Transition Metal High‐Entropy Nanozyme: Multi‐Site Orbital Coupling Modulated High‐Efficiency Peroxidase Mimics

Abstract Strong substrate affinity and high catalytic efficiency are persistently pursued to generate high‐performance nanozymes. Herein, with unique surface atomic configurations and distinct d‐orbital coupling features of different metal components, a class of highly efficient MnFeCoNiCu transition metal high‐entropy nanozymes (HEzymes) is prepared for the first time. Density functional theory calculations demonstrate that improved d‐orbital coupling between different metals increases the electron density near the Fermi energy level (E F) and shifts the position of the overall d‐band center with respect to E F, thereby boosting the efficiency of site‐to‐site electron transfer while also enhancing the adsorption of oxygen intermediates during catalysis. As such, the proposed HEzymes exhibit superior substrate affinities and catalytic efficiencies comparable to that of natural horseradish peroxidase (HRP). Finally, HEzymes with superb peroxidase (POD)‐like activity are used in biosensing and antibacterial applications. These results suggest that HEzymes have great potential as new‐generation nanozymes.


Introduction
Nanozymes are explicitly defined as a collection of nanomaterial-based artificial enzyme mimics, and they have gradually evolved as alternatives to natural enzymes due to their outstanding environmental tolerance, recyclability, and long-term stability. [1]][12] Of note, multimetallic nanozymes have recently attracted considerable attention due to their unique cocktail effects.][15] However, most conventional design concepts involving doping, heterojunctions, or a combination of both are trial-and-error strategies, and the limited component space for these empirical approaches together with the lack of theoretical guidance have hindered diversification and catalytic performance improvements of the nanozymes. [8]herefore, guided by predictive models of catalytic activity, such as the d-band model and e g -occupancy, the development of multimetallic nanocatalytic platforms with a wide range of elements would create a new revolution in nanozymes. [7,16,17]o resolve these issues, we introduced the concept of high entropy into the development of high-performance nanozymes, which integrate state-of-the-art high-entropy alloys with intrinsic enzyme-like active sites.[23][24] Unlike conventional alloys, HEAs have defined content boundaries (5-35%) for the principal elements (≥ 5 species), but no particular element dominates. [25,26]In an ideal HEA, the formation enthalpy of the compound is overcome by the dramatic increase in configurational entropy induced by the mixing of the multiple components, facilitating the formation of stable single-phase solid solutions rather than intermetallic compounds. [25,27,28]Accordingly, they tend to form multicomponent single phases with face-centered cubic (FCC), body-centered cubic, and hexagonal-close-packed structures, in which the internal atoms are randomly distributed. [18,29]Therefore, HEAs are also referred to as complex solid solutions. [30]In theory, confinement of the atoms with disparate properties and atomic sizes in the same lattice would lead to noticeable lattice distortions and synergistic effects, which normally generate a definite structure-property relationship for the catalyst. [19]More specifically, the abundant and varied atomic sites in HEAs optimize the geometric and electronic configurations of the reactive surface, which in turn affects the adsorption energies of substrates or intermediates and ultimately the catalytic activity.In addition, the strategy of tuning HEAs to nanostructures enables amplification of the size effect and consequently improvement of their catalytic performance. [20,31,32]As such, mainstream nano-HEAs with tailored components and structures are of interest in energy-related fields, especially in electrocatalysis, due to their superior catalytic performance and stability. [23,24,33]ecent studies have highlighted the emergence of high-entropy materials with POD-like or oxidase (OXD)-like activity, which can be exploited for efficient nanozyme-based antitumor and antibacterial therapies. [34,35]Additionally, nanotransition metals are important multimetallic nanozymes due to their easy accessibility, low costs, and excellent catalytic performance.Theoretically, the relatively explicit and easy-to-tune d-band structures of transition metals are potentially advantageous for the design and structure-property relationship studies of HEzymes, which suggests that effort is still needed to fill this research gap. [11,14,15]n this work, we report the first transition metal high-entropy POD mimic with strong substrate affinity and high catalytic efficiency.The proposed HEzymes contain five transition metals with similar atomic radii, which frequently serve as the catalytic active sites of redox nanozymes, including manganese, iron, cobalt, nickel, and copper. [1,3]DFT calculations offer insight into the extraordinary POD-like activity of the HEzymes.The strong interactions among the d electrons of the elements in the HEA NPs optimize the electronic distribution around the E F of the bulk material, which enhances the adsorption of intermediates at the surface-active sites while increasing the efficiency of electron transfer during the catalytic process, thereby enhancing the performance of the nanocatalyst.As a proof of concept, the HEsymes were used as biosensors and in biomedicine.In addition to guaranteeing both selectivity and accuracy of the colorimetric platform for the target, the HEzymes also achieved efficient elimination of multidrug-resistant bacteria and biofilm eradication by triggering ROS outbreaks.Our findings advance the systematic understanding of the structure-property relationships between the electronic structure and catalytic performance of nanozymes and provide an original paradigm for engineering high-performance HEzymes.

