Silver-doped full-transition metal Prussian blue derivative with high antibacterial activity as well as preparation method and application of silver-doped full-transition metal Prussian blue derivative

Abstract

The invention discloses a silver-doped full transition metal Prussian blue derivative with high antibacterial activity and a preparation method and application thereof, and the preparation method comprises the following steps: S1, dissolving Zn (NO3) 2.6 H2O, Cu (NO3) 2.3 H2O, Ni (NO3) 2.6 H2O, Mn (NO3) 2.4 H2O, Na3C6H5O7. 2H2O and AgNO3 in deionized water, and stirring to obtain a solution A; s2, dissolving K3 [Fe (CN) 6] in deionized water, and stirring to obtain a solution B; and S3, slowly pouring the solution B into the solution A, standing at room temperature, centrifuging, removing the supernatant, repeatedly washing the precipitate with deionized water and absolute ethyl alcohol, and carrying out vacuum drying. The silver-doped PBA composite nano material Ag (at) ZnCuNiMn-PBA is synthesized through a one-step coprecipitation method, and the silver-doped PBA composite nano material Ag (at) ZnCuNiMn-PBA has great application potential and commercial value in the fields of in-vivo and in-vitro antibiosis, tumor resistance and the like.

Description

Technical Field

[0001] The present invention relates to the field of antibacterial nanomaterials, and more particularly to a silver-doped all-transition metal Prussian blue derivative with high antibacterial activity, and a preparation method and application thereof. Background Art

[0002] Prussian blue (PB) is a Fe 2+ and Fe 3+ Nanomaterials formed by combining cyano groups with coordinated bonds. By introducing other transition metal ions during the synthesis process to replace some of the iron ions, a series of materials with different chemical properties are produced, known as Prussian blue analogs (PBAs). PBAs have attracted widespread attention from researchers due to their large specific surface area, tunable component structure, and high reactivity.

[0003] There are currently some reports on the antibacterial use of PBA. Duan et al. (Duan C, Liu X, Tian G, et al. A one-stone-two-birds strategy for cellulose dissolution, regeneration, and functionalization as a photocatalytic composite membrane for wastewater purification. International Journal of Biological Macromolecules, 2024, 274: 133317) constructed a multifunctional regenerated cellulose composite membrane decorated with Prussian blue analog (ZnPBA) microspheres for wastewater purification. The membrane achieved separation efficiencies of 99.4% and 99.2% for Escherichia coli and Staphylococcus aureus, respectively, demonstrating excellent treatment efficiency and reusability, providing a simple and efficient method for efficient wastewater purification. Liu et al. (Xie, B.; Sun, B.; Gao, T.; Ma, Y.; Yin, G.; Zuo, P. Recent Progress of Prussian Blue Analogues as Cathode Materials for Nonaqueous Sodium-Ion Batteries. Coordination Chemistry Reviews 2022, 460, 214478) reported a self-assembled zinc-doped Prussian blue analogue, named zinc hexacyanoferrate nanocatalyst (ZnPBA NCs). This catalyst has broad-spectrum antibacterial activity and can kill more than 85% of bacteria at a concentration of 100 μg / ml. It also has antioxidant catalytic activity against various ROS and related cytokine storms. The mouse bacterial pneumonia model was then used to verify that ZnPBA NCs can eliminate bacterial infection and largely alleviate the oxidative stress and inflammation caused by infection, indicating that this material has potential in preventing and treating related diseases caused by various bacterial infections.In order to further improve the antibacterial efficiency, a literature report designed a gold and silver filled PBA nanomaterial (Zhou, S.; Guo, X.; Huang, H.; Huang, X.; Zhou, X.; Zhang, Z.; Sun, G.; Cai, H.; Zhou, H.; Sun, P. Triple-Function Au-Ag-Stuffed Nanopancakes for SERS Detection, Discrimination, and Inactivation of Multiple Bacteria.Anal.Chem.2022,94(15),5785–5796), Au@Ag was synthesized by selective silver etching particle growth, and then potassium ferrocyanide was added to the system to form unstable PBA, and then 4-mercaptophenylboronic acid with dual functions was introduced to form an optimal surface enhanced Raman scattering (SERS) enhancement and high silver loading antibacterial drug delivery sandwich nanostructure. This nanomaterial can not only highly sensitively distinguish and detect three bacteria (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa), but also achieve synergistic bactericidal effects. However, the step-by-step synthesis strategy has problems such as a complex preparation process.

[0004] Therefore, there is an urgent need to develop a PBA derivative with simple synthesis methods and excellent antibacterial properties to provide an efficient nanoplatform for bacterial infection. Summary of the Invention

[0005] The purpose of the present invention is to provide a silver-doped full-transition metal Prussian blue derivative with high antibacterial activity and its preparation method and application, so as to solve the problems of complex synthesis steps and low antibacterial efficiency of antibacterial nanomaterials in the prior art.

[0006] In order to solve the above problems, the present invention adopts the following technical solutions:

[0007] According to the first aspect of the present invention, a method for preparing a silver-doped all-transition metal Prussian blue derivative with high antibacterial activity is provided, comprising the following steps: S1: dissolving appropriate amounts of Zn(NO3)2·6H2O, Cu(NO3)2·3H2O, Ni(NO3)2·6H2O, Mn(NO3)2·4H2O, Na3C6H5O7·2H2O and AgNO3 in 50-200 mL of deionized water, and stirring to obtain solution A; S2: dissolving an appropriate amount of K3[Fe(CN)6] in 50-200 mL of deionized water, and stirring to obtain solution B; S3: slowly pouring solution B into solution A, allowing to stand at room temperature, removing the supernatant after centrifugation, repeatedly washing the precipitate with deionized water and anhydrous ethanol, respectively, and vacuum drying to obtain the product.

[0008] Preferably, in step S1, the total content of Zn, Cu, Ni and Mn in solution A is 2 mmol.

[0009] Preferably, the amount of Zn(NO3)2·6H2O is 0.2-0.8 mmol, the amount of Cu(NO3)2·3H2O is 0.2-0.8 mmol, the amount of Ni(NO3)2·6H2O is 0.8-1.2 mmol, and the amount of Mn(NO3)2·4H2O is 0.01-0.8 mmol.

[0010] Preferably, the amount of Na3C6H5O7·2H2O used is 2 to 3 mmol, and the amount of AgNO3 used is 0.1 to 1.0 mmol.

