Shape-controllable and kinetically miscible Copper-Palladium bimetallic nanozymes with enhanced Fenton-like performance for biocatalysis.

Bimetallic nanozymes have been emerging as essential catalysts due to their unique physicochemical properties from the monometallics. However, the access to optimize catalytic performance is often limited by the thermodynamic immiscibility and also heterogeneity. Thus, we present a one-step coreduction strategy to prepare the miscible Cu-Pd bimetallic nanozymes with controllable shape and homogeneously alloyed structure. The homogeneity is systematically explored and luckily, the homogeneous introduction of Cu successfully endows Cu-Pd bimetallic nanozymes with enhanced Fenton-like efficiency. Density functional theory (DFT) theoretical calculation reveals that Cu-Pd bimetallic nanozymes exhibit smaller d-band center compared with Pd nanozymes. Easier adsorption of H2O2 molecular contributed by the electronic structure of Cu significantly accelerate the catalytic process together with the strong repulsive interaction between H atom and Pd atom. In vitro cytotoxicity and intracellular ROS generation performance reveal the potential for in vivo biocatalysis. The strategy to construct kinetically miscible Cu-Pd bimetallic nanozymes will guide the development of bimetallic catalysts with excellent Fenton-like efficiency for biocatalytic nanomedicine.

Introduction

Bimetallic nanoparticles have attracted broad attentions due to the unique catalytic properties in various fields, especially in chemical sensing, heterogeneous catalysis, and nanomedicine [[1], [2], [3]]. Different from monometallic nanoparticles, bimetallic nanoparticles always present unique multifunctional performances due to the synergistic effects among of corresponded monometallic analogues [4]. For instance, Cu–Ni bimetallic nanoparticles have been obtained via nonequilibrium synthetic strategy and exhibit enhanced C2+ product Faradaic efficiencies (∼76%), which is ∼20% higher than that of monometallic Cu [5]. Yang and coworkers have precisely explored the activity of Au–Cu bimetallic nanoparticles as a function of their composition and demonstrated that uniform Au3Cu bimetallic nanoparticles exhibit highest catalytic activity, while pure Cu nanoparticles owned the lowest overall activity [6]. Compared to monometallic materials, the design and development of bimetallic nanoparticles greatly optimize the functionality and broaden the applications.

Due to the fact that various factors like size, morphology, component, and nanoscale arrangement will influence the physicochemical properties, many research interests have been paid to deliberately identify the relationship between enhanced catalytic performances and structures in order to expand the practical applications in the field of catalysis [7,8]. However, there still remains two fundamental challenges: the limited access to obtain homogeneously alloyed bimetallic nanozymes due to the thermodynamic immiscibility and the difficulty to identify and optimize the catalytic performance because of the heterogeneity in bimetallic catalysts [5]. Traditional preparation methods such as seed-mediated growth, galvanic replacement, concurrent thermal decomposition, and impregnation tend to yield bimetallic nanozymes with unfavorable structures, broad size distributions, inhomogeneous alloying, and other heterostructures [2,9]. New methods like surface plasmon resonance [10] and pulsed laser ablation [11,12] always require complex processes and extreme conditions. Although J. R. Regalbuto and coworkers have successfully obtained highly dispersed, well-alloyed bimetallic ∼1 ​nm-diameter nanoparticles via strong electrostatic adsorption, the strategy dependents on the oppositely charged oxide or carbon supports [7]. Thus, it is of great significance to construct bimetallic nanozymes with homogeneously alloyed structure and optimized catalytic properties [13].

Herein, to optimize the Fenton-like performance of Pd-based nanozymes, in this study, a one-step coreduction strategy was employed to prepare miscible Cu–Pd bimetallic nanozymes (alliums-like CuPd3 nanozyme, and concave rhombic dodecahedral-like Cu3Pd) with controllable shape and homogeneously alloyed structure. The synthetic procedure lasts only 2 ​h under 110 ​°C by using Na2PdCl4 and CuCl2 as precursors in the presence of hexadecylamine (HDA) and glucose. We firstly systematically investigated the morphologies, structures, and atomically spatial states of as-synthesized Cu–Pd bimetallic nanozymes. Then the optimal catalytic activities, kinetics, and ROS products were carefully evaluated by adjust the molar ratio of precursors. To disclose the mechanism of optimal Fenton-like efficiency, the density functional theory (DFT) theoretical calculation was performed to explore the total reaction energy, density of state, and d-band center. Finally, the in vitro cytotoxicity and intracellular ROS generation performance were evaluated to reveal the potential for in vivo biocatalysis. It is believed that this research will guide the development of homogeneous Cu–Pd bimetallic nanozymes with excellent Fenton-like efficiency for biocatalytic nanomedicine.

