Preparation method and application of CoFeCu-LDH nano-enzyme

Abstract

The invention discloses a preparation method and application of CoFeCu-LDH nano-enzyme, and the preparation method comprises the following steps: S1, dissolving soluble inorganic salts of Co < 2 + >, Fe < 3 + > and Cu < 2 + > in deionized water to prepare a solution A; s2, adjusting the pH value of the solution A to a set range by using an alkaline solution B; and S3, carrying out a stirring reaction under set conditions, washing redundant inorganic salt with deionized water and ethanol after the reaction is finished, and drying to obtain the CoFeCu-LDH nano-enzyme. The CoFeCu-LDH material prepared by the preparation method disclosed by the invention can provide multienzyme mimic activities such as POD (Potential of Hydrogen) and GPx (Glutathione Phosphate When the pH value of the CoFeCu-35% is 6.5, the specific activity of POD is 0.01 U mg <-1 >, the specific activity of OXD is 0.005 U mg <-1 >, the specific activity of GPx is 0.18 U mg <-1 >, and the specific activity of GSHOx is 0.039 U mg <-1 >. Therefore, the CoFeCu-LDH nano material with the POD activity, the OXD activity, the GPx activity and the GSHOx activity has potential application in the antibacterial field.

Description

Technical Field

[0001] The present invention belongs to the fields of the preparation of two-dimensional metal nanomaterials and biomedicine, and specifically relates to a preparation method and application of CoFeCu-LDH nanozyme. Background Art

[0002] Incurable bacterial infections and the increasing multi-drug resistance pose a huge threat to public health, resulting in high mortality and heavy economic burden. Nanozymes have become highly potential antibacterial materials due to their catalytic functions similar to natural enzymes and rapid bactericidal effects. However, most peroxidase (POD) or oxidase (OXD) nanomaterials usually face efficacy limitations during antibacterial applications. This is mainly because the microenvironment at the site of bacterial infection usually has an abnormally high expression of glutathione (GSH) level, which is caused by anaerobic glycolysis and significantly reduces the oxidative killing ability of nanozymes. Therefore, developing novel nanozymes with combined POD or OXD activity and GSH consumption function will contribute to obtaining better antibacterial effects.

[0003] Layered double hydroxides (LDH) are two-dimensional layered materials composed of positively charged metal hydroxide layers and interlayer anions. Due to their rich chemical composition, simple preparation method, strong structural stability, and good biocompatibility, they show great application potential in biomedical fields such as drug delivery, bioimaging, and cancer treatment. At the same time, their characteristics of low cost, easy large-scale synthesis, and little environmental impact further enhance the value of LDH in practical applications. Therefore, it is expected to develop nanozymes with multi-enzyme mimicking activities such as POD or OXD, and glutathione peroxidase (GPx) or glutathione oxidase (GSHOx) by adjusting the metal element composition in LDH. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a preparation method and application of CoFeCu-LDH nanozyme.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] In the first aspect, the present invention provides a preparation method of CoFeCu-LDH nanozyme, including the following steps:

[0007] S1. Add Co 2+, Fe 3+ , Cu 2+ The soluble inorganic salts of are dissolved in deionized water to prepare solution A;

[0008] S2. Use alkaline solution B to adjust the pH of solution A to the set range;

[0009] S3. Stir and react under set conditions. After the reaction is completed, wash the excess inorganic salts with deionized water and ethanol, and obtain CoFeCu-LDH nanozyme after drying.

[0010] Further, in step S1, in terms of the molar ratio of metal ions, Co 2+ , Fe 3+ , Cu 2+ The addition amount of the soluble inorganic salts of is, (Co 2+ + Cu 2+ ):Fe 3+ = 3:1.

[0011] Further, in step S1, the soluble inorganic salts of Co 2+ , Fe 3+ , Cu 2+ are selected from nitrates, sulfates and chlorides soluble in water.

[0012] Further, in step S2, the set range of pH is 8-11.

[0013] Further, in step S2, alkaline solution B is prepared by dissolving an excessive amount of intercalated anion salt and base in deionized water, wherein,

[0014] The intercalated anion salts include at least one of Na2CO3 and K2CO3;

[0015] The base includes at least one of NaOH and KOH.

