Atomically dispersed golds on degradable zero-valent copper nanocubes augment oxygen driven Fenton-like reaction for effective orthotopic tumor therapy

Wang, Liu-Chun; Chang, Li-Chan; Chen, Wen-Qi; Chien, Yi-Hsin; Chang, Po-Ya; Pao, Chih-Wen; Liu, Yin-Fen; Sheu, Hwo-Shuenn; Su, Wen-Pin; Yeh, Chen-Hao; Yeh, Chen-Sheng

doi:10.1038/s41467-022-35515-8

Download PDF

Article
Open access
Published: 15 December 2022

Atomically dispersed golds on degradable zero-valent copper nanocubes augment oxygen driven Fenton-like reaction for effective orthotopic tumor therapy

Nature Communications volume 13, Article number: 7772 (2022) Cite this article

4547 Accesses
12 Citations
Metrics details

Subjects

Abstract

Herein, we employ a galvanic replacement approach to create atomically dispersed Au on degradable zero-valent Cu nanocubes for tumor treatments on female mice. Controlling the addition of precursor HAuCl₄ allows for the fabrication of different atomic ratios of Au_xCu_y. X-ray absorption near edge spectra indicates that Au and Cu are the predominant oxidation states of zero valence. This suggests that the charges of Au and Cu remain unchanged after galvanic replacement. Specifically, Au_0.02Cu_0.98 composition reveals the enhanced •OH generation following O₂ → H₂O₂ → •OH. The degradable Au_0.02Cu_0.98 released Cu⁺ and Cu²⁺ resulting in oxygen reduction and Fenton-like reactions. Simulation studies indicate that Au single atoms boot zero-valent copper to reveal the catalytic capability of Au_0.02Cu_0.98 for O₂ → H₂O₂ → •OH as well. Instead of using endogenous H₂O₂, H₂O₂ can be sourced from the O₂ in the air through the use of nanocubes. Notably, the Au_0.02Cu_0.98 structure is degradable and renal-clearable.

Fabrication of an Au-doped Cu/Fe oxide-polymer core–shell nanoreactor with chemodynamic and photodynamic dual effects as potential cancer therapeutic agents

Article Open access 04 November 2022

Magnesium galvanic cells produce hydrogen and modulate the tumor microenvironment to inhibit cancer growth

Article Open access 28 April 2022

Design of a two-dimensional interplanar heterojunction for catalytic cancer therapy

Article Open access 03 May 2022

Introduction

The successful preparations from the groups of Flytzani-Stephanopoulos and Zhang have given the unforeseen demonstration on single-atom catalysts (SACs) and have brought nanocatalysis into atomic age^1,2. SACs possess atomically dispersed structures and well-defined coordination architectures, making them useful in both heterogeneous and homogeneous catalysis^3,4,5. Taking advantage of the endogenous H₂O₂ in the tumoral microenvironment, SACs have emerged as a form of nanocatalytic medicine used to perform chemodynamic therapy following •OH generation. In addition to the well-known Fenton or Fenton-like actions, SACs also show enzyme mimicking activity, e.g. a peroxidase (POD)-like reaction, to produce •OH in tumor catalytic therapy. That is, these SACs are viewed as nanozymes, revealing catalytic activity with enzyme-like properties due to their unique electronic structures and their specific coordination environments to catalyze versatile reactions^6,7,8. Undoubtedly, SACs promote nanozymes at the atomic level and thus provide potential workarounds for current inherent limitations.

Previous studies typically used Fe single-atom or peroxidase mimicking catalytic activity to generate •OH for single-atom-based tumor catalytic therapy. Importantly, the studies all used H₂O₂ from the tumor microenvironment (endogenous H₂O₂) to perform tumor catalytic therapy. However, because of the cellular redox homeostasis, the intratumoral H₂O₂ level is usually below the threshold (ca.100 µM), which limits the therapeutic effect of •OH. Thus, alternative approaches have been employed to boost the availability of reactive oxygen species (ROS) for tumor catalytic treatments. For example, parallel catalytic behavior was found to simultaneously generate superoxide ion (O₂•⁻) and •OH following peroxidase-like catalytic activity⁹. A co-catalysis approach was designed by taking MoS₂ support as a co-catalyst to accelerate the conversion of Fe³⁺ to Fe²⁺ for Fenton reactions¹⁰. Integrated cascade reactions have also been proposed based on Fenton- and peroxidase-like activities to concurrently generate •OH and O₂•⁻¹¹. Recently, a MOF-derived flower-like structure was created to provide 3D accessibility of active sites to boost •OH following peroxidase-like activity¹². Apart from the use of endogenous H₂O₂, recently copper hexacyanoferrate forming the single-site nanozyme was fabricated to process glutathione-oxidase, resulting in H₂O₂ formation accompanying the conversion of Cu²⁺ to Cu⁺ for the subsequent Fenton-like reaction leading to •OH¹³. In this work, the single-atom Au on the zero-valent Cu nanocubes was fabricated to effectively reduce O₂ to form H₂O₂ for the following •OH production (Fig. 1a).

**Fig. 1: Characterization of Au/CuO nanocubes.**

A general approach to creating atomically dispersed metal typically uses a heterogeneous active metal as a single-atom anchored on support following bottom-up methods, such as adsorption following impregnation and co-precipitation, or top-down methods using pyrolysis to conduct evaporation process for the generation of single-atom sites^14,15. On the other hand, galvanic replacement is a process that follows a redox reaction to gradually replace one metal with another metal ion with a higher reduction potential to construct alloyed nanostructures, thereby creating diverse morphologies, shapes, and sizes. Galvanic replacement can thus be viewed as a hybrid bottom-up and top-down approach. This reaction process has great potential to achieve single-atom site formation by controlling either the number of metal ions added (higher reduction potential) or the reaction period of galvanic replacement. However, few reports have described the use of galvanic replacement to manipulate SACs and are largely limited to the catalysis of hydrogenation or glucose oxidation from Cu/Pt_(SA), Cu/Pd_(SA), Ni/Pt_(SA), and Pd/Au_(SA)^{16,17,18,19,20,21}. Here, we use a galvanic replacement reaction to fabricate the Au single atoms on zero-valent copper nanocubes. The reduction potentials of copper (+0.522 V, Cu⁺/Cu; +0.341 V, Cu²⁺/Cu) are more negative than that (+0.695 V) of O₂/H₂O₂, making zero-valent Cu thermodynamically feasible to reduce oxygen to H₂O₂. Therefore, the zero-valent Cu can be an O₂ activator for •OH production. Specifically, the resulting Au_0.02Cu_0.98 composition reveals the enhanced •OH generation, making it a promising chemodynamic agent, following O₂ → H₂O₂ → •OH. The degradable Au_0.02Cu_0.98 released Cu⁺ and Cu²⁺ resulting in oxygen reduction and Fenton-like reactions. Instead of using endogenous H₂O₂, H₂O₂ can be supplied from the O₂ under aerobic conditions. Simulation has indicated that the Au single atom facilitates zero-valent copper to reveal the catalytic property of Au_0.02Cu_0.98 composition for O₂ → H₂O₂ → •OH as well. Furthermore, the Au_0.02Cu_0.98 structure is degradable under acidic conditions, which favors the excretory system through urinary metabolism when applied in tumoral treatments.

Results and discussion

Characterization of Au/Cu⁰ nanocubes

Hydrophobic-based Cu nanocubes were prepared and implemented by oil/water phase transformation with cetyltrimethylammonium bromide (CTAB) surfactant and polyvinylpyrrolidone (PVP) treatments in a water phase. The characteristic results of Cu nanocubes are shown in detail in Fig. 1b, c and Supplementary Fig. 1. The Cu nanocubes have a surface plasmonic resonance (SPR) absorption band at 580 nm (Fig. 1b) and an edge length of 50 nm (Fig. 1c and Supplementary Fig. 1a). The XRD results and electron diffraction pattern show a well-defined crystalline structure for the Cu nanocube corresponding to a face-centered cubic (fcc) crystal structure with (111), (200) and (220) faces (Supplementary Fig. 1b–e). The various ratios of Au/Cu⁰ nanocubes were synthesized through a galvanic replacement reaction by tuning the addition of precursor HAuCl₄⁻. The oxidized Cu atoms were replaced by Au⁰ atoms owing to the different redox potential between Cu²⁺/Cu (0.34 V vs. the standard hydrogen electrode [SHE]) and AuCl₄⁻/Au (0.99 V vs. SHE). The SPR bands of Au/Cu⁰ nanocubes gradually red-shifted to 590 nm following the increase of Au atoms, Au_0.02Cu_0.98 → Au_0.05Cu_0.95 → Au_0.1Cu_0.9 → Au_0.5Cu_0.5 (Fig. 1b). The fixed concentration of Cu nanocubes (10⁴ ppm, 100 μL) was used for galvanic replacement reaction accompanied with 50 mM HAuCl₄ in the volumes of 5, 10, 20, 40, 60, 80, and 100 μL, respectively. A 5 μL of HAuCl₄ obtained Au_0.02Cu_0.98. The ratios of different compositions for nanocubes are determined by AA measurements. The corresponding TEM images of the nanocubes can be seen in Fig. 1d–g. The morphology change could be observed beginning from solid to the rough surface and then turning into a cage-like structure as the Au atoms increased. For comparison, Au nanocubes with similar edge lengths²² were also fabricated for further studies (Supplementary Fig. 2). Au nanocubes have an SPR band at 535 nm (Fig. 1b). It is noted that the composition and morphology of Au_xCu_y could be further transferred into alloyed nanocages (Au_0.75Cu_0.25), nanoframes (Au_0.8Cu_0.2), and the completed replacement yielding Au nanoparticles with the addition of the volumes of HAuCl₄ in 60, 80, and 100 μL, respectively. The characteristic results including the TEM images, XRD patterns, and HR-TEM images were shown in Supplementary Fig. 3 to Fig. 5. The alloyed nanocages (Au_0.75Cu_0.25) and nanoframes (Au_0.8Cu_0.2) could be obviously observed by TEM images and the corresponding XRD patterns were assigned to fcc crystal structure individually followed JCPDS cards 01-071-5023 and JCPDS cards 01-072-5241. In addition, the HR-TEM images and their related electron diffractions indicate the arrangements of (200) and (110).

