Bimetallic Cu/Zn Single‐Atom Nanozyme with Superoxide Dismutase‐Like Activity

Abstract

The superoxide ion (O^2•−), a radical species, is significant in chemical and biological systems. Nanozymes, enzyme‐mimicking nanomaterials, have been developed to replicate superoxide dismutase (SOD), which counters O^2•−. Traditionally, nano ceria (CeO₂) is used for SOD mimicry due to its Ce³⁺/Ce⁴⁺ cycling ability, but issues like toxicity, biodistribution, aggregation, and specificity hinder practical use. Single‐atom nanozymes (SANs) offer a solution, with metal centers mimicking natural metal‐based enzymes. A Cu/Zn bimetallic SAN is synthesized, structurally resembling natural SOD and exhibiting comparable activity. Its performance is assessed by capturing superoxide radicals and inhibiting Nitro‐blue tetrazolium (NBT) photoreduction to blue Formazan. The Cu/Zn‐SAN shows a half‐maxima inhibitory concentration (IC₅₀) of 0.115 µg mL⁻¹ and a catalytic activity of 7820 U mg⁻¹, compared to 4264 U mg⁻¹ for the natural SOD enzyme. Unlike many dual‐metal nanozymes with multiple ROS activities, Cu/Zn‐SAN selectively mimics SOD activity with no detectable oxidase or peroxidase‐like behavior. Additionally, its performance in cigarette smoke extract demonstrates its practical relevance and biological safety. These findings highlight its potential for reducing oxidative stress in cardiovascular, inflammatory, and neurodegenerative diseases, as well as applications in cosmetic anti‐aging products and skin protection, offering a promising alternative to traditional nanozymes.

A novel Cu/Zn dual‐site single‐atom nanozyme is synthesized via a PVP‐assisted MOF‐derived strategy, mimicking natural CuZn‐superoxide dismutase enzyme with high selectivity. The catalyst exhibits robust superoxide dismutation activity without undesired oxidase or peroxidase functions and effectively scavenges reactive oxygen species in complex environments such as cigarette smoke extract, highlighting its promise for real‐world antioxidant applications.

1. Introduction

Natural enzymes are biological macromolecules that can catalyze a variety of biochemical events in living things and serve important roles in biological systems.^{[ 1} , ^{2 ]} However, the expensive cost and low stability of natural enzymes prohibit their widespread application.^{[ 3 ]} So far, nanozymes have gained a lot of interest and are predicted to replace conventional protein‐based enzymes because of their affordability, great stability, and ease of manufacture.^{[ 4} , ⁵ , ⁶ , ^{7 ]} Superoxide dismutase (SOD),^{[ 8} , ⁹ , ¹⁰ , ^{11 ]} catalase (CAT),^{[ 12} , ¹³ , ^{14 ]} oxidase (OXD),^{[ 15} , ¹⁶ , ¹⁷ , ^{18 ]} peroxidase (POD),^{[ 19} , ²⁰ , ²¹ , ²² , ²³ , ^{24 ]} Laccase (LAC)^{[ 25} , ²⁶ , ^{27 ]} and other multi‐enzymatic nanomaterials have been widely employed in anti‐inflammatory, antibacterial, cancer treatment, and biomarker applications.^{[ 28} , ²⁹ , ³⁰ , ³¹ , ^{32 ]} SOD‐like nanozymes are probably the least explored category among the four.

Natural SOD enzymes are generally categorized into three types based on the metal co‐factor: Cu/Zn SOD for humans and eukaryotes, Fe‐ or Mn‐ SOD in mitochondria and chloroplasts, and Ni‐ SOD in prokaryotes and marine cyanobacteria.^{[ 33 ]} For a long time, the SOD‐mimicking activity has been exclusively presented by ceria nanoparticles (CeO₂ NPs).^{[ 34} , ³⁵ , ^{36 ]} This is due to their remarkable ability to eliminate free radicals by binding oxygen reversibly and changing the surface of the NPs between the reduced Ce³⁺ form and the oxidized Ce⁴⁺ form.^{[ 37 ]} However, their potential toxicity, biodistribution, aggregation, and relatively low specificity limit their real‐life application.

