Ferroxy artificial enzyme as well as preparation method and application thereof

Abstract

The invention discloses an iron-oxygen-based artificial enzyme as well as a preparation method and application thereof. The method comprises the following steps: preparing a chelating layer on a basement membrane; mixing ferric chloride hexahydrate, ferrous sulfate heptahydrate, histidine and a solvent to obtain a mineralized precursor solution, and removing oxygen in the mineralized precursor solution; the mineralization precursor solution is spread on the chelating layer to form a solution layer, the mineralization precursor solution of the solution layer is subjected to a mineralization reaction in an alkaline environment, and a mineral layer is generated on the chelating layer. According to the method, the histidine is added into the mineralization precursor solution, the mineralization process of the mineralization precursor solution is intervened, more ferrihydrite and delta-iron oxyhydroxide stably exist in a mineral layer, meanwhile, the histidine on the surfaces of various mineral crystals also creates a chiral microenvironment, and a chiral microenvironment is formed. And the mineral layer realizes the synchronous improvement of the activities of multiple enzymes such as CAT-like, POD-like and SOD-like, and is beneficial to the multifunctionality of the iron-based artificial enzyme in different scenes.

Description

Technical Field

[0001] This invention relates to the field of functional materials, specifically to an ferrooxy artificial enzyme, its preparation method, and its application. Background Technology

[0002] Natural enzymes found in living organisms require a mild environment to exert their catalytic effects, which limits their application in engineering. In recent years, researchers have developed a series of "artificial enzymes" with micro- and nanostructures to mimic the activities of natural enzymes. Their matrix materials include metal-organic frameworks, layered double hydroxides, noble metal nanoparticles, transition metal oxides, and carbon-based nanomaterials, each exhibiting different enzyme-like activities, such as oxidase-like, peroxidase-like, catalase-like, superoxide dismutase-like, and hydrolase-like activities.

[0003] Ferroxyl-based artificial enzymes have been extensively studied due to their excellent catalytic activity, low cost, and good stability. As a transition metal, iron can participate in reactions in either electrophilic or nucleophilic forms. For example, in the peroxidase-like activity mechanism, the active species adsorbs onto iron sites to form intermediates, ultimately promoting the conversion of H2O2 into highly reactive hydroxyl radicals.

[0004] Currently, by introducing different heteroatoms, highly active sites can be precisely designed and the electronic structure of materials can be controlled at the atomic level, effectively enhancing the catalytic activity of ferrooxy artificial enzymes. However, this existing atomic doping strategy requires precise and complex control methods, which limits the application of ferrooxy artificial enzymes to some extent. More importantly, existing strategies can only target the enhancement and adjustment of one type of enzyme activity, and cannot simultaneously address the activities of other types of enzymes. This is because there is an unavoidable negative correlation between the various enzyme-like activities of ferrooxy artificial enzymes. Specifically, under acidic conditions, peroxidase-like (POD-like) and superoxide dismutase-like (SOD-like) activities depend on the divalent iron (Fe2+) in the iron oxide crystal. 2+ ), Fe 2+ It is the reaction center for capturing relevant active species and undergoing intermediate transformation; while under near-neutral conditions, catalase-like activity depends on ferric iron (Fe) in the iron oxide crystal. 3+ Therefore, it can be seen that the surface regulation of ferrooxy artificial enzymes leads to changes in the iron valence state, causing different enzyme activities to improve or deteriorate in opposite directions, which is not conducive to the multifunctionality of ferrooxy artificial enzymes, resulting in limited application range and single function. Summary of the Invention

[0005] One object of the present application is to provide a preparation method of ferrioxamine artificial enzyme, which uses histidine to intervene in the mineralization process to achieve the purpose of regulating the crystal form of ferrioxamine mineral, so that the mineral layer has a stable existing multi-phase ferrioxamine mineral composite; at the same time, the histidine combined on the surface of the mineral makes the ferrioxamine artificial enzyme realize the synchronous improvement of multiple enzyme-like activities, breaks the negative correlation effect of enzyme-like activity of ferrioxamine mineral under the limitation of iron valence, enriches the functionality of ferrioxamine artificial enzyme, and expands its application range.

