CuN3 / Cu3 artificial metalloenzyme with precise coordination control and preparation method of CuN3 / Cu3 artificial metalloenzyme

Abstract

The invention discloses a CuN3 / Cu3 artificial metalloenzyme with precise coordination control and a preparation method of the CuN3 / Cu3 artificial metalloenzyme. The CuN3 / Cu3 artificial metalloenzyme with precise coordination control is reasonably designed by combining an excellent atomic structure and an electron coordination environment of a monatomic catalyst. The enzyme simulates the active center of multi-copper oxidase, and shows high-performance catalytic activity on oxygen reduction reaction in a chemically favorable environment (half-wave potential E1 / 2 is equal to 0.86 V). Advanced characterization and density functional theory calculation are utilized, the synergistic effect of CuN3-Cu3 sites in an enzyme sample mode is systematically studied, it is revealed that the change of the energy level of a d orbit reduces the potential barrier of the reaction, and the coupling interaction between CuN3 monatomic and a Cu3 cluster enhances adsorption and desorption of an oxygen intermediate, so that the reaction kinetics is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of artificial enzymes, and particularly relates to a CuN3 / Cu3 artificial metal enzyme with precise coordination control and a preparation method thereof. BACKGROUND

[0002] Advanced artificial enzymes are the primary requirement for biomimetic catalysis, which can overcome the inadaptability of natural enzymes in practical applications, including low stability, high cost and difficulty in storage. By combining the physical and chemical properties of nanomaterials with the catalytic ability of natural enzymes, artificial enzymes can adjust catalytic functions, enhance stability, and achieve other application capabilities. Multi-copper oxidases (MCOs) are important metal enzymes in nature, which are used for energy conversion of oxygen molecules in respiration and metabolism processes. MCOs can reduce O2 to H2O through the synergistic effect of multi-nuclear Cu, without forming other oxygen-containing substances. At present, by using adsorption, covalent cross-linking and other methods, the performance of MCOs in the oxygen reduction reaction (ORR) under partial neutral conditions is close to Pt. For example, laccase almost does not have overpotential (20 mV), and can reach diffusion-limited behavior at an overpotential of 70 mV. However, natural enzymes have serious problems such as complex operation and insufficient stability, which limit their application in actual ORR environments. In recent years, artificial metal enzymes (ArMs) simulating MCOs have been developed to reproduce the activity of MCOs in a chemically favorable environment for use in energy conversion systems involving ORR.

[0003] By coordination or geometric engineering of atoms, metal factor active sites with substrate activation and electron transfer ability can be synthesized to reproduce the catalytic activity of enzymes. The original ArMs mimicking MCOs use single-nuclear Cu as the active site, and porphyrin, phthalocyanine and other aromatic N-donors as ligands. For example, Langerman et al. reported the use of Cu(tmpa) (tmpa = tris(2-pyridylmethyl)amine) as an ORR catalyst, and they found that the flexibility of the tmpa ligand made the Cu center adopt a tetrahedral coordination geometry, thereby enhancing the binding affinity for O2, making it one of the highest ORR active single-nuclear catalysts to date. However, the mechanism of synthesizing compounds to reproduce MCOs is not clear, and their conductivity is impaired due to excessive reliance on the flexibility of the ligand. The catalytic activity of multi-nuclear Cu catalysts is theoretically higher than that of single-nuclear copper catalysts; however, their stability on the electrode surface is relatively low. Gentil et al. developed a binuclear Cu complex with a side chain pyrene group, which was successfully immobilized on the surface of carbon nanotubes. This innovative method makes it possible to construct a platinum-free H2 / air fuel cell, with a power density of 0.15 mW cm -2And it runs stably. In recent years, Cu single atoms fixed on carbon carriers have shown significant catalytic activity for ORR, while nitrogen-modified Cu single atom interfaces enhance their ability to adsorb intermediates and improve the thermodynamics of the ORR process. Using only atomically dispersed metal sites to simulate the active center of an enzyme obviously deviates from the principles that control enzyme catalysis. The design of high-activity ArMs is still in its infancy, and its development still faces great challenges. SUMMARY

[0004] An object of the present application is to provide a CuN3 / Cu3 artificial metal enzyme with precise coordination control and a preparation method thereof.

[0005] The CuN3 / Cu3 artificial metal enzyme with precise coordination control provided by the present application is composed of a nitrogen-rich carbon substrate and an anchoring CuN3 monatomic coordination with a three-atomic Cu (Cu3) cluster.

[0006] Further, the CuN3 / Cu3 artificial metal enzyme is prepared by a method comprising the following steps:

[0007] 1) Mixing bovine serum albumin (BSA), melamine and a soluble copper salt, and adding ascorbic acid under alkaline conditions with a pH of 12±0.2 for reduction reaction to obtain a product BSA-melamine-Cu;

[0008] 2) Mixing the BSA-melamine-Cu with copper phthalocyanine (CuPc), uniformly grinding into blue particles, and pyrolyzing in a nitrogen atmosphere to obtain the CuN3 / Cu3 artificial metal enzyme, named BCMC.

[0009] In the step 1) of the above method, the soluble copper salt can be CuCl2.

[0010] In the step 1) of the above method, the mass ratio of the BSA, melamine, CuCl2, ascorbic acid is 1:(9.95-10.05):(0.05±0.15):(0.45-0.55).

[0011] In the step 1) of the above method, the alkaline conditions can be achieved by adjusting the alkali solution. The alkali solution can be NaOH solution (the concentration can be 2M).

[0012] In the step 1) of the above method, the reaction conditions of the reduction reaction are stirring at 78±2℃ for 30 min, specifically stirring at 78℃ for 30 min.

[0013] According to one embodiment of the present application, the specific steps of the above method step 1) are as follows: dissolving BSA and melamine in double-distilled water; then, adding CuCl2 aqueous solution, stirring at room temperature for 30 min; then, quickly adding 2M NaOH solution (2.4 mL), adjusting the pH to 12, and the color immediately changes from light blue to deep purple; then stirring the solution at 78°C for another 30 min; then adding ascorbic acid, stirring the mixture at 78°C for 1 h, and the solution gradually presents transparent brown color. After cooling, the solution is centrifuged and named as BSA-melamine-Cu.

[0014] In the above method step 2), the mass ratio of BSA-melamine-Cu to copper phthalocyanine (CuPc) is 10:(0.95-1.05).

[0015] In the above method step 2), the pyrolysis reaction is carried out in a tubular furnace; the reaction conditions of the pyrolysis reaction are as follows: heating the mixture to 800±10°C at a rate of 5±0.02°C per minute, and keeping at this temperature for 2±0.1h.

[0016] In the above method, bovine serum albumin (BSA) is selected as the scaffold for limiting the growth of Cu atoms. Initially, Cu 2+ is converted into Cu + by reduction of ascorbic acid, and is incorporated into the protein scaffold. Then, more dispersed Cu sites are generated by pyrolysis of copper phthalocyanine (CuPc), in the process, melamine is converted into pyridine-N carbon nanoframe. At this stage, due to the presence of abundant N-containing functional groups (such as pyrrolic-N, pyridinic-N), the monodisperse Cu sites avoid agglomeration to form nanoparticles during pyrolysis, and generate thermodynamically more stable Cu3 clusters, thereby forming a BCMC coexisting with Cu single atom and Cu3 cluster configurations.

[0017] Another object of the present application is to provide the application of the above CuN3 / Cu3 artificial metal enzyme.

[0018] The application provided by the present application is the application of CuN3 / Cu3 artificial metal in preparing an oxygen reduction reaction (ORR) catalyst.

[0019] Further, the ORR catalyst is used for oxygen reduction reaction under alkaline conditions.