Materials and methods

Materials

Sodium tetrachloropalladate (II) (Na2PdCl4), cupric chloride dehydrates (CuCl2·2H2O), glucose, and hexadecylamine (HDA) were purchased from Sigma-Aldrich LLC. Ethanol and deionized water were obtained from Beijing Chemical Industry Group Corporation Co., Ltd. Copper (II) phthalocyanine (CuPc) was brought from Zancheng (Tianjin) Technology Co., Ltd. TMB (3,3′,5,5′-Tetramethylbenzidine) Single-Component Substrate Solution, CCK-8 Cell Proliferation and Cytotoxicity Assay Kit, and 2′,7′-Dichlorofluorescin Diacetate (DCFH-DA) fluorescent probe were obtained from Solarbio Life Sciences & Technology Co., Ltd. All chemicals were employed without further purification.

Characterization

Transmission electron microscopy (TEM, HT-7700, Hitachi) and high-resolution transmission electron microscopy (HRTEM, JEM-2100F, JEOL) with energy dispersive X-ray (EDX) spectroscopy were used to explore the morphology, crystal structure, and element components of as-synthesized Pd and Cu–Pd bimetallic nanozymes. The size distribution and zeta potential in aqueous solution were measured on Malvern Zetasizer Nano ZS. The aberration-corrected high-angle annular dark-field scanning TEM (AC HAADF-STEM, FEI Titan Cubed G2 300) was performed to obtain the high-resolution AC HAADF-STEM images of Pd and Cu–Pd bimetallic nanozymes at 300 ​keV. X-ray diffractometer (XRD, D/max-2550, Rigku) was used to analyze the crystal structure of powder. XPS spectra of samples were measured on the X-Ray Photoelectron Spectroscopy (250XI, Thermo Fisher Scientific). The electron spin resonance (ESR) spectra were trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in aqueous solution with Cu3Pd, CuPd3, and Pd (10 ​μg/mL). The intracellular GSSG level was measured by GSH and GSSG Assay Kit.

Preparation of Cu–Pd bimetallic nanozymes

Hovenia acerba-like Pd nanozymes: 2.67 ​mL Na2PdCl4 aqueous solution (122.4 ​mM) and 200 ​mg HDA were added into 7 ​mL ultrapure (UP) water in a 20 ​mL Wheaton Sample Vial. The mixture was magnetically stirred overnight at ambient temperature for complete dissolution. Then, the solution was transferred into a 100 ​mL round-bottom flash and another 1 ​mL glucose aqueous solution (75.7 ​mM) was added. The system was heated to 110 ​°C for 2 ​h under magnetic stirring. After cooling down to room temperature, the product was washed with aqueous ethanol solution (20 ​vol%) for four times and redispersed in 2 ​mL UP water for further use.

Cu–Pd bimetallic nanozymes: Following the same strategy as Pd nanozymes, alliums-like CuPd3 nanozymes were prepared by adding 0.42 ​mL CuCl2 H2O aqueous solution (100 ​mM) at the beginning, and concave rhombic dodecahedral-like Cu3Pd nanozymes were prepared by synchronously adding 1.24 ​mL CuCl2 H2O aqueous solution (100 ​mM).

Fenton-like efficiency and kinetics measurement

In order to evaluate the Fenton-like efficiency and kinetics performance, TMB Single-Component Substrate Solution was employed to detect the generated hydroxide radical (OH) as protocol in our previous study. For testing Cu–Pd bimetallic nanozymes concentration-dependent kinetics, 0.5 ​mL TMB Single-Component Substrate Solution and 0.5 ​mL UP water were mixed in a 48-well plate firstly. Then Cu–Pd bimetallic nanozymes were added for a final concentration of 500, 200, 100, and 50 ​μg/mL quickly. The time-dependent absorbance value at 652 ​nm was detected in a Microplate Reader (Varioskan LUX, Thermo Scientific). The UV–vis spectra of above mixture were measured when the reaction lasts for 0.5 ​h. Besides, for measuring H2O2 concentration-dependent kinetics, additional H2O2 was added into the TMB Single-Component Substrate Solution for a final H2O2 concentration of 40, 30, 20, 10, 5, 1 ​mM. The time-dependent absorbance value at 652 ​nm was detected immediately after Pd and Cu–Pd bimetallic nanozymes were added (Pd: 300 ​μg/mL, CuPd3: 300 ​μg/mL, Cu3Pd: 150 ​μg/mL). Meanwhile, the UV–vis spectra of Cu–Pd bimetallic nanozyme in various TMB/H2O2 solution (H2O2 concentration: 40, 30, 20, 10, 5, 1 ​μM) were measured when the reaction lasts for 0.5 ​h.