Synchrotron powder X-ray diffraction (XRD) measurements were performed to verify the face-centered cubic (FCC) crystal structure of the Au/Cu⁰ nanocubes. Details of the material characterization experiments are given in the Supporting Information. As shown in Fig. 1h, the characteristic peaks in the XRD patterns of Au/Cu⁰ nanocubes can be attributed to Au (COD ID. 1100138) and Cu (COD ID. 4105040) in the Fm-3m cubic phase. The sharp peaks for Cu nanoparticles indicate larger crystal sizes, while the broadened peaks are indexed to gold-rich crystalline structures with smaller grain sizes. The grain size was calculated based on the peak width according to the Scherrer equation (Supplementary Table 1). The grain sizes of the gold-rich crystalline structures of the Au/Cu⁰ nanocubes are close to 3.5 nm, while the copper nanoparticles are between 35–39 nm. The Rietveld refinement method was used to evaluate the crystalline phases and their quantities. The composition of Au/Cu⁰ is close to its nominal composition (Supplementary Table 2 and Supplementary Fig. 6). The XRD peak area at 40° with an index of Au(111) shows that the integrated peak areas in these Au/Cu⁰ nanocubes decrease with Au content. By controlling the concentration of Au, the diffraction intensity of the gold-rich crystalline structures decreases, and an Au_0.02Cu_0.98 in particular becomes very weak. In fact, high-resolution TEM clearly shows that single Au atoms are on the surface of the Cu⁰ nanocubes in Au_0.02Cu_0.98, which will be shown later.

Although atomic-resolution TEM can provide direct visualization of single-atom composition, it only provides information about a specific local area. In contrast, X-ray absorption spectroscopy (XAS) is element-sensitive and can be used to measure microcrystalline and amorphous materials with a large number of samples. XAS can be divided into X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS). XANES is subjected to the measured elemental oxidation states and their local symmetries. In contrast, EXAFS can detect chemical bond distances (~0.01 Å level), coordination numbers, and structural disorders to detect the detailed local structure of elements. Unlike distinguishable single-atom composition, indistinguishable single-atom composition usually consists of two metallic elements with similar atomic numbers, or the two have close X-ray absorption energies. Thus, the characterization of such materials is very challenging^23,24,25. We use a silicon drift detector (SDD) to collect energies at the Au Lα(9713 eV) in a 20 eV window, which allows for the elimination of fluorescence from Cu Kα, Kβ, and other scattered X-rays. The XANES spectra of Au L₃-edge and Cu K-edge of Au/Cu⁰ nanocubes are respectively shown in Fig. 1i and j. Overlaid XANES spectra of Au/Cu⁰ nanocubes and metal foils indicate that Au and Cu are still the predominant oxidation states of zero valences. This suggests that the charges of Au and Cu are not changed after galvanic replacement. The Fourier-transformed EXAFS at the Au L₃-edge and Cu K-edge with model fits are, respectively, shown in Fig. 1k and l. The detailed fitting results are summarized in Supplementary Table 3.

As shown in Fig. 1k and Table 1, the EXAFS spectrum of the Au L₃-edge shows that the Au/Cu⁰ nanocubes exhibit coordination shells at 2.64–2.7 Å, which is attributed to the Au–Cu bond in the first shell, while the second shell is the Au–Au bond at 2.82 Å. On the other hand, Fig. 1l and Table 1 show that the first shell of the Cu–Cu bond is located at 2.54 Å. The absence of Cu–Au coordination shells in the Cu K-edge EXAFS is due to the large Cu core nanocrystalline signal overshadowing the weak Cu–Au signal. This indicates that the galvanic replacement between Au atoms and Cu nanocubes is easily accomplished, leading to the formation of Au_0.02Cu_0.98 nanocubes. The coordination numbers (CN) of Au_0.02Cu_0.98 bonded to Cu and Au with Au as the central atom are, respectively, 4.73 and 6.87. These findings indicate the formation of Au atoms anchored on the surface of Au_0.02Cu_0.98 nanocubes. The bond distance of Au–Cu (2.67 Å) is longer than that of Cu–Cu (2.54 Å) and shorter than that of Au–Au (2.82 Å), which may create tensile-strained Cu atoms coordinated to Au atoms, thus boosting its chemical reactivity²⁶.

Table 1 EXAFS fitting results of Au L₃_edge and Cu K_edge for Au/Cu⁰ nanocubes^a

Full size table

After an integral structural characterization in XRD, XANES, and EXAFS measurements, the Au_0.02Cu_0.98 specifically appeared to be an atomically dispersed Au onto Cu⁰ nanocube. Therefore, we further explore the features of Au_0.02Cu_0.98 using HR-TEM and aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (AC-HAADF-STEM). As seen in Fig. 1m–o, the compositions of Au and Cu in Au_0.02Cu_0.98 could be, respectively, identified as 2.5% and 97.5% by EDX-mapping measurements, which is consistent with the AA determination. The Au_0.02Cu_0.98 reveals a well-fined crystalline with a lattice spacing of 0.18 nm corresponding to the (200) plane (Fig. 1p–r). Importantly, the AC-HAADF-STEM with the atomic resolution confirms atomically Au atoms (marked by red circles) bearing onto the Cu nanocube, as seen in Fig. 1r and the magnified view of Fig. 1r in Supplementary Fig. 7.

Self-supplying H₂O₂ from aerobic O₂ and Fenton-like reaction

Since the zero-valent Cu has been verified for the as-prepared nanocubes, the reduction potentials of copper (+0.522 V, Cu⁺/Cu; +0.341 V, Cu²⁺/Cu) are more negative than that (+0.695 V) of O₂/H₂O₂, making it possible for zero-valent Cu to thermodynamically reduce oxygen (O₂) to H₂O₂. A colorimetric method was used to measure H₂O₂ generation from the oxidation reaction between H₂O₂ and KMnO₄ by the observation of the drop of the absorption band at 525 nm contributed from KMnO₄ (Fig. 2a). The absorbance intensity of 525 nm decreased, indicating H₂O₂ generation, showing a different degree of color faded in colloidal solutions from the Au/Cu⁰ and pure Cu nanocubes, but no change in the Au nanocubes. The quantitative analysis of intensity from the response of H₂O₂ is summarized in Fig. 2b. The Au_0.02Cu_0.98 reveals superior H₂O₂ generation compared to other nanocubes. Next, we inspect whether the H₂O₂ generation is associated with aerobic or anaerobic conditions. A control examination is conducted to incubate Au_0.02Cu_0.98 nanocubes with KMnO₄ under N₂-filled (anaerobic) conditions. After 7 days of storage, we observed no change to color and absorption intensity at 525 nm, consistent only with the KMnO₄ (Fig. 2c and Supplementary Fig. 8). Therefore, the H₂O₂ is in situ generated from the O₂ in the aerobic environment. The quantitative results of H₂O₂ were shown in Fig. 2d. The H₂O₂ generation was evaluated by hydrogen peroxide assay kit in different compositions of Au_xCu_y (Cu, Au_0.02Cu_0.98, Au_0.05Cu_0.95, Au_0.1Cu_0.9, Au_0.5Cu_0.5, and Au). The largest H₂O₂ generation was calculated to be 122 μM in Au_0.02Cu_0.98 in 10 min of reaction. The amount of H₂O₂ in Au_0.02Cu_0.98 is higher than the endogenous H₂O₂ in tumoral microenvironments (~100 µM) and can be generated in the presence of O₂. The Au_0.02Cu_0.98, which has the greatest capability to form H₂O₂, was then chosen to demonstrate time- and concentration-dependence (Fig. 2e and Supplementary Fig.9). Because of the generation of H₂O₂, Cu is a potential candidate to process Fenton-like reaction for •OH formation. The •OH generation was identified by the increased fluorescence emission (λ_em = 425 nm) of the terephthalic acid (TPA) probe in different pH values (Fig. 2f). More •OH can be produced by Au_0.02Cu_0.98 than other groups of Au/Cu⁰ nanocubes. Specifically, a significant enhancement can be observed in acid conditions. The •OH increased as a function of Au_0.02Cu_0.98 concentration at pH 5.5 (Fig. 2g). The amplitude of the electron spin resonance (ESR) signal evidenced more •OH generation in Au_0.02Cu_0.98 among the Au/Cu⁰ nanocubes (Fig. 2h). According to the above results, the Au_0.02Cu_0.98 could act as a promising chemodynamic agent following O₂ → H₂O₂ → •OH reactions.

