Benchmarking analytical parameters of chemiluminescent superoxide systems for robust quantification of graphene oxide...

Three chemiluminescent (CL) systems for superoxide anion radical (SAR) generation were evaluated using two probes: luminol (Lum), responsive to detecting a broad range of reactive oxygen species (ROS), and lucigenin (Luc), most selective to SAR. The Co(II)/H₂O₂ system, in both Lum- and Luc-based assays, showed good repeatability and reproducibility (s_r < 15%), suitable for routine screening, whereas the enzymatic system using xanthine/xanthine oxidase (Xa/XO) provided higher SAR generation selectivity but markedly poorer reproducibility (s_r > 20%). An operational threshold for detectable CL response changes was estimated for the Lum/Co(II)/H₂O₂ system, whereas the Luc/Xa/XO system is suitable for selective same-day SOD-referenced comparative assays. Thirty aqueous graphene oxide dispersions (aqGO), including pristine (unpurified) and purified by a dialysis bag membrane, were studied in these CL-based assays. The CL intensity decreased by 50-70% in the presence of pristine aqGO and by 80-90% with purified samples, resulting in an operational detectable-response threshold of 1-2 mg L^-1 depending on aqGO type under the Lum/Co(II)/H₂O₂ protocol. Compared to SOD, aqGO exhibits antioxidant activity up to 10⁶ times less efficiently. The obtained results show the advantages and limitations of each CL system for SAR-based antioxidant/prooxidant assessment and validate the strong modulatory role of GO. A practical quantitative criterion of inhibitory activity based on half-suppression of CL intensity (or integrated area) is proposed to evaluate antioxidant and prooxidant activity across a batch of GO samples. An approach to estimate the concentration of accessible sorption-active oxygen-containing sites is proposed. A metrological scheme for Co(II)/H₂O₂ systems in both probes, standardized assessment of the "antioxidant activity" and nanozyme-like behavior of graphene-based materials, suitable for interlaboratory comparison and batch quality control, is outlined.