X-ray absorption spectrum measurement

For X-ray absorption spectrum measurement, Cu–Pd bimetallic nanozyme powders were prepared via lyophilization strategy. Cu K-edge X-Ray absorption spectra (XAS) data of Cu–Pd bimetallic nanozyme were collected on beam line Si(111) crystal monochromators at the BL11B beamlines at the Shanghai Synchrotron Radiation Facility (SSRF) (Shanghai, China). X-ray absorption near-edge structure (XANES) was observed between two scans taken for a specific sample and extended X-ray absorption fine structure (EXAFS) data were performed by in transmission mode. The data were analyzed by the software of Athena. Wavelet Transform (WT) was carried out with hamaFortran software.

DFT theoretical calculation

The structural files of Cu (111) and Pd (111) were downloaded from AMCSD (American Mineralogist Crystal Structure Database). The substitutional atoms were set, and three metal-organic models were created by GaussView5.0, which had 16 transition metal atoms and two hydrogen peroxide molecules. The optimizations of the systems were carried out by using PBE method and LANL2TZ basis set as employed in Gaussian 16 package. Then the equilibrations in a constant ensemble (NPT) for a duration of 2 ps and the molecular dynamics (MD) simulations with 200 ps in a canonical ensemble (NVT) were performed by using GFN2-XTB method in the CP2K package, of which the temperature was 310 ​K and the pressure was 1 ​bar. Density of States (DOS) analysis has also been obtained by MULTIWFN 3.8 program, and d-band center was calculated to study the adsorption between the transition metal surface and hydrogen peroxide (H2O2).

In vitro cytotoxicity evaluation

To evaluate the in vitro cytotoxicity of as-synthesized Pd and Cu–Pd bimetallic nanozyme, 4T1 murine mammary cancer cell line was chosen as model. In detail, cells in exponential phase were seeded in a 96-well plate with a concentration of 5000/well. 12 h later, the medium was replaced with fresh medium (various Pd and Cu–Pd bimetallic nanozyme: 400, 200, 100, 50, 25, 12.5, 6.25, 0 ​μg/mL). After another 24 ​h incubation, the cell viability of 4T1 cells was measured by cell count kit-8 (CCK-8).

Intracellular hydroxide radical detection

DCFH-DA fluorescent probe was employed to detect the intracellular OH catalyzed by Pd and Cu–Pd bimetallic nanozymes. In general, 4T1 cells in exponential phase were seeded in a 96-well plate with a concentration of 5000/well. 12 h later, the medium was replaced with fresh medium with Pd and Cu–Pd bimetallic nanozymes concentration of 25 ​μg/mL. After incubation for another 6 ​h, the Pd and Cu–Pd bimetallic nanozymes were removed thoroughly and the cells were stained with DCFH-DA probe. Finally, confocal laser scanning microscopy (FV3000, Olympus) was used to image the cells with excitation/emission wavelength: 504/529 ​nm.

Statistical analysis

Results in this study were presented as mean values ​± ​SD, and the statistical difference was calculated by two-tailed student's t-test. ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001.

Results and discussion

Synthesis and characterization of Pd and Cu–Pd bimetallic nanozymes

As illustrated in Fig. 1a, the one-step coreduction strategy was introduced to prepare the hovenia acerba-like Pd nanozyme and Cu–Pd bimetallic nanozymes (alliums-like CuPd3 nanozyme, and concave rhombic dodecahedral-like Cu3Pd) for optimized Fenton-like efficiency (catalyzing H2O2 into hydroxide radical (OH)). Generally, Na2PdCl4 and CuCl2·2H2O were employed as metal precursors and glucose was taken as reductant during the one-pot preparation procedures in the presence of hexadecylamine (HDA). By adjusting the molar ratio of Pd and Cu precursor, homogeneously alloyed Cu–Pd bimetallic nanozymes with different morphology and structure were obtained. As shown in Fig. 1b, the transmission electron microscopy (TEM) image of Pd nanozyme presents regular and homogeneous hovenia acerba-like structure with a narrow size distribution. When 1/3 ​M ratio of Cu/Pd precursor was added, the obtained CuPd3 nanozyme exhibits agminated and pyknotic alliums-like structure (Fig. 1c). Furthermore, a concave rhombic dodecahedral-like structure (Fig. 1d) appears when the molar ratio of Cu/Pd precursor increases to 3. High-resolution transmission electron microscopy (HRTEM) images of Pd and Cu–Pd bimetallic nanozymes clearly demonstrate the fine crystal structure with periodic fringe spaces (Fig. 1e–g). As presented in Fig. 1e, the d-spacing value is measured to be 1.935 ​Å, which is consistent with the d-spacing value for (200) of pure Pd nanozyme. Meanwhile, the d-spacing of 2.071 ​Å, and 2.078 ​Å for CuPd3 nanozyme (Fig. 1f) reveals the lattice plane of Cu (111) and Pd (200), respectively. The same lattice planes are also detected for Cu3Pd nanozyme at d-spacing of 2.078 ​Å and 1.948 ​Å, indicating the fine crystal structure of Cu–Pd bimetallic nanozymes. As-synthesized Pd and Cu–Pd bimetallic nanozymes show excellent dispersibility and stability in water, phosphate buffer saline (PBS), and DMEM medium (Fig. S1). The size distribution obtained by dynamic light scattering (Fig. S2) is consistent with TEM images and all of them demonstrate negative zeta potential (Fig. S3). Besides, X-ray diffraction (XRD) was performed to explore the crystal structure of as-prepared Cu–Pd bimetallic nanozymes. The XRD pattern in Fig. S4a shows the characteristic peaks of Pd at about 40.1° for (111), 46.7° for (200), and 68.1° for (220), which is consistent with the standard PDF card (PDF#46–1043). For the samples were loaded by a transparent glass slide, the strong peak at about 21.6° is responsible for the (111) of silicon dioxide (PDF#27–0605). The XRD pattern of CuPd3 (Fig. S4b) and Cu3Pd (Fig. S4c) nanozymes present obvious diffraction peak shifting to high angle positions, indicating the increasing doping amount of Cu in Pd lattice. Meanwhile, the characteristic peaks of (111), (200), and (220) of Cu at about 43.3°, 50.4°, and 74.1° appear (PDF#04–0836), demonstrating that cubic crystal system of Cu become the main phase. It is well known that Cu and Pd have the same crystal system (cubic), similar space group (Fm-3m and Fd-3m), and approximately equal atomic diameter (1.28 ​Å and 1.37 ​Å), which making it easy to form the homogenous Cu–Pd bimetallic nanozymes [5]. According to the Cu–Pd binary phase diagrams (Fig. S5), there are two superlattices (L12 and B2) for the low-temperature ordered phases [14,15]. And the L12 structure is the ordered phase Cu3Pd with a composition range of homogeneity.

Fig. 1

Synthesis and basic characterization of Pd and Cu–Pd bimetallic nanozymes. (a) Schematical illustration of Pd and Cu–Pd bimetallic nanozymes synthetic procedure and Fenton-like reaction activity. (b–d) TEM images of Pd (b), CuPd3 (c), and Cu3Pd (d). (e–g) HRTEM images of Pd (e), CuPd3 (f), and Cu3Pd (g).

In order to further study the atomic spatial information of Cu and Pd (single-atom or intermetallic compound), we tested the Pd and Cu–Pd nanozymes using the high-angle-annular-dark-field scanning transmission electron microscopy (HAADF-STEM) (Fig. 2a–c). Compared with the HAADF-STEM images of pure Pd nanozyme (Fig. 2a), there is no evident nanoparticle and metallic cluster in both CuPd3 (Fig. 2b) and Cu3Pd (Fig. 2c) nanozymes, indicating the homogenous alloying of Cu and Pd, which is consistent with the results of element mapping. Meanwhile, none small bright/dark dots appear in the aberration-corrected HAADF-STEM (AC-HAADF-STEM) images with sub-Angstrom resolution of CuPd3 (Fig. 2b) and Cu3Pd (Fig. 2c) nanozymes, demonstrating the spacing state of Cu and Pd atom in lattice is not single-atom, but intermetallic compound. The simultaneously obtained energy-dispersive X-ray spectroscopy (EDS) spectra (Figs. S6a–c) confirms the presence of Cu and Pd in CuPd3 and Cu3Pd nanozymes. To intuitively verify the successful synthesis of Pd and Cu–Pd bimetallic nanozymes, the EDS mapping on HRTEM was carried out (Fig. 2d–f). The results clearly exhibit the pure Pd nanozyme and the homogenous distribution of Cu and Pd in both CuPd3 and Cu3Pd nanozymes. Furthermore, the atomic ratio of Cu/Pd in Cu–Pd bimetallic nanozymes is qualitatively verified by the EDS mapping, which are 1:3.4 for CuPd3 and 3.4:1 for Cu3Pd nanozyme (Fig. S6d&e).

Fig. 2

(a–f) AC-HAADF-STEM images of Pd (a), CuPd3 (b), and Cu3Pd (c). (d–f) Corresponding element mapping (HADDF image, Cu, Pd, merge image) of Pd (d), CuPd3 (e), and Cu3Pd (f). (g) Atomistic simulation scheme describes the formation of kinetically trapped homogeneous Cu–Pd bimetallic nanozymes.