**Fig. 2: H₂O₂, and ‧OH generation from Au/CuO nanocubes.**

The degradable nature of the Au_0.02Cu_0.98 nanocubes

Since we have observed the reactions of O₂ → H₂O₂ → •OH, the zero-valent Cu (Cu⁰) is likely oxidized to Cu⁺ and Cu²⁺. The nanocubes may release oxidized copper ions resulting in the dissolution of structures. The stability of Au_0.02Cu_0.98 nanocubes showing superior H₂O₂ and •OH generation was then monitored for 7 days under H₂O, PBS (pH 7), and PBS (pH 5.5) (Fig. 3a). Under PBS, nanocubes already started to decompose at day 0. Higher acidity resulted in faster dissolution, with nanocubes nearly disintegrating completely after 1-day incubation. Under an H₂O environment, the structures also showed apparent dissolution after 3-day incubation. This self-decomposing behavior makes the Au_0.02Cu_0.98 nanocubes a promising renal-clearable agent when applied in in vivo for tumor studies. We further used XPS measurements to inspect the signal of Cu²⁺ formation by incubation of Au_0.02Cu_0.98 nanocubes in PBS (pH 7) after 1 day. The intensity of Cu²⁺ increased post-1 day given the evidence of Cu⁰ oxidized to Cu²⁺(Fig. 3b). On the one hand, the degradable nature under acidic conditions favors excretion through urinary metabolism. On the other hand, the self-decomposing behavior may also cause oxidative stress in the course of the blood circulation in vivo because of •OH generation. To avoid the self-generation of H₂O₂ during blood circulation, the stearic acid (SA) molecule was then used to modify the surface of the nanocubes. The modification of SA retained the structure of Au_0.02Cu_0.98 nanocubes resulting in the dispersed colloidal solutions (Fig. 3c). A layer of SA coated on the nanocube can be clearly seen (inset of Fig. 3c). FTIR analysis provided evidence of SA on the surface of Au_0.02Cu_0.98 nanocubes (Supplementary Fig. 10). To further support the successful SA coating on nanocubes, the generation of H₂O₂ (Fig. 3d) and •OH (Fig. 3e) was, respectively, conducted using KMnO₄ treatment and TPA fluorescence probe. Neither a drop in intensity from the KMnO₄ treatment nor fluorescence from the TPA probe was detected from Au_0.02Cu_0.98@SA. The generation of H₂O₂ and •OH was successfully suppressed after SA modification. In addition, the Au_0.02Cu_0.98@SA nanocubes exhibited excellent stability and retained their morphology features under different solution conditions (Fig. 3a). No dissolution of the nanocubes was seen after 7-day incubation.

**Fig. 3: The stability and self-decomposition behavior in Au_0.02Cu_0.98 and Au_0.02Cu_0.98@SA under different conditions (H₂O, PBS at pH = 5.5 and 7).**

Simulation analysis for oxidase and Fenton-like reactions

To gain more insight into the mechanisms by which Au/Cu⁰ nanocubes proceed with the reactions of O₂ → H₂O₂ → •OH, density functional theory (DFT) was carried out to calculate the reaction barriers of hydrogen peroxide formation via O₂ hydrogenation on Au/Cu⁰ nanocubes with different Au/Cu ratios, including pure Cu, Au_0.02Cu_0.98, Au_0.5Cu_0.5, and pure Au. The (100) surface model was considered for these Au/Cu⁰ nanocubes, and the surfaces were pre-covered with hydrogen atoms. The water dehydrogenation reaction to produce the hydrogen atoms was also calculated. As shown in Supplementary Table 4 and Supplementary Fig. 11, the first dehydrogenation barriers of H₂O are similar on pure Cu, Au_0.02Cu_0.98, and Au_0.5Cu_0.5, but they are much larger on pure Au. The second dehydrogenation barriers of the OH group on pure Cu and Au_0.02Cu_0.98 are smaller than those on Au_0.5Cu_0.5 and pure Au. It reveals that the hydrogen formation rates on Cu and Au_0.02Cu_0.98 are faster than those on Au_0.5Cu_0.5 and pure Au. Besides, we have also calculated the adsorption energies of the H₂O molecule, OH group, H atom, and O atom on all possible active sites at the Cu(100), Au_0.02Cu_0.98(100), Au_0.5Cu_0.5(100), and Au(100) surfaces, as shown in Supplementary Figs. 11–15 and Supplementary Tables 4–8. The intermediates in the reaction mechanisms were carried out using the most stable adsorption structures to proceed. All the adsorption energies, activation energies, and reaction energies were considered using the solvent effect.

Supplementary Figs. 16, 17 and Supplementary Table 9 show the side-on and end-on configurations of O₂ molecules on all the possible active sites at Cu(100), Au_0.02Cu_0.98(100), Au_0.5Cu_0.5(100), and Au(100) surfaces. According to the calculated adsorption energies, all the results show that the side-on configuration is more stable than the end-on configuration for the O₂ molecule. The calculated most stable adsorption energies of O₂ on pure Cu, Au_0.02Cu_0.98, Au_0.5Cu_0.5, and pure Au are, respectively, −2.12, −2.00, −1.16, and −0.42 eV. These results indicate that the interaction of O₂ to the surface is proportional to the number of copper atoms, meaning that the gold atoms would reduce the activity of the copper atoms in the alloys. In addition, as shown in Fig. 4a, the calculated hydrogenation barriers from O₂ to OOH and OOH to H₂O₂ on pure Cu are 1.32 and 1.29 eV, respectively. For the Au_0.02Cu_0.98, there are two different reaction pathways via two chemical environments: (1) with the Au atom and (2) without the Au atom (Fig. 4b). The hydrogenation barriers of two-step reactions in pathway 1 are 0.75 and 0.76 eV, while those in pathway 2 are 0.98 and 0.99 eV. For both pathways, the activation energy of the hydrogenation reaction on the Au_0.02Cu_0.98 is smaller than that of Cu. For the Au_0.5Cu_0.5, the two hydrogenation barriers for forming the H₂O₂ molecule from O₂ are 1.15 and 0.61 eV (Fig. 4c). Figure 4d shows the hydrogenation barriers from O₂ to OOH and OOH to H₂O₂ are, respectively, 0.48 and 0.74 eV on pure Au. Although the activation energies are small on both Au_0.5Cu_0.5 and pure Au, the first hydrogenation barrier is larger than the adsorption energy of the O₂ molecule. That is, the reaction of the first hydrogenation on these two surfaces is more difficult, resulting in the relatively poor reactivity of O₂ hydrogenation on both Au_0.5Cu_0.5 and pure Au.

**Fig. 4: Simulation analysis in Cu, Au, and different Au/CuO.**

Furthermore, the reaction energies between the H₂O₂ desorption and the production of OH radicals were also performed. For the production of •OH radical, we carried out a Fenton-like reaction as follows: \({{{{{{\rm{H}}}}}}}_{2}{{{{{{\rm{O}}}}}}}_{2\left({{{{{\rm{a}}}}}}\right)}\to \,{{{{{{\rm{OH}}}}}}}_{({{{{{\rm{a}}}}}})}^{-}+\,\bullet {{{{{{\rm{OH}}}}}}}_{({{{{{{\rm{aq}}}}}}})}\,\); one •OH radical can form in the solvent via desorption and the other OH group would adsorb on the surface to form OH⁻ anion. The OH⁻ + •OH can be clarified by the projected density of states (PDOS). As shown in Supplementary Fig. 18, the peaks of the adsorbed OH group have no spin-splitting property, while the gas-phase OH group possesses the spin-splitting character. This reveals that the electron configuration of the adsorbed OH group is fully-filled, but the electron configuration of the gas-phase OH group is half-filled. Thus, the adsorbed OH and the gas-phase OH group can be regarded as OH anion and •OH radical, respectively. On the pure Cu, the desorption energy of H₂O₂ is 0.35 eV while the reaction energy of •OH radical is −0.03 eV. For the Au_0.02Cu_0.98, the desorption energy of H₂O₂ is 0.34 eV, whereas the reaction energy of •OH radical formation is −0.01 eV. The •OH radical formation energies are less than those of H₂O₂ desorption on both pure Cu and Au_0.02Cu_0.98, indicating that the •OH radical formation readily occurs on these two nanocubes. Contrarily, the desorption energy of H₂O₂ is smaller than that of •OH radical formation on either Au_0.5Cu_0.5 or pure Au, reflecting that the •OH radical production on these two Au/Cu⁰ nanocubes is thermodynamically unfavorable. Overall, DFT results reveal that the kinetic effect of H₂O₂ formation on Au_0.02Cu_0.98 is faster than on other compositions of nanocubes, and the thermodynamic effect of •OH radical production is favorable on Au_0.02Cu_0.98. All the calculated reaction barriers and reaction energies of O₂ hydrogenation to H₂O₂ and •OH formation are listed in Table 2. The reactivity of •OH radical production on Au_0.02Cu_0.98 is superior to pure Cu, Au_0.5Cu_0.5, and pure Au.

