A nano-silver-copper suspension and its in-situ autocatalytic reduction preparation method

Abstract

This invention discloses a nano-silver-copper suspension and its in-situ autocatalytic reduction preparation method. The method described in this invention does not add a reducing agent, but relies solely on surfactants to achieve the in-situ autocatalytic reduction of silver and copper ions. The synthesized nano-silver-copper suspension has a concentration of 50 μg / ml to 200 μg / ml, containing both nano-silver and nano-copper particles. The content, by mass fraction, is 93.33% to 95.23% silver and 6.67% to 4.76% copper. The in-situ autocatalytic reduction method of this invention is green and environmentally friendly, with a simple operation process. The prepared nano-silver-copper particles have advantages such as uniform particle size, good dispersibility in aqueous solution, and the ability to remain suspended for a long time without aggregation and sedimentation. The nano-silver-copper suspension exhibits excellent bactericidal ability and good in vitro biocompatibility, and can be used as a broad-spectrum antibacterial agent in wound disinfection, surgical dressings, and in vivo medical devices.

Description

Technical Field

[0001] This invention relates to a nano-silver-copper suspension and its in-situ autocatalytic reduction preparation method, belonging to the field of antibacterial and sterilization using inorganic nano-metal materials. Background Technology

[0002] Antibiotics are the most frequently used and widely applied antibacterial agents to address the harm that pathogenic bacteria and other microorganisms pose to human health. However, due to problems such as antibiotic resistance caused by overuse and excessive dosage, antibiotics are now strictly controlled in various countries. Furthermore, antibiotics themselves have drawbacks such as instability due to high-temperature inactivation and sensitivity to the pH of the surrounding environment, which also limits their application scenarios. To address the aforementioned antibiotic crisis and achieve a clean and hygienic living environment, it is necessary to develop novel inorganic nanomaterial antibacterial materials as alternatives, gradually reducing dependence on antibiotics.

[0003] Silver and copper have a long history of antibacterial use. Modern scientific research has found that silver and copper exhibit excellent bactericidal capabilities against more than 300 types of bacteria, proving them to be among the most promising broad-spectrum antibacterial agents, comparable to antibiotics. Nanoparticle processing of silver and copper can increase their specific surface energy and area, promoting the release of more active ions and further enhancing their antibacterial effect. It also reduces the amount needed, lowering the high cost associated with their precious metal properties. However, nanoparticle processing cannot be achieved through bulk mechanical crushing. Currently, the typical preparation method for nano-silver and nano-copper is the chemical reduction method, which uses reducing agents such as sodium hypophosphite, hydrazine hydrate, and sodium borohydride to reduce soluble salt solutions of silver and copper. However, this method is not perfect. The main problems it exposes are concentrated on the reducing agent, such as sodium borohydride, which reacts too quickly with silver and copper ions. The resulting nanoparticles are unstable and easily aggregate into secondary large particles or even settle, further affecting their antibacterial properties and long-term stability. In addition, hydrazine hydrate itself is toxic, and its addition to the reaction system raises questions about its biosafety, which limits the use of the reaction products in biomedicine. Summary of the Invention

[0004] To address the aforementioned shortcomings of chemical reduction methods, the present invention aims to provide a method for preparing nano-silver-copper suspensions via in-situ autocatalytic reduction. The nanoparticles synthesized by this method are small and relatively uniform in size, exhibit good stability in suspension systems, demonstrate excellent antibacterial properties against Escherichia coli and Staphylococcus aureus, and show no cytotoxicity against mouse fibroblasts (L929). The nano-silver-copper suspension of the present invention can be used for antibacterial and sterilization applications in skin wounds, surgical dressings, and in vivo medical devices.

[0005] The in-situ autocatalytic reduction autocatalytic method proposed in this invention is characterized by the absence of a reducing agent; instead, it utilizes surfactants to reduce silver and copper ions. Furthermore, surfactants are common additives in pharmaceuticals and food products, thus reducing biosafety risks from a raw material perspective. A second characteristic is the use of a mixture of binary (A+B) nonionic surfactants.