Syntheses and Characterizations of MnFeCoNiCu HEzymes
MnFeCoNiCu HEA NPs were fabricated via a low-temperature oil phase synthetic strategy. [23,33]Manganese(II) acetylacetonate (Mn(acac) 2 ), iron(III) acetylacetonate (Fe(acac) 3 ), cobalt(III) acetylacetonate (Co(acac) 3 ), nickel(II) acetylacetonate (Ni(acac) 2 ) and copper acetylacetonate (Cu(acac) 2 ) were used as the metal precursors, glucose as the reductant, and cetyltrimethylammonium bromide (CTAB) and oleylamine (OAm) as solvents and structure inducers.A brown, finely dispersed mixture of the raw materials was formed through ultrasonication.As the temperature of the oil bath increased, the mixture turned black, possibly due to the coordination and coreduction of the five metals.Subsequently, the single-phase HEA was formed after diffusion and rearrangement of the atoms (Figure 1a). [33]To reveal the crystal lattice parameters and morphology of HEA NPs, X-ray diffraction (XRD), transmission electron microscope (TEM), high-resolution TEM (HRTEM), and scanning electron microscopy (SEM) were used to characterize the prepared HEA NPs.As shown in Figures S1 and S2 (Supporting Information), the synthesized HEA were nanoparticles with an average diameter of 20.55 nm.The XRD pattern for the HEA NPs indicated a FCCstructure, and the peaks at 44.10°and 50.33°were attributed to the (111) and (200) facets of CuNi (JCPDS No. 47-1406), respectively (Figure 1b).A representative HRTEM image of the HEA NPs exhibited an average lattice spacing of 0.182 nm, which corresponded to the (200) facet of CuNi, illustrating a single FCC phase in agreement with the XRD result (Figure 1c).Of note, the fast Fourier transform (FFT) patterns of selected regions are displayed in Figure c 1 -c 3 and suggest that the alloy had a (010)-oriented FCC structure.The average lattice spacings determined from the FFT patterns of selected regions varied from 1.755 to 1.827 Å, which implied lattice distortions in the alloy phase (Figure 1d; Figure S3, Supporting Information). [36]To investigate the effects of the reagents on the morphology of the alloy, TEM images of different products prepared under various reagent combinations are presented in Figures S4-S7 (Supporting Information).Clearly, CTAB or STAB serves to direct the alloy to form uniformly dispersed nanoparticles, while glucose functions as the reductant to control the size and morphology of the alloy. [33,37]Furthermore, HEA NPs fabricated with CTAB display a more uniform distribution of size dimensions compared to those prepared with STAB, resulting in a more consistent shape and morphology distribution.
The energy dispersive spectroscopy (EDS) maps and line scans of the HEA NPs showed that the five metallic elements were homogeneously distributed in the nanoparticles, which provides evidence for the successful synthesis of the MnFeCoNiCu quinary HEA NPs (Figure 1e; Figures S8 and S9a, Supporting Information).Likewise, the HEA NPs had 2 values for the major peaks in the XRD patterns that were consistent with those for the pure constituent metals forming FCC phases (Figure 1f).Obviously, the diffraction peaks for the HEA NPs were broadened, and their positions were slightly shifted compared with those of the pure metals, confirming the formation of the nanometer-scale alloy. [24]ccording to the quantitative results of the inductively coupled plasma optical emission spectroscopy (ICP-OES) and X-ray photoelectron spectra (XPS) (Figure 1e; Figure S9b, Supporting Information), the atomic ratio for Mn, Fe, Co, Ni and Cu was 12: 32: 9: 23: 24.The configurational entropy of MnFeCoNiCu nanoparticles was calculated as 1.52 R, surpassing the boundary of 1.5 R separating high-entropy and medium-entropy alloys.Therefore, MnFeCoNiCu nanoparticles are classified as members of high-entropy materials.Then, XPS was employed to analyze the surface states of the HEA NPs. Figure S10 (Supporting Information) displays the 2p XPS spectra obtained for the 2p edges of Mn, Fe, Co, Ni, and Cu.Of note, the five metals in the HEA NPs all showed mixed valence states, including metallic and oxidized states. [23,37]The Fourier transform-infrared (FT-IR) spectrum of the HEA NPs showed peaks at 1485.19 and 3016.66 cm −1 , which arose from CTAB, and these disappeared or were significantly attenuated, indicating that the residual surfactant on the surfaces of the particles had been removed (Figure S11, Supporting Information).In addition, the test photographs illustrated that the prepared HEA NPs were dispersed and remained stable during storage in aqueous solutions (Figure S12, Supporting Information).