[0016] Further, in step S3, the set conditions include that the reaction temperature is 25-100 °C and the reaction time is 1-24 hours.

[0017] In the second aspect, the present invention also provides a CoFeCu-LDH nanozyme prepared by the above-mentioned preparation method.

[0018] In the third aspect, the present invention also provides the application of the above-mentioned CoFeCu-LDH nanozyme in antibacterial.

[0019] The size of the CoFeCu-35% nanomaterial prepared by the method of the present invention is about 166 nm. The CoFeCu-35% nanozyme can provide POD and GPx activities, can significantly catalyze H2O2 to produce reactive oxygen species (ROS) for antibacterial purposes, and can also consume GSH to solve the antioxidant effect of the bacterial microenvironment.

[0020] After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art:

[0021] (1) In the present invention, the preparation of nanoscale CoFeCu-LDH is realized by the water bath method for the first time, and the synthesis method is simple, the particle size is small, and the energy consumption is low;

[0022] (2) The CoFeCu-LDH material prepared by the present invention can provide multi-enzyme mimicking activities such as POD and GPx. Among them, for CoFeCu-35% at pH = 6.5, the specific activity of POD is 0.01 U mg -1 , the specific activity of OXD is 0.005 U mg-1, the specific activity of GPx is 0.18 U mg -1 , and the specific activity of GSHOx is 0.039 U mg -1 . Therefore, such CoFeCu-LDH nanomaterials with POD, OXD, GPx, and GSHOx activities have potential applications in the antibacterial field. Description of the Drawings

[0023] The drawings, as a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation to the present invention. Obviously, the drawings in the following description are only some embodiments, and those of ordinary skill in the art can obtain other drawings according to these drawings without creative efforts. In the drawings:

[0024] Figure 1 are the XRD patterns of CoFe-LDH, CoFeCu-5%, CoFeCu-10%, CoFeCu-20%, CoFeCu-35% and CoFeCu-50% in the embodiments of the present invention.

[0025] Figure 2 are the XRD patterns of CoFeCu-65% and Cu:Fe = 3:1 in the embodiments of the present invention.

[0026] Figure 3SEM images of (a) CoFe-LDH, (b) CoFeCu-5%, (c) CoFeCu-10%, (d) CoFeCu-20%, (e) CoFeCu-35% and (f) CoFeCu-50% in the embodiments of the present invention.

[0027] Figure 4 EDS analysis and elemental distribution maps of CoFe-LDH in the embodiments of the present invention.

[0028] Figure 5 EDS analysis and elemental distribution maps of CoFeCu-5% in the embodiments of the present invention.

[0029] Figure 6 EDS analysis and elemental distribution maps of CoFeCu-10% S in the embodiments of the present invention.

[0030] Figure 7 EDS analysis and elemental distribution maps of CoFeCu-20% in the embodiments of the present invention.

[0031] Figure 8 EDS analysis and elemental distribution maps of CoFeCu-35% in the embodiments of the present invention.

[0032] Figure 9 EDS analysis and elemental distribution maps of CoFeCu-50% in the embodiments of the present invention.

[0033] Figure 10 Hydrodynamic diameter distribution maps of CoFeCu-35% in the embodiments of the present invention.

[0034] Figure 11 AFM height profile maps of CoFeCu-35% in the embodiments of the present invention.

[0035] Figure 12 a is the TEM image of CoFeCu-35% in the embodiments of the present invention.

[0036] Figure 12 b is the HRTEM image of CoFeCu-35% in the embodiments of the present invention.

[0037] Figure 12 c is the SAED image of CoFeCu-35% in the embodiments of the present invention.

[0038] Figure 12 d-i are the elemental distribution maps of CoFeCu-35% in the embodiments of the present invention.

[0039] Figure 13a is the oxidation ability of CoFeCu-35% in the 0.2 M NaAc / HAc buffer solution with different pH values to 3,3′,5,5′-tetramethylbenzidine (TMB) in the embodiments of the present invention. The experimental conditions were 10 μg mL -1 CoFeCu-35%, 1 mM TMB, 1 mM H2O2, and the reaction time was 5 min.