Besides the elemental components measurement, the X-ray photoelectron spectroscopy (XPS) was performed to further investigate the surface chemical composition of Pd and Cu–Pd bimetallic nanozymes. As shown in Figs. S7a–c, none peak is detected for Cu 2p spectrum, but the narrow-scan Pd 3d spectra exhibits characteristic high (343.2 ​eV) and low (337.8 ​eV) energy regions that assigning to the 3d3/2 and 3d5/2 regions. For CuPd3 nanozyme, the Cu 2p spectra appears and is divided into two regions (high energy region of 952.3 ​eV and low energy region of 932.5 ​eV), which are attributed to 2p1/2 and 2p3/2 (Figs. S8a–c). Meanwhile, the divided regions of Pd 3d remain (Fig. S8c) similar with that of Pd nanozyme. With the increase of Cu amount, Cu3Pd nanozyme exhibits more obvious Cu 2p peak in XPS survey spectra (Fig. S9a&b). Importantly, the peaks of narrow-scan Cu 2p spectra at Cu 2p3/2 shows the Cu(0) (931.9 ​eV)/Cu(I) (933.5 ​eV) species (Fig. S9b), indicating the partial Cu oxidation on the surface. The narrow-scan Pd 3d spectra of Cu3Pd nanozyme is similar with that of CuPd3 nanozyme (Fig. S9c), revealing the similar atomic state. Besides, the zero-valent metal peaks for the Cu 2p spectra in both CuPd3 and Cu3Pd nanozymes and Pd 3d spectra in all samples further confirming the homogenous alloying of Cu–Pd bimetallic nanozymes [16]. Based on above results, we speculate that both Cu and Pd atom are kinetically trapped in bimetallic lattice, as shown in Fig. 2g. Because the thermodynamic equilibrium state eliminates the phase segregation of Cu or Pd, homogeneity of Cu–Pd bimetallic nanozymes is obtained during the coreduction process, which has been verified by the AC-HAADF-STEM images.

XAS measurements for coordination structure

For further authenticating the kinetically trapped structure in Fig. 2g, X-ray absorption spectroscopy (XAS) was performed to investigate the precise coordination structure of the Cu–Pd bimetallic nanozymes. The Cu K-edge X-ray absorption near-edge structure (XANES) of both CuPd3 (Fig. 3a) and Cu3Pd (Fig. 3b) bimetallic nanozymes exhibit that the pre-edge peaks are located close to Cu-foil and far away from CuPc, indicating that the average valence of Cu in both CuPd3 and Cu3Pd nanozymes is Cu(0), which is consistent with the results of XPS spectra. Furthermore, the κ3-weighted Fourier-transformed extended X-ray absorption fine structure spectra (FT-EXAFS) of both CuPd3 (Fig. 3c) and Cu3Pd (Fig. 3d) bimetallic nanozymes show that the major peak locates at about 2.25 ​Å, which means that the existence of Cu atom in lattice is not single-atom state. That is also verified by none significant peaks appear at about 1.56 ​Å as that in CuPc. Furthermore, the wavelet transform (WT) of the EXAFS plot (Fig. 3e&f) is employed to examine the atomic configuration via the information of κ- and R-spaces. As shown in Fig. 3e, both the centers of CuPd3 nanozyme and Cu-foil are located at κ-space (about 7.95 ​Å−1) and R-space (about 2.25 ​Å), which are attributed by the Cu–Cu scattering signal [17,18]. The WT image of CuPc presents a center at κ-space (about 6.25 ​Å−1) and R-space (about 1.02 ​Å), revealing the typical signal of Cu–N coordinated center. In contrast to the WT image of Cu-foil and CuPc, it can be concluded that the Cu atoms in CuPd3 nanozyme are Cu–Cu pair, a form of miscible bimetallic phase. Similarly, the WT-EXAFS of Cu3Pd nanozyme (Fig. 3f) shows an average bond distance of 2.26 ​Å, indicating the typical Cu–Cu pair. All of the results confirm the as-speculated homogenously alloyed atomic arrangement structure of Cu–Pd bimetallic nanozymes (Fig. 2g).

Fig. 3

X-ray absorption evaluation for Cu–Pd bimetallic nanozymes. (a, b) XANES and magnified pre-edge XANES spectra taken at Cu K-edge of CuPd3 (a) and Cu3Pd (b). (c, d) Fourier transform of Cu-edge EXAFS of CuPd3 (c) and Cu3Pd (d) in R-space. (e, f) Wavelet Transform image at the Cu-edge of CuPd3 (e) and Cu3Pd (f). Cu-foil and CuPc were taken as reference.