Table 2 Calculated reaction barriers (E_a in eV) and reaction energies (ΔE in eV) for elementary reactions of O₂ hydrogenation to H₂O₂ on the Au/Cu⁰ nanocubes

Full size table

Figure 4e–h shows the electron localization function (ELF) diagrams of the pure Cu, Au_0.02Cu_0.98, Au_0.5Cu_0.5, and pure Au. The electrons locate around the hollow position between the copper atoms on the pure Cu (Fig. 4e), reflecting the strong activity of the hollow site for the pure Cu. On the other hand, the electron localization around the hollow site becomes weaker while the electrons begin to accumulate more to the gold atoms from Au_0.02Cu_0.98 to Au_0.5Cu_0.5 (Fig. 4b and c). For the pure Au (Fig. 4d), the ELF plot displays the electron localization around the gold atoms rather than at the hollow region. Since the electronegativity of Au is larger than the Cu, the charge of the Au atoms can become more negative in the Au/Cu⁰ nanocubes. According to previous theoretical studies^27,28, increased negative charge distribution of Au atoms will reduce the activity of bimetallic systems. As there is small amount of Au atoms in Au_0.02Cu_0.98, the activity of Au_0.02Cu_0.98 is anticipated to slightly decrease, thus facilitating the reactivity of the O₂ hydrogenation to H₂O₂. As a reference, our aforementioned XANES measurements indicate the charges of Au and Cu do not change after galvanic replacement. Generally, the association reaction between the reactant (i.e. O₂ here) and hydrogen atoms would be enhanced as the binding activity gradually decreases between the catalyst (i.e. Cu here) and both reactant and hydrogen atoms²⁸. That is, the small amount of Au atoms in Au_0.02Cu_0.98 reduces Cu activity in oxygen binding, which in turn strengthens the hydrogenation reaction. In comparison with pure Cu, the adsorption activities of O₂ and H atoms are weakened by small amounts of Au atoms in Au_0.02Cu_0.98 and then the catalytic property of O₂ reduction to H₂O₂ is enhanced at the Au_0.02Cu_0.98. As the concentrations of Au atoms in the Au/Cu⁰ nanocubes gradually increase to the Au_0.5Cu_0.5 and pure Au, the binding strengths to O₂ are significantly decreased. However, this change weakens the overall reactivity of both Au_0.5Cu_0.5 and pure Au because the hydrogenation barriers exceed the adsorption energy of the O₂ on both Au_0.5Cu_0.5 and pure Au. Therefore, although the binding strength to O₂ can be decreased on both Au_0.5Cu_0.5 and pure Au, the large amount of Au atoms limits, rather than facilitates, the reaction.

The mechanisms by which of the degradable Au/Cu⁰ nanocubes to trigger the self-supply of H₂O₂ followed by the Fenton-like reaction are then proposed to perform CDT. As shown in Fig. 1a, the different ratios of Au/Cu⁰ nanocubes could be synthesized through galvanic replacement. Once Au_0.02Cu_0.98 nanocubes proceed the endocytosis, the nanocubes can act as catalysts for oxygen hydrogenation and the subsequent Fenton-like reaction. Concurrently, Au_0.02Cu_0.98 begins to oxidize from Cu⁰ to Cu⁺ and/or Cu²⁺ (Cu → Cu⁺ + e⁻ → Cu²⁺ + 2e⁻). Evidence for the presence of Cu⁺ in cells will be provided later using a CopperGreen dye, a Cu⁺ indicator. The released electrons are then used to reduce O₂ to H₂O₂ under acidic conditions (O₂ + 2H⁻ + 2e⁻ → H₂O₂). Therefore, the catalytic reactions of Au_0.02Cu_0.98 and the formation of the released Cu⁺ or Cu²⁺ simultaneously contribute to the generation of H₂O₂ and •OH. The released copper ions cause the nanocubes to decompose, which favor metabolic excretion.

In vitro evaluation

For in vitro studies, all Cu, Au/Cu⁰, and Au nanocubes were modified with SA. MTT assay was used to investigate the cell viability of HepG2-Red-FLuc hepatocellular carcinoma cells incubated with Cu@SA, Au/Cu⁰@SA, and Au@SA nanocubes where Au_0.02Cu_0.98@SA outperformed other groups in killing cancer cells (Fig. 5a). Fluorescence staining experiments in live and dead cells also demonstrated the efficacy of Au_0.02Cu_0.98@SA in damaging cancer cells (Fig. 5b). Decreased cell survival rate was observed as a function of Au_0.02Cu_0.98@SA concentrations (Fig. 5c). Flow cytometry analysis demonstrated the increased late-apoptosis after 24 h incubation in Au_0.02Cu_0.98@SA nanocubes (Fig. 5d). The cells incubated with Au_0.02Cu_0.98@SA have a relatively higher late apoptotic ratio (39.09%) compared to other groups (cell only: 0%, Cu@SA: 16.43%, Au_0.05Cu_0.95@SA: 14.24%, Au_0.1Cu_0.9@SA: 5.5%, Au_0.5Cu_0.5@SA: 0.17%, and Au@SA: 0.42%). The decomposition of nanocubes was again monitored by the morphological change when nanocubes were cultured with cancer cells as a function of time (Fig. 6a). TEM images displayed the structural disintegration of Au_0.02Cu_0.98@SA nanocubes with cells as a function of time as compared with nanocubes with no cell incubation. CopperGreen dye with green emission was used to verify the presence of oxidized Cu⁺ ions in cells (Fig. 6b). In addition, hydrogen peroxide assay kit and APF fluorescence agent were, respectively, used to confirm the signals of H₂O₂ and •OH produced from Au_0.02Cu_0.98@SA in cells (Fig. 6c, d). Contrarily, the very weak fluorescence signals were seen in anoxic condition (2% O₂) corresponding to the low amount of H₂O₂ and •OH generation, indicating the support of the O₂ → H₂O₂ → •OH reactions under Au_0.02Cu_0.98@SA treatment (Supplementary Fig. 19). No hemolysis and damage in vascular endothelial cells were observed from Au_0.02Cu_0.98@SA (Fig. 5e, f), which ensures safety in the course of blood circulation.

**Fig. 5: In vitro studies of Cu@SA, Au@SA, and Au/Cu0@SA nanocubes.**

**Fig. 6: In vitro studies of Cu@SA, Au@SA, and Au/Cu0@SA nanocubes.**

In vivo animal studies against tumors

To validate the in vivo biosafety of nanocubes, the C57BL/6 mice were intravenously administered with 600 ppm Au_0.02Cu_0.98@SA (in sterilized PBS) and continuously observed for 7 days. There was no significant difference in the body weight, blood biochemical index (for liver and kidney functions), and histological morphology (heart, lung, liver, spleen, kidney) between the nanocubes group and PBS control group, which suggests our Au_0.02Cu_0.98@SA nanocubes do not have acute toxicity. (Supplementary Figs. 20, 21 and Fig. 7a). In vivo biodistribution indicates that, among major organs, nanocubes accumulation concentrates in the liver, but gradually decreased as a function of time. However, the Cu content in urine increased post-24 h (Fig. 7b), which is associated with the decomposition of Au_0.02Cu_0.98@SA to release copper ions. The amount of copper accumulation in urine was 10 times greater than that of the PBS post-24 h, indicating the clearance effect. The inset of Fig. 7b has removed liver results to clarify the enhanced accumulation in urine.

**Fig. 7: In vivo anti-tumor activity of the mice with HepG2-Red-FLuc orthotopic tumors.**

The in vivo antitumor efficacy was established in the HepG2-Red-FLuc orthotopic hepatocellular carcinoma xenograft mice. The tumor-bearing mice were randomly treated with one dose of 600 ppm nanocubes including Au@SA, Au_0.02Cu_0.98@SA, Au_0.5Cu_0.5@SA, and Cu@SA as well as PBS (as control) through intravenous administration, respectively. The Cu@SA but not Au@SA shows the anti-tumor effect observed from the IVIS system (Fig. 7c), which is consistent with the in vitro study. Surprisingly, the one-dose treatment with Au_0.02Cu_0.98@SA exhibited more remarkable antitumor efficacy, showing significant inhibition of tumor growth, than the Cu@SA. On the other hand, the different atomic ratio of Au_0.5Cu_0.5@SA cannot suppress tumor growth, indicating the significant efficacy of Au_0.02Cu_0.98@SA (Fig. 6c). The tumoral sizes (Supplementary Fig. 22a) and the total flux by IVIS (Fig. 6d) of the remaining hepatocellular carcinoma treated with different nanocubes were analyzed after mice sacrificed. Among the treatments, Au_0.02Cu_0.98@SA was confirmed to possess the best anti-tumor effect with tumor nearly suppressed. We also examined the morphology change in tumors, and found that Au_0.02Cu_0.98@SA treatment induced more tumor necrosis (area outside the white dots enclosed region in each picture) than other treated groups (Supplementary Fig. 22b). Moreover, the abundant nuclear expression of γ-H2AX (DNA damage marker) and cytoplasmic expression of cleaved caspase-3 (apoptosis cell death marker) were observed within the tumor region because of the excessive ROS production by Au_0.02Cu_0.98@SA treatment in tumors (Fig. 6e). Taken together, these results indicate the excellent efficacy of Au_0.02Cu_0.98@SA for antineoplastic therapy in hepatocellular carcinoma. Evaluation of the clinical potential is in progress.