[0040] Figure 13 b is the absorbance spectrum of TMB in different reaction systems containing 0.2 M NaAc / HAc buffer (pH = 3.5), 1 mM TMB, with or without the addition of 10 μg mL -1 LDH, with or without the addition of 1 mM H2O2 in the embodiments of the present invention.

[0041] Figure 13 c-d are the Michaelis-Menten curves of CoFeCu-35% to H2O2 and TMB substrates in the embodiments of the present invention.

[0042] Figure 13 e is the determination of the POD specific activity (U mg -1 ) of CoFeCu-35% at different pH values in the embodiments of the present invention. One nanozyme activity unit (U) is defined as the amount of nanozyme that catalyzes 1 μmol of product per minute.

[0043] Figure 13 f is the absorbance spectrum of GSH after different reaction times in different reaction systems containing 0.2 M NaAc / HAc buffer (pH = 7.5) and 0.2 mM GSH in the embodiments of the present invention, with the addition of 10 μg mL -1 of LDH, with or without the addition of 0.2 mM H2O2. The indicator used was DTNB.

[0044] Figure 13 g-h are the Michaelis-Menten curves of CoFeCu-50% to H2O2 and GSH substrates in the embodiments of the present invention.

[0045] Figure 13 i is the determination of the GPx specific activity (U mg -1 ) of CoFeCu-50% at different pH values in the embodiments of the present invention.

[0046] Figure 14 a is the absorbance spectrum of TMB measured in different reaction systems containing 0.2 M NaAc / HAc buffer (pH = 3.5) and 1 mM TMB in the embodiments of the present invention, with or without the addition of 20 μg mL -1 of LDH, and the reaction time was 5 min.

[0047] Figure 14 b is the time-dependent absorption spectrum of CoFeCu-20% reacting with different concentrations of TMB reaction substrate recorded at 652 nm in the embodiment of the present invention.

[0048] Figure 14 c is the Michaelis-Menten curve of CoFeCu-20% against TMB substrate in the embodiment of the present invention.

[0049] Figure 15 a-c are the OXD specific activities (U mg -1 ) determinations of CoFeCu-20% at different pH values in the embodiment of the present invention.

[0050] Figure 16 is the absorbance spectrum of GSH after reacting for different times with or without adding 20 μg mL -1 LDH in a reaction system containing 0.2 M NaAc / HAc buffer (pH = 7.5) and 0.2 mM GSH in the embodiment of the present invention.

[0051] Figure 17 are the (a) absorption spectra and (b) Michaelis-Menten curves of CoFeCu-50% against different concentrations of GSH reaction substrate after reacting for 0 and 20 minutes in the embodiment of the present invention. The test conditions are adding 20 μg mL -1 CoFeCu-50% in 2 mL of 0.2 M NaAc / HAc buffer (pH = 7.5), and the GSH concentration ranges from 0 to 0.2 mM.

[0052] Figure 18 are the GSHOx specific activities (U mg -1 ) determinations of CoFeCu-50% at different pH values in the embodiment of the present invention.

[0053] Figure 19 are the digital colony photos of Staphylococcus aureus after being exposed to different treatments in the embodiment of the present invention.

[0054] Figure 20 a is the digital photo of the agar plate showing the bacterial colonies of Staphylococcus aureus after being exposed to different concentrations of CoFe-LDH and CoFeCu-LDH in the embodiment of the present invention: (I) 10 μg / mL, (II) 25 μg / mL, and (III) 50 μg / mL of CoFe-LDH, CoFeCu-5%, CoFeCu-10%, CoFeCu-20%, CoFeCu-35%, and CoFeCu-50%.

[0055] Figure 20b is the digital photo of Staphylococcus aureus colonies after 25 μg / mL CoFe-LDH, CoFeCu-5%, CoFeCu-10%, CoFeCu-20%, CoFeCu-35% and CoFeCu-50% in the embodiments of the present invention were respectively exposed for different times (IV) 1 hour, (V) 2 hours and (VI) 4 hours.

[0056] Figure 20 c is the measurement of the fluorescence intensity of ROS in Staphylococcus aureus cells treated with CoFe-LDH, CoFeCu-5%, CoFeCu-10%, CoFeCu-20%, CoFeCu-35% and CoFeCu-50% in the embodiments of the present invention.

[0057] Figure 20 d is the SEM image of Staphylococcus aureus before treatment in the embodiments of the present invention. Scale bar = 2 μm.

