CN114669317B - Nano-enzyme with multistage enzyme-linked reaction performance, and preparation method and application thereof - Google Patents
Nano-enzyme with multistage enzyme-linked reaction performance, and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000000203 mixture Substances 0.000 claims abstract description 20
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- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 12
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- 239000002243 precursor Substances 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims abstract description 6
- 239000010949 copper Substances 0.000 claims description 59
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 11
- XTVVROIMIGLXTD-UHFFFAOYSA-N copper(II) nitrate Chemical group [Cu+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XTVVROIMIGLXTD-UHFFFAOYSA-N 0.000 claims description 5
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 12
- 238000010523 cascade reaction Methods 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 239000002086 nanomaterial Substances 0.000 abstract description 3
- 239000012567 medical material Substances 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 description 83
- 239000004366 Glucose oxidase Substances 0.000 description 23
- 108010015776 Glucose oxidase Proteins 0.000 description 23
- 229940116332 glucose oxidase Drugs 0.000 description 23
- 235000019420 glucose oxidase Nutrition 0.000 description 23
- 238000012360 testing method Methods 0.000 description 23
- 102000003992 Peroxidases Human genes 0.000 description 19
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- 229910021641 deionized water Inorganic materials 0.000 description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 11
- 239000008103 glucose Substances 0.000 description 11
- 239000000758 substrate Substances 0.000 description 11
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 10
- 241000191967 Staphylococcus aureus Species 0.000 description 10
- 108040007629 peroxidase activity proteins Proteins 0.000 description 10
- 108010046301 glucose peroxidase Proteins 0.000 description 9
- 230000031700 light absorption Effects 0.000 description 8
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 7
- 238000004321 preservation Methods 0.000 description 7
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 6
- 230000003197 catalytic effect Effects 0.000 description 6
- 229910052573 porcelain Inorganic materials 0.000 description 6
- 238000000502 dialysis Methods 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
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- 229910052786 argon Inorganic materials 0.000 description 3
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- 238000001291 vacuum drying Methods 0.000 description 3
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
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- 229910001882 dioxygen Inorganic materials 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000004627 transmission electron microscopy Methods 0.000 description 2
- AYLDJQABCMPYEN-UHFFFAOYSA-N (4-azaniumylphenyl)-diethylazanium;sulfate Chemical compound OS(O)(=O)=O.CCN(CC)C1=CC=C(N)C=C1 AYLDJQABCMPYEN-UHFFFAOYSA-N 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
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- 239000003593 chromogenic compound Substances 0.000 description 1
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- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
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- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 238000003837 high-temperature calcination Methods 0.000 description 1
- LELOWRISYMNNSU-UHFFFAOYSA-N hydrogen cyanide Chemical compound N#C LELOWRISYMNNSU-UHFFFAOYSA-N 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002064 nanoplatelet Substances 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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- 230000004083 survival effect Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- DKZXTOPFCDDGGX-UHFFFAOYSA-N tri-s-triazine Chemical group C1=NC(N23)=NC=NC2=NC=NC3=N1 DKZXTOPFCDDGGX-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J27/00—Catalysts comprising the elements or compounds of halogens, sulfur, selenium, tellurium, phosphorus or nitrogen; Catalysts comprising carbon compounds
- B01J27/24—Nitrogen compounds
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION 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
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION 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
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/16—Heavy metals; Compounds thereof
- A01N59/20—Copper
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J23/00—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00
- B01J23/70—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the iron group metals or copper
- B01J23/72—Copper
-
- B01J35/39—
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B21/00—Nitrogen; Compounds thereof
- C01B21/06—Binary compounds of nitrogen with metals, with silicon, or with boron, or with carbon, i.e. nitrides; Compounds of nitrogen with more than one metal, silicon or boron
- C01B21/0605—Binary compounds of nitrogen with carbon
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
- C01P2002/84—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by UV- or VIS- data
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/04—Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM
Abstract
The invention discloses a nano enzyme with multistage enzyme-linked reaction performance, and a preparation method and application thereof, and relates to the technical field of medical materials. The nano enzyme is Cu/g-C 3 N 4 By modifying Cu to g-C 3 N 4 And obtaining the nano-sheet. The preparation method comprises the following steps: combining a Cu metal precursor with g-C 3 N 4 Adding the nano-sheets into water, mixing and stirring uniformly, and drying to obtain a mixture; and calcining the mixture in an inert atmosphere, cooling, washing and drying to obtain the nano enzyme with the multi-stage enzyme-linked reaction performance. The invention realizes the enzyme cascade reaction and proves that the enzyme cascade reaction has effective sterilization performance. Expands the research scope of nano enzyme materials and g-C 3 N 4 The application field of the nano material provides a novel multifunctional nano enzyme material.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to nano-enzyme with multistage enzyme-linked reaction performance, and a preparation method and application thereof.
