Real-time neurochemical sensing of hydrogen peroxide production in brain tissue using advanced nanostructured Au microelectrodes functionalized with a ruthenium purple film

Real-time monitoring of H₂O₂ is essential for understanding its role in neurological physio(path)ology. In this study, we developed an advanced electrochemical sensor utilizing nanostructured gold wire microelectrodes functionalized with a Ruthenium Purple (RP) film, a polynuclear transition metal hexacyanoferrate analog of the prototypical Prussian Blue (PB). The RP film exhibits enhanced stability in neutral buffer solutions and reduced interference from biologically relevant cations such as Na⁺, Mg²⁺, and Ca²⁺. By integrating gold nanoparticles onto a nanoporous gold surface, we significantly increased the electroactive surface area of the microwire (ρ=26.1 ± 10.2). The electrodeposition of a thin RP film (4.23 ± 1.2 nm) resulted in a highly selective H₂O₂ sensor, operating at an optimal working potential of -0.05 V vs. Ag/AgCl, with a linear detection range of 0.5–500 µM, sensitivity of 1.18 ± 0.37 µA µM^-1 cm^-2, and a low limit of detection (LOD) of 66.6 ± 34.9 nM. Adding a Nafion® layer enhanced the sensor's operational stability, maintaining optimal performance over three hours under physiological conditions of pH and ion concentration. We validated the sensor's capability to monitor transient changes in H₂O₂ concentration in brain tissue by assessing its response to exogenously applied H₂O₂. Furthermore, we demonstrated that glutamate receptor activation in hippocampal slices induces a rapid and transient increase in extracellular H₂O₂ levels, underscoring the sensor's potential for studying oxidative stress in neurobiological contexts.