[0058] Figure 20 e is the SEM image showing Staphylococcus aureus after treatment with CoFeCu-35% and H2O2 in the embodiments of the present invention. Scale bar = 2 μm.

[0059] Figure 21 It is the schematic diagram of the planar structure of CoFeCu-LDH nanozyme and the schematic diagram of the antibacterial effect provided by the present invention.

[0060] It should be noted that these drawings and text descriptions are not intended to limit the scope of the concept of the present invention in any way, but to illustrate the concept of the present invention to those skilled in the art by referring to specific embodiments. Detailed Embodiments

[0061] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0062] The present invention provides a new CoFeCu-LDH prepared by a water bath method, which can provide enzyme activities such as POD, OXD, GPx and GSHOx in a weak acid environment, and thus has potential applications in antibacterial.

[0063] In this embodiment, the LDH material with POD, OXD, GPx and GSHOx activities is the CoFeCu-LDH powder material, the raw materials are Co(NO3)2·6H2O, Fe(NO3)3·9H2O and Cu(NO3)2·xH2O, and the inorganic salts are Na2CO3 and NaOH. These raw materials can all be obtained through commercial channels and other means.

[0064] The chemical formula of the LDH nanozyme prepared by the method of the present invention is CoFeCu - x% (x% represents the molar percentage of Cu element in all metal elements, x = 1 - 60). As an example, the synthesis method of CoFeCu - 35% is as follows:

[0065] Dissolve 0.8 mmol of Co(NO3)2·6H2O, 0.7 mmol of Cu(NO3)2·xH2O and 0.5 mmol of Fe(NO3)3·9H2O in 100 mL of deionized water to form solution A. Then dissolve 12.5 mmol of NaOH and 2.5 mmol of Na2CO3 in 50 mL of deionized water to form solution B. Then slowly add solution B to solution A while adjusting the pH to 10. Stir the resulting mixture at 65 °C for 12 hours under continuous stirring.

[0066] Subsequently, centrifuge and wash the precipitate 4 times with deionized water and 2 times with ethanol.

[0067] Finally, dry the sample in an oven at 60 °C to obtain CoFeCu - 35% powder.

[0068] For other samples, while keeping other preparation conditions unchanged, only the molar amounts of metal nitrates are different. Specifically:

[0069] The molar composition of CoFe - LDH is Co(NO3)2·6H2O (1.5 mmol) and Fe(NO3)3·9H2O (0.5 mmol);

[0070] For CoFeCu - 5%, the molar composition is Co(NO3)2·6H2O (1.4 mmol), Fe(NO3)3·9H2O (0.5 mmol) and Cu(NO3)2·xH2O (0.1 mmol);

[0071] For CoFeCu - 10%, the molar composition is Co(NO3)2·6H2O (1.3 mmol), Fe(NO3)3·9H2O (0.5 mmol) and Cu(NO3)2·xH2O (0.2 mmol);

[0072] For CoFeCu - 20%, the molar composition is Co(NO3)2·6H2O (1.1 mmol), Fe(NO3)3·9H2O (0.5 mmol) and Cu(NO3)2·xH2O (0.4 mmol);

[0073] For CoFeCu-50%, the molar composition is Co(NO3)2·6H2O (0.5mmol), Fe(NO3)3·9H2O (0.5mmol) and Cu(NO3)2·xH2O (1mmol).

[0074] It should be noted that in the above preparation process, the Co source, Fe source, and Cu source include but are not limited to inorganic salts such as nitrates, sulfates, and chlorides soluble in water. In terms of the molar ratio of metal ions, Co 2+ , Fe 3+ , Mn 2+ , Cu 2+ The amount of soluble inorganic salt added is (Co 2+ +Cu 2+ ):Fe 3+ =3:1;

[0075] Solution B includes excess intercalation anion salt and base, the intercalation anion is selected from any one of Na2CO3 and K2CO3 or a combination of two thereof, and the base is selected from any one of NaOH and KOH or a combination of two or more thereof.