A promising development in the realm of nanozymes, single‐atom nanozymes (SANs) with atomically distributed metal active sites have recently come to light because of their high activity, stability, and ability to employ the highest possible utilization of metal atoms.^{[ 1} , ³ , ³⁸ , ³⁹ , ⁴⁰ , ^{41 ]} Some trials have been delivered already to synthesize SANs that mimic the action of natural SOD enzyme that rely on one metal co‐factor such as Fe‐SAN,^{[ 42 ]} Mn‐SAN,^{[ 43 ]} and Cu‐SAN.^{[ 44 ]} However, our work offers a systematic investigation of a Cu/Zn single‐atom nanozyme with high SOD‐like activity and exclusive selectivity, which has not been fully addressed in prior reports. In recent years, SANs have attracted considerable attention for their high atomic utilization and enzyme‐like behavior in diverse catalytic systems.^{[ 44} , ^{45 ]} However, most SANs reported to date are based on single‐metal active sites or form multi‐metal clusters that lack atomic‐level control. The Cu/Zn‐SAN developed in this study is distinct in that it features atomically dispersed dual active sites, where Cu and Zn are independently stabilized within a nitrogen‐doped carbon matrix. This bimetallic architecture enables selective SOD‐like catalysis, without undesired oxidase or peroxidase side reactions, representing a rare example of a SAN with exclusive enzymatic selectivity. Furthermore, we demonstrate the nanozyme's robust activity in complex environments, including real‐world application in superoxide removal from cigarette smoke. While this dual‐site approach offers advantages in selectivity and catalytic efficiency, further computational and spectroscopic work is needed to fully unravel the Cu–Zn synergistic mechanism.

While several Cu/Zn‐based nanozymes have been previously explored as SOD mimics, most display broad ROS activity including oxidase or peroxidase‐like functions, which can complicate biological applications due to non‐specific ROS modulation. Moreover, few studies have systematically examined dual‐metal SANs in complex oxidative environments like cigarette smoke extract. In this Article, a copper/zinc bimetallic SAN with excellent SOD activity was developed by controlling the isolated copper and zinc active sites and precisely coordinating nitrogen (N) to mimic the action of natural SOD enzyme with higher efficiency and stability. The SAN was used to eliminate the superoxide radicals among other radicals in cigarette smoke. While recent studies have explored Cu/Zn‐MOFs for AD treatment via ROS scavenging,^{[ 46 ]} this work diverges by designing a carbon‐supported Cu/Zn SAN with atomic‐level precision to mimic natural Cu/Zn‐SOD. Unlike MOF‐based systems, this SAN exhibits unparalleled SOD‐like selectivity (no OXD/POD activity) and exceptional catalytic efficiency (IC50 = 0.115 µg mL⁻¹, activity = 7820 U mg⁻¹). This fundamental advance expands applications beyond dermatology to oxidative stress management in diverse pathologies and bolster the potential application of Cu/Zn SAN in skin protection and cosmetic anti‐aging products, as well as in the reduction of oxidative stress in inflammatory, neurological, and cardiovascular diseases.

2. Results

2.1. Synthesis and Characterization of Cu/Zn‐SAN

The preparation process of Cu/Zn dual SAN was carried out following the method published by Q. Chen et al.^{[ 47 ]} with a few modifications as illustrated in Figure 1A. First, the octahedral‐shaped Cu/Zn‐MOF precursor was synthesized in the presence of polyvinylpyrrolidone (PVP) to modulate the morphology of Cu/Zn‐MOF. PVP is adsorbed on the surface of the MOF crystal nucleus during the growth, thus reducing the “face‐to‐face stacking” between MOF layers and inducing the anisotropic growth of MOF. Next, the Cu/Zn was obtained by direct pyrolysis of the Cu/Zn‐MOF precursor and dicyandiamide (DCD) in an alumina boat at 300 °C for 2h under flowing nitrogen in a tube furnace, maintaining a heating rate of 5 °C min⁻¹ and then at 550 °C for another 4 h.

Figure 1.

A) Illustration of the synthesis process of Cu/Zn‐SAN. B&C) TEM images showing the octahedral‐shaped morphology of Cu/Zn‐MOF. Scale bar 0.5 µm and 20 nm, respectively, and D) STEM analysis for Cu/Zn‐SAN showing the single atoms as the bright dots. Scale bar 2 nm.