[0006] The method described in this invention is achieved through the following technical solution: no reducing agent is added to the reaction system; the in-situ autocatalytic reduction of silver and copper ions is accomplished solely by a surfactant. Specifically, it includes the following steps:

[0007] (1) Dissolve silver nitrate, copper nitrate and surfactant in deionized water to prepare aqueous solutions.

[0008] (2) Add the aqueous solutions of silver nitrate and copper nitrate dropwise to the continuously stirred aqueous solution of surfactant and stir for 0.5 h to make a mixed solution; the surfactant is a mixture of binary (A+B) nonionic surfactants. The A type surfactant realizes the reduction of metal ions; the B type surfactant is used to ensure the stability and dispersibility of silver and copper nanoparticles in suspension.

[0009] (3) The mixed solution is heated to carry out an in-situ autocatalytic reduction reaction. The reaction temperature is controlled at 60℃~140℃ and the reaction time is 3h~8h.

[0010] (4) After the reaction is complete, the mixture is allowed to cool naturally to room temperature to obtain the reaction product: nano silver copper suspension.

[0011] Preferably, in step (1) of the present invention, the mass concentration of silver nitrate is 73.5 μg / ml to 299.5 μg / ml, and the mass concentration of copper nitrate is 9.8 μg / ml to 28.2 μg / ml.

[0012] Preferably, the surfactant added in this invention is 400 to 1000 times the total mass of silver nitrate and copper nitrate.

[0013] Preferably, the surfactant of the present invention is a mixture of binary A+B nonionic surfactants, wherein the A-component nonionic surfactant is polyvinyl alcohol, and the B-component nonionic surfactant is one of polyoxyethylene sorbitan fatty acid ester, sucrose fatty acid monoester, alkyl glycoside (APG), and polyethylene glycol monooctylphenyl ether, wherein the A-component surfactant is 93.5% to 99.5% and the B-component surfactant is 0.5% to 6.5%.

[0014] The polyoxyethylene sorbitan fatty acid esters described in this invention are commonly known as the Tween series, such as Tween 20, 21, 40, 40, 60, 80, etc.

[0015] The polyethylene glycol monooctylphenyl ether described in this invention is commonly known as the Triton series, such as Triton 45, 100, etc.

[0016] Another objective of this invention is to provide a nano-silver-copper suspension with a concentration of 50 μg / ml to 200 μg / ml, containing both nano-silver particles and nano-copper particles. The content of both, by mass fraction, is 93.33% to 95.23% for silver and 6.67% to 4.76% for copper. The nano-silver-copper suspension prepared by this invention exhibits excellent antibacterial and bactericidal abilities under the synergistic effect of nano-silver, nano-copper, and polyvinyl alcohol. Furthermore, it uses non-toxic raw materials and does not contain any reducing agents. The nano-silver-copper suspension has no in vitro cytotoxicity, good safety, and can be applied to wound disinfection, surgical dressings, and in vivo medical devices.

[0017] Preferably, the size of the nano-silver copper particles of the present invention is 10nm to 200nm, and the structure formed includes one or more of the following: core-shell, Janus, and bimetal.

[0018] The nano-silver-copper suspension described in this invention exhibits excellent bactericidal activity against pathogenic strains such as Escherichia coli and Staphylococcus aureus, with a minimum inhibitory concentration (MIC) of no more than 6.25 ppm and a minimum bactericidal concentration (MBC) of no more than 3.125 ppm, through the synergistic effect of silver, copper, and polyvinyl alcohol. Furthermore, it does not contain any reducing agents and uses non-toxic raw materials, thus exhibiting no cytotoxicity to mouse fibroblasts (L929) and demonstrating good in vitro biosafety. It can be used as a broad-spectrum antibacterial agent in wound disinfection, surgical dressings, and in vivo medical devices.