[0076] Figure 1 The X-ray diffraction (XRD) patterns of CoFe-LDH series materials with different copper doping amounts (CoFeCu-5% to CoFeCu-50%) are shown. The characteristic diffraction peaks of all samples (such as (003), (006), and (012) crystal planes) are highly consistent with the standard CoFe-LDH card (JCPDS 50-0235), indicating that copper doping does not destroy the main structure of layered double hydroxide (LDH).

[0077] Figure 2 The XRD patterns of samples with high copper doping (65%) and Cu:Fe=3:1 were studied. When the copper content exceeds 65% of the total metal molar amount, the (003) and (006) crystal plane diffraction peaks disappear, and the characteristic peaks of CuO (JCPDS 48-1548) appear, indicating that the material transforms from LDH phase to metal oxide phase. This result confirms that the copper doping amount must be strictly controlled below 65% to maintain the layered structure of LDH, which provides a theoretical basis for the selection of doping ratios in subsequent experiments.

[0078] Figure 3 The morphology of CoFe-LDH with different copper doping amounts was characterized by scanning electron microscopy (SEM). All samples showed a typical two-dimensional nanosheet structure with uniform sheet thickness. Among them, when the copper doping amount was ≤35%, the lateral size of the nanosheet was stable at about 200nm.

[0079] Figures 4 - 9The compositional homogeneity of the materials was systematically analyzed using energy-dispersive X-ray spectroscopy (EDS) and elemental mapping techniques. The atomic ratios of Co, Fe, and Cu in each sample showed little deviation from the preset stoichiometric ratios, and the elemental maps indicated that the three metal elements were uniformly dispersed within the nanosheet region, with no evidence of local enrichment or phase separation observed. This demonstrated that copper ions were successfully incorporated into the LDH lamellar structure via isomorphous substitution, forming a stable solid solution structure.

[0080] Figure 10 The hydrodynamic diameter distribution of CoFeCu-35% in aqueous solution was determined by dynamic light scattering (DLS). The material exhibited a unimodal distribution in the dispersed state, with the main peak located at 166 nm and the size range concentrated in 100 - 300 nm. No micron-sized aggregates (>1 μm) were detected, indicating that the material had good colloidal stability and no significant aggregation, which was beneficial for in vitro and in vivo experiments.

[0081] Figures 11 - 12 The nanostructural characteristics of CoFeCu-35% were further verified by combining atomic force microscopy (AFM) and transmission electron microscopy (TEM). The AFM height profile showed that the thickness of the nanosheets was approximately 45 nm ( Figure 11 ), which, together with the lateral size (100 - 300 nm) observed by TEM, indicated the two-dimensional nature of the material with a high aspect ratio. The lattice fringes with a spacing of 0.245 nm in the high-resolution TEM (HRTEM) image corresponded to the (104) crystal plane of CoFe-LDH ( Figure 12 b), and the diffraction rings of the (104) and (0012) crystal planes shown by selected area electron diffraction (SAED) ( Figure 12 c) confirmed the polycrystalline nature of the material. The elemental maps ( Figure 12 d-i) showed the uniform distribution of Co, Fe, Cu, O, and C elements.

[0082] Figure 13 The peroxidase (POD) activity of CoFeCu-LDH and its regulatory mechanism were systematically studied from a - e. Under acidic conditions with a pH of 3.5, the POD specific activity of CoFeCu-35% reached a peak value (0.92 U mg-1) ( Figure 13 a, e), and thus, we further evaluated the POD activity of different samples under the optimal pH conditions. Figure 13b shows the color development of TMB under different reaction conditions in this example. No color change was observed in the TMB + H2O2 group, indicating that H2O2 alone cannot catalyze the oxidation of TMB. Compared with CoFe-LDH, the introduction of Cu significantly enhanced the color reaction between H2O2 and TMB, and the POD activity increased significantly. In particular, when the copper doping amount reached 35%, its POD activity reached the peak, and excessive doping (50%) led to a decrease in activity instead. Steady-state kinetic analysis showed ( Figure 13 c-d), the Michaelis constants (K m ) of CoFeCu-35% for H2O2 and TMB were 0.064×10 -3 M and 3.82×10 -3 M respectively, indicating that the material has a significantly higher affinity for H2O2 than for TMB. Figure 13 e shows the specific activity of CoFeCu-35% at different pH values in this example. The specific activities of CoFeCu-35% at pH 3.5 and 6.5 were 0.92 U mg -1 and 0.010 U mg -1 respectively. Under weak acidic conditions (pH 6.5), although the POD specific activity of CoFeCu-35% decreased significantly (0.010 U mg-1), it still maintained a certain POD activity, indicating its ability to generate ROS in a weak acid environment and can be used for antibacterial.