As shown by transmission electron microscopy (TEM) images, an octahedron‐shaped MOF was obtained while no nanoparticles (NPs) were observed (Figure 1B,C). Similarly, high‐angle annular dark field scanning tunneling electron microspore (HAADF‐STEM) results of the annealed product indicate the atomic dispersion of Cu and Zn single atoms over the Carbon sheet (Figure 1D; Figure S1, Supporting Information). Energy Dispersive Spectroscopy (EDS) mapping illustrates the homogenous dispersibility of Cu, Zn, C, and N throughout the construction of the SAN (Figure 2 ). Brunauer–Emmett–Teller analysis (BET) revealed that the SAN had a porous structure with a large surface area of 983.4 m² g⁻¹, and the average pore diameter was 2.3 nm, as calculated by the Horvath–Kawazoe method. The XRD pattern of the Cu/Zn‐MOF precursor (Figure S2, Supporting Information) shows sharp reflections matching those of the previously reported Cu/Zn‐MOF^{[ 46 ]} and HKUST‐1 MOF,^{[ 48 ]} indicating that the Cu/Zn‐MOF likely adopts a similar framework structure. Minor peak shifts were observed due to Zn incorporation and the well‐defined and ordered precursor framework supports controlled pyrolysis and retention of porosity, which is beneficial for forming well‐dispersed single atoms in the derived catalyst. This supports its role as a suitable template for the subsequent pyrolysis process to generate Cu/Zn‐SAN.

Figure 2.

HAADF‐STEM image of the Cu/Zn‐SAN; scale bar: 10 nm, and the corresponding EDS elemental mapping showing B) C, C) N, D) Cu, and E) Zn.

X‐ray photoelectron spectroscopy (XPS) was used to inspect the binding states of Cu, Zn, C, and N on the SAN. The N 1S spectrum showed the existence of oxidized N (403.8 eV), graphite N (401.5 eV), pyrrolic N (400.7 eV), Metal‐N (398.1 eV), and pyridine N (397.8 eV) (Figure 3A). The detailed proportions of each species are summarized in Table S1 (Supporting Information). The C 1S spectrum fitted well with the peaks at 287.8 eV (C‐N), 285.5 eV (C═N), and 284.5 eV (C═C) (Figure 3B). The Zn 2P spectra displayed two strong peaks at 1042.6 and 1019.9 eV for Zn 2P_1/2 and Zn 2P_3/2, respectively, showing that Zn¹ species are abundant in Zn‐SAN (Figure 3C). Similarly, the Cu 2P spectrum exhibited two prominent peaks at 951.6 and 932.1 eV for the Cu 2P_1/2 and Cu 2P_3/2, respectively, implying that Cu¹ species are predominant in the Cu/Zn‐SAN (Figure 3D). In accordance with XPS analysis, the elemental composition of Cu, Zn, N, O, and C was estimated to be 12.27%, 8.42%, 19.13%, 2.95%, and 57.23%, respectively (Figure S31, Supporting Information). The initial Cu:Zn precursor molar ratio was 10:1 to offset the preferential loss of Cu during pyrolysis and ensure a final atomic Cu:Zn ratio close to 1.5:1 in the resulting Cu/Zn‐SAN. Attempts to adjust the initial precursor ratio resulted in lower catalytic activities, supporting the choice of this synthesis condition.

Figure 3.

XPS spectra of A) N 1S, B) C 1S, C) Zn 2p, and D) Cu 2P of the Cu/Zn‐SAN.

2.2. Atomic Structure Analysis

Extended X‐ray absorption fine structure spectroscopy (EXAFS) was used to investigate the coordination environment of Cu/Zn‐SAN. The Fourier‐transformed magnitudes of the Cu K‐edge EXAFS and the Zn K‐edge EXAFS are shown in Figure 4A,B. The Cu/Zn‐SAN showed a major peak at 1.48 Å in the of the Cu K‐edge EXAF, which comes in complete agreement with the Cu‐N peak in copper phthalocyanine attributed to the Cu and N atoms backscattering.^{[ 49 ]}

Figure 4.