We used galvanic replacement to fabricate different compositions of zero-valent Cu nanocubes which generated H₂O₂ and •OH by taking O₂ from air under aerobic conditions. Specifically, Au_0.02Cu_0.98 outperformed other Au/Cu⁰ nanocubes in the production of H₂O₂ and •OH. The degradable Au_0.02Cu_0.98 released Cu⁺ and Cu²⁺ resulting in oxygen reduction and Fenton-like reactions. Simulation analysis also revealed the catalytic capability of Au_0.02Cu_0.98 with the facilitation of Au for O₂ → H₂O₂ → •OH reactions.

Methods

Chemicals

All reagents were in analytical purity and used without further purification. Ethanol (C₂H₅OH, 99.9%) was purchased from J.T. Baker. Aminophenyl fluorescein solution (APF, C₂₆H₁₇NO₅, 98%) was acquired from Life Technologies. A hydrogen peroxide assay kit was acquired from abcam. CopperGreen dyes were acquired from Merck. Copper(I) bromide (CuBr, 98%), Octadecylamine (CH₃(CH₂)₁₇NH₂, 99%), trioctylphosphine oxide ([CH₃(CH₂)₇]₃PO, 90%), Oleylamine (CH₃(CH₂)₇CH=CH(CH₂)₇CH₂NH₂, 90%), cetyltrimethylammonium bromide (C₁₉H₄₂BrN), Polyvinylpyrrolidone (PVP, (C₆H₉NO)_n, MW = 55,000), hydrogen peroxide solution (H₂O₂, 30%), Sodium bromide (NaBr, 99.5%), ascorbic acid (C₆H₈O₆, 99%), cetyltrimethylammonium chloride (C₁₉H₄₂ClN, 25%), Sodium borohydride (NaBH₄, 99%) and 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT, C₁₈H₁₆BrN₅S, 97.5%) were bought from Sigma-Aldrich. Water was obtained by using a Millipore direct-Q deionized water system throughout all studies.

Preparation of Cu nanocubes

A total of 0.05 g of CuBr, 0.08 g of octadecylamine (ODA), and 1 g of trioctylphosphine oxide (TOPO) were mixed in 20 mL of oleylamine. Subsequently, the reaction apparatus was filled with argon, heated at 20 °C/min in a heating jacket, and maintained at 300 °C for 10 min until the Cu nanoparticles (NPs) grown into nanocubes. The reaction solution was cooled to room temperature and centrifuged at 6200 × g for 5 min. After the supernatant was removed, the nanocubes were washed three times using a toluene solution. Finally, Cu nanocubes were collected and stored in oleylamine.

Preparation of Au/Cu⁰ nanocubes

The cetyltrimethylammonium bromide (CTAB) and polyvinylpyrrolidone (PVP) solutions emulsified the Cu nanocubes from the oil phase to the water phase. And then, Cu nanocubes acted as a sacrificial template of the galvanic replacement reaction, and HAuCl₄ solution was used as the precursor of the reaction. First, the Cu nanocubes were distributed in a 100-μL toluene solution with a concentration of 10,000 ppm. Subsequently, 10 mL of CTAB and PVP solution was added and shaken evenly, and the Cu nanocubes were emulsified from the oil phase to the water phase and dispersed in the aqueous solution. Various volumes of HAuCl₄ solution with a molar concentration of 50 mM were added to systematically alter the composition of the Au/Cu⁰ nanocubes. The Au/Cu⁰ nanocubes with an Au:Cu ratio of 0.02:0.98, 0.05:0.95, 0.1:0.9, and 0.5:0.5 were obtained after 5, 10, 20, and 40 μL of HAuCl₄ were added, respectively. The alloyed Au_xCu_y nanocages (Au_0.75Cu_0.25), nanoframes (Au_0.8Cu_0.2), and completed replacement Au nanoparticles were obtained after 60, 80, and 100 μL of 50 mM HAuCl₄ treatments, respectively.

Preparation of Au nanocubes

Gold seeds were prepared first by volume of 10 mL H₂O containing 1325 μL cetyltrimethylammonium chloride (CTAC) (25%), 500 μL of 5 mM HAuCl₄ solution, and 450 μL of 0.02 M NaBH₄. Next, two vials were labeled A and B. A growth solution was prepared in each of the two vials. First, both vials with 10 mL H₂O contained 1325 μL CTAC (25%), 500 μL of 5 mM HAuCl₄ solution, 10 μL of 0.01 M NaBr, and 90 μL of 0.04 M ascorbic acid. Next, 25 μL of the seed solution was added to the solution in vial A for 10 min. Then 25 μL of the solution in vial A was transferred to vial B and stirred for another 15 min. Finally, Au nanocubes were collected and stored in H₂O.

Structural characterization in XRD

The crystal structures were determined by synchrotron X-ray diffraction analysis (the incident X-ray wavelength of 0.7749 Å) using a large Debye–Scherrer camera in BL01C2 at the Taiwan Light Source (TLS) of the National Synchrotron Radiation Research Center in which the electron storage ring was operating at 1.5 GeV and 360 mA under top-up injection. Powder X-ray diffraction patterns of samples have been collected by transmission type. The powder samples are sealed in two Scotch tapes in the glove box of Ar atmospheres for preventing oxidization from the air. Two-dimensional powder X-ray diffraction patterns were recorded by the MAR345 imaging plate. One-dimensional PXRD profiles were integrated from the 2D patterns by GSAS-II software²⁹. The XRD patterns were calibrated by the CeO₂ standard and altered to that with a wavelength of 1.5406 Å for easy comperes. Rietveld refinement was applied to analyze crystalline phases by using GSAS software³⁰.

X-ray absorption spectroscopy analysis

X-ray absorption spectroscopy including X-ray absorption near edge spectra (XANES) and extended X-ray absorption fine structure (EXAFS) at Cu K-edge and Au L₃-edge were collected in transmission type at BL01C1 (TLS) and TPS 44A at Taiwan Photon Source (TPS). The scan range was kept in an energy range of 8700–9800 eV for Cu K-edge and 11,719–12,719 eV for Au L₃-edge for transmission type. Due to the low Au loading, the Au L₃-edge of Au_0.02Cu_0.98 was measured in fluorescence mode at TPS 44A. Since Cu has a fluorescence line near that of Au L Lα and there is a large loading of Cu, an energy-resolved fluorescence spectrometer using seven-element silicon drift detectors (SDD) was required to detect the weak Au fluorescence signal without saturating the detector with fluorescence form copper²⁵. Subtracting the baseline of pre-edge and normalizing that of post-edge obtained the spectra using Athena software. EXAFS analysis was conducted using Fourier transform on k³-weighted EXAFS oscillations to evaluate the contribution of each shell to the Fourier transform peak and fitted by using Artemis software³¹.

Computational details

All periodic DFT calculations in this study were performed with the generalized gradient approximation (GGA) of Perdew–Burke–Ernzerhof (PBE)³² change-correlation functional employing Vienna ab initio simulation program (VASP)^33,34,35,36. The projector augmented wave (PAW) method^37,38,39 is applied to describe the electron core interactions. The Kohn–Sham orbitals are expanded in a plane-wave basis set with a kinetic energy cutoff of 400 eV. Spin polarization calculation was also involved in the calculation. To carry out the dispersion correction, we performed DFT-D3 functional in our calculation⁴⁰. The convergence threshold was set to be 10⁻⁵ eV for the total electronic energy in the self-consistent loop. The atomic positions were relaxed using either the conjugate gradient algorithm until the x, y, and z-components of unconstrained atomic force were smaller than 2 × 10⁻² eV/Å.

All Au/Cu⁰ nanocubes systems were modeled by the FCC cubic unit cell, including pure Cu, Au_0.02Cu_0.98, Au_0.5Cu_0.5, and pure Au. The (100) slab model was considered for these Au/Cu⁰ nanocubes by using (3 × 3) lateral supercells with a vacuum space of 15 Å between the slab and its periodic replicas. The atomic layers in the slab in this study were constructed with six atomic layers models where the bottom three layers are fully fixed, as shown in Supplementary Fig. S23. The Brillouin zone was sampled with Monkhorst–Pack mesh⁴¹ to be (10 × 10 × 10) and (5 × 5 × 1) for the Au/Cu⁰ nanocubes unit cell and supercell, respectively. In addition, the Climbing Image Nudged Elastic Band (CI-NEB) method^42,43,44 was applied for finding transition states and the minimum energy path of all reactions. The adsorption energy (E_ads) of the species with the surfaces is defined by the formula: E_ads = E_mole./sur.–E_mole.–E_sur., where E_sur. is the total energy of the Au/Cu⁰ nanocubes, E_mole. is the total energy of a molecule in gas-phase, and E_mole./sur. is the total energy of the Au/Cu⁰ nanocubes together with the adsorbate. Furthermore, to realistically simulate the effect of the catalytic environment in the solution, we considered the implicit solvent model by using vaspsol^45,46. The implicit solvent effect of water was adopted in this work, where the corresponding dielectric constant of water is 78.3553.

To verify our computational settings, we have compared our results with some references regarding O₂ adsorption on the pure Cu and Au surface, including both experimental and theoretical reports. Experimentally, Cruickshank et al. reported that both O atom and OH⁻ could be chemisorbed on the 4-fold hollow site on the Cu(100) surface⁴⁷. Compared with our calculated results, the adsorption of the O₂ molecule on the 4-fold hollow site via side-on configuration is also the most stable adsorption structure of O₂ on the Cu(100) surface. Our results also reveal that the most stable adsorption structures of the OH group and O atom are all on the 4-fold hollow site on the Cu(100) surface. Thus, the trends of our computational results are in good agreement with the experimental observations. Theoretically, Gómez et al. have calculated that the most stable adsorption geometries of O₂, O, OH, and H₂O species on the Cu(100) surface are the hollow, hollow, hollow, and top sites, respectively⁴⁸. These results also have similar trends to our results.

For pure Au surface, Kim and Gewirth have observed the oxygen molecular adsorption on Au(100) surface via the SERS study⁴⁹. In addition, some theoretical studies have reported that the most stable adsorption site on Au(100) surface for oxygen molecules is at the hollow site^49,50. A recent DFT study by Oguz et al. showed that the adsorption energy of O₂ on the hollow site at Au(100) surface with PBE functional and DFT-D3 correction is −0.36 eV⁵⁰, which is similar to our results. Hence, according to these comparisons, our computational setting is reliable.

SA modified Au/Cu⁰ nanocubes

Briefly, Au/Cu⁰ nanocubes solution (1000 ppm) in 0.5 ml ethanol was mixed with 0.5% SA and sonicated for 5 min. Then, 1 mL of water was added to the mixture and sonicated for another 10 min, followed by centrifugation, and washing with water to remove any excess SA. The obtained pellet was suspended and diluted based on the requirements of the experiments.

Evaluation of H₂O₂ generation

For H₂O₂ detection, a colorimetric analysis in the presence of H₂O_2. KMnO₄ was dissolved in an aqueous solution containing H₂SO₄. The mixture was treated with Au/Cu⁰ nanocubes for 10 min. Subsequently, the UV−vis spectra were measured from 400 to 650 nm. The H₂O₂ quantitative of Au/Cu⁰ nanocubes was measured by using a probe hydrogen peroxide assay kit. The calibration curve was prepared in serial dilutions of 300 μM H₂O₂. The 20 ppm of Au/Cu⁰ nanocubes were mixed with a hydrogen peroxide assay kit for 10 min. Then, the fluorescence was measured at the emission wavelength of 510 nm (excitation wavelength of 490 nm) using a spectrofluorometer. The hydrogen peroxide assay kit solution alone in PBS was also carried out as a control group.

Evaluation of •OH generation capability

The •OH generation of Au/Cu⁰ nanocubes reacted with different pH of PBS were measured by using a probe terephthalic acid. The 20 ppm of Au/Cu⁰ nanocubes at various pH were mixed with terephthalic acid (0.1 M) for 10 min. Then, the terephthalic acid fluorescence was measured at an emission wavelength of 425 nm (excitation wavelength of 315 nm) using a spectrofluorometer. The terephthalic acid solution alone in PBS was also carried out as a control group.

Detection of •OH by ESR

The generation of •OH was evaluated by an ESR spectrometer using a DMPO spin-trapping adduct. During the experiments, the solutions included 100 mM DMPO and nanocubes. All mixtures were dispersed in PBS (0.1 M). The solutions were then aspirated into quartz capillaries for ESR analysis.

Stability analysis

For the stability examination, the Au_0.02Cu_0.98 nanocubes and Au_0.02Cu_0.98@SA nanocubes were individually dispersed in phosphate-buffered saline (PBS) (pH 7), PBS (pH 5), and deionized water in the Eppendorf tubes and incubated at 37 °C and observed for 7 days. For the structural stability of nanocubes at days 0, 1, 3, 5, and 7, the solutions were centrifuged, washed thrice with deionized water, and observed under TEM.

Cell culture

HepG2-Red-FLuc (Human hepatocellular carcinoma cell line) cells were maintained in MEM supplemented with 10% FBS and the antibiotics penicillin/streptomycin All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. HUV-EC-C cells (endothelial cell line) were cultured in F-12k containing EGCS (0.03 mg/mL), heparin (0.1 mg/mL), and fetal bovine serum (FBS, 10%) in the incubator at 37 °C and 5% CO₂. The anoxic condition was operated on by maintaining gas concentration at 2% O₂. HepG2-Red-FLuc cells were obtained from PerkinElmer (Product No. BW134280).

In vitro cytotoxicity test

The standard methyl thiazolyltetrazolium (MTT) assay was utilized to assess the toxicity of Au/Cu⁰ nanocubes in the HepG2-Red-FLuc hepatocellular carcinoma cell line. The cells were cultured in 96-well plates (1 × 10⁵ cells/well) for 24 h in complete media prior to treatment with various Cu concentrations in nanocubes at 37 °C under 5% CO₂ for 24 h. After washing the cells with PBS buffer, the fresh media containing MTT reagent (0.5 mg mL⁻¹) was added and incubated for another 4 h. Then, the medium was then substituted with DMSO to dissolve the resulting formazan. The absorbance was measured at a wavelength of 540 nm by using an ELISA reader.

Live and dead cells assay

The dyes of propidium iodide (PI) and Calein-AM were used to stain the dead and live cells, respectively. The HepG2-Red-FLuc cancer cells were seeded in 96-well plates (8000 cells/per well) and incubated for 24 h, followed by the treatments of medium alone (blank) or 100 ppm of Au/Cu⁰ nanocubes for another 24 h. After treatments, the cells were gently rinsed twice and further stained by PI and Calein-AM following the standard process. The distribution of dead and live cells was observed by laser scanning confocal microscope.

Flow cytometry assay

HepG2-Red-FLuc hepatocellular carcinoma cells were seeded in a 6-cm culture dish with a population of 5 × 10⁵ cells and incubated overnight. Cells were then treated with Au/Cu⁰ nanocubes at 100 ppm. Control cells (culture medium only as negative control and 2 μM Thapsigargin as positive control) were also included in this experiment. After 24 h, cells were washed twice with PBS (phosphate buffer saline) and were later detached by trypsinisation. Then, the cells were harvested and washed with PBS. Cells were then re-suspended in 500 μL of 1X annexin V binding buffer. Next, 10 μL of annexin-V(FITC) and 10 μL of propidium iodide were added to cells. Cells were incubated at room temperature for 15 min and analyzed by flow cytometry. Cell populations were initially gated using forward scatter and side scatter plots of the cell-only samples. The gate was set to remove dead cells and aggregates of cells and then applied to all samples (a figure exemplifying the gating strategy is provided in Supplementary Fig. 24).

In vitro Cu⁺ detection

The HepG2-Red-FLuc cancer cells were seeded in eight-well plates (10,000 cells/per well) and incubated for 24 h, followed by the treatments of CopperGreen dyes with the Au_0.02Cu_0.98@SA nanocubes for another 4 and 24 h. The cells were treated by the medium as the control group. And then, the cells were gently rinsed twice for further observation by laser scanning confocal microscope

In vitro H₂O₂ detection

The HepG2-Red-FLuc cancer cells were seeded in eight-well plates (10,000 cells/per well) and incubated for 24 h, followed by the treatments of Hydrogen Peroxide Assay Kit with the Au_0.02Cu_0.98@SA nanocubes for another 4 and 24 h. The cells were treated by the medium as the control group. And then, the cells were gently rinsed twice for further observation by laser scanning confocal microscope

In vitro •OH detection

The HepG2-Red-FLuc cancer cells were seeded in eight-well plates (10000 cells/per well) and incubated for 24 h, followed by the treatments of APF dyes (5 mM) with the Au_0.02Cu_0.98@SA nanocubes for another 4 and 24 h. The cells were treated by the medium as the control group. And then, the cells were gently rinsed twice for further observation by laser scanning confocal microscope

Hemolysis analysis

The 2% red blood cells were prepared in deionized water (positive control group), PBS (negative control group), and PBS containing with 20 ppm Au_0.02Cu_0.98@SA nanocubes. These solutions were stood at dark for 1 h. And then, the solutions were centrifugated at 6200×g for 5 min to evaluate the hemolysis condition.

Biosafety in vivo

Animal care was provided in accordance with the Laboratory Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of National Cheng Kung University (NCKU). All animal treatments and surgical procedures were performed in accordance with the guidelines of NCKU Laboratory Animal Center (IACUC No. 111177). The experimental mice were housed in cages (three to five mice in each cage) at 22–23 °C and 55 ± 10% humidity with 13 h/11 h light/dark cycle. C57BL/6 mice (6–8 weeks of age, female) were treated with 100 μL sterilized PBS or 100 μL 600 ppm Au_0.02Cu_0.98@SA (in sterilized PBS) through intravenous administration. The body weight of each group was recorded daily. After 7 days post-treatment, the blood was collected for biochemical analysis and normal organs (heart, lung, spleen, liver, and kidney) were gathered for H&E staining.

Hematoxylin and eosin (H&E) staining

The samples of tumors and other normal organs (heart, lung, spleen, liver, and kidney) were paraffin-embedded and then sliced into 5 μM thickness. The sections were deparaffinized, rehydrated, washed in PBS, and stained with hematoxylin solution for 3 min. After washing in tap water, the sections were stained with eosin solution for 1 min. Finally, the sections were immersed in ethanol and xylene and mounted for evaluation. The sections were observed under a microscope Olympus BX51 (Olympus, Japan), and three different fields for each group were taken.

