CN113582216B

CN113582216B - Cuprous sulfide nano antibacterial agent and preparation method and application thereof

Info

Publication number: CN113582216B
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: 2023-12-19
Anticipated expiration: 2041-07-01
Also published as: CN113582216A

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 the copper nano particles and an etchant to prepare the cuprous sulfide nano antibacterial agent; wherein the average particle diameter of the copper nano particles is 2-6nm, the valence state of copper element in the copper nano particles contains positive bivalent, and the etchant is at least one selected from 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 nano particles, 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 even cause more and more deaths in developing countries and in less developed areas, and the need for antimicrobial materials is even more urgent. In recent years, bacterial infection has indeed been inhibited to some extent due to the widespread use of antibiotic drugs, but at the same time, another new problem has been derived, namely the emergence of many drug-resistant superbacteria. So-called superbacteria, which are more harmful. For example, the number of deaths caused by "superbacteria" in the united states can reach 18000 per year, exceeding 16000 deaths in 2005 in the united states due to aids; in the last two years, 313 people in France have developed VRE urinary tract or digestive tract infections, 3 of which die due to drug free treatment-! Therefore, the design and development of antibacterial materials superior to antibiotics has been elusive.

In recent years, various antibacterial agents have been developed. Such as photocatalytic antibacterial agents, metal oxide antibacterial agents, silver-based antibacterial agents, copper-based antibacterial materials, and the like. The copper antibacterial material has the most application prospect. There are many methods for preparing copper-based antibacterial agents, which are roughly classified into a solid phase method and a gas phase method. The solid phase method is to change particles with larger particle size into particles with smaller particle size through ball milling by a physical method, and the solid phase method can produce products in quantity, but the prepared particles with larger particle size and unavoidable introduction of impurities are produced, and the particle size distribution of the prepared nano particles is uneven; the gas phase method has high purity and narrow particle size distribution compared with the solid phase method, but has higher requirements on equipment.

Disclosure of Invention

The invention aims to provide a cuprous sulfide nano antibacterial agent, a preparation method and application thereof, wherein the cuprous sulfide nano antibacterial agent has excellent antibacterial performance and dye degradation performance, and the preparation method has the advantages of simple procedures and simplicity and convenience in operation, so that the antibacterial agent can be widely applied to the antibacterial field and the dye degradation field.

In order to achieve the above purpose, 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 the copper nano particles and an etchant to prepare the cuprous sulfide nano antibacterial agent; wherein the average particle diameter of the copper nano particles is 2-6nm, the valence state of copper element in the copper nano particles contains positive bivalent, and the etchant is at least one selected from 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 application of the cuprous sulfide nano-antibacterial agent in catalyzing dye degradation and inhibiting bacteria.

In the above technical scheme, whatThe valence state of copper element in the copper nano-particle contains positive monovalent and positive divalent, and the reduced GSH can make Cu in the copper nano-particle ²⁺ Continuing to reduce to Cu ⁺ Cu, i.e. Cu ²⁺ +GSH＝Cu ⁺ +gssh, thereby completing etching such that the copper nanoparticle particle size becomes small.

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

As can be seen from fig. 11A and 11B, the analysis result of the Cu2p orbit of the cuprous sulfide nano-antibacterial agent in example 1 is: there was no characteristic peak around 942eV, which also indicates no Cu ²⁺ Is present. Two strong peaks at 952.7eV and 933.0eV belong to Cu2p1/2 and Cu2p3/2, respectively. Due to Cu ⁰ And Cu ⁺ The oxidation state of copper was further confirmed by auger spectroscopy, which is very close to the binding energy of (c). As shown in FIG. 11B, the Cu (LMM) peak at 570.9eV indicates that the CuNPs-G, i.e., the cuprous sulfide nano-antibacterial agent is composed of Cu ⁺ Is composed of the components.

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 from fig. 12, the average particle size of the copper nano-particles in mixture 1 in example 1 was about 5nm, thereby also demonstrating that GSH etching can reduce the particle size of the copper nano-particles. 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 diameter 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 discovers that the cuprous sulfide nano-antibacterial agent can directly catalyze the degradation of methylene blue and also can directly catalyze PTA to generate fluorescence on the premise of not adding hydrogen peroxide, because the cuprous sulfide nano-antibacterial agent is mainly composed of positive monovalent copper, the positive monovalent copper can catalyze dissolved oxygen or a small amount of hydrogen peroxide to generate active oxygen (Reactive oxygen Species, ROS), and the strong oxidizing property and high toxicity of ROS can destroy oxidized proteins and DNA, so that bacteria apoptosis is caused. Experimental results prove that the cuprous sulfide nano-particles prepared by the method have good antibacterial effects 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 regulated by the size of copper nano-particles.

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

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

Drawings

The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:

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

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

FIG. 5A is an ultraviolet photograph of the degradation of methylene blue of test example 2;

FIG. 5B is a graph showing the time of degradation of methylene blue upon addition of cuprous sulfide nanoantimicrobial and hydrogen peroxide;

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

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

FIG. 7B is a visual graph of the minimum inhibitory concentration of the cuprous sulfide nano-antimicrobial agent against E.coli in application example 1;

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

FIG. 9A is a graph showing the results of an enzyme-labeled method for measuring a growth curve of a cuprous sulfide nano-antimicrobial agent against Staphylococcus aureus in application example 3;

FIG. 9B is a graph showing the experimental results of the measurement of the growth curve of the cuprous sulfide nano-antibacterial agent in application example 3 against the E.coli by the enzyme-labeled method;

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

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

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

FIG. 11B is a graph showing the Auger spectrum of the copper sulfide nano-antimicrobial agent of 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 specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

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 the copper nano particles and an etchant to prepare the cuprous sulfide nano antibacterial agent; wherein the average particle diameter of the copper nano particles is 2-6nm, the valence state of copper element in the copper nano particles contains positive bivalent, and the etchant is at least one selected from reduced glutathione, cysteine and serine.

In the present invention, the amounts of the copper nanoparticles and the etchant are not particularly limited, but in order to make the cuprous sulfide nano-antibacterial agent have more excellent antibacterial properties, it is preferable that the weight ratio of the copper nanoparticles to the etchant is 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 satisfies at least the following conditions: the reaction temperature is 15-35 ℃ and the reaction time is 0.5-1.5h.

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

In the present invention, the copper nanoparticle may be commercially available or may be prepared by itself, and in order to effectively adjust the particle size of the copper nanoparticle, the preparation method preferably further includes the preparation of the copper nanoparticle: 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 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 solution contains positive divalent copper ions after being dissolved in water, but is preferably selected from CuCl from the viewpoint of difficulty in dispersion ₂ 、Cu(NO ₃ ) ₂ And CuSO ₄ At least one of them.

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 ensure the particle size of the copper nanoparticles, it is preferable that the second contact reaction satisfies at least the following conditions: the pH is 7-9, the reaction temperature is 15-35 ℃, and the reaction time is 20-40min; the particle size of the copper nano particles can be effectively controlled by controlling the pH value, and when the pH value is 7, the average particle size of the obtained copper nano particles is 5-6nm; when the pH is 8, the average particle size of the obtained copper nano particles is 3-4nm; 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 not particularly limited, but for convenience of operation, preferably, the pH of the second contact reaction is adjusted by adding an alkaline agent; such as with 1mol/L NaOH solution.

In the above embodiment, in order to further increase the yield of the copper nanoparticles, it is preferable that the second contact reaction is performed in a solvent, such as a reaction in 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 this basis, in order to further control the yield and the particle size of the copper nanoparticles, it is preferable that the addition sequence of each material in the preparation process of the copper nanoparticles is as follows: the reducing agent was dissolved in water and the pH was adjusted, followed by slowly adding the aqueous reducing agent solution to the aqueous copper source solution in a syringe dropwise manner to effect the reaction.

The particle size of the prepared cuprous sulfide nano-antibacterial agent can be controlled by the above method, and can vary within a wide range, but from the aspect of antibacterial effect, it is preferable that the average particle size of the cuprous sulfide nano-antibacterial agent is 1-3nm.

The invention will be described in detail below by way of examples. Reduced glutathione (reduced GSH) is a commercially available product from the company aladine. Staphylococcus aureus is purchased from the Beijing microorganism strain preservation center, and escherichia coli is purchased from the Beijing microorganism strain preservation center. LB solid medium is prepared by the self-operation of laboratory (the configuration components mainly comprise sodium chloride, agar, peptone and yeast powder). The buffer solution is PBS (prepared by the self of a laboratory, and the configuration components mainly comprise sodium dihydrogen phosphate, potassium dihydrogen phosphate and sodium hydroxide).

Example 1

Step one: 0.023mmol (4 mg) of ascorbic acid AA was weighed and dissolved in 2mL of water to obtain an ascorbic acid solution, 1mol/L NaOH solution was added to the ascorbic acid solution until the pH of the ascorbic acid solution was close to 14 (so that the pH of the finally obtained mixture 1 was 7), and then the ascorbic acid solution was slowly dropped into 3mL of CuCl by syringe dropwise addition ₂ Solution (concentration 4.2mg/mL, containing 0.093mmol CuCl) ₂ ) In the above, the mixture was stirred at 25℃for 30 minutes to prepare a mixture 1.

Step two: 0.7mL of the prepared mixture 1 (10.2 mg of copper-containing nanoparticles were weighed on an electronic balance after drying by centrifugal washing) was added to 4mL of a reduced GSH aqueous solution (50 mmol/L, containing 61.4mg of reduced GSH) and stirred at 25℃and white precipitate appeared slowly with stirring for a total of 1h. After the reaction, it was centrifugally washed, centrifuged at 8000rpm for 10min, then washed with ethanol (75 vol%) for 3 times, the precipitate was collected, and then freeze-dried to obtain a cuprous sulfide nano-antibacterial agent, which was finally stored in a-20 ℃ refrigerator for long-term use.

Example 2

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

Example 3

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

Example 4

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

Example 5

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

Detection example 1

1) The cuprous sulfide nano-antibacterial agent obtained in example 1 was subjected to transmission electron microscope characterization by means of HT-7700 transmission electron microscope (Hitachi, japan), and the characterization result is shown in FIG. 1, from which it is clear that the cuprous sulfide nano-antibacterial agent with a particle size of about 2nm was finally obtained.

2) The cuprous sulfide nano-antimicrobial agent prepared in example 1 was characterized by Dynamic Light Scattering (DLS) measurement of size distribution (ALV/CGS-8 f, germany), and the characterization result is shown in fig. 2, from which it is known that the average particle size of the particles is about 2nm and is highly dispersed.

Characterization of the products of examples 2-5 by the same method as in 1) -2) showed that the average particle size of the product of example 2 was 3-4nm, the average particle size of the product of example 3 was about 2nm, the average particle size of the product of example 4 was 1-2nm, and the average particle size of the product of example 5 was about 1 nm.

3) The Zeta potential characterization of the cuprous sulfide nano-antibacterial agent prepared in the example 1 is carried out by a ZS90 nano-particle size potential analyzer, the characterization result is shown in figure 3, and the figure shows that the Zeta potential is negative, thus indicating that the particle stability is good.

4) The cuprous sulfide nanoantimicrobial (CuNCs) prepared in example 1 was infrared characterized by infrared spectrometer (IR-211R-21), and the characterization result is shown in fig. 4, from which it is known that the bonding of the cuprous sulfide nanoantimicrobial through Cu-S bond has been successfully synthesized.

5) The copper sulfide nano-antibacterial agent prepared in the example 1 is subjected to Cu2p electron XPS characterization by an X-ray photoelectron spectrometer (Thermo Scientific K-Alpha), the characterization results are shown in fig. 11A and 11B, and the chemical valence of Cu element in the copper sulfide nano-antibacterial agent is +1 by graph analysis.

Detection example 2

1) Preparation of 10ug/ml of Methylene Blue (MB) solution, 50mmol/L of Glutathione (GSH) solution, 10mmol/L of hydrogen peroxide water (H) ₂ O ₂ ) A solution. To a buffer solution of different pH 2mL, 0.5mg of CuNPs (the product of step one in example 1), 0.2mL of LGSH solution, 0.2mL of H were optionally added ₂ O ₂ At least one of the solution and 0.04mL of MB solution. After various time periods, the hydroxyl radical-mediated degradation of methylene blue was measured by the change in absorbance, as shown in fig. 5A and 5B; in FIG. 5A, control refers to a blank (with water as the blank), and CuNPs refers to the addition of only CuNPs, H ₂ O ₂ Refers to adding only H ₂ O ₂ The solution, cuNPs-G, refers to the simultaneous addition of CuNPs and GSH solution, cuNPs-G+H ₂ O ₂ Refers to the simultaneous addition of CuNPs, GSH solution and H ₂ O ₂ Solution, cuNPs+H ₂ O ₂ Refers to the simultaneous addition of CuNPs and H ₂ O ₂ A solution.

As can be seen from 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 has good degradation effect, and can degrade methylene blue in the absence of hydrogen peroxide, but has poor degradation effect; as can be seen in FIG. 5B, cuNPs-G+H was added within 30min ₂ O ₂ A certain amount of methylene blue can be degraded.

2) For oxidation of terephthalic acid PTA, 5mmol/L PTA solution of PTA finger was added to 10mL of 2mmol/L NaOH solution, then 0.05mL of PTA solution was added to buffer solutions of different pH values, followed by 0.05mL of H ₂ O ₂ And 0.5mg of at least one of the cuprous sulfide nano-antibacterial agents (Cu-G) prepared in example 1. After 10min, the fluorescence intensity at 440nm under excitation at 312nm wavelength was measured, and the result is shown in FIG. 6, cu-G+H ₂ O ₂ Refers to the simultaneous addition of Cu-G and H ₂ O ₂ Solution, control refers to the blank (i.e., water as the blank), cu-G refers to the addition of Cu-G, H alone ₂ O ₂ Refers to adding only H ₂ O ₂ Solution, control, H ₂ O ₂ And 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 itself can cause PTA to generate fluorescence, and the fluorescence generated by catalyzing PTA in the presence of hydrogen peroxide is highest, i.e., the catalytic activity is best, in a slightly acidic environment with ph=5.0.

According to the detection, the cuprous sulfide nano-antibacterial agent is mainly positive monovalent copper, and the positive monovalent copper can catalyze dissolved 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 tested in the same manner as in 1) -2), and the results showed that the products of examples 2-5 were tested in a comparable manner to the products of example 1.

Application example 1

Trace broth dilution culture experiment

1) The frozen gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (escherichia coli) were removed from the-80 ℃ environment, streaked on LB solid medium with an agar concentration of 1.5 wt%, and the streaked plates were cultured in a constant temperature incubator at 37 ℃ for 24 hours.

2) Two 5ml LB liquid culture media are taken, single colonies are respectively picked from the two plates by an inoculating loop and inoculated into the liquid culture media, and the liquid culture media are cultured in a constant temperature shaking table at 37 ℃ until OD600 apprxeq 0.8.

3) In an ultra clean bench, the total reaction system was 2mL, 20. Mu.L of bacterial liquid was diluted in an appropriate multiple, and the drug (cuprous sulfide nano-antimicrobial agent in example 1) with a mother liquor concentration of 120. Mu.g/mL was diluted in a multiple ratio with a culture medium, and the control was bacterial liquid without drug.

4) Placing the test tubes in a shaking table, reacting at 37 ℃ and 180rpm for 24 hours, and finally shaking the bacterial liquid to take pictures, wherein in the figure 7A, the test tubes are numbered 0, 1, 2, 3, 4, 5, 6, 7 and 8 from left to right in sequence as shown in the figures 7A and 7B. Wherein, the test tube No. 0 represents a staphylococcus aureus which is not treated with a medicine (i.e., cuprous sulfide nano-antimicrobial agent), the test tube No. 1 represents a reaction mixture formed by applying 60ug/mL of cuprous sulfide nano-antimicrobial agent to a staphylococcus aureus, the test tube No. 2 represents a reaction mixture formed by applying 30ug/mL of cuprous sulfide nano-antimicrobial agent to a staphylococcus aureus, the test tube No. 3 represents a reaction mixture formed by applying 15ug/mL of cuprous sulfide nano-antimicrobial agent to a staphylococcus aureus, the test tube No. 4 represents a reaction mixture formed by applying 7.5ug/mL of cuprous sulfide nano-antimicrobial agent to a staphylococcus aureus, the test tube No. 5 represents a reaction mixture formed by applying 3.75ug/mL of cuprous sulfide nano-antimicrobial agent to a staphylococcus aureus, the test tube No. 6 represents a reaction mixture formed by applying 1.88ug/mL of cuprous sulfide nano-antimicrobial agent to a staphylococcus aureus, the test tube No. 7 represents a reaction mixture formed by applying 0.94ug/mL of cuprous sulfide nano-antimicrobial agent to a staphylococcus aureus, and the test tube No. 8 represents a reaction mixture formed by applying 0.47ug/mL of cuprous sulfide nano-antimicrobial agent to a staphylococcus aureus. The final clarity of each tube was ordered as No. 1 >2 >3 >4 >5 >6 >7 >8 >0, (wherein each tube was yellow in color).

In fig. 7B, the test tubes are numbered 0, 1, 2, 3, 4, 5, 6, 7, and 8 in this order from left to right. Wherein, the test tube No. 0 represents the E.coli which is not treated with the drug (i.e., cuprous sulfide nano-antibacterial agent), the test tube No. 1 represents the reaction mixture formed by allowing 60ug/mL of cuprous sulfide nano-antibacterial agent to act on the E.coli, the test tube No. 2 represents the reaction mixture formed by allowing 30ug/mL of cuprous sulfide nano-antibacterial agent to act on the E.coli, the test tube No. 3 represents the reaction mixture formed by allowing 15ug/mL of cuprous sulfide nano-antibacterial agent to act on the E.coli, the test tube No. 4 represents the reaction mixture formed by allowing 7.5ug/mL of cuprous sulfide nano-antibacterial agent to act on the E.coli, the test tube No. 5 represents the reaction mixture formed by allowing 3.75ug/mL of cuprous sulfide nano-antibacterial agent to act on the E.coli, the test tube No. 6 represents the reaction mixture formed by allowing 1.88ug/mL of cuprous sulfide nano-antibacterial agent to act on the E.coli, the test tube No. 7 represents the reaction mixture formed by allowing 0.94ug/mL of cuprous sulfide nano-antibacterial agent to act on the E.coli, and the test tube No. 8 represents the reaction mixture formed by allowing 0.47ug/mL of cuprous sulfide nano-antibacterial agent to act on the E.g. The final clarity of each tube was ordered as No. 1 >2 >3 >4 >5 >6 >7 >8 >0, (wherein each tube was 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 obvious antibacterial effect on staphylococcus aureus and escherichia coli.

Application example 2

Oxford cup test (antibacterial circle)

1) Two 5ml LB liquid culture media are taken, single colonies are respectively picked from the two plates by an inoculating loop and inoculated into the liquid culture media, and the liquid culture media are cultured in a constant temperature shaking table at 37 ℃ until OD600 apprxeq 0.8.

2) The mixed culture medium is adopted, namely, bacterial liquid with proper dilution multiple is added into 100mL of warm solid culture medium, then mixed evenly, poured into 3 culture mediums rapidly, and after being cooled, 4 oxford cups are placed respectively, and the contact is tight by light pressure.

3) The drug (cuprous sulfide nanoantimicrobial in example 1, mother liquor concentration 120 ug/mL) was diluted 3-fold with sterile phosphate buffer (PBS, ph=7). The first oxford cup was used as a non-dosing control, and the remaining 3 were each aspirated with 200 μl of the drug solution, 3 replicates per strain. After the medicines are added, the flat plate is placed in an ultra-clean workbench and blown for 10min to diffuse the medicines, the flat plate is covered on the flat plate and is cultured in a 37 ℃ incubator, and the experimental result is observed after 24 hours.

As shown in fig. 8, wherein s.aureus represents staphylococcus aureus, 12mm represents the diameter of a bacteriostasis zone formed after 250ug/mL of cuprous sulfide nano-antibacterial agent reacts with staphylococcus aureus, 17mm represents the diameter of a bacteriostasis zone formed after 500ug/mL of cuprous sulfide nano-antibacterial agent reacts with staphylococcus aureus, and 26mm represents the diameter of a bacteriostasis zone formed after 1mg/mL of cuprous sulfide nano-antibacterial agent reacts with staphylococcus aureus; e.coli represents Escherichia coli, blank represents Blank control, 7mm represents the diameter of a bacteriostasis zone formed after 250ug/mL of cuprous sulfide nano-antibacterial agent acts on Escherichia coli, 11mm represents the diameter of a bacteriostasis zone formed after 500ug/mL of cuprous sulfide nano-antibacterial agent acts on Escherichia coli, 16mm represents the diameter of a bacteriostasis zone formed after 1mg/mL of cuprous sulfide nano-antibacterial agent acts on Escherichia coli, and therefore, the effect of medicines with different concentrations on two bacteria is obvious, the concentration of the medicines is increased, and the diameter of the bacteriostasis zone is obviously increased.

Application example 3

Growth curve experiment

2) The medicine (cuprous sulfide nano-antibacterial agent in example 1) is diluted by the addition of the culture medium in a multiple ratio, the diluted medicine liquid and the bacterial liquid with proper multiple of dilution are uniformly mixed, and then are added into a 96-well plate, 200 mu L of the system is adopted, and 6 groups of parallel experiments are carried out on each concentration. The 96-well plates were then sealed with plate membranes and reacted in a shaker at 37℃and 120rpm, reading data in a microplate reader every 2h, and reacting for 24h.

3) The obtained data are screened and integrated, and the growth curves of bacteria are plotted in an Origin, as shown in fig. 9A and 9B, and the data result shows that the cuprous sulfide nano-antibacterial agent has excellent antibacterial effect on staphylococcus aureus and escherichia coli in a certain concentration range (0.47-60 mug/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 results of the product of example 1.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

1. A preparation method of a cuprous sulfide nano antibacterial agent, which is characterized by comprising the following steps: carrying out a first contact reaction on the copper nano particles and an etchant to prepare the cuprous sulfide nano antibacterial agent; wherein the weight ratio of the copper nano particles to the etchant is 1:3-1:12, the average particle diameter of the copper nano particles is 2-6nm, the valence state of copper element in the copper nano particles contains positive monovalent and positive divalent, and the etchant is at least one of reduced glutathione and cysteine; the first contact reaction satisfies at least the following conditions: the reaction temperature is 15-35 ℃ and the reaction time is 0.5-1.5h;

the first contact reaction is carried out in a solvent;

the preparation method further comprises the preparation of copper nano particles: carrying out a second contact reaction on a copper source and a reducing agent under neutral or alkaline conditions; wherein the copper source is selected from、/>And->The reducing agent is selected from at least one of ascorbic acid AA, L-cysteine and hydrazine hydrate, and the molar ratio of the copper source to the reducing agent is 3:1-6:1;

the second contact reaction satisfies at least the following conditions: the pH is 7-9, the reaction temperature is 15-35 ℃, and the reaction time is 20-40min;

the pH of the second contact reaction is adjusted by adding an alkaline reagent;

the second contact reaction is carried out in a solvent.

2. A cuprous sulfide nano-antibacterial agent prepared by the preparation method of claim 1.

3. A cuprous sulfide nano antibacterial agent as claimed in claim 2 wherein the average particle diameter of the cuprous sulfide nano antibacterial agent is 1-3nm.

4. Use of a cuprous sulfide nano-antimicrobial agent as claimed in claim 2 or 3 for catalyzing dye degradation and for inhibiting bacteria.