Ethical statementAll animal experiments were performed in accordance with the published guidelines of the CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety (Institute of High Energy Physics and National Center for Nanoscience and Technology; Approval ID: IHEPLLSC-2023-52). All mice were housed in a specific pathogen-free environment on a 12/12 h light-dark cycle with the standard conditions: Temperature, 20-25 °C; Relative humidity, 40-70%. All research was carried out according to relevant guidelines and regulations. The maximal tumor size permitted by the Animal Experiment Administration Committee of the Institute of High Energy Physics and National Center for Nanoscience and Technology is 15 mm in diameter, and no mice exceeded this criterion in this work.MaterialsCopper (II) chloride hydrate (CuCl2·H2O, 99 + %,), ascorbic acid (AA, 99 + %), copper nitrate hydrate (Cu(NO3)2·xH2O, 99 + %), zinc nitrate hexahydrate (Zn(NO3)2·6H2O, 99 + %), 2-methylimidazole (98%), and glacial acetic acid (99%) were purchased from Alfa Aesar. Copper(I) acetate (CuCH3COO, 97%) and Tween-80 were purchased from Sigma-Aldrich. Other chemical reagents, including sodium hydroxide solution, potassium chloride, methanol, and ethanol, were supplied from Beijing Chemical Reagent Co. All reagents were used without further purification.Synthesis of CuN3-SAzyme0.7275 g of Cu(NO3)2·xH2O and 0.8210 g of 2-methylimidazole were dissolved in absolute methanol (30 mL), respectively. Upon mixing with each other, 500 g of KCl was added in the resulting solution. The precipitate was collected by centrifugation (7162 g, 5 min) and annealed at 850 °C for 2 h under argon flow. Finally, the sample was washed with H2O and H2SO4 solution several times. Similarly, Cu-free N-doped carbon support was prepared in the absence of Cu(NO3)2·xH2O.Synthesis of CuN4-SAzymeFirst, ZIF-8 was synthesized. 2.6260 g of 2-MI and 2.2380 g of Zn(NO3)2·6H2O were dissolved in absolute methanol (30 mL), respectively, and then mixed with each other at room temperature. The resulting precipitate was centrifuged (7,162 g, 5 min) and washed with absolute methanol several times. After dried in vacuum at 70 °C overnight, the ZIF-8 powder was placed in a tube furnace and heated at 900 °C for 3 h under flowing argon gas. The product was cooled down to room temperature naturally (named carbon nanoparticles, CNs).Second, 0.0100 g of Cu(NO3)2·xH2O and 2.0000 g of urea were dissolved in absolute methanol (30 mL) and then mixed the resulting NCs (200 mg). When the mixture was ultra-sonicated for 1 h and then stirred at room temperature for 6 h, the resulting precipitate was centrifuged (7162 g, 5 min) and washed with absolute methanol several times. The dried powder was transferred into a ceramic boat and placed in a tube furnace to be heated at 700 °C for 3 h under flowing argon gas, the sample was naturally cooled down to room temperature and washed with H2O and H2SO4 several times.Synthesis of Cu2O nanozymeTypically, 1 mmol CuCl2·H2O was dissolved in the deionized water (100 mL) under vigorous stirring. The obtained solution was placed into an oil bath and heated at 50 oC for 0.5 h, followed by the addition of 2 M NaOH solution (10 mL). After stirring at 50 oC for another 0.5 h, 0.6 M AA solution (10 mL) was added dropwise (3 mL min−1) in the dark brown solution. This red mixture was aged at 50 oC for 3 h and finally cooled down to room temperature naturally. The resulting red precipitate was collected by centrifugation (13,400 g, 5 min) and washed with deionized water several times to remove the residuals. The precipitate was re-dispersed in deionized water and the concentration of this dispersion was measured by inductively coupled plasma mass spectrometry (ICP-MS, Thermo-X7, USA).Synthesis of CuO nanozymeIn brief, 1 mmol of CuCH3COO was dispersed in the deionized water (50 mL) under sonication and 0.25 mL of glacial acetic acid was added dropwise (0.25 mL min−1) in the resulting solution. After heating to 100 oC under vigorous stirring, 1 M NaOH solution (5 mL) was added to form the black precipitate. This black precipitate was separated by centrifugation (13,400 g, 5 min) and washed with deionized water several times. The concentration of this dispersion was measured by ICP-MS.CharacterizationTransmission electron microscopy images and elemental mapping were obtained from a Tecnai G2 F20 microscope operated at 200 kV (FEI, USA) and equipped with an energy-dispersive X-ray analysis system. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) was performed on a JEOL-ARM300F microscope equipped with a spherical aberration corrector at 200 keV. X-ray photoelectron spectroscopy (XPS) analysis was performed on a VG Multilab 2000 instrument (Thermo Fisher). X-ray diffraction (XRD) patterns were obtained from a D8 ADVANCE apparatus (Bruker, Germany; Cu Kα, 0.15406 nm). Inductively coupled plasma optical emission spectroscopy (ICP-OES) was conducted using an Agilent 720ES apparatus. X-ray absorption fine structure (XAFS) spectra at Cu K-edge were acquired at the 1W1B station in Beijing Synchrotron Radiation Facility (BSRF) and the BL11B station in Shanghai Synchrotron Radiation Facility (SSRF).XAFS measurement and analysisThe Cu K-edge XAFS data were recorded in the fluorescence mode, using Cu foil and CuOx nanozymes as references. All spectra were collected under ambient conditions. The extended Cu K-edge EXAFS data were processed using the ATHENA module in the IFEFFIT software packages. To obtain k3-weighted EXAFS spectra, the post-edge background was subtracted from the overall absorption and then normalized to the edge-jump step. These k3-weighted χ(k) data of Cu K-edge were Fourier transformed into real (R) space using a Hanning window (dk = 1.0 Å−1) to isolate the EXAFS contributions from various coordination shells. Quantitative structural parameters around the central atoms were derived through least-squares curve fitting, performed with the ARTEMIS module of the IFEFFIT software packages, using the following EXAFS equation:$${{{{\rm{\chi }}}}}\left({{{{\rm{k}}}}}\right)={\sum}_{j}\frac{{N}_{j}{S}_{0}{F}_{j}(k)}{k{R}_{j}^{2}}\exp \left[-2{k}^{2}{\sigma }_{j}^{2}\right]\exp \left[\frac{-2{R}_{j}}{\lambda \left(k\right)}\right]\sin [2k{R}_{j}+{\phi }_{j}(k)]$$
(1)
where, S02 is the amplitude reduction factor, Fj(k) is the effective curved-wave backscattering amplitude, Nj is the number of neighbors in the jth atomic shell, Rj is the distance between the X-ray absorbing central atom and the atoms in the jth atomic shell (backscatterer), λ is the mean free path in Å, ϕj(k) is the phase shift (including the phase shift for each shell and the total central atom phase shift), σj is the Debye-Waller parameter of the jth atomic shell (variation of distances around the average Rj). The functions Fj(k), λ, and ϕj(k) were calculated with the ab initio code FEFF8.2. The additional details for the EXAFS simulations are given below. The coordination numbers of the model samples were set to their nominal values, and the obtained S02 was kept constant in subsequent fittings. However, the internal atomic distances (R), Debye-Waller factor (σ2), and the edge-energy shift (ΔE0) were allowed to run freely.Measurement of photothermal effect of CuN3-SAzymeThe aqueous dispersions (1 mL) of CuN3-SAzyme with different concentrations were exposed to 808 nm NIR light (1.00 W cm−2) for 10 min. At each interval time, the temperature and infrared thermal images were recorded by the thermal camera (FLIR Therma CAM E40). To evaluate the influence of power density on the temperature change, 50 μg mL−1 of the aqueous dispersions of CuN3-SAzyme were illuminated with 808 nm NIR light with the power density ranging from 0.25 W cm−2 to 2.00 W cm−2 and the temperature was recorded by the same protocol as described above. To calculate the photothermal conversion efficiency of CuN3-SAzyme, 25 μg mL−1 of the aqueous dispersion of CuN3-SAzyme was illuminated with 808 nm NIR light at the power density of 1.00 W cm−2 for 750 s and then cooled naturally for 1500 s, respectively. 