Nanozyme Catalytic Performance of the HEzymes
Inspired by the structure and catalytic pathway of natural HRP, the development of POD-like enzymes involving transition metal elements as protagonists has proven to be efficient and economical. [38]Herein, the enzyme-like activity of HEA NPs was systematically investigated by using 3,3′,5,5′tetramethylbenzidine (TMB), TMB as the chromogenic substrate.As shown in Figure 2a, transition metal-based POD mimics catalyzed the decomposition of H 2 O 2 , resulting in the color rendering of the chromogenic substrates.Specifically, oxidation reaction of 2,2′-Azino-bis (3-ethylbenzothiazoline-6sulfonic acid) diammonium salt (ABTS), o-phenylenediamine (OPD) and TMB were catalyzed by the HEA NPs in the absence of H 2 O 2 , and the typical peaks (ABTS: 415 nm, OPD: 431 nm, TMB: 652 nm) for the colored products were observed in the UV-vis spectra (Figure 2b).A series of pH-, temperature-and material concentration-dependent tests was performed to optimize the catalytic parameters.As shown in Figure 2c, noticeable POD-like activity was observed for the HEA NPs and compared to the OXD-like activity, and the optimal pH and temperature were determined to be ≈3.0 and 70 °C, respectively (Figure 2d).Additionally, the oxidation of TMB was also strongly dependent on the concentration of the HEA NPs under the given conditions (Figure 2e).To evaluate the enzymatic activities of the POD mimics, a steady-state kinetic study was carried out to obtain the rate constants of the HEAs by varying the concentration of H 2 O 2 with a constant concentration of TMB or vice versa.Typical Michaelis-Menten curves and Lineweaver-Burk double reciprocal plots were fitted to obtain the Michaelis constant (K m ) and maximum reaction rate (V max ) of the HEAs, and the calculated parameters are listed in Table S1 (Figure 2f,g; Figures S13  and S14, Supporting Information).As expected, the HEAs with a defined morphology exhibited higher POD-like activity and improved kinetic parameters compared to aggregated irregular alloys, which was possibly caused by exposure to more active sites on the surface (Figure S15, Supporting Information). [19,20]Compared with the nanozymes addressed in previous studies, the HEA NPs exhibited superior affinities and catalytic efficiencies for substrates (K m = 0.07 and 0.6 mm for TMB and H 2 O 2 ; K cat /K m = 1.74 × 10 12 and 5.38 × 10 11 for TMB and H 2 O 2 , respectively; Table S2, Supporting Information).These results suggested that HEA NPs had remarkably higher POD-like activity than HEA bulk (specific activity: 109.65 U mg −1 for HEA NPs and 43.48 U mg −1 for HEA bulk) 0and exhibited acceptable catalytic stability during long-term preservation at a wide range of temperatures and pHs (Figure 2h; Figure S16, Supporting Information).
The catalytic mechanism of the HEA POD mimic was investigated with ESR spectroscopy, and strong ESR signals for • OH, • O 2 − , and 1 O 2 were detected when the HEA NPs were mixed with H 2 O 2 (Figure 2i).Therefore, the ESR spectra indicated that the HEA NPs catalyzed the decomposition of H 2 O 2 to produce reactive oxygen species (ROS) such as • OH, • O 2 − , and 1 O 2 , which is consistent with the mechanism hypothesized for metallic POD mimics. [39]In contrast, only weak signals of  S17, Supporting Information), which suggests that HEA NPs only exhibit limited OXD-like activity compared to their PODlike activity.This finding is consistent with the results of the pH optimization.

DFT Calculations for POD-Like Activity of the HEA NPs
First-principles DFT calculations were used to investigate the POD catalytic performance of the HEA NPs.It is well known that the catalytic efficiency of a nanozyme mainly depends on its surface atomic composition and structure, which generally have significant impacts on adsorption or electron transport during catalysis. [18,29]Therefore, a lattice model with the FCC random phase for a relatively Fe-, Ni-and Cu-enriched surface was proposed based on the ICP-OES, HRTEM, and XRD results (Figure 3a).From the lattice parameters revealed by the XRD and HRTEM characterization, it was inferred that the CuNi bimetallic solid solution phase could well be an integral matrix or constituent phase of the HEA crystal.Also, the abundant Fe sites on the surface of HEA NPs are indispensable for their excellent POD-like activity.Hence, in this section, the CuNi bimetallic alloy and FeCuNi trimetallic alloys were selected for comparison of the electronic structures and catalytic mechanisms. [40]The partial projected density of states (PDOSs) for the HEA NPs, CuNi, and FeCuNi are presented in Figure 3b,c and Figure S18 (Supporting Information) to illustrate their electronic structures.Apparently, the Cu, Fe, and Ni sites were electron-rich, and HEA exhibited a higher electron abundance at the E F due to the contributions of Mn, Fe, and Co (Figure S19, Supporting Information).Distinct d-orbital overlaps among the different metals were clearly observed, demonstrating that the elements in the alloy were strongly bonded to each other.On the other hand, the d-electron complementation effect amplified the synergistic effect of the d-electrons from different metal sites.In brief, in the alloy lattice, the internally paired d-electrons from the late transition metals were likely redistributed into empty or halffilled vacant d-orbitals of the early transition metals, resulting in strong d-orbital coupling. [37,41]Thus, the efficiency for site-to-site electron transfer of the alloy between the constituent metals was potentially increased, which in turn activated the peroxide substrate with the POD mimics. [24]The Cu-3d band showed a sharp peak at −2.95 eV, which was the furthest position from the E F , indicating that Cu served as an electron reservoir to maintain the valence balance of the HEsymes during catalysis.It is clear that the Mn-3d band spanned the E F with high electron density and acted as an electron consumption center during catalysis, making it easier to transfer electrons from the alloy surface to the adsorbed substrate.Additionally, the Fe, Co, and Ni 3d orbitals were located in the middle and exhibited broad bands, both of which contributed to the stabilization of the intermediates and reduction of the energy barrier for electron transfer during the redox reaction.
