CN114392773B - Cu/Au/Pt-MOFs composite material with enhanced peroxidase activity and preparation method and application thereof - Google Patents

Cu/Au/Pt-MOFs composite material with enhanced peroxidase activity and preparation method and application thereof Download PDF

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CN114392773B
CN114392773B CN202111646310.7A CN202111646310A CN114392773B CN 114392773 B CN114392773 B CN 114392773B CN 202111646310 A CN202111646310 A CN 202111646310A CN 114392773 B CN114392773 B CN 114392773B
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composite material
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CN114392773A (en
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丁萍
伍翩
开天瀚
何兴侯
龚芳洁
陈翠梅
王丹琪
黄瑞雪
张竞文
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Central South University
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    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J31/00Catalysts comprising hydrides, coordination complexes or organic compounds
    • B01J31/16Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
    • B01J31/18Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes containing nitrogen, phosphorus, arsenic or antimony as complexing atoms, e.g. in pyridine ligands, or in resonance therewith, e.g. in isocyanide ligands C=N-R or as complexed central atoms
    • B01J31/1805Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes containing nitrogen, phosphorus, arsenic or antimony as complexing atoms, e.g. in pyridine ligands, or in resonance therewith, e.g. in isocyanide ligands C=N-R or as complexed central atoms the ligands containing nitrogen
    • B01J31/181Cyclic ligands, including e.g. non-condensed polycyclic ligands, comprising at least one complexing nitrogen atom as ring member, e.g. pyridine
    • B01J31/1825Ligands comprising condensed ring systems, e.g. acridine, carbazole
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01N59/00Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/16Heavy metals; Compounds thereof
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/16Heavy metals; Compounds thereof
    • A01N59/20Copper
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2531/00Additional information regarding catalytic systems classified in B01J31/00
    • B01J2531/02Compositional aspects of complexes used, e.g. polynuclearity
    • B01J2531/0238Complexes comprising multidentate ligands, i.e. more than 2 ionic or coordinative bonds from the central metal to the ligand, the latter having at least two donor atoms, e.g. N, O, S, P
    • B01J2531/0241Rigid ligands, e.g. extended sp2-carbon frameworks or geminal di- or trisubstitution
    • B01J2531/025Ligands with a porphyrin ring system or analogues thereof, e.g. phthalocyanines, corroles
    • BPERFORMING OPERATIONS; TRANSPORTING
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    • B01J2531/00Additional information regarding catalytic systems classified in B01J31/00
    • B01J2531/10Complexes comprising metals of Group I (IA or IB) as the central metal
    • B01J2531/16Copper
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a Cu/Au/Pt-MOFs composite material with enhanced peroxidase activity, which belongs to the technical field of nano mimic enzyme and medical antibacterial, and mainly comprises Cu-TCPP (Fe) and Cu/Au/PtNPs growing on the surface of the Cu-TCPP (Fe) in situ. It utilizes the catalase activity of Cu/Au/PtNPs and Cu-TCPP (Fe), has synergistic double simulated enzyme activity, greatly improves the catalase-like activity, and has low concentration H 2 O 2 Under the existing conditions, the bactericidal composition has strong killing effect on various bacteria including common infectious bacteria such as staphylococcus aureus, salmonella and the like, and also has excellent killing effect on pseudomonas aeruginosa which is easy to form a biological membrane. The invention also discloses a preparation method of the Cu/Au/Pt-MOFs composite material and application of the Cu/Au/Pt-MOFs composite material in preparation of an antibacterial agent.

Description

Cu/Au/Pt-MOFs composite material with enhanced peroxidase activity and preparation method and application thereof
Technical Field
The invention belongs to the technical field of nano mimic enzyme and medical antibiosis, and particularly relates to a Cu/Au/Pt-metal organic framework (Cu/Au/Pt-MOFs) composite material with peroxidase activity, a preparation method thereof and application thereof in the field of antibiosis.