[0006] The present application is realized by the following technical solutions:

[0007] A preparation method of ferrioxamine artificial enzyme, comprising the following steps:

[0008] S1: preparing a chelating layer on a substrate film;

[0009] S2: mixing ferric chloride hexahydrate, ferrous sulfate heptahydrate, histidine and a solvent to obtain a mineralization precursor solution, and removing oxygen in the mineralization precursor solution;

[0010] S3: spreading the mineralization precursor solution on the chelating layer to form a solution layer, and performing a mineralization reaction on the mineralization precursor solution in the solution layer under an alkaline environment to generate a mineral layer on the chelating layer.

[0011] In step S1 of the technical solution, the substrate film can be selected according to actual needs. For example, in some preferred embodiments, polyether sulfone can be used as the substrate film. Polyether sulfone has high temperature stability (180 °C), acid and alkali resistance (tolerance range pH 1-14, including the pH change in the mineralization reaction process), and excellent tensile, compression and creep resistance. In one or more embodiments, polyvinylidene fluoride (PVDF), cellulose derivative film, polysulfone (PSF) and heteronaphthalene biphenyl polyether sulfone (PPES) and other materials can also be used as the substrate film.

[0012] After the substrate film is cleaned, a chelating layer is prepared on the substrate film. The chelating layer can use a polymer with phenolic hydroxyl and amine groups to exhibit hydrophilicity and adsorbability by the synergistic effect of phenolic hydroxyl and amine groups. The hydrophilicity is conducive to the contact between the chelating layer and the mineralization precursor solution, while the adsorbability can chelate iron ions, so that the subsequently formed mineral layer can be more stably connected to the chelating layer. In some preferred embodiments, the chelating layer is a polydopamine layer. In one or more embodiments, the chelating layer can also use polyvinyl alcohol-dopamine graft copolymer (OPVA-DA), dopamine-polyethylene glycol derivative (DA-PEG) and other materials.

[0013] In some preferred embodiments, in step S1, dopamine hydrochloride solution is added to the substrate membrane until the dopamine hydrochloride solution covers the surface of the substrate membrane, and a base solution is added to the dopamine hydrochloride solution to form a polydopamine layer on the substrate membrane, which serves as the chelating layer. In one or more embodiments, the base solution can be sodium hydroxide or potassium hydroxide to increase the alkalinity of the dopamine hydrochloride solution so that the dopamine hydrochloride polymerizes on the substrate membrane to form a polydopamine layer.

[0014] In step S2, ferric chloride hexahydrate, ferrous sulfate heptahydrate, and histidine are added to a solvent to obtain a mineralization precursor solution, and oxygen in the mineralization precursor solution is removed or the oxygen content in the mineralization precursor solution is reduced during preparation. In some preferred embodiments, in step S2, ferric chloride hexahydrate and ferrous sulfate heptahydrate are dissolved in a solvent to obtain a mixed solution, inert gas is introduced into the mixed solution, and then histidine is added to the mixed solution to obtain a mineralization precursor solution, and inert gas is introduced into the mineralization precursor solution.

[0015] In one or more embodiments, the histidine is L-histidine and / or D-histidine.

[0016] In step S3, the mineralization precursor solution is added to the chelating layer, and after the mineralization precursor solution naturally spreads on the chelating layer, a solution layer covering the upper surface of the chelating layer is formed. The solution layer has a thickness of millimeters, for example, 0.3-1.0 mm. Subsequently, the mineralization precursor solution in the solution layer initiates a mineralization reaction in an alkaline environment, for example, by contacting with ammonia gas, and finally forms a mineral layer on the surface of the chelating layer.

[0017] In the technical solution, by adding histidine to the mineralization precursor solution, the crystal form of the iron-oxy mineral in the mineralization process is controlled. Specifically, as the alkalinity of the mineralization precursor solution gradually increases, the iron ions in the mineralization precursor solution are first converted into amorphous iron oxide precursors, and continue to convert towards the final highly crystalline magnetite phase, and in the process of conversion, a series of iron-oxy mineral intermediates are formed, such as ferrihydrite and δ-hydroxy iron oxide. After adding histidine to the mineralization precursor solution, the imidazole group and carboxyl group of histidine rely on their ability to complex metal ions to interact with iron ions in the mineralization precursor solution. Due to this strong complexation, the conversion process of mineralization is intervened, and chiral histidine binds to specific crystal planes of mineral intermediates, promoting the next step of crystal transformation to proceed in a different direction, ultimately obtaining a stable multi-phase iron-oxy mineral complex. That is, the addition of histidine makes the mineral layer not only have the final magnetite, but also have the intermediate ferrihydrite and δ-hydroxy iron oxide. The different active crystal planes brought by the rich crystal forms expand the potential of the mineral layer to exhibit a wide range of enzyme-like activities, and can exhibit catalase-like (CAT-like), peroxidase-like (POD-like), and superoxide dismutase-like (SOD-like) activities.

