CN116688987A - Biological catalyst for producing ROS, preparation method and application thereof - Google Patents

Biological catalyst for producing ROS, preparation method and application thereof Download PDF

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CN116688987A
CN116688987A CN202310940456.5A CN202310940456A CN116688987A CN 116688987 A CN116688987 A CN 116688987A CN 202310940456 A CN202310940456 A CN 202310940456A CN 116688987 A CN116688987 A CN 116688987A
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ros
iron
biocatalyst
vanadium
producing
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CN116688987B (en
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程冲
黄豪举
赵长生
文琴龙
耿巍
吴熙政
马田
李玲
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Sichuan University
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K33/00—Medicinal preparations containing inorganic active ingredients
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    • 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
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
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    • A62D3/30—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by reacting with chemical agents
    • A62D3/38—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by reacting with chemical agents by oxidation; by combustion
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    • B01J23/70—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the iron group metals or copper
    • B01J23/76—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the iron group metals or copper combined with metals, oxides or hydroxides provided for in groups B01J23/02 - B01J23/36
    • B01J23/84—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the iron group metals or copper combined with metals, oxides or hydroxides provided for in groups B01J23/02 - B01J23/36 with arsenic, antimony, bismuth, vanadium, niobium, tantalum, polonium, chromium, molybdenum, tungsten, manganese, technetium or rhenium
    • B01J23/847—Vanadium, niobium or tantalum or polonium
    • B01J23/8472—Vanadium
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
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    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
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    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Abstract

The invention relates to a biological catalyst for producing ROS, a preparation method and application thereof, and belongs to the field of catalysts. The invention provides a preparation method of a biocatalyst for producing ROS, which comprises the following steps: taking vanadium-containing substances and iron-containing substances as raw materials, and preparing an iron-vanadium precursor by adopting a hydrothermal conversion method; then the obtained iron vanadium precursor is subjected to heat treatment at 350-450 ℃ to prepare the biocatalyst for producing the ROS; wherein the molar ratio of the vanadium-containing substance to the iron-containing substance is 1:10 to 3. The invention utilizes the alpha-Fe with excellent biocompatibility 2 O 3 Dispersing V monoatoms, thereby constructingROS-producing biocatalyst V-Fe having unique V-O-Fe atomic structure 2 O 3 The method comprises the steps of carrying out a first treatment on the surface of the The bactericide has excellent capability of producing OH in the process of killing bacteria, thereby realizing better bacterial sterilization effect.

Description

Biological catalyst for producing ROS, preparation method and application thereof
Technical Field
The invention relates to a biological catalyst for producing ROS, a preparation method and application thereof, and belongs to the field of catalysts.
Background
Bacterial antibiotic resistance has become one of the major public health threats in the 21 st century. In order to cope with such serious threat to human health, efforts have been made to explore nanomaterials with effective bactericidal therapeutic effects as alternatives to conventional antibiotics. The concept of "capture and kill" includes the design of spike-like nanostructures to enhance interactions between nanomaterials and pathogenic bacteria (referred to as "capture") and the construction of peroxidase-like (POD) active sites to produce reactive oxygen species sterilization (referred to as "kill"), which achieves more efficient bacterial killing than traditional ROS-producing antimicrobial nanomaterials.
However, the antibacterial effect is not yet satisfactory due to the complex ROS environment in POD systems. Specifically, because of the problems of high energy barrier, complex reaction path, low efficiency and the like in the formation process of ROS, OH and O can exist in a POD-like system 2 - 、 1 O 2 And the like. Among these, with superoxide anion radical (. O) 2 - -0.33V vs. SHE) and singlet oxygen 1 O 2 +1.88V vs. SHE) is the most oxidizing hydroxyl radical (.oh, +2.38V vs. SHE) and can lead to excessive oxidation of the substrate to CO in organic synthesis 2 And H 2 O, or disruption of intracellular ROS-antioxidant balance, results in irreversible damage to healthy cells.