1 mL of pure deionized water was set as a control sample. To assess the photothermal stability under 808 nm NIR light (1.00 W cm−2), the aqueous dispersion of CuN3-SAzyme was illuminated with five heating/cooling (600 s/600 s) cycles. The stability of CuN3-SAzyme in the physiological solutions was also detected by monitoring the heating profiles of CuN3-SAzyme after storage for various days.Measurement of enzyme-like activities of CuNx-SAzymes and CuOx nanozymesThe peroxidase-like property was detected by colorimetric assays. In a typically procedure, 3.6 μL of aqueous suspension of CuNx-SAzyme (1.0 mg mL−1), 30 μL of TMB solution (6 mM), and 22.5 μL of H2O2 solution (400 mM) were mixed into HAc-NaAc buffer solution (10 mM, pH 3.54) with a final volume of 180 μL. The catalytic oxidation of TMB (ox-TMB) was studied by monitoring the absorbance of ox-TMB (652 nm, ε = 39,000 M−1 cm−1) using a microplate spectrophotometer (Multiskan MK3, Thermo Fisher Scientific, USA). As for the kinetic data towards TMB substrate, TMB solutions with different concentrations were added into HAc-NaAc buffer solutions containing CuNx-SAzyme (3.6 μL, 1 mg mL−1) and H2O2 (22.5 μL, 400 mM), finally obtaining the mixtures with the final volume of 180 μL. The kinetic data towards H2O2 substrate was obtained using the method as described above except the addition of H2O2 solution with different concentrations into HAc-NaAc buffer solutions containing CuNx-SAzyme (3.6 μL, 1.0 mg mL−1) and TMB (30 μL, 6 mM). The kinetic data were calculated using the following typical Michaelis-Menten equation,$$v=\frac{{{{{{\rm{V}}}}}}_{\max }\cdot [S]}{{K}_{M}+[S]}$$
(2)
where v is the initial velocity, [S] is the concentration of the substrate, KM is the Michaelis-Menten constant, and Vmax is the maximal reaction velocity. The specific activities (U mg−1) were measured by monitoring the absorbance of the mixture (the total volume was 180 μL) containing 60 μL of H2O2 solution (9 M), 30 μL of TMB solution (6 mM), and various concentrations of CuNx-SAzyme. In particular for catalase-like enzymatic activity, the concentration of generated O2 was detected by a specific oxygen electrode (JPSJ-605F, INESA). Meanwhile, the external field-enhanced peroxidase-like performance was detected using the same protocol besides the introduction of X-/γ-ray and 808 nm NIR light (1.00 W cm−2).Evaluation of the stability of CuN3-SAzymeTo assess the influence of temperature and pH on the enzymatic-like performance, the absorbance of the mixture (the total volume was 180 μL) containing 3.6 μL of aqueous suspension of CuNx-SAzyme (1.0 mg mL−1), 30 μL of TMB solution (6 mM), and 22.5 μL of H2O2 solution (400 mM) was recorded in the range of temperature from 7 °C to 87 °C and pH value from 2.16 to 12.04. For the assessment of radio-resistance, the aqueous suspensions of CuN3-SAzyme were irradiated by X-/γ-ray with the radiation dose ranging from 1 Gy to 500 Gy, and then the enzymatic-like activity was measured using the protocol as described above.Evaluation of cytotoxicityLuciferase-transfected murine breast carcinoma cell line (4T1-Luc), mouse osteosarcoma cell line (K7M2), and mouse embryonic fibroblast cell line (3T3) were obtained from Peking Union Medical College Hospital. In particular, 4T1-Luc cell line was authenticated using Short Tandem Repeat (STR) analysis on Apr 15th 2024. The scientific justification demonstrated that 4T1-Luc cells are not cross-contaminated or otherwise misidentified cell lines (Supplementary Table 5). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) containing 10% Fetal Bovine Serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) in 5% CO2 at 37 °C. When seeded in the 96-well plates for 24 h (6,000 cells/well for 4T1-Luc cells; 6,000 cells/well for K7M2 cells; 6,000 cells/well for 3T3 cells), cultured cells were co-incubated with CuN3-SAzyme, CuN4-SAzyme, CuO nanozyme or Cu2O nanozyme at various concentrations for 24 h (consistent with the content of copper in CuN3-SAzyme). The standard Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay was performed to determine the cytotoxicity via recording the absorbance at 450 nm using the microplate spectrophotometer. To evaluate photothermal cytotoxicity, CuN3-SAzyme-treated cells were further illuminated with 808 nm NIR light at various power densities for 10 min and the cell viability was measured using the same protocol as described above.Live/dead cell staining assayTo further evaluate the photothermal killing