Background
Diseases caused by bacterial infections are one of the global public health problems. Statistically, millions of people die each year from bacterial infections. Antibiotics are the traditional method of treating infectious bacteria, but abuse of antibiotics leads to increasing bacterial resistance, even the appearance of "superbacteria". Therefore, the search for new antibacterial agents is urgently needed. In recent years, a series of antibacterial materials such as nano silver are used for killing bacteria due to unique antibacterial mechanisms. These antibacterial materials can react with the biomolecules of the bacteria, change their structures, affect the biological functions of the bacteria, and ultimately lead to bacterial death. Although the above antibacterial materials have shown great potential as alternatives to antibiotics, their clinical use is still limited due to their potential biotoxicity and low antibacterial efficiency. Therefore, the development of nano materials with low biotoxicity and high antibacterial activity is still urgent.
The nanometer mimic enzyme is a nanometer material with a similar reaction mechanism to natural enzyme, and has attracted great attention in antibacterial treatment. In particular, functional nanoparticles with peroxidase-like properties may convert H 2 O 2 Converted into hydroxyl radicals (. OH) which are more toxic to bacteria, thereby killing bacteria. Co-V mixed metal oxide, pd-Fe modified graphite nanosheet, copper sulfide nanoparticle and the like have been applied to H 2 O 2 Their application is still subject to the susceptibility of nanoparticles to aggregation and the use of high concentrations of H 2 O 2 The limit of (2). Therefore, the development of a nano material with stronger peroxidase activity and better dispersibility to improve the antibacterial performance of the nano material is still needed, and the maximization of the enzyme-like activity of the nano material by adjusting the form and the composition of the nano material is an effective way for developing a better antibacterial agent.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and defects mentioned in the background technology, and provide a Cu/Au/PtNPs-metal organic framework (Cu/Au/Pt-MOFs) composite material with enhanced peroxidase activity, a preparation method thereof and application thereof in preparing an antibacterial agent, and the composite material is applied to antibacterial treatment of common infectious bacteria such as staphylococcus aureus, pseudomonas aeruginosa, salmonella and the like.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a Cu/Au/Pt-MOFs composite material with enhanced peroxidase activity mainly comprises a metal organic framework composite material Cu-TCPP (Fe) and Cu/Au/Pt multi-metal nanoparticles (Cu/Au/PtNPs) growing on the surface of the Cu/Au/Pt multi-metal nanoparticles in situ.
In the above Cu/Au/Pt-MOFs composite material, the mass ratio of the Cu-TCPP (Fe) to the Cu/Au/Pt polymetallic nanoparticles is preferably 1 (0.1-10) (more preferably 1.57).
Preferably, in the Cu/Au/Pt multi-metal nanoparticles, the mass ratio of Cu to Au to Pt is 1 (0.1-50) to (0.1-50) (more preferably 1.
The metal organic framework Materials (MOFs) are organic-inorganic hybrid porous crystalline materials formed by self-assembling metal ions or metal clusters as nodes and organic ligands as connectors through the nodes and the connectors. The porphyrin-metal framework material (TCPP) with the porphyrin compound as the organic connector not only has a spatial structure and functions with large specific surface area and easy regulation, but also shows good peroxidase-like activity. In addition, noble metal nanoparticles, such as gold (Au), platinum (Pt), etc., also have peroxidase-like activity due to quantum size effect and quantum confinement effect. The Cu/Au/Pt NPs and Cu-TCPP (Fe) nanometer materials with peroxidase activity are combined to cooperate with the peroxidase activities of the two types to synthesize a Cu/Au/Pt-MOFs composite material with enhanced peroxidase activity as a bactericide to realize low H 2 O 2 Can kill bacteria efficiently under the condition.
Based on a general inventive concept, the invention also provides a preparation method of the Cu/Au/Pt-MOFs composite material, which comprises the following steps: dispersing Cu-TCPP (Fe) in ultrapure water, adding CuSO under magnetic stirring 4 Stirring the mixture continuously after the trisodium citrate is added, and dripping KBH 4 Then stirring is continued, HAuCl is added dropwise 4 、K 2 PtCl 4 Then the stirring is continued to be carried out,and centrifuging after stirring, washing the precipitate with ultrapure water, centrifuging and collecting to obtain the Cu/Au/Pt-MOFs composite material.
