CN114452386A - Preparation method and application of gold-copper bimetallic nano-enzyme composite material - Google Patents
Preparation method and application of gold-copper bimetallic nano-enzyme composite material Download PDFInfo
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/242—Gold; Compounds thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of nano materials, and discloses a preparation method and application of a gold-copper bimetal nano enzyme composite material. According to the invention, a mixed solution of chloroauric acid and copper chloride is added into a lysozyme fiber solution, and after the mixed solution is kept stand to be fully mixed, a freshly prepared sodium borohydride solution is added to be used as a reducing agent, so that the gold-copper bimetal nano enzyme composite material is obtained. In the gold-copper bimetallic nano-enzyme composite material, lysozyme fibers are uniform in size, and small-size gold-copper nano-particles are uniformly distributed on the surfaces of the lysozyme fibers in high density, so that the catalytic activity of peroxidase-like enzymes can be remarkably enhanced under near-infrared light radiation, and the aim of high-efficiency sterilization is fulfilled.
Description
Technical Field
The invention belongs to the technical field of nano materials, and relates to a gold-copper bimetallic nano-enzyme composite material and application thereof in catalysis/photothermal antibiosis, in particular to a method for achieving efficient antibiosis by taking gold-copper bimetallic composite nano-particles (Au @ Cu) as a catalyst and improving the efficiency of catalyzing hydrogen peroxide through enhancing the activity of peroxidase under the irradiation of near infrared light (NIR).
Background
The nano enzyme is a nano material with similar enzyme activity, is used as a substitute of natural enzyme, and has great interest because of the obvious advantages of the nano enzyme compared with the natural enzyme, such as simple synthesis, adjustable catalytic activity, good stability in severe environment and the like. The diversity of the functions of the nano materials endows the nano enzyme with various functions, so that the nano enzyme is widely researched in the field of biomedicine and is mainly applied to biomolecule detection, biosensors, antibiosis, immunoassay, cancer diagnosis and treatment, environmental monitoring and the like. Generally, peroxidase mimetics can specifically catalyze the conversion of hydrogen peroxide to highly toxic Reactive Oxygen Species (ROS), such as hydroxyl radicals (. OH), which attack the cell membranes of microorganisms at weakly acidic sites of infection. The nano biological catalysis system taking the nano enzyme as the core has the activity of various oxidoreductases, and can adjust the level of ROS according to the conditions of pH and the like, so that various super drug-resistant germs can be killed quickly and the biomembrane can be eliminated based on the principle.
Similar to natural enzymes, the activity of nanoenzymes can be modulated by a variety of factors, such as pH, temperature, ambient environment, and metal ions. In addition, the activity of the nanoenzyme can also be adjusted by changing the physicochemical properties and the structure of the nanoenzyme, and typical nanoscale factors such as size, morphology, surface modification and valence state, composition and configuration of an active center and the like significantly influence the activity of the nanoenzyme. Therefore, research and development of a novel nano enzyme material which has good bacteria binding capacity and enhanced catalytic activity are significant.
Disclosure of Invention
The invention aims to overcome the defects of low catalytic efficiency and weak interaction with bacteria of gold-based nano-particles in the prior art, provides a gold-copper nano-particle composite material taking lysozyme as a template and uses the gold-copper nano-particle composite material for catalytic antibiosis; the gold-copper nanoparticle composite material synthesized by the method has the advantages of low consumption, high photo-thermal enhanced catalysis efficiency, strong interaction with bacteria and capability of efficiently killing the bacteria. According to the invention, the size and distribution of gold-copper nanoparticles are regulated and controlled by using the lysozyme fiber as a template, so that the catalytic efficiency is effectively improved, the lysozyme fiber has good interaction with bacteria, and the purpose of high-efficiency sterilization can be achieved.
The technical scheme of the invention is as follows:
the invention firstly provides biomacromolecule lysozyme fibers (LNFs) as a template, and can regulate and control the high-density distribution of metal particles on the fibers to obtain metal nanoparticles with smaller sizes, and the bimetallic nanoparticles can be uniformly modified on the surfaces of the lysozyme fibers. The preparation method comprises the following steps:
and (2) adding a certain proportion of a mixed solution of chloroauric acid and copper chloride into the synthesized lysozyme fiber solution, standing for more than 30min to fully mix the lysozyme fiber solution and the mixed solution, and adding a freshly prepared sodium borohydride solution serving as a reducing agent to obtain the gold-copper bimetallic nano-enzyme composite material, namely the LNFs @ Au/Cu composite material.
Wherein, the volume ratio of the lysozyme fiber solution, the mixed solution of chloroauric acid and copper chloride and the sodium borohydride solution is 1: 1: 1, wherein the concentration of the lysozyme fiber solution is 5mg/mL, the total concentration of metal ions in the mixed solution of chloroauric acid and copper chloride is 0.15mM, and the concentration of the sodium borohydride solution is 0.01 mM.
Further, the preparation method of the lysozyme fiber in the test is as follows:
preparing 10mL of hydrochloric acid solution with the concentration of 1M, and adding 0.015g of glycine to prepare solution A; preparing 1mL of glacial acetic acid solution with the concentration of 1mM, and adding 0.1396g of choline chloride to prepare solution B; 0.01g of lysozyme is taken and added with 4750 mu LA solution and 250 mu L B solution for dissolution, the mixture is stirred and reacted for 5 hours in an oil bath kettle at 70 ℃, and after the reaction is finished, the mixture is centrifuged at 12000rpm and washed twice by ultrapure water for 20min each time.
In the gold-copper bimetallic nano-enzyme composite material prepared by the invention, the lysozyme fiber is uniform in size, and the small-size gold-copper nano-particles are uniformly distributed on the surface of the lysozyme fiber in high density, so that the catalytic activity of peroxidase-like enzyme can be obviously enhanced under the radiation of near infrared light, and the aim of high-efficiency sterilization is fulfilled.
The invention also provides a method for catalyzing/photo-thermal sterilizing the gold-copper bimetallic nano-enzyme composite material (LNFs @ Au/Cu), which comprises the following steps:
(1) the LNFs @ Au/Cu composite material solvent is replaced by 0.1M sodium acetate-acetic acid buffer solution with the pH value of 5.5;
(2) and (2) placing the bacterial suspension into the LNFs @ Au/Cu composite material treated in the step (1), adding hydrogen peroxide with a certain concentration, standing for a while, reacting for a while under the irradiation of near infrared light, diluting with a phosphate buffer solution, taking the diluted suspension, placing the suspension into a Luria Bertani solid culture medium, culturing at 37 ℃ for 12 hours, and calculating the colony count.
Wherein the volume ratio of the bacterial suspension to the LNFs @ Au/Cu composite material is 1: 9;
further, the concentration of the LNFs @ Au/Cu composite material is 70-80 mu g/ml, and the concentration of the bacterial suspension is 108one/mL.
After addition of hydrogen peroxide, the final concentration of hydrogen peroxide was 200. mu.M.
The near infrared light irradiation conditions are as follows: irradiating with power of 2W for 10min, wherein the wavelength of the near-infrared light is 808 nm;
the dilution is 10000 times.
The bacteria is one of pseudomonas aeruginosa, salmonella, escherichia coli or staphylococcus aureus.
Compared with the prior art, the invention has the following beneficial effects:
currently, antibiotics are commonly used as sterilization methods, but tend to cause bacterial resistance. The gold-copper bimetallic nano-enzyme composite material prepared by the invention has small gold-copper nano-particle size and is uniformly modified on the surface of lysozyme fiber (as shown in figure 1), so that the distribution density of metal active centers is improved, and the catalytic effect is enhanced. Meanwhile, the lysozyme fiber not only serves as a biological template, but also can effectively adhere bacteria, has strong interaction with the bacteria, can be combined on the surface of the bacteria (as shown in figure 2), and promotes the Fenton-like reaction of hydrogen peroxide due to the existence of LNFs @ Au/Cu nano-enzyme, so that the generation efficiency of hydroxyl radicals is improved, and the sterilization can be effectively realized. Under the irradiation of near infrared light, the photo-thermal effect can be utilized, meanwhile, the photo-thermal temperature can improve the catalytic activity, the hydrogen peroxide with lower concentration is used to achieve better sterilization effect (as shown in figure 3), the bacterial resistance cannot be caused, and the method is a novel high-efficiency, green and antibacterial method.
Drawings
FIG. 1 is a TEM image of LNFs @ Au/Cu composite.
FIG. 2 is a TEM image of the action of LNFs @ Au/Cu composite with Staphylococcus aureus.
FIG. 3 is a graph of the sterilization effect of LNFs @ Au/Cu composite materials in different sterilization modes.
Detailed Description
The technology of the present invention will be further explained with reference to the drawings and examples.
Example 1:
preparation of lysozyme fiber:
10mL of a hydrochloric acid solution having a concentration of 1M was prepared, and 0.015g of glycine was added to prepare a solution A. 1mL of a 1mM glacial acetic acid solution was prepared, and 0.1396g of choline chloride was added to prepare a solution B. 0.01g of lysozyme was dissolved by adding 4750. mu.LA solution and 250. mu. L B solution. The reaction was stirred in an oil bath kettle at 70 ℃ for 5 h. After the reaction was completed, the reaction mixture was washed twice with 12000rpm centrifugal ultrapure water for 20min each. 8ml of the final lysozyme fiber aqueous solution was obtained.
Preparing an LNFs @ Au/Cu nano enzyme composite material:
and (3) taking 800 mu L of lysozyme fiber solution, adding 800 mu L of chloroauric acid and copper chloride solution, reacting for 30min, adding 800 mu L of freshly prepared sodium borohydride solution, and quickly reducing to obtain the metal nanoparticle composite material.
Wherein, the concentration of the lysozyme fiber is 5mg/mL, the total concentration of the metal salt solution is 0.15mM, and the concentration of the sodium borohydride solution is 0.01 mM.
LNFs @ Au/Cu nano enzyme composite material catalysis/photo-thermal synergistic sterilization:
mu.L of Staphylococcus aureus suspension was placed in a volume of 180. mu.L of 80. mu.g mL–1In the LNFs @ Au/Cu, 200 mu M hydrogen peroxide exists in the system, the system is kept stand for 10min, under the irradiation of near infrared light with the power of 2W and the wavelength of 808nm,after 10min of irradiation, the reaction is continued for 10min, and finally, the solution is diluted by 10000 times by phosphate buffer solution, 100 mu L of the diluted suspension is put into Luria Bertani solid culture medium and cultured for 12h at 37 ℃, and the colony number is calculated. The survival rates of the obtained bacteria are shown in Table 1.
Example 2:
in the same manner as in example 1, LNFs @ Au/Cu photothermal sterilization was performed by changing only the ratio of the gold copper salt solution in the preparation step of the gold copper nanoenzyme composite, and the survival rate of the obtained bacteria is shown in table 1. The result shows that the sterilizing efficiency of LNFs @ Au/Cu is increased along with the increase of the added amount of copper, but the sterilizing efficiency is reduced due to the further increase of the copper content, which is attributable to the fact that the addition of higher amount of copper promotes the size of LNFs @ Au/Cu metal nanoparticles to be larger, the near-infrared absorption is reduced, and the photo-thermal conversion efficiency is reduced.
TABLE 1 influence of different copper additions on LNFs @ Au/Cu photothermal sterilization
| Copper addition (Au to Cu molar ratio) | Bacterial survival Rate (%) |
| 1：0 | 65.13 |
| 4：1 | 28.24 |
| 3：1 | 27.5 |
| 2：1 | 56.21 |
| 1：1 | 76.3 |
| 0：1 | 100 |
Example 3:
in the same way as in example 1, the ratio of the gold copper salt solution in the preparation step of the gold copper nanoenzyme composite material was changed, LNFs @ Au/Cu catalyzed hydrogen peroxide sterilization was performed, and the survival rate of the obtained bacteria is shown in table 2. Therefore, the LNFs @ Au/Cu catalytic sterilization efficiency is increased along with the increase of the added amount of copper, but the sterilization efficiency is reduced due to the further increase of the copper content, and the size of the LNFs @ Au/Cu metal nano particles is increased and the catalytic activity is reduced due to the addition of higher copper amount.
TABLE 2 Effect of different copper additions on LNFs @ Au/Cu catalytic sterilization
| Copper addition (Au to Cu molar ratio) | Bacterial survival Rate (%) |
| 1：0 | 87.26 |
| 4：1 | 68.13 |
| 3：1 | 61.34 |
| 2：1 | 73.46 |
| 1：1 | 91.74 |
| 0：1 | 100 |
Example 4:
in the same way as in example 1, only the radiation power of near infrared light in the photothermal sterilization step of the LNFs @ Au/Cu nanoenzyme composite material is changed, and the survival rate of the obtained bacteria is shown in Table 3. It follows that as the near-infrared radiation power increases, the photothermal sterilization efficiency gradually increases, which is attributable to the fact that higher radiation power is favorable for the photothermal agent to generate higher heat.
TABLE 3 Effect of different NIR light radiation powers on photothermal sterilization
Example 5:
in the same way as in example 1, the hydrogen peroxide concentration in the catalytic sterilization step of the LNFs @ Au/Cu nano-enzyme composite material is only changed, and the survival rate of the obtained bacteria is shown in Table 4. It follows that higher hydrogen peroxide concentrations can improve sterilization efficiency.
TABLE 4 Effect of different Hydrogen peroxide concentrations on catalytic Sterilization
| H2O2Concentration (μ M) | Bacterial survival Rate (%) |
| 50 | 89.12 |
| 100 | 64.05 |
| 200 | 51.75 |
| 300 | 38.48 |
| 400 | 22.41 |
Example 6:
in the same manner as in example 1, under the final reaction conditions, the concentration of LNFs @ Au/Cu was 80. mu.g/ml, the concentration of hydrogen peroxide was 200. mu.M, and the bacterial survival rate was shown in Table 5 by the catalytic and photothermal co-sterilization method. Therefore, the photo-thermal has an enhancement effect on catalytic sterilization.
TABLE 5 Sterilization effects of different types of sterilization
| Type of sterilization | Bacterial survival Rate (%) |
| Photo-thermal sterilization | 27.8 |
| Catalytic sterilization | 60.36 |
| Catalytic/photothermal |
0 |
Example 7:
in the same way as in example 1, only the bacteria in the synergistic sterilization step of the LNFs @ Au/Cu nano-enzyme composite material are changed to be pseudomonas aeruginosa, escherichia coli and salmonella respectively, and the survival rates of the obtained bacteria are shown in Table 6. It can be seen that under the same conditions, LNFs @ CuS all kill a variety of bacteria.
| Bacterial types | Bacterial survival Rate (%) |
| |
0 |
| |
0 |
| |
0 |
| |
0 |
The results of comparative examples 1, 2 and 3, changing the preparation parameters of gold-copper nanoparticles, had important effects on photo-thermal and catalytic sterilization efficiency.
FIG. 1 is a TEM image of LNFs @ Au/Cu nanoenzyme, which shows that Au/Cu nanoparticles are relatively uniform in size and are relatively uniformly loaded on lysozyme fibers.
FIG. 2 is a TEM image of the interaction of LNFs @ Au/Cu nanoenzyme with Staphylococcus aureus, which shows that LNFs @ Au/Cu has good interaction with bacteria.
FIG. 3 shows that the LNFs @ CuS nanoenzyme has a sterilization effect by utilizing different mechanisms of photo-thermal and catalysis, which indicates that photo-thermal has a certain enhancement effect on catalysis, and the photo-thermal and the catalysis are synergistic to kill bacteria efficiently.
Claims (10)
1. The preparation method of the gold-copper bimetallic nano-enzyme composite material is characterized by comprising the following steps:
and (2) adding a mixed solution of chloroauric acid and copper chloride in a certain proportion into the lysozyme fiber solution, standing to fully mix the lysozyme fiber solution and the mixed solution, and adding a freshly prepared sodium borohydride solution serving as a reducing agent to obtain the gold-copper bimetallic nano-enzyme composite material, namely the LNFs @ Au/Cu composite material.
2. The method according to claim 1, wherein the volume ratio of the lysozyme fiber solution, the mixed solution of chloroauric acid and copper chloride and the sodium borohydride solution is 1: 1: 1, wherein the concentration of the lysozyme fiber solution is 5mg/mL, the total concentration of metal ions in the mixed solution of chloroauric acid and copper chloride is 0.15mM, and the concentration of the sodium borohydride solution is 0.01 mM.
3. The method according to claim 1, wherein the standing time is 30min or more.
4. The method of claim 1, wherein the lysozyme fiber is prepared by the method comprising:
preparing 10mL of hydrochloric acid solution with the concentration of 1M, and adding 0.015g of glycine to prepare solution A; preparing 1mL of glacial acetic acid solution with the concentration of 1mM, and adding 0.1396g of choline chloride to prepare solution B; 0.01g of lysozyme is taken and added with 4750 mu L A solution and 250 mu L B solution for dissolution, the mixture is stirred and reacted for 5 hours in an oil bath kettle at 70 ℃, and after the reaction is finished, the mixture is centrifuged at 12000rpm and washed twice by ultrapure water, and each time lasts for 20 min.
5. The Au-Cu bimetallic nano-enzyme composite material prepared by the preparation method according to any one of claims 1 to 4, wherein in the Au-Cu bimetallic nano-enzyme composite material, lysozyme fibers are uniform in size, and Au-Cu nano-particles are uniformly distributed on the surfaces of the lysozyme fibers in a high density.
6. Use of the gold-copper bimetallic nano-enzyme composite material as defined in claim 5 for catalytic/photothermal sterilization.
7. The use according to claim 6, characterized by the specific steps of:
(1) the LNFs @ Au/Cu composite material solvent is replaced by 0.1M sodium acetate-acetic acid buffer solution with the pH value of 5.5;
(2) and (2) placing the bacterial suspension into the LNFs @ Au/Cu composite material treated in the step (1), adding hydrogen peroxide with a certain concentration, standing for a while, reacting for a while under the irradiation of near infrared light, diluting with a phosphate buffer solution, taking the diluted suspension, placing the suspension into a Luria Bertani solid culture medium, culturing at 37 ℃ for 12 hours, and calculating the colony count.
8. The use according to claim 7, wherein in step (2), the volume ratio of the bacterial suspension to the LNFs @ Au/Cu composite is 1: 9; wherein the concentration of the LNFs @ Au/Cu composite material is 70-80 mu g/ml, and the concentration of the bacterial suspension is 108one/mL.
9. The use according to claim 7, wherein in step (2), the final concentration of hydrogen peroxide after addition of hydrogen peroxide is 200 μ M.
10. The use according to claim 7, wherein, in step (2),
the near infrared light irradiation conditions are as follows: irradiating with power of 2W for 10min, wherein the wavelength of the near-infrared light is 808 nm;
the dilution is 10000 times;
the bacteria is one of pseudomonas aeruginosa, salmonella, escherichia coli or staphylococcus aureus.
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