Fenton-like activity evaluation of Pd and Cu–Pd bimetallic nanozymes

The Fenton-like reaction activity of as-synthesized Pd and Cu–Pd bimetallic nanozymes were systematically explored by 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric assays. The TMB could be efficiently catalyzed into oxidized TMB (oxTMB) by the Fenton-like reaction products (hydroxide racial: ∙OH) and generated oxTMB exhibits a characteristic absorption peak at about 652 ​nm, which is proportional to the amount of ∙OH [19]. Firstly, the Fenton-like reaction kinetics of Pd and Cu–Pd bimetallic nanozymes was investigated with various concentrations (Fig. 4a–c). As shown in Fig. 4a, although Pd nanozyme exhibits weak catalytic activity below 200 ​μg/mL, time-dependent oxTMB accumulation under 500 ​μg/mL shows significant enhancement, indicating the possible Fenton-like reaction catalyzed by Pd nanozyme. Compared with Pd nanozyme, CuPd3 nanozyme reveals efficient catalytic activity (Fig. 4b). All the substrates (TMB/H2O2) were consumed by 200 ​μg/mL CuPd3 nanozyme within about 20,000 ​s. And when the concentration was increased to 500 ​μg/mL, the reaction time would shorten to 11,000 ​s. Surprisingly, Cu3Pd nanozyme presents highest Fenton-like efficiency for the reaction time is only about 8000 ​s with low concentration of 100 ​μg/mL (Fig. 4c). Meanwhile, the kinetic curve of Pd and Cu–Pd bimetallic nanozymes with same concentration (200 ​μg/mL) further clearly confirms the results that the introduction of Cu will efficiently optimize the Fenton-like reaction performance of Cu–Pd bimetallic nanozymes (Fig. 4d). Meanwhile, Electron spin resonance (ESR) was applied to detect the hydroxide radical trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in aqueous solution. Compared with alone H2O2 group, no significant OH yield is detected for Pd ​+ ​H2O2 group with concentration of 10 ​μg/mL (Fig. 4e). However, the strongly characteristic ESR spectra of spin adduct DMPO/OH are observed in both CuPd3 ​+ ​H2O2 and Cu3Pd ​+ ​H2O2 groups, indicating the successful generation of OH via Fenton-like reaction. Meanwhile, Cu3Pd ​+ ​H2O2 group exhibits the strongest peak intensity compared with Pd and CuPd3, which is consistent with the results of kinetics assay. UV–vis absorbance value at 652 ​nm of TMB/H2O2 solutions after incubation with Cu–Pd bimetallic nanozymes (200, 150, 100, 50 ​μg/mL) for 1.5 ​h (Fig. 4f) demonstrates the concentration-dependent catalysis process and enhancement of Fenton-like efficiency via Cu-introduction, which is further confirmed by the UV–vis spectra in Fig. S10. And the photographs of TMB/H2O2 solutions after incubation with Cu–Pd bimetallic nanozymes for 1.5 ​h (Fig. 4g) intuitively illustrate the difference of reaction activity. The substrate concentration is one of the main variables to influence the activity of nanozyme [20,21]. Thus, the Fenton-like efficiency of Cu–Pd bimetallic nanozymes was evaluated in various H2O2 concentration system (Fig. 4h). With the increase of H2O2 concentration, all Cu–Pd bimetallic nanozymes present excellent catalytic response. However, compared with Pd, CuPd3 and Cu3Pd demonstrate faster reaction under higher H2O2 concentration system (Fig. S11). The catalytic kinetics were also measured to compare the catalytic activity of Pd and Cu–Pd bimetallic nanozymes in different substrate environment (Fig. 4i, Fig. S12). The results show that higher substrate concentration will bring faster reaction efficiency, indicating that substrate do not influence the activity of nanozymes. Meanwhile, the kinetics results under 40 ​μM ​H2O2 concentration increasingly confirms the enhanced Fenton-like property of Cu–Pd bimetallic nanozymes (Fig. S13). All above results reveal that as-synthesized Cu–Pd bimetallic nanozymes possess excellent Fenton-like reaction performance, and the reaction efficiency is effectively enhanced via increasing level of Cu.

Fig. 4

Fenton-like reaction activity and kinetics performance of Cu–Pd bimetallic nanozymes. (a-c) Time-dependent UV–vis absorbance of Cu3Pd (a), CuPd3 (b), and Pd (c) in TMB/H2O2 solution with various concentrations (500, 200, 100, 50 ​μg/mL). (d) Time-dependent UV–vis absorbance of Cu–Pd bimetallic nanozymes in TMB/H2O2 solution with concentration of 200 ​μg/mL (e) ESR spectra of hydroxide radical trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in aqueous solution with Cu3Pd, CuPd3, and Pd (10 ​μg/mL). (f) UV–vis absorbance value at 652 ​nm of TMB/H2O2 solutions after incubation with Cu–Pd bimetallic nanozymes (200, 150, 100, 50 ​μg/mL) for 1.5 ​h. (g) Photographs of TMB/H2O2 solutions after incubation with Cu–Pd bimetallic nanozymes (500, 200, 100, 50 ​μg/mL) for 1.5 ​h. (h) UV–vis absorbance value at 652 ​nm of TMB/H2O2 solutions (H2O2 concentration: 40, 30, 20, 10, 5, 1 ​mM) after incubation with Cu–Pd bimetallic nanozymes for 0.5 ​h. (i) Time-dependent UV–vis absorbance of Cu3Pd in various TMB/H2O2 solution (H2O2 concentration: 40, 30, 20, 10, 5, 1 ​μM).