efficacy, 4T1-Luc cells were seeded in 6-weel plates for 24 h (200,000 cells/well) and treated with of CuN3-SAzyme at different concentrations. After co-incubation for 6 h, these treated cells were illuminated with 808 nm NIR light at different power densities for 10 min. Following co-incubation for another 18 h, these cells were stained with calcein AM (CA, Beyotime, China) and propidium iodide (PI, Beyotime, China) for 20 min at 37 °C in the dark. Finally, the fluorescent images were collected using an inverted fluorescence microscope (Olympus X73, Tokyo, Japan).Colony formation assayTo evaluate the inhibition effects, 4T1-Luc cells seeded in 6-well plates (2,000 cells/well) were incubated with CuN3-SAzyme. After incubation for 6 h, these treated cells were irradiated with 808 nm NIR light (1.00 W cm−2) for 10 min and/or X-ray (6 Gy). Following incubation for another 18 h, these cells were continued to be cultured in fresh DMEM medium for 7 days. Subsequently, formed clones were fixed with 4% paraformaldehyde (Innochem, China) for 10 min and stained by Giemsa Staining Solution (Beyotime, China) for 30 min. Finally, clones were counted and used to plot the survival fraction. To further evaluate the radiosensitization efficiency of CuN3-SAzyme, 4T1-Luc cells were incubated with CuN3-SAzyme at different concentrations for 6 h and then irradiated by X-ray with different doses for 10 min and/or 808 nm NIR light (1.00 W cm−2) for another 10 min. Following incubation for 7 days, the number of clones were obtained using the same protocol as described above to plot the survival fraction.Detection of intracellular ROSTo detect the level of intracellular ROS, 4T1-Luc cells were seeded in the confocal dish (200,000 cells/well) and treated with CuN3-SAzyme at various concentrations for 6 h. When irradiated with 808 nm NIR light (1.00 W cm−2) for 10 min and/or X-ray (6 Gy) for another 10 min, these treated cells were co-stained with 2’,7’-dichlorofluorescin diacetate (DCFH-DA, Beyotime, China) and the nuclei probe 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime, China) for 20 min at 37 °C in the dark. The fluorescent images were recorded using a fluorescence confocal microscope (A1/LSM-Kit, Nikon/PicoQuant GmbH, Japan/Germany).Evaluation of intracellular mitochondrial membrane potentialTo detect the change of intracellular mitochondrial membrane potential (MMP), 4T1-Luc cells (200,000 cells/well) were treated with CuN3-SAzyme at different concentrations for 6 h and then irradiated with 808 nm NIR light (1.00 W cm−2) for 10 min and/or X-ray (6 Gy) for another 10 min. After incubation for 12 h, these treated cells were incubated with JC-1 working solution (10 μg mL−1) for 20 min. Finally, the change of intracellular MMP was monitored by recording the fluorescent images using the fluorescence confocal microscope.Evaluation of intracellular DNA damageTo detect the intracellular DNA damage, CuN3-SAzyme-treated cells (20,000 cells/well) were irradiated with 808 nm NIR light (1.00 W cm−2) for 10 min and/or X-ray (6 Gy) for another 10 min. After incubation for 12 h, these cells were fixed with 4% paraformaldehyde for 10 min and treated with 0.25% Triton X-100 to enhance the permeability of cell membranes. Following the blocking with 1% bovine serum albumin, these treated cells were labeled by γ-H2AX mouse monoclonal antibody overnight and secondary antibody of Cy3-conjugated sheep anti-mouse for 2 h. The intracellular DNA damage was monitored by recording the fluorescent images using the fluorescence confocal microscope.Evaluation of cytochrome c releaseIn detail, 4T1-Luc cells were pre-seeded in 24-well plates (20,000 cells/well) and then treated with CuN3-SAzyme. After incubation for 6 h, these cells were irradiated with 808 nm NIR light (1.00 W cm−2) for 10 min and/or X-ray (6 Gy). After another 12 h, these treated cells were stained by MitoTracker Red CMXRos for 30 min and fixed with 4% paraformaldehyde for another 10 min. Subsequently, 0.5% Triton X-100 was used to permeabilize the cell membrane. When further blocked by 1% bovine serum albumin for 1 h at room temperature and labeled with Cyt c antibody (1:1000) overnight at 4  °C, these cells