[0019] Experiments showed that the nano-silver-copper suspension could be stored statically for more than 6 months without agglomeration of the nano-silver-copper particles; the minimum inhibitory concentration (MIC) of the suspension against Escherichia coli and Staphylococcus aureus was no higher than 6.25 ppm, and the minimum bactericidal concentration (MBC) was no higher than 3.125 ppm; the relative growth rate (RGR) of cells co-cultured with mouse fibroblasts (L929) for 24 h in vitro was 84.3%–111.6%, with toxicity grades of 0 and 1, and no cytotoxicity. The nano-silver-copper suspension of this invention is superior to existing commercial nano-copper and nano-silver products and can also be used for antibacterial and sterilization applications on skin wounds, surgical dressings, and in vivo medical devices.

[0020] The beneficial effects of the present invention are as follows:

[0021] (1) The original in-situ autocatalytic reduction method of this invention does not add a reducing agent in the entire reaction system. It only relies on surfactants to complete the in-situ autocatalytic reduction of silver ions and copper ions. It has the characteristics of being green and environmentally friendly, simple in operation, economical and practical. It can reduce the threat to biosafety from the raw materials. The resulting suspension product can be directly applied to various disinfection and sterilization scenarios, which is significantly better than some commercial products that first centrifuge and separate silver and copper particles and then prepare them into suspensions.

[0022] (2) The binary nonionic surfactant selected in this invention, on the one hand, utilizes the type A surfactant to reduce metal ions and also provides antibacterial effect for the nano silver copper suspension; on the other hand, the excellent dispersing effect of the type B surfactant ensures that the silver copper nanoparticles can remain stable in the suspension for a long time without agglomeration and sedimentation; it can compensate for or even improve the stability loss of the reaction system caused by the PVA reaction; compared with common reducing agents, PVA has a weak reducing ability and a slow reduction reaction rate, which results in smaller nanoparticle size, thereby further enhancing its antibacterial performance.

[0023] (3) In preparing the nano-silver-copper suspension, this method allows for the control of the suspension concentration and relative silver and copper content by adjusting the concentration and volume of the silver nitrate and copper nitrate aqueous solutions, as well as the in-situ autocatalytic reduction reaction temperature and time. It also allows for the control of nanoparticle size and the silver-copper composite structure by adjusting the concentration of the silver nitrate and copper nitrate aqueous solutions, the amount of surfactant, and the in-situ autocatalytic reduction reaction temperature and time. Furthermore, it allows for the regulation of the dispersibility and stability of the nano-silver-copper product in the suspension by adjusting the combination type and relative content of type A and type B surfactants. The nano-silver-copper suspension can be stored statically for more than 6 months without agglomeration or sedimentation of the nano-silver-copper particles.

[0024] (4) The nano silver-copper suspension of the present invention can effectively kill pathogenic bacteria by utilizing the synergistic enhancement effect of silver, copper and polyvinyl alcohol in antibacterial activity. Under the disc diffusion method, the nano silver-copper suspension of 50-200 μg / ml produces an inhibition zone diameter of 0.63cm-1.15cm against Escherichia coli and 0.85cm-1.35cm against Staphylococcus aureus.

[0025] (5) The nano-silver-copper suspension of the present invention exhibits excellent antibacterial properties, with a minimum inhibitory concentration (MIC) of no more than 6.25 ppm and a minimum bactericidal concentration (MBC) of no more than 3.125 ppm against Escherichia coli. It also has a minimum inhibitory concentration (MIC) of no more than 6.25 ppm and a minimum bactericidal concentration (MBC) of no more than 3.125 ppm against Staphylococcus aureus.

[0026] (6) The relative growth rate (RGR) of the nano-silver copper suspension of the present invention after co-culturing with mouse fibroblasts (L929) in vitro for 24 h was 84.3% to 111.6%. According to ISO10993, its toxicity level was determined to be grade 0 and grade 1, with no cytotoxicity, and it showed good in vitro biological safety. Attached Figure Description

[0027] Figure 1 (a) is the X-ray energy spectrum (EDX) of the silver-copper nanoparticles in the silver-copper nanoparticle suspension obtained in Example 1; Figure 1 (b) is its full spectrum under X-ray energy spectrum (mapping).