Figure 3d-f depicts the spin up/down d-orbitals and d-band centers of elements in the HEA NPs and CuNi.According to the d-band center theory and Sabatier principle, a rational upshift of the d-band center position relative to the E F generally enhances the bond strengths between the metal atoms and oxygen intermediates, thus facilitating the POD-like activity of the nanozyme. [17,42,43]For spin-polarized transition metal active sites, such as those of Mn, Fe, and Co, the spin-down d-orbitals usually occupy higher energy levels relative to the E F due to their lower occupancy levels, and they are more likely to be involved in intermediate adsorption. [44,45]Taking into account the delocalization and the strong interactions of the d-orbital electrons in the alloy, the overall d-band center was calculated as a descriptor.CuNi showed a low d-band center (spin-down, −1.412 eV) owing to the high occupancy of the Cu and Ni 3d orbitals, which normally disfavors the adsorption of oxygenated intermediates. [11,46]Compared with CuNi, the contribution of Fe sites results in a noticeable enrichment of d-electrons near the E F for FeCuNi, which is particularly prominent in the spin-down electron distribution.As such, the d-band center of FeCuNi displays a pronounced upshift (−0.885 eV) relative to the E F when compared to CuNi (Figures S20 and S21, Supporting Information).Likewise, the spin-down d-band center of the HEA bulk was explicitly balanced at a higher position of −0.995 eV with respect to the E F because the Co and Mn 3d orbitals with higher d-band centers overlapped with the Cu and Ni 3d orbitals.The upshift of the d-band center relative to the E F described above facilitated the adsorption and stabilization of the oxygen intermediates and may well be considered an index for the reliable catalytic performance of the HEA NPs. [17,41] conclusion, the d-electron structure of the HEA NPs was regulated by different constituent metals, which guaranteed efficient and stable POD-like activity.
DFT was also used to study the POD-like mechanism on the HEA surface, and the reaction model was constructed with the (111) lattice plane of the alloy (Figure 4a).As shown in Figure 4b, there were two main pathways for the degradation of H 2 O 2 in the

Application of the HEzymes in Biosensing and Biomedicine
As practical and versatile biosensors, nanozymes are employed in rapid analyses of ascorbic acid (AA), H 2 O 2, and glucose (Figure 5a).Food antioxidants, such as AA, polyphenols, and glutathione (GSH), inhibit the oxidation of TMB due to their reducibility. [49]Therefore, the HEA/H 2 O 2 /TMB system is sensitive to the antioxidants in the sample, which is reflected by an obvious decrease in the intensity of the absorption peak at 652 nm (Figure 5b; Figure S24a, Supporting Information).As shown in Figure S24b,c (Supporting Information), the ∆absorbance of the HEA/H 2 O 2 /TMB system at 652 nm showed an excellent linear relationship with AA concentration over the range of 40 to 800 μm and a favorable correlation coefficient (R 2 = 0.9970), and the detection limit of the method was 28.59 μm for the 3S/N (signal/noise) equation.H 2 O 2 is an essential substrate, and it often serves as a ROS supplier for the chromogenic reactions of POD mimics.On the other hand, GOX catalyzes the oxidation of glucose to produce H 2 O 2 , which can be employed as the substrate for TMB oxidation (Figure 5a). [50]As shown in Figure 5c and Figure S25 (Supporting Information), the results demonstrated that the linear range and the LOD (S/N = 3) for the H 2 O 2 detection assay were 40-400 μm and 15.29 μm, respectively, and the data showed a high correlation coefficient (R 2 = 0.9970).Likewise, a good linear relationship (R 2 = 0.9966) between the glucose concentration, ranging from 20 to 160 μm, and the ab-sorbance of the system at 652 nm is depicted in Figure 5d and Figure S26 (Supporting Information), and the LOD (S/N = 3) was 4.00 μm.The effects of potential interferents on the chromogenic reaction were not apparent except for those of alanine, histidine, and arginine, which indicated a satisfactory selectivity for the assay (Figure 5e).Moreover, the established colorimetric platform for the detection of TAC and glucose was applied to beverage samples and vitamin C tablets.The results were essentially consistent with the specifications for the food samples, confirming the practicality of the TAC method (Figure S27 and Table S3, Supporting Information).Assay stability and recycling tests demonstrated the excellent reusability and reliability of the HEsymes (Figure S28, Supporting Information).In conclusion, the HEsymes-based colorimetric platform is useful for measurements of TAC, H 2 O 2, and glucose (Tables S4 and S5, Supporting Information).POD mimics are indispensable mediators in recent studies on nanozyme-based antibacterial and antitumor therapies, whose POD-like activity exerts a decisive influence on therapeutic efficacy. [39,47,51]Dispersion tests revealed that HEA NPs with superior POD-like activity exhibited favorable dispersibility in common aqueous matrices, suggesting that HEA NPs hold the potential to be utilized for the development of efficient antibacterial therapeutics in physiological conditions (Figure S29, Supporting Information).Hence, HEsymes have been employed for combating four representative pathogenic bacteria (Gramnegative: Escherichia coli O157:H7 (E. coli O157:H7), Salmonella enteritidis (S. enteritidis).Gram-positive: methicillin-resistant Staphylococcus aureus (MRSA) and Listeria monocytogenes (L.monocytogenes)) by catalyzing H 2 O 2 to generate excess ROS and mediate membrane disruption, which is lethal to bacteria (Figure 6a).