CN113582216A

CN113582216A - Cuprous sulfide nano antibacterial agent and preparation method and application thereof

Info

Publication number: CN113582216A
Application number: CN202110750649.5A
Authority: CN
Inventors: 何月珍; 尹曼悦; 孙健
Original assignee: Anhui Normal University
Current assignee: Anhui Normal University
Priority date: 2021-07-01
Filing date: 2021-07-01
Publication date: 2021-11-02
Anticipated expiration: 2041-07-01
Also published as: CN113582216B

Abstract

The invention discloses a cuprous sulfide nano antibacterial agent and a preparation method and application thereof, wherein the preparation method comprises the following steps: carrying out a first contact reaction on copper nanoparticles and an etching agent to prepare the cuprous sulfide nano antibacterial agent; the average particle size of the copper nanoparticles is 2-6nm, the valence state of copper element in the copper nanoparticles contains positive bivalence, and the etching agent is selected from at least one of reduced glutathione, cysteine and serine. The cuprous sulfide nano antibacterial agent has excellent antibacterial performance and dye degradation performance, and meanwhile, the preparation method has the advantages of simple process and simplicity and convenience in operation, so that the antibacterial agent can be widely applied to the antibacterial field and the dye degradation field.

Description

Cuprous sulfide nano antibacterial agent and preparation method and application thereof

Technical Field

The invention relates to copper nanoparticles, in particular to a cuprous sulfide nano antibacterial agent and a preparation method and application thereof.

Background

Bacterial infections have become a major threat to human health and have even led to the death of an increasing number of people in developing countries and less developed regions, and the need for antimicrobial materials is therefore at hand. In recent years, the problem of bacterial infection has indeed been suppressed to some extent by the widespread use of antibiotic drugs, but at the same time a new problem has emerged, namely the emergence of many drug-resistant superbacteria. So-called superbacteria, which are more harmful. For example, the number of deaths annually in the united states due to "superbacteria" can reach 18000, which is more than 16000 deaths due to aids in the united states in 2005; over the past two years, 313 of france had developed VRE urinary or digestive tract infections, with 3 dying due to lack of medication. Therefore, the design and development of antibacterial materials superior to antibiotics have been diligent.

In recent years, various antibacterial agents have been emerging. Such as photocatalytic antibacterial agents, metal oxide antibacterial agents, silver-based antibacterial agents, copper-based antibacterial materials, and the like. The copper-based antibacterial material has the most application prospect. At present, the methods for preparing the copper-based antibacterial agent are many and are roughly divided into a solid phase method and a gas phase method. The solid phase method is to change the particles with larger particle size into the particles with smaller particle size through ball milling by a physical method, although the solid phase method can be used for mass production of products, the prepared particles have larger particle size and inevitably introduce impurities, and the particle size distribution of the prepared nano particles is not uniform; compared with the solid phase method, the gas phase method has the advantages of high product purity and narrow particle size distribution, but has higher requirements on equipment.

Disclosure of Invention

The invention aims to provide a cuprous sulfide nano antibacterial agent, and a preparation method and application thereof.

In order to realize the aim, the invention discloses a preparation method of a cuprous sulfide nano antibacterial agent, which comprises the following steps: carrying out a first contact reaction on copper nanoparticles and an etching agent to prepare the cuprous sulfide nano antibacterial agent; the average particle size of the copper nanoparticles is 2-6nm, the valence state of copper element in the copper nanoparticles contains positive bivalence, and the etching agent is selected from at least one of reduced glutathione, cysteine and serine.

The invention also provides a cuprous sulfide nano antibacterial agent which is prepared by the preparation method.

The invention further provides an application of the cuprous sulfide nano antibacterial agent in catalyzing dye degradation and inhibiting bacteria.

In the above technical solution, the valence state of the copper element in the copper nanoparticles contains positive one and positive two, and the reduction GSH can convert the Cu in the copper nanoparticles²⁺Continuously reducing the solution into Cu⁺I.e. Cu²⁺+GSH＝Cu⁺+ GSSH, thereby completing the etching and reducing the particle size of the copper nanoparticles.