[0011] In order to optimize the amount of Ag added to Ag@ZnCuNiMn-PBA, different molar amounts of AgNO3 (0.1, 0.3 and 0.5 mmol) were used to synthesize Ag@ZnCuNiMn-PBA during the preparation process, and three different products Ag 0.1 @ZnCuNiMn-PBA,Ag 0.3 @ZnCuNiMn-PBA and Ag 0.5 @ZnCuNiMn-PBA,

[0012] Their antibacterial activities were determined by plate counting method, and the results showed that Ag 0.1 The MBC value of @ZnCuNiMn-PBA is 16μg / mL, Ag 0.3 The MBC value of @ZnCuNiMn-PBA dropped to 8μg / mL, and when the concentration of AgNO3 continued to increase to 0.5mmol, Ag 0.5 The MBC value of @ZnCuNiMn-PBA remains unchanged. Therefore, according to a preferred embodiment of the present invention, the amount of AgNO3 used is 0.3 mmol.

[0013] Preferably, in step S2, 1-5 mmol K3[Fe(CN)6] is dissolved in 50-200 mL deionized water.

[0014] Preferably, in step S3, the mixture is allowed to stand at room temperature for 2 to 24 hours to ensure sufficient time for particle nucleation and growth. The longer the standing time, the more intense the particle aggregation and the larger the particle size.

[0015] Preferably, in step S1 and step S2, the stirring time is 5 to 60 minutes. Studies have shown that too short a stirring time may lead to uneven mixing, while too long a stirring time is not conducive to the nucleation and growth of particles.

[0016] According to a preferred embodiment of the present invention, to synthesize Ag@ZnCuNiMn-PBA, 0.4 mmol Zn(NO3)2·6H2O, 0.5 mmol Cu(NO3)2·3H2O, 1 mmol Ni(NO3)2·6H2O, 0.1 mmol Mn(NO3)2·4H2O, 2.25 mmol Na3C6H5O7·2H2O, and 0.1-0.5 mmol AgNO3 were dissolved in 50 mL of deionized water and stirred for 10 minutes to obtain solution A. Then, 2 mmol K3[Fe(CN)6] was dissolved in 50 mL of deionized water and stirred for 10 minutes to obtain solution B. Solution B was slowly poured into solution A, allowed to stand at room temperature for 24 hours, and centrifuged to remove the supernatant. The precipitate was repeatedly washed with deionized water and anhydrous ethanol. The precipitate was vacuum dried overnight.

[0017] According to a second aspect of the present invention, there is provided a silver-doped all-transition metal Prussian blue derivative with high antibacterial activity prepared according to the above preparation method.

[0018] In addition to maintaining the original cubic morphology of PBA, the Ag@ZnCuNiMn-PBA prepared according to the present invention also uniformly loads many spherical ultra-small dots on the surface of the material. These dots are silver nitrate nanoparticles (AgNPs) formed by in situ reduction. Subsequent HAADF-STEM analysis further proves that the dots are completely consistent with the Ag element distribution in the elemental map.

[0019] According to a third aspect of the present invention, there is provided a use of a silver-doped all-transition metal Prussian blue derivative in the preparation of a medicament for preventing and treating bacterial infection.

[0020] Preferably, the bacteria include: Gram-negative bacteria represented by Escherichia coli (such as Salmonella, Pseudomonas aeruginosa, Helicobacter pylori, etc.), and Gram-positive bacteria represented by Staphylococcus aureus (such as Bacillus subtilis, Streptococcus pyogenes, lactic acid bacteria, etc.).

[0021] As described in the background section of this invention, problems such as drug resistance caused by the overuse of antibiotics pose a serious challenge to the control of bacterial infections, necessitating the development of a new broad-spectrum antimicrobial agent. This invention investigates the antimicrobial activity of different transition metal salts (zinc, copper, cobalt, nickel, and manganese) to synthesize Prussian blue derivatives (PBAs) with different metal components, aiming to screen for an all-transition metal PBA with high antimicrobial activity. By comparing the antibacterial properties of a series of ternary (CuCo-PBA, CuNi-PBA, ZnCu-PBA, and NiCo-PBA), quaternary (ZnCuNi-PBA), quinary (ZnCuNiMn-PBA), and high-entropy PBAs (ZnCuNiCoMn-PBA) against Escherichia coli, the transition metal combinations ZnCu-PBA, ZnCuNi-PBA, and ZnCuNiMn-PBA with excellent antimicrobial activity were obtained.

[0022] To enhance their antibacterial properties, silver-doped PBA composite nanomaterials were synthesized via a simple one-step coprecipitation method. The inhibition zone diameters of ZnCu-PBA and ZnCuNi-PBA increased from 13.4 mm to 20.6 mm (Ag@ZnCu-PBA) and 13.6 mm to 24.4 mm (Ag@ZnCuNi-PBA), respectively. Transmission electron microscopy revealed that silver nanoparticles aggregated at the ZnCuNi-PBA interface. To address this aggregation issue, we achieved in situ reduction and uniform distribution of silver nanoparticles at the ZnCuNiMn-PBA interface by manipulating the metal components. Density functional theory (DFT) calculations revealed that ZnCuNiMn-PBA exhibited a higher density of states and a d-band center closer to the Fermi level than ZnCuNi-PBA, indicating that ZnCuNiMn-PBA possesses a stronger adsorption capacity for intermediates during synthesis, allowing for uniform adsorption of silver nanoparticles at the ZnCuNiMn-PBA interface. TEM elemental analysis images show that six metal elements, zinc, copper, nickel, manganese, iron and silver, are evenly distributed on the tetrahedron of Ag@ZnCuNiMn-PBA, proving that the material was successfully synthesized.

[0023] The minimum bactericidal concentrations of Ag@ZnCuNiMn-PBA for Escherichia coli and Staphylococcus aureus were 8 and 4 μg / mL, respectively. Moreover, the corresponding SEM images showed that the cell membranes of Escherichia coli and Staphylococcus aureus were severely damaged, the intracellular cytoplasmic components were obviously leaking, and the bacterial morphology was severely shrunk and destroyed. Electron paramagnetic resonance (EPR) confirmed the presence of two active oxygen molecules, hydroxyl radicals and singlet oxygen, produced by Ag@ZnCuNiMn-PBA during the bactericidal process. Further study of its bactericidal mechanism showed that Ag@ZnCuNiMn-PBA would quickly release Ag after contact with bacteria. +, while triggering the production of ROS and inducing oxidative stress, thereby producing a strong antibacterial effect.