[54] The corresponding results demonstrated that the HEA NPs/H 2 O 2 system exhibited remarkable broad-spectrum antibacterial effects against both Gram-positive and Gramnegative bacteria, and it was also more potent in killing Grampositive bacteria (Figure 6b; Figures S30-S34 and Table S6, Supporting Information).][57] To further investigate the bactericidal mechanism of the HEA NPs/H 2 O 2 antibacterial system, MRSA was used as a model strain for the measurement of biochemical indicators after different treatments.The live/dead bacterial staining reveals that the HEA/H 2 O 2 groups exhibit the most effective synergistic bactericidal effect (Figure S35, Supporting Information).The membrane permeability test and the MDA assay demonstrated that bacterial death caused by the HEA/H 2 O 2 system arose from peroxidative damage to the bacterial membrane and was correlated with the concentration of the HEA NPs (Figure 6c; Figure S36a,b, Supporting Information).Meanwhile, the virulence of MRSA, such as the hemolytic capacity and plasma coagulase activity, explicitly declined after the treatment with HEA/H 2 O 2 , which was mainly due to the death of MRSA organisms caused by ROS attack (Figure S36c,d, Supporting Information).The GSH depletion capacity of the HEA/H 2 O 2 system disrupted the antioxidant system of the bacteria, thereby enhancing the bactericidal capacity (Figure S37, Supporting Information).DCFH-DA staining demonstrated that both the dramatic elevation of bacterial endogenous ROS levels and the membrane damage mediated by ROS outbreaks were closely correlated with the bactericidal mechanism of the POD mimetic enzymes (Figure S38, Supporting Information).The morphologies of the bacteria after the application of different therapies are displayed in Figure 6d.In contrast to the control group, the attack of ROS for the HEA/H 2 O 2 group caused nonnegligible wrinkling and destruction of the bacterial surface.As a consequence, irreversible damage and changes in the permeabilities of the bacterial membranes occurred, leading to leakage of the endogenous contents and triggering bacterial death.The biocompatibility evaluation suggested that the HEA NPs exhibited desirable hemocompatibility and cytocompatibility, which can be used for the elimination of drug-resistant bacteria (Figure S39 and Table S7, Supporting Information).
Multidrug-resistant bacterial infections, especially those accompanied by biofilm formation, present considerable challenges for wound therapy. [58]The formation of multidrugresistant bacterial biofilms inhibits the permeation of antibiotics and conventional bactericides and facilitates the horizontal transfer of pathogenic and drug-resistance genes among bacteria. [59]n particular, extracellular DNA (eDNA) is an integral part of the bacterial biofilm matrix, and it is capable of cross-linking with extracellular polysaccharides to create a "shelter" for the bacteria. [59,60]Herein, the HEA NPs with POD-like activity, acted as protagonists and used H 2 O 2 as a weapon to provoke an outbreak of ROS that damaged the organism and lysed the biofilm (Figure 6e).As shown in Figure 6f and Figures S40 and S41 (Supporting Information), due to the synergy of the HEA NPs and H 2 O 2 , MRSA biofilm formation was significantly inhibited, and the concentration of eDNA in the biofilm was remarkably lower than that in the control group.These results indicated that ROS burst-mediated nanocatalysis could be adopted as an efficient and safe countermeasure to biofilm formation by drug-resistant bacteria.

Conclusion
We have developed a class of novel HEzymes with multiple tailored active sites and tuned electronic structures.The strong electron interactions among the constituent metals enhanced the catalytic efficiency, as manifested by the regulated electron distribution of the bulk near the E F and the rational shift of the d-band center of the bulk relative to the E F .With a POD nanocatalyst as a model, detailed analyses indicated that the proposed HEA POD mimic with an FCC crystal structure exhibited remarkable binding affinity and catalytic efficiency comparable to those of natural enzymes.The d-electron complementary effect and synergy endowed the HEzymes with significantly higher catalytic efficiencies than conventional nanozymes.Moreover, HEzymes exhibited desirable performance in target compound detection, drug-resistant bacteria elimination, and bacterial biofilm eradication.In general, this work is intended to provide an original paradigm for the exploitation of high-performance nanozymes with enhanced catalytic activities and to make HEAs a resourcerich and versatile platform for the effective design and application of nanozymes.
Synthesis of MnFeCoNiCu HEA NPs: MnFeCoNiCu HEA NPs were synthesized according to previous reports with a slight modification. [23,33,37]TAB (90 mg) was added into a flask containing OAm (5 mL).After ultrasonication for 30 min, Mn(acac) 2 (31 mg), Fe(acac) 3 (42 mg), Co(acac) 3 (9 mg), Ni(acac) 2 (9 mg) and Cu(acac) 2 (10 mg) were successively added.Then the mixture was sonicated with glucose for 2 h to obtain a homogeneous solution.After that, nitrogen was injected into the flask and the solution was heated to 220 °C for 6 h under magnetic stirring in an oil bath.The mixture was rapidly cooled to room temperature and the black products were collected by centrifugation and washed three times with an ethanol/cyclohexane mixture (v/v: 9:1).Finally, the precipitates were lyophilized and dispersed in distilled water for further experiments.In addition, other reference materials in the work were synthesized under the same conditions and processes as the proposed approach, except for the different formulations of the ingredients.