In addition, care should be takenWherein the diffusion coefficient of OH is 3.0.+ -. 0.2X10 -10 m 2 ·s -1 Ratio H 2 O 2 （1.1±0.1×10 -10 m 2 ·s -1 ) And. O 2 - （2.9±0.3×10 -12 m 2 ·s -1 ) Higher and therefore more oxidizing power to organic materials. Therefore, based on the oxidative sterilization mechanism of ROS, we believe that OH is theoretically superior to O 2 - And 1 O 2 has better sterilization capability. Therefore, designing peroxidases with high-efficiency.OH-producing ability is an effective strategy for enhancing antibacterial effect.
Fe has not been adopted in the prior art 2 O 3 A relevant report that a biological catalyst (peroxidase mimic) capable of producing ROS with high-efficiency producing OH can be prepared by loading V single atoms on a carrier.
Disclosure of Invention
In view of the above drawbacks, the present invention is directed to an alpha-Fe 2 O 3 The carrier is doped with V atoms to construct the ROS-producing biocatalyst (V-Fe) with the V-O-Fe atomic structure 2 O 3 ) The method comprises the steps of carrying out a first treatment on the surface of the It has excellent OH-producing capacity [ V-Fe ] in the process of killing bacteria 2 O 3 V of (2) max （1.07×10 -6 M·s -1 ) And TON (22.38 s) -1 ) The values are V respectively 2 O 5 Is approximately 2 times and 10 times, and has excellent OH efficiency]Thereby realizing better bacterial sterilization effect (V-Fe 2 O 3 Is about 128 mug.mL -1 ，V 2 O 5 The minimum inhibitory concentration of (C) is about 256. Mu.g.mL -1 ). Research has shown that strong electron coupling between vanadium and iron results in more electrons being in V-Fe 2 O 3 * OOH forms a bond state, resulting in stronger OOH adsorption and lower energy barrier, so that the constructed V-O-Fe atomic structure can produce strong oxidative. OH with ultra-high efficiency in POD system, and the V-Fe obtained by the invention has high-efficient production of. OH capability 2 O 3 Realizes better bacterial sterilization effect to resist drug-resistant bacteria and biological membranes, and verifies the material energy and vancomic through a rabbit wound infection modelThe mycin realizes similar effect of resisting bacterial infection and accelerating wound healing, and has good biocompatibility.
The technical scheme of the invention is as follows:
the first technical problem to be solved by the invention is to provide a preparation method of a biocatalyst for producing ROS, which comprises the following steps: taking vanadium-containing substances and iron-containing substances as raw materials, and preparing an iron-vanadium precursor by adopting a hydrothermal conversion method; then the obtained iron vanadium precursor is subjected to heat treatment at 350-450 ℃ to prepare the biocatalyst for producing the ROS; wherein the molar ratio of the vanadium-containing substance to the iron-containing substance is 1:10 to 3.
Preferably, the molar ratio of the vanadium-containing material to the iron-containing material is 1:5 to 3.
In the hydrothermal conversion method, the temperature is 110 to 130 ℃ (preferably 120 ℃), and the heating time is 6 to 8 hours (preferably 6 hours).
Further, the heat treatment time is 3-4 h. Preferably, the heat treatment temperature is 400 ℃.
Further, the vanadium-containing material is selected from: VOSO 4 ·xH 2 O、VO(acac) 2 、NH 4 VO 3 Or NaVO 3 . The substance is mainly a V atom that provides a catalytically active site.
Further, the iron-containing material is selected from: feCl 3 ·6H 2 O、Fe(NO 3 ) 3 Or Fe (Fe) 2 (SO 4 ) 3 。
The second technical problem to be solved by the invention is to provide a biological catalyst for producing ROS, wherein the catalyst is prepared by adopting the preparation method.
Further, in the biocatalyst, vanadium single atoms are uniformly loaded on Fe 2 O 3 In (C) is denoted as V-Fe 2 O 3 。
Further, the biocatalyst has POD activity.
Further, the biocatalyst has haloperoxidase-like (HPO) activity.
The third technical problem to be solved by the invention is to provide the application of the biological catalyst for producing ROS in preparing materials for resisting bacterial infection by oxidative degradation of organic pollutants.
The fourth technical problem to be solved by the invention is to point out V-Fe 2 O 3 Use in a biocatalyst for the production of ROS, said V-Fe 2 O 3 The preparation method comprises the following steps: taking vanadium-containing substances and iron-containing substances as raw materials, and preparing an iron-vanadium precursor by adopting a hydrothermal conversion method; then the obtained iron vanadium precursor is subjected to heat treatment at 350-450 ℃ to prepare the biocatalyst for producing the ROS; wherein the molar ratio of the vanadium-containing substance to the iron-containing substance is 1:10 to 3.
