CN114191562B - Preparation method and application of double-enzyme-activity antibacterial material - Google Patents

Preparation method and application of double-enzyme-activity antibacterial material Download PDF

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CN114191562B
CN114191562B CN202111568394.7A CN202111568394A CN114191562B CN 114191562 B CN114191562 B CN 114191562B CN 202111568394 A CN202111568394 A CN 202111568394A CN 114191562 B CN114191562 B CN 114191562B
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molybdate
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glucose oxidase
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CN114191562A (en
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牛玉生
许元红
张彦芳
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Qingdao University
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    • A—HUMAN NECESSITIES
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • 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
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K38/44—Oxidoreductases (1)
    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • A—HUMAN NECESSITIES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G49/00—Compounds of iron
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/01—Particle morphology depicted by an image
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/01—Particle morphology depicted by an image
    • C01P2004/03—Particle morphology depicted by an image obtained by SEM
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/01—Particle morphology depicted by an image
    • C01P2004/04—Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • 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 preparation and application of antibacterial materials, and relates to a preparation method and application of a double-enzyme-activity antibacterial material, which comprises the following specific process steps: dispersing ferric molybdate in acetate buffer solution with pH=4, adding glucose oxidase, stirring at normal temperature of 200-300 r/min for 10 minutes, centrifuging, collecting precipitate, freeze-drying to obtain a double-enzyme-activity antibacterial material Fe of ferric molybdate loaded glucose oxidase 2 (MoO 4 ) 3 @ GOx; based on Fe 2 (MoO 4 ) 3 GOx has excellent peroxidase and glucose oxidase activities, can reduce injury of exogenous hydrogen peroxide to tissues in antibacterial application, and starvation therapy generated by glucose oxidase can cooperate with active oxygen generated by ferric molybdate to realize efficient antibacterial; the preparation method is simple, has high-efficiency antibacterial performance, is environment-friendly and pollution-free in the whole process, and has wide application prospect.

Description

Preparation method and application of double-enzyme-activity antibacterial material
Technical field:
the invention belongs to the technical field of preparation and application of antibacterial materials, and relates to Fe with double enzyme activities 2 (MoO 4 ) 3 According to the preparation method and application of the @ GOx material, ferric molybdate is synthesized by a hydrothermal method, glucose oxidase is loaded in an electrostatic adsorption mode, and the aim of efficient bacteriostasis is achieved by cooperation of starvation therapy generated by the glucose oxidase and active oxygen generated by the ferric molybdate, so that the material has a good bacteriostasis effect and is harmless to a human body.
The background technology is as follows:
diabetes is a disease currently prevalent worldwide, with an increasing number of patients each year, and has become one of the most prominent public health threats affecting the health of about 4.25 million people worldwide. Diabetic foot ulcers, also known as diabetic limb gangrene, are one of the most serious chronic complications of diabetes and are also an important cause of disability and death for diabetics. Foot ulcers in diabetics are caused by peripheral neuropathy, insufficient blood supply to the lower limb arteries, bacterial infection, and the like. Among them, bacterial infection is an important factor affecting wound healing, and antibiotic treatment often cannot play an effective bactericidal role, but can cause appearance of drug-resistant bacteria, and aggravate illness.
Therefore, it has been urgent to find antibacterial materials having high sterilization efficiency and inhibiting the development of bacterial resistance. The nanometer enzyme is a nanometer material with enzyme catalysis, and can simulate natural enzyme to generate toxic active oxygen for sterilization, and has peroxidase-like (such as V 2 O 5 、Co 3 O 4 Two-dimensional (2D) Co-MOF can convert exogenous hydrogen peroxide into toxic hydroxyl radicals (· OH) to treat bacterial infections. However, in practical wound disinfection applications, the addition of exogenous hydrogen peroxide inevitably causes secondary pain to the infected tissue, and the added hydrogen peroxide is easily decomposed, and the addition amount is not easily controlled, which is very troublesome in practical application.
Studies have shown that blood glucose concentrations at the wounds of diabetic feet are higher than in healthy people. Since glucose in high concentration in the wound is a nutrient for bacteria, it is more favorable for bacterial reproduction, further deteriorating the wound. Thus, if the nutrient source of the bacteria at the wound can be broken down, the growth rate of the bacteria gradually decreases in the nutrient deficient environment. Glucose oxidase is an endogenous oxidoreductase that catalyzes the production of hydrogen peroxide and gluconic acid from glucose. Glucose oxidase has been widely used as a starvation therapy for cancer in the last few years, because it consumes not only energy and nutrients (glucose and oxygen) but also hydrogen peroxide at the catalytic site. Fortunately, glucose oxidase can produce endogenous hydrogen peroxide, thus solving the problem of exogenous addition. However, the bacteriostatic effect of single glucose oxidase is not ideal, and a double-enzyme active bacteriostatic material which is simple in step and does not need to add hydrogen peroxide exogenously is necessary to be sought.