were co-labeled with Alexa Fluor 488 secondary antibody and nuclei probe DAPI. Finally, the fluorescent images were recorded using the fluorescence confocal microscope.Western blot analysis apoptosis-related proteinsAfter different treatments, 4T1-Luc cells (1,000,000 cells/well) were collected and lysed with radioimmunoprecipitation assay buffer (50 mM Tris-HCl containing 1% NP-40, 1 mM EDTA, 0.1% SDS, 150 mM NaCl, supplemented with 1 mM PMSF, pH 7.50) on ice for 30 min. The whole protein of cells was obtained by centrifugation at 4 °C for 15 min. The concentration of whole protein was quantified by the BCA Protein Assay Kit (Beyotime, China). Subsequently, the apoptosis-related proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted on nitrocellulose membranes. The primary antibody was incubated with target protein at 4 °C overnight. After using Tris-buffer saline (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1‰ Tween-20) to wash the nitrocellulose membranes three times, the secondary antibody was incubated for 2 h. The protein marker and target protein undergo electrophoresis on the same gel under identical conditions. The molecular weight of target protein of interest by simply comparing the position of its band on the gel with those of the PageRuler prestained protein marker (Catalog number. 26616, Thermo Fisher Scientific, US). The target protein visualization was performed by enhanced chemiluminescence (ECL, Beyotime, P0018).In vitro apoptosis analysis4T1-Luc cells were pre-seeded in 6-well plates (200,000 cells/well) and incubated with CuN3-SAzyme for 6 h and sequentially irradiated with 808 nm NIR light (1.00 W cm−2) for 10 min and/or X-ray (6 Gy). After another 24 h, these treated cells were collected and stained by annexin V-FITC (AV, Dojindo, Japan) and propidium iodide (PI, Dojindo, Japan) at 37 °C. Finally, the apoptosis cells were quantified by flow cytometry (BD Accuri C6, USA).Cellular uptake pathway of CuN3-SAzymeTypically, 4T1-Luc cells were seeded in 6-well plates (200,000 cells/well) and incubated with various inhibitors including filipin III (7.5 µM), chlorpromazine (50 µM), cytochalasin D (5 µM), and wortmannin (5 µM) for 30 min. Then, FITC-labeled CuN3-SAzyme was added and incubated for another 2 h. Finally, cells were washed three times with phosphate buffer saline (PBS, pH 7.40) and analyzed immediately using flow cytometry.Hemolysis assayFresh mouse blood was obtained from BALB/c mice (6-8 weeks old, female) and washed with PBS three times to collect mouse blood cells (RBCs). The resulting RBCs were re-dispersed in aqueous suspensions of CuN3-SAzyme with different concentrations. The deionized water was used as the corresponding positive control and PBS (pH 7.40) was used as the negative control. After incubation for 4 h, the specimens were centrifuged at 2990 g for 5 min and the absorbance of the supernatant was detected using the microplate spectrophotometer. The hemolysis ratios of CuN3-SAzyme were calculated by the following equation:$${{{{\rm{Hemolysis\; ratio}}}}}=\frac{{{{{\rm{OD}}}}}({{{{\rm{test}}}}})-{{{{\rm{OD}}}}}({{{{\rm{negative\; control}}}}})}{{{{{\rm{OD}}}}}({{{{\rm{positive\; control}}}}})-{{{{\rm{OD}}}}}({{{{\rm{negative\; control}}}}})}$$
(3)
Evaluation of enhanced radio-enzymatic therapyBALB/c mice (4-6 weeks old, female) were provided by Beijing HFK Bioscience Co., Ltd. To establish the 4T1 bear tumor model, 4T1 cells suspended in PBS were subcutaneously injected into the right flank of the mice. Tumor volumes were calculated by the following equation:$${{{{\rm{V}}}}}=\frac{L\times {W}^{2}}{2}$$
(4)