[0028] Figure 2 This is a transmission electron microscope (TEM) image of the silver-copper nanoparticles in the silver-copper nanoparticle suspension obtained in Example 2.

[0029] Figure 3 The images show a comparison of optical photographs of the nano-silver-copper suspension obtained in Example 3 before and after it has been left to stand for 6 months.

[0030] Figure 4 This is an optical photograph of the antibacterial ring of the nano-silver-copper suspension obtained in Example 4, tested using the disk diffusion method.

[0031] Figure 5 This is an optical photograph of the minimum inhibitory concentration (MIC) of the nano-silver copper suspension obtained in Example 5, tested using the broth dilution method.

[0032] Figure 6 The relative cell proliferation (RGR) is the result of co-culturing the nano-silver copper suspension obtained in Example 7 with mouse fibroblasts (L929) in vitro for 24 hours. Detailed Implementation

[0033] The following embodiments are a series of detailed descriptions of the material characteristics and preparation methods of the present invention, and should not be construed as limiting the claims of the present invention. It should also be noted that any substitutions and improvements made without departing from the concept of the present invention are within the scope of protection of the present invention. Unless otherwise specified, the raw materials used in the present invention are all commercially available conventional products, with the lowest grade being analytical grade.

[0034] Example 1

[0035] A nano-silver-copper suspension prepared by in-situ autocatalytic reduction method has a concentration of 50 μg / ml and contains both nano-silver and nano-copper particles. The relative contents of the two particles, by mass fraction, are 95.23% silver and 4.76% copper. The specific preparation steps are as follows:

[0036] (1) Dissolve silver nitrate, copper nitrate and surfactant in deionized water to prepare aqueous solutions; wherein, the binary nonionic surfactant is a mixture of polyvinyl alcohol (PVA) and sucrose fatty acid monoester, and the content of the two by mass fraction is 93.5% for polyvinyl alcohol (PVA) and 6.5% for sucrose fatty acid monoester; the mass concentration of silver nitrate after dissolution is 74.9 μg / ml; the mass concentration of copper nitrate is 9.8 μg / ml; the amount of surfactant is 1000 times the total amount of silver nitrate and copper nitrate.

[0037] (2) Add the aqueous solutions of silver nitrate and copper nitrate dropwise to the continuously stirred aqueous solution of surfactant, and then stir for 0.5 h to make a mixed solution.

[0038] (3) The mixed solution was heated to carry out an in-situ autocatalytic reduction reaction. The reaction temperature was controlled at 140℃ and the reaction time was 3h.

[0039] (4) After the reaction is complete, the mixture is allowed to cool naturally to room temperature to obtain the reaction product: nano silver copper suspension.

[0040] The nano-silver-copper suspension was centrifuged, and the separated solid particles were washed and vacuum dried to obtain the solid particle product. X-ray energy dispersive spectroscopy was used to characterize it, and the results are as follows: Figure 1 As shown in (EDX). Figure 1 (a) It can be seen that the particulate matter only exhibits Ag and Cu in its energy spectrum. Considering the solid properties of the nanoparticles, the product can be considered to be nano-silver-copper metal. Furthermore, a full-spectrum mapping analysis of the material was performed, and the results are as follows: Figure 1 As shown in (b), the full spectrum results show that copper particles are concentrated on the surface of silver particles, indicating that the two particles form a core-shell structure with copper on the outside and silver on the inside during reduction.

[0041] Example 2

[0042] A nano-silver-copper suspension prepared by in-situ autocatalytic reduction method has a concentration of 200 μg / ml and contains both nano-silver and nano-copper particles. The relative contents of the two particles, by mass fraction, are 93.33% silver and 6.67% copper. The specific preparation steps are as follows:

[0043] (1) Dissolve silver nitrate, copper nitrate, and surfactant separately in deionized water to prepare aqueous solutions; wherein, the binary nonionic surfactant is a mixture of polyvinyl alcohol (PVA) and polyoxyethylene sorbitan fatty acid ester (commercial product Tween-80), and the content of both by mass fraction is 99.5% for polyvinyl alcohol (PVA) and 0.5% for Tween-80; the mass concentration of silver nitrate after dissolution is 296.8 μg / ml; the mass concentration of copper nitrate is 27.3 μg / ml; and the amount of surfactant used is 400 times the total amount of silver nitrate and copper nitrate.