In the above preparation method, preferably, the preparation method of Cu-TCPP (Fe) comprises the steps of: adding Cu (NO) 3 ) 2 ·3H 2 Dissolving O, TCPP (Fe), trifluoroacetic acid and polyvinylpyrrolidone in a mixture of DMF and ethanol, carrying out ultrasonic treatment, carrying out heating reaction, washing the obtained product, centrifuging and collecting to obtain Cu-TCPP (Fe).
More preferably, the Cu (NO) 3 ) 2 ·3H 2 The molar ratio of O, TCPP (Fe) and trifluoroacetic acid is 1; in the mixture of DMF and ethanol, the volume ratio of DMF to ethanol is 3; the ultrasonic treatment time is 5-30min; the heating reaction is carried out at the temperature of 50-150 ℃ for 8-24h; the resulting product was dark brown, washed twice with ethanol and centrifuged at 5000-15000rpm at 15-30 ℃ for 5-30min to collect Cu-TCPP (Fe).
In the above production method, preferably, the final concentration of the Cu-TCPP (Fe) solution is 0.1-10mg/mL (more preferably 1 mg/mL); the KBH 4 The concentration of the active component is 5-100mM, and the addition amount is 0.5-5mL; the HAuCl 4 In a concentration of 0.01-1M, the HAuCl 4 、K 2 PtCl 4 The addition amount of (B) is 10-200. Mu.L.
Preferably, the stirring is continued for 10-40min, the centrifugation is performed at 5000-15000rpm for 5-30min, and the obtained Cu/Au/Pt-MOFs composite material is dispersed in ultrapure water.
Based on a general inventive concept, the invention also provides application of the Cu/Au/Pt-MOFs composite material in preparation of an antibacterial agent, wherein the antibacterial agent comprises the Cu/Au/Pt-MOFs composite material and H 2 O 2
Preferably, the antibacterial object of the antibacterial agent is any one or more of Staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (Pseudomonas aeruginosa) and salmonella (salmonella).
Preferably, the antimicrobial agent contains H 2 O 2 Has a final concentration of 50-300 μ M and contains Cu/Au/Pt-OD of MOFs composite material 350 =0.5-6。
More preferably, it is 10 for each ml concentration 6 CFU/mL bacterial solution, and the dosage of the antibacterial agent is more than 70 mu L.
The technical principle of the invention is as follows (as shown in fig. 1):
metal-organic frameworks (MOFs) are a class of crystalline materials with abundant pores and large specific surface area. Compared with the traditional 3D bulk MOFs,2D MOFs, such as Cu-TCPP (Fe), co-TCPP (Fe), zn-TCPP (Fe) and the like, have larger specific surface area and more active sites on the surface, and have important significance for catalysis. Cu-TCPP (Fe) is used as a metal node, TCPP (Fe) is used as an organic ligand to synthesize MOFs with a 2D lamellar structure, the MOFs have strong peroxidase-like activity, and Cu with low toxicity is used, so that the Cu-TCPP/MOFs are more beneficial to application of biomedicine. The gold material has local Surface Plasmon Resonance (SPR), a photothermal conversion effect and catalytic capability on a plurality of important oxidation reactions, the yield is stable, and the gold material has good biocompatibility; the copper can make up the defects of high cost and relatively low thermal conductivity of the gold nanoparticles; platinum has strong catalytic properties. The characteristics of three metals are combined, a large amount of Cu/Au/PtNPs are uniformly loaded on Cu-TCPP (Fe) by adopting an in-situ growth method, and the Cu/Au/Pt NPs and the peroxidase activity of the Cu-TCPP (Fe) are cooperated to synthesize the Cu/Au/Pt-MOFs composite material with the enhanced peroxidase activity. The Cu/Au/Pt-MOFs composite material can efficiently catalyze H 2 O 2 Decomposing to generate superoxide radical such as OH. Therefore, even at low concentrations of H 2 O 2 Under the condition, cu/Au/Pt-MOFs composite material and H 2 O 2 The system can also generate a large amount of free radicals, thereby effectively killing common infectious bacteria such as staphylococcus aureus, pseudomonas aeruginosa, salmonella and the like.
H can also be catalyzed by Cu/Au/PtNPs-MOFs 2 O 2 The principle that TMB is oxidized to be changed from colorless to blue can be realized by the colorimetry of an ultraviolet-visible spectrophotometer 2 O 2 The quantitative determination of (3); cys inhibits the Cu/Au/PtNPs-MOFs against H 2 O 2 And TMBThe color of the TMB is not changed by the catalytic oxidation reaction of (1), and the Cys can be quantitatively determined by ultraviolet-visible spectrophotometer colorimetry. In addition, the H is generated based on the oxidation of glucose by glucose oxidase 2 O 2 By reaction with glucuronic acid, H produced is detected 2 O 2 The content reflects the glucose content in the sample. And this feature is compared with H in the present invention 2 O 2 The detection system is combined, and the quantitative detection of the glucose is realized.
Compared with the prior art, the invention has the following beneficial effects:
1. the Cu/Au/Pt-MOFs composite material utilizes the catalase activity of Cu/Au/Pt NPs and Cu-TCPP (Fe), has synergistic double simulated enzyme activity, and greatly improves the catalase-like activity.
2. The Cu/Au/Pt-MOFs composite material of the invention is prepared at low concentration H 2 O 2 Under the existing conditions, the bactericidal composition has strong killing effect on various bacteria including common infectious bacteria such as staphylococcus aureus, salmonella and the like, and also has excellent killing effect on pseudomonas aeruginosa which is easy to form a biological membrane.
3. The Cu/Au/Pt-MOFs composite material can also catalyze H 2 O 2 Oxidizing TMB, and combining with ultraviolet-visible spectrophotometer to realize H pair color comparison 2 O 2 Cys and glucose, simple operation, high speed, low cost, high sensitivity and low detection limit.
4. The preparation method of the Cu/Au/Pt-MOFs composite material is simple in preparation process, easy to operate and environment-friendly.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a Cu/Au/Pt-MOFs based synergy of H 2 O 2 The sterilization application schematic diagram;
FIG. 2 is a schematic diagram of the synthesis of Cu/Au/Pt-MOFs;
FIG. 3 is transmission electron microscopy (A-C) and elemental mapping (D-I) of Cu/Au/Pt-MOFs;
FIG. 4 is a graph of Cu/Au/PtNPs, cu-TCPP (Fe), and Cu/Au/Pt-MOFs catalyzing H 2 O 2 Ultraviolet-visible absorption spectrum of oxidized TMB;
FIG. 5 is the steady state kinetics of Cu/Au/Pt-MOFs;
FIG. 6 is a diagram of Cu/Au/Pt-MOFs-H 2 O 2 The bactericidal effect of the reaction system on the salmonella is preliminarily explored;
FIG. 7 is a graph of a difference H 2 O 2 Concentration vs. Cu/Au/Pt-MOFs-H 2 O 2 Influence of the bactericidal effect of the reaction system;
FIG. 8 is a graph of different Cu/Au/Pt-MOFs dose vs. Cu/Au/Pt-MOFs-H 2 O 2 Influence of the bactericidal effect of the reaction system;
FIG. 9 is a diagram of Cu/Au/Pt-MOFs-H under optimum conditions 2 O 2 The reaction system has the bactericidal effect on salmonella, staphylococcus aureus and pseudomonas aeruginosa.
Detailed Description
In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example (b):
a Cu/Au/Pt-MOFs composite material with enhanced peroxidase activity is composed of Cu-TCPP (Fe) and Cu/Au/PtNPs growing in situ on the surface of the Cu-TCPP (Fe) and the Cu/Au/Pt polymetallic nanoparticles, wherein the mass ratio of the Cu to the Au to the Pt in the Cu/Au/Pt polymetallic nanoparticles is 1.
1. The preparation method of Cu/Au/Pt-MOFs is shown in FIG. 2 and comprises the following specific steps:
0.012g of Cu (NO) 3 ) 2 ·3H 2 O (0.01 mmoL), 50 μ L trifluoroacetic acid (1.0M) and 50.0mg polyvinylpyrrolidone are dissolved in a mixture of 60ml dmdmaf and ethanol (V: V = 3. 0.022g of TCPP (Fe) (0.005 mmoL) was dissolved in a mixed solution of 20mLDMF and ethanol (V: V = 3. Then, the solution was sonicated for 10min, placed in a reaction kettle and heated to 80 ℃ and held for 15h. The resulting dark brown product was washed twice with ethanol and collected by centrifugation at 8000rpm at 25 ℃ for 10min. Finally, the obtained Cu-TCPP (Fe) is redissolved by 3mL of ethanol to obtain a Cu-TCPP (Fe) solution.