[0083] Figure 13 f shows that in the buffer solution with pH 7.5 in this example, after 20 minutes of reaction in the GSH group, the absorbance values at 412 nm showed no significant difference, indicating that GSH itself has no auto-oxidation activity. However, after adding CoFeCu-50%, the absorbance decreased slightly, indicating that this sample has a certain GSHOx activity. It should be noted that the GSH content in the GSH + H2O2 group decreased more significantly, indicating that H2O2 has an oxidation effect on GSH. After adding CoFeCu-50%, the absorbance at 412 nm decreased further significantly, indicating its GPx activity. In addition, all the samples used in this example showed a certain level of GPx activity, but the strength of GPx activity was proportional to the amount of Cu introduced.

[0084] Figure 13 g-h shows the steady-state kinetics analysis of CoFeCu-50% for H2O2 and GSH in this example. The K M values with H2O2 and GSH as substrates were 0.67×10 -3 M and 0.044×10 -3 M respectively. This indicates that CoFeCu-50% has a significantly higher affinity for GSH than for H2O2.

[0085] Figure 13i represents the GPx specific activity of CoFeCu-50% in this example at different pH values. The GPx specific activities of CoFeCu-50% at pH 6.5 and 7.5 are 0.16 U mg -1 and 0.18 U mg -1 .

[0086] Figure 14 a represents the OXD activity of different samples in this example. The results show that the introduction of Cu significantly improves the OXD activity of CoFe-LDH. When the molar percentage of Co introduced is 20%, its OXD activity reaches the highest.

[0087] Figure 14 b-c represents the steady-state kinetic analysis of CoFeCu-20% on the TMB substrate in this example, and its K M value is 0.24×10 -3 M.

[0088] Figure 15 a-c represents the specific activities of CoFeCu-20% at pH 3.5 and 6.5 in this example, which are 0.19 U mg -1 and 0.005 U mg -1 . Therefore, it can be inferred that CoFeCu-20% can utilize the ability to generate ROS using O2 in a weak acid environment for antibacterial purposes.

[0089] Figure 16 This represents the comparison of the GSHOx activities of the samples used in a buffer solution at pH 7.5 in this example. The results show that, similar to the GPx activity, the strength of the GSHOx activity is also proportional to the amount of Cu introduced.

[0090] Figure 17 This represents the analysis of the GSH steady-state kinetics of CoFeCu-50% in this example. The K M value with GSH as the substrate is 0.0038×10 -3 M, indicating its potential for ultrasensitive GSH detection.

[0091] Figure 18 This represents the GSHOx specific activities of CoFeCu-50% at different pH values in this example. The GPx specific activities of CoFeCu-50% at pH 6.5 and 7.5 are 0.045 U mg -1 and 0.039 U mg -1 .

[0092] Experimental process for anti-Staphylococcus aureus: Prepare a Staphylococcus aureus suspension (ATCC27853, 10 7(CFU / mL) was used for subsequent experiments. According to whether compound treatment was carried out, the bacterial suspension was divided into an experimental group and a control group. 200 μL of Staphylococcus aureus suspension was added to sodium acetate buffer (pH = 6.5) to make the final volume reach 1 mL. The experimental group was respectively exposed to compounds at concentrations of 10, 25, 50 μg / mL and H2O2 at a concentration of 1 mM. The control group was divided into three types: pure compound (25 μg / mL), pure H2O2, and untreated bacterial suspension. After thorough mixing, the samples were incubated in a shaking incubator at 37 °C and 220 rpm for 1, 2, 4 hours. Subsequently, 100 μL of each sample was diluted 10 times and evenly spread on Mueller-Hinton (MH) agar plates and incubated overnight at 37 °C. Colony formation was observed and recorded.