The invention comprises the following steps:
the invention aims to overcome the defects of the prior art and provide a preparation method and application of a double-enzyme-activity antibacterial material of ferric molybdate loaded glucose oxidase, wherein the ferric molybdate has excellent peroxidase property, and the ferric molybdate loaded glucose oxidase can undergo cascade reaction in an electrostatic adsorption mode, so that starvation therapy generated by the glucose oxidase and active oxygen generated by the ferric molybdate cooperate to efficiently inhibit bacteria.
In order to achieve the above purpose, the invention provides a preparation method of a double-enzyme active antibacterial material of ferric molybdate loaded glucose oxidase, which comprises the following specific process steps:
iron molybdate (Fe) 2 (MoO 4 ) 3 ) Dispersing in acetate buffer solution with pH=4, adding glucose oxidase, stirring at normal temperature of 200-300 r/min for 10min, centrifuging, collecting precipitate, and freeze drying to obtain double enzyme activity antibacterial material (Fe) with ferric molybdate loaded glucose oxidase 2 (MoO 4 ) 3 @GOx)。
Further, the mass ratio of the ferric molybdate to the glucose oxidase is 1:1.
further, the centrifugal speed is 5000-8000 r/min, and the centrifugal time is 5-10 min.
Further, the freeze drying time is 24-36 h; freezing temperature is-40 to-50 ℃.
Further, the preparation method of the iron molybdate comprises the following steps:
(1) Iron salt and molybdate are respectively dissolved into two parts of deionized water with the same volume, and the molar ratio of the iron salt to the molybdate is 1:1 dissolving, dropwise adding a molybdate solution into an iron salt solution, adjusting the pH value of a final reaction system to be 2.5-4 by using sodium hydroxide, stirring at normal temperature of 200-300 r/min for 30min, transferring into a 100mL reaction kettle, and reacting for 8-15 h at 150-200 ℃;
(2) Taking out the reacted sample, centrifuging for 5-10 min at 5000-10000 r/min, removing supernatant, collecting precipitate, adding ultrapure water, centrifuging repeatedly until the supernatant is colorless, collecting precipitate, freeze drying for 24-48 h to obtain Fe 2 (MoO 4 ) 3 。
Further, the ferric salt is Fe 2 (NO 3 ) 3 The method comprises the steps of carrying out a first treatment on the surface of the The molybdate is Na 2 MoO 4 。
Fe prepared by the invention 2 (MoO 4 ) 3 GOx can decompose glucose to produce hydrogen peroxide and gluconic acid, reduce nutrient substances of bacteria, produce starvation therapy, and inhibit bacterial growth; under the condition of not adding hydrogen peroxide, the hydrogen peroxide generated by decomposition of the upper-level reaction can be converted into toxic hydroxyl free radicals, and double damage is caused to bacteria by cooperating with starvation therapy.
The invention also provides Fe with the double enzyme activity 2 (MoO 4 ) 3 Application of @ GOx as antibacterial material, specifically Fe 2 (MoO 4 ) 3 Application of @ GOx as antibacterial material for treating wound.
Compared with the prior art, the invention utilizes the electrostatic adsorption method to adsorb the natural glucose oxidase (GOx) on the surface of the ferric molybdate to obtain the material (Fe) with two enzyme properties 2 (MoO 4 ) 3 @ GOx); based on Fe 2 (MoO 4 ) 3 GOx has excellent peroxidase and glucose oxidase activities, can reduce damage to tissues caused by exogenous hydrogen peroxide in bacteriostasis application, and reduces complicated steps of additional addition, the double-enzyme active material can spontaneously perform cascade reaction, and starvation therapy generated by glucose oxidase can cooperate with active oxygen generated by ferric molybdate to perform efficient antibiosis, so that bacteriostasis by using the double-enzyme system is very important in significance and application prospect; the preparation method is simple, the preparation equipment is easy to obtain, the preparation process is simple, the antibacterial performance is high, the whole process is green and environment-friendly, no pollution is caused, and the application prospect is wide.
Description of the drawings:
FIG. 1 shows the double enzyme activity Fe according to the present invention 2 (MoO 4 ) 3 Schematic diagram of bacteriostasis principle of @ GOx.