1mL of a Cu-TCPP (Fe) solution (1 mg/mL) was dissolved in 10mL of ultrapure water. Under magnetic stirring, 36 μ LCuSO was added 4 75 μ L of trisodium citrate, and stirring for 10min. 1.5mLKBH is added dropwise 4 (25 mM) and stirring continued for 10min. 75 μ L of HAuCl was added dropwise 4 (0.1M),75μL K 2 PtCl 4 After that, stirring was continued for 20min. After stirring, the mixture was centrifuged at 10000rpm for 10min. After washing the precipitate with ultrapure water, it was collected by centrifugation at 12000rpm for 10min. Finally, the resulting Cu/Au/Pt-MOFs were dispersed in 1mL of ultrapure water, at which point the OD of the solution at 350nm was 5.28.
The transmission electron microscope and element distribution diagram of the obtained Cu/Au/Pt-MOFs are shown in FIG. 3, and it can be seen that Cu/Au/PtNPs are uniformly grown on Cu-TCPP (Fe) with a lamellar structure, and the elements of Cu, au, pt and Fe are uniformly distributed.
2. Peroxidase activity validation of Cu/Au/Pt-MOFs
By H 2 O 2 TMB catalytic oxidation model the peroxidase activity of Cu/Au/Pt-MOFs was studied. To 145. Mu.L of acetic acid-sodium acetate solution (NaOAc, 0.01M, pH 5.5) were added 20. Mu.L of TMB (6 mmol/L) and 10. Mu.L of H, respectively 2 O 2 (10 mmol/L) and25 mu L of Cu/Au/Pt-MOFs or Cu/Au/PtNPs or Cu-TCPP (Fe), standing at room temperature for 10min for reaction, and measuring the absorption spectrum of the reaction system in the range of 550-750nm by using an ultraviolet-visible spectrophotometer.
The results are shown in FIG. 4, TMB and TMB + H 2 O 2 Almost no absorption in the 550-750nm range; only when Cu/Au/Pt-MOFs or Cu/Au/PtNPs or Cu-TCPP (Fe) is added to TMB + H 2 O 2 When the system is in the system, the system changes from colorless to obvious blue and has a maximum absorption peak at 652 nm; shows that Cu/Au/Pt-MOFs, cu/Au/Pt NPs and Cu-TCPP (Fe) all have peroxidase activity. But the color change of Cu/Au/Pt-MOFs is most obvious, and the absorbance is strongest; indicating that the Cu/Au/Pt-MOFs have enhanced peroxidase-like activity compared to the single Cu/Au/Pt NPs or Cu-TCPP (Fe).
3. Steady state kinetic analysis of Cu/Au/Pt-MOFs
Kinetic experiments were carried out on Cu/Au/Pt-MOFs containing 25. Mu.L of the above prepared Cu/Au/Pt-MOFs, with different concentrations of TMB and H 2 O 2 NaOAc (0.01M, pH 5.5). The mixed solution was incubated at room temperature for 1min, and the ultraviolet-visible absorption spectrum at a wavelength of 652nm was measured. By means of a double reciprocal curve (Lineweaver-Burkplot) 1/V = Km/Vm (1/[ S ]]+ 1/Km) calculating the Michaelis constant, where V is the initial velocity, V m Is the maximum reaction rate, [ S ]]Is the substrate concentration, K m Is the mie constant.
FIG. 5 shows a typical Michaelis-Menten curve, table 1 is a V calculated using the Lineweaver-Burk curve max And K m In comparison with horseradish peroxidase (HRP) and other reported nano-mimic enzymes, the Cu/Au/Pt-MOFs are directed against H 2 O 2 And TMB has a lower K m Values, indicating the Cu/Au/Pt-MOFs vs. H 2 O 2 And TMB has higher affinity.
