Modular hybrid silica–hydrogel sensors enable high-resolution extracellular pH sensing in tumor and healthy cell models

Monitoring subtle changes in pH within cell cultures provides crucial insights into cell metabolism and the tumor microenvironment. Fluorescence-based optical sensors are particularly powerful because they allow real-time, non-invasive tracking of these variations. In this study, we present a new class of ratiometric fluorescent sensors built on biocompatible silica microparticles, a material well known for its safety, stability, and low cost. By combining silica with hydrogel layers, we created hybrid particles that can be covalently linked to different fluorescent probes, enabling robust and tunable sensing platforms. We systematically compared three probe-functionalized designs. Among the tested designs, hybrid particles incorporating acrylate-polyethylene glycol-amine and 2-aminoethyl methacrylate hydrogel layers loaded with SNARF-1 and FITC/Cy3 exhibited superior sensitivity and stability, as confirmed by both bulk fluorimetric analyses and single-particle confocal imaging. To bridge toward biologically relevant applications, these sensors were embedded into electrospun nanofiber scaffolds that mimic the structure of the native extracellular matrix. Using human keratinocyte and melanoma cultures, time-lapse confocal imaging revealed distinct extracellular pH signatures, with tumor cells displaying a more acidic environment. These results demonstrate how hybrid silica-hydrogel microparticles can be tailored into versatile sensing platforms for high-resolution extracellular pH monitoring in complex cellular models, paving the way for advanced applications in biomedical research and diagnostics. • Synthesis of biocompatible, hydrophilic, hybrid silica-hydrogel microparticles enabling covalent linking of both electrophilic and nucleophilic molecules. • Comparative characterization of three ratiometric fluorescent pH sensors developed by integrating SNARF-1, FITC, and HPTS fluorophores into modular hybrid microparticles. • Superior pH-sensing performance demonstrated by SNARF-1 and FITC/CY3 particles functionalized with acrylate-PEG-amine and AEMA. • Real-time confocal imaging on sensor-loaded electrospun nanofibers reveals differential acidification dynamics in melanoma vs. healthy keratinocyte cultures.