One-Pot and Gram-Scale Synthesis of Fe-Based Nanozymes with Tunable O₂ Activation Pathway and Specificity Between Associated Enzymatic Reactions

Nanozymes have recently gained attention for their low cost and high stability. However, unlike natural enzymes, they often exhibit multiple enzyme-like activities, complicating their use in selective bioassays. Since H₂O₂ and O₂ are common substrates in these reactions, controlling their activation-and thus reaction specificity-is crucial. Recent advances in tuning the chemical state of cerium have enabled control over H₂O₂ activation pathways for tunable peroxidase/haloperoxidase-like activities. In contrast, the control of O₂ activation on an element in oxidase/laccase nanozymes and the impact of its chemical state on these activities remains unexplored. Herein, a facile one-pot method is presented for the gram-scale synthesis of Fe-based nanozymes with tunable compositions of Fe₃O₄ and Fe₃C by adjusting preparation temperatures. The Fe₃O₄-containing samples exhibit superior laccase-like activity, while the Fe₃C-containing counterparts demonstrate better oxidase-like activity. This divergent O₂ activation behavior is linked to their surface Fe species: the abundant reactive Fe²⁺ in Fe₃O₄ promotes laccase-like activity via Fe³⁺-superoxo formation, whereas metallic Fe in Fe₃C facilitates OH radical generation for oxidase-like activity. Controlled O₂ activation pathways in these Fe-based nanozymes demonstrate improved sensitivity in the corresponding biomolecule detection, which should inform the design of nanozymes with enhanced activity and specificity.