FIG. 2 shows iron molybdate and Fe prepared in example 1 of the present invention 2 (MoO 4 ) 3 A characterization electron microscope image of @ GOx, wherein A is a scanning electron microscope image of iron molybdate; b is a high-resolution transmission electron microscope image of the iron molybdate; c is Fe 2 (MoO 4 ) 3 Scanning electron microscope of @ GOxA drawing.
FIG. 3 is a diagram of Fe according to the present invention 2 (MoO 4 ) 3 Schematic of peroxidase and glucose oxidase property detection at GOx and schematic of active oxygen type detection, wherein, A is Fe 2 (MoO 4 ) 3 Detecting peroxidase properties of @ GOx; b is Fe 2 (MoO 4 ) 3 Detecting the glucose oxidase property of @ GOx; panel C is the enzyme cascade Fe 2 (MoO 4 ) 3 ESR detection results of hydroxyl radical generated by @ GOx.
FIG. 4 is a diagram of example 3Fe according to the present invention 2 (MoO 4 ) 3 Photo graph of in vitro bacteriostasis experiment result of @ GOx.
FIG. 5 is a diagram of example 4Fe according to the present invention 2 (MoO 4 ) 3 Photo graph of in vivo bacteriostasis test results of @ GOx.
FIG. 6 is a chart showing the process of example 5 according to the present invention 2 (MoO 4 ) 3 Schematic of the results of major organs of mice treated with @ GOx.
FIG. 7 is a diagram of example 5Fe according to the present invention 2 (MoO 4 ) 3 Schematic of cytotoxicity test results of @ GOx.
The specific embodiment is as follows:
the invention will now be described in more detail with reference to the following examples and with reference to the accompanying drawings.
Example 1:
fe according to the present example 2 (MoO 4 ) 3 The preparation method of the @ GOx double-enzyme-activity antibacterial material comprises the following specific process steps:
iron molybdate (Fe) 2 (MoO 4 ) 3 ) 1.0mg of the iron molybdate-loaded glucose oxidase double-enzyme activity antibacterial material (Fe) is obtained by dispersing 1mg of the iron molybdate-loaded glucose oxidase double-enzyme activity antibacterial material in acetate buffer solution with pH=4, adding 1mg of glucose oxidase, stirring at normal temperature of 200r/min for 10 minutes, centrifuging at rotation speed of 5000r/min for 5 minutes, collecting precipitate, and freeze-drying at-40 ℃ for 24 hours 2 (MoO 4 ) 3 @GOx)。
The preparation method of the iron molybdate according to the embodiment comprises the following steps:
(1) Iron salt andthe molybdate is dissolved in two parts of deionized water with the same volume respectively, and the mol ratio of the ferric salt to the molybdate is 1:1.5, dropwise adding the molybdate solution into the ferric salt solution, adjusting the pH value of a final reaction system to be=3 by using sodium hydroxide, stirring at normal temperature of 200r/min for 30min, transferring into a 100mL reaction kettle, and reacting for 15h at 150 ℃; the ferric salt is Fe 2 (NO 3 ) 3 The method comprises the steps of carrying out a first treatment on the surface of the The molybdate is Na 2 MoO 4 。
(2) Taking out the reacted sample, centrifuging at 5000r/min for 10min, removing supernatant, collecting precipitate, adding ultrapure water, centrifuging repeatedly until supernatant is colorless, collecting precipitate, and lyophilizing for 24 hr to obtain Fe 2 (MoO 4 ) 3 。
In this example, fe was prepared by using a scanning electron microscope and a high-resolution transmission electron microscope pair 2 (MoO 4 ) 3 Characterization was performed, and the results are shown in FIG. 2A and FIG. 2B, fe 2 (MoO 4 ) 3 Has a pattern multi-layer structure, good dispersibility, average diameter of 15+/-4.75 mu m, large specific surface area, more active sites and stronger negative potential, so that the glucose oxidase is more effectively loaded. Scanning electron microscope for preparing Fe 2 (MoO 4 ) 3 Characterization by @ GOx, results are shown in FIG. 2C, where Fe is seen from the figure 2 (MoO 4 ) 3 The morphology of @ GOx is uniform.
Then the prepared double-enzyme-activity antibacterial material is subjected to enzyme activity characterization, and the figures 3A and 3B respectively show Fe 2 (MoO 4 ) 3 Excellent peroxidase and glucose oxidase properties @ GOx. Fe (Fe) 2 (MoO 4 ) 3 GOx Electron Spin Resonance (ESR) was performed to further verify the form of active oxygen. As shown in FIG. 3C, fe 2 (MoO 4 ) 3 Characteristic peaks 1:2:2:1 appear at @ GOx, illustrating Fe 2 (MoO 4 ) 3 The peroxidase at GOx is capable of converting hydrogen peroxide into toxic hydroxyl radicals.