The invention has the beneficial effects that:
alpha-Fe with excellent biocompatibility for use herein 2 O 3 Dispersing V monoatoms, thereby constructing a unique V-O-Fe atomic structure. By injecting charge into the V-3d track, V-Fe 2 O 3 * The bonding state of OOH is obviously enhanced, so that the key intermediate of the OOH, which generates the OH reaction, is stabilized, and the sterilization effect is enhanced by efficiently generating the OH. Designed thorn-shaped V-Fe 2 O 3 Can promote wound healing of drug-resistant bacterial infection rapidly and has excellent biocompatibility, and can be compared with the treatment effect of vancomycin which is a common antibiotic.
Drawings
FIG. 1 is an SEM image of a composite material according to various V-Fe raw material ratios under a heat treatment at 400 ℃, wherein (a) Fe 2 O 3 ； (b) V-Fe 2 O 3 -1:10； (c) V-Fe 2 O 3 -1:5； (d) V-Fe 2 O 3 -1:3； (e) V 2 O 5 The method comprises the steps of carrying out a first treatment on the surface of the The scale bars in the figures are all 1 μm.
FIG. 2 shows the POD-like activity test results of a composite material prepared by heat treatment at 400℃in accordance with the different V-Fe raw material ratios, wherein (a) TMB solution is prepared in H 2 O 2 Ultraviolet visible spectrum after co-incubation with three different proportions of synthetic materials in the presence of the fluorescent dye; (b) A plot of POD-like enzyme activity results of the samples was characterized by absorbance of 650 nm.
FIG. 3 shows the V starting material: fe raw material = 1:5, but changing the heat treatment temperature to synthesize the POD-like enzyme test result of the material, whichIn (a) TMB solution in H 2 O 2 A graph of uv-vis spectrum after co-incubation with a material synthesized by varying the heat treatment temperature in the presence, (b) a graph of POD-like enzyme activity of the sample characterized by absorbance of 650 nm.
Fig. 4 (a) (b) is a graph of high angle annular dark field scanning transmission electron microscope (HAADF-STEM) imaging results.
FIG. 5 is V-Fe 2 O 3 X-ray diffraction pattern of the material and the control.
FIG. 6 is V-Fe 2 O 3 A TEM image of material (a); (b) an HR-TEM image; (c) EDX results plot.
FIG. 7 is V-Fe 2 O 3 Aberration correcting high angle annular dark field scanning transmission electron microscopy (AC-HAADF-STEM) images of the material (a-b); (c-D) 3D reconstruction of atomic resolution AC-HAADF-STEM; (e-h) atomic resolution energy dispersive X-ray (EDX) images.
Fig. 8: (a) a K-edge XANES spectral plot of V; (b) By means of absorption threshold (E 0 ) Calculating a valence state result diagram of the V atom; (c) Fourier Transform (FT) k 3 Weighting the EXAFS spectrum and a corresponding fitting result diagram; (d-f) Wavelet Transform (WT) map.
Fig. 9: (a) a high resolution XPS map of V2 p; (b) high resolution XPS map of O1 s.
Fig. 10: (a) a POD-like enzyme test result graph of a sample and a comparison sample; (b) a Machaelis-Menton kinetic plot; (c) Material kinetic parameters (V) fitted by kinetic curves max , TON, K m ) Results graph.
Fig. 11: (a) Detection of V-Fe by radical quenching experiments 2 O 3 A plot of ROS species results generated; (b) Detection of V by radical quenching experiments 2 O 5 A plot of ROS species results generated.
Fig. 12: (a) Detecting an OH signal result graph generated in the sample by EPR; (b) Detection of generated O in sample by EPR 2 - Signal diagram.
Fig. 13: (a) a plot of the OH results of the TA probe test samples; (b) Detection of sample by HE Probe. O 2 - Results graph.
Fig. 14: (a) CB solution in H 2 O 2 An ultraviolet-visible spectrum after incubation with the material in the presence of the fluorescent dye; (b) HPO-like enzyme with CB as chromogenic substrate detection material can produce HClO.