Atomic structure analysis of Cu/Zn‐SAN showing FT‐EXAFS spectra at the R‐space of A) Cu and B) Zn in the Cu/Zn‐SAN, and K‐edge XANES spectra of C) Cu and D) Zn in the Cu/Zn‐SAN.

Moreover, The Cu and N atoms backscattering led to another peak at 1.52 Å. The prominent peak of Cu‐Cu at 2.22 Å in the Cu‐foil was not observed in the Cu/Zn‐SAN, indicating that most of the loaded metal existed as isolated atoms.^{[ 50 ]} The expected Cu–Cu backscattering peak at ∼2.5 Å is notably weak. This can be attributed to the use of nano‐sized CuO with low crystallinity, the fluorescence detection mode, and possible disorder in the second coordination shell, all of which can attenuate higher‐shell signals in EXAFS. To provide a more accurate reference for the oxidation state of Cu, the EXAFS spectrum of Cu₂O was added (Figure 4A). The similarity in spectral features between Cu/Zn‐SAN and Cu₂O further supports the presence of Cu in a Cu(I)‐like coordination environment. Notably, no significant second‐shell peaks were observed at ∼3 Å in either the Cu or Zn K‐edge EXAFS spectra. This suggests the absence of strong Cu–Zn or Zn–Cu backscattering and indicates that Cu and Zn atoms are likely atomically dispersed and independently coordinated to nitrogen atoms, rather than forming a fixed linear arrangement.

For the Zn K‐edge EXAF, Cu/Zn‐SAN exhibited a prominent peak at 1.52 Å, which aligned perfectly with the Zn‐N peak observed for zinc phthalocyanine. This peak is ascribed to the backscattering of Zn and N atoms.^{[ 51 ]} The small peak of Zn‐Zn observed at 2.35 Å in the Cu/Zn‐SAN spectrum was far more prominent in the Zn‐foil spectrum, suggesting that the majority of the metal present was in the form of individual atoms.^{[ 52 ]}

The coordination configuration was then investigated using quantitative EXAFS curve‐fitting analysis. By fitting the EXAFS at the Cu K‐edge and Zn K‐edge, the structural parameters and quantitative chemical configuration of Cu and Zn atoms were obtained (Table S2, Supporting Information). Based on the quantitative fitting analysis, the coordination numbers of Cu and Zn was determined to be ≈4 for both metals. The Cu K‐edge X‐ray absorption near edge structure (XANES) shown in Figure 4C reveals that the absorption edge of Cu/Zn‐SAN closely aligns with that of CuO, suggesting that the majority of Cu atoms in Cu/Zn‐SAN are present in a Cu(II)‐like oxidation state. This observation suggests that coordination with nitrogen atoms in the SAN structure effectively stabilizes the reduced Cu species. The Cu/Zn‐SAN showed a peak at 8,992 eV arising from the electron transfer between Cu and N. Since the transition between 1S and the unoccupied 4P states produces the Cu K‐edge white‐line peak, higher peak intensity means more electron transfer between the metal center and coordination atoms.

Moreover, the Zn K‐edge XANES of Cu/Zn‐SAN shows an absorption edge that is slightly shifted relative to Zn foil and ZnPC, suggesting a distinct electronic structure and coordination environment for Zn, likely due to its atomically dispersed bonding with nitrogen atoms (Figure 4D). Electron transport between Zn and N caused Cu/Zn‐SAN to peak at 9663 eV, which is different from Zn‐O and Zn‐N at 9669 and 9673 eV, respectively. A higher peak intensity indicates more electron transport between the coordination atoms and the metal center since the Zn K‐edge white‐line peak is the result of the transition between the unoccupied 4P states and 1S. Results for Feffit for a simple 1‐shell fit to a spectrum from Cu/Zn SAN in the R‐space are shown in Figure S4 (Supporting Information). The results indicate how well the theoretical model matches the experimental data.

These findings suggest potential electronic interactions between Cu and Zn centers within the Cu/Zn‐SAN structure, which could underlie the observed enhanced SOD‐like activity. Such bimetallic synergistic effects have been proposed in previous studies of Cu/Zn‐based systems.

The observed shifts in the Cu and Zn K‐edge XANES spectra, along with coordination environment changes identified by EXAFS, suggest electronic modulation and potential synergy between Cu and Zn sites. While no direct Cu–Zn scattering was detected, these spectral features imply indirect interactions through shared nitrogen coordination and electronic coupling.