Biochemical analysis

The mice’s blood was obtained from the heart, and then heparin sodium was added immediately. The collected blood samples were centrifuged at 100 × g for 10 min to obtain the serum. The serum samples were used for the blood biochemistry analysis for alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (T-Bil), blood urea nitrogen (BUN), creatine (CREA), and uric acid (UA) expression by a FUJI DRI-CHEM 4000i (FUJIFILM).

Biodistribution in vivo

Female SCID mice aged 4–7 weeks were obtained from the laboratory animal center, National Cheng Kung University, Taiwan. The Au_0.02Cu_0.98@SA nanocubes (600 ppm in Cu concentration, 100 µL) were intravenously injected into the SCID healthy mice (n = 3 per group). The control group with only PBS was also examined. The major organs of the mice (heart, liver, spleen, lungs, and kidneys) and urine were harvested and weighed and then the Cu content was analyzed by ICP-AES.

Establishment of HepG2-Red-FLuc orthotopic hepatocellular carcinoma model

NOD-SCID mice (6–8 weeks, female) were anesthetized using an intraperitoneal injection of Zoletil 100 (Virbac) and put in a face-up position. Then, 2 × 10⁶ HepG2-Red-Fluc cells in a solution containing 10 μL of PBS and 10 μL of Basement Membrane Matrix (BD) were surgically implanted into either right or left lobe of the liver using BD Insulin Syringes 30 G 3/10cc (BD). The wound was sutured using CT204 Chromic Catgut (20 mm, 75 cm, UNIK SURGICAL SUTURES MFG. CO.) as well as NC193 Monofilament Nylon (19 mm, 45 cm, UNIK SURGICAL SUTURES MFG. CO.) and the mice were allowed to rest until they fully recover. The tumor growth of HepG2-Red-FLuc hepatocellular carcinoma cells were monitored by the IVIS imaging system (Caliper Life Sciences). The maximal tumor burden permitted by Institutional Animal Care and Use Committee of NCKU was the weight of tumor should not exceed 10% of body weight and without ascites formation. All the experimental mice with orthotopic hepatocellular carcinoma were sacrifice before reaching the above standard.

IVIS imaging system and quantification

Mice were anesthetized with a mixture of oxygen and isoflurane and intraperitoneally injected with 100 μL of d-luciferin (catalog # 122796, Caliper Life Sciences). Later at 10 min, mice underwent imaging with the Xenogen IVISR Spectrum Noninvasive Quantitative Molecular Imaging System (Caliper Life Sciences) and analyzed with Living Image 4.7.3 (PerkinElmer, USA). After mice sacrifice, the liver organ was then collected and subjected to IVIS detection ex vivo.

Immunohistochemistry (IHC) staining

The tumor samples were paraffin-embedded and then sliced into 5 μm thicknesses. The sections were deparaffinized, rehydrated, incubated with phospho-histone H2A.X (Ser139) antibody (1:400 dilution, #9718, Cell Signaling Technology) or cleaved caspase-3 (Asp175) antibody (#9661, Cell Signaling Technology), and then stained by an ABC peroxidase standard staining kit (Thermo Fisher Scientific) containing biotinylated affinity-purified goat anti-rabbit IgG (1:1000 dilution, 32054, Thermo Fisher Scientific) and a DAB peroxidase (HRP) substrate kit (Vector Laboratories) according to the manufacturer’s protocol. Finally, the sections were observed under a microscope Olympus BX51 (Olympus) with three different fields taken for each group.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data generated that support the findings of this study are present in the article and supplementary information. Besides. Source data are provided with this paper.

References

Fu, Q., Saltsburg, H. & Flytzani-Stephanopoulos, M. Active nonmetallic Au and Pt species on ceria-based water–gas shift catalysts. Science 301, 935–938 (2003).
Article ADS CAS Google Scholar
Qiao, B. et al. Single-atom catalysis of CO oxidation using Pt1/FeOx. Nat. Chem. 3, 634–641 (2011).
Article CAS Google Scholar
Yang, X.-F. et al. Single-atom catalysts: a new frontier in heterogeneous catalysis. Acc. Chem. Res. 46, 1740–1748 (2013).
Article Google Scholar
Hannagan, R. T., Giannakakis, G., Flytzani-Stephanopoulos, M. & Sykes, E. C. H. Single-atom alloy catalysis. Chem. Rev. 120, 12044–12088 (2020).
Article CAS Google Scholar
Guo, W., Wang, Z., Wang, X. & Wu, Y. General design concept for single‐atom catalysts toward heterogeneous catalysis. Adv. Mater. 33, 2004287 (2021).
Article CAS Google Scholar
Huang, L., Chen, J., Gan, L., Wang, J. & Dong, S. Single-atom nanozymes. Sci. Adv. 5, eaav5490 (2019).
Zhang, H., Lu, X. F., Wu, Z. P. & Lou, X. W. D. Emerging multifunctional single-atom catalysts/nanozymes. ACS Cent. Sci. 6, 1288–1301 (2020).
Article CAS Google Scholar
Jiao, L. et al. When nanozymes meet single‐atom catalysis. Angew. Chem. 132, 2585–2596 (2020).
Article ADS Google Scholar
Lu, X. et al. Bioinspired copper single‐atom catalysts for tumor parallel catalytic therapy. Adv. Mater. 32, 2002246 (2020).
Article CAS Google Scholar
Yang, J., Yao, H., Guo, Y., Yang, B. & Shi, J. Enhancing tumor catalytic therapy by co‐catalysis. Angew. Chem. 134, e202200480 (2022).
Google Scholar
Zhu, Y. et al. Stimuli‐responsive manganese single‐atom nanozyme for tumor therapy via integrated cascade reactions. Angew. Chem. Int. Ed. 60, 9480–9488 (2021).
Article CAS Google Scholar
Xing, Y. et al. Flower‐like nanozymes with large accessibility of single atom catalysis sites for ROS generation boosted tumor therapy. Adv. Funct. Mater. 32, 2111171 (2022).
Wang, D. et al. Self‐assembled single‐site nanozyme for tumor‐specific amplified cascade enzymatic therapy. Angew. Chem. 133, 3038–3044 (2021).
Article ADS Google Scholar
Li, Z., Wang, D., Wu, Y. & Li, Y. Recent advances in the precise control of isolated single-site catalysts by chemical methods. Natl Sci. Rev. 5, 673–689 (2018).
Article CAS Google Scholar
Shen, L., Ye, D., Zhao, H. & Zhang, J. Perspectives for single-atom nanozymes: advanced synthesis, functional mechanisms, and biomedical applications. Anal. Chem. 93, 1221–1231 (2020).
Article Google Scholar
Lucci, F. R. et al. Selective hydrogenation of 1, 3-butadiene on platinum–copper alloys at the single-atom limit. Nat. Commun. 6, 1–8 (2015).
Article ADS Google Scholar
Marcinkowski, M. D. et al. Pt/Cu single-atom alloys as coke-resistant catalysts for efficient C–H activation. Nat. Chem. 10, 325–332 (2018).
Article CAS Google Scholar
Zhang, X. et al. Platinum–copper single atom alloy catalysts with high performance towards glycerol hydrogenolysis. Nat. Commun. 10, 1–12 (2019).
Article ADS Google Scholar
Boucher, M. B. et al. Single atom alloy surface analogs in Pd0.18Cu15 nanoparticles for selective hydrogenation reactions. Phys. Chem. Chem. Phys. 15, 12187–12196 (2013).
Article CAS Google Scholar
Peng, Y. et al. Pt single atoms embedded in the surface of Ni nanocrystals as highly active catalysts for selective hydrogenation of nitro compounds. Nano Lett. 18, 3785–3791 (2018).
Article ADS CAS Google Scholar
Zhang, H., Watanabe, T., Okumura, M., Haruta, M. & Toshima, N. Catalytically highly active top gold atom on palladium nanocluster. Nat. Mater. 11, 49–52 (2012).
Article ADS Google Scholar
Wu, H. L. et al. A comparative study of gold nanocubes, octahedra, and rhombic dodecahedra as highly sensitive SERS substrates. Inorg. Chem. 50, 8106–8111 (2011).
Article CAS Google Scholar
Zhang, T., Walsh, A. G., Yu, J. & Zhang, P. Single-atom alloy catalysts: structural analysis, electronic properties and catalytic activities. Chem. Soc. Rev. 50, 569–588 (2021).
Article CAS Google Scholar
Sun, G. et al. Breaking the scaling relationship via thermally stable Pt/Cu single atom alloys for catalytic dehydrogenation. Nat. Commun. 9, 1–9 (2018).
Article ADS Google Scholar
Pao, C. W. et al. The new X-ray absorption fine-structure beamline with sub-second time resolution at the Taiwan Photon Source. J. Synchrotron Radiat. 28, 930–938 (2021).
Article CAS Google Scholar
Luo, S. et al. A tensile‐strained Pt–Rh single‐atom alloy remarkably boosts ethanol oxidation. Adv. Mater. 33, 2008508 (2021).
Article CAS Google Scholar
Li, H.-J. & Ho, J.-J. Theoretical calculations on the oxidation of CO on Au55, Ag13Au42, Au13Ag42, and Ag55 clusters of nanometer size. J. Phys. Chem. C 116, 13196–13201 (2012).
Article CAS Google Scholar
Wu, S.-Y. & Ho, J.-J. Adsorption, dissociation, and hydrogenation of CO₂ on WC(0001) and WC–Co alloy surfaces investigated with theoretical calculations. J. Phys. Chem. C 116, 13202–13209 (2012).
Article CAS Google Scholar
Toby, B. H. & Von Dreele, R. B. GSAS-II: the genesis of a modern open-source all purpose crystallography software package. J. Appl. Crystallogr. 46, 544–549 (2013).
Article CAS Google Scholar
Larson, A. C. & Von Dreele, R. B. General Structure Analysis System. Los. Alamos National Laboratory Report LAUR 86-748 (1994).
Ravel, B. & Newville, M. ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron Radiat. 12, 537–541 (2005).
Article CAS Google Scholar
Perdew, J. P., Burke, K. & Ernzerhof, M. Generalized gradient approximation made simple. Phys. Rev. Lett. 77, 3865 (1996).
Article ADS CAS Google Scholar
Kresse, G. & Hafner, J. Ab initio molecular dynamics for liquid metals. Phys. Rev. B 47, 558 (1993).
Article ADS CAS Google Scholar
Kresse, G. & Hafner, J. Ab initio molecular-dynamics simulation of the liquid-metal–amorphous-semiconductor transition in germanium. Phys. Rev. B 49, 14251 (1994).
Article ADS CAS Google Scholar
Kresse, G. & Furthmüller, J. Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis set. Phys. Rev. B 54, 11169 (1996).
Article ADS CAS Google Scholar
Kresse, G. & Furthmüller, J. Efficiency of ab-initio total energy calculations for metals and semiconductors using a plane-wave basis set. Comput. Mater. Sci. 6, 15–50 (1996).
Article CAS Google Scholar
Blöchl, P. E. Projector augmented-wave method. Phys. Rev. B 50, 17953 (1994).
Article ADS Google Scholar
Kresse, G. & Joubert, D. From ultrasoft pseudopotentials to the projector augmented-wave method. Phys. Rev. B 59, 1758 (1999).
Article ADS CAS Google Scholar
Grimme, S., Antony, J., Ehrlich, S. & Krieg, H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. J. Chem. Phys. 132, 154104 (2010).
Article ADS Google Scholar
Grimme, S., Ehrlich, S. & Goerigk, L. Effect of the damping function in dispersion corrected density functional theory. J. Comput. Chem. 32, 1456–1465 (2011).
Article CAS Google Scholar
Monkhorst, H. J. & Pack, J. D. Special points for Brillouin-zone integrations. Phys. Rev. B 13, 5188 (1976).
Article ADS MathSciNet Google Scholar
Henkelman, G. & Jónsson, H. Improved tangent estimate in the nudged elastic band method for finding minimum energy paths and saddle points. J. Chem. Phys. 113, 9978–9985 (2000).
Article ADS CAS Google Scholar
Henkelman, G., Uberuaga, B. P. & Jónsson, H. A climbing image nudged elastic band method for finding saddle points and minimum energy paths. J. Chem. Phys. 113, 9901–9904 (2000).
Article ADS CAS Google Scholar
Sheppard, D., Terrell, R. & Henkelman, G. Optimization methods for finding minimum energy paths. J. Chem. Phys. 128, 134106 (2008).
Article ADS Google Scholar
Mathew, K., Sundararaman, R., Letchworth-Weaver, K., Arias, T. A. & Hennig, R. G. Implicit solvation model for density-functional study of nanocrystal surfaces and reaction pathways. J. Chem. Phys. 140, 084106 (2014).
Article ADS Google Scholar
Mathew, K., Kolluru, V. S. C., Mula, S., Steinmann, S. N. & Hennig, R. G. Implicit self-consistent electrolyte model in plane-wave density-functional theory. J. Chem. Phys. 151, 234101 (2019).
Article ADS Google Scholar
Cruickshank, B. J., Sneddon, D. D. & Gewirth, A. A. In situ observations of oxygen adsorption on a Cu(100) substrate using atomic force microscopy. Appl. Surf. Sci. 281, 308–314 (1993).
Article Google Scholar
Gómez, Ed. V., Amaya-Roncancio, S., Avalle, L. B. & Gimenez, M. C. DFT Study of adsorption and diffusion of H₂O and related species on Cu(100) surfaces. Surf. Sci. 714, 121920 (2021).
Article Google Scholar
Kim, J. & Gewirth, A. A. Mechanism of oxygen electroreduction on gold surfaces in basic media. J. Phys. Chem. B 110, 2565–2571 (2006).
Article CAS Google Scholar
Oguz, I. C., Mineva, T. & Guesmi, H. The effect of Pd ensemble structure on the O₂ dissociation and CO oxidation mechanisms on Au–Pd(100) surface alloys. J. Chem. Phys. 148, 024701 (2018).
Article ADS Google Scholar