TABLE 1 different Nano-simulated enzyme kinetic parameters (K) m And V max ) Comparison of
Figure BDA0003445287660000061
4. Cu/Au/Pt-MOFs-H 2 O 2 Antibacterial system exploration
1. And (3) bacterial culture: inoculating the preserved salmonella strain into a beef extract peptone liquid culture medium, and performing shake culture at 37 ℃ for 18-24h. Centrifuging 1mL of bacterial solution at 5000rpm for 8min, discarding the supernatant, resuspending the bacteria with 0.9% physiological saline, and diluting the bacteria concentration to 10 by adopting multiple dilution 6 CFU/mL, spare.
2. The antibacterial effect is preliminarily researched: 1mL of 10 6 CFU/mL bacterial solution, 100. Mu.L Cu/Au/Pt-MOFs (OD) was added 350 = 5.28) and a final concentration of 100 μ M H 2 O 2 After shaking treatment at 37 deg.C for 2h, 100. Mu.L of the treated bacterial liquid was applied to a solid medium, and 100. Mu.L of 10 cells were applied without any treatment 6 Coating a flat plate with CFU/mL bacterial liquid as a control group, and culturing at 37 ℃ for 18-24h; and taking a picture of the flat plate by adopting a full-automatic colony counter.
The preliminary bactericidal effect is shown in FIG. 6, except that Cu/Au/Pt-MOFs and H are contained simultaneously 2 O 2 The treated group (2) had no obvious bacterial growth, and the other three groups had colonies; illustrates 100. Mu.M H alone 2 O 2 Or 100 mu L of Cu/Au/Pt-MOFs can not effectively kill bacteria; only when Cu/Au/Pt-MOFs and H 2 O 2 And can effectively kill bacteria when existing at the same time.
3、H 2 O 2 Effect of concentration on antibacterial effect:
100 μ L of the above synthesized Cu/Au/Pt-MOFs (OD) 350 = 5.28) and final concentrations of 50, 100, 200, 300 μ M of H, respectively 2 O 2 Add to 1mL of 10 6 The mixture was shaken at 37 ℃ for 2 hours in CFU/mL bacterial solution. Respectively taking 100 μ L of bacterial liquid, coating on solid culture medium, and simultaneously taking 100 μ L of 10 μ L without any treatment 6 And (3) coating a plate with CFU/mL bacterial liquid as a control group, culturing for 18-24h in an incubator at 37 ℃, and photographing the plate by using a full-automatic colony counter.
H 2 O 2 The effect of concentration on the bactericidal effect is shown in FIG. 7, with H at 50-300. Mu.M 2 O 2 All have good sterilization effect, and are particularly remarkableH at biologically relevant concentrations (50-100. Mu.M) 2 O 2 Next, cu/Au/Pt-MOFs-H 2 O 2 The reaction system also has remarkable antibacterial effect, and H which is about two orders of magnitude higher than other required uses 2 O 2 Compared with the nanometer material, the Cu/Au/Pt-MOFs are more beneficial to practical application because of high concentration of H 2 O 2 To some extent, can damage healthy cells.
4. Influence of the dosage of Cu/Au/Pt-MOFs on the antibacterial effect:
10, 30, 50, 70, 100. Mu.L of the above-synthesized Cu/Au/Pt-MOFs (OD) 350 = 5.28) were added to a solution containing 50 μ M H 2 O 2 10 of 6 After shaking treatment at 37 ℃ for 2h in CFU/mL bacterial solution, 100. Mu.L of bacterial solution was applied to solid medium and 100. Mu.L of 10 cells were applied without any treatment 6 CFU/mL bacterial solution coated plate as a control group, and cultured in an incubator at 37 ℃ for 18-24h.
The effect of the dosage of Cu/Au/Pt-MOFs on the bactericidal effect is shown in FIG. 8, and the dosage of Cu/Au/Pt-MOFs increases, so that the dosage of Cu/Au/Pt-MOFs-H 2 O 2 The more obvious the antibacterial effect of the system is, when the dosage of Cu/Au/Pt-MOFs is more than 70 muL, almost no bacterial colony grows on the flat plate, so that the dosage of Cu/Au/Pt-MOFs in the subsequent experiment is 70 muL.