As can be seen from fig. 10A and 10B, the XPS characterization result of the Cu element of the copper nanoparticles in the mixture 1 in example 1 is: cu⁺The binding energy of the characteristic peak of 2P1/2 is 952.1eV, Cu⁺The binding energy of the characteristic peak of 2P3/2 is 932.4eV, Cu²⁺The binding energy of the characteristic peak of 2P1/2 is 953.9eV, Cu²⁺The binding energy of the characteristic peak of 2P3/2 is 933.9eV, which indicates that the valence univalent and bivalent of the Cu element in the CuNPs are both available; in addition, the strong satellite peaks at 941.2eV, 943.9eV, and 962.3eV are all Cu²⁺。

As can be seen from fig. 11A and 11B, the analysis result of the Cu2p orbital of the cuprous sulfide nano antibacterial agent in example 1 is: there was no characteristic peak around 942eV, indicating the absence of Cu²⁺Is present. The two strong peaks at 952.7eV and 933.0eV belong to Cu2p1/2 and Cu2p3/2, respectively. Due to Cu⁰And Cu⁺The binding energies of the copper and the copper oxidation state are further confirmed by Auger spectroscopy. As shown in FIG. 11B, the Cu (LMM) peak at 570.9eV indicates that the CuNPs-G, cuprous sulfide nano-antimicrobial agent is composed of Cu⁺The components are as follows.

As can be seen from the analysis of fig. 1 and 2, the average particle size of the cuprous sulfide nano antibacterial agent in example 1 was about 2nm, and as can be seen from fig. 12, the average particle size of the copper nanoparticles in mixture 1 in example 1 was about 5nm, thus also demonstrating that the etching of GSH can reduce the particle size of the copper nanoparticles. The particle size of the cuprous sulfide nano antibacterial agent is further reduced relative to the copper nano particles, and the inventor knows through experiments that: the smaller the particle size of the nano copper particles is, the stronger the antibacterial performance of the nano copper particles is, so that the antibacterial performance of the cuprous sulfide nano antibacterial agent is further ensured.

In addition, the inventor finds that the cuprous sulfide nano antibacterial agent prepared by the invention can directly catalyze the degradation of methylene blue and can also directly catalyze PTA to generate fluorescence on the premise of not adding hydrogen peroxide, because the cuprous sulfide nano antibacterial agent mainly contains positive cuprous which can catalyze dissolved oxygen or a small amount of hydrogen peroxide to generate active oxygen (ROS), and because of the strong oxidizing property and high toxicity of ROS, oxidized protein and DNA can be damaged to cause the apoptosis of bacteria. Experimental results prove that the cuprous sulfide nano particles prepared by the method have good antibacterial effect on staphylococcus aureus and escherichia coli.

Meanwhile, the cuprous sulfide nano antibacterial agent has uniform size and high dispersibility, and the size of particles can be adjusted by the size of copper nanoparticles.

Finally, the raw materials and experimental equipment required by the preparation method are simple, the cost is low, the application prospect is wide, and the popularization of the cuprous sulfide nano antibacterial agent is easy.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a transmission electron microscope image of the cuprous sulfide nano-antimicrobial prepared in example 1;

FIG. 2 is a graph showing the particle size distribution of the cuprous sulfide nano-antimicrobial agent prepared in example 1;

FIG. 3 is a Zeta potential diagram of the cuprous sulfide nano-antimicrobial prepared in example 1;

FIG. 4 is an infrared image of the cuprous sulfide nano-antimicrobial prepared in example 1;

FIG. 5A is a photograph of the ultraviolet light at the time of degrading methylene blue in test example 2;

FIG. 5B is a graph of the time to degrade methylene blue with the addition of cuprous sulfide nano-antimicrobial and hydrogen peroxide;

FIG. 6 is a fluorescence diagram during oxidation of PTA in detection example 2;

FIG. 7A is a visual chart of the minimum inhibitory concentration of the cuprous sulfide nano-antibacterial agent in application example 1 to Staphylococcus aureus;

FIG. 7B is a visual chart of the minimum inhibitory concentration of the cuprous sulfide nano-antibacterial agent in application example 1 to Escherichia coli;

FIG. 8 is a photograph of the zone of inhibition after the cuprous sulfide nano-antimicrobial of application example 1 has reacted with Staphylococcus aureus and Escherichia coli, respectively;

FIG. 9A is a graph showing the experimental results of the growth curve measured by the enzyme-linked immunosorbent assay of the cuprous sulfide nano antibacterial agent in application example 3 on Staphylococcus aureus;

FIG. 9B is a graph showing the experimental results of the growth curve of the cuprous sulfide nano-antibacterial agent in application example 3 measured by an enzyme-linked immunosorbent assay on Escherichia coli;

FIG. 10A is a graph showing the X-ray photoelectron spectroscopy (XPS) characteristics of the copper nanoparticles in mixture 1 of example 1 with respect to the Cu element at 925-950 eV;

FIG. 10B is a graph showing the X-ray photoelectron spectroscopy (XPS) characteristics of the copper nanoparticles in the mixture 1 in example 1 with respect to the Cu element at 950-965 eV;

FIG. 11A is a graph of the characterization of X-ray photoelectron spectroscopy (XPS) of the cuprous sulfide nano antibacterial agent of example 1 with respect to Cu element at 925-965 eV;

FIG. 11B is a graph showing the Auger spectrum of the cuprous sulfide nano antibacterial agent in example 1 with respect to Cu element at 560-580 eV;

FIG. 12 is a transmission electron micrograph of copper nanoparticles in mixture 1 of example 1.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The invention discloses a preparation method of a cuprous sulfide nano antibacterial agent, which comprises the following steps: carrying out a first contact reaction on copper nanoparticles and an etching agent to prepare the cuprous sulfide nano antibacterial agent; the average particle size of the copper nanoparticles is 2-6nm, the valence state of copper element in the copper nanoparticles contains positive bivalence, and the etching agent is selected from at least one of reduced glutathione, cysteine and serine.

In the present invention, the amount of the copper nanoparticles and the etchant is not particularly limited, but in order to make the cuprous sulfide nano antimicrobial agent have more excellent antimicrobial property, the weight ratio of the copper nanoparticles to the etchant is preferably 1:3 to 1: 12.

In the present invention, the conditions of the first contact reaction are not particularly limited, but in order to allow the cuprous sulfide nano antibacterial agent to have a higher yield, it is preferable that the first contact reaction at least satisfies the following conditions: the reaction temperature is 15-35 ℃, and the reaction time is 0.5-1.5 h.

In the present invention, in order to obtain a higher yield of the cuprous sulfide nano antibacterial agent, it is preferable that the first contact reaction is performed in a solvent, such as an aqueous solution, and the solvent can be added separately, or the reactants can be dispersed in the solvent and then mixed.

In the present invention, the copper nanoparticles may be commercially available or may be prepared by themselves, and in order to effectively adjust the particle size of the copper nanoparticles, the preparation method preferably further comprises: carrying out a second contact reaction on a copper source and a reducing agent under neutral or alkaline conditions; wherein the reducing agent is at least one selected from the group consisting of ascorbic acid AA, L-cysteine and hydrazine hydrate.

In the above embodiment, the kind of the copper source is not particularly limited as long as the divalent and cationic copper ions are present in the solution after dissolving in water, but it is preferable that the copper source is selected from CuCl in view of difficulty in dispersion₂、Cu(NO₃)₂And CuSO₄At least one of (1).

In the above embodiment, the amount of the copper source and the reducing agent is not particularly limited, and in order to reduce the copper source as much as possible, the molar ratio of the copper source to the reducing agent is preferably 3:1 to 6: 1.

In the above embodiment, the conditions of the second contact reaction may also be selected within a wide range, but in order to further improve the yield and secure the particle size of the copper nanoparticles, it is preferable that the second contact reaction at least satisfies the following conditions: the pH value is 7-9, the reaction temperature is 15-35 ℃, and the reaction time is 20-40 min; wherein, the particle size of the copper nanoparticles can be effectively controlled by controlling the pH value, and when the pH value is 7, the average particle size of the obtained copper nanoparticles is 5-6 nm; when the pH value is 8, the average particle size of the obtained copper nanoparticles is 3-4 nm; when the pH is 9, the average particle diameter of the obtained copper nanoparticles is about 2 nm.

In the present invention, the manner of adjusting the pH is also not particularly limited, but for convenience of operation, it is preferable that the pH of the second contact reaction is adjusted by adding an alkaline agent; for example, the adjustment is carried out by using a 1mol/L NaOH solution.

In the above embodiment, in order to further improve the yield of the copper nanoparticles, it is preferable that the second contact reaction is performed in a solvent, such as an aqueous solution; similarly, the solvent may be added separately, or the reactants may be dispersed in the solvent and then mixed for reaction.

On the basis, in order to further control the yield and the particle size of the copper nanoparticles, preferably, the addition sequence of the materials in the preparation process of the copper nanoparticles is as follows: the reducing agent is dissolved in water, the pH value is adjusted, and then the reducing agent aqueous solution is slowly added into the copper source aqueous solution in a dropping mode through a syringe for reaction.

The particle size of the prepared cuprous sulfide nano antibacterial agent can be regulated and controlled by the method, and can be changed in a wide range, but the average particle size of the cuprous sulfide nano antibacterial agent is preferably 1-3nm in view of antibacterial effect.

The present invention will be described in detail below by way of examples. Reduced glutathione (reduced GSH) is commercially available from Aladdin. Staphylococcus aureus was purchased from Beijing microbial strain preservation center, and Escherichia coli was purchased from Beijing microbial strain preservation center. The LB solid medium is prepared by the laboratory (the preparation components mainly comprise sodium chloride, agar, peptone and yeast powder). The buffer solution is PBS (prepared by laboratory and mainly comprises sodium dihydrogen phosphate, potassium dihydrogen phosphate and sodium hydroxide).

Example 1

The method comprises the following steps: 0.023mmol (4mg) of ascorbic acid AA is weighed out and dissolved in 2mL of water to obtain ascorbic acid solution, 1mol/L NaOH solution is added to the ascorbic acid solution until the pH of the ascorbic acid solution is close to 14 (so that the pH of the finally obtained mixture 1 is 7), and then the ascorbic acid solution is slowly dropped into 3mL of CuCl in a syringe dropping manner₂Solution (concentration 4.2mg/mL, containing 0.093mmol of CuCl₂) And the mixture is stirred and reacted for 30min at 25 ℃ to prepare a mixture 1.

Step two: 0.7mL of the prepared mixture 1 (washed by centrifugation, dried and then weighed on an electronic balance to obtain 10.2mg of copper-containing nanoparticles) was added to 4mL of an aqueous solution of reduced GSH (50mmol/L, containing 61.4mg of reduced GSH), and stirred at 25 ℃ to gradually form a white precipitate with stirring for 1 hour. After the reaction is finished, the product is centrifugally washed, is centrifuged at 8000rpm for 10min, is washed for 3 times by using ethanol (75 vol%), is collected and precipitated, is frozen and dried to obtain the cuprous sulfide nano antibacterial agent, and is finally stored in a refrigerator at the temperature of-20 ℃ for long-term use.

Example 2

The procedure is as in example 1, except that in step one, the ascorbic acid solution is adjusted in pH by means of a 1mol/L NaOH solution until the ascorbic acid solution is close to pH 14 (so that the pH of the finally obtained mixture 1 is 8).

Example 3

The procedure is as in example 1, except that in step one, the ascorbic acid solution is adjusted in pH by means of a 1mol/L NaOH solution until the ascorbic acid solution is close to pH 14 (so that the pH of the finally obtained mixture 1 is 9).

Example 4

The procedure was as in example 1, except that in step two, the amount of aqueous reduced GSH solution (50mmol/L) was 2mL (containing 30.7mg of reduced GSH).

Example 5

The procedure was as in example 1, except that in step two, the amount of aqueous reduced GSH solution (50mmol/L) was 6mL (containing 92.1mg of reduced GSH).

Detection example 1

1) The cuprous sulfide nano antibacterial agent prepared in example 1 is characterized by a transmission electron microscope (Hitachi, Japan), and the characterization result is shown in figure 1, so that the cuprous sulfide nano antibacterial agent with the particle size of about 2nm is finally obtained.

2) The cuprous sulfide nanoantimicrobial agent prepared in example 1 was characterized by its particle size distribution by measuring the size distribution by Dynamic Light Scattering (DLS) (ALV/CGS-8F, Germany), and the characterization results are shown in fig. 2, from which it can be seen that the particles had an average particle size of about 2nm and were highly dispersed.

The products of examples 2 to 5 were characterized in the same manner as in 1) to 2), and the results showed that the average particle size of the product of example 2 was 3 to 4nm, the average particle size of the product of example 3 was about 2nm, the average particle size of the product of example 4 was 1 to 2nm, and the average particle size of the product of example 5 was about 1 nm.

3) Zeta potential characterization is carried out on the cuprous sulfide nano antibacterial agent prepared in the example 1 by a ZS90 type nano particle size potential analyzer, the characterization result is shown in figure 3, and the Zeta potential is known to be negative, which shows that the particle stability is good.

4) The cuprous sulfide nano-antibacterial agent (CuNCs) prepared in example 1 was subjected to infrared characterization by an infrared spectrometer (IR-211R-21), and the characterization result is shown in FIG. 4, which shows that the cuprous sulfide nano-antibacterial agent has been successfully synthesized through bonding of Cu-S bond.

5) Cu2p electronic XPS characterization of the cuprous sulfide nano antibacterial agent prepared in example 1 was performed by an X-ray photoelectron spectroscopy (Thermo Scientific K-Alpha), and the characterization results are shown in fig. 11A and 11B, and the valence of Cu element in the cuprous sulfide nano antibacterial agent is +1 through the graph analysis.

Detection example 2

1) Preparation of 10ug/ml Methylene Blue (MB), 50mmol/L Glutathione (GSH), 10mmol/L hydrogen peroxide (H)₂O₂) And (3) solution. To 2mL of buffer solution at different pH, 0.5mg of CuNPs (product of step one in example 1), 0.2mL of LGSH solution, 0.2mL of H were selectively added₂O₂At least one of the solution and 0.04mL of MB solution. After different time periods, the hydroxyl radical-guided degradation of methylene blue was measured by the change in absorbance, and the results are shown in fig. 5A and 5B; in FIG. 5A, Control refers to blank Control (water is used as blank Control), CuNPs refers to addition of CuNPs only, H₂O₂Means that only H is added₂O₂The solution CuNPs-G refers to the simultaneous addition of CuNPs and GSH solution, CuNPs-G + H₂O₂Means that CuNPs, GSH solution and H are added simultaneously₂O₂Solution, CuNPs + H₂O₂Refers to the simultaneous addition ofCuNPs and H₂O₂And (3) solution.

As shown in fig. 5A, CuNPs-G (i.e., the cuprous sulfide nano antimicrobial agent in example 1) can degrade methylene blue in the presence of hydrogen peroxide, and the degradation effect is good, and can degrade methylene blue in the absence of hydrogen peroxide, but the degradation effect is poor; as can be seen from FIG. 5B, CuNPs-G + H was added within 30min₂O₂A certain amount of methylene blue can be degraded.

2) For the oxidation of PTA, 5mmol/L PTA solution as PTA is added to 10mL of 2mmol/L NaOH solution, then 0.05mL of PTA solution is added to the buffer solution at different pH values, followed by 0.05mL of H₂O₂And 0.5mg of at least one of the cuprous sulfide nano antimicrobial (Cu-G) prepared in example 1. After 10min, the fluorescence intensity at 440nm under excitation at a wavelength of 312nm was measured, and the results are shown in FIG. 6, Cu-G + H₂O₂Means that Cu-G and H are added simultaneously₂O₂Solution, Control refers to the blank (i.e., water as the blank), Cu-G refers to the addition of Cu-G only, H₂O₂Means that only H is added₂O₂Solution, Control, H₂O₂And the pH of the buffer solution in the curve represented by Cu-G was 7.4.

As can be seen from fig. 6, the cuprous sulfide nano antimicrobial material can cause PTA to generate fluorescence by itself, and the fluorescence generated by catalyzing PTA in the presence of hydrogen peroxide is the highest, i.e., the catalytic activity is the best, in a slightly acidic environment with a pH of 5.0.

According to the detection, the cuprous sulfide nano antibacterial agent mainly comprises the monovalent copper which can catalyze and dissolve oxygen or a small amount of hydrogen peroxide and can generate Reactive Oxygen Species (ROS), so that the cuprous sulfide nano antibacterial agent has excellent catalytic activity; in the presence of hydrogen peroxide, the cuprous sulfide nano antibacterial agent can catalyze the hydrogen peroxide to generate more ROS, so that the catalytic performance of the cuprous sulfide nano antibacterial agent is further improved.

The products of examples 2-5 were examined in the same manner as in 1) -2), and the results showed that the examination results of the products of examples 2-5 were comparable to that of example 1.

Application example 1

Trace broth dilution culture experiment

1) The gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (escherichia coli) which are frozen and stored are taken out from the environment of minus 80 ℃, plate streaking is carried out on LB solid culture medium with agar concentration of 1.5 weight percent, and the streaked plate is cultured in a constant temperature incubator at 37 ℃ for 24 hours.

2) Two 5ml LB liquid culture medium, using inoculating loop to pick single colony from two plates, inoculating in liquid culture medium, culturing in constant temperature shaker at 37 deg.C until OD600 is about 0.8.

3) In the clean bench, the total reaction volume is 2mL, 20 μ L of the bacterial solution is diluted by an appropriate amount, and the drug (cuprous sulfide nano antibacterial agent in example 1) with the mother liquor concentration of 120 μ g/mL is diluted by a multiple ratio by the culture medium, and the control is the bacterial solution without the drug.

4) The test tubes were placed in a shaker, reacted at 37 ℃ and 180rpm for 24 hours, and finally shaken to homogenize the bacterial solution and photographed as shown in fig. 7A and 7B, wherein in fig. 7A, the test tubes are numbered as 0, 1, 2, 3, 4, 5, 6, 7 and 8 in sequence from left to right. Wherein, test tube 0 represents staphylococcus aureus which is not treated by medicament (namely cuprous sulfide nano antibacterial agent), test tube 1 represents reaction mixture formed by applying 60ug/mL of cuprous sulfide nano antibacterial agent to staphylococcus aureus, test tube 2 represents reaction mixture formed by applying 30ug/mL of cuprous sulfide nano antibacterial agent to staphylococcus aureus, test tube 3 represents reaction mixture formed by applying 15ug/mL of cuprous sulfide nano antibacterial agent to staphylococcus aureus, test tube 4 represents reaction mixture formed by applying 7.5ug/mL of cuprous sulfide nano antibacterial agent to staphylococcus aureus, test tube 5 represents reaction mixture formed by applying 3.75ug/mL of cuprous sulfide nano antibacterial agent to staphylococcus aureus, and test tube 6 represents reaction mixture formed by applying 1.88ug/mL of cuprous sulfide nano antibacterial agent to staphylococcus aureus Compound, test tube No. 7 represents the reaction mixture formed by 0.94ug/mL of the cuprous sulfide nanoantimicrobial agent acting on staphylococcus aureus, and test tube No. 8 represents the reaction mixture formed by 0.47ug/mL of the cuprous sulfide nanoantimicrobial agent acting on staphylococcus aureus. The final clarity of each tube is ordered as 1 >2 >3 >4 >5 >6 >7 >8 >0 (where each tube is yellow in color).

In fig. 7B, the test tubes are numbered 0, 1, 2, 3, 4, 5, 6, 7 and 8 in sequence from left to right. Wherein, test tube No. 0 represents Escherichia coli which is not treated with a drug (i.e. cuprous sulfide nano antibacterial agent), test tube No. 1 represents a reaction mixture formed by allowing 60ug/mL of cuprous sulfide nano antibacterial agent to act on Escherichia coli, test tube No. 2 represents a reaction mixture formed by allowing 30ug/mL of cuprous sulfide nano antibacterial agent to act on Escherichia coli, test tube No. 3 represents a reaction mixture formed by allowing 15ug/mL of cuprous sulfide nano antibacterial agent to act on Escherichia coli, test tube No. 4 represents a reaction mixture formed by allowing 7.5ug/mL of cuprous sulfide nano antibacterial agent to act on Escherichia coli, test tube No. 5 represents a reaction mixture formed by allowing 3.75ug/mL of cuprous sulfide nano antibacterial agent to act on Escherichia coli, test tube No. 6 represents a reaction mixture formed by allowing 1.88ug/mL of cuprous sulfide nano antibacterial agent to act on Escherichia coli, test tube 7 represents the reaction mixture formed by applying 0.94ug/mL of the cuprous sulfide nano antibacterial agent to Escherichia coli, and test tube 8 represents the reaction mixture formed by applying 0.47ug/mL of the cuprous sulfide nano antibacterial agent to Escherichia coli. The final clarity of each tube is ordered as 1 >2 >3 >4 >5 >6 >7 >8 >0 (where each tube is yellow in color).

The higher the clarity, the better the bacteriostatic effect. According to the clarity of the bacterial liquid, the cuprous sulfide nano antibacterial agent has an obvious antibacterial effect on staphylococcus aureus and escherichia coli.

Application example 2

Oxford cup test (bacteriostasis ring)

1) Two 5ml LB liquid culture medium, using inoculating loop to pick single colony from two plates, inoculating in liquid culture medium, culturing in constant temperature shaker at 37 deg.C until OD600 is about 0.8.

2) A mixed culture medium is adopted, namely bacterial liquid diluted by a proper amount is added into 100mL of warm solid culture medium and then mixed uniformly, the mixture is poured into 3 culture media rapidly, 4 Oxford cups are placed in each culture medium after cooling, and the culture media are tightly contacted by light pressure.

3) The drug substance (cuprous sulfide nano-antibacterial agent in example 1, mother liquor concentration of 120ug/mL) was diluted in sterile phosphate buffer (PBS, pH 7) at 3 concentrations in double. The first Oxford cup was used as a control without drug, and the remaining 3 were aspirated separately by 200 μ L of liquid, 3 replicates for each bacterium. After adding the medicine, the flat plate is placed in an ultra-clean workbench to blow for 10min so that the medicine is diffused, the flat plate cover is covered to be cultured in an incubator at 37 ℃, and the experimental result is observed after 24 h.

As shown in fig. 8, s.aureus represents staphylococcus aureus, 12mm represents the diameter of the zone of inhibition formed after 250ug/mL of the cuprous sulfide nano antibacterial agent reacts with staphylococcus aureus, 17mm represents the diameter of the zone of inhibition formed after 500ug/mL of the cuprous sulfide nano antibacterial agent reacts with staphylococcus aureus, and 26mm represents the diameter of the zone of inhibition formed after 1mg/mL of the cuprous sulfide nano antibacterial agent reacts with staphylococcus aureus; coli represents escherichia coli, Blank represents Blank control, 7mm represents the diameter of a bacteriostatic zone formed after 250ug/mL of cuprous sulfide nano antibacterial agent reacts with escherichia coli, 11mm represents the diameter of a bacteriostatic zone formed after 500ug/mL of cuprous sulfide nano antibacterial agent reacts with escherichia coli, and 16mm represents the diameter of a bacteriostatic zone formed after 1mg/mL of cuprous sulfide nano antibacterial agent reacts with escherichia coli.

Application example 3

Growth curve experiment

2) The drug (cuprous sulfide nano antibacterial agent in example 1) was diluted in multiple by adding the culture medium, the diluted drug solution was mixed with the bacterial solution diluted by an appropriate amount, and the mixture was added to a 96-well plate in a system of 200. mu.L for 6 parallel experiments at each concentration. Then, the 96-well plate is sealed with a sealing plate membrane, and the reaction is carried out in a shaker at 37 ℃ and 120rpm, and the data is read in a microplate reader every 2h for 24 h.

3) The obtained data are screened and integrated, and the growth curves of the bacteria obtained by mapping in Origin are shown in fig. 9A and fig. 9B, and the data result shows that the cuprous sulfide nano antibacterial agent has excellent antibacterial effect on staphylococcus aureus and escherichia coli within a certain concentration range (0.47-60 mu g/mL).

The products of examples 2 to 5 were examined in the same manner as in application examples 1 to 3, and the results showed that the examination results of the products of examples 2 to 5 were comparable to the examination result of the product of example 1.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. The preparation method of the cuprous sulfide nano antibacterial agent is characterized by comprising the following steps: carrying out a first contact reaction on copper nanoparticles and an etching agent to prepare the cuprous sulfide nano antibacterial agent; the average particle size of the copper nanoparticles is 2-6nm, the valence state of copper element in the copper nanoparticles contains positive bivalence, and the etching agent is selected from at least one of reduced glutathione, cysteine and serine.

2. The preparation method of claim 1, wherein the weight ratio of the copper nanoparticles to the etchant is 1:3-1: 12.

3. The production method according to claim 1, wherein the first contact reaction satisfies at least the following condition: the reaction temperature is 15-35 ℃, and the reaction time is 0.5-1.5 h;

preferably, the first contact reaction is carried out in a solvent.

4. The production method according to any one of claims 1 to 3, wherein the production method further comprises production of copper nanoparticles: carrying out a second contact reaction on a copper source and a reducing agent under neutral or alkaline conditions; wherein the reducing agent is at least one selected from the group consisting of ascorbic acid AA, L-cysteine and hydrazine hydrate.

5. The method of claim 4, wherein the copper source is selected from CuCl₂、Cu(NO₃)₂And CuSO₄At least one of (1).

6. The production method according to claim 4, wherein the molar ratio of the copper source to the reducing agent is 3:1 to 6: 1.

7. The production method according to claim 4, wherein the second contact reaction satisfies at least the following condition: the pH value is 7-9, the reaction temperature is 15-35 ℃, and the reaction time is 20-40 min;

preferably, the pH of the second contact reaction is adjusted by adding an alkaline agent;

preferably, the second contact reaction is carried out in a solvent.

8. A cuprous sulfide nano antibacterial agent characterized by being prepared by the preparation method of any one of claims 1 to 7.

9. A cuprous sulfide nano antimicrobial according to claim 8, wherein the average particle size of the cuprous sulfide nano antimicrobial is 1-3 nm.

10. Use of a cuprous sulfide nanoantimicrobial agent as claimed in claim 8 or 9 to catalyze dye degradation and inhibit bacteria.