2.3. SOD‐Like Activity

The nitro‐blue tetrazolium (NBT) experiment was carried out to verify SOD‐like activity of Cu/Zn‐SAN. NBT is known to be photo‐reduced by superoxide radicals to form the blue formazan product with a sharp absorption at 560 nm. However, in presence of the SOD enzyme, the superoxide radical is scavenged, and the absorption peak is, hence, reduced.^{[ 53 ]} The same observation was spotted in presence of Cu/Zn‐SAN with the absorption peak at 560 nm being reduced with the increasing concentration of the Cu/Zn‐SAN (Figure S5, Supporting Information). When the concentration of Cu/Zn‐SAN was 2 µg mL⁻¹, the maximum inhibition percentage of the NBT reduction reaction under the assay conditions was about 81.5%, and reached about 98% when the catalyst's concentration was 10 µg mL⁻¹ (Figure 5A). On the other side, when the natural SOD enzyme was used, the removal efficiency was about 57% with 2 µg mL⁻¹ of the enzyme and reached a maximum of 67% when the enzyme's concentration was 4 µg mL⁻¹ (Figure 5B). No more significant enhancement in efficiency was recorded when the concentration of the SOD enzyme increased beyond 4 µg mL⁻¹.

Figure 5.

O₂ ^•− removal rate as a function of the concentration of A) Cu/Zn‐SAN and B) SOD enzyme using NBT indicator. C) The inhibition of NBT reduction reaction with the increasing concentration of Cu/Zn‐SAN (Black) and SOD enzyme (Red). D) EPR spectra of Xanthine (2mM)/Xanthine oxidase (3 U mL⁻¹) in DMPO in presence (Red) and absence (Black) of the Cu/Zn‐SAN.

The inhibitory concentration of 50% (IC₅₀) for both the natural SOD enzyme and the Cu/Zn‐SAN were also evaluated to determine the concentration at which the catalyst would inhibit the reaction by 50%. The IC₅₀ of SOD was found to be 0.195 µg mL⁻¹ whereas that of the Cu/Zn‐SAN was 0.115 µg mL⁻¹ with a catalytic activity of 7820 U mg⁻¹ compared to 4264 U mg⁻¹ for the natural SOD enzyme as quantified using a commercial SOD assay kit (WST‐1) (Figure 5C). This indicates that Cu/Zn‐SAN effectively captures the superoxide radical and inhibits NBT photoreduction, demonstrating catalytic performance comparable to that of the natural SOD enzyme on a mass basis. However, we acknowledge that due to differences in active metal content, direct comparison of catalytic efficiency must be interpreted with caution. For better context, the catalytic activity of Cu/Zn‐SAN (7820 U mg⁻¹) was compared to that of previously reported nanozymes (Table S3, Supporting Information).

Besides, electron paramagnetic resonance (EPR) was used to monitor the superoxide radical produced during the reaction. The Xanthine/Xanthine oxidase system was used to generate the superoxide radical in the presence of 5,5‐Dimethyl‐1‐Pyrroline‐N‐Oxide (DMPO) as the spin trap. The DMPO/OOH EPR adduct showed four lines with an equal relative intensity, which sharply decreased in presence of Cu/Zn‐SAN due to the elimination of superoxide radicals (Figure 5D).

Moreover, the relative activity of the Cu/Zn‐SAN was checked regularly over two months, and no significant depletion in its performance was observed (Figure S6, Supporting Information). The Cu/Zn‐SAN also showed excellent stability under harsh acidic (1.0 m HCl) and basic (1.0 m NaOH) media (Figure S7, Supporting Information). The nanozyme's reusability can also be highlighted by its ability to maintain ≈95% of its relative activity after 5 cycles (Figure S8, Supporting Information). These findings demonstrate that SANs catalyzing enzyme‐like reactions may exhibit essential properties of natural enzymes, such as quick kinetics, productivity, and high activity under identical reaction conditions.