Background
The nano-enzyme has high-efficiency and unique biological catalytic activity, has the characteristics of high catalytic efficiency, stability, economy and large-scale preparation, and is widely researched in the fields of sterilization, tissue repair, tumor treatment, biological detection and the like. Nanomaterial-derived artificial enzymes have been extensively studied to mimic and replace proteases. Glucose oxidase (GOx) and horseradish peroxidase (HRP) are commonly used in enzyme cascade catalytic reactions and can be used for monitoring blood glucose in human body, and the principle is that GOx can oxidize glucose into H 2 O 2 And gluconic acid, H 2 O 2 The glucose level can be detected by colorimetry by reaction with chromogenic substrate under the action of HRP. The disadvantage of using GOx and HRP to achieve this cascade is that both are less stable as natural proteases and the production costs are higher. Some reported artificial enzymes have GOx or HRP functions, but few artificial enzymes with GOx and HRP functions are reported. The artificial enzyme with glucose oxidase and peroxidase functions can improve the efficiency of two catalytic reactions and increase the atom availability. Therefore, the development of artificial enzymes having both glucose oxidase and peroxidase functions has wide economic and research values.
Disclosure of Invention
The invention aims to provide a nano-enzyme with multi-stage enzyme-linked reaction performance, a preparation method and application thereof, so that the nano-enzyme has GOx and peroxidase functions at the same time, can be used for cascade reaction of glucose oxidase and peroxidase, and has effective sterilization performance.
In order to achieve the above object, the present invention provides the following solutions:
one of the technical proposal of the invention is that the multi-stage ELISANano-enzyme with reactivity, wherein the nano-enzyme is Cu/g-C 3 N 4 By modifying Cu to g-C 3 N 4 And obtaining the nano-sheet.
Further, the Cu/g-C 3 N 4 Cu and g-C in the middle 3 N 4 The mass ratio of (2) is 1:10.
the second technical scheme of the invention is that the preparation method of the nano-enzyme with the multistage enzyme-linked reaction performance comprises the following steps:
combining a Cu metal precursor with g-C 3 N 4 Adding the nano-sheets into water, mixing and stirring uniformly, and drying to obtain a mixture;
and calcining the mixture in an inert atmosphere, cooling, washing and drying to obtain the nano enzyme with the multi-stage enzyme-linked reaction performance.
Further, the Cu metal precursor copper nitrate.
Further, the Cu metal precursor and the g-C 3 N 4 The mass ratio of the nano-sheets is 1:10
Further, the calcining specifically comprises: heating to 130-150deg.C at 9-11deg.C/min, maintaining for 8-10 hr, and heating to 500-600deg.C at 8-9deg.C/min, and maintaining for 1-2 hr.
Further, the g-C 3 N 4 The preparation method of the nano-sheet comprises the following steps:
step 1, calcining dicyandiamide to obtain a block g-C 3 N 4 ；
Step 2, the block g-C 3 N 4 Dissolving in anhydrous sulfuric acid to obtain a mixture, diluting the mixture to obtain a clear solution, adding the clear solution into anhydrous ethanol, stirring, dialyzing to neutrality, and drying to obtain the g-C 3 N 4 A nano-sheet.