[0018] In the technical solution, by adding histidine to the mineralization precursor solution, the crystal form of the iron-oxy mineral in the mineralization process is controlled. Specifically, as the alkalinity of the mineralization precursor solution gradually increases, the iron ions in the mineralization precursor solution are first converted into amorphous iron oxide precursors, and continue to convert towards the final highly crystalline magnetite phase, and in the process of conversion, a series of iron-oxy mineral intermediates are formed, such as ferrihydrite and δ-hydroxy iron oxide. After adding histidine to the mineralization precursor solution, the imidazole group and carboxyl group of histidine rely on their ability to complex metal ions to interact with iron ions in the mineralization precursor solution. Due to this strong complexation, the conversion process of mineralization is intervened, and chiral histidine binds to specific crystal planes of mineral intermediates, promoting the next step of crystal transformation to proceed in a different direction, ultimately obtaining a stable multi-phase iron-oxy mineral complex. That is, the addition of histidine makes the mineral layer not only have the final magnetite, but also have the intermediate ferrihydrite and δ-hydroxy iron oxide. The different active crystal planes brought by the rich crystal forms expand the potential of the mineral layer to exhibit a wide range of enzyme-like activities, and can exhibit catalase-like (CAT-like), peroxidase-like (POD-like), and superoxide dismutase-like (SOD-like) activities.

[0019] In the technical solution, the relative concentration of histidine determines the number of action sites between histidine and transformed crystals, resulting in different intervention degrees, and finally obtaining mineral complexes with different contents of ferrihydrite, δ-hydroxy iron oxide and magnetite; at the same time, the relative concentration of histidine also determines the content of histidine on the surface of the mineral, thereby affecting the chiral microenvironment on the surface of the mineral, causing the difference in the amplitude of the synchronous promotion of multiple enzyme-like activities. As can be seen, by adjusting the concentration of histidine in the mineralization precursor solution, multiple enzyme-like activities can be efficiently and conveniently controlled, the control is more flexible, and the technical solution can be used in various application scenarios and has wide popularization value.

[0020] As a preferred embodiment of the concentration of histidine in the present application, in step S2, the concentration of histidine in the mineralization precursor solution is 10-20 mM. It is found through experiments that when the concentration of histidine in the mineralization precursor solution is 10-20 mM, the activities of CAT-like, POD-like and SOD-like can be simultaneously promoted compared with the mineralization precursor solution without adding histidine. Preferably, when the concentration of histidine is 20 mM, the three enzyme-like activities are all obviously increased.

[0021] Further, in step S2, the molar ratio of the ferric chloride hexahydrate to the ferrous sulfate heptahydrate is 1:1-3:1. The molar ratio of the ferric chloride hexahydrate to the ferrous sulfate heptahydrate determines the order and content of different crystal forms of intermediates, and as a preferred embodiment, the molar ratio of the ferric chloride hexahydrate to the ferrous sulfate heptahydrate in the technical solution is set to 1:1-3:1. In some more preferred embodiments, the molar ratio of the ferric chloride hexahydrate to the ferrous sulfate heptahydrate is 2:1.

[0022] Further, in step S3, a pressing plate is placed on the solution layer, the pressing plate has a hydrophobic surface, the hydrophobic surface contacts the upper surface of the solution layer, and the solution layer contacts the ammonia gas through the side surface of the solution layer, and the ammonia gas provides the alkaline environment for the mineralization precursor solution of the solution layer.

[0023] In the technical solution, the mineralization process of the mineralization precursor solution in the solution layer is carried out in a limited space. Specifically, when the mineralization precursor solution infiltrates the chelating layer, a pressing plate is placed on the spreading surface of the solution layer, that is, on the upper surface of the solution layer. The lower surface of the pressing plate is a hydrophobic layer, which reduces the adhesion of the mineralization precursor solution to the pressing plate. The pressing plate extrudes the solution layer under its own gravity, promotes the movement of the mineralization precursor solution to the edge of the chelating layer, and finally ensures that the edge of the solution layer is flush with the edge of the chelating layer, and the mineralization precursor solution is uniformly distributed on the chelating layer.