Example 2:
fe according to the present example 2 (MoO 4 ) 3 The preparation method of the @ GOx double-enzyme-activity antibacterial material comprises the following specific process steps:
iron molybdate (Fe) 2 (MoO 4 ) 3 ) 1.0mg of the iron molybdate-loaded glucose oxidase double-enzyme activity antibacterial material (Fe) is dispersed in acetate buffer solution with pH=4, 1.0mg of glucose oxidase is added, stirring is carried out for ten minutes at normal temperature of 300r/min, then centrifugation is carried out for 10 minutes at the rotating speed of 8000r/min, sediment is collected, freeze drying is carried out for 36 hours at-50 ℃, and the iron molybdate-loaded glucose oxidase double-enzyme activity antibacterial material (Fe 2 (MoO 4 ) 3 @GOx)。
The preparation method of the iron molybdate according to the embodiment comprises the following steps:
(1) Iron salt and molybdate are respectively dissolved into two parts of deionized water with the same volume, and the molar ratio of the iron salt to the molybdate is 1:1.5, dropwise adding the molybdate solution into the ferric salt solution, adjusting the pH value of a final reaction system to be=3 by using sodium hydroxide, stirring at normal temperature 300r/min for 30min, transferring into a 100mL reaction kettle, and reacting at 200 ℃ for 8h; the ferric salt is Fe 2 (NO 3 ) 3 The method comprises the steps of carrying out a first treatment on the surface of the The molybdate is Na 2 MoO 4 。
(2) Taking out the reacted sample, centrifuging at 10000r/min for 5min, removing supernatant, collecting precipitate, adding ultrapure water, centrifuging repeatedly until supernatant is colorless, collecting precipitate, and lyophilizing for 24 hr to obtain Fe 2 (MoO 4 ) 3 。
Example 3:
the present example relates to Fe 2 (MoO 4 ) 3 Application experiment of @ GOx in vitro bacteriostasis Fe prepared in example 1 2 (MoO 4 ) 3 Application of GOx to in vitro antibacterial experiments single colony resistant bacteria (resistant E.coli and resistant Staphylococcus aureus) on solid LB medium were inoculated into 50mL of sterile liquid LB medium (containing tryptone (0.5 g), yeast extract (0.25 g) and NaCl (0.5 g)), and then suspensions of the resistant bacteria were incubated overnight at 37℃at 180r/min on a rotary shaker; the bacteria were then diluted to 10 with sterile PBS 6 CFU/mL, the bacterial solution (100. Mu.l) obtained was mixed with 200. Mu.l of a mass concentration of 60. Mu.g.mL -1 Fe of (2) 2 (MoO 4 ) 3 Incubation of aqueous solution @ GOx with 700. Mu.l PBS buffer for 30min at 37 ℃Then, 50 microliters of the mixed solution is uniformly coated on a solid LB culture medium, the solid LB culture medium is placed in an incubator at 37 ℃ for culturing for 12 hours, and the bacterial colony number is counted by a CFU method; parallel control experiments were performed using PBS as a blank control, with iron molybdate or glucose oxidase alone to treat bacteria. The measurement results are shown in FIG. 4, which shows that GOx or Fe alone is used 2 (MoO 4 ) 3 Compared with 46.2% -59.404% of sterilization rate to escherichia coli and staphylococcus aureus, fe 2 (MoO 4 ) 3 The @ GOx groups are 98.396% and 98.776%, respectively, which shows that the synergistic effect of the ferric molybdate and the glucose oxidase exerts a stronger antibacterial effect.
Example 4:
the present example is Fe 2 (MoO 4 ) 3 The application experiment of the @ GOx in the aspect of in vivo bacteriostasis is specifically as follows: fe prepared in example 1 2 (MoO 4 ) 3 Application of GOx to in vivo antibacterial experiments mice were divided into 4 groups of 3 after infection with MRSA (methicillin resistant Staphylococcus aureus) by creating a model of wound infection of mice using male and female hybrid DBDB mice (six weeks, 30-34 g, 12 total) and circular skin lesions of approximately l cm in diameter were made on the backs of all mice. Each group receives different therapeutic drugs, namely PBS and Fe 2 (MoO 4 ) 3 +H 2 O 2 、GOx、Fe 2 (MoO 4 ) 3 @ GOx; and the whole treatment course was set to 10 days, and photographs of the wound were collected every 2 days, and the results are shown in fig. 5. As can be seen from FIG. 5, fe 2 (MoO 4 ) 3 The wound treated by the @ GOx group is the smallest and the healing effect is the most obvious, indicating that Fe 2 (MoO 4 ) 3 The @ GOx has obvious antibacterial property on drug-resistant bacteria and can promote the rapid healing of wounds of mice.
