Photoelectrochemical bioimaging of redox events for 3D cultured cells via hematite-based light-addressable electrodes

Light-addressable electrochemistry offers a contact-free route to map chemical activity with spatial precision. Bioimaging in three-dimensional (3D) cultures remains challenging under operating conditions dominated by water oxidation. This work establishes hematite (Fe₂O₃) light-addressable electrodes that operate under visible light to image redox events in complex microenvironments. Hydrothermally synthesized hematite films were structurally and photoelectrochemically characterized, revealing a visible absorption edge and favorable band energetics for driving biomolecule oxidation at comparatively low bias. Photoelectrochemical performance was quantified by linear sweep voltammetry, cyclic voltammetry, and chronoamperometry under full-field and focused illumination. Using ferrocenemethanol as a model analyte, bias windows were identified that suppress background oxidation and enable analysis via differential photocurrent. For hydrogen peroxide (H₂O₂), calibration under full-field illumination yielded a limit of detection of 0.74 μM (R² = 0.96); focused-spot operation preserved spatial resolving power with a limit of detection (LOD) of 2.6 μM, sufficient for biological measurements. A glass-masking evaluation quantified spatial resolution with a full width at half maximum of 120 ± 22 μm, and stability tests showed no significant degradation over 100 illumination cycles. The photoelectrochemical imaging resolved diffusion barriers and concentration gradients around MCF-7 spheroids. The system also quantified localized, enzyme-generated H₂O₂ using glucose oxidase beads with catalase controls that confirmed the reaction mechanism. Importantly, phorbol 12-myristate 13-acetate stimulation enabled time-resolved visualization of H₂O₂ release from tumor spheroids at micromolar levels. These results position hematite-based photoelectrochemical imaging as a practical, selective, and noninvasive approach for time-dependent chemical mapping in 3D cell models and related bioanalytical applications.