Standardized assays for evaluating superoxide dismutase-like and catalase-like activities of nanozymes

Antioxidant nanozymes are nanomaterials with superoxide dismutase-like or catalase-like activities. They have emerged as promising therapeutics for oxidative stress-related disorders. However, the lack of standardized quantitative assays for activity characterization has limited cross-study reproducibility and direct comparison of reported performance. Here this protocol provides step-by-step procedures for the quantitative characterization of the superoxide dismutase-like and catalase-like activities of nanozymes. Superoxide dismutase-like activity is quantified via a self-prepared xanthine/xanthine oxidase/water-soluble tetrazolium salt-1 system, in which nanozymes catalyze the dismutation of superoxide anions (O₂^•-), thereby suppressing formazan formation. By tuning O₂^•- generation through controlled xanthine oxidase concentrations, the protocol enables the determination of Michaelis-Menten kinetics for the short-lived O₂^•-. Catalase-like activity is quantified by monitoring hydrogen peroxide (H₂O₂) decomposition at 240 nm via ultraviolet-visible spectrophotometry, which yields specific activity and kinetic constants, whereas a complementary 3,3',5,5'-tetramethylbenzidine oxidation assay excludes interference from peroxidase-like reactions. Representative nanozymes spanning carbon-based, metal oxide, noble metal and single-atom systems are used as case studies to demonstrate the robustness and versatility of this protocol. Functional validation in cellular models using flow cytometry further demonstrates the practical applicability of reactive oxygen species scavenging. This protocol enables reliable and reproducible evaluation of antioxidant nanozymes, facilitating direct comparison across materials, elucidation of structure-activity relationships and rational optimization for biomedical applications. The complete procedure can be performed by researchers with standard training in biochemistry and flow cytometric analysis within 10-12 h.