[0020] Further, the ORR catalyst can be used for cathode oxygen reduction reaction in metal-air batteries or fuel cells.

[0021] Further, the metal-air battery can be a zinc-air battery, preferably an alkaline zinc-air battery.

[0022] Another object of the present application is to provide an air cathode.

[0023] The air cathode provided by the present application is prepared from the CuN3 / Cu3 artificial metal enzyme provided by the present application.

[0024] Further, the air cathode is coated on the current collector from the solution containing the CuN3 / Cu3 artificial metal enzyme.

[0025] Further, the current collector can be carbon paper, carbon cloth, air diffusion layer or nickel mesh.

[0026] Further, the loading amount of the CuN3 / Cu3 artificial metal enzyme on the current collector is 1.0±0.05 mg cm-2. -2 .

[0027] Another object of the present application is to provide a zinc-air battery (ZABs).

[0028] The zinc-air battery provided by the present application comprises an anode, a cathode and an electrolyte, wherein the cathode is the air cathode provided by the present application.

[0029] Further, the anode is a zinc electrode, such as zinc foil.

[0030] Further, the electrolyte is an alkaline electrolyte, specifically 6.0 M KOH solution.

[0031] The present application further provides a self-powered sensing system (SPSSs).

[0032] The self-powered sensing system (SPSSs) provided by the present application comprises an anode, a biological cathode and an electrolyte, wherein the anode is a zinc electrode, and the biological cathode is GDH-BCMC@GCE.

[0033] Further, the electrolyte is a triacetate solution with a pH value of 7.

[0034] Further, the preparation method of the GDH-BCMC@GCE comprises the following steps: dropping a BCMC catalyst solution onto the surface of a glassy carbon electrode (GCE) to prepare a BCMC@GCE; then, adding a FAD-dependent glucose dehydrogenase (FAD-GDH) solution and a Nafion solution onto the surface of the BCMC@GCE electrode, and drying to obtain a GDH-BCMC@GCE.

[0035] Further, the mass ratio of the BCMC catalyst and the FAD-dependent glucose dehydrogenase (FAD-GDH) can be 1:(3.5-3.7), and the dried electrode is packaged with 0.05% Nafion (sigma).

[0036] According to an embodiment of the present application, the concentration of the solution of the BMC catalyst is 5 mg mL -1 , the concentration of the solution of the FAD-dependent glucose dehydrogenase (FAD-GDH) is 20 mg mL -1 , and the concentration of the solution of Nafion is 0.05%.

[0037] The ratio of the amount of use of the solution of the BMC catalyst, the solution of the FAD-dependent glucose dehydrogenase (FAD-GDH), and the solution of Nafion can be 5.6 μL:5 μL:2 μL.

[0038] More specifically, the glassy carbon electrode (GCE) can be a glassy carbon electrode (GCE) with a diameter of 3 mm.

[0039] The present application provides an artificial metalloenzyme (ArMs) model, which shows high activity of ORR by simulating the spatial structure of the active center of MCOs. Advanced spectroscopy and electron microscopy techniques are used to systematically characterize the ArMs, and the results show that the ArMs is composed of a nitrogen-rich carbon substrate and an anchoring Cu monomer (CuN3) coordinated with a trimer Cu (Cu3) cluster (denoted as BMC), which is consistent with the spatial structure of the active center of MCOs. The electrochemical experiments of BMC show that, compared with the reversible hydrogen electrode (RHE), BMC has a high half-wave potential (E 1 / 2 ) of 0.86 V, and the catalytic current retention rate is 73% in alkaline medium for 120 h, which is better than that of the commercial Pt / C catalyst (0.86 V, 58%), indicating that BMC has high ORR activity. In-situ Fourier transform infrared spectroscopy (FTIR) results confirm the existence of effective ORR on the surface of the BMC functionalized electrode, and the significant change in the bond strength of the active center oxygen-containing intermediate is conducive to the activation and cleavage of O-O bond. Density functional theory (DFT) further calculation shows that the introduction of Cu3 cluster adjusts the electronic structure of CuN3, resulting in a decrease in the energy barrier of water molecule reaction and an acceleration of the reaction kinetics involving the transformation of oxygen-containing intermediates. This synergistic effect is similar to the catalytic mechanism observed in the ORR process of the active center of natural MCOs, which ultimately improves the catalytic ORR activity. In addition, the feasibility of BMC is verified in zinc-air batteries (ZABs) and self-powered sensing systems (SPSSs), which provide a peak power density of 101 mW cm -2 and an excellent detection range of 0.1-13 mM, respectively. This work not only reveals a new method to adjust the atomic position and electronic structure to enhance the ORR activity of ArMs, but also provides a basic understanding of the synergistic electrocatalytic mechanism from MCOs to ArMs. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1Schematic diagram for the preparation principle and morphology characterization of BCMC; a, Schematic diagram for the preparation of BCMC; b, SEM of BCMC; c-d, TEM; e, HAADF-STEM image of BCMC, Figure 1 The yellow and red circles in e represent Cu clusters and Cu single atoms, respectively; f-g, EDX mapping images of BCMC.

[0041] Figure 2 SEM images of BCMC at different magnifications.

[0042] Figure 3 TEM images and EDX mapping images of BCMC at different magnifications.

[0043] Figure 4 Intensity distribution diagram corresponding to the HAADF-STEM image of BCMC.

[0044] Figure 5 SEM images of BCMCH at different magnifications.

[0045] Figure 6 N2adsorption / desorption isotherm and pore size distribution curve of the catalyst.

[0046] Figure 7 TEM images and EDX mapping images of BCMCH at different magnifications.

[0047] Figure 8 XRD pattern of the catalyst.

[0048] Figure 9 Raman spectrum of the catalyst.

[0049] Figure 10 XPS survey spectrum of the catalyst.

[0050] Figure 11 Structure analysis of BCMC; a, High-resolution Cu 2p; b, N 1s XPS spectrum; c, Normalized Cu k-edge XANES spectrum; d, Fourier-transform k3-weighted Cu k-edge EXAFS spectra of BCMC, BCMCH, Cu2O, CuO, CuPc and Cu foil; e, Wavelet transform; f, Experimental and fitted curves of EXAFS spectra of BCMCH and g, BCMC in R space.

[0051] Figure 12 Wavelet transform of (a) BCMC, (b) BCMCH, (c) CuPc, (d) Cu foil, (e) CuO, (f) Cu2O.

[0052] Figure 13EXAFS spectra in k-space and fitting curves of (a) Cu2O, (b) CuO, (c) CuPc, (d) Cu foil.

[0053] Figure 14 EXAFS spectra in r-space and fitting curves of (a) Cu2O, (b) CuO, (c) CuPc, (d) Cu foil.

[0054] Figure 15 Electrocatalytic ORR performance of catalysts. a, LSV curves of synthetic catalysts and Pt / C in 0.1 M KOH, b, LSV curves of BCMC at different rotation rates. c, E onset ,E1 / 2, limiting current density (J L ), kinetic current density (J k ), and the radar plot of the number of transferred electrons in 0.1 M KOH. d, Mass activity of BCMC, BCMCH, and Pt / C at 0.75 V (left) and 0.80 V (right). e, Tafel slope. f, H2O2 selectivity and number of electrons transferred of samples. g, Before and after 5000 potential cycles (0.7-1.0 V vs. RHE). h, Relative retention i-t curves of BCMC and Pt / C in 0.1 M KOH, where the concentration of Cu in electrolyte at different times is shown.

[0055] Figure 16 LSV curves of BCMC and BCMCH in 0.1 M KOH.