Characterization of MnFeCoNiCu HEA NPs: TEM and HRTEM images were characterized by a JEM 2100F (JEOL, Japan) at an accelerating voltage of 200 kV.EDS was obtained by PV97-617300-ME (AMETEK, US).Powder XRD patterns were recorded on an X-ray diffractometer (D8 Advance, Bruker, Germany).XPS were obtained by an Axis Ultra DLD instrument (Kratos Analytical, UK) equipped with an Al K X-ray source (1486.6 eV).ICP-OES (720ES, Agilent, US) was used to determine the compositions of HEA NPs.FT-IR spectra were obtained on the wavenumber range of 400-4000 cm −1 with a Vetex70 (Bruker Corp, Germany) instrument using the KBr pellet method.All UV-visible absorption spectra were recorded using a UV-vis spectrophotometer (UV-2550, Shimadzu, Japan).A microplate reader (Multiskan MK3, Thermo Fisher Scientific, US) was used to measure the absorption at 652 nm of all test groups.
The configurational entropy of the high-entropy alloy was defined by the following equation: (∆S configuration >1.5 R: high-entropy; 1.0-1.5Rmiddle (or medium)-entropy; <1.0R low-entropy class.)In this work, ∆S configuration for MnFeCoNiCu NPs = 1.52 R, so HEA NPs are classified as high-entropy materials.
Enzyme-Like Activity of MnFeCoNiCu HEA NPs: The OXD-and PODlike activity of HEA NPs were determined by TMB colorimetric assays. [61]n a typical test, TMB (1 mm) and H 2 O 2 (10 mm) were added into pH 3.0 HAc-NaAc buffer (0.1 m) containing 6 μg mL −1 HEA NPs and absorbances of the mixtures at 652 nm were recorded at room temperature (reaction time = 10 min).Meanwhile, the OXD-like activity of HEA NPs was determined without the addition of H 2 O 2 .
To explore the optimal conditions of the enzyme-like activity of HEA NPs, a range of pH values (2.0 to 7.0) and temperature (20 to 80 °C) for the reaction were tested to investigate the oxidation of TMB.Moreover, the time-dependent kinetics were also conducted to optimize the incubation time and enzyme concentration of the reaction.
Enzyme Determination of Specific Activity: Briefly, a series of concentrations of HEA NPs were mixed with H 2 O 2 (1 m) and TMB (0.5 mg mL −1 ) in 2 mL HAc-NaAc buffer (pH 3.0, 0.1 m), and the initial reaction rate of the solution was calculated from the absorbance variations of TMB (∆A/∆t).A linear fit curve of the nanozyme concentration to the initial rate of reaction was performed to determine its specific activity.
ESR Measurement: An Electron spin-resonance (ESR) spectroscopy spectrometer was utilized to measure the generation of hydroxyl radicals ( • OH), superoxide anion ( • O 2-), and singlet oxygen ( 1 O 2 ).Briefly, the tests were conducted under the following conditions including H 2 O 2 , DMPO, and HEA NPs.All mixtures were dispersed in pH 3.0 HAc-NaAc buffer (0.1 m).The solutions were subjected to ESR analysis immediately after reacting for 10 min at room temperature.
Colorimetric Detection of Ascorbic Acid: Ascorbic acid was used as a representative antioxidant to establish an indirect method for the total antioxidant capacity of samples.HEA NPs (6 μg mL −1 ) and H 2 O 2 (20 mm) were added into pH 3.0 HAc-NaAc buffer (0.1 m).Then, TMB (1.0 mm) with varying concentrations of ascorbic acid was added to the mixture.The absorption spectra of the systems were measured after incubation for 10 min at room temperature.
TAC Detection in Food Samples: Several commercial beverages and two vitamin C tablets were preprocessed to fit the determination range of the AA detection assay, which included dissolution and dilution.After that, the same protocol for AA detection was applied to the TAC detection of samples.Therefore, the TAC of two vitamin C tablets was evaluated to validate the accuracy and feasibility of the AA detection assay mentioned above.
Colorimetric Detection of H 2 O 2 : HEA NPs (6 μg mL −1 ) and TMB (1.0 mm) were added into pH 3.0 HAc-NaAc buffer (0.1 m) containing varying concentrations of H 2 O 2 .The absorption of the determination system at 652 nm was recorded after incubation for 10 min.
Colorimetric Detection of Glucose: HAc-NaAc buffer (pH 3.0, 0.1 m) containing GOX (1.0 mg mL −1 ), HEA NPs (6 μg mL −1 ) TMB (1.0 mm), and glucose with various concentrations was incubated for 15 min at 37 °C.Subsequently, the absorption of the solution at 652 nm was recorded at room temperature.The protocol above was also applied to the glucose detection of food samples.Above all, the concentration of all samples was adjusted to the linear range of the glucose detection assay.
Selectivity Test of the Detection Assay: To assess the selectivity for AA, H 2 O 2, and glucose in the detection assay, certain substances that commonly occur in food samples were added into the detection system, with concentrations of these interference factors or times that of AA.The absorbances of all test groups at 652 nm were recorded at room temperature after incubation for 10 min.
Preparation of Bacterial Suspensions: Four representative pathogenic in isoamyl acetate.Finally, the samples were dried by supercritical fluid desiccation and observed with a scanning electron microscope (SEM, S-4800 FE-SEM, Hitachi, Japan).Bacterial Virulence Test: Briefly, 500 μL of sterile saline was injected into a penicillin vial containing lyophilized rabbit plasma.Then, 300 μL of the treated bacterial suspension collected in each antibacterial activity test group was injected into the prepared rabbit plasma and mixed well.The penicillin vials were incubated at 37 °C, and the results were observed within 6 h.Similarly, the different treated bacterial suspensions were mixed with red blood cells washed with PBS (pH 7.4) and co-incubated at 37 °C for 5 h.Finally, the samples were centrifuged (2500 rpm, 10 min), and the supernatant was retained and its absorbance was measured at 540 nm.