[0024] The main point of the present invention is that it overcomes the disadvantages of poor antibacterial performance of traditional single-metal or bimetallic PBA materials by systematically regulating the types of transition metals; then, in order to further improve the antibacterial activity of PBA, silver-doped PBA composite nanoparticles are synthesized, which overcomes the disadvantages of silver nanoparticles being easy to aggregate and unable to be evenly loaded on the PBA interface.

[0025] The present invention also lies in the fact that the antimicrobial activity of several transition metals, Zn, Cu, Ni, and Mn (copper > cobalt > nickel > zinc > manganese), was screened. Based on the preference for Cu, which has strong antimicrobial activity, a series of binary PBAs were synthesized, including CuCo-PBA, CuNi-PBA, ZnCu-PBA, and NiCo-PBA, with the strongest performance, ZnCu-PBA, being selected. On this basis, the types of metals were gradually increased to synthesize quaternary and high-entropy PBAs, with ZnCuNi-PBA showing the strongest antimicrobial performance. Subsequent silver-doping experiments were conducted to prepare composite nanomaterials. While the antimicrobial performance was further enhanced, the nanosilver aggregated and mixed with the PBA. To address this issue, the PBA components were finally manipulated to achieve uniform distribution of nanosilver at the interface of the PBA block. An overly smooth PBA interface would prevent the nanosilver particles from being loaded, so manganese doping was necessary to increase the interface roughness and facilitate silver adsorption. It is through this series of screening that the present invention ultimately produced a Prussian blue derivative (PBA) with excellent antimicrobial activity.

[0026] The silver-doped all-transition metal Prussian blue derivative with high antibacterial activity provided by the present invention has the following significant beneficial effects compared with the prior art:

[0027] 1) The present invention develops an ultra-small AgNPs-modified Ag@ZnCuNiMn-PBA with highly effective antibacterial properties. This material realizes the in situ reduction and uniform distribution of AgNPs on its surface, and HADDF images show that the average particle size of the reduced AgNPs is 3.6 nm, which also gives the carrier ZnCuNiMn-PBA higher antibacterial properties.

[0028] 2) Antibacterial experiments showed that Ag@ZnCuNiMn-PBA exhibited MBC values ​​of 8 μg / mL against Escherichia coli and 4 μg / mL against Staphylococcus aureus, significantly lower than those reported in the literature. Scanning electron microscopy (SEM) images demonstrated that Ag@ZnCuNiMn-PBA possessed a strong ability to destroy bacterial cell membranes, with virtually no bacteria surviving at the MBC concentration.

[0029] 3) Further research on the bactericidal mechanism of Ag@ZnCuNiMn-PBA found that the material rapidly releases Ag after contact with bacteria. + , while triggering the production of ROS and inducing oxidative stress, resulting in a potent antibacterial effect. These results demonstrate the potential of Ag@ZnCuNiMn-PBA as a multifunctional nanomaterial for antibacterial therapy and wound healing, providing a promising alternative to traditional antibiotic therapy.

[0030] In summary, according to the present invention, a silver-doped all-transition metal Prussian blue derivative with high antibacterial activity, its preparation method and application, a silver-doped PBA composite nanomaterial is synthesized by a simple one-step co-precipitation method. The research results show that the Prussian blue derivative and its composite nanomaterial, as candidates for antibacterial treatment, have great application potential and commercial value in the fields of in vitro and in vivo antibacterial and anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 shows images of colonies formed by Escherichia coli and Staphylococcus aureus after exposure to different concentrations of transition metal nitrates;

[0032] Figure 2 Atomic force microscopy images of CuCo-PBA (A), CuNi-PBA (B), ZnCu-PBA (C), and NiCo-PBA are shown, with scanning size scales of 200 nm and 400 nm, and color scales of -30-120 nm, -20-150 nm, 0-230 nm, and -90-350 nm; images of inhibition zones formed after E. coli was exposed to different PBAs, (E) CuCo-PBA, CuNi-PBA, ZnCu-PBA, and NiCo-PBA;

[0033] Figure 3 Atomic force microscopy images of ZnCuNi-PBA (A) and ZnCuNiCoMn-PBA (B) are shown, with scanning size scales of 200 nm and 300 nm, and color scales of -30-220 nm and -370-400 nm; images of inhibition zones formed after E. coli was exposed to different PBAs, (E) ZnCuNi-PBA and ZnCuNiCoMn-PBA;

[0034] Figure 4 Atomic force microscopy images of Ag@ZnCuNi-PBA (A) and Ag@ZnCu-PBA (B) are shown, with scanning size scales of 200 nm and 300 nm, and color scales of -10-150 nm and -50-200 nm; images of inhibition zones formed after E. coli was exposed to different PBAs, (C) for Ag@ZnCuNi-PBA and Ag@ZnCu-PBA, respectively;

[0035] Figure 5 Scanning electron microscopy (A) and transmission electron microscopy (B) images of Ag@ZnCuNi-PBA are shown, with scanning scales of 500 nm and 50 nm.

[0036] Figure 6 Atomic force microscopy images of ZnCuNiCo-PBA (A) and Ag@ZnCuNiCo-PBA (BC) are shown. Scanning size scales are 240 nm, 300 nm, and 480 nm, and color scales are 0-120 nm, 0-220 nm, and -20-200 nm. Images of inhibition zones formed after E. coli were exposed to different PBAs. (D) is for ZnCuNiCo-PBA and Ag@ZnCuNiCo-PBA, respectively.

[0037] Figure 7 (A) and (B) TEM images of Ag@ZnCuNiMn-PBA and their corresponding HAADF-STEM (C) and elemental distribution map (D) are shown. Scanning size scale: 20 nm.

[0038] Figure 8 High-resolution XPS scanning spectra of Ag@ZnCuNiMn-PBA are shown, Ag (A), Zn (B), Cu (C), Ni (D), Mn (E), and Fe (F);

[0039] Figure 9 The images of colonies formed by E. coli after exposure to different concentrations of Ag@ZnCuNi-PBA and Ag@ZnCuNiMn-PBA are shown;

[0040] Figure 10 The colony images of Ag@ZnCuNiMn-PBA prepared with different molar amounts of silver nitrate after incubation with Escherichia coli are shown, Ⅰ, 0.1mmol; Ⅱ, 0.3mmol; Ⅲ, 0.5mmol;

[0041] Figure 11 Figure 3 shows the growth curves of Escherichia coli (A) and Staphylococcus aureus (B) in MH broth containing different concentrations of Ag@ZnCuNiMn-PBA solutions. The optical density at 600 nm reflects the concentration of bacteria in the culture medium. Figure 3 shows the colony images of Escherichia coli (C) and Staphylococcus aureus (D) after exposure to different concentrations of Ag@ZnCuNiMn-PBA solutions.