Furthermore, the activity of the Cu/Zn‐SAN was maintained in the range of 24 to 100 °C, while the natural SOD did not possess the same thermal stability and was deactivated at temperatures higher than 60 °C temperature (Figure S9, Supporting Information). Also, the activity of Cu/Zn‐SAN was not much affected by the pH change unlike the natural SOD whose activity is limited by maintaining a pH between 8 and 10 (Figure S10, Supporting Information). However, when the pH was lower than 4, both the natural SOD and Cu/Zn‐SAN did not function properly.

To evaluate the robustness of Cu/Zn‐SAN under biologically relevant conditions, its SOD‐like activity was tested after incubation in 10% fetal bovine serum (FBS) and 1 m NaCl for 24 h. As shown in Figure S11 (Supporting Information), the nanozyme maintained 88.5% and 93.4% of its initial catalytic activity, respectively. These results demonstrate the excellent stability of Cu/Zn‐SAN in complex chemical environments, making it suitable for biomedical and environmental applications.

Kinetic studies of SOD‐like activity were conducted by varying the Xanthine's concentration between 0.05 and 0.5 mm while keeping Cu/Zn‐SAN concentrations constant. The apparent kinetic parameters (derived from NBT inhibition curves) were an apparent Vmax of 7.56 mm min⁻¹ and an apparent Km of 0.99 mm (Figure S12, Supporting Information). These values are provided for comparative purposes only, as indirect methods do not measure true enzymatic rates. A more accurate second‐order rate constant (k) was calculated at the IC50 (0.115 µg mL⁻¹) using the relationship k × [Cu/Zn‐SAN] = kNBT × [NBT], where kNBT is the rate constant for NBT reduction by O₂ ^•−.

Although direct computational modeling could not be performed due to current technical constraints, we propose a mechanism for the Cu/Zn synergistic effect based on experimental findings and literature reports. It is hypothesized that Cu atoms primarily act as the active centers for superoxide radical binding and electron transfer, while Zn atoms stabilize the surrounding coordination environment and facilitate charge delocalization. This division of labor enhances both the catalytic efficiency and selectivity of the Cu/Zn‐SAN. Similar synergistic behaviors between Cu and Zn have been reported in related bimetallic systems.^{[ 46 ]} Future density functional theory (DFT) studies will be undertaken to validate this mechanistic hypothesis.

2.4. OXD‐ and POD‐Like Activities

The OXD‐ and POD‐like activities were inspected by testing the ability of Cu/Zn‐SAN to oxidize TMB in the absence and presence of H₂O₂, respectively. Generally, TMB can be oxidized by H₂O₂ in presence of a catalyst to give the blue oxidation product that shows a peak at 652 nm under UV/Vis spectrophotometer. As depicted in Figures S13 and S14 (Supporting Information), The Cu/Zn‐SAN could not oxidize TMB under optimal conditions, neither in the absence not the presence of H₂O₂. This shows that the Cu/Zn‐SAN does not possess any OXD‐ or POD‐like activities, unlike the previously reported Cu‐SAN.^{[ 18 ]} This observation proves the function of the Zn metal in the structural integrity of the Cn/Zn‐SAN, which endowed the Cu/Zn SAN with high selectivity towards the SOD. Importantly, the Cu/Zn‐SAN exhibited no detectable OXD or POD activity, highlighting its superior enzymatic selectivity compared to previously reported SANs with multi‐enzymatic behaviors.

2.5. O₂ ^•− Elimination in Cigarette Smoke

Cigarette smoke was drawn from commercial cigarettes into PBS buffer using a pump attached to a Buchner flask in a Shisha‐like setup (Figure S15 and Video S1, Supporting Information). The resulting solution was labeled as the radical‐containing system, and the NBT assay was followed according to the proposed methodology. The colored product was monitored at 560 nm in the presence and the absence of the Cu/Zn SAN. As shown in Figures S16 and S17 (Supporting Information), 2 µg mL⁻¹ of the Cu/Zn‐SAN was able to eliminate 90.7% of the ROS in the cigarette smoke extract verifying the exceptional SOD‐like activity of the Cu/Zn‐SAN.