Fig. 15: in vitro antibacterial experiment (a) material and comparative sample antibacterial capability test result diagram; (b) a sterilization capability test result graph of the material and the comparison sample; (c) And (3) a graph of colony counting results of the agar plates after the sample and bacteria are treated together.
Fig. 16: SEM images of MRSA samples after different treatments; the scale bar represents 1 μm.
Fig. 17: live and dead fluorescent staining results of bacteria after different treatments.
Fig. 18: graph of MRSA biofilm results using Alexa Fluor 647 labelling after different treatments.
Fig. 19: animal experiments prove that the antibacterial infection of the material accelerates the wound healing effect: (a) photograph of a 13 day infected wound; (b) a plot of wound colony counts after material treatment; (c) Hematoxylin-eosin (HE) staining profile after 13 days of wound recovery; (d) Masson staining pattern after 13 days of wound recovery; (e) CD31 staining pattern after 13 days of wound recovery.
Fig. 20: V-Fe 2 O 3 Is a biosafety test of (2): (a) staining of visceral HE sections of rabbits after 13 days of wound recovery; (b) Sample at H 2 O 2 Live/dead cell staining after incubation with Human Umbilical Vein Endothelial Cells (HUVECs) in the presence.
Detailed Description
The invention utilizes the alpha-Fe with excellent biocompatibility 2 O 3 Dispersing V monoatoms, thereby constructing a unique V-O-Fe atomic structure. By injecting charge into the V-3d track, V-Fe 2 O 3 * The bonding state of OOH is obviously enhanced, so that the key intermediate of the OH reaction of OOH is stabilized, and the sterilization effect is finally enhanced. Designed V-Fe 2 O 3 Can promote wound healing of bacterial infection rapidly and has excellent biocompatibility, and can be compared with the treatment effect of vancomycin which is a common antibiotic.
The following describes the invention in further detail with reference to examples, which are not intended to limit the invention thereto.
Example 1
FeCl is added 3 ·6H 2 O（405 mg/1.498 mmol）、Na 2 SO 4 (205 mg/1.44 mmol) and VOSO 4 ·xH 2 O (48 mg/0.3 mmol, V: fe molar ratio=1:5) was dissolved in deionized water and stirred for 10 minutes to uniformly distribute the metal ions. The resulting solution was then transferred to a 100 mL teflon lined stainless steel autoclave and held at 120 ℃ for 6 hours; after cooling to room temperature, the precursor was collected by centrifugation and washed 3 times with deionized water and ethanol; after drying in air at 60 ℃ overnight, the prepared V-Fe precursor was transferred to a ceramic boat, placed in a tube furnace, and then heated at 400 ℃ at a heating rate of 2 ℃ per minute under an air atmosphere for 3h to prepare the final material.
Examples 2 to 3
The specific preparation process is the same as in example 1, except that VOSO 4 ·xH 2 The amount of O added was 81 mg/0.499 mmol (V: fe=1:3) (example 2); VOSO 4 ·xH 2 The amount of O added was 24 mg/0.15 mmol (V: fe=1:10) (example 3).
Comparative example 1
In the same manner as in example 1, feCl alone 3 ·6H 2 O was used as a raw material, and the obtained material was used as comparative example 1.
Comparative example 2
In the same manner as in example 1, only VOSO was used 4 ·xH 2 O was used as a raw material, and the obtained material was used as comparative example 2.
Comparative examples 3 to 4
The specific preparation process was the same as in example 1, except that the obtained precursor was heat-treated at 200℃for 3 hours (comparative example 3) and at 600℃for 3 hours (comparative example 4), respectively, under an air atmosphere.
Structure and performance characterization
FIG. 1 is an SEM image of a synthetic material according to various V-Fe raw material ratios under a heat treatment at 400 ℃, (a) Fe 2 O 3 , (b) V-Fe 2 O 3 -1:10， (c) V-Fe 2 O 3 -1:5，(d) V-Fe 2 O 3 -1:3，(e) V 2 O 5 The method comprises the steps of carrying out a first treatment on the surface of the As can be seen from fig. 1, when the ratio of V raw material to Fe raw material is greater than 1:5, the thorn-shaped structure of the material disappears; FIG. 2 shows the results of POD-like activity test of synthetic materials according to different V-Fe raw material ratios under heat treatment at 400 ℃, (a) shows TMB solution in H 2 O 2 The uv-vis spectrum after co-incubation with three different ratios of synthetic materials in the presence, (b) graph is a graph of POD-like enzyme activity of the sample characterized by absorbance of 650 nm; as can be seen from fig. 2, when the ratio of V raw material to Fe raw material is greater than 1: the POD-like activity of the material is not obviously improved at the time of 5.