Example 5:
the present example is Fe 2 (MoO 4 ) 3 Application experiment of GOx in biotoxicity test the experimental cells were mouse L929 cells, and the cells were individually enriched with 100. Mu.l Fe at different concentrations (0,10,20,30,45,50,60. Mu.g/mL) 2 (MoO 4 ) 3 Incubating the 7 mixtures of aqueous solution @ GOx and 150. Mu.l DMEM solution for 24 hours, then adding 10mL of 5mg/mL MTT aqueous solution, respectively, and continuing to pass 5% CO 2 And incubating at 37deg.C for 4 hr, dissolving the crystals with 100 microliters of dimethyl sulfoxide, measuring absorbance of each well at 490nm of ELISA, and measuring the results without Fe as shown in FIG. 7 2 (MoO 4 ) 3 The cell activity incubated with GOx solution was set to 100% and the results showed that Fe was present at different concentrations (10,20,30,45,50,60. Mu.g/mL) respectively 2 (MoO 4 ) 3 After incubation with aqueous solution @ GOx, the cell activity was maintained at 99% or more. In addition, PBS and Fe with a mass-volume concentration of 60. Mu.g/mL were used, respectively 2 (MoO 4 ) 3 The results of treatment of the major organs (liver, heart, lung, spleen and kidney) of mice with aqueous solution @ GOx, as shown in FIG. 6, showed no significant damage or tissue abnormality to the liver, heart, lung, spleen and kidney, demonstrating that Fe was prepared 2 (MoO 4 ) 3 The @ GOx double-enzyme activity antibacterial material has good biocompatibility and low cytotoxicity.

Claims (7)

1. The preparation method of the double-enzyme-activity antibacterial material is characterized by comprising the following specific process steps of: dispersing ferric molybdate in acetate buffer solution with pH=4, adding glucose oxidase, stirring at normal temperature of 200-300 r/min for 10 minutes, centrifuging, collecting precipitate, freeze-drying to obtain a double-enzyme-activity antibacterial material Fe of ferric molybdate loaded glucose oxidase 2 (MoO 4 ) 3 @GOx；
The iron molybdate has a pattern multilayer structure, good dispersibility, average diameter of 15+/-4.75 mu m, large specific surface area, more active sites and stronger negative potential.
2. The preparation method of the double-enzyme-activity antibacterial material according to claim 1, wherein the mass ratio of the ferric molybdate to the glucose oxidase is 1:1.
3. the method for preparing a double-enzyme-activity antibacterial material according to claim 1, wherein the centrifugal speed is 5000-8000 r/min and the centrifugal time is 5-10 min.
4. The method for preparing the double-enzyme-activity antibacterial material according to claim 1, wherein the freeze-drying time is 24-36 hours; freezing temperature is-40 to-50 ℃.
5. The method for preparing the double-enzyme-activity antibacterial material according to claim 1, wherein the method for preparing the iron molybdate is as follows:
(1) Iron salt and molybdate are respectively dissolved into two parts of deionized water with the same volume, and the molar ratio of the iron salt to the molybdate is 1:1.5 dissolving the catalyst, dropwise adding a molybdate solution into an iron salt solution, adjusting the pH value of a final reaction system to be 2.5-4 by using sodium hydroxide, stirring at normal temperature of 200-300 r/min for 30min, transferring the mixture into a 100mL reaction kettle, and reacting for 8-15 h at 150-200 ℃;
(2) Taking out the reacted sample, centrifuging for 5-10 min at 5000-10000 r/min, removing supernatant, collecting precipitate, adding ultrapure water, centrifuging repeatedly until the supernatant is colorless, collecting precipitate, freeze drying for 24-48 h to obtain Fe 2 (MoO 4 ) 3 。
6. The method for preparing a double-enzyme-activity antibacterial material according to claim 5, wherein the iron salt is Fe 2 (NO 3 ) 3 The method comprises the steps of carrying out a first treatment on the surface of the The molybdate is Na 2 MoO 4 。
7. The bacteriostatic material Fe with double enzyme activities obtained by the preparation method according to any one of claims 1-6 2 (MoO 4 ) 3 @GOx。
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