where L (mm) is the tumor long dimension and W (mm) is the tumor width.When the tumor volume reached a size of about 100 mm3, the mice were randomly divided into several groups (n = 5). After intratumoral injection of CuN3-SAzyme dispersed in 5% glucose solution (volume: 50 µL; dose: 5 mg kg−1 and 10 mg kg−1), mice were irradiated with 808 nm NIR light (1.00 W cm−2) for 10 min and/or X-ray (3 Gy or 6 Gy). Tumor volume and body weight of mice were measured every other day. At the end of treatment period, the tumors from different groups were exfoliated, weighed, and fixed with 4% polyoxymethylene (v/v). The exfoliated tumors were sliced to hematoxylin and eosin (H&E, Abcam, UK) staining, immunohistochemical analysis of Ki-67 (Beyotime, China) and γ-H2AX (Beyotime, China).Evaluation of biosafety of CuN3-SAzymeBALB/c mice (6-8 weeks old, female) were subcutaneously injected by CuN3-SAzyme with various doses. At each interval time, treated mice were sacrificed and major organs including the heart, liver, spleen, lung, and kidney were dissected and harvested. To prepare the ICP-MS samples, the harvested organs were weighed and mixed with nitric acid (65%, m/m) and incubated at 60 °C for 72 h. After dilution and filtration, the biodistribution of Cu element was measured by ICP-MS and calculated as Cu percentage over administrated dose per gram of tissues.In vivo photoacoustic imaging4T1-bearing BALB/c nude mice (5–6 weeks old, male) were anesthetized by inhalation of isoflurane/air for 5 min. Subsequently, 25 μL of the s4T1-bearing BALB/c nude mice (5–6 weeks old, male)uspension of CuN3-SAzyme (4 mg mL−1) was intratumorally injected. The mice were illuminated by 808 nm NIR light (1.00 W cm−2) for 10 min. The photoacoustic images were collected and analyzed on a multispectral optoacoustic tomography (MSOT, iThera Medical 128, Germany) system (750 nm to 900 nm).Computational detailsThe structural relaxations, energy calculations, and electronic structure analyzes were performed using the Vienna ab initio simulation package (VASP)62,63. The interactions between ionic cores and valence electrons were considered by using the projector augmented wave (PAW) method64, and the exchange-correlation interactions were described by the optPBE-vdW functional65,66. The energy cutoff for the plane-wave basis sets was set to 500 eV. Both CuN3 and CuN4 units were embedded in a 5 × 5 graphene unit cell. The vacuum space between consecutive slabs along the vertical direction was set to 14 Å. A 3 × 3 × 1 Monkhorst-Pack grid was utilized for the first Brillouin zone sampling67. The atomic coordinates were relaxed until the maximum force was less than 0.03 eV Å−1. All structures were visualized using the program VESTA68.Under irradiation, X-ray excites electrons in the inner layers of constituent atoms, causing not only radiative but also non-radiative transitions. The non-radiative transitions could induce a drastic energetic stimulation and lead to further ionization of the matter. Considering that it is rather complex to directly simulate the structural evolution of matter under X-ray radiation from first principles, we focused on whether CuN3-SAzyme can maintain its stability under drastic energy fluctuations (equivalent to being in a hot environment of thousands of Kelvin) and when one to several electrons are ionized, by using extensive ab initio molecular dynamics (AIMD) simulations. The AIMD simulations were performed using the CP2K package69. The unit cell used here is nine times (3 × 3) the size of the unit cell used in the VASP calculations, containing a total of 414 C atoms, 27 N atoms, and 9 Cu atoms. The exchange-correlation interactions were described by the PBE functional70, and the empirical scheme (D3) developed by Grimme et al. 71 was employed for the dispersion correction. We used the optimized double-ξ Gaussian basis sets72 and an auxiliary plane wave basis set (energy cutoff: 500 Ry)73 to expand wavefunctions. The core electrons were treated with the scalar relativistic norm-conserving pseudopotentials74 and the number of valence electrons adopted for C, N, and Cu were 4, 5, and 11, respectively. Only the Γ point was used for the first Brillouin zone sampling. The canonical (NVT) ensemble was adopted in the AIMD calculations with a time step of 1 fs. The Nosé-Hoover thermostats75,76 were employed here and the corresponding temperature was set to a range between 1773 K and 3273 K (equivalent to 1,500 °C and 3000 °C, respectively), to simulate potential drastic energy fluctuations that the CuN3-SAzyme may experience under X-ray irradiation.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Enhancing radiation-resistance and peroxidase-like activity of single-atom copper nanozyme via local coordination manipulation
