CN111217401A - Copper-cobalt-sulfur nano enzyme material, preparation method and antibacterial application thereof - Google Patents
Copper-cobalt-sulfur nano enzyme material, preparation method and antibacterial application thereof Download PDFInfo
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Abstract
The invention discloses a copper-cobalt-sulfur nano enzyme material, a preparation method and an antibacterial application thereof, wherein the preparation method of the nano enzyme material comprises the following steps: (1) fully dissolving biocompatible macromolecules into a solvent to obtain a uniform solution; (2) under the condition of stirring, adding CuCl2.2H2O、CoCl2.6H2O and thiourea are added toAdding ethylenediamine into the solution obtained in the step (1), and stirring to fully mix the ethylenediamine into a clear solution; (3) and (3) taking the clear solution obtained in the step (2), reacting at the temperature of 160-180 ℃, cooling, centrifuging, washing and drying to obtain the copper-cobalt-sulfur nano enzyme material. The copper-cobalt-sulfur nano enzyme obtained by the invention has activity of simulating peroxidase, and can catalyze hydrogen peroxide to generate hydroxyl radicals under the condition of physiological pH. The preparation method of the copper-cobalt-sulfur nano enzyme is simple and easy to implement, has good dispersibility and good antibacterial performance, and can be applied to treatment of bacterial infection.
Description
Technical Field
The invention belongs to the technical field of nano enzyme, and particularly relates to a copper-cobalt-sulfur nano enzyme material, a preparation method and an antibacterial application thereof.
Background
Bacterial infections are a serious health hazard to humans, causing disease and death in millions of people each year, while bacterial resistance associated with biofilms often leads to more serious persistent infectious diseases. The increasing problem of antibiotic resistance forces the search for new antibacterial strategies. With the development of nanotechnology, people find that inorganic nano materials have the advantages of no drug resistance, broad-spectrum antibacterial property, good stability and the like in the antibacterial aspect. The mechanism widely studied at present for inorganic nanomaterial antibacterial is to affect the function of bacteria by generating Reactive Oxygen Species (ROS) or inducing oxidative stress, thereby destroying structures such as lipid, protein and DNA of the bacteria, and causing bacterial death.
Nanoenzymes refer to a class of nanomaterials that have catalytic activity similar to that of natural enzymes. The reported enzyme-like activities of nanomaterials include oxidases, peroxidases, hydrogen peroxide, superoxide dismutase, and the like. Compared with natural enzymes, the nano-enzyme has a series of advantages of simple preparation, low cost, stable property, easy preservation, ecological friendliness and the like. More than 50 kinds of nano materials are reported to have enzyme-like activity, such as gold nano, Fe3O4，V2O5Graphene, and the like. Nanoenzymes with peroxidase activity are used in the antibacterial field because they catalyze the production of active oxygen. For example, Fe3O4 nanoenzyme with peroxidase activity can catalyze hydrogen peroxide to generate high-activity hydroxyl radicals, thereby effectively killing bacteria and inhibiting the growth of a biological membrane. Graphitic nano-particles, V, having similar enzymatic activities2O5And MoS2Etc. are also used for antibacterial. However, most of nano-enzymes have the optimal activity, the pH value is 3-5, and the catalytic activity is low under the condition of neutral physiological pH, so that the application in the biomedical field is limited. Therefore, there is a need to develop a nano-enzyme antibacterial material that can exert optimal activity under physiological pH conditions.
Copper-based nanomaterials, including copper oxide and copper sulphide, are extensively developed for use in the antibacterial field, where the antibacterial mechanisms include the release of copper ions and the induction of oxidative stress. While the use of catalytic properties of copper nanoparticles for antimicrobial applications has largely been overlooked.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the technical problems, the invention provides a copper-cobalt-sulfur nano enzyme material, a preparation method and an antibacterial application thereof. The invention synthesizes the copper-cobalt-sulfur nano enzyme protected by biocompatible macromolecules, and utilizes the high-efficiency peroxidase activity of the copper-cobalt-sulfur nano enzyme under the physiological pH condition to carry out antibiosis and inhibit the growth of a biological film.
The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a preparation method of a copper-cobalt-sulfur nano enzyme material comprises the following steps:
(1) fully dissolving biocompatible macromolecules into a solvent to obtain a uniform solution;
(2) under the condition of stirring, adding CuCl2.2H2O、CoCl2.6H2Adding O and thiourea into the solution obtained in the step (1), then adding ethylenediamine, and stirring to fully mix into a clear solution;
(3) and (3) taking the clear solution obtained in the step (2), reacting at the temperature of 160-180 ℃, cooling, centrifuging, washing and drying to obtain the copper-cobalt-sulfur nano enzyme material.
Preferably, in step (1), the biocompatible macromolecule is selected from dextran, polyvinylpyrrolidone, polyethylene glycol or bovine serum albumin; preferably dextran, having a molecular weight of one of 50 ten thousand, 7 ten thousand and 1 ten thousand.
Preferably, in step (1), the solvent is selected from water or ethylene glycol, and the concentration of the biocompatible macromolecule in the obtained homogeneous solution is 15-18 mg/mL.
Preferably, in the step (2), the reaction of CuCl2.2H2O、CoCl2.6H2Adding O and thiourea into the solution obtained in the step (1), wherein the CuCl2.2H2O、CoCl2.6H2The mol ratio of O to thiourea is 1:2 (4-12), and CuCl2.2H2O、CoCl2.6H2The total concentration of O and thiourea is 1.75-3.75 mol/L.
Preferably, in the step (2), after the ethylenediamine is added, the concentration of the ethylenediamine is 0.3-0.45%.
Preferably, in the step (3), the reaction time at 160-180 ℃ is 6-24 hours.
The copper-cobalt-sulfur nano enzyme material prepared by the preparation method.
The copper-cobalt-sulfur nano enzyme material is applied as an antibacterial agent.
The copper-cobalt-sulfur nano enzyme material is applied to the preparation of medicaments for treating scald infection.
The pH range of the copper nano material in the Fenton reaction is wide, so that the copper nano material is suitable for catalyzing and generating active oxygen to be applied to the field of biomedical antibiosis. In addition, the metal sulfide has higher conductivity than metal oxide, and has better catalytic activity. And the bimetal nanometer has richer oxidation-reduction property than the single metal nanometer, and is more beneficial to catalyzing to generate active oxygen for antibiosis.
Has the advantages that: compared with the prior art, the invention obtains the biocompatible macromolecule-protected copper-cobalt-sulfur nano-particles by a simple and easy solvothermal method, and the biocompatible macromolecule-protected copper-cobalt-sulfur nano-particles have good dispersibility in water, stable properties and good biocompatibility. The composition of the bimetallic sulfide enables the copper-cobalt-sulfur nano material to have excellent peroxidase catalytic activity, and compared with single-metal copper sulfide and cobalt sulfide, the copper-cobalt-sulfur nano material can catalyze hydrogen peroxide to generate hydroxyl radicals with strong oxidizing property more efficiently under neutral physiological conditions, so that the copper-cobalt-sulfur nano material has a remarkable antibacterial effect, can effectively inhibit the growth of a biological membrane, and has an application prospect in the field of bacterial infection as a novel antibacterial material.
Drawings
FIG. 1 is a scanning electron microscope image of copper cobalt sulfur nanoenzyme according to example 1 of the present application;
FIG. 2 is a transmission electron microscope image of copper cobalt sulfide nanoenzyme according to example 1 of the present application;
FIG. 3 is an X-ray diffraction pattern of a copper cobalt sulfide nanoenzyme according to example 1 of the present application;
FIG. 4 is a schematic diagram showing peroxidase activity of copper cobalt sulfide nanoenzyme according to example 1 of the present application;
FIG. 5 shows the paramagnetic resonance spectrum of the copper cobalt sulfide nanoenzyme according to example 1 of the present application;
FIG. 6 shows the results of Escherichia coli resistance by the Cu-Co-S nanoenzyme according to example 1 of the present application;
FIG. 7 shows the results of the Cu-Co-S nanoenzyme against Staphylococcus aureus in example 1 of the present application;
FIG. 8 shows the MRSA resistance results of copper cobalt sulfide nanoenzyme according to example 1 of the present application;
FIG. 9 is a scanning electron microscope image of the copper cobalt sulfide nanoenzyme inhibiting the growth of MRSA biofilm according to example 1 of the present application;
FIG. 10 shows the results of treating scald infection in mice with CuCoS nanoenzyme according to example 1 of the present application.
Detailed Description
The technical solution of the present invention is further described in detail by the following specific examples.
Example 1 preparation of copper cobalt sulphur nanoenzyme:
0.5g of dextran (molecular weight: 50 ten thousand) was dissolved in 30mL of ultrapure water, and the solution was magnetically stirred for 30 minutes to dissolve the dextran sufficiently. Then 0.25mmol of CuCl was added2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 100 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing the reaction product for three times by using water and ethanol respectively, and performing freeze vacuum drying to finally obtain 150mg of black powder of the copper-cobalt-sulfur nano-particles, wherein the yield is 54.4%. XRD analysis shows that the obtained product is copper cobalt sulfur (CuCo)2S4) (see FIG. 3), the size distribution was uniform, and the average particle diameter was 30nm (see FIGS. 1 and 2).
Example 2 preparation of copper cobalt sulphur nanoenzyme:
0.5g of dextran (molecular weight: 7 ten thousand) was dissolved in 30mL of ultrapure water, and the solution was magnetically stirred for 30 minutes to dissolve the dextran sufficiently. Then 0 is added.25mmol of CuCl2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 100 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 3 preparation of copper cobalt sulphur nanoenzyme:
0.5g of dextran (molecular weight: 1 ten thousand) was dissolved in 30mL of ultrapure water, and the solution was magnetically stirred for 30 minutes to dissolve the dextran sufficiently. Then 0.25mmol of CuCl was added2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 100 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 4 preparation of copper cobalt sulphur nanoenzyme:
0.5g of polyethylene glycol (molecular weight: 1 ten thousand) was dissolved in 30mL of ultrapure water, and the solution was magnetically stirred for 30 minutes to dissolve the polyethylene glycol sufficiently. Then 0.25mmol of CuCl was added2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 100 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 5 preparation of copper cobalt sulphur nanoenzyme:
0.5g of polyvinylpyrrolidone (molecular weight: 1 ten thousand) was dissolved in 30mL of ultrapure water,stirring by magnetic force for 30 minutes to fully dissolve the polyvinylpyrrolidone. Then 0.25mmol of CuCl was added2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 100 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 6 preparation of copper cobalt sulphur nanoenzyme:
bovine serum albumin (molecular weight: 1 ten thousand) (0.5 g) was dissolved in 30mL of ultrapure water, and the solution was magnetically stirred for 30 minutes to dissolve the albumin sufficiently. Then 0.25mmol of CuCl was added2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 100 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 7 preparation of copper cobalt sulphur nanoenzyme:
0.5g of dextran (molecular weight: 50 ten thousand) was dissolved in 30mL of ethylene glycol, and the solution was magnetically stirred for 30 minutes to dissolve the dextran sufficiently. Then 0.25mmol of CuCl was added2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 100 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 8 preparation of copper cobalt sulphur nanoenzyme:
0.5g of dextran (molecular weight: 50 ten thousand) was dissolved in 30mL of ultrapure water, and the solution was magnetically stirred for 30 minutes to dissolve the dextran sufficiently. Then 0.25mmol of CuCl was added2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 100 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 12 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 9 preparation of copper cobalt sulphur nanoenzyme:
0.5g of dextran (molecular weight: 50 ten thousand) was dissolved in 30mL of ultrapure water, and the solution was magnetically stirred for 30 minutes to dissolve the dextran sufficiently. Then 0.25mmol of CuCl was added2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 150 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 10 preparation of copper cobalt sulphur nanoenzyme:
0.5g of polyvinylpyrrolidone (molecular weight: 1 ten thousand) was dissolved in 30mL of ethylene glycol, and the mixture was magnetically stirred for 30 minutes to sufficiently dissolve the polyvinylpyrrolidone. Then 0.25mmol of CuCl was added2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 100 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 11 preparation of copper cobalt sulphur nanoenzyme:
0.5g of bovine serum albumin (molecular weight: 1 ten thousand) was dissolved in 30mL of ethylene glycol, and the mixture was magnetically stirred for 30 minutes to dissolve the albumin sufficiently. Then 0.25mmol of CuCl was added2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 100 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 12 preparation of copper cobalt sulphur nanoenzyme:
0.5g of dextran (molecular weight: 50 ten thousand) was dissolved in 30mL of ultrapure water, and the solution was magnetically stirred for 30 minutes to dissolve the dextran sufficiently. Then 0.25mmol of CuCl was added2.2H2O, 0.5mmol of CoCl2.6H2O, and 2.5mmol of thiourea, and magnetically stirring for 30 minutes to obtain a mixed solution. Then 150 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 13 preparation of copper cobalt sulphur nanoenzyme:
0.25mmol of CuCl2.2H2O, 0.5mmol of CoCl2.6H2O, and 1.5mmol of thiourea were dissolved in 30mL of ultrapure water, and magnetically stirred for 30 minutes to obtain a mixed solution. Then 100 mul of ethylenediamine is slowly added dropwise, and the mixture is fully stirred and evenly mixed to obtain reaction liquid. The reaction solution was poured into a 50 ml Teflon reaction kettle, transferred to an oven, and reacted at 160 ℃ for 20 hours. And naturally cooling the reaction product to room temperature, performing centrifugal separation, washing with water and ethanol for three times respectively, and performing freeze vacuum drying to finally obtain the copper-cobalt-sulfur nano-particles.
Example 14 peroxidase Activity assay of copper cobalt sulphur nanoenzymes:
(1) and (3) determining the copper-cobalt-sulfur nanoenzyme by using the chemiluminescence condition of a substrate luminol of the peroxidase. The copper-cobalt-sulfur nanoenzyme prepared in the example (1), luminol and hydrogen peroxide are respectively added into a phosphate buffer solution with pH of 7.4, and the chemiluminescence signal intensity of the luminol is tracked and detected in real time. Nanoenzymes and luminol, hydrogen peroxide and luminol were used as control experiments. As can be seen from fig. 4, the copper-cobalt-sulfur nanoenzyme can effectively catalyze luminol to emit light in the presence of hydrogen peroxide, which indicates that the copper-cobalt-sulfur nanoenzyme has peroxidase activity.
(2) And detecting the condition of hydroxyl free radicals generated by catalysis of the copper-cobalt-sulfur nano enzyme by using electron paramagnetic resonance. The copper-cobalt-sulfur nanoenzyme prepared in example (1), a radical scavenger BMPO and hydrogen peroxide were added to a phosphate buffer solution of ph7.4, and a signal of a radical was immediately detected. Nanoenzymes and BMPO, hydrogen peroxide and BMPO were used as control experiments. As shown in fig. 5, the detected signal is a hydroxyl radical, the single copper cobalt sulfur nanoenzyme can generate a weak hydroxyl radical, and the signal of the hydroxyl radical is significantly enhanced in the presence of hydrogen peroxide, which indicates that the copper cobalt sulfur nanoenzyme can catalyze the hydrogen peroxide to generate a large amount of hydroxyl radicals.
Example 15 evaluation of antibacterial Properties of copper cobalt Sulfur nanoenzyme:
monoclonal escherichia coli (e.coli), staphylococcus aureus (s.au), and methicillin-resistant staphylococcus aureus (MRSA) were each cultured in LB liquid medium at 37 ℃ for 12 hours at 180 rpm. Then taking the bacterial liquid to dilute with LB according to the ratio of 1:100, continuing to culture for 2 hours, and diluting the bacteria to 10 by using PBS buffer solution when OD600nm reaches 0.77CFU/mL. Then, the copper-cobalt-sulfur nanoenzyme (100ug/mL) prepared in example (1), hydrogen peroxide (2mM) and bacteria were mixed, and after 30 minutes of action, the mixture was diluted to a certain gradient, 100uL of the mixture was applied to the surface of LB agar plate medium, and the plate was inverted and placed in a 37 ℃ incubator to culture for 12 hours, and the colony growth on the plate was observed and counted. Copper cobalt sulfur nanoenzyme and bacteria, hydrogen peroxide and bacteria, PBS and bacteria were used as controls.
The statistical results are shown in fig. 6,7 and 8, and in PBS, the copper-cobalt-sulfur nanoenzyme has significant antibacterial performance in the presence of hydrogen peroxide, and has significant killing effects on e. While the hydrogen peroxide with low concentration and the single copper-cobalt-sulfur nano enzyme have no obvious antibacterial action.
Example 16 evaluation of biofilm inhibition by copper cobalt sulphur nanoenzymes:
methicillin-resistant Staphylococcus aureus (MRSA) was cultured in LB liquid medium at a rotation speed of 180rpm at 37 ℃ for 12 hours. Then, the bacterial suspension was diluted 100-fold with LB, and cultured for 2 hours, and when OD600nm reached 0.7, the bacteria were diluted 100-fold with LB liquid medium containing 0.1% glucose, and transferred to a 24-well plate. Then the round glass sheets are fixed by self-made iron wire clamps and vertically placed into a pore plate with bacteria culture solution, and the biological membrane is cultured and grown in a constant temperature incubator at 37 ℃. Every 12 hours, the glass plate with the biofilm grown thereon was taken out and added into PBS containing the copper-cobalt-sulfur nanoenzyme (100g/mL) prepared in example (1) and hydrogen peroxide (2mM) for 1 hour, and then taken out and washed, and the culture was continued in new LB. After 72 hours, the glass slide was removed and the biofilm was fixed with PBS buffer containing 2.5% glutaraldehyde for 12 hours. Then dehydrating the mixture by using an ethanol/water gradient mixed solution, drying the gold spraying solution, and observing the growth condition of the biological membrane by using a scanning electron microscope. As shown in fig. 9, the copper cobalt sulfur nanoenzyme can significantly inhibit the growth of MRSA biofilm in the presence of hydrogen peroxide, while the low-concentration hydrogen peroxide and the copper cobalt sulfur nanoenzyme alone have no significant inhibitory effect.
Example 17 evaluation of the Performance of copper-cobalt-sulfur nanoenzyme for treating Scald infection
(1) Establishing a mouse scald wound model: after acclimation for one week, 32 Balb/c male mice, 6-8 weeks old, were randomly divided into 4 groups. After shaving the back, the cylindrical copper blank, which was preheated by the alcohol burner, was placed on the exposed skin of the back of the mouse and allowed to contact the skin for 5 seconds before being removed to form a scald wound of 1cm in diameter. Subsequently, 20. mu.l × 10 was added dropwise to the scald wound7CFU/mL MRSA, infection for 2 days.
(2) Evaluating the treatment performance of the copper-cobalt-sulfur nano enzyme: four groups of mice were treated with sterile PBS (II) H2O2(2mM)(Ⅲ)CuCo2S4(100μg/mL)(Ⅳ)CuCo2S4+H2O2Wounds were treated twice in the morning and evening, bandaged with 3M sterile dressing, and rats were weighed on day 02614 after wound formation and photographs of the wounds were taken. At the same time, the wound tissue is taken as H&E staining, analyzing and evaluating the wound healing condition.
As shown in figure 10, the copper-cobalt-sulfur nanoenzyme can obviously promote wound healing and inhibit inflammatory reaction in the presence of hydrogen peroxide, and has a good effect of treating scald infection. While the hydrogen peroxide with low concentration and the single copper-cobalt-sulfur nano enzyme have no obvious treatment effect.
Claims (9)
1. The preparation method of the copper-cobalt-sulfur nano enzyme material is characterized by comprising the following steps of:
(1) fully dissolving biocompatible macromolecules into a solvent to obtain a uniform solution;
(2) under the condition of stirring, adding CuCl2.2H2O、CoCl2.6H2Adding O and thiourea into the solution obtained in the step (1), then adding ethylenediamine, and stirring to fully mix into a clear solution;
(3) and (3) taking the clear solution obtained in the step (2), reacting at the temperature of 160-180 ℃, cooling, centrifuging, washing and drying to obtain the copper-cobalt-sulfur nano enzyme material.
2. The method for preparing the copper-cobalt-sulfur nanoenzyme material according to claim 1, wherein in the step (1), the biocompatible macromolecule is selected from dextran, polyvinylpyrrolidone, polyethylene glycol or bovine serum albumin; preferably dextran, having a molecular weight of one of 50 ten thousand, 7 ten thousand and 1 ten thousand.
3. The method for preparing the copper-cobalt-sulfur nanoenzyme material according to claim 1, wherein in the step (1), the solvent is selected from water or ethylene glycol, and the concentration of the obtained biocompatible macromolecule in the solution is 15-18 mg/mL.
4. Copper cobalt sulphur according to claim 1The preparation method of the nano enzyme material is characterized in that in the step (2), CuCl is added2.2H2O、CoCl2.6H2Adding O and thiourea into the solution obtained in the step (1), wherein the CuCl2.2H2O、CoCl2.6H2The mol ratio of O to thiourea is 1:2 (4-12), and CuCl2.2H2O、CoCl2.6H2The total concentration of O and thiourea is 1.75-3.75 mol/L.
5. The method for preparing the copper-cobalt-sulfur nanoenzyme material according to claim 1, wherein in the step (2), after the ethylenediamine is added, the concentration of the ethylenediamine is 0.3-0.45%.
6. The method for preparing copper-cobalt-sulfur nanoenzyme material as claimed in claim 1, wherein in the step (3), the reaction time at 160-180 ℃ is 6-24 hours.
7. The copper-cobalt-sulfur nanoenzyme material prepared by the preparation method of any one of claims 1 to 6.
8. Use of the copper cobalt sulphur nanoenzyme material of claim 7 as an antibacterial agent.
9. The use of the copper cobalt sulphur nanoenzyme material of claim 7 in the preparation of a medicament for the treatment of scald infection.
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