[0093] Figure 19 This is the colony plate diagram after different treatment groups in this example. The results show that sodium acetate and H2O2 itself have no antibacterial effect. Pure CoFeCu-35% and CoFeCu-50% also have no obvious effect, indicating that OXD and GSHOx activities are not reflected in this system. However, after adding H2O2, CoFeCu-35% and CoFeCu-50% show significant antibacterial effects, which are mainly attributed to POD and GPx activities.

[0094] Figure 20 a shows the effect of different concentrations of LDH on antibacterial performance in the examples of the present invention. LDH after the introduction of Cu shows obvious antibacterial effect at 10 μg / mL, while CoFe-LDH needs to reach 50 μg / mL to show obvious antibacterial effect.

[0095] Figure 20 b shows the effect of different action times on antibacterial performance in the examples of the present invention. LDH after the introduction of Cu shows obvious antibacterial effect after acting for 1 hour, while CoFe-LDH needs to act for 4 hours to show obvious antibacterial effect.

[0096] Figure 20 c shows the measurement of the fluorescence intensity of ROS in Staphylococcus aureus cells treated with LDH in the examples of the present invention. The results show that the production of high levels of ROS is the reason for antibacterial. The introduction of Cu significantly enhances the consumption of GSH in the bacterial microenvironment, so that the ROS produced by POD is not consumed by GSH. At the same time, the introduction of Cu can also enhance the POD activity of CoFe-LDH. Therefore, all CoFeCu-LDH show stronger fluorescence intensity than CoFe-LDH.

[0097] Figure 20d-e are SEM images of Staphylococcus aureus before and after treatment with CoFeCu-35% and H2O2 in the embodiments of the present invention. The strain treated with sodium acetate was used as a control, showing a uniform and complete grape-like morphology without obvious damage to the bacterial cell membrane. Under the action of CoFeCu-35% + H2O2, obvious damage to the cell membrane occurred, leading to the collapse of the overall structure.

[0098] These results indicate that CoFeCu-LDH has strong antibacterial effects and is expected to become a new option for clinical antibacterial treatment.

[0099] The present invention is the first to discover a CoFeCu-35% nanomaterial with multiple enzyme activities of POD, OXD, GPx, and GSHOx. Especially the POD and GPx enzyme activities can effectively induce trace H2O2 to generate ROS and consume GSH molecules, showing broad application prospects in the field of antibacterial treatment.

[0100] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0101] The above is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Although the present invention has been disclosed as above with a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art of this patent can make some changes or modifications to equivalent embodiments by using the technical content prompted above within the scope of the technical solution of the present invention. The implementation schemes in the above embodiments can also be further combined or replaced. However, as long as the content does not depart from the technical solution of the present invention, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the present invention.

Claims

1. A preparation method of CoFeCu-LDH nanozyme, characterized in that, It includes the following steps: S1. Dissolve the soluble inorganic salts of Co 2+ , Fe 3+ , Cu 2+ in deionized water to prepare Solution A; S2. Adjust the pH of solution A to a set range using alkaline solution B; S3. Stir and react under set conditions. After the reaction ends, wash the excess inorganic salts with deionized water and ethanol, and obtain CoFeCu-LDH nanozyme after drying.

2. The preparation method according to claim 1, wherein: In step S1, in terms of the molar ratio of metal ions, the addition amount of soluble inorganic salts of Co 2+ , Fe 3+ , Cu 2+ is such that (Co 2+ +Cu 2+ ): Fe 3+ = 3:

1.

3. The preparation method according to claim 1, characterized in that: In step S1, Co 2+ , Fe 3+ , Cu 2+ The soluble inorganic salts of are selected from nitrates, sulfates and chlorides soluble in water.

4. The preparation method according to claim 1, characterized in that: In step S2, the set range of pH is 8-11.

5. The preparation method according to claim 1, wherein: In step S2, alkaline solution B is prepared by dissolving an excessive amount of intercalated anion salt and base in deionized water, wherein, The intercalated anion salt includes at least one of Na2CO3 and K2CO3; The base includes at least one of NaOH and KOH.

6. The preparation method according to claim 1, characterized in that: In step S3, the set conditions include that the reaction temperature is 25-100 °C and the reaction time is 1-24 hours.

7. A CoFeCu-LDH nanozyme prepared by the preparation method according to any one of claims 1-6.

8. The application of the CoFeCu-LDH nanozyme according to claim 7 in antibacterial.

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