[0042] Figure 12 CLSM and SEM images of Escherichia coli (A) and Staphylococcus aureus (B) after incubation with Ag@ZnCuNiMn-PBA solution are shown;

[0043] Figure 13 The electron paramagnetic resonance results of Ag@ZnCuNiMn-PBA are shown;

[0044] Figure 14 The release of silver ions from Ag@ZnCuNiMn-PBA solution over time is shown;

[0045] Figure 15 Shown are the mouse wound infection healing experiment, representative wound photos and wound healing traces of different groups after treatment with ZnCuNiMn-PBA and Ag@ZnCuNiMn-PBA, respectively. DETAILED DESCRIPTION

[0046] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional in the art, or according to the experimental methods recommended by the instrument and equipment manufacturers. The reagents and materials used in the examples are commercially available unless otherwise specified.

[0047] The present invention first systematically studied the antimicrobial properties of different transition metal nitrates (zinc, copper, cobalt, nickel, and manganese) using minimum bactericidal concentrations (MBCs). Then, by adjusting the type of nitrate added, PBA derivatives with different compositions, including ternary, quaternary, and high-entropy PBAs, were synthesized, ultimately yielding a PBA carrier with optimal antimicrobial properties. Furthermore, using PBA as the carrier material and sodium citrate as the reducing agent, a simple one-step coprecipitation method was used to achieve in situ reduction and efficient loading of silver nitrate onto the PBA carrier, resulting in an ultra-small AgNPs-loaded PBA composite nanomaterial (Ag@ZnCuNiMn-PBA) with excellent antimicrobial properties.

[0048] Furthermore, the present invention also uses density functional theory (DFT) calculations to analyze the mechanism of inconsistent distribution of nanosilver particles on different PBA carriers ZnCuNiMn-PBA and ZnCuNi-PBA. Then, its morphology was characterized by TEM, SEM and AFM. The antibacterial properties of Ag@ZnCuNiMn-PBA and Ag@ZnCuNi-PBA against Staphylococcus aureus and Escherichia coli were compared by growth curve and plate colony method, and the damage of Ag@ZnCuNiMn-PBA to bacterial cell membrane was evaluated by SEM, and the killing ability of bacteria was verified by live and dead bacterial dyes.

[0049] To further explore the antibacterial mechanism, the present invention first used electron paramagnetic resonance (EPR) to verify the type of reactive oxygen species generated, and also used inductively coupled plasma optical emission spectrometry (ICP-OES) to measure the release of silver ions over time. The biosafety of Ag@ZnCuNiMn-PBA was evaluated by cytotoxicity and hemolysis experiments, and then the ability of Ag@ZnCuNiMn-PBA to eliminate bacteria and promote wound healing in vivo was evaluated by a mouse wound infection healing experiment. This study provides a promising new material for solving the problem of easy aggregation of silver particles, eliminating bacterial infections and promoting wound healing.

[0050] 1. Experimental Methods

[0051] 1.1 Preparation of ternary and quaternary PBA

[0052] To synthesize CuZn-PBA, 1.0 mmol of Cu(NO₃)₂·3H₂O, 1.0 mmol of Zn(NO₃)₂·6H₂O, and 2.25 mmol of Na₃C₆H₅O₇·2H₂O were dissolved in 50 mL of deionized water and stirred for 10 minutes to obtain Solution A. Next, 2 mmol of K₃[Fe(CN)₆]₆ was dissolved in 50 mL of deionized water and stirred for 10 minutes to obtain Solution B. Solution B was slowly poured into Solution A and allowed to stand at room temperature for 24 hours. After centrifugation, the supernatant was removed and the precipitate was repeatedly washed with deionized water and anhydrous ethanol, respectively, and then dried under vacuum overnight. The synthesis procedures for other ternary PBAs, including CuCo-PBA, CuNi-PBA, and CoNi-PBA, were similar to those for CuZn-PBA, except that the nitrate in Solution A was replaced with a component of the target product.

[0053] To synthesize ZnCuNi-PBA, 1.0 mmol Cu(NO3)2·3H2O, 1.0 mmol Zn(NO3)2·6H2O, 0.4 mmol Ni(NO3)2·6H2O, and 2.25 mmol Na3C6H5O7·2H2O were dissolved in 50 mL of deionized water and stirred for 10 minutes to obtain Solution A. Next, 2 mmol K3[Fe(CN)6] was dissolved in 50 mL of deionized water and stirred for 10 minutes to obtain Solution B. Solution B was slowly poured into Solution A and allowed to stand at room temperature for 24 hours. After centrifugation, the supernatant was removed and the precipitate was repeatedly washed with deionized water and anhydrous ethanol, then dried under vacuum overnight. The synthesis process for the other quaternary PBAs was similar to that of ZnCuNi-PBA.

[0054] 1.2 Preparation of ZnCuNiMn-PBA and Ag@ZnCuNiMn-PBA

[0055] To synthesize ZnCuNiMn-PBA, 0.4 mmol Zn(NO3)2·6H2O, 0.5 mmol Cu(NO3)2·3H2O, 1 mmol Ni(NO3)2·6H2O, 0.1 mmol Mn(NO3)2·4H2O, and 2.25 mmol Na3C6H5O7·2H2O were dissolved in 50 mL of deionized water and stirred for 10 minutes to obtain solution A. Subsequently, 2 mmol K3[Fe(CN)6] was dissolved in 50 mL of deionized water and stirred for 10 minutes to obtain solution B. Solution B was slowly poured into solution A and allowed to stand at room temperature for 24 hours. After centrifugation, the supernatant was removed and the precipitate was repeatedly washed with deionized water and anhydrous ethanol, then dried in vacuum overnight.