[0024] Next, in the ammonia environment, the pressing plate blocks the upper surface of the solution layer from contacting the ammonia, while the lower surface of the solution layer is the chelating layer, so that the ammonia in the environment can only gradually contact the mineralization precursor solution through the side surface of the solution layer, thereby forming an alkali gradient on the solution layer, which gradually changes from the edge to the center. With the gradual increase of the alkali, the mineralization precursor solution at the edge starts the mineralization reaction earlier, while the mineralization precursor solution at the center mineralizes later, thereby effectively slowing down the speed of the mineralization process, making the mineral intermediate transform more slowly, allowing the histidine to stably bind to the special crystal surface, thereby stabilizing more metastable mineral crystal phases. Finally, the mineralization precursor solution in the solution layer is completely reacted, and a highly consistent and uniformly distributed mineral layer is obtained.

[0025] Further, in step S3, the solution layer is placed in a closed space, and the closed space has an ammonia water solution therein, and the ammonia water solution volatilizes ammonia gas for contacting the solution layer at room temperature.

[0026] In the technical solution, the substrate film, the chelating layer, the solution layer and the pressing plate are placed in a closed container. The container has an ammonia water solution therein, and the ammonia water solution does not contact the solution layer. The ammonia gas naturally volatilized from the ammonia water solution contacts the side surface of the solution layer through the gap between the pressing plate and the chelating layer, thereby gradually starting the mineralization reaction of the mineralization precursor solution at each position on the solution layer.

[0027] In one or more embodiments, the ammonia water solution is a dilute ammonia water solution with a mass fraction of 1% to 5%.

[0028] In one or more embodiments, the mineralization reaction lasts for 18 to 24 hours at room temperature.

[0029] Another object of the present application is to provide an iron-oxygen artificial enzyme, which comprises a substrate film, a chelating layer and a mineral layer connected in sequence, and the mineral layer comprises ferrihydrite, δ-hydroxy ferric oxide and magnetite, and the surface of the mineral layer has histidine.

[0030] In the technical solution, based on the chiral histidine mineralized iron source, the mineral layer formed on the chelating layer has stable ferrihydrite, δ-hydroxy ferric oxide and magnetite three crystal forms due to the intervention of histidine in the mineralization process, which provides conditions for the iron-oxygen artificial enzyme to exhibit catalase activity, peroxidase-like activity and superoxide dismutase-like activity. At the same time, the histidine on the surface of the three crystal forms creates a chiral microenvironment, which can improve the affinity of the surface to the reactant species, the charge transfer behavior of the surface and the desorption effect of the intermediate product in the related enzyme catalysis process, realize the integration and synchronous improvement of multiple enzyme-like activities, and break the negative correlation effect of the enzyme-like activity of the iron-oxygen mineral under the limitation of the iron valence state.

[0031] Further, the area of the mineral layer is 10 to 50 mm 2When the mineralization area is too large, the ammonia gradient will cause too large an alkalinity difference between the edge and the center of the precursor solution, making it difficult to complete the entire mineralization process at the center, and the iron source will be depleted at the edge, resulting in a defect of no ore in the center. Therefore, in the technical solution, the area of the mineral layer is 10-50mm 2 In some preferred embodiments, multiple ferrioxamine artificial enzymes can be spliced to obtain the desired area.

[0032] Another object of the present application is to provide a wound dressing based on any of the foregoing ferrioxamine artificial enzymes, which takes advantage of the characteristic of the mineral layer having multiple enzyme-like activities, and has strong application potential in efficient bacterial killing and efficient wound healing.

[0033] In the technical solution, the mineral layer realizes the synchronous improvement of CAT-like, POD-like and SOD-like triple enzyme-like activities, which is beneficial to the multifunctionality of the ferrioxamine artificial enzyme in different scenarios. For example, the enhancement of peroxidase-like activity can convert H2O2 into a large amount of hydroxyl radicals, which can be used for efficient bacterial killing, and the cascade of superoxide dismutase-like activity and catalase-like activity can significantly remove H2O2 and superoxide anion radicals and other active oxygen, promoting the healing and recovery of wounds.