[0056] Figure 17 LSV curves of catalysts under different conditions; (a) temperature, (b) CuPc usage, (c) CuCl2 usage.

[0057] Figure 18 ORR polarization curves and corresponding K-L plots at different potentials; (a) MC at different rotation rates in 0.1 M KOH, (b) BCC, (c) BCM, (d) BC / MC, (e) BCMC, (f) BCMCH.

[0058] Figure 19 HAADF-STEM image of BCMC after ADT showing Cu clusters (yellow circles) and Cu single atoms (red circles).

[0059] Figure 20 CV curves of (a) MC, (b) BCC, (c) BCM, (d) BC / MC, (e) BCMC at different scan rates, 1.01-1.11 V vs. RHE, f) Relationship between scan rate and current density of catalysts.

[0060] Figure 21 Theoretical analysis for single-atom cluster co-adsorption. In situ FTIR spectra of BCMC during ORR at (a) different reduction potentials and (b) 0.6 V vs. RHE under 0.1 M KOH bias. c, Comparison of peak maximum position change with time (inset: peak at 1420 cm -1 around). d, Complete 4e - ORR pathway. ORR free energy diagram of CuN4, CuN3, and CuN3-Cu3 at (e) U = 0 V and (f) U = 1.23 V. g, Differential charge density of CuN4, CuN3, and CuN3-Cu3. h, Calculated Cu 3d orbital PDOS curves of CuN4, CuN3, and CuN3-Cu3. i, Cu 3d and O 2p PDOS of CuN4, CuN3, and CuN3-Cu3. j, Geometric optimization of OOH adsorption configurations on CuN4, CuN3, and CuN3-Cu3. k, Geometric optimization of OH adsorption configurations on CuN3 and CuN3-Cu3. 1, Formation energy of adsorbed OH on CuN3 and CuN3-Cu3 as a function of Cu-O bond distance.

[0061] Figure 22 In situ FTIR spectra of BCMC during ORR at different reduction potentials under 0.1 M KOH.

[0062] Figure 23 In situ FTIR spectra of BCMC during ORR CA at 0.6 V vs. RHE under 0.1 M KOH.

[0063] Figure 24 Model structures of (a) CuN4, (b) CuN3, (c) CuN3-Cu3.

[0064] Figure 25 Adsorption configurations of each elementary step on CuN4.

[0065] Figure 26 Adsorption configurations of each elementary step on CuN3.

[0066] Figure 27 Adsorption configurations of each elementary step on CuN3-Cu3-S1.

[0067] Figure 28 Adsorption configurations of each elementary step on CuN3-Cu3-S2.

[0068] Figure 29 PDOS of Cu d and N p orbitals in CuN4, CuN3, and CuN3-Cu3.

[0069] Figure 30 Applications of BCMC in ZABs and SPSS. a) Schematic diagram of a ZAB equipped with BCMC. b) OCV of ZABs based on BCMC and commercial Pt / C. c) Discharge polarization curves and corresponding power density curves of ZABs. d) Rate performance tests of ZABs at different current densities. e) Current density at 10 mA cm⁻¹ -2 The constant current discharge curve at time f, (A)J max (B)P max (C) Specific capacity, (D) 10mA cm -2 Discharge time, (E)OCV radar plot. g, Schematic diagram of SPSS. h, OCV response of SPSS to different glucose concentrations. i, Linear relationship curve between OCV response and glucose concentration. j, OCV response of SPSS with 5 mM glucose and 1 mM AA, BSA, DA, Lac, UA added. k, Power density response of SPSS to different glucose concentrations. l, P max The linear relationship curve between the reaction and glucose concentration.

[0070] Figure 31 The relative current of BCMC@GCE(a) and Pt / C@GCE(b) to a 1mM interference when a potential of -0.3V is applied.

[0071] Figure 32 (a) SPSS response to OCV of different glucose concentrations; (b) linear relationship curve of OCV response with glucose concentration.

[0072] Figure 33 (a) Power density response of SPSS to different glucose concentrations; (b) Linear relationship curve between Pmax response and glucose concentration. Detailed Implementation

[0073] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0074] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0075] All solvents used in the experiments were reagent grade. Bovine serum albumin (BSA, 98%), copper chloride dihydrate (CuCl2, biological reagent), ascorbic acid (AA, 99%), and melamine (99%) were purchased from Sigma-Aldrich (Beijing, China). Sodium hydroxide (NaOH, 97%), copper phthalocyanine (II) (CuPc, 90%), dopamine hydrochloride (DA, 98%), lactate (Lac, 90%), and uric acid (UA, 99%) were purchased from Aladdin (Shanghai, China). Sulfuric acid (H2SO4, 95%) was purchased from China National Pharmaceutical Group Corporation (Beijing, China). FAD-dependent glucose dehydrogenase (FAD-GDH, 900 U mg) -1 Purchased from Swkisui (Japan). D(+)-glucose (99%) purchased from Acros (Shanghai, China). Commercial Pt / C catalyst purchased from Johnson Matthey, model 20 wt.%.

[0076] The physical characterization methods in the following embodiments are as follows: The morphology of the samples at accelerating voltages of 20 kV and 200 kV was observed using scanning electron microscopy (SEM, Gemini-300) and transmission electron microscopy (TEM, FEI Talos F200X). Energy-dispersive X-ray spectroscopy (EDX) was performed using FEI Talos F200X TEM. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images were captured using a JEM-ARM300 at 300 kV. The catalyst was analyzed for composition using an Agilent ICP-OES inductively coupled plasma atomic emission spectrometer. Cu Kα radiation was used in an A Libra diffractometer. The scan rate is 10°min. -1 (Bruker D8Advance, Germany) The crystal phase of the catalyst was identified by powder X-ray diffraction (XRD). Raman spectra were obtained using a LabRAM HR800 system equipped with a 10 mW argon laser source at a wavelength of 532 nm. The surface area and pore size distribution were characterized using the Brunauer-Emmet-teller (BET) method, based on N2 adsorption isotherms measured on an ASAP 2460 instrument. X-ray photoelectron spectroscopy (XPS) analysis was performed using a Thermo Escalab 250XI system. X-ray absorption fine structure (XAFS) data were analyzed using the standard procedure in the ATHENA module of the IFEFFIT software package. In-situ electrochemical Fourier transform infrared spectroscopy (FTIR) studies were performed using a Bruker Invenios FTIR spectrometer. A CaF2 hemispherical window was used, with the working electrode placed 1 mm above the window. Measurement parameters were 0.4 cm⁻¹. -1Resolution and 64 scans.

[0077] The electrochemical measurement methods in the following examples are as follows: Electrochemical measurements were performed using a CHI760E workstation (Shanghai Chenhua Instruments, China) equipped with a three-electrode system. The oxygen reduction reaction (ORR) was evaluated in 0.1M KOH electrolyte. The experimental setup included a working electrode (RDE or RRDE), a reference electrode (Ag / AgCl), and a counter electrode (Pt wire), all of which were calibrated before measurement. To prepare the catalyst ink, 5 mg of catalyst and 20 μL of 5% Nafion were added to 1 mL of a solution containing 250 μL of isopropanol and 750 μL of water, thoroughly mixed, and sonicated for 30 min. The uniform catalyst ink was dropped onto a polished RDE with a diameter of 3 mm or an RRDE with a diameter of 4 mm, resulting in a catalyst loading density of approximately 0.39 mg / cm³. -2 Pt / C catalyst ink (metal weight percentage approximately 20%) was prepared using the same method. Cyclic voltammetry scans were performed on all tested catalysts at 0.2 V to -1.0 V using a reversible hydrogen electrode (RHE) at a scan rate of 50 mV s⁻¹, in the presence of the aforementioned KOH electrolyte. -1 This is used for initial activation purposes. During these scans, the working electrode potential is maintained at 5 mV / s. -1 The cathode was scanned at a speed varying from 400 rpm to 1600 rpm. The catalyst performance was evaluated using linear sweep voltammetry (LSV). All potentials were converted to RHE using the Nernst equation:

[0078] E RHE =E (Ag / AgCl) +E 0(Ag / AgCl) +0.0591*pH#(1)

[0079] At different potentials (J) -1 vs.ω -1 / 2 The Koutecky-Levich plot of the catalyst was analyzed below. Using the Koutecky-Levich equation, the number of transferred electrons (n) can be determined by calculating the slope of the optimal linear regression line:

[0080]

[0081]

[0082] J K =nFkC o #(4)

[0083] In the formula, J is the measured current density, J K and J LJL, Jlim, and Jdiffare the kinetic and diffusion limiting current densities, ω is the angular velocity, n is the number of transferred electrons, F is the Faraday constant, C is the bulk concentration of O2, v is the kinematic viscosity of the electrolyte, and k is the electron transfer rate constant. The kinetic current density of the reaction is calculated as follows: o Jkin = FkCv

[0084]

[0085] The yield of HO2 - and the number of electron transfers in the RRDE experiment are determined by the following equations:

[0086]

[0087]

[0088] where N = 0.37 represents the ring collection efficiency, I R and I D are the ring and disk currents, respectively.

[0089] In the non-faradaic region, the electrochemically active area (ECSA) of the catalyst was evaluated by measuring the double layer capacitance (C dl ) at different scan rates, as ECSA is directly proportional to Cdl. The long-term stability of the catalyst was investigated by accelerated durability testing (ADT) and chronoamperometric response tests. ADT was performed by cyclic potential scanning between 0.6 and 1.0 V, while chronoamperometry was performed at 0.7 V, with a rotation scan rate of 1600 rpm, relative to RHE.

[0090] The calculation method is as follows:

[0091] All calculations were performed using spin-polarized density functional theory (DFT) implemented in the Vienna ab initio simulation package (VASP). The generalized gradient approximation (GGA) in the form of the Perdew-Burke-Ernzerhof (PBE) functional was used to account for the exchange-correlation interactions, and the DFT-D3 semi-empirical correction method was used to describe the dispersion interactions. The projector augmented wave method was used to handle the core-valence electron interactions with an energy cutoff of 500 eV for the plane wave basis. The geometry optimization was performed using a 2 x 2 x 1 Monkhorst-Pack k-point grid for the first Brillouin zone, and the electronic structure calculations were performed using a 5 x 5 x 1 k-point grid. The force and energy convergence tolerances for the geometry optimization were set to and 10 -5 eV, respectively. The distance in the z-direction (perpendicular to the interface) in the vacuum region was set to to avoid interactions between adjacent layers.

[0092] Example 1, Synthesis and Characterization of Catalysts

[0093] Synthesis of catalyst:

[0094] The preparation of BCMC is shown in the schematic diagram as Figure 1 a detailed preparation method is as follows:

[0095] BSA (500 mg) and melamine (5 g) were dissolved in 250 mL of double distilled water. Subsequently, 5 ml of CuCl2 aqueous solution (containing 53.6 mg of CuCl2) was added and stirred at room temperature for 30 min. Subsequently, 2M NaOH solution (2.4 mL) was quickly added to adjust the pH to 12, and the color immediately changed from light blue to deep purple. Then the solution was stirred at 78°C for another 30 min. Subsequently, ascorbic acid (264.2 mg) was added, and the mixture was stirred at 78°C for 1 h, and the solution gradually showed a transparent brown color. After cooling, the solution was centrifuged and named BSA-melamine-Cu. Then the product was mixed with CuPc at a mass ratio of 10:1, uniformly ground into blue particles, and placed in a tubular furnace containing nitrogen. Then the mixture was heated to 800°C at a rate of 5°C per minute, and kept at this temperature for 2 h. The resulting sample was named BCMC.

[0096] Briefly, bovine serum albumin (BSA) was chosen as a scaffold to limit the growth of Cu atoms. Initially, Cu 2+ was converted to Cu + by reduction of ascorbic acid and incorporated into the protein scaffold. Subsequently, more dispersed Cu sites were generated by pyrolysis of copper phthalocyanine (CuPc), in which melamine was converted to pyridine-N carbon nanoframes. At this stage, due to the presence of abundant N-containing functional groups (such as pyrrolic-N, pyridinic-N), monodisperse Cu sites avoid agglomeration to form nanoparticles during pyrolysis, generating thermodynamically more stable Cu3 clusters, thus forming BCMC with coexistence of Cu single atoms and Cu3 cluster configurations.

[0097] Synthesis of BCMCH: BCMC was dispersed in an excess of 1M H2SO4 solution at 80°C and continuously stirred for 6 h. After the separation was completed by deionized water washing, the resulting material was named BCMCH.

[0098] Synthesis of BC: BC was synthesized according to previous work, reference as follows: X. Wang, Q. Chen, Y. Zhu, K. Wang, Y. Chang, X. Wu, W. Bao, T. Cao, H. Chen, Y. Zhang, H. Qin, Signal Transduct Target Ther 2023, 8, 277. 5 ml of CuCl2 aqueous solution (containing 53.6 mg of CuCl2) was added to 250 mL of double distilled water containing 500 mg of BSA. Then it was stirred at room temperature for 30 min. Subsequently, 2.4 mL of 2M NaOH solution was quickly added to adjust the pH to 12, and the color immediately changed from light blue to deep purple. It was heated at 78 °C for 30 min. Subsequently, 264.2 mg of ascorbic acid was added, and it was stirred at 78 °C for 1 h, during which the solution gradually appeared translucent brown. After cooling, the solution was concentrated and separated with a dialysis bag, and named BC.

[0099] Synthesis of BCC: BC and CuPc were mixed in a mass ratio of 10:1, mixed well and ground into blue particles of uniform size. These particles were then loaded into a tube furnace and placed in a nitrogen atmosphere. The furnace was raised to 800 °C at a rate of 5 °C min

[0100] Synthesis of BCM: BC and melamine were mixed in a mass ratio of 1:10, mixed well, and then loaded into a tube furnace containing nitrogen. The mixture was then heated to a temperature of 800 °C at a rate of 5 °C min

[0101] Synthesis of BC / MC: BC, melamine and CuPc were mixed in a mass ratio of 1:10:1.1, then ground into uniform blue particles. The resulting mixture was loaded into a tube furnace under a nitrogen atmosphere and subjected to a heat treatment process with a temperature reaching 800 °C at a rate of 5 °C min -1 ) and maintained at this temperature for 2 h, after which the sample was designated as BC / MC.

[0102] Synthesis of MC: Melamine and CuPc were mixed in a mass ratio of 10:1, then processed into blue particles of uniform size, and loaded into a tube furnace under a nitrogen atmosphere. The mixture was then heated at 800 °C at a rate of 5 °C min -1 ) and maintained for 2 h. The resulting sample was named MC.

[0103] Structural characterization:

[0104] Figure 1 b-d and Figure 2BCMC were characterized by field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). The pyrolysis process resulted in the formation of a highly integrated structure composed of porous carbon and graphene-like sheets. The presence of the porous structure can facilitate efficient mass transport during the ORR process, reducing the diffusion distance at the electrode / electrolyte interface, thus improving the kinetics of the catalytic reaction. In addition, the graphene-like sheet structure can significantly increase the specific surface area (SSA) of the BCMC, thus promoting the exposure of the initial buried sites within the carbon matrix, leading to an increased density of accessible active sites upon contact with the electrolyte. The formation of BCMC involved the utilization of an abundant N source and C source with a large surface area to facilitate the immobilization of Cu clusters and individual Cu atoms. No identifiable metal nanocrystals were observed in the high-magnification TEM images of BCMC Figure 3 ). The presence of irregular carbon lattices indicates the formation of amorphous carbon structures with low crystal defects during the thermal annealing process. These defects can promote the transport and diffusion of gases and cause changes in local electron density, thus providing catalytic reaction centers.