5. Cu/Au/Pt-MOFs-H 2 O 2 Sterilizing effect of reaction system on salmonella, staphylococcus aureus and pseudomonas aeruginosa
1. And (3) bacterial culture: respectively inoculating the preserved salmonella strain, the preserved staphylococcus aureus strain and the preserved pseudomonas aeruginosa strain into a beef extract peptone liquid culture medium, and carrying out shake culture at 37 ℃ for 18-24h. Centrifuging 1mL of bacterial solution at 5000rpm for 8min, discarding the supernatant, resuspending the bacteria with 0.9% physiological saline, and diluting the bacteria concentration to 10 by adopting multiple dilution 6 CFU/mL, spare.
2.Cu/Au/Pt-MOFs-H 2 O 2 The reaction system has the bactericidal effect on salmonella, staphylococcus aureus and pseudomonas aeruginosa:
respectively taking 1mL of the solution with the concentration of 10 6 CFU/mL Salmonella, staphylococcus aureus and Pseudomonas aeruginosa70 μ L of Cu/Au/Pt-MOFs (OD) was added to the bacterial solution 350 = 5.28) and a final concentration of 50 μ M H 2 O 2 After shaking treatment at 37 deg.C for 2h, 100. Mu.L of the treated bacterial liquid was applied to a solid medium, and 100. Mu.L of 10 cells were applied without any treatment 6 Coating a flat plate with CFU/mL bacterial liquid as a control group, and culturing at 37 ℃ for 18-24h; and taking a picture of the flat plate by adopting a full-automatic colony counter. Cu/Au/Pt-MOFs-H 2 O 2 The bactericidal effect of the system on salmonella, staphylococcus aureus and pseudomonas aeruginosa is shown in figure 9, and Cu/Au/Pt-MOFs and H 2 O 2 Can effectively kill salmonella, staphylococcus aureus and pseudomonas aeruginosa in a neutral medium, and shows that Cu/Au/Pt-MOFs-H 2 O 2 The system has good broad-spectrum bactericidal activity on gram-negative enteric bacilli, gram-positive staphylococci and gram-negative bacilli which are easy to form a difficult-to-treat biological membrane.
6. Cu/Au/Pt-MOFs catalyzed H 2 O 2 Effect of oxidizing TMB
1. Catalysis of Cu/Au/Pt-MOFs by H 2 O 2 Oxidation of TMB to achieve assay H 2 O 2 The operation steps are as follows:
mu.L of TMB (6 mmol/L), 25. Mu.L of LCu/Au/Pt-MOFs and 10. Mu.L of H at different concentrations were added to 145. Mu.L of NaOAc buffer solution (0.01M, pH 5.5) 2 O 2 And standing the solution at room temperature for 10min, and measuring the absorption spectrum of the reaction system in the range of 550-750nm by using an ultraviolet-visible spectrophotometer.
Through experiments, the following results are found: the absorbance is in the range of 0-1200. Mu.M with H 2 O 2 The concentration increases with increasing; absorbance at 652nm with H in the range of 10 μ M to 800 μ M 2 O 2 The concentration is in a linear relation; and the method is used for detecting H 2 O 2 Has a lower detection limit.
2. Catalysis of Cu/Au/Pt-MOFs by H 2 O 2 Oxidation of TMB to determine Cys was carried out as follows:
to 135. Mu.L of a buffer solution of NaOAc (0.01M, pH 5.5) were added 20. Mu.L of TMB (6 mmol/L) and 10. Mu.L of H 2 O 2 (20mmol/L)、25 mu L of Cu/Au/PtNPs-MOFs and 10 mu L of Cys solution with different concentrations are stood for reaction at room temperature for 15min, and then an ultraviolet-visible spectrophotometer is used for measuring the absorption spectrum of the reaction system in the range of 550-750 nm.
Through the experiment, the following results are found: absorbance decreases with increasing Cys concentration in the range of 0-700 μ M; in the range of 0 μ M to 300 μ M, the absorbance at 652nm is linear with Cys concentration; and the method has a lower detection limit for detecting Cys.