[0056] To synthesize Ag@ZnCuNiMn-PBA, 0.4 mmol Zn(NO₃)₂·6H₂O, 0.5 mmol Cu(NO₃)₂·3H₂O, 1 mmol Ni(NO₃)₂·6H₂O, 0.1 mmol Mn(NO₃)₂·4H₂O, 2.25 mmol Na₃C₆H₅O₇·2H₂O, and 0.1–0.5 mmol AgNO₃ were dissolved in 50 mL of deionized water and stirred for 10 minutes to obtain Solution A. Subsequently, 2 mmol K₃[Fe(CN)₆]₆ was dissolved in 50 mL of deionized water and stirred for 10 minutes to obtain Solution B. Solution B was slowly poured into Solution A and allowed to stand at room temperature for 24 hours. After centrifugation, the supernatant was removed and the precipitate was repeatedly washed with deionized water and anhydrous ethanol. The precipitate was then vacuum-dried overnight.

[0057] 1.3 Morphological and structural characterization

[0058] The morphology of the material was analyzed using a transmission electron microscope. 1 mg of the material was ultrasonically dispersed in 1 mL of anhydrous ethanol. The solution was dropped onto a copper mesh covered with a carbon support film and dried at room temperature overnight to obtain the test sample. A transmission electron microscope was used to observe the size and morphology of the material and analyze its structure. The morphology of the material was analyzed using a scanning electron microscope. First, the conductive tape was stuck on the sample stage, and then the material was evenly sprinkled on the conductive tape. After blowing off the excess sample with an ear bulb, the material was placed in a scanning electron microscope to observe the morphology of the material and take a picture. The morphology and size of the material were analyzed using an atomic force microscope. 1 mg of nanoparticles were ultrasonically dispersed in 1 mL of deionized water. A pipette was used to draw 10 μL of the ultrasonically dispersed material and drop it on a mica sheet. It was dried at room temperature overnight, and the nanomaterial was imaged and analyzed using an atomic force microscope.

[0059] 1.4 Study on the antibacterial properties of Ag@ZnCuNiMn-PBA

[0060] The antibacterial properties of the material were tested using Gram-negative Escherichia coli (BL21) and Gram-positive Staphylococcus aureus (ATCC 6538) as model bacteria. The revived bacterial suspension was inoculated into MH broth the night before and cultured at 37°C and 220 rpm for 16 hours. The suspension was diluted to 6×10 6 CFU / mL for subsequent antibacterial experiments.

[0061] (1) Punching method

[0062] 100 μL of the diluted bacterial solution was inoculated onto a plate culture medium and evenly spread with a spreading rod. A sterile Oxford cup was then gently pressed vertically onto the surface of the plate culture medium to create even holes. Excess agar was removed and 100 μL of ZnCuNiMn-PBA solution of varying concentrations was added to the wells. The plate was then incubated at 220 rpm and 37°C for 16 hours. The inhibition diameters of the ZnCuNiMn-PBA at varying concentrations were measured with a ruler to provide a preliminary assessment of the material's antibacterial properties. The experiment was repeated three times to improve the accuracy of the results.

[0063] (2) Growth curve method

[0064] 100 μL of ZnCuNiMn-PBA solution of different concentrations and 100 μL of diluted bacterial solution were added to a test tube containing 5 mL of MH broth medium. The test tube was then incubated at 220 rpm and 37 °C for 3 h. Sterile water and kanamycin (50 μg / mL) were set as the control group. The optical density (OD) of the bacterial solution was measured at 600 nm every 2 h using a UV-spectrophotometer to monitor bacterial growth. In addition, in order to avoid the influence of the sample on the measured value in the culture bottle, the OD measured each time was 600 Subtract the OD of the sample in the culture medium without bacterial solution under the same conditions. 600 The experiment was repeated three times to improve the accuracy of the experimental results.

[0065] (3) Plate count method

[0066] 100 μL of ZnCuNiMn-PBA solution of different concentrations and 100 μL of diluted bacterial solution were added to a test tube containing 5 mL of MH broth medium. The test tube was then incubated at 220 rpm and 37°C for 3 hours. Sterile water and kanamycin (50 μg / mL) were used as the control group. A certain amount of bacterial solution was taken from each test tube and diluted 10 times. 100 μL of the diluted bacterial solution was inoculated onto the plate culture medium, spread evenly with a spreading rod, and placed in a 37°C constant temperature incubator for 16 hours. The experiment was repeated three times to improve the accuracy of the experimental results.

[0067] 1.5 Morphological observation of bacteria

[0068] To prepare bacterial samples for SEM imaging, first, fresh bacterial suspension (6 × 10 6 CFU / mL) were incubated with ZnCuNiMn-PBA and Ag@ZnCuNiMn-PBA for 12 hours. Each bacterial suspension was then centrifuged at 5000 rpm for 3 minutes. The resulting bacteria were evenly distributed in a 2.5% glutaraldehyde solution and fixed overnight. Subsequently, the cells were dehydrated using a gradient of ethanol (30%, 50%, 70%, 80%, 90%, and 100%). Finally, the cells were gold-sprayed and observed and photographed using a scanning electron microscope.

[0069] 1.6 Determination of reactive oxygen free radicals

[0070] The release of reactive oxygen species (ROS) determines the antibacterial and antitumor properties of a material. The ability of ZnCuNiMn-PBA and Ag@ZnCuNiMn-PBA to generate hydroxyl radicals (·OH) was investigated using reduced methylene blue (MB), which reacts with hydroxyl radicals (·OH) to form blue. TMB (10 μg / mL) and H2O2 (100 mM) were added to ZnCuNiMn-PBA and Ag@ZnCuNiMn-PBA solutions, respectively. The absorbance at 650 nm was measured at different times using a UV-spectrophotometer to determine the generation of ·OH.

[0071] DPBF is a singlet oxygen-indicating fluorescent probe that is sensitive to singlet oxygen ( 1 O2) has high specificity and is irreversibly oxidized to form endoperoxides after reaction. Based on this characteristic, it is used to study the generation of ZnCuNiMn-PBA and Ag@ZnCuNiMn-PBA. 1 DPBF (10 μL, 1 mg / mL) was added to ZnCuNiMn-PBA and Ag@ZnCuNiMn-PBA solutions, and the absorption intensity of the solution at 420 nm was measured at different times using a UV-spectrophotometer to determine the 1 The production of O2.