Hemolytic Test: The sterile defibrinated sheep blood was centrifuged (2500 rpm, 10 min) to collect the red blood cells.Afterward, the red blood cells were washed and resuspended with PBS (pH 7.4).The red blood cell suspensions were added to a 96-well plate and incubated with different concentrations of HEA NPs.0.1% Triton X-100 was used as the positive control and PBS as the negative control.All groups were incubated at 37 °C for 4 h.Then, the plate was centrifuged (2500 rpm, 10 min), and the supernatant of each well was taken to measure its absorbance at 540 nm.
Cytotoxicity Measurement of HEA NPs: Briefly, after resuscitation, 3T3 cells were transferred into 96-well plates and co-incubated with various concentrations of HEA NPs at 37 °C and 5% CO 2 for 4 h.Subsequently, MTT (5.0 mg mL −1 ) solution was added to each well and incubated for 4 h.DMSO was used to dissolve the produced formazan and the absorbance of the solution was measured at 570 nm.
Bacterial Biofilm Formation Inhibition Activity: To simulate the initial formation of biofilm under physiological conditions, 1 mL MRSA suspension was fully mixed with 1 mL LB broth (containing 1% glucose), and the mixture was subsequently added dropwise to the surface of sterile cell culture dishes and cultured at 37°C overnight.Then, HEA NPs (40 μg mL −1 ) and H 2 O 2 of different concentrations (250, 500, and 750 μm) were added, and all samples were incubated at 37 °C for 24 h.PBS was then used to thoroughly rinse off the suspended residues.The formed biofilms were stained with FDA (200 μg mL −1 ) and the excess dye was washed with PBS.The results were analyzed by a spinning disk confocal microscope (Revolution WD, Andor, UK).
Cv Staining For Assessment of the Biomass of Bacterial Biofilms: First, 500 μL of the prepared MRSA suspension was added into 500 μL liquid LB broth in a 24-well plate and incubated at 37 °C for 12 h.After that, different test groups were conducted in the wells: (II) HEA NPs (40 μg mL −1 ), (III) H 2 O 2 (750 μm), (IV) HEA NPs + H 2 O 2 .After incubation at 37 °C for 24 h, the plate was washed with PBS three times, and 400 μL of methanol was added to immobilize the bacterial biofilm for 20 min.Next, 400 μL of 0.1% CV solution was added to stain the biofilm for 15 min.The redundant dye was gently washed with PBS, and then a 33% acetic acid aqueous solution was added after the water was completely dry.Lastly, the absorbance of each well at 595 nm was measured.
Inhibition of Extracellular DNA in Bacterial Biofilms: The same procedure used in CV staining was employed to form MRSA biofilms of different treatments.Then, the bacterial extracellular DNA inside the biofilm was extracted following the instructions of the Ezup Column Bacteria Genomic DNA Purification Kit without wall-breaking treatment of the bacterial cells.Ultimately, the extracellular DNA of the bacterial biofilm was determined by a Nano-200 (Aosheng Instrument Co., Ltd., China), and guaranteed that the ratio of A 260 /A 280 was ≈1.8 while the ratio of A 260 /A 230 was located at 2.0-2.2.
Density Functional Theory Computational Method: In this work, the Vienna Ab Initio Package (VASP) [65,66] was employed to perform all the density functional theory (DFT) calculations within the generalized gradient approximation (GGA) using the PBE [67] formulation.The projected augmented wave (PAW) potentials [68,69] were chosen to describe the ionic cores and take valence electrons into account using a plane wave basis set with a kinetic energy cutoff of 400 eV.Partial occupancies of the Kohn−Sham orbitals were allowed using the Gaussian smearing method and a width of 0.05 eV.The electronic energy was considered selfconsistent when the energy change was smaller than 10 −5 eV.A geometry optimization was considered convergent when the force change was <0.02 eV Å −1 .Grimme's DFT-D3 methodology [70] was used to describe the dispersion interactions.
Based on the results of previous experiments, the equilibrium lattice constant of FCC (face-centered cubic) Mn 0.125 Fe 0.313 Co 0.083 Ni 0.229 Cu 0.250 unit cell was optimized to be a = 3.4884 Å.It was then used to construct a (111) surface model with p(3×2√3) periodicity in the x and y directions and four atomic layers in the z-direction separated by a vacuum layer in the depth of 15 Å in order to separate the surface slab from its periodic duplicates.During structural optimizations, a 4 × 3 × 1 k-point grid in the Brillouin zone was used for k-point sampling, and the bottom two atomic layers were fixed while the top two were allowed to relax.
The free energy of a gas phase molecule or an adsorbate on the surface was calculated by the equation G = E + ZPE − TS, where E is the total energy, ZPE is the zero-point energy, T is the temperature in kelvin (298.15K is set here), and S is the entropy.
Statistical Analysis: All data were obtained from at least three independent experiments and presented as mean ± standard deviation (SD).The sample size for each analysis was indicated in the corresponding figure caption.The Student's t-test was used to evaluate the difference between the means of the two groups (significance level: *p<0.05,**p<0.01,***p<0.001).The one-way ANOVA and one-sided Tukey's multiple comparison tests were performed to evaluate the differences among the means of three or more groups (significance level: *p<0.05,**p<0.01,***p<0.001).All statistical analyses in this work were performed using Minitab 18.0 software.

Figure 1 .
Figure 1.Synthesis and characterizations of the MnFeCoNiCu HEA NPs.a) Schematic illustration showing the formation process of the HEA NPs.b) XRD pattern and crystal structure (inset).c) HRTEM image.The inset diagram shows the atomic arrangement.c 1--c 3 are the corresponding FFT patterns along the [010] zone axis for Zone 1-Zone 3. d) Integrated intensity profiles for pixels in Zone 1-3 of the HRTEM image of the HEA NPs.e) HAADF-STEM-EDS spectrum and metallic element contents obtained by ICP-OES.f HEA NPs versus pure metals in the XRD patterns.

Figure 2 .
Figure 2. Peroxidase-like activity and mechanism of the HEA NPs.a) Schematic diagram of the POD-like activity evaluations of the HEA NPs with OPD, ABTS, and TMB.b) UV-vis absorption spectra and visual colors of different chromogenic reactions (OPD: yellow line, ABTS: green line, and TMB: blue line).c) OXD-like and POD-like activities of the HEA NPs at different pH values.d) POD-like activity of the HEA NPs at different temperatures.Data are presented as the mean ± SD (standard deviation) (n = 3 independent samples).e) Time-dependent absorbance spectra with different concentrations of HEA NPs.f-g) Michaelis-Menten curves of the HEA NPs with different concentrations of H 2 O 2 and TMB, respectively.The data are presented as the mean ± SD (n = 3 independent samples).h) Specific activity of the HEA NPs.i) ESR spectra showing 1 O 2 , • OH and • O 2 − formed from H 2 O 2 .The data are presented as the mean ± SD (n = 3 independent samples).

Figure 3 .
Figure 3. DFT calculation of the electron distribution and structural configuration.a) 3D atomic model showing the crystal structure of the HEA NPs.b,c) PDOSs of the HEA NPs and CuNi, respectively.d) Calculated PDOSs and d-band centers (including spin-up and spin-down) for each element and the bulk HEA NPs and CuNi.e,f) d-band center comparisons for the individual elements and the bulk HEA NPs and CuNi, respectively.

Figure 4 .
Figure 4. Theoretical calculations of the POD-like mechanism of the HEA NPs.a) Scheme showing the surface reactivity of the HEA NPs during POD-like activity.The red and white balls represent O and H, respectively.b) Different reaction pathways for the decomposition of H 2 O 2 to generate • OH on the HEA NPs with optimization.The white, red, blue, orange, yellow, pink, and green balls represent H, O, Mn, Fe, Co, Ni, and Cu atoms, respectively.c,d) PDOSs and partial enlargement for O* and OH* adsorption on the HEA NPs.e,f) PDOSs and partial magnification of 2OH* adsorption.g) Optimized free energy profiles for H 2 O 2 decomposition along different pathways on the CuNi, FeCuNi, and HEA NPs.

Figure 5 .
Figure 5. Use of the HEA NPs for Biosensing.a) Mechanism for the proposed detection method based on the POD-like activity of the HEA NPs (GOX PBD code: 1GAL, [https://www.rcsb.org/structure/1GAL]).b) UV-vis absorption spectra and visual colors of the HEA/H 2 O 2 /TMB system with different AA concentrations.Incubation conditions: 10 min at room temperature.c) UV-vis absorption spectra and visual colors of the HEA/TMB system with different H 2 O 2 concentrations.Incubation conditions: 10 min at room temperature.d) UV-vis absorption spectra and visual colors of the GOX/HEA/TMB system with different glucose concentrations.Incubation conditions: 15 min at 37 °C.e) Selectivity analysis for the HEA/TMB chromogenic system.

Figure 6 .
Figure 6.Use of the HEA NPs for Biomedical Applications.a) Principle for ROS-mediated inactivation of drug-resistant bacteria based on the HEA NPs.b) Determination of the bactericidal capacity of the HEA/H 2 O 2 system with the MTT assay.The data are presented as the mean ± SD (n = 3 independent samples).c) Leakage of bacterial intracellular proteins.The data are presented as the mean ± SD (n = 3 independent samples).d) Typical SEM images of MRSA after different treatments.e) HEA nanozyme-mediated ROS generation inhibits MRSA biofilms at low H 2 O 2 concentrations.f) 3D CLSM images of biofilms formed by MRSA with different treatments.One-way ANOVA and one-sided Tukey's multiple comparison tests were performed to evaluate the differences in the means of the groups (significance level: **p<0.01,***p<0.001).
Kinetics Studies of MnFeCoNiCu HEA NPs: A steady-state kinetics assay of HEA NPs was performed in pH 3.0 HAc-NaAc buffer (0.1m) by changing the concentrations of H 2 O 2 (5.0 to 70 mm) with a fixed concentration of TMB (1.0 mm) or changing the concentrations of TMB (0.1 to 1.0 mm) while maintaining the concentration of H 2 O 2 (20 mm).The Michaelis-Menten constant was calculated using Lineweaver-Burk plots of the double reciprocal of the Michaelis-Menten equation v = v max ×[S]/(K m +[S]), where v is the initial velocity, v max is the maximal reaction velocity, [S] is the concentration of substrate, and the K m is the Michaelis constant.