DFT theoretical calculation for enhanced Fenton-like performance

The above optimized Fenton-like efficiency of Cu–Pd bimetallic nanozymes compared with Pd nanozyme inspires us to disclose the catalytic mechanism. Therefore, we performed the molecular dynamic simulation in a canonical ensemble (NVT) by using GFN2-XTB method in the CP2K package. Density of States (DOS) analysis has also been obtained by MULTIWFN 3.8 program, and d-band center was calculated to study the adsorption between the transition metal surface and H2O2. Fig. 5a–f shows the critical intermediate structures for the generation of ·OH from H2O2 catalyzed by Cu–Pd bimetallic nanozymes. The initial H2O2 molecular will absorb on the Pd and Cu–Pd bimetallic nanozymes and then dissociate into ·OH and OH− homogeneously. Thereafter, one ·OH will react with H2O2 molecular to produce OOH− and H2O. Finally, the Cu–Pd bimetallic nanozymes will return to the original state via desorption of H2O molecular [22]. Along this reaction routine, the total energy diagrams of Cu–Pd bimetallic nanozymes was calculated and depicted in Fig. 5b,d,f. It is obviously that Cu3Pd system presents the largest total energy decrease (0.648 ​eV) compared with that of CuPd3 (0.448 ​eV) and Pd (0.443 ​eV), indicating the highest Fenton-like efficiency, which is consistent with previous experimental results. To further disclose catalytic mechanism, the partial density of states (pDOS) is explored to analyze the d-band of Cu–Pd bimetallic nanozymes. As shown in Fig. 5g–I, and Table 1, Pd nanozyme has a larger absolute pDOS value (14.633 ​eV) than that of CuPd3 (10.041 ​eV) and Cu3Pd (9.366 ​eV) bimetallic nanozymes, indicating the weaker interaction between Pd nanozyme with H2O2. Meanwhile, the order of d-band center is Cu3Pd (−3.302 ​eV) ​< ​CuPd3 (−1.871 ​eV) ​< ​Pd (−1.490 ​eV), demonstrating the easier absorption of H2O2 on the transition metal surface of Cu3Pd then both CuPd3 and Pd nanozymes. Our previous study has demonstrated that Pd atom has strong repulsive interaction to H atom in H2O2 due to the electronic structure [23]. Herein, we speculate the easier absorption effect for CuPd3 and Cu3Pd is contributed by the optimized electronic structure of Cu compared with Pd nanozyme. Therefore, contributing by the optimized electronic structure by Cu to absorb H2O2 molecular and largest total energy decrease to produce ·OH, Cu3Pd nanozyme displays highest Fenton-like efficiency than both CuPd3 and Pd nanozymes.

Fig. 5

Density Functional Theory (DFT) theoretical calculation for the Fenton-like efficiency of Cu–Pd bimetallic nanozymes. (a-f) The critical intermediate structures and corresponding total energy diagram of Fenton-like reaction path catalyzed by Pd (a, b), CuPd3 (c, d) and Cu3Pd (e, f) nanozymes. (g-i) The pDOS of d-band center analysis of Pd (g), CuPd3 (h) and Cu3Pd (i) nanozymes.

Table 1

The corresponding data of d-band center analysis from DFT theoretical calculation.

eV	Pd	CuPd3	Cu3Pd
tDOS	−13.679	−9.806	−8.828
pDOS	−14.633	−10.041	−9.366
HOMO level	−13.143	−8.170	−6.064
d-band center	−1.490	−1.871	−3.302