3. Conclusion

In summary, a Cu/Zn dual single‐atom nanozyme with well‐dispersed active centers was synthesized to mimic the natural SOD enzyme. The Cu/Zn SAN showed higher activity in suppressing the photoreduction of NBT by removing the superoxide radical with a maximum inhibition percentage of the NBT reduction reaction of ≈98%. The catalytic selectivity of Cu/Zn‐SAN was evaluated through EPR and NBT assays, which consistently indicated the specific dismutation of superoxide radicals. No oxidase or peroxidase activity was detected under identical conditions, highlighting the selective enzyme‐mimicking behavior of Cu/Zn‐SAN. This behavior contrasts with many previously reported nanozymes that exhibit multiple ROS‐related activities and underscores the importance of atomic‐level control over active site architecture in achieving enzymatic specificity. The SAN was stable under harsh acidic and basic media and maintained its stability for over two months. In addition to its catalytic performance, the Cu/Zn‐SAN exhibited excellent operational stability in protein‐rich and high‐salinity environments. Based on its SOD‐like activity, the Cu/Zn SAN was used to eliminate the superoxide radical in cigarettes smoke with more than 90% efficiency. The proposed synergistic roles of Cu and Zn in the SAN structure, supported by XANES/EXAFS data and literature analysis, provide a framework for understanding the high catalytic performance observed in this study. Compared to previously reported Cu/Zn‐based nanozymes that mimic natural SOD activity, our system exhibits exclusive SOD‐like activity with no detectable oxidase or peroxidase function, verified under physiologically relevant conditions and is applicable in complex environments such as cigarette smoke extract, demonstrating its practical potential for superoxide detoxification. Also, the synthesis approach involves a PVP‐assisted Cu/Zn‐MOF precursor, which leads to atomic‐level dispersion of both metal centers. These elements position Cu/Zn‐SAN as a uniquely selective and practical nanozyme within the broader field of dual‐metal SACs. This suggests that the Cu/Zn SAN can be utilized in the diagnosis and treatment of diseases associated with superoxide radicals like cardiovascular diseases (e.g. hypertension and atherosclerosis), inflammatory diseases (e.g. rheumatoid arthritis), and neurodegenerative diseases (e.g. Alzheimer and Parkinson).

4. Experimental Section

Synthesis of Cu/Zn MOF

The Cu/Zn MOF was synthesized by dissolving 1 g of PVP in 50 mL of a mixture of DMF and ethanol (4:1). Afterward, 0.1 mmol of Zn(NO₃)₂. 6H₂O and 1 mmol of Cu(NO₃)₂. 3 H₂O was added and mixed with the solution followed by the stepwise addition of 1 mL Trifluoroacetic acid until the addition caused no more fumes. A solution of 0.84 g Trimesic acid in 50 mL of a mixture of DMF and ethanol (4:1) was added to the aforementioned solution. The resulting solution was autoclaved in a capped Teflon reactor for 1 h at 150 °C. Then, the light‐blue precipitate was collected, washed multiple times with ethanol, and then left to dry at 80 °C. A relatively high Cu:Zn molar ratio (10:1) was used intentionally to account for the lower incorporation efficiency of Cu compared to Zn during MOF formation and subsequent pyrolysis, aiming to achieve a final balanced Cu/Zn ratio in the SAN material.

Synthesis of Cu/Zn SAN

The Cu/Zn‐SAN was synthesized by mixing 0.2 g of the Cu/Zn MOF with 2.0 g of DCD in an alumina boat and heat at 300 °C for 2 h under flowing nitrogen in a tube furnace, maintaining a heating rate of 5 °C min⁻¹ and then at 550 °C for another 4 h. After cooling, the Cu/Zn SAN was collected directly for further use. Cu‐SAN and Zn‐SAN were synthesized according to previous work and used as controls.^{[ 54 ]}