[0044] (2) Add the above aqueous solutions of silver nitrate and copper nitrate dropwise to the continuously stirred aqueous solution of surfactant, and stir for 0.5 h to prepare a mixed solution.

[0045] (3) The mixed solution was heated to carry out an in-situ autocatalytic reduction reaction. The reaction temperature was controlled at 60℃ and the reaction time was 8h.

[0046] (4) After the reaction is complete, the mixture is allowed to cool naturally to room temperature to obtain the reaction product: nano silver copper suspension.

[0047] The nano-silver-copper suspension was centrifuged, and the separated solid particles were washed and vacuum dried to obtain nano-silver-copper particles. These particles were characterized using transmission electron microscopy (TEM), and the results are as follows: Figure 2 As shown; by Figure 2 It can be seen that the particle size of the silver-copper product is 10μm to 45μm, and the particle size uniformity is good; it can also be preliminarily determined that the silver and copper form a bimetallic structure.

[0048] Example 3

[0049] A nano-silver-copper suspension prepared by in-situ autocatalytic reduction method has a concentration of 150 μg / ml and contains both nano-silver and nano-copper particles. The relative contents of the two particles, by mass fraction, are 94.70% silver and 5.30% copper. The specific preparation steps are as follows:

[0050] (1) Dissolve silver nitrate, copper nitrate, and surfactant separately in deionized water to prepare aqueous solutions; wherein, the binary nonionic surfactant is a mixture of polyvinyl alcohol (PVA) and polyethylene glycol monooctylphenyl ether (commercial product Triton-45) with a content of 97.5% PVA and 2.5% Triton-45 by mass fraction; the mass concentration of silver nitrate after dissolution is 197.4 μg / ml; the mass concentration of copper nitrate is 20.1 μg / ml; and the amount of surfactant used is 620 times the total amount of silver nitrate and copper nitrate.

[0051] (2) Add the above aqueous solutions of silver nitrate and copper nitrate dropwise to the continuously stirred aqueous solution of surfactant, and stir for 0.5 h to prepare a mixed solution.

[0052] (3) The mixed solution was heated to carry out an in-situ autocatalytic reduction reaction. The reaction temperature was controlled at 120℃ and the reaction time was 4.5h.

[0053] (4) After the reaction is complete, the mixture is allowed to cool naturally to room temperature to obtain the reaction product: nano silver copper suspension. It is characterized by transmission electron microscopy (TEM) and analyzed to be a silver copper bimetallic structure.

[0054] Example 4

[0055] A nano-silver-copper suspension prepared by in-situ autocatalytic reduction method has a concentration of 100 μg / ml and contains both nano-silver and nano-copper particles. The relative contents of the two particles, by mass fraction, are 95.1% silver and 4.90% copper. The specific preparation steps are as follows:

[0056] (1) Dissolve silver nitrate, copper nitrate and surfactant in deionized water to prepare aqueous solutions; wherein, the binary nonionic surfactant is a mixture of polyvinyl alcohol (PVA) and alkyl glycoside (APG), and the content of both by mass fraction is 96.5% for polyvinyl alcohol (PVA) and 3.5% for Tween-80; the mass concentration of silver nitrate after dissolution is 149.6 μg / ml; the mass concentration of copper nitrate is 14.5 μg / ml; the amount of surfactant used is 800 times the total amount of silver nitrate and copper nitrate.

[0057] (2) Add the above aqueous solutions of silver nitrate and copper nitrate dropwise to the continuously stirred aqueous solution of surfactant, and stir for 0.5 h to prepare a mixed solution.

[0058] (3) The mixed solution was heated to carry out an in-situ autocatalytic reduction reaction. The reaction temperature was controlled at 80℃ and the reaction time was 5.5h.