[0034] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0035] 1. By adding histidine to the mineralization precursor solution, the present application intervenes in the mineralization process of the mineralization precursor solution, and more stable goethite and delta-hydroxy ferric oxide exist in the mineral layer. At the same time, the histidine on the surface of various mineral crystals also creates a chiral microenvironment, so that the mineral layer realizes the synchronous improvement of CAT-like, POD-like and SOD-like multiple enzyme-like activities, which is beneficial to the multifunctionality of the ferrioxamine artificial enzyme in different scenarios.

[0036] 2. The present application can adjust the content of different crystal forms of minerals in the mineral layer by the concentration of histidine, and then efficiently and conveniently regulate the multiple enzyme-like activities, which is more flexible and can be used in various application scenarios.

[0037] 3. By mineralizing the solution in a confined space, the present application can obtain a mineral layer with uniform distribution and high consistency. At the same time, ammonia gradually diffuses in the solution layer to start the mineralization reaction, which can effectively slow down the speed of the mineralization process, so that the mineral intermediates are converted more slowly, and the histidine can be stably combined with special crystal faces to stabilize more metastable mineral crystal phases. BRIEF DESCRIPTION OF DRAWINGS

[0038] The accompanying drawings, which are included to provide a further understanding of the embodiments of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:

[0039] Figure 1 A flow chart of the preparation method of the ferrioxo artificial enzyme in the embodiment of the application is shown;

[0040] Figure 2 A scanning electron microscope image of the ferrioxo artificial enzyme 2 in the embodiment of the application is shown;

[0041] Figure 3 A transmission electron microscope-selected area electron diffraction image of the mineral layer of the ferrioxo artificial enzyme 2 in the embodiment of the application is shown;

[0042] Figure 4 X-ray photoelectron spectroscopy of the iron element of the mineral layer of the ferrioxo artificial enzymes 1-4 in the embodiment of the application is shown;

[0043] Figure 5 The intensity of three kinds of enzyme activities of the ferrioxo artificial enzymes 1-4 in the embodiment of the application is shown. DETAILED DESCRIPTION

[0044] In order to make the objects, technical solutions and advantages of the application clearer, further detailed description will be given to the application in combination with embodiments and drawings, the schematic embodiments of the application and the description thereof are only used to explain the application, and do not limit the application.

[0045] All raw materials of the application are not particularly limited in source, and can be purchased on the market or prepared according to conventional methods well known to those skilled in the art. All raw materials of the application are not particularly limited in purity, and the application preferably adopts analytical purity or conventional purity requirements in the field of artificial enzymes. The trade name and abbreviation of all raw materials of the application belong to conventional trade names and abbreviations in the art, and each trade name and abbreviation is clear and explicit in the field of its relevant use. Those skilled in the art can purchase or prepare them by conventional methods according to the trade name, abbreviation and corresponding use.

[0046] The expression of the substituent group is not particularly limited, and the expression well known to those skilled in the art is adopted. Those skilled in the art can correctly understand the meaning thereof according to the expression based on common sense.

[0047] In addition, the term "connection" used herein can be direct connection or indirect connection via other groups without special description.

[0048] Example 1:

[0049] As Figure 1A method for preparing an iron-oxygen artificial enzyme is shown, comprising the following steps:

[0050] S1: preparing a chelating layer on a substrate film;

[0051] S2: mixing ferric chloride hexahydrate, ferrous sulfate heptahydrate, histidine, and a solvent to obtain a mineralization precursor solution, and removing oxygen in the mineralization precursor solution;

[0052] S3: spreading the mineralization precursor solution on the chelating layer to form a solution layer, and performing a mineralization reaction on the solution layer in an alkaline environment to generate a mineral layer on the chelating layer.

[0053] In some embodiments, the substrate film is cleaned with deionized water, and then dried with nitrogen. Then, the substrate film is laid flat, and a chelating solution is added dropwise thereon. In some preferred embodiments, taking the polydopamine layer as the chelating layer, in step S1, a dopamine hydrochloride solution is added dropwise to the substrate film until the dopamine hydrochloride solution covers the surface of the substrate film. An alkali solution is added to the dopamine hydrochloride solution to generate a polydopamine layer on the substrate film.

[0054] In some preferred embodiments, in step S2, the molar ratio of the ferric chloride hexahydrate to the ferrous sulfate heptahydrate is 1:1 to 3:1.

