The Understanding and Regulation of H₂O₂ Activation over Metal Oxide-based Nanozymes

Abstract

Since Fe₃O₄ was reported to mimic horseradish peroxidase (HRP) with comparable activity (2007), countless peroxidase nanozymes have been developed for a wide range of applications from biological detection assays to disease diagnosis and biomedicine development. However, researchers have recently argued that Fe₃O₄ has no peroxidase activity because surface Fe(III) cannot oxidize tetramethylbenzidine (TMB) in the absence of H₂O₂ (cf. Horseradish Peroxidase (HRP)). This motivated us to investigate the origin of transition metal oxides as peroxidase mimetics. The redox between their surface Mⁿ⁺ (oxidation) and H₂O₂ (reduction) was found to be the key step generating OH radicals, which oxidize not only TMB for color change but other H₂O₂ to produce HO₂ radicals for Mn+ regeneration. This mechanism involving free OH and HO₂ radicals is distinct from that of HRP with a radical retained on the Fe-porphyrin ring. Most importantly, it also explains the origin of their catalase-like activity (i.e., the decomposition of H₂O₂ into H₂O and O₂). Because the production of OH radicals is the rate-limiting step, the poor activity of Fe₃O₄ compared with Cu₂O can be attributed to the slow redox of Fe(II) with H₂O₂, which is two orders of magnitude slower than the most active Cu(I) among common transition metal oxides. The difference in the peroxidase-like activity of Fe₃O₄ and Cu₂O also reflects on their sensitivity in the coupled glutathione (GSH) detection.

Based on the understanding of mechanism, we further reviewed the definition of descriptors used for peroxidase mimicking. And we found that the disagreement on the peroxidase-like activity of Fe₃O₄ NPs (cf. HRP) in the literature was attributed to the use of different denominator (concentration of active Fe sites [Fe] or particles [P]) for the V_max obtained from Michaelis-Menten kinetics. Surprisingly, the distribution and chemical state of Fe species were found to be very different on single- and polycrystalline Fe₃O₄ nanoparticles with the latter bearing not only a higher Fe(II)/Fe(III) ratio but a more reactive Fe(II) species at surface grain boundaries. This accounts the unexpected gap in the catalytic constant (k_cat) observed for this material in the literature.

In light of the discovery that Cu₂O exhibits exceptional peroxidase-like activity among transition metal-oxide based nanozyme, further research based on Cu₂O nanozyme is required. Recent studies have shown that altering the exposed facet of Cu₂O alters the surface chemistry at the nano level and can significantly change its surface catalytic properties. Although the facet-dependent catalysis of Cu₂O has been widely reported, few study regarding their enzyme-like activity. Therefore, Cu₂O cube with (100) facet, octahedron with (111) facet and rhombic dodecahedra (RD) with (110) facet were synthesized and then evaluated in terms of their activity in mimicking horseradish peroxidase (HRP). Their peroxidase-like activity was then obtained in the order of RD > octahedron > cube by the oxidation of indicator, tetramethylbenzidine (TMB), in the presence of H₂O₂. Since GSH can reduce the oxidized TMB (blue color) to colorless TMB, the RD shape with highest peroxidase-like activity indeed provides highest GSH detection sensitivity.

Although many literatures focus on enhancing the enzyme mimicking activity of nanozyme, the improvement of specificity is also imperative as poor specificity has severely limited their applications. For example, CeO₂ activates H₂O₂ and displays peroxidase (POD)-like, catalase (CAT)-like, and haloperoxidase (HPO)-like activities. Since they unavoidably compete for H₂O₂ affecting its utilization in the target application, the precise manipulation of reaction specificity is thus imperative. Herein, we showed that one can simply achieve this by manipulating H₂O₂ activation pathway on pristine CeO₂ in well-defined shapes. This is because the coordination and electronic structures of Ce sites vary with CeO₂ surfaces that (100) and (111) surfaces display unprecedented specificity towards POD-/CAT-like and HPO-like activity, respectively. The antibacterial results suggest that the latter surface can well utilize H₂O₂ to kill bacteria (cf. the former), which is promising for antibiofouling applications.

These works highlight the importance of understanding the mechanism and definition of descriptors. Besides, the significance of surface control even for pristine nanozymes in catalytic correlation is well demonstrated. Thus, it provides insights into further nanozyme design for improved activity, reaction specificity, and high substrate utilization in the target application and beyond.

Date of Award	2 Aug 2023
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Yung-kang PENG (Supervisor)