[0059] (4) After the reaction is complete, the mixture is allowed to cool naturally to room temperature to obtain the reaction product: nano silver copper suspension. It is characterized by transmission electron microscopy (TEM) and analyzed to be a silver copper bimetallic structure.

[0060] Example 5

[0061] A nano-silver-copper suspension prepared by in-situ autocatalytic reduction method has a concentration of 50 μg / ml and contains both nano-silver and nano-copper particles. The relative contents of the two particles, by mass fraction, are 94.0% silver and 6.0% copper. The specific preparation steps are as follows:

[0062] (1) Dissolve silver nitrate, copper nitrate, and surfactant separately in deionized water to prepare aqueous solutions; wherein, the binary nonionic surfactant is a mixture of polyvinyl alcohol (PVA) and polyoxyethylene sorbitan fatty acid ester (commercial product Tween-60), the content of which, by mass fraction, is 98.5% for polyvinyl alcohol (PVA) and 1.5% for Tween-60. After dissolution, the mass concentration of silver nitrate is 73.9 μg / ml; the mass concentration of copper nitrate is 8.9 μg / ml; the amount of surfactant used is 1000 times the total amount of silver nitrate and copper nitrate.

[0063] (2) Add the above aqueous solutions of silver nitrate and copper nitrate dropwise to the continuously stirred aqueous solution of surfactant, and stir for 0.5 h to prepare a mixed solution.

[0064] (3) The mixed solution was heated to carry out an in-situ autocatalytic reduction reaction. The reaction temperature was controlled at 90℃ and the reaction time was 4h.

[0065] (4) After the reaction is completed, the mixture is allowed to cool naturally to room temperature to obtain the reaction product: nano-silver copper suspension. The product is characterized by transmission electron microscopy (TEM) and it is determined to be a core-shell structure with copper on the outside and silver on the inside.

[0066] Example 6

[0067] A nano-silver-copper suspension prepared by in-situ autocatalytic reduction method has a concentration of 75 μg / ml and contains both nano-silver and nano-copper particles. The relative contents of the two particles, by mass fraction, are 94.3% silver and 5.7% copper. The specific preparation steps are as follows:

[0068] (1) Dissolve silver nitrate, copper nitrate, and surfactant separately in deionized water to prepare aqueous solutions; wherein, the binary nonionic surfactant is a mixture of polyvinyl alcohol (PVA) and polyethylene glycol monooctylphenyl ether (commercial product Triton-100), the content of which, by mass fraction, is 95.5% for polyvinyl alcohol (PVA) and 4.5% for Tween-60. After dissolution, the mass concentration of silver nitrate is 111.2 μg / ml; the mass concentration of copper nitrate is 12.7 μg / ml; and the amount of surfactant used is 900 times the total amount of silver nitrate and copper nitrate.

[0069] (2) Add the above aqueous solutions of silver nitrate and copper nitrate dropwise to the continuously stirred aqueous solution of surfactant, and stir for 0.5 h to prepare a mixed solution.

[0070] (3) The mixed solution was heated to carry out an in-situ autocatalytic reduction reaction. The reaction temperature was controlled at 110℃ and the reaction time was 6h.

[0071] (4) After the reaction is complete, the mixture is allowed to cool naturally to room temperature to obtain the reaction product: nano silver copper suspension. It is characterized by transmission electron microscopy (TEM) and analyzed to be a silver copper bimetallic structure.

[0072] Example 7

[0073] A nano-silver-copper suspension prepared by in-situ autocatalytic reduction method has a concentration of 125 μg / ml and contains both nano-silver and nano-copper particles. The relative contents of the two particles, by mass fraction, are 93.38% silver and 6.62% copper. The specific preparation steps are as follows:

[0074] (1) Dissolve silver nitrate, copper nitrate, and surfactant separately in deionized water to prepare aqueous solutions; wherein, the binary nonionic surfactant is a mixture of polyvinyl alcohol (PVA) and polyoxyethylene sorbitan fatty acid ester (TWEEN-20), and the content of both by mass fraction is 94.5% for polyvinyl alcohol (PVA) and 5.5% for TWEEN-60. After dissolution, the mass concentration of silver nitrate is 183.6 μg / ml; the mass concentration of copper nitrate is 24.6 μg / ml; and the amount of surfactant used is 600 times the total amount of silver nitrate and copper nitrate.