[0105] The atomic structure of BCMC was further investigated by high-angle annular dark-field scanning TEM (HAADF-STEM) measurements. As shown in FIG. 6a, Figure 1 a clear number of isolated and dispersed bright spots can be seen, which can be attributed to individual Cu atoms marked by red circles. In addition, a larger concentration of bright spots representing copper atom clusters marked by yellow circles was also observed. In combination with the corresponding intensity profile of the HAADF-STEM image of BCMC Figure 4 , the coexistence of mononuclear and polynuclear Cu sites on the carbon support was demonstrated. Energy dispersive X-ray spectroscopy (EDS) elemental mapping Figure 1 f,g) confirmed the uniform distribution of C, N, and Cu elements, while indicating that there was no significant regional enrichment of Cu elements. In addition, the observed distribution was consistent with the distribution of N elements. These preliminary results indicate that the prepared BCMC catalyst contains highly exposed Cu single atoms and Cu clusters coexisting on the porous N-doped carbon.

[0106] As a comparison, BCMC was immersed in 1 M H2SO4 aqueous solution at 80 °C for 6 h to remove the Cu clusters inside BCMC, denoted as BCMCH. The SEM image of BCMCH Figure 5 ) was very similar to that of BCMC, both retaining the porous carbon structure and graphene-like sheets. However, a significant decrease in Cu content was observed in this region. As shown in Table 1, by inductively coupled plasma (ICP) emission spectrometer analysis, it was determined that the mass loading of Cu in BCMC and BCMCH was 16.18 wt% and 5.86 wt%, respectively, indicating partial removal of Cu. N2adsorption / desorption tests were performed to evaluate the porosity and SSA of the samples.

[0107] Table 1 shows the elemental contents of BCMC and BCMCH based on ICP analysis.

[0108]

[0109] According to the Brunauer-Emmet-Teller method, the SSAs of BCMC and BCMCH were calculated to be 128.03 and 124.42 m, respectively. 2 g -1 ( Figure 6 The pore size distribution curves obtained using the Barrett-Joyner-Halenda model clearly demonstrate the presence of mesoporous structures, which enhance electrocatalytic ORR performance by promoting mass transfer and exposing atomic sites. EDS results from BCMCH (…) Figure 7 The uniform distribution of C, N, and Cu indicates that the acid etching process effectively removed the Cu clusters. X-ray diffraction (XRD) patterns of the catalyst show no diffraction peaks corresponding to Cu crystals detected in BCMC and BCMCH. Figure 8 As shown. The broad peak at approximately 26° can be attributed to the (002) crystal plane of the graphitic carbon. Therefore, combined with the TEM results, it can be concluded that the carbon matrix obtained by thermal annealing is characterized by low crystallinity, local defects, and structural distortion. Meanwhile, the Raman spectrum of the catalyst ( Figure 9 The figure shows that at 1580cm -1 and 1340cm -1 There are two distinct carbon peaks at this point, corresponding to the G and D bands respectively. The intensity ratio (I) is significantly increased. D / I G This further confirms the widespread existence of internal defects in carbon matrices.

[0110] XPS was performed to elucidate the chemical composition, bonding, and valence state of the obtained materials. Figure 10 The main difference lies in the variation in Cu atom distribution caused by different forms of Cu addition. C, N, and O elements are prominent in all the different materials, and Cu 2p and Auger peaks are widely observed. However, variations in diffraction intensity indicate differences between different contents. High-resolution Cu 2p spectra ( Figure 11 a) indicates that the dominant oxidation state of Cu, rather than the nonmetallic state, is observed in BCMCH. Instead, Cu exists in three different valence states in BCMC materials: Cu... 2+ (934.3 and 954.8 eV), Cu + (932.0 and 952.0 eV) and Cu 0(932.5 and 952.5 eV). The high-resolution N1s spectra of BCMC and BCMCH can be divided into four distinct types: pyridine-N (398.6 eV), Cu-N (399.8 eV), pyridine-N (400.8 eV), and graphite-N (401.8 eV). Figure 11 b) Quantitative analysis of the nitrogen (N) content in BCMC and BCMCH was performed. As shown in Table 2, the N content in both BCMC and BCMCH exceeds 18 at.%, thereby improving carrier conductivity and providing sufficient anchoring sites for stable Cu single atoms. Furthermore, most of these N atoms are pyridine-N, which not only increases the number of active centers but also simplifies the modification of electronic properties. These results confirm that Cu and N materials are doped into the carbon framework.

[0111] Table 2. Elemental contents of BCMC and BCMCH based on XPS analysis.

[0112]

[0113]

[0114] X-ray absorption spectroscopy was used to characterize the complex details of the electronic structure and microchemical coordination environment of Cu atoms in the sample. For example, X-ray absorption near-edge structure (XANES) spectroscopy... Figure 11 As shown in c), the Cu k-edge of an element identifies changes in valence state through variations in absorption intensity. The Cu k-edge absorption positions in BCMC and BCMCH are located between CuO and Cu₂O, indicating that the oxidation state of Cu atoms in the BCMC and BCMCH samples is intermediate between CuO and Cu₂O. + and Cu 2+ between. Figure 11 The orange arrow in c indicates the oxidation state of Cu atoms in BCMC and Cu + The relationship is closer, while BCMCH tends to favor Cu. 2+ The Fourier transform extended EXAFS spectra of the samples were acquired sequentially, such as... Figure 11 As shown in d. BCMC and BCMCH in There is a distinct peak at each location, similar to the CuPc spectrum, indicating the presence of Cu-N(O) scattering. BCMC shows a peak at approximately [missing value]. A distinct second intensity peak was observed at this location, corresponding to the Cu-Cu bonds in the copper foil. This observation indicates that monodisperse Cu atoms and Cu atom clusters coexist in BCMC. Compared to Cu-N bonds, the diffraction peaks of Cu-Cu bonds in BCMC are slightly smaller, suggesting that monodisperse Cu atoms and Cu atom clusters are in equilibrium in number.

[0115] Due to the limited resolution of Cu-N(O) in R-space, wavelet transform (WT) EXAFS analysis in k-space was needed to identify backscattering atoms. The highest intensity of the highest scattering path of BCMC and BCMCH was about As shown in Figure 11 e and Figure 12 This observation distinguishes them from CuO and Cu2O, indicating that Cu atoms in BCMC and BCMCH exhibit typical Cu-N chemical coordination. WT plot of BCMC shows the presence of Cu-Cu scattering path around , indicating the presence of both Cu atom clusters and monodispersed Cu atoms in BCMC. The coordination configuration of Cu atoms in BCMC and BCMCH was studied using quantitative least square method. As shown in Figure 2 f,g, Figure 13 , 14 and Table 3, EXAFS fitting shows that the coordination number of Cu-N in BCMCH is 4.4 with a corresponding bond length of indicating the formation of Cu-N4 configuration with Cu atom coordinated with 4 N atoms. Compared with BCMCH, the coordination environment of Cu in BCMC is 2.1 coordination bonds with Cu and 3.4 coordination bonds with N, which is speculated to be CuN3-Cu3 configuration. The above structures and compositions indicate that BCMC has the potential to predict high performance ORR. Under the influence of Cu3 cluster, the valence state of Cu is similar to Cu + , compared with Cu 2+ , Cu3 cluster is considered to be a key active site in ORR. This preference is due to the fact that Cu 2+ completely occupies d xz and d yz orbitals, which is not conducive to bonding with O2. Cu + -N3 structure in BCMC sample simultaneously promotes the transfer of electrons to oxygen molecules through antibonding orbitals, thereby promoting the rapid activation of O2, leading to oxygen dissociation and continuous electrochemical reduction to OH - .

