty nanocavities, endowing them with selective mass transport capabilities. Inspired by these, solid-state nanochannels have been integrated into functional materials for a broad range of applications, such as energy conversion, filtration, and nanoelectronics, and here we extend these to electrochemical biosensors coupled to mass transport control elements. Specifically, we report hierarchically organized structures with block copolymers on tyrosinase-modified two-electrode nanopore electrode arrays (BCP@NEAs) as stimulus-controlled electrochemical biosensors for alkylphenols. A polystyrene-<i>b</i>-poly(4-vinyl)pyridine (PS-<i>b</i>-P4VP) membrane placed atop the NEA endows the system with potential-responsive gating properties, where water transport is spatially and temporarily gated through hydrophobic P4VP nanochannels by the application of appropriate potentials. The reversibility of hydrophobic voltage-gating makes it possible to capture and confine analyte species in the attoliter-volume vestibule of cylindrical nanopore electrodes, enabling redox cycling and yielding enhanced currents with amplification factors >100× when operated in a generator-collector mode. The enzyme-coupled sensing capabilities are demonstrated using nonelectroactive 4-ethyl phenol, exploiting the tyrosinase-catalyzed turnover into reversibly redox-active quinones, then using the quinone-catechol redox reaction to achieve ultrasensitive cycling currents in confined BCP@NEA sensors giving a limit-of-detection of ∼120 nM. The mass transport controlled sensing platform described here is relevant to the development of enzyme-coupled multiplex biosensors for sensitive and selective detection of biomarkers and metabolites in next-generation point-of-care devices.

The development of metal-free, carbon-based electrocatalysts derived from biomass waste has attracted significant attention due to their low cost along with excellent activity, selectivity, and stability for the oxygen reduction reaction (ORR).In this study, a nitrogen (N)-doped porous carbon catalyst was synthesized from mandarin peel waste (MPW) via a simple thermal treatment method.The optimized electrocatalyst, N-doped MPW-derived catalyst-2 (NMPC-2), exhibits a hierarchically porous structure with a high surface area, facilitating efficient access of reactants to active sites.NMPC-2 demonstrates outstanding ORR activity and stability comparable to that of commercial Pt/C, maintaining excellent performance even over 40,000 cycles of accelerated stability testing.Furthermore, its application in a zinc-air battery shows a high peak power density and stable discharge characteristics.This work presents a simple yet effective strategy for developing biowaste-derived ORR electrocatalysts through a sustainable and practical approach.

Potentiometric ion-selective electrode (ISE) sensors are powerful electrochemical tools used in various applications in different fields, including the biological, clinical, and environmental fields, owing to their high selectivity, sensitivity, simplicity, and versatility. This review highlights recent advancements in ionophore-based polymeric ISE sensors over the past five years, with a particular focus on progress at the micro- and nanoscales. After discussing the conventional ISE configuration and its general operational principles, we explore the notable advancements in terms of the key ion-selective membrane components, such as ionophores, and other techniques combined with ISEs. These advancements have significantly improved the sensing performances and expanded the practical applications. We also examine the progress in the field of miniaturized solid-contact microelectrodes and the incorporation of novel functional materials for efficient ion-to-electron transduction. Miniaturized solid-state ISEs provide low limits of detection with reduced sample volume requirements, extended stability, and rapid response times. When combined with scanning electrochemical microscopy, ion-selective microelectrodes enable highly spatially resolved ion analyses. The integration of solid-contact ISEs into compact, portable, wearable devices has advanced the field of wearable on-body ISE sensors. Finally, we briefly introduce the development of ion-selective optode sensors as promising optical sensors based on ionophores that are particularly advantageous for cellular imaging.

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.