3. Cu/Au/Pt-MOFs by catalysis of H 2 O 2 Oxidizing TMB to realize the determination of glucose, and the operation steps are as follows:
adding 10 mu L of 8mg/mL glucose oxidase solution into 290 mu L of glucose solution with different concentrations, mixing uniformly, and putting into a metal bath for incubation for 15min; then, 135. Mu.L of the above hatching solution was added to 135. Mu.L of NaOAc buffer solution (0.01M, pH 5.5) containing 20. Mu.L of TMB (6 mmol/L) and 25. Mu.L of Cu/Au/Pt NPs-MOFs, and after allowing to stand at room temperature for reaction for 15 minutes, the absorbance of the reaction system at 652nm was measured using an ultraviolet-visible spectrophotometer.
Through experiments, the following results are found: the absorbance at 652nm increases with increasing glucose concentration; in the range of 0 to 800. Mu.M, the absorbance at 652nm is linear with the glucose concentration; and the method has lower detection limit for detecting glucose.

Claims (4)

1. A Cu/Au/Pt-MOFs composite material with enhanced peroxidase activity is characterized by mainly comprising a metal organic framework composite material Cu-TCPP (Fe) and Cu/Au/Pt multi-metal nanoparticles growing on the surface in situ;
the mass ratio of the Cu-TCPP (Fe) to the Cu/Au/Pt multi-metal nano particles is 1 (0.1-10);
in the Cu/Au/Pt multi-metal nano particles, the mass ratio of Cu to Au to Pt is 1 (0.1-50) to 0.1-50;
the Cu/Au/Pt-MOFs composite material is prepared by the following method: dissolving 1mL of Cu-TCPP (Fe) solution with the concentration of 1mg/mL in 10mL of ultrapure water; under magnetic stirring, 36. Mu.L of CuSO was added 4 75 mu.L of trisodium citrate, and stirring the mixture for 10 minutesmin; 1.5mL of KBH with a concentration of 25mM was added dropwise 4 Then, stirring for 10 min; 75 μ L of 0.1M HAuCl was added dropwise 4 ,75μL K 2 PtCl 4 Then, stirring for 20 min; centrifuging at 10000rpm for 10min after stirring; washing the precipitate with ultrapure water, centrifuging at 12000rpm for 10min, and collecting to obtain the Cu/Au/Pt NPs-MOFs composite material.
2. A method for preparing the Cu/Au/Pt-MOFs composite material according to claim 1, comprising the steps of: dissolving 1mL of Cu-TCPP (Fe) solution with the concentration of 1mg/mL in 10mL of ultrapure water; under magnetic stirring, 36. Mu.L of CuSO was added 4 Stirring 75 mu L of trisodium citrate for 10 min; 1.5mL of KBH with a concentration of 25mM was added dropwise 4 Then, stirring for 10 min; 75 μ L of 0.1M HAuCl was added dropwise 4 ,75μL K 2 PtCl 4 Then, stirring for 20 min; centrifuging at 10000rpm for 10min after stirring; washing the precipitate with ultrapure water, centrifuging at 12000rpm for 10min, and collecting to obtain the Cu/Au/Pt NPs-MOFs composite material.
3. Use of the Cu/Au/Pt-MOFs composite material according to claim 1 or the Cu/Au/Pt-MOFs composite material prepared by the preparation method according to claim 2 in preparation of an antibacterial agent, wherein the antibacterial agent comprises the Cu/Au/Pt-MOFs composite material and H 2 O 2 (ii) a The antibacterial object of the antibacterial agent is staphylococcus aureus(Staphylococcus aureus)Pseudomonas aeruginosa(Pseudomonas aeruginosa)And Salmonella(salmonella)Any one or more of the bacteria; the antibacterial agent contains H 2 O 2 The final concentration of the composite material is 50-300 mu M, and the OD of the composite material containing Cu/Au/Pt-MOFs 350 =0.5-6。
4. Use according to claim 3, characterized in that the concentration per ml is 10 6 CFU/mL bacterial liquid, and the dosage of the antibacterial agent is more than 70 muL.
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