[0055] In some preferred embodiments, in step S3, a pressing plate is placed on the solution layer, the pressing plate has a hydrophobic surface, the hydrophobic surface contacts the upper surface of the solution layer, the solution layer is placed in a closed space, the closed space has an ammonia solution therein, the ammonia solution volatilizes ammonia gas at room temperature for contacting the solution layer, the solution layer contacts the ammonia gas through the side surface of the solution layer, and the ammonia gas provides the alkaline environment for the mineralization precursor solution of the solution layer.

[0056] Embodiment 2:

[0057] In this embodiment, an iron-oxygen artificial enzyme 1 is prepared.

[0058] (1) 152 milligrams of dopamine hydrochloride is added to 10 milliliters of deionized water, and ultrasonic treatment is performed for 10 minutes until the dopamine hydrochloride is completely dissolved. Meanwhile, a substrate film is cleaned with deionized water and dried with nitrogen. The substrate film is selected from polyether sulfone.

[0059] (2) The dried polyether sulfone substrate film is laid flat, and a dopamine hydrochloride solution is added dropwise to the surface of the substrate film using a dropper until the surface of the substrate film is covered with the dopamine hydrochloride solution. Then, a sodium hydroxide solution is added, and the dopamine is polymerized on the surface of the substrate film. This process is allowed to stand for 5 minutes.

[0060] (3) The substrate film with the polydopamine layer is cleaned with deionized water for three times, and then dried with nitrogen for later use.

[0061] (4) Preparation of mineralized precursor solution, specifically, 432 mg of ferric chloride hexahydrate and 222 mg of ferrous sulfate heptahydrate were dissolved in 10 mL of deionized water, followed by nitrogen gas for 10 minutes to remove oxygen, then 15.52 mg of L-histidine (10 mM) was added, followed by nitrogen gas for 10 minutes to remove oxygen again;

[0062] (5) The prepared mineralized precursor solution was evenly added to the surface of the dry polydopamine layer, and waited for about 1 minute for the mineralized precursor solution to naturally infiltrate the surface. Then a hydrophobic pressing plate was used to contact the spreading surface of the mineralized precursor solution and was squeezed until the outer edge of the mineralized precursor solution was at the same vertical height as the edge of the substrate film, and this state was maintained in a closed container;

[0063] (6) 10 mL of 2% dilute ammonia solution was added to the closed container, and ammonia gas was naturally volatilized at room temperature, then dissolved in the mineralized precursor solution to increase the pH, and start the mineralization reaction, which continued for 20 hours at room temperature;

[0064] (7) The volatilized ammonia water solution was removed, and the mineralized substrate film was naturally dried in an air circulating environment, then washed with deionized water several times to ensure that the surface salt crystal impurities were completely dissolved, then naturally dried again, and finally obtained the iron-oxygen-based artificial enzyme with a chiral mineral layer.

[0065] Example 3:

[0066] Example 3 prepared an iron-oxygen-based artificial enzyme 2. The preparation method and reaction conditions were basically the same as those of Example 2, except that in step (4), 31.04 mg of L-histidine was added to the mineral precursor solution, so that the concentration of L-histidine in the mineral precursor solution was 20 mM.

[0067] Example 4:

[0068] Example 4 prepared an iron-oxygen-based artificial enzyme 3. The preparation method and reaction conditions were basically the same as those of Example 2, except that in step (4), 62.08 mg of L-histidine was added to the mineral precursor solution, so that the concentration of L-histidine in the mineral precursor solution was 40 mM.

[0069] Comparative Example:

[0070] The preparation method and reaction conditions of the iron-oxygen-based artificial enzyme 4 of the comparative example were basically the same as those of Example 2, except that in step (4), no L-histidine was added to the mineral precursor solution, and the concentration of L-histidine in the mineral precursor solution was 0 mM.

[0071] Example 5:

[0072] In this embodiment, the ferroxane artificial enzyme 2 is characterized. Figure 2 A scanning electron microscope image of the ferroxane artificial enzyme 2 is shown. Figure 3 A transmission electron microscopy-selected area electron diffraction image of the mineral layer of the ferroxane artificial enzyme 2 is shown, from which it can be seen that the crystal form of the mineral layer includes magnetite, ferrihydrite and δ-hydroxy ferric oxide.

[0073] Figure 4 X-ray photoelectron spectroscopy of the iron element of the mineral layer of the ferroxane artificial enzymes 1-4 is shown. As can be seen from the figure, with the increase of the concentration of L-histidine, the proportion of divalent iron (mainly provided by magnetite) in the final mineral layer gradually decreases, while the proportion of ferrihydrite and δ-hydroxy ferric oxide increases.