[0075] (2) Add the above aqueous solutions of silver nitrate and copper nitrate dropwise to the continuously stirred aqueous solution of surfactant, and stir for 0.5 h to prepare a mixed solution.

[0076] (3) The mixed solution was heated to carry out an in-situ autocatalytic reduction reaction. The reaction temperature was controlled at 120℃ and the reaction time was 3.5h.

[0077] (4) After the reaction is completed, the mixture is allowed to cool naturally to room temperature to obtain the reaction product: nano-silver copper suspension. The product is characterized by transmission electron microscopy (TEM) and it is determined to be a core-shell structure with copper on the outside and silver on the inside.

[0078] To investigate the properties of the nano-silver-copper suspension prepared in this invention, the nano-silver-copper suspension obtained in Example 1 was randomly selected for testing. The specific results are analyzed below:

[0079] The nano-silver-copper suspension was allowed to stand for 6.5 months, and its stability was recorded using optical photography. The photographs are shown below. Figure 3 As shown; by Figure 3The prepared fresh nano-silver-copper suspension is light yellow and uniformly colored. After standing for 6.5 months, the color, uniformity and transmittance of the suspension did not change significantly, indicating that the suspension has good stability. The nano-silver-copper particles can remain stable in the aqueous solution for more than 6 months without significant agglomeration and sedimentation. The nano-silver-copper suspensions obtained in Examples 2 to 7 have similar properties to those in Example 1, and can all remain stable in the aqueous solution for more than 6 months without significant agglomeration and sedimentation.

[0080] With a bacterial suspension concentration of 1.5 × 10⁻⁶ 6 Using *E. coli* strains at CFU / ml as the target strain, the above-mentioned nano-silver copper suspension was tested for antibacterial and antimicrobial activity using the disc diffusion method. The results are as follows: Figure 4 As shown; by Figure 4 As shown, on MB culture plates coated with bacterial suspension, inhibition zones appeared only around the drug sensitivity strips containing the nano-silver-copper suspension. Measurements showed that the diameter of the inhibition zone was above 95 mm, significantly higher than the lower limit (6 mm) for determining antibacterial activity, indicating that the prepared nano-silver-copper suspension possesses good antibacterial properties and has a killing effect on Escherichia coli. The nano-silver-copper suspensions obtained in Examples 2-7 showed similar performance to those in Example 1; measurements showed that the diameter of the inhibition zone was above 95 mm in all cases.

[0081] With a bacterial suspension concentration of 1.5 × 10⁻⁶ 8 Using *Staphylococcus aureus* at CFU / ml as the target strain, the minimum inhibitory concentration (MIC) of the prepared nano-silver-copper suspension was determined using the broth dilution method. Optical photographs of the stained wells are shown below. Figure 5 As shown; by Figure 5 It can be seen that the initial concentration of the first well from the left was 50 μg / ml, and the concentration became 0.0244 μg / ml when diluted to the last well from the right. In the blank group with only bacterial suspension added, regardless of the concentration, it could be stained blue-purple by thiazolyl blue staining agent. However, after adding nano silver copper suspension, some wells were not stained, indicating that the bacteria contained therein had been killed. Therefore, the maximum minimum inhibitory concentration of the product can be determined to be 3.125 μg / ml. Multiple parallel results were concentrated at 1.5625 μg / ml. The performance of the nano silver copper suspension obtained in Examples 2 to 7 was similar to that in Example 1.

[0082] Using Escherichia coli and Staphylococcus aureus as target strains, the minimum inhibitory concentration (MIC) of the prepared nano-silver-copper suspension against the two pathogenic strains was tested using the broth dilution method. The antibacterial properties were compared with those of nano-silver suspension and nano-copper suspension prepared using the same preparation method and parameters. The results are summarized in Table 1.