Further, the calcining treatment in step 1 specifically includes: heating from 20deg.C to 500-600deg.C at uniform speed in air atmosphere for 3-5 hr, and maintaining the temperature in air atmosphere for 3-5 hr; naturally cooling to room temperature after heat preservation is finished, and grinding to obtain a block g-C 3 N 4 。
Further, the block g-C in step 2 3 N 4 The mass volume ratio of the anhydrous sulfuric acid to the anhydrous ethanol is 1-2g:10-20mL:30-40mL; the stirring time is 16-20h; the dialysis specifically comprises the following steps: dialyzing in deionized water to neutrality by using a dialysis bag with membrane cutoff of 3500 kDa; the drying temperature is 50-60 ℃.
In the third technical scheme of the invention, the nano-enzyme with the multi-stage enzyme-linked reaction performance is applied to multi-stage enzyme-linked reaction and sterilization.
The technical conception of the invention is as follows:
by H 2 Gaseous reagents for the production of H 2 O 2 Is a common way, however, the photoinduction of H by proton-coupled electron transfer to molecular oxygen 2 O 2 Is a superior choice in the future because it does not require H 2 Gaseous reagents and no environmental pollution. The main problem in this process is the need to reduce the H produced as much as possible 2 O 2 And selectively effecting the transfer of both electrons to molecular oxygen. As a unique two-dimensional polymer metal-free semiconductor, graphitic carbon nitride (g-C 3 N 4 ) Consisting of tri-s-triazine units bridged with tertiary nitrogen, for H 2 O 2 Has lower adsorption energy and can be used for effectively inhibiting H 2 O 2 Ideal material for in situ decomposition. g-C 3 N 4 The chemical functional groups and electronic properties of (a) can be altered by modification, which makes it H-producing 2 O 2 Is a promising material for the same. Despite good biocompatibility of g-C 3 N 4 Peroxidase-mimicking nanoenzymes have also been used for glucose detection, but there are no reports on enzymatic tandem cascade and sterilization using GOx-mimicking behavior for glucose colorimetric detection. g-C 3 N 4 The light absorption capacity of the fluorescent lamp is weaker and is generally smaller than 420nm, and the fluorescent lamp has important research significance for improving the visible light absorption capacity. The invention constructs Cu modified g-C 3 N 4 The nano-sheet improves the light absorption range and realizes the effective absorption of lightAnd (5) collecting. The nano-enzyme material can effectively realize the dual-function performance of glucose oxidase and peroxidase, realize the cascade reaction of the glucose oxidase and the peroxidase, kill staphylococcus aureus and have high-efficiency sterilization capability.
The invention discloses the following technical effects:
(1) The invention is realized by modifying Cu to g-C 3 N 4 Obtaining nano enzyme Cu/g-C on the nano sheet 3 N 4 The glucose oxidase and peroxidase activities of the strain are respectively studied, the enzyme cascade reaction is realized, and the strain has effective sterilization performance. Expands the research scope of nano enzyme materials and g-C 3 N 4 The application field of the nano material provides a novel multifunctional nano enzyme material.
(2) The nano enzyme Cu/g-C prepared by the invention 3 N 4 Low toxicity and good biocompatibility.
(3) The nano enzyme Cu/g-C of the invention 3 N 4 The preparation method is simple, and can be obtained in large quantity through high-temperature calcination.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the nano-enzyme Cu/g-C prepared in example 1 3 N 4 Transmission electron microscopy images of (2);
FIG. 2 shows the nano-enzyme Cu/g-C prepared in example 1 3 N 4 Is a light absorption capacity test chart of (1);
FIG. 3 shows the nano-enzyme Cu/g-C prepared in example 1 3 N 4 As a glucose oxidase test chart; wherein, the left graph shows the test of different oxydols (H) under different glucose concentrations 2 O 2 ) The generated ultraviolet visible graph, the right graph is an enzyme activity graph；
FIG. 4 shows the nano-enzyme Cu/g-C prepared in example 1 3 N 4 As a graph for testing the bactericidal effect of glucose oxidase;
FIG. 5 shows the nano-enzyme Cu/g-C prepared in example 1 3 N 4 A test chart using TMB as a substrate as a peroxidase; wherein the left graph shows the test Cu/g-C under different TMB concentrations 3 N 4 Catalytic oxidation H 2 O 2 The right graph is an enzyme activity curve graