CN114452386B - 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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- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/34—Copper; 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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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- 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 bimetallic nano enzyme composite material. According to the invention, chloroauric acid and copper chloride mixed solution are added into lysozyme fiber solution, standing is carried out to fully mix, and then freshly prepared sodium borohydride solution is added as a reducing agent, so that the gold-copper bimetallic nano-enzyme composite material is obtained. In the gold-copper bimetallic nano-enzyme composite material, lysozyme fibers are uniform in size, small-size gold-copper nano-particles are uniformly distributed on the surfaces of the lysozyme fibers in a high density, and under the irradiation of near infrared light, the catalytic activity of the peroxidase-like enzyme can be remarkably enhanced, so that the aim of high-efficiency sterilization is fulfilled.
Description
Technical Field
The invention belongs to the technical field of nano materials, relates to a gold-copper bimetal nano enzyme composite material and application of the gold-copper bimetal nano enzyme composite material in catalysis/photo-thermal antibiosis, and particularly relates to a method for achieving efficient antibiosis by taking gold-copper bimetal composite nano particles (Au@Cu) as a catalyst and improving hydrogen peroxide catalysis efficiency through enhancing peroxidase activity under near infrared light (NIR) irradiation.
Background
The nano enzyme is a nano material with similar enzyme activity, and is used as a substitute of natural enzyme, and great interest is brought to people due to obvious advantages compared with the natural enzyme, such as simple synthesis, adjustable catalytic activity, good stability under severe environment and the like. The diversity of the functions of the nano material endows the nano enzyme with multiple functions, so that the nano enzyme is widely researched in the biomedical field and is mainly applied to biomolecule detection, biosensors, antibiosis, immunoassay, cancer diagnosis and treatment, environmental monitoring and the like. In general, peroxidase mimics, which can specifically catalyze the conversion of hydrogen peroxide to highly toxic Reactive Oxygen Species (ROS), such as hydroxyl radicals (·oh), attack the cell membrane of microorganisms at weak acid sites of infection. The nano biological catalytic system taking nano enzyme as a core has various oxidoreductase activities, and can adjust the level of ROS according to conditions such as pH, so that various super drug-resistant pathogens can be killed rapidly and biological films can be cleared based on the principle.
Similar to natural enzymes, the activity of nanoenzymes can be regulated by a variety of factors, such as pH, temperature, ambient environment, and metal ions. In addition, the activity of the nano-enzyme can be regulated by changing the physicochemical properties and structure of the nano-enzyme, and typical nano-scale 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 nano-enzyme. Therefore, a new nano enzyme material is researched and developed, so that the nano enzyme material has better bacterial binding capacity and enhanced catalytic activity, and has important significance.
Disclosure of Invention
The invention aims to overcome the defects of low catalytic efficiency and weak interaction with bacteria of gold-based nano-enzyme in the prior art, and provides a gold-copper nano-particle composite material taking lysozyme as a template and using the gold-copper nano-particle composite material for catalytic antibiosis; the gold-copper nanoparticle composite material synthesized by the method has the advantages of small dosage, high photo-thermal enhancement catalytic efficiency, strong interaction with bacteria and capability of killing bacteria efficiently. According to the invention, the size and the distribution of gold-copper nano particles are regulated and controlled by using lysozyme fibers as templates, so that the catalytic efficiency is effectively improved, the bacteria-bacteria interaction is good, and the aim of high-efficiency sterilization can be achieved.
The technical scheme of the invention is as follows:
the invention firstly provides a biological macromolecule lysozyme fiber (LNFs) as a template, and can regulate and control the high density distribution of metal particles on the fiber to obtain metal nano particles with smaller size, wherein the bimetal nano particles can be uniformly modified on the surface of the lysozyme fiber. The preparation method comprises the following steps:
and (3) adding a mixed solution of chloroauric acid and copper chloride in a certain proportion into the synthesized lysozyme fiber solution, standing for more than 30min to fully mix, and adding a freshly prepared sodium borohydride solution 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 chloroauric acid and copper chloride mixed solution to 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 0.01mM.
Further, the preparation method of the lysozyme fiber is as follows:
preparing 10mL of hydrochloric acid solution with the concentration of 1M, and adding 0.015g of glycine to prepare solution A; 1mL of glacial acetic acid solution with the concentration of 1mM is prepared, and 0.1396g of choline chloride is added to prepare a solution B; 0.01g of lysozyme is taken, 4750 mu LA solution and 250 mu L B solution are added for dissolution, the reaction is stirred in an oil bath at 70 ℃ for 5 hours, and after the reaction is completed, the reaction is washed twice with 12000rpm centrifugal ultrapure water for 20 minutes each time.
In the gold-copper bimetallic nano-enzyme composite material prepared by the invention, the lysozyme fiber is uniform in size, small-size gold-copper nano-particles are uniformly distributed on the surface of the lysozyme fiber in a high density, and the catalytic activity of the peroxidase-like enzyme can be remarkably enhanced under the irradiation of near infrared light, so that the aim of high-efficiency sterilization is fulfilled.
The invention also provides a method for catalyzing/photo-thermally sterilizing the gold-copper bimetallic nano-enzyme composite material (LNFs@Au/Cu), which comprises the following steps:
(1) The lnfs@au/Cu composite solvent was replaced with sodium acetate-acetic acid buffer solution at ph= 5.5,0.1M;
(2) And (3) 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 certain time, reacting for a period of time under the irradiation of near infrared light, diluting with phosphate buffer solution, placing the diluted suspension into Luria Bertani solid culture medium, culturing at 37 ℃ for 12 hours, and calculating the colony number.
Wherein, the volume ratio of the bacterial suspension to the LNFs@Au/Cu composite material is 1:9, a step of performing the process;
further, the concentration of the LNFs@Au/Cu composite material is 70-80 mug/ml, and the concentration of the bacterial suspension is 10 8 And each mL.
After the addition of hydrogen peroxide, the final concentration of hydrogen peroxide was 200. Mu.M.
The conditions of the near infrared light irradiation are as follows: the power is 2W, the irradiation is carried out for 10min, and the wavelength of near infrared light is 808nm;
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, the use of antibiotics is a common sterilization method, but is prone to 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 (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 can be used as a biological template, but also can effectively adhere to bacteria, has strong interaction with the bacteria, can be combined on the surface of the bacteria (shown in a figure 2), promotes the Fenton-like reaction of hydrogen peroxide due to the existence of LNFs@Au/Cu nano-enzyme, improves the generation efficiency of hydroxyl free radicals, and can effectively sterilize. 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 better sterilization effect can be achieved by using the hydrogen peroxide with lower concentration (as shown in the figure 3), bacterial resistance can not be caused, and the method is a novel efficient and green antibacterial method.
Drawings
FIG. 1 is a TEM image of LNFs@Au/Cu composites.
FIG. 2 is a TEM image of the effect of LNFs@Au/Cu composites on Staphylococcus aureus.
Fig. 3 is a graph of sterilization effects of lnfs@au/Cu composites of different sterilization modes.
Detailed Description
The technology of the present invention will be further described with reference to the accompanying drawings and examples.
Example 1:
preparation of lysozyme fiber:
10mL of a 1M hydrochloric acid solution was prepared, and 0.015g of glycine was added thereto to prepare a solution A. 1mL of glacial acetic acid solution having a concentration of 1mM was prepared, and 0.1396g of choline chloride was added to prepare a solution B. 0.01g of lysozyme was taken and dissolved in 4750. Mu.LA solution and 250. Mu. L B solution. The reaction was stirred in an oil bath at 70℃for 5h. After completion of the reaction, the reaction mixture was washed twice with ultrapure water at 12000rpm for 20 minutes each. 8ml of a final lysozyme fiber aqueous solution was obtained.
Preparation of LNFs@Au/Cu nano enzyme composite material:
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 rapidly 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 0.01mM.
LNFs@Au/Cu nano enzyme composite material catalysis/photo-thermal synergistic sterilization:
mu.L of the Staphylococcus aureus suspension was placed in 180. Mu.L at a concentration of 80. Mu.g mL –1 In LNFs@Au/Cu, 200 mu M hydrogen peroxide exists in the system, the system is kept stand for 10min, after irradiation for 10min under the irradiation of near infrared light with the power of 2W and the wavelength of 808nm, the system continues to act for 10min, finally, 10000 times of phosphate buffer solution is diluted, 100 mu L of diluted suspension is taken and put into Luria Bertani solid culture medium, and the culture is carried out for 12h at 37 ℃, so as to calculate the colony number. The bacterial viability obtained is shown in Table 1.
Example 2:
as in example 1, the LNFs@Au/Cu photo-thermal sterilization was performed by changing only the ratio of the gold-copper salt solution in the preparation step of the gold-copper nano enzyme composite material, and the obtained bacterial viability was shown in Table 1. The result shows that the sterilization efficiency of LNFs@Au/Cu is increased along with the increase of the addition amount of copper compared with the unmodified gold nanoparticle material, but the sterilization efficiency is reduced due to the fact that the addition of the higher copper amount promotes the increase of the size of LNFs@Au/Cu metal nanoparticles, the near infrared absorption is reduced, and the photo-thermal conversion efficiency is reduced.
TABLE 1 effects of different copper additions on photo-thermal sterilization of LNFs@Au/Cu
| Copper addition (molar ratio of Au to Cu) | Bacterial viability (%) |
| 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:
as in example 1, the LNFs@Au/Cu catalytic hydrogen peroxide sterilization was performed by changing only the ratio of gold-copper salt solution in the preparation step of the gold-copper nano enzyme composite material, and the obtained bacterial viability is shown in Table 2. It can be seen that the catalytic sterilization efficiency of LNFs@Au/Cu increases with increasing copper addition, but the sterilization efficiency decreases with further increasing copper content, which can be attributed to the fact that the higher copper addition promotes the larger size of LNFs@Au/Cu metal nanoparticles and decreases the catalytic activity.
TABLE 2 catalytic sterilizing Effect of different copper addition levels on LNFs@Au/Cu
| Copper addition (molar ratio of Au to Cu) | Bacterial viability (%) |
| 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:
as in example 1, only the power of near infrared light radiation in the photo-thermal sterilization step of LNFs@Au/Cu nano-enzyme composite material was changed, and the obtained bacterial viability was shown in Table 3. It can be seen that the photo-thermal sterilization efficiency gradually increases with the increase of the near infrared radiation power, which is attributable to the fact that higher radiation power is favorable for the photo-thermal agent to generate higher heat.
TABLE 3 Effect of different near infrared radiation powers on photo-thermal sterilization
Example 5:
as in example 1, the hydrogen peroxide concentration in the catalytic sterilization step of the LNFs@Au/Cu nanoenzyme composite material was only changed, and the obtained bacterial viability is shown in Table 4. It follows that higher hydrogen peroxide concentrations can increase the sterilization efficiency.
TABLE 4 Effect of different hydrogen peroxide concentrations on catalytic sterilization
| H 2 O 2 Concentration (mu M) | Bacterial viability (%) |
| 50 | 89.12 |
| 100 | 64.05 |
| 200 | 51.75 |
| 300 | 38.48 |
| 400 | 22.41 |
Example 6:
as in example 1, under the final reaction conditions, LNFs@Au/Cu concentration was 80. Mu.g/ml and hydrogen peroxide concentration was 200. Mu.M, and sterilization was performed by a catalytic, photo-thermal synergistic sterilization method, and the obtained bacterial viability was shown in Table 5. Therefore, the photo-thermal has an enhancement effect on catalytic sterilization.
TABLE 5 Sterilization effects of different sterilization types
| Type of sterilization | Bacterial viability (%) |
| Photo-thermal sterilization | 27.8 |
| Catalytic sterilization | 60.36 |
| Catalytic/photo-thermal synergistic sterilization | 0 |
Example 7:
as in example 1, only LNFs@Au/Cu nano-enzyme composite material was changed, and the bacteria in the synergistic sterilization step were Pseudomonas aeruginosa, escherichia coli and Salmonella respectively, and the obtained bacterial survival rates are shown in Table 6. It can be seen that under the same conditions, LNFs@CuS kills a variety of bacteria.
| Bacterial type | Bacterial viability (%) |
| Staphylococcus aureus | 0 |
| Pseudomonas aeruginosa | 0 |
| Coli bacterium | 0 |
| Salmonella bacteria | 0 |
The results of comparative examples 1, 2 and 3, in which the preparation parameters of gold-copper nanoparticles were changed, had an important effect on the photo-thermal and catalytic sterilization efficiency.