[0116] Table 3 Structural parameters of different samples determined by EXAFS fitting

[0117]

[0118]

[0119] Example 2, Electrochemical performance of catalyst and catalytic mechanism

[0120] Linear sweep voltammetry (LSV) was used to evaluate the ORR activity of samples in alkaline environment. As can be seen from Figure 15 a, the onset potential (E oneset ) and half-wave potential (E1 / 2 The values ​​are 0.96V and 0.86V respectively, which are higher than... Figure 16 The BCMCH values ​​shown (0.91V, 0.74V) were also higher than those of other control samples with different Cu contents. Figure 17 These values ​​are comparable to those of commercial Pt / C catalysts (1.01V, 0.86V). The dynamic current density (J / L) of BCMC was calculated based on the LSV curve. k The value is 39.42 mA cm. -2 It is superior to BCMCH and commercial Pt / C catalysts, such as Figure 15 As shown in c. Furthermore, at potentials of 0.75V and 0.80V, the mass activity of BCMC (0.083 A mg) -1 It is superior to BCMC (0.069Amg). -1 ),like Figure 15 As shown in d. To elucidate the kinetics of ORR, the Tafel slope of the catalyst was meticulously determined. Notably, the Tafel slope of BCMC is 68 mV dec. -1 Lower than Pt / C catalyst ((104mV dec) -1 This highlights the superior ORR kinetic properties of BCMC. Figure 15 e). The Koutecky-Levich (KL) diagram is used to illustrate the ORR mechanism. For example... Figure 18 As shown, the ORR catalyzed by the BCMC catalyst underwent a four-electron transfer process. To characterize the number of electrons transferred (n) and selectivity of the BCMC catalyst in the ORR process, a rotating ring-disk electrode (RRDE) measurement was performed. Figure 15 As can be seen from f, HO2 is generated within the potential window of 0.2-0.8V. - The yield remained consistently below 2%, with a calculated n value of 3.98, thus confirming its specific selectivity for ORR. The stability of the electrocatalyst is a critical factor for practical applications and must be evaluated in conjunction with its performance. As expected, BCMC exhibited significant stability, achieving a stable ET after 5000 rigorous accelerated durability tests (ADT) within a potential window of 0.7–1.0 V at 0.1 MKOH. 1 / 2 The reduction of only 15mV is far lower than the potential window of commercially available Pt / C (40mV) under the same conditions. Figure 15 g). Ampere measurement further demonstrates that BCMC has good stability ( Figure 15h). Notably, the current decay of the BMCs was about 20% after 100 h of evaluation, while the commercial Pt / C catalyst showed an immediate and significant drop in activity of more than 42% after 22 h of evaluation. By collecting electrolyte samples every 24 h for ICP analysis, the presence of potential Cu leakage in the electrolyte was positively confirmed. It can be seen that the Cu content gradually increased Figure 15 h), which can be attributed to the dissolution of unstable Cu monomers or Cu clusters in the BMCs. HAADF-STEM studies Figure 19 showed that Cu monomers and Cu cluster structures within the BMCs remained even after 5000 cycles in the alkaline environment. This indicates that the active sites maintained a consistent loading level without any significant aggregation. The structural integrity can have contributed to the observed continuous and stable superior performance during the ORR process. The catalytic efficiency of the active sites was evaluated by the determination of the electrochemically active surface area (ECSA). The BMCs in the study showed an increase in ECSA compared to the control samples, as shown in Figure 20 , indicating an enhancement in the presence of active sites on the surface of the BMCs. The results suggest that the BMCs precisely replicate the spatial arrangement of active centers in MCOs, which significantly outperforms atomically dispersed metal centers for the catalysis of the ORR.

[0121] Synergistic mechanism between cluster and monatomic sites

[0122] To understand the catalytic mechanism of the BMCs, Fourier-transform infrared spectroscopy (FTIR) was utilized to identify the corresponding stretching frequencies of intermediates formed during the ORR process. Figure 21 a and Figure 22 shows the potential-dependent in-situ FTIR spectra during the ORR process, the peak at 1245 cm -1 slowly appears as the overpotential increases, indicating the oscillation of the adsorbate OOH* on the catalyst surface, and the peak position shifts significantly as the overpotential is applied, indicating a rapid interface transition and O2 protonation. The peaks at 1420 cm -1 and 1080 cm -1 correspond to the O-O vibration of adsorbed oxygen molecules O2. As shown in Figure 21 b, c, the peak position corresponding to the O-O stretching mode of O2 adsorption shifts as the polarization time increases, indicating a change in the strength of the O-O bond during the ORR process, and the electronic feedback from Cu to the O2 active site weakens the O-O bond. The weaker the O-O bond, the easier it is for O-O to cleave, thus promoting the 4e - transfer mechanism of the ORR. In addition to these ORR intermediate peaks, the peaks at 1560 cm -1 and 1610 cm -1The peaks at the interface are replaced by anions with increased concentration at the interface, thus expelling water at the electrode / electrolyte interface, indicating that the ORR process is effective. The peaks at 3000-3800 cm -1 The peaks at the interface are replaced by anions with increased concentration at the interface, thus expelling water at the electrode / electrolyte interface, indicating that the ORR process is effective. The peaks at 3000-3800 cm Figure 23 The peaks at the interface are replaced by anions with increased concentration at the interface, thus expelling water at the electrode / electrolyte interface, indicating that the ORR process is effective. The peaks at 3000-3800 cm 47,48 The peaks at the interface are replaced by anions with increased concentration at the interface, thus expelling water at the electrode / electrolyte interface, indicating that the ORR process is effective. The peaks at 3000-3800 cm

[0123] The effect of the interaction between atomic clusters and single atoms on catalytic performance was studied using DFT. Based on the XANES fitting data, CuN4, CuN3, and CuN3-Cu3 structure models were established Figure 24 The energy changes of the five basic steps of the ORR process were calculated Figure 21 d). The Gibbs free energy distribution of the ORR at CuN4, CuN3, and CuN3-Cu3 sites (where S1 represents a single atom adsorption site and S2 represents a cluster adsorption site) was calculated, and the adsorption configuration of each basic step is shown in Figure 25-28 At U = 0 V Figure 21 e), the energy paths of CuN4, CuN3, and CuN3-Cu3-S1 samples are all downhill, indicating that these processes are spontaneous exothermic processes. At the cluster adsorption site of CuN3-Cu3-S2, the energy barrier for removing the hydroxyl radical (*OH) is 0.68 eV, which may be due to the excessive adsorption strength of the cluster on the intermediate. According to the research of Noskov et al., when there is no detailed kinetic data, the largest free energy difference in the reaction mechanism can be used as an indicator of the activation energy barrier of the rate-determining step. The calculation results show that at 1.23 V, the initial proton transfer process (*OO→*OOH) is the rate-determining step (RDS) Figure 21 f). Notably, the energy barrier of CuN4 is 1.23 eV, while the defective CuN3 has a lower energy barrier of 0.73 eV. This observation indicates that the CuN3 structure has better thermodynamic ORR performance compared to CuN4. The free energy diagram of the CuN3-Cu3 model shows that the addition of Cu clusters significantly reduces the energy barrier of the *OO→*OOH transition on the CuN3 site to 0.7 eV, indicating that the Cu3 cluster enhances the protonation of *OO, thus improving the ORR activity of the CuN3-Cu3 system. In addition, the visualization of atomic differential charge density provides a more direct description of the influence of cluster participation on the electronic distribution around the Cu single atom. As Figure 21As shown in g, the aggregation of clusters causes the electron delocalization between Cu monomers, thus changing their electron density. This leads to the transfer of electrons from Cu atoms to the coordinated N atoms and intermediates, thus increasing the active sites for ORR.

[0124] In addition, the influence of Cu clusters on the electronic structure of Cu monomers was studied using the partial density of states (PDOS). As shown in h, the distribution of d-electrons between Cu monomers in the CuN3-Cu3 structure is significantly different from that observed in the CuN4 and CuN3 frameworks. As shown in i, the d-band center of Cu atoms in CuN3-Cu3 shifts to -2.01 eV compared to -2.88 eV and -2.09 eV for CuN4 and CuN3, respectively. Based on the d-band center theory, CuN3-Cu3 has a stronger adsorption ability for oxygen-containing intermediates due to its d-band center close to the Fermi level. The change in the electronic structure of Cu monomers affects the adsorption energy of oxygen-containing intermediates due to the distribution, thus affecting the ORR barrier. The significant overlap of Cu 3d and O 2p orbitals in the CuN3-Cu3 group, as shown in the PDOS plot in j, indicates the formation of Cu-O bonds. This interaction can be mediated by stronger σ bonds between the d orbitals of Cu on CuN3-Cu3 and the p orbitals of O in O2, which increases the adsorption of *OO and promotes the cleavage of O-O bonds. The study found that the O-O bond length in the *OOH group was elongated in the presence of Cu clusters, thus increasing the possibility of bond breaking. As shown in k and l, the O-O bond length of CuN3-Cu3 was measured to be 1.32 A, which is longer than that of CuN4 (1.28 A) and CuN3 (1.27 A). Figure 21 Figure 29 Figure 21 Figure 21 Figure 21

[0125] The comparison of the ORR catalytic performance and stability of the BMCM and the biological catalyst is shown in Tables 4 and 5 below.

[0126] Table 4 Comparison of ORR catalytic performance of BMCM and biological catalyst

[0127]

[0128] ​​​​​​​​

[0129] Table 5 Stability comparison of BCMC with biocatalysts

[0130]

[0131] References:

[0132] 1. S. Gentil, J. K. Molloy, M. Carriere, A. Hobballah, A. Dutta, S. Cosnier, W. J. Shaw, G. Gellon, C. Belle, V. Artero, F. Thomas, A. Le Goff, Joule 2019, 3, 2020-2029.

[0133] 2. K. Iwase, K. Kamiya, M. Miyayama, K. Hashimoto, S. Nakanishi, ChemElectroChem 2018, 5, 805-810.

[0134] 3. F. He, L. Mi, Y. Shen, X. Chen, Y. Yang, H. Mei, S. Liu, T. Mori, Y. Zhang, J. Mater. Chem. A 2017, 5, 17413-17420.

[0135] 4. Y. P. Kharwar, S. Mandal, K. Ramanujam, J. Electrochem. Soc. 2019, 166, F3193.

[0136] 5. E. C. Tse, D. Schilter, D. L. Gray, T. B. Rauchfuss, A. A. Gewirth, Inorg. Chem. 2014, 53, 8505-8516.6. H. Wu, H. Li, X. Zhao, Q. Liu, J. Wang, J. Xiao, S. Xie, R. Si, F. Yang, S. Miao, X. Guo, G. Wang, X. Bao, Energy Environ. Sci. 2016, 9, 3736-3745.

[0137] 7. H. Yu, A. Fisher, D. Cheng, D. Cao, ACS Appl. Mater. Interfaces 2016, 8, 21431-21439.

[0138] 8. S. H. Ma, Z. Han, K. Y. Leng, X. J. Liu, Y. Wang, Y. T. Qu, J. B. Bai, Small 2020, 16, 2001384.

[0139] 9. H. M. Liu, Q. Jin, L. Z. Meng, H. F. Gu, X. Liang, Y. Fan, Z. Li, F. Zhang, H. P. Rong, J. T. Zhang, Nanoscale 2023, 15, 13459-13465.

[0140] 10. M. M. Tong, F. F. Sun, Y. Xie, Y. Wang, Y. Q. Yang, C. G. Tian, L. Wang, H. G. Fu, Angew. Chem.-Int. Edit. 2021, 60, 14005-14012.

[0141] 11. H. Shang, X. Zhou, J. Dong, A. Li, X. Zhao, Q. Liu, Y. Lin, J. Pei, Z. Li, Z. Jiang, D. Zhou, L. Zheng, Y. Wang, J. Zhou, Z. Yang, R. Cao, R. Sarangi, T. Sun, X. Yang, X. Zheng, W. Yan, Z. Zhuang, J. Li, W. Chen, D. Wang, J. Zhang, Y. Li, Nat. Commun. 2020, 11, 3049.

[0142] 12. L. Zong, K. Fan, W. Wu, L. Cui, L. Zhang, B. Johannessen, D. Qi, H. Yin, Y. Wang, P. Liu, L. Wang, H. Zhao, Adv. Funct. Mater. 2021, 31, 2104864.

[0143] Example 3, Application of BCMC in Zinc-air batteries (ZABs) and self-powered sensing systems (SPSSs)

[0144] Inspired by the good ORR activity and stability of BCMC, the practicality of BCMC in ZABs and SPSSs was evaluated.

[0145] Assembly and testing of zinc-air batteries:

[0146] The air cathode was composed of either BCMC or Pt / C, and the anode was composed of zinc foil. The BCMC or Pt / C was dispersed in a mixed solution of water and isopropanol (volume ratio 3:1) at 5 mg / mL, and then a certain amount was coated on the surface of carbon paper so that the total mass of catalyst loaded on the carbon paper was 1.0 mg cm -2 . A 6.0 M KOH solution was used as the electrolyte.

[0147] Figure 30 a shows the alkaline ZABs assembled by zinc anode and air cathode functionalized with BCMC. In comparison, the ZAB was assembled with the air cathode functionalized with commercial Pt / C catalyst. The BCMC-based ZAB Figure 30 b can be seen that the open-circuit voltage (OCV) of the ZAB using BCMC is 1.41 V, which is very close to the performance of the commercial Pt / c ZAB (1.43 V), and when the two ZABs are connected in series, the OCV can easily reach 2.83 V, which can meet the power supply needs of small electrical equipment. The ZAB equipped with BCMC catalyst can provide a high power density of 101 mW cm -2 at a current density of 150.4 mA cm -2 , which is superior to the benchmark commercial Pt / C catalyst, which has a power density of 73.8 mW cm -2 at a current density of 117.4 mA cm -2 . The rate capability of the ZABs was evaluated by operation at different current densities. As shown in Figure 30 d, the BCMC-based ZAB generates voltages of 1.33, 1.31, 1.29 and 1.24 V at discharge current densities of 1.0, 3.0, 5.0 and 10 mA cm -2 , respectively. At the same time, when the current density gradually decreases from 10 mA cm -2 to 1.0 mA cm -2 , the battery voltage can be restored to the initial value, and the speed of recovery to the OCV (1.41 V) at the end of discharge is particularly fast, which confirms its impressive rate performance and stability. The specific capacity of the BCMC-based ZAB is as high as 775.5 mAh g Zn -1 at a current density of 10 mA cm-2, which is significantly better than that of the commercial Pt / c-based ZAB (720.1 mAh g Zn -1 ). These results show that BCMC has good ORR activity and stability and is a promising candidate for ORR applications, and it is possible to replace the commercial Pt / C catalyst.

