Luminescent CeO₂:Eu³⁺ nanocrystals for robust in situ H₂O₂ real-time detection in bacterial cell cultures

Hydrogen peroxide (H₂O₂) quantification in biomedicine is valuable as inflammation biomarker but also in assays employing enzymes that generate or consume H₂O₂ linked to a specific biomarker. Optical H₂O₂ detection is typically performed through peroxidase-coupled reactions utilizing organic dyes that suffer, however, from poor stability/reproducibility and also cannot be employed in situ in dynamic complex cell cultures to monitor H₂O₂ levels in real-time. Here, we utilize enzyme-mimetic CeO₂ nanocrystals that are sensitive to H₂O₂ and study the effect of H₂O₂ presence on their electronic and luminescent properties. We produce and dope with Eu³⁺ these particles in a single-step by flame synthesis and directly deposit them on Si and glass substrates to fabricate nanoparticle layers to monitor in real-time and in situ the H₂O₂ concentrations generated by Streptococcus pneumoniae clinical isolates. Furthermore, the small CeO₂:Eu³⁺ nanocrystals are combined in a single-step with larger, non-responsive Y₂O₃:Tb³⁺ nanoparticles during their double-nozzle flame synthesis to engineer hybrid luminescent nanoaggregates as ratiometric robust biosensors. We demonstrate the functionality of these biosensors by monitoring their response in the presence of a broad range of H₂O₂ concentrations in vitro from S. pneumoniae, highlighting their potential for facile real-time H₂O₂ detection in vitro in cell cultures.