FIG. 1 is a TEM image of LNFs@Au/Cu nanoenzyme, illustrating that Au/Cu nanoparticles are relatively uniform in size and relatively uniformly loaded on lysozyme fibers.
FIG. 2 is a TEM image of the effect of LNFs@Au/Cu nanoenzymes on Staphylococcus aureus, demonstrating that LNFs@Au/Cu interact well with bacteria.
Fig. 3 shows the sterilizing effect of LNFs@CuS nano-enzyme by utilizing different mechanisms of photo-thermal and catalysis, which shows that the photo-thermal has a certain reinforcing effect on catalysis, and the two have synergistic effect on killing bacteria with high efficiency.
Claims (8)
1. The preparation method of the gold-copper bimetallic nano enzyme composite material is characterized by comprising the following steps:
adding a lysozyme fiber solution into a mixed solution of chloroauric acid and copper chloride, standing to fully mix the mixture, and adding a freshly prepared sodium borohydride solution as a reducing agent to obtain a gold-copper bimetallic nano-enzyme composite material, namely an LNFs@Au/Cu composite material;
the volume ratio of the lysozyme fiber solution to the chloroauric acid and copper chloride mixed solution to 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 was 0.15mM, and the concentration of sodium borohydride solution was 0.01mM.
2. The method according to claim 1, wherein the standing time is 30 minutes or longer.
3. The preparation method of claim 1, wherein the preparation method of the lysozyme fiber comprises the following steps:
preparing 10mL of hydrochloric acid solution with the concentration of 1M, and adding 0.015g of glycine to prepare solution A; 1mL of glacial acetic acid solution with the concentration of 1mM is prepared, and 0.1396g of choline chloride is added to prepare a solution B; 0.01g of lysozyme is taken, 4750 mu L A solution and 250 mu L B solution are added for dissolution, the reaction is stirred in an oil bath at 70 ℃ for 5 hours, and after the reaction is completed, the reaction is washed twice with 12000rpm centrifugal ultrapure water for 20 minutes each time.
4. The gold-copper bimetallic nano-enzyme composite material prepared by the preparation method according to any one of claims 1-3, wherein in the gold-copper bimetallic nano-enzyme composite material, lysozyme fibers are uniform in size, and gold-copper nano-particles are uniformly distributed on the surfaces of the lysozyme fibers.
5. The use of the gold-copper bimetallic nano-enzyme composite material as set forth in claim 4 for preparing a catalytic/photo-thermal bactericide, which is characterized by comprising the following specific steps:
(1) The lnfs@au/Cu composite solvent was replaced with sodium acetate-acetic acid buffer solution at ph= 5.5,0.1M;
(2) And (3) placing the bacterial suspension in the LNFs@Au/Cu composite material treated in the step (1), adding hydrogen peroxide, standing for a while, reacting for a period of time under the irradiation of near infrared light, diluting with phosphate buffer solution, placing the diluted suspension in Luria Bertani solid medium, culturing at 37 ℃ for 12h, and calculating the colony number.
6. The use according to claim 5The method is characterized in that in the step (2), the volume ratio of the bacterial suspension to the LNFs@Au/Cu composite material is 1:9, a step of performing the process; wherein the concentration of the LNFs@Au/Cu composite material is 70-80 mug/ml, and the concentration of the bacterial suspension is 10 8 And each mL.
7. The method according to claim 5, wherein in step (2) the final concentration of hydrogen peroxide is 200. Mu.M after the hydrogen peroxide has been added.
8. The method according to claim 5, wherein in step (2),
the conditions of the near infrared light irradiation are as follows: the power is 2W, the irradiation is carried out for 10min, and the wavelength of near infrared light is 808 and nm;
the dilution is 10000 times of dilution;
the bacteria is one of Pseudomonas aeruginosa, salmonella, escherichia coli or Staphylococcus aureus.
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