[0148] To further promote the application of BCMC-based ZABs in human smart healthcare, a SPSS was developed for the detection of glucose as a target substance. The glucose concentration in the human body environment is an important indicator of the health status of patients, especially for individuals showing symptoms related to diabetes.

[0149] Assembly and testing of self-powered sensing system:

[0150] Under the condition of room temperature drying, 5.6 μL of 5 mg mL -1 BCMC catalyst (solvent is a mixed solution of water and isopropanol with a volume ratio of 3:1) was dropped onto the surface of a glassy carbon electrode (GCE) with a diameter of 3 mm to prepare BCMC@GCE. Subsequently, 5 μL of 20 mg mL -1 FAD-GDH was added to the electrode surface, and 2 μL of 0.05% Nafion solution was added to prevent FAD-GDH from falling off after drying at room temperature. This resulted in GDH-BCMC@GCE. Finally, a self-powered sensing system (SPSS) was assembled with GDH-BCMC@GCE and zinc foil as the cathode and anode, respectively, and a triacetic acid solution with a pH value of 7 as the electrolyte.

[0151] As shown in Figure 30 g, the SPSS is composed of a zinc anode, while the biological cathode is designed by immobilizing FAD-dependent glucose dehydrogenase (FAD-GDH) on the surface of BCMC. When the anode and cathode are connected, zinc is oxidized at the anode, and the generated electrons are transferred from the external circuit to the cathode, where oxygen is catalytically reduced to water by BCMC. In the presence of glucose, FAD-GDH catalyzes the oxidation of glucose and the reduction of oxygen, generating gluconolactone and hydrogen peroxide, respectively. This can change the cathode potential due to the decrease in oxygen concentration, thereby affecting the OCV of ZABs. By taking advantage of the competitive inhibition effect of the oxygen concentration on the electrode surface, the SPSS can induce a corresponding voltage / power decrease. First, the robustness of BCMC to various potential interferences present in human body fluids was confirmed. As shown in Figure 31 , the BCMC electrode showed excellent performance in the presence of common interferences such as ascorbic acid (AA) and bovine serum albumin (BSA), with a maximum current attenuation of only 20%. In contrast, when glucose was added, a commercial Pt / C catalyst showed a current interference loss of about 100%. BCMC has excellent anti-interference ability, which may be due to the reduced adsorption / electrooxidation tendency of biomarkers on the surface of BCMC. After SPSS assembly using stable BCMC, the glucose concentration was described by changing the OCV signal. Figure 30hThe OCV response of SPSS at different glucose concentrations (0.1-13 mM) is shown. Due to the sensitivity of BCMC to oxygen, there is a good linear correlation between the OCV response and the logarithm of glucose concentration (0.1-12 mM), which meets the requirements of human application, with a detection threshold of 0.07 mM Figure 30 iAs shown in Figure 32 , the data of multiple evaluations have high reproducibility with little difference. Importantly, the interference of common interfering substances in body fluids such as AA, BSA, dopamine (DA), lactic acid (Lac) and uric acid (UA) can be ignored Figure 30 jThe results show that SPSS has good selectivity for target analytes, indicating its feasibility for integration into smart devices for target substance analysis in human blood and sweat samples. Similar to OCV, the power output of SPSS also changes with the change of glucose concentration. As shown in Figure 30 kand Figure 33 , there is a commendable linear correlation between the peak output power of SPSS and the logarithm of glucose concentration. This relationship can be divided into two different parts Figure 30 lcorresponding to the fluctuations of glucose concentration in the ranges of 0.1-4.0 mM and 5.0-12 mM, respectively. These findings show that SPSS integrated with BCMC-based ZAB has excellent glucose response capability, showing flexibility to the influence of various interfering agents and delineating different ranges of glucose concentration changes. Despite the use of competitive inhibition mechanism, the output voltage / power of SPSS is still sufficient to maintain the operation of the minimum circuit, which is crucial in the field of human smart medical applications.

[0152] In summary, by simulating the catalytic active center of MCOs, a new artificial metalloenzyme BCMC with dual ORR sites of CuN3 monatomic and Cu3 cluster was developed, which has better catalytic ORR activity than Cu monatomic and commercial Pt / C catalyst. Through the combination of in situ FTIR and DFT calculation, the synergistic effect between CuN3 monatomic and Cu3 cluster was revealed. The Cu monatomic site changes the d-orbital energy level, reduces the reaction barrier, while the Cu3 cluster enhances the adsorption and desorption of oxygen intermediates, improves the reaction kinetics. In addition, BCMC as a highly efficient ORR catalyst has been verified in ZAB and SPSS systems, showing high power density, long-term stability and high sensitivity to target substance glucose in complex environment. This work reports the preparation of atomically precise biomimetic ArM, and has a clear understanding of the ORR mechanism, providing a new principle for the design of ORR catalysts in the fields of metal-air batteries, fuel cells, human smart medical technology, etc.

[0153] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a specific example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. A CuN3 / Cu3 artificial metalloenzyme, which is composed of nitrogen-enriched carbon substrate and anchoring CuN3 monatomic coordination coordinated with Cu3 clusters.

2. The CuN3 / Cu3 artificial metalloenzyme according to claim 1, wherein: The CuN3 / Cu3 artificial metalloenzyme is prepared according to a method comprising the following steps: 1) mixing bovine serum albumin, melamine and soluble copper salt, and adding ascorbic acid under alkaline conditions with pH of 12±0.2 to perform reduction reaction to obtain product BSA-melamine-Cu; 2) mixing the BSA-melamine-Cu with copper phthalocyanine, uniformly grinding into blue particles, and performing pyrolysis in nitrogen atmosphere to obtain the CuN3 / Cu3 artificial metalloenzyme, which is named as BCMC.

3. The CuN3 / Cu3 artificial metalloenzyme according to claim 2, wherein: In the step 1), the soluble copper salt is CuCl2. The feeding mass ratio of the BSA, melamine, CuCl2 and ascorbic acid is 1:(9.95-10.05):(0.05±0.15):(0.45-0.55).

4. The CuN3 / Cu3 artificial metalloenzyme according to claim 2 or 3, characterized in that: In the step 1), the alkaline conditions are achieved by adjusting with alkali solution; the alkali solution is NaOH solution.

5. The CuN3 / Cu3 artificial metalloenzyme according to any one of claims 2-4, wherein: In the step 1), the reaction conditions of the reduction reaction are stirring at 78±2℃ for 30 min.

6. The CuN3 / Cu3 artificial metalloenzyme according to any one of claims 2-5, wherein: In the step 2), the feeding mass ratio of BSA-melamine-Cu and copper phthalocyanine is 10:(0.95-1.05).

7. The CuN3 / Cu3 artificial metalloenzyme according to any one of claims 2-6, wherein: In the step 2), the pyrolysis reaction is performed in a tubular furnace; the reaction conditions of the pyrolysis reaction are heating the mixture to 800±10℃ at a rate of 5±0.02℃ per minute, and maintaining at the temperature for 2±0.1h.

8. Use of the CuN3 / Cu3 artificial metalloenzyme according to any one of claims 1-7 in preparation of an oxygen reduction reaction (ORR) catalyst.

9. Use according to claim 8, characterized in that: The ORR catalyst is used for oxygen reduction reaction under alkaline conditions.

10. Use according to claim 8 or 9, characterized in that: The ORR catalyst is used for cathode oxygen reduction reaction in metal-air battery or fuel cell. Further, the metal-air battery is zinc-air battery, preferably alkaline zinc-air battery.

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