Characterization

A JEOL JEM‐2010F Field Emission Electron Microscope, a 200 kV class analytical TEM was used to record HR‐TEM. A JEOL JSM‐6701F Field Emission Scanning Electron Microscope as a 30 kV class analytical SEM was used to obtain SEM images. HAADF‐STEM was done using a double spherical aberration electron JEOL JEM‐2100F microscope operating at 200 kV. XRD (Bruker D8 Advance diffractometer with Cu Kα radiation (λ = 1.54056 Å) at = 40 KV & 40mA) was used to acquire the crystal structure and phase purity. XPS measurements were carried out using a Kratos Axis Ultra DLD x‐ray spectrometer with a monochromatic aluminum Kα X‐ray source (energy = 1486.7 eV). HORIBA Jobin Yvon Modular Raman Microscope equipped with a 514 nm (10 to 50 mW) laser source for excitation was used for Raman spectra. To check the nature of the produced reactive oxygen species (ROS), JEOL FA200 EPR spectrometer was used in the frequency range of 8–10 GHz (X‐Band). Agilent Cary 60 UV/Vis spectrophotometer was used for all absorbance measurements. Quantachrome Autosorb iQ C‐XR with both physisorption and chemisorption capabilities was used for BET analysis at ambient temperature to confirm the porous structure of the catalyst and calculate its surface area. The sample was degassed at 200 °C for 12 h before adsorption measurements. The XAS measurements were performed at the BL14W1 station in Shanghai Synchrotron Radiation Facility (SSRF), China. The spectra were recorded in fluorescent mode using a Si (311) double‐crystal monochromator and a 32‐element Canberra/XIA Ge solid‐state detector. The electron storage ring is operated at 3.5 GeV, and the data was processed using Demeter Athena and Artemis software, according to the standard procedures.

Evaluation of the SOD‐Like Activity

To study the SOD‐like performance of Cu/Zn SAN, a colorimetric method of the inhibitory effect of photoreduction of NBT to the blue formazan was used. The superoxide radical was produced by mixing 0.25 mM xanthine and xanthine oxidase (0.05 UmL⁻¹) in phosphate buffer solution (PBS) (50 mM, pH = 7.5). The mixture included 100 µm NBT, and different concentrations of Cu/Zn SAN (0–10 µg mL⁻¹). Then, the mixture solutions were placed under fluorescent lamp for 10 min, and then the absorbance was measured at 560 nm immediately. A control experiment was conducted in the same way replacing the Cn/Zn SAN with different concentrations of the natural SOD enzyme (0–5 µg mL⁻¹).

The NBT assay measures inhibition efficiency rather than direct catalytic turnover. Thus, the apparent Km and Vmax values in Figure S12 (Supporting Information) are estimates for cross‐nanozyme comparison and should not be interpreted as classical enzymatic parameters. The SOD‐like kinetic parameters were determined using an NBT inhibition assay. The reaction rate of formazan formation was monitored at 560 nm and converted to molar units using an extinction coefficient of 12 800 M⁻¹ cm⁻¹. The substrate concentration was approximated by varying the initial xanthine concentration (0.05–0.5 mM), which regulates the steady‐state generation of superoxide anion via xanthine oxidase. Thus, the calculated Vmax and Km values are apparent values reflecting the enzyme‐like activity under these indirect assay conditions.

For a more rigorous kinetic analysis, the second‐order rate constant (k) was calculated at IC₅₀ (0.115 µg mL⁻¹) using the equation k × [Cu/Zn‐SAN] = k_NBT × [NBT], where k_NBT is the known rate constant for NBT reduction by O₂ ^•− (5.94 × 10⁴ M⁻¹ s⁻¹). With the measured IC₅₀ of 0.115 µg mL⁻¹, this gives k = 2.17 × 10⁸ M⁻¹s⁻¹, compared to 2.0 × 10⁹ M⁻¹s⁻¹ for the natural SOD.^{[ 55 ]}

Evaluation of the OXD‐ and POD‐Like Activities

The OXD‐ and POD‐ like activities of the Cu/Zn SAN were evaluated based on the catalyst's performance in the oxidation reactions of 3,3′,5,5′‐tetramethylbenzidine (TMB). Typically, 50 µL of the dispersed Cu/Zn SAN (100 mg L⁻¹) in acetate buffer (pH = 3.0, 100 mM) was allowed to react with 100 µL of TMB (0.5 mM) in absence and presence of H₂O₂, and the absorbance was monitored at 652 nm. A control sample was prepared in the same way without the addition of Cu/Zn SAN.

Conflict of Interest

The authors declare no conflict of interest.

Supporting information

Supporting Information

Supplemental Video 1

Hamed E. M., Fung F. M., and Li S. F. Y., “Bimetallic Cu/Zn Single‐Atom Nanozyme with Superoxide Dismutase‐Like Activity.” Small 21, no. 36 (2025): 21, e03879. 10.1002/smll.202503879

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.