In vitro evaluation of Fenton-like efficiency

As one of the tumor-specific treatments, chemodynamic therapy (CDT) mediated by both Fenton or Fenton-like reaction in tumor microenvironment (TME) has exhibited various advantages such as high selectivity, deep tissue penetration, and excellent sensitivity [[24], [25], [26]]. It is well known that the intracellular H2O2 level in tumor cells (about 50–100 ​μM) is higher than that of normal cells [27,28]. Therefore, the optimized Fenton-like efficiency of Cu–Pd bimetallic nanozymes inspire us to investigate in vitro antitumor effect. 4T1 murine mammary cancer cell line was chosen as model to coincubation with various concentrations of Pd and Cu–Pd bimetallic nanozymes (400, 200, 100, 50, 25, 12.5, 6.25, 0 ​μg/mL) for 24 ​h. The relative cell viabilities were evaluated by Cell Count Kit-8 (CCK-8) and the results were shown in Fig. 6a–f. Cu3Pd (Fig. 6a), CuPd3 (Fig. 6b), and Pd (Fig. 6c) nanozymes demonstrate the concentration-dependent cytotoxicity to 4T1 cells with corresponding IC50 value of 59.55 ​μg/mL, 68.18 ​μg/mL, and 95.82 ​μg/mL, respectively. As comparison, Cu3Pd and CuPd3 nanozymes have 1.6 and 1.4 folds cytotoxicity lower than Pd nanozymes, which is agreement with the kinetic results (Fig. 4). When incubation 4T1 cells with Cu3Pd, CuPd3, and Pd nanozymes with concentration of 100 ​μg/mL for 24 ​h, there are about 88.55%, 78.43%, and 44.15% cells were killed compared with Control group (Fig. 6d). And the cytotoxic difference among of them is remarkable due to the different Fenton-like efficiency. Once the concentration raises to above 200 ​μg/mL, practically all cells are unable to survival in both Cu3Pd and CuPd3 group (Fig. 6e), indicating the optimized Fenton-like performance in tumor microenvironment. It is notable that 29.75% of cells are still alive even incubating with 400 ​μg/mL Pd nanozymes, demonstrating the low biocatalytic performance and also good biocompatibility (Fig. 6f). Meanwhile, the biocompatibility of as-synthesized Pd and Cu–Pd bimetallic nanozymes were evaluated on mouse fibroblast cell line (L929), and the results (Fig. S14) show that few toxicities are observed even under 400 ​μg/mL nanozyme treatment. Furthermore, to verify the Fenton-like reaction catalyzed by Cu–Pd bimetallic nanozymes in 4T1 cells, 2′-7'dichlorofluorescin diacetate (DCFH-DA) which is a cell-permeant reagent fluorogenic dye that tests peroxyl, hydroxyl, and other ROS activity was employed to intuitively evaluate the generated ·OH [29]. After incubation with Cu–Pd bimetallic nanozymes (25 ​μg/mL) for 6 ​h, a few green signals are detected in Pd group compared with Control group (Fig. 6g, Fig. S15), indicating the successful generation of ·OH. Both CuPd3 and Cu3Pd groups exhibit obviously enhanced green signal, demonstrating higher ·OH level contributed by the enhanced Fenton-like efficiency, which is consistent with cell viability results. It is well-known that higher ·OH generation will cause the intracellular oxidative stress, which then upregulate the GSSG level [29]. It is clear that Cu–Pd bimetallic nanozymes treated 4T1 cells produce higher GSSG level compared with Pd nanozyme treated group (Fig. S16), which confirms the higher level of ·OH. All above results reveal the optimized Fenton-like efficiency of Cu–Pd bimetallic nanozymes compared with Pd nanozymes and great potential as agents for cancer chemodynamic treatment.

Fig. 6

In vitro cytotoxicity of Cu–Pd bimetallic nanozymes. (a-f) The cell viability of 4T1 cells after incubation with Pd and Cu–Pd bimetallic nanozymes (400, 200, 100, 50, 25, 12.5, 6.25, 0 ​μg/mL) for 24 ​h. (g) Fluorescent images of 4T1 cells after incubation with Pd and Cu–Pd bimetallic nanozymes (25 ​μg/mL) for 6 ​h and staining with DCFH-DA probe.

Conclusions

In summary, we presented a one-step coreduction strategy to prepare the miscible Cu–Pd bimetallic nanozymes with controllable shape and homogeneously alloyed structure to avoid the immiscibility and heterogeneity. The homogeneity is systematically explored through HRTEM, AC-HAADF-STEM with sub-Angstrom resolution, XPS, and XAS. Wavelet Transform images at the Cu-edge clearly demonstrate the homogenously alloyed structure of Cu–Pd bimetallic nanozymes via the κ-space and R-space. TMB colorimetric assays exhibit homogeneous introduction of Cu successfully endows Cu–Pd bimetallic nanozymes with enhanced Fenton-like efficiency, especially for Cu3Pd nanozymes. Further density functional theory (DFT) theoretical calculation reveals that Cu–Pd bimetallic nanozymes exhibit smaller d-band center compared with Pd nanozymes. Easier adsorption of H2O2 molecular contributed by the electronic structure of Cu significantly accelerate the catalytic process together with the strong repulsive interaction between H atom and Pd atom. In vitro cytotoxicity and intracellular ROS generation performance are evaluated to reveal the potential for in vivo biocatalysis. The strategy to construct kinetically miscible Cu–Pd bimetallic nanozymes will guide the development of bimetallic catalysts with excellent Fenton-like efficiency for biocatalytic nanomedicine.

Credit author statement

Wensheng Xie: Conceptualization, Methodology, Investigation, Writing - review & editing. Genpei Zhang: Methodology, Calculation, Validation. Zhenhu Guo: Methodology, Investigation, Validation. Hongye Huang: Methodology, Investigation, Validation. Jielin Ye: Methodology, Investigation, Validation. Xiaohan Gao: Methodology, Investigation, Validation. Kai Yue: Writing - review & editing. Yen Wei: Conceptualization, Supervision, Writing - review & editing. Lingyun Zhao: Conceptualization, Supervision, Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.