Figures 3a-b are V starting materials: as a result of the POD-like enzyme test using three heat treatment temperature synthetic materials at a ratio of Fe raw material=1:5, it was found that the material synthesized at 400 ℃ (V-Fe 2 O 3 -400) has the most excellent POD-like activity.
FIG. 4 (a) (b) high angle annular dark field scanning transmission electron microscope (HAADF-STEM) imaging results; further illustrates the V-Fe obtained by the invention 2 O 3 Is in the shape of thorn-shaped particles.
FIG. 5 shows the X-ray diffraction results of the obtained material and the comparative sample, illustrating V-Fe 2 O 3 (example 1, hereinafter not specifically noted, V-Fe 2 O 3 All obtained in example 1) and Fe 2 O 3 Diffraction peak positions of (C) and hematite (PDF#87-1164: alpha-Fe) 2 O 3 ) Match with V-Fe 2 O 3 The peak intensity of (C) is obviously weakened, indicating that V-Fe 2 O 3 Inherits Fe 2 O 3 And has a significant lattice damage. V-Fe in FIG. 6a 2 O 3 The material's Transmission Electron Microscope (TEM) further demonstrates the material's thorn-like microparticle morphology, and FIG. 6b is a high resolution transmission electron microscope (HR-TEM) result, illustrating the material's crystal structure as α -Fe with different exposed crystal planes 2 O 3 The crystal, FIG. 6c is an energy dispersive X-ray (EDX) spectrum thereof, showing that the material contains three uniformly distributed elements of V, O and Fe. Furthermore, we studied V-Fe by aberration-correcting high-angle annular dark field scanning transmission electron microscope (AC-HAADF-STEM) characterization 2 O 3 The atomic phase structure of (a) is shown in FIG. 7, and FIGS. 7 (a-b) further demonstrate that the material has a different exposed crystal plane of alpha-Fe 2 O 3 A crystal; FIGS. 7 (c-D) 3D reconstruction of an atomic resolution AC-HAADF-STEM image demonstrates that the incorporated V atoms replace alpha-Fe 2 O 3 Fe atoms in the crystal lattice, thereby constructing a V-O-Fe atomic structure; FIG. 7 (e-h) atomic resolution energy dispersive X-ray (EDX) spectroscopy further demonstrates alpha-Fe 2 O 3 The V, O and Fe are uniformly distributed in the crystal at the atomic level.
We performed X-ray absorbing near edge structure (XANES) and extended X-ray absorbing fine structure (EXAFS) to investigate V-Fe 2 O 3 Coordination chemistry of the V site. FIG. 8 (a) V shows V-Fe in K-edge XANES spectrum 2 O 3 The front edge of the middle V is positioned between the V foil and the V 2 O 5 Between, indicating that the oxidation state of the V species is lower than V 2 O 5 V of (2) 5+ The method comprises the steps of carrying out a first treatment on the surface of the FIG. 8 (b) shows the absorption threshold (E 0 ) To calculate the valence of the V atom, and the result shows that the oxidation state of V is 3.99; FIG. 8 (c) is a Fourier Transform (FT) k 3 Weighted EXAFS spectra and FIG. 8 (d-f) are Wavelet Transform (WT) images of the corresponding samples; in WT images, V-Fe 2 O 3 And V 2 O 5 The scattering peak is shown to be located at 1.0-2.0 a, k.apprxeq.5.0 a -1 Proving V-Fe 2 O 3 The first coordination shell of V is an O atom and the result of the EXAFS fit reveals a V-O bond length of 1.70 a and a coordination number of 2.