[0083] Table 1. Statistical comparison of the minimum inhibitory concentrations (MICs) of nano-silver and copper suspensions against two materials and two bacterial strains.

[0084]

[0085] As shown in Table 1, compared with nano-silver and nano-copper suspensions, the prepared nano-silver and copper suspensions exhibited lower minimum inhibitory concentrations (MICs) against both Escherichia coli and Staphylococcus aureus. This indicates that the material has a certain broad-spectrum antibacterial ability. More importantly, it confirms that there is a synergistic antibacterial enhancement effect between nano-silver and copper, which can further enhance the antibacterial and bacteriostatic ability of the material, thereby reducing the amount of precious metals used and achieving a better cost performance.

[0086] Using mouse fibroblasts (L929) as the model cell, the effect of the suspension on cell proliferation was determined by testing the relative growth rate (RGR) of L929 cells after co-culturing with a silver-copper nanoparticle suspension for 24 hours, thereby assessing its cytotoxicity. The test results are shown in Figure 6. Figure 6 It can be seen that after L929 cells were co-cultured with the nano silver copper suspension for 24 hours, the relative cell proliferation rate became 84.3%. According to the ISO10993 judgment criteria, the toxicity level was 1, indicating that the nano silver copper suspension had no cytotoxicity and showed good in vitro cell biocompatibility. The nano silver copper suspensions obtained in Examples 2 to 7 had similar performance to those in Example 1.

[0087] The above tests were conducted using Example 1 as an example. The performance of the nano-silver copper suspensions obtained in Examples 2 to 7 is similar to that in Example 1.

Claims

1. A method for in-situ autocatalytic reduction preparation of a nano-silver-copper suspension, characterized in that, The in-situ autocatalytic reduction of silver and copper ions is achieved solely through surfactants without the addition of a reducing agent in the reaction system. This process includes the following steps: (1) Dissolve silver nitrate, copper nitrate and surfactant in deionized water to prepare aqueous solutions; the surfactant is a mixture of binary A+B nonionic surfactants; (2) Add aqueous solutions of silver nitrate and copper nitrate dropwise to an aqueous solution of surfactant that is continuously stirred, and then stir for 0.5 h to prepare a mixed solution; (3) The mixed solution is heated to carry out an in-situ autocatalytic reduction reaction, and the reaction temperature is controlled at 60°C. o C ~ 140 o C, reaction time is 3 h ~ 8 h; (4) After the reaction is complete, the mixture is allowed to cool naturally to room temperature to obtain the reaction product: nano silver copper suspension; The suspension has a concentration of 50 μg / ml to 200 μg / ml and contains both silver nanoparticles and copper nanoparticles. The content of both, by mass fraction, is 93.33% to 95.23% for silver and 6.67% to 4.76% for copper. The A-component nonionic surfactant is polyvinyl alcohol; the B-component nonionic surfactant is one of polyoxyethylene sorbitan fatty acid ester, sucrose fatty acid monoester, alkyl glycoside, and polyethylene glycol monooctylphenyl ether; the content of both is by mass fraction; the A-component surfactant is 93.5% to 99.5%, and the B-component surfactant is 0.5% to 6.5%; the mass of surfactant added is 400 to 1000 times the total mass of silver nitrate and copper nitrate.

2. The in-situ autocatalytic reduction preparation method of the nano-silver-copper suspension according to claim 1, characterized in that: In step (1), the mass concentration of silver nitrate is 73.5 μg / ml ~ 299.5 μg / ml, and the mass concentration of copper nitrate is 9.8 μg / ml ~ 28.2 μg / ml.

3. The in-situ autocatalytic reduction preparation method of the nano-silver-copper suspension according to claim 1, characterized in that: The nano-silver copper particles have a size of 10nm to 200nm, and the resulting structures include one or more of the following: core-shell structure, Janus structure, and bimetallic structure.

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