[0074] Example 5:

[0075] In this embodiment, the intensity of three kinds of enzyme-like activities of the ferroxane artificial enzymes 1-4 is detected. The test results are shown in Figure 5 After the mineralization reaction gradually started by ammonia in a confined space, the ferroxane artificial enzyme 4 without the addition of L-histidine has a crystal phase composition mainly composed of magnetite, and itself shows a certain intensity of CAT-like, POD-like and SOD-like activities, and the three kinds of enzyme-like activities show a negative correlation effect under the limitation of iron valence state, theoretically, the ratio of divalent iron / trivalent iron in the crystal gradually decreases, resulting in the decrease of POD-like and SOD-like activities, and the increase of CAT-like activity.

[0076] After the addition of 10-20 mM of L-histidine in the mineralization precursor solution, although the ratio of divalent iron / trivalent iron in the crystal gradually decreases, as shown in Figure 5 the three kinds of enzyme-like activities break the negative correlation effect of enzyme-like activities, and the three kinds of enzyme-like activities are simultaneously improved. In particular, the three kinds of enzyme-like activities of the ferroxane artificial enzyme 2 are greatly improved compared with the ferroxane artificial enzyme 4. With the continuous increase of L-histidine, although the activities of POD-like and SOD-like can be greatly improved, the activity of CAT-like is obviously decreased. Therefore, in a more preferred embodiment, the concentration of histidine in the mineralization precursor solution is set to 10-20 mM.

[0077] The above specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing an iron-based artificial enzyme, characterized by, Includes the following steps: S1: Prepare a chelation layer on the basement membrane; S2: Mix ferric chloride hexahydrate, ferrous sulfate heptahydrate, histidine, and solvent to obtain a mineralization precursor solution, and remove oxygen from the mineralization precursor solution; S3: The mineralization precursor solution spreads on the chelating layer to form a solution layer, and the mineralization precursor solution in the solution layer undergoes a mineralization reaction in an alkaline environment to generate a mineral layer on the chelating layer.

2. The method for preparing a ferrooxygenated artificial enzyme according to claim 1, characterized in that, In step S1, a dopamine hydrochloride solution is dropped onto the basement membrane until the dopamine hydrochloride solution covers the surface of the basement membrane. An alkaline solution is then added to the dopamine hydrochloride solution to generate a polydopamine layer on the basement membrane. The polydopamine layer serves as the chelating layer.

3. The method for preparing a ferrooxygenated artificial enzyme according to claim 1, characterized in that, In step S2, the concentration of histidine in the mineralization precursor solution is 10-20 mM.

4. The method for preparing a ferrooxygenated artificial enzyme according to claim 1, characterized in that, In step S2, the molar ratio of ferric chloride hexahydrate to ferrous sulfate heptahydrate is 1:1 to 3:

1.

5. A method of preparing an iron-based artificial enzyme according to claim 3 or 4, characterized in that, In step S2, ferric chloride hexahydrate and ferrous sulfate heptahydrate are dissolved in a solvent to obtain a mixed solution. An inert gas is introduced into the mixed solution, and then histidine is added to the mixed solution to obtain a mineralization precursor solution. An inert gas is then introduced into the mineralization precursor solution.

6. The method for preparing a ferrooxygenated artificial enzyme according to claim 1, characterized in that, In step S3, a pressure plate is placed on the solution layer. The pressure plate has a hydrophobic surface that contacts the upper surface of the solution layer. The solution layer contacts the ammonia gas through its side surface. The ammonia gas provides the alkaline environment for the mineralization precursor solution of the solution layer.

7. The method for preparing a ferrooxygenated artificial enzyme according to claim 6, characterized in that, In step S3, the solution layer is placed in a closed space containing an ammonia solution, which volatilizes at room temperature to release ammonia gas that comes into contact with the solution layer.

8. An iron-based artificial enzyme, characterized in that, It includes a basement membrane, a chelating layer and a mineral layer connected in sequence, wherein the mineral layer includes ferrohydrite, δ-hydroxy iron oxide and magnetite, and the surface of the mineral layer has histidine.

9. The ferroxyl artificial enzyme according to claim 9, characterized in that, The mineral layer has an area of 10 to 50 mm 2 .

10. A wound dressing, characterized in that, The wound dressing includes a ferrooxygenated artificial enzyme as described in claim 8 or 9.

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