V-Fe was studied by X-ray photoelectron Spectrometry (XPS) 2 O 3 Valence and electron transfer of (a). FIG. 9 (a) V-Fe 2 O 3 V2 p of (2) 3/2 Peak binding energy 516.14 eV, ratio V 2 O 5 (517.00 eV) 0.86 eV lower; meanwhile, FIG. 9 (b) V-Fe 2 O 3 The binding energy of medium V is lower than V 2 O 5 Binding energy of V, and V-Fe 2 O 3 Wherein the binding energy of O is lower than V 2 O 5 Fe (Fe) 2 O 3 The binding energy of O in the material indicates that Fe exists in the material and is transferred to V through O.
FIG. 10 (a) is a graph showing the verification of V-Fe by using a typical 3, 5-Tetramethylbenzidine (TMB) colorimetric method 2 O 3 Has optimal POD-like enzyme activity compared with the comparison sample. FIG. 10 (b) shows the calculation of the maximum initial velocity (V) from the Michaelis-Menten curve max ) Michaelis constant (K) m ) And turnover number (TON, maximum number of substrate molecules converted per active catalytic site). FIG. 10 (c) calculated V-Fe 2 O 3 V of (2) max （1.07×10 -6 M·s -1 ) And TON (22.38X10) -3 s -1 ) The values are almost V 2 O 5 2 times and 10 times, and K m Has a low value (V-Fe) 2 O 3 K of (2) m 4.58×10 -3 M，V 2 O 5 K of (2) m The value was 9.76X10 -3 M), showing V-Fe 2 O 3 For H 2 O 2 The molecules are more effective in catalytic activation and affinity.
The ROS species generated in the system are also detected on this basis. Free radical quenching experiments were performed to identify 1 O 2 ,. OH and O 2 - . FIG. 11 (a) detection of V-Fe by radical quenching experiments 2 O 3 The type of ROS produced, (b) detection of V by free radical quenching experiments 2 O 5 The type of ROS generated; FIG. 11 shows that OH is V-Fe 2 O 3 Absolute main ROS, while V in POD system of (C) 2 O 5 Mainly produce O 2 - And a small amount of. OH. Notably, no detection was made in either system 1 O 2 . FIG. 12 (a) Electron Paramagnetic Resonance (EPR) assay also demonstrates V-Fe using 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) as a specific spin trap reagent 2 O 3 The main ROS in the system is. OH, and little. O is detected 2 - While FIG. 12 (b) V 2 O 5 System generated. O 2 - The amount is relatively higher thanOH. In addition, use of O 2 - The specific probes, hydrogen Ethylidine (HE) and. OH specific probe, further demonstrated V-Fe for phthalic acid (TA) 2 O 3 Excellent OH-producing ability. In V form 2 O 5 For comparison, the increase in absorption at about 435 nm in the TA fluorescence spectrum of FIG. 13, a, and the decrease in absorption at about 610 nm in the HE fluorescence spectrum of FIG. 13b also confirm V-Fe 2 O 3 OH yield increases and inhibits O in the system 2- And (5) generating. In addition to excellent. OH catalytic production, V-Fe 2 O 3 Also has excellent activity of Haloperoxidase (HPO), and FIG. 14a, b shows that V-Fe is detected by using azure (CB) as reagent 2 O 3 Can also be at H 2 O 2 Catalytic Cl in the presence of - Oxidation to HClO.
In vitro antibacterial experiments were performed with methicillin-resistant staphylococcus aureus (MRSA) as an example. FIG. 15 (a) is a graph showing the optical density value (OD) at 600 nm 600 ) The Minimum Inhibitory Concentration (MIC) detected indicates that V-Fe 2 O 3 At about 128. Mu.g.mL -1 Can completely inhibit bacterial growth at the concentration of (2) and the comparative sample V 2 O 5 Then it is required to be 256 mug.mL -1 The same effect can be achieved at the concentration of (3). In addition Fe 2 O 3 Almost no obvious antibacterial effect is achieved. To quantitatively evaluate bactericidal capacity, figure 15 (b, c) study viability of MRSA incubated with five different systems by agar plate counting. V-Fe 2 O 3 Bacterial survival of the group was near 0%, while V 2 O 5 Group and Fe 2 O 3 The bacterial survival of the groups was about 41% and 82.3%, respectively, demonstrating the excellent bactericidal effect of the material.