Download references

Acknowledgements

C.-S.Y. appreciates the financial support provided by the Ministry of Science and Technology, Taiwan (109-2113-M-006-011-MY3). W.-P.S. appreciates the financial support provided by the Ministry of Science and Technology or National Science and Technology Council, Taiwan (109-2314-B-006-084-MY3; 111-2321-B-006-011; 111-2314-B-006-004). Y.-H.C. appreciate the financial support provided by the Ministry of Science and Technology, Taiwan (MOST 111-2113-M-035-003-MY2). H.-S.S. appreciates the TLS 01C1 and TPS44A beamline stuffs Dr. Ting-Shan Chan, Dr. Tai-Sing Wu, Dr. Jeng-Lung Chen for their helps in XAS data collection. C.-H.Y. appreciates the financial support provided by the Ministry of Science and Technology, Taiwan (109-2113-M-035-003-MY3).

Author information

Authors and Affiliations

Department of Chemistry, National Cheng Kung University, Tainan, 701, Taiwan
Liu-Chun Wang, Wen-Qi Chen & Chen-Sheng Yeh
Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan
Li-Chan Chang, Yin-Fen Liu & Wen-Pin Su
Department of Materials Science and Engineering, Feng Chia University, Taichung, 40724, Taiwan
Yi-Hsin Chien & Chen-Hao Yeh
National Synchrotron Radiation Research Center, Hsinchu, 30076, Taiwan
Po-Ya Chang, Chih-Wen Pao & Hwo-Shuenn Sheu
Departments of Oncology and Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan
Wen-Pin Su

Authors

Liu-Chun Wang
View author publications
You can also search for this author in PubMed Google Scholar
Li-Chan Chang
View author publications
You can also search for this author in PubMed Google Scholar
Wen-Qi Chen
View author publications
You can also search for this author in PubMed Google Scholar
Yi-Hsin Chien
View author publications
You can also search for this author in PubMed Google Scholar
Po-Ya Chang
View author publications
You can also search for this author in PubMed Google Scholar
Chih-Wen Pao
View author publications
You can also search for this author in PubMed Google Scholar
Yin-Fen Liu
View author publications
You can also search for this author in PubMed Google Scholar
Hwo-Shuenn Sheu
View author publications
You can also search for this author in PubMed Google Scholar
Wen-Pin Su
View author publications
You can also search for this author in PubMed Google Scholar
Chen-Hao Yeh
View author publications
You can also search for this author in PubMed Google Scholar
Chen-Sheng Yeh
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

C.-S.Y. conceived the research. C.-S.Y., Y.-H.C., H.-S.S., W.-P.S. and L.-C.W. designed experiments, interpreted data, and wrote manuscript. L.-C.W. designed experiments, materials preparation and characterization and cells studies. H.-S.S., P.-Y.C. and C.-W.P. conducted Synchrotron powder X-ray diffraction, XANES and EXAFS measurements. C.-H.Y. performed Density Functional Theory (DFT) calculation studies. W.-P.S., L.-C.C. and Y.-F.L. conducted animal studies. W.-Q.C. prepared materials and characterization and cells imaging.

Corresponding authors

Correspondence to Hwo-Shuenn Sheu, Wen-Pin Su, Chen-Hao Yeh or Chen-Sheng Yeh.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary infromation

Peer Review File

Reporting Summary

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Wang, LC., Chang, LC., Chen, WQ. et al. Atomically dispersed golds on degradable zero-valent copper nanocubes augment oxygen driven Fenton-like reaction for effective orthotopic tumor therapy. Nat Commun 13, 7772 (2022). https://doi.org/10.1038/s41467-022-35515-8

Download citation

Received: 30 August 2022
Accepted: 08 December 2022
Published: 15 December 2022
DOI: https://doi.org/10.1038/s41467-022-35515-8

This article is cited by

Emerging single-atom nanozymes for catalytic biomedical uses
- Shuangfei Cai
- Wei Zhang
- Rong Yang
Nano Research (2023)
Natural keratin-based Fe-S1N3 single atom catalyst for insights into the coordination regulation effect of Fenton-like catalysis with high efficiency
- Zhiyi Sun
- Yujuan Wei
- Shuo Wang
Nano Research (2023)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.