[0072] 2. Results and Discussion

[0073] 2.1 Screening of metal salt components of Prussian blue derivatives

[0074] In order to screen the metal components of Prussian blue derivatives (PBA) with excellent antibacterial activity, the antibacterial properties of different transition metal nitrates (zinc, copper, cobalt, nickel and manganese) were first studied using Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) as representative strains. Figure 1As shown in the figure, fresh bacterial cultures in the logarithmic growth phase were mixed with various concentrations of transition metal nitrates for 3 hours, then plated on agar plates. After incubation at 37°C for 16 hours, bacterial growth was observed. The plate image shows that copper ions have the strongest antibacterial properties among transition metal nitrates, followed by cobalt, nickel, and zinc, while manganese ions have the weakest antibacterial properties. Therefore, transition metal salts with strong antibacterial properties are preferred when preparing PBAs.

[0075] In order to obtain the combination of PBAs with the best antibacterial properties, a series of PBAs with different transition metal components, including ternary, quaternary and high entropy PBAs, were prepared by changing the type of transition metal nitrate added during the synthesis process. Figure 2 As shown in Figure A, various ternary PBAs with different combinations were first synthesized, including CuCo-PBA, CuNi-PBA, CuZn-PBA, and CoNi-PBA. Their morphologies were characterized by AFM. Figure 3 The results showed that the PBAs prepared from different transition metal salts had different sizes, but all maintained the classic cubic morphology of PBA. The antibacterial properties of these four PBAs against Escherichia coli were preliminarily evaluated using the punching method. Figure 2 The inhibition zone size of A was found to be the largest for CuZn-PBA against E. coli, with a diameter of 13.4 mm. The inhibition zone size of CuNi-PBA was 12.1 mm, while CuCo-PBA and CoNi-PBA showed no inhibition zone, indicating that PBA containing copper and zinc components has the strongest antibacterial properties. Quaternary ZnCuNi-PBA and high-entropy ZnCuNiCoMn-PBA were further synthesized by adding more transition metal elements to CuZn-PBA. The antibacterial properties of these materials were also compared using the punching method. Figure 3 Figures A and B show the cubic morphology of ZnCuNi-PBA and ZnCuNiCoMn-PBA. Figure 3 Figure C shows that the inhibition zone diameter of ZnCuNi-PBA is 13.6 mm, which shows strong antibacterial activity, but high-entropy ZnCuNiCoMn-PBA has no inhibition zone. In summary, ZnCu-PBA and ZnCuNi-PBA were selected for subsequent experiments.

[0076] In order to further improve the antibacterial properties of the material, attempts were made to dope silver into the PBA material. Figure 4 As shown in Figures A and B, the cubic morphology of the material remains almost unchanged before and after the addition of silver, indicating that the addition of AgNPs does not affect the lattice morphology of PBA. Figure 4The inhibition zone results for C show that the antibacterial properties of the material were significantly improved after the addition of silver. The diameter of the inhibition zone of silver-doped ZnCuNi-PBA (Ag@ZnCuNi-PBA, 24.4 mm) was significantly larger. Although the antibacterial effect of Ag@CuZn-PBA (20.6 mm) was also improved compared to CuZn-PBA (13.4 mm), it was smaller than that of Ag@ZnCuNi-PBA, indicating that Ag@ZnCuNi-PBA has a superior antibacterial effect. Figure 5 The SEM results show that Ag@ZnCuNi-PBA presents a uniform cubic morphology, which is consistent with the general morphology and structure of the TEM image. However, the distribution of AgNPs on ZnCuNi-PBA is very insufficient, and there is agglomeration phenomenon, which may affect the antibacterial performance of AgNPs to some extent.

[0077] 2.2 Preparation and Characterization of Ag@ZnCuNiMn-PBA

[0078] Considering that the agglomeration of AgNPs will seriously affect its antibacterial performance, in order to solve this problem, by regulating the components of PBA carrier, Co was added during the synthesis of Ag@ZnCuNi-PBA. 2+ The amount of silver nitrate used in the synthesis process was also regulated. Figure 6 A in the figure is the AFM image of ZnCuNiCo-PBA, Co 2+ The addition of did not affect the original cubic shape of PBA. Figure 6 B and C are AFM images of Ag@ZnCuNiCo-PBA formed after the addition of 0.1 and 0.3 mmol silver nitrate, respectively. Figure 6 The inhibition zone results of D showed that Co 2+ After addition, the generated ZnCuNiCo-PBA itself has no inhibition zone, while the inhibition zone diameters of Ag@ZnCuNiCo-PBA synthesized with 0.1 and 0.3 mmol silver nitrate are 11.0 mm and 17.6 mm, respectively, and the antibacterial effect is not as good as Ag@ZnCuNi-PBA (24.4 mm).

[0079] Then we tried adding Mn 2+ , Ag@ZnCuNi-PBA and Ag@ZnCuNiMn-PBA were synthesized.

[0080] TEM images of Ag@ZnCuNiMn-PBA are shown in Figure 2. Figure 7 As shown in A, in addition to maintaining the original cubic shape, a number of ultra-small spherical points are evenly loaded on the surface of the material. Figure 6These dots were also observed in the magnified TEM image of Figure B. To prove that these dots were AgNPs, the Ag@ZnCuNiMn-PBA was further characterized by HAADF-STEM analysis ( Figure 7 ). The image clearly shows that the dots are completely consistent with the distribution of Ag elements in the elemental map. Figure 7 Figure D also shows the distribution of Zn, Ni, Mn, Fe, and Cu elements, which is consistent with the shape of Ag@ZnCuNiMn-PBA, proving the successful synthesis of Ag@ZnCuNiMn-PBA.

[0081] X-ray photoelectron spectroscopy (XPS) can reflect the elemental composition and chemical valence state of Ag@ZnCuNiMn-PBA. Figure 8 As shown in A, Ag was observed in Ag@ZnCuNiMn-PBA. 0 The XPS spectrum of Ag 3d shows different peaks at 368.2 and 374.1 eV, which can be attributed to Ag 0 Ag 3d 5 / 2 and Ag 3d 3 / 2 orbital, which also proves the successful introduction of AgNPs. In addition, according to the elemental valence analysis of Ag@ZnCuNiMn-PBA, the XPS spectrum of Zn 2p shows that zinc exists in the form of divalent ions ( Figure 8 Middle B), Figure 8 C in the middle indicates that Cu exists in monovalent and divalent forms. Figure 8 DF in the middle shows that the three elements, tungsten, manganese and iron, exist in the material in the form of divalent and trivalent valence states in Ag@ZnCuNiMn-PBA.

[0082] The lowest concentration of nanoparticles that completely kills the tested bacteria is called the minimum bactericidal concentration (MBC). Figure 9 As shown in the figure, the MBC value of Ag@ZnCuNiMn-PBA is 8μg / mL, while the MBC value of Ag@ZnCuNi-PBA is 16μg / mL, indicating that the bactericidal ability of PBA matrix against E. coli is significantly enhanced after doping with a small amount of manganese, which may be attributed to the uniform distribution of nanosilver at the PBA interface. In order to optimize the proportion of silver content in Ag@ZnCuNiMn-PBA, different molar amounts of AgNO3 (0.1, 0.3 and 0.5 mmol) were used to prepare three products Ag@ZnCuNiMn-PBA. 0.1 @ZnCuNiMn-PBA、Ag 0.3 @ZnCuNiMn-PBA and Ag 0.5@ZnCuNiMn-PBA, their antibacterial activities were determined by plate count method. The three products Ag@ZnCuNiMn-PBA solutions were mixed with fresh bacterial suspension and cultured on MH broth agar medium. After culturing at 37 ° C for 16 hours, the colonies on the surface of the culture medium were as follows Figure 10 As shown, 0.1mmol AgNO3 prepared Ag 0.1 The MBC value of @ZnCuNiMn-PBA is 16μg / mL; when the amount of AgNO3 increases to 0.3mmol, Ag 0.3 @ZnCuNiMn-PBAMBC value dropped to 8μg / mL. When the concentration was further increased to 0.5mmol, Ag 0.5 The MBC value of @ZnCuNiMn-PBA remains unchanged. Therefore, the more economical combination Ag is selected. 0.3 @ZnCuNiMn-PBA, that is, 0.3mmol AgNO3 was used to prepare the Ag@ZnCuNiMn-PBA required for subsequent experiments.

[0083] In order to detect the minimum inhibitory concentration (MIC), the growth curves of Ag@ZnCuNiMn-PBA with different concentrations were measured using the optical density at a wavelength of 600 nm (OD600). Figure 11 The MIC values ​​of Ag@ZnCuNiMn-PBA against Escherichia coli and Staphylococcus aureus were 8 and 4 μg / mL, respectively, and the MBC value against Staphylococcus aureus was 4 μg / mL, which is superior to previously reported silver-containing nanomaterials (as shown in Table 1). Antibacterial experiments showed that Ag@ZnCuNiMn-PBA has strong broad-spectrum antibacterial activity.

[0084] Table 1. Comparison of MIC values ​​of Ag@ZnCuNiMn-PBA with reported silver-doped nanomaterials

[0085] 2.3 Evaluation of the antibacterial properties of Ag@ZnCuNiMn-PBA in vitro

[0086] In order to investigate the damage caused by Ag@ZnCuNiMn-PBA to Escherichia coli and Staphylococcus aureus, Figure 12CLSM and SEM images of bacteria treated with a control group, ZnCuNiMn-PBA, and Ag@ZnCuNiMn-PBA, respectively, are shown. SYTO 9 / PI was used for dual staining of live and dead bacteria. SYTO 9 can label all bacteria (live and dead), while PI can only penetrate dead bacteria with damaged cell membranes. Therefore, live bacteria only have SYTO 9 dye, which exhibits green fluorescence. Dead bacteria, on the other hand, have both SYTO 9 and PI dyes. The addition of PI reduces the fluorescence intensity of SYTO 9 dye, resulting in red fluorescence in dead bacteria. The CLSM image of the control group shows strong green fluorescence, indicating healthy bacterial growth and the absence of dead bacteria. The corresponding SEM image is consistent with the confocal microscope results, showing that both E. coli and S. aureus maintain smooth, intact membranes. E. coli cells exhibit a complete rod-shaped morphology, while S. aureus exhibits a round, plump spherical shape. In the ZnCuNiMn-PBA group, partial red fluorescence was observed, indicating the presence of some dead bacteria. At the same time, SEM images showed slight damage to the bacterial cell membrane surface, with wrinkles and small pits. In the Ag@ZnCuNiMn-PBA experimental group, the full red fluorescence under the microscope indicated the presence of almost no live bacteria. The corresponding SEM images showed that the cell membranes of both Escherichia coli and Staphylococcus aureus were severely damaged, with the entire bacterial morphology showing severe atrophy and destruction, and obvious leakage of intracellular cytoplasmic components. These results indicate that the antibacterial effect of Ag@ZnCuNiMn-PBA is significantly superior to that of ZnCuNiMn-PBA.

[0087] 2.4 Investigation of the antibacterial mechanism of Ag@ZnCuNiMn-PBA

[0088] Electron paramagnetic resonance (EPR) analysis was used to verify the types of reactive oxygen species (ROS) generated by Ag@ZnCuNiMn-PBA during the sterilization process. Figure 13, when Ag@ZnCuNiMn-PBA is combined with DMPO, a characteristic signal with a relative intensity of 1:2:2:1 is observed, proving the generation of hydroxyl radicals (·OH). ·OH is a very active free radical with extremely high oxidizing ability. It can react rapidly with all biological molecules in bacterial cells, including sugars, proteins, nucleic acids and lipids. When ·OH reacts with biological molecules, it will capture hydrogen atoms from these molecules to form new free radicals, triggering a series of chain reactions, causing serious damage to the structure and function of the biological molecules. For example, it can cause lipid peroxidation on the bacterial cell membrane, destroying the integrity of the cell membrane; it can also attack the bacterial DNA, causing DNA chain breaks and base damage, causing the bacteria to lose their ability to reproduce and survive. Similarly, when Ag@ZnCuNiMn-PBA is combined with TEMP, a characteristic signal with a relative intensity of 1:1:1:1 is observed, proving that singlet oxygen ( 1 O2) generation. 1 O2 is an oxygen molecule in an excited state. It has strong oxidizing properties and can react with various biological molecules in bacterial cells, such as unsaturated fatty acids, proteins and nucleic acids. 1 When O2 reacts with unsaturated fatty acids on the bacterial cell membrane, it triggers lipid peroxidation, which destroys the structure and function of the cell membrane and causes the leakage of intracellular substances, thereby inhibiting bacterial growth or causing bacterial death. 1 O2 can also directly act on the nucleic acid of bacteria, causing the breakage of nucleic acid chains or damage to bases, interfering with the transmission and replication of bacterial genetic information, and thus achieving the purpose of sterilization.

[0089] In addition, in order to explore the antibacterial mechanism of the material, the Ag content of Ag@ZnCuNiMn-PBA in aqueous solution was determined by inductively coupled plasma optical emission spectrometry (ICP-OES). + Release situation. Figure 14 The results show that Ag@ZnCuNiMn-PBA has a relatively sharp Ag + Release behavior. + The rapid release of Ag@ZnCuNiMn-PBA enables it to exhibit rapid and effective antibacterial activity when in contact with bacteria. After the acute release phase, Ag@ZnCuNiMn-PBA continues to release Ag at a relatively slow rate over the next 36 hours. + Ag@ZnCuNiMn-PBA released 13.97 μg / mL of Ag after 48 hours. + This indicates that during the antibacterial process, Ag@ZnCuNiMn-PBA not only kills bacteria by producing ROS, but also produces Ag + The release of the two together achieves the effect of killing bacteria.

[0090] To study the in vivo antibacterial effect of Ag@ZnCuNiMn-PBA, a S. aureus-infected mouse back skin wound model was established. After the skin wound model was established in BALB / c mice, S. aureus bacteria were injected into the wound surface to establish a wound infection model. The next day, the mice were randomly divided into three groups: control group, ZnCuNiMn-PBA group, and Ag@ZnCuNiMn-PBA experimental group. Wound healing ( Figure 15 ) showed that on the 5th day, the wounds of the mice in the Ag@ZnCuNiMn-PBA experimental group had shown obvious healing effects. On the 14th day, the wounds of the mice in the Ag@ZnCuNiMn-PBA experimental group had returned to a smooth state, with hair growing, similar to the skin of normal mice. On the 11th day, the wounds of the mice in the ZnCuNiMn-PBA experimental group showed more obvious healing. This shows that both ZnCuNiMn-PBA and Ag@ZnCuNiMn-PBA materials showed the effect of promoting wound healing and good biocompatibility on damaged skin, but the wound healing speed of each group was different. For example, on the 7th day, the wound healing rate of the Ag@ZnCuNiMn-PBA group was 75.31%, while the wound healing rate of the ZnCuNiMn-PBA experimental group was 24.67%. In comparison, the healing effect of the Ag@ZnCuNiMn-PBA group was better. The wound healing rate in the control group on the third day was -26.38%, indicating that the lack of antimicrobial therapy in the control group led to an increase in the size of the S. aureus-infected wound. These results further demonstrate that Ag@ZnCuNiMn-PBA possesses excellent antimicrobial properties, effectively inhibiting S. aureus growth and promoting wound healing.

[0091] References [1]Feng, L.; Liu, Y.; Chen, Y.; Xiang, Q.; Huang, Y.; Liu, Z.; Materials2023,12(22),2203201. [2]Xie,W.;Chen,J.;Cheng,X.;Feng,H.;Zhang,X.;Zhu,Z.;Dong,S.;Wan,Q.;Pei,X.;Wang,J.Multi-Mechanism Antibacterial Strategies Enabled by SynergisticActivity of Metal–Organic Framework-Based Nanosystem for Infected TissueRegeneration.Small2023,19(14),2205941. [3]Li,Y.J.;Gao,Z.G.;Zhang,Y.;Chen,F.H.;An,P.J.;Wu,H.S.;You,C.Q.;Sun,B.W.MOF-Shielded and Glucose-Responsive Ultrasmall Silver Nano-Factory forHighly-Efficient Anticancer and Antibacterial Therapy.Chemical EngineeringJournal2021,416,127610.. [4]Kim,D.;Park,K.W.;Park,J.T.;Choi,I.Photoactive MOF-DerivedBimetallic Silverand Cobalt Nanocomposite with Enhanced AntibacterialActivity.ACS Appl.Mater.Interfaces2023,15(19),22903–22914. [5]Yang,Y.;Wu,X.;He,C.;Huang,J.;Yin,S.;Zhou,M.;Ma,L.;Zhao,W.;Qiu,L.;Cheng,C.;Zhao,C.Metal–Organic Framework / Ag-Based Hybrid Nanoagents for Rapidand Synergistic Bacterial Eradication.ACS Appl.Mater.Interfaces2020,12(12),13698–13708.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Various modifications are possible. Any simple, equivalent changes and modifications made in accordance with the claims and description of the present invention are within the scope of protection of the patent claims. Anything not fully described in this invention is conventional technology.

Claims

1. A method for preparing a silver-doped all-transition metal Prussian blue derivative with high antibacterial activity, characterized in that: The following steps are involved: S1: Dissolve appropriate amounts of Zn(NO3)2·6H2O, Cu(NO3)2·3H2O, Ni(NO3)2·6H2O, Mn(NO3)2·4H2O, Na3C6H5O7·2H2O, and AgNO3 in 50-200 mL of deionized water and stir to obtain solution A; S2: Dissolve an appropriate amount of K3[Fe(CN)6] in 50-200 mL of deionized water and stir to obtain solution B; S3: Slowly pour solution B into solution A, let it stand at room temperature, centrifuge and remove the supernatant, wash the precipitate repeatedly with deionized water and anhydrous ethanol respectively, and vacuum dry to obtain.

2. The preparation method according to claim 1, characterized in that In step S1, the total content of Zn, Cu, Ni, and Mn in solution A is 2 mmol.

3. The preparation method according to claim 2, characterized in that The amount of Zn(NO3)2·6H2O used is 0.2~0.8mmol, the amount of Cu(NO3)2·3H2O used is 0.2~0.8mmol, the amount of Ni(NO3)2·6H2O used is 0.8~1.2mmol, and the amount of Mn(NO3)2·4H2O used is 0.01~0.8mmol.

4. The preparation method according to claim 1, characterized in that The amount of Na3C6H5O7·2H2O used is 2 to 3 mmol, and the amount of AgNO3 used is 0.1 to 1.0 mmol.

5. The preparation method according to claim 1, characterized in that In step S2, 1-5 mmol K3[Fe(CN)6] is dissolved in 50-200 mL deionized water.

6. The preparation method according to claim 1, characterized in that In step S3, the mixture is allowed to stand at room temperature for 2 to 24 hours.

7. The preparation method according to claim 1, characterized in that In step S1 and step S2, the stirring time is 5 to 60 minutes.

8. A silver-doped all-transition metal Prussian blue derivative with high antibacterial activity obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the silver-doped all-transition metal Prussian blue derivative according to claim 8 in the preparation of a medicament for preventing and treating bacterial infection.

10. The use according to claim 9, characterized in that The bacteria include: Gram-negative bacteria represented by Escherichia coli, and Gram-positive bacteria represented by Staphylococcus aureus.

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