FIG. 16 is a further view of V-Fe by SEM imaging results 2 O 3 Irreversible damage to bacteria. V-Fe can be seen 2 O 3 The spike nanostructures of (2) are capable of penetrating the bacterial lipid membrane and firmly capturing the bacteria, and visible deformation and collapse of the bacterial morphology indicates severe damage to the bacteria captured by the material. However, with V 2 O 5 The treated bacteria showed only lipid membrane uptakeShrinkage and slight damage. These results indicate that V-Fe 2 O 3 In situ efficient. OH formation helps to enhance bacterial killing. At the same time Fe 2 O 3 No obvious damage to bacteria. Furthermore, FIG. 17 shows that V-Fe was confirmed by live-dead fluorescent staining 2 O 3 Can cause much higher bacterial kill than the rest of the control. The present invention thus far proves that V-Fe 2 O 3 The high-efficiency OH production capability brings excellent sterilization effect.
In addition, planktonic bacteria tend to form biofilms on wound surfaces, acting as barriers to penetration of materials, increasing the difficulty of combating drug-resistant bacteria. As shown in fig. 18, the extracellular matrix in MRSA biofilm was labeled with dextran Alexa Fluor 647, which showed Fluor 647 signals in CLSM images. V (V) 2 O 5 And V-Fe 2 O 3 The weaker Fluor 647 signal in (B) indicates that MRSA biofilm is substantially eradicated by the ROS produced, while V-Fe 2 O 3 +H 2 O 2 The obvious enhancement of the PI signals of the group indicates that only V-Fe 2 O 3 Can kill MRSA embedded in bacterial biofilm.
Further evaluate V-Fe using rabbit wound infection model 2 O 3 The application of the composition in resisting drug-resistant bacteria (MRSA) infection and accelerating wound healing. Fig. 19 (a) shows the change in wound area size by photograph. On day 4, control group and H 2 O 2 The group showed severe deterioration due to the proliferation of MRSA, whereas V-Fe 2 O 3 + H 2 O 2 And vancomycin treated wounds showed a significant reduction in wound area. After 13 days, V-Fe 2 O 3 +H 2 O 2 Both groups and vancomycin groups recovered, while control groups and H 2 O 2 The treatment group had poor wound recovery, skin abscess and severe inflammation. FIG. 19 (b) shows the number of colonies on each group of wound surfaces after material treatment by the agar plate count method, thereby determining V-Fe 2 O 3 Is effective in eradicating bacteria in vivo.
In addition, fig. 19 (c) study the inflammation of each group after 13 days of healing by hematoxylin-eosin (HE) staining. Wherein neutrophils were in control group and H 2 O 2 Treatment of a large number of wounds indicates severe inflammation in the wound area; at the same time, V-Fe 2 O 3 +H 2 O 2 The group showed similar conditions to vancomycin and the normal group, with a significant decrease in inflammatory cells. We also used Masson trichromatic staining of fig. 19 (d) to confirm newly formed collagen fibers, which was taken as an indicator of wound recovery. V-Fe 2 O 3 +H 2 O 2 And vancomycin group showed collagen fiber content similar to normal rabbit skin, far superior to control group and H 2 O 2 Treatment groups. New blood vessels continue to coexist with wound recovery, fig. 19 (e) endothelial cells are labeled with the aid of CD31 staining to show newly formed blood vessels during the healing process of the wound area. In control group and H 2 O 2 Less CD31 positive signal was detected in the group, while V-Fe 2 O 3 And vancomycin groups showed increased CD31 expression, indicating better wound recovery results.
FIG. 20 (a) results of staining HE sections of rabbit viscera 13 days after wound treatment, demonstrating V-Fe 2 O 3 The material has no obvious damage to animal viscera. FIG. 20 (b) sample at H 2 O 2 Live/dead cell staining images after incubation with Human Umbilical Vein Endothelial Cells (HUVECs) in the presence indicated that V-Fe 2 O 3 +H 2 O 2 Some damage to mammalian cells will occur, but the damage will recover after 48 hours, and V 2 O 5 Irreversible damage to the cells can occur. The above experiments demonstrate the extraordinary biocompatibility of the materials.
Taken together, the invention is characterized in that alpha-Fe 2 O 3 The carrier constructs a unique V-O-Fe atomic structure with strong electron coupling through V-atom doping, and the catalyst V-Fe is prepared 2 O 3 Which has excellent OH-producing ability (V-Fe 2 O 3 V of (2) max （1.07×10 -6 M·s -1 ) And TON (22.38X10) -3 s -1 ) The values are V respectively 2 O 5 Near 2-fold and 10-fold, and has excellent OH efficiency), thereby achieving better bacterial sterilizationEffect (V-Fe) 2 O 3 Is about 128 mug.mL -1 ，V 2 O 5 The minimum inhibitory concentration of (C) is about 256. Mu.g.mL -1 ). Research has shown that strong electron coupling between vanadium and iron results in more electrons being in V-Fe 2 O 3 * OOH forms a bond state, resulting in stronger OOH adsorption and lower energy barrier, so that the constructed V-O-Fe atomic structure can produce strong oxidative. OH with ultra-high efficiency in POD system, and due to its high efficient production of. OH capability, the V-Fe is designed 2 O 3 The material can achieve similar antibacterial infection acceleration wound healing effect with vancomycin through verification of a rabbit wound infection model, and has good biocompatibility.

Claims (10)

1. A method for preparing a biocatalyst for producing ROS, the method comprising: taking vanadium-containing substances and iron-containing substances as raw materials, and preparing an iron-vanadium precursor by adopting a hydrothermal conversion method; then the obtained iron vanadium precursor is subjected to heat treatment at 350-450 ℃ to prepare the biocatalyst for producing the ROS; wherein the molar ratio of the vanadium-containing substance to the iron-containing substance is 1:10 to 3.
2. The method for preparing a biocatalyst for producing ROS according to claim 1, wherein the molar ratio of the vanadium-containing substance to the iron-containing substance is 1:5 to 3.
3. The method for preparing the biological catalyst for producing the ROS according to claim 1 or 2, wherein the temperature is 110-130 ℃ and the heating time is 6-8 hours in the hydrothermal conversion method.
4. A method of preparing a ROS-producing biocatalyst according to claim 1 or 2, wherein the vanadium-containing material is selected from the group consisting of: VOSO 4 ·xH 2 O、VO(acac) 2 、NH 4 VO 3 Or NaVO 3 The method comprises the steps of carrying out a first treatment on the surface of the And/or:
the iron-containing material is selected from: feCl 3 ·6H 2 O、Fe(NO 3 ) 3 Or Fe (Fe) 2 (SO 4 ) 3 。
5. A ROS producing biocatalyst, characterized in that it is produced by the preparation method according to any of claims 1-4.
6. The ROS producing biocatalyst of claim 5, wherein the vanadium single atoms are homogeneously supported in Fe 2 O 3 In (C) is denoted as V-Fe 2 O 3 。
7. The ROS producing biocatalyst of claim 6, wherein the biocatalyst has POD activity; and/or:
the biocatalyst has HPO activity.
8. Use of a ROS producing biocatalyst according to any of claims 5-7 for the oxidative degradation of organic contaminants for the preparation of a material for the treatment of bacterial infections.
9.V-Fe 2 O 3 Use in a biocatalyst for the production of ROS, characterized in that the V-Fe 2 O 3 The preparation method comprises the following steps: taking vanadium-containing substances and iron-containing substances as raw materials, and preparing an iron-vanadium precursor by adopting a hydrothermal conversion method; then the obtained iron vanadium precursor is subjected to heat treatment at 350-450 ℃ to prepare the biocatalyst for producing the ROS; wherein the molar ratio of the vanadium-containing substance to the iron-containing substance is 1:10 to 3.
10. V-Fe according to claim 9 2 O 3 Use in a biocatalyst for the production of ROS, characterized in that the molar ratio of vanadium-containing substances to iron-containing substances is 1:5 to 3; and/or:
in the hydrothermal conversion method, the temperature is 110-130 ℃, and the heating time is 6-8 hours; and/or:
the vanadium-containing material is selected from: VOSO 4 ·xH 2 O、VO(acac) 2 、NH 4 VO 3 Or NaVO 3 The method comprises the steps of carrying out a first treatment on the surface of the And/or:
the iron-containing material is selected from: feCl 3 ·6H 2 O、Fe(NO 3 ) 3 Or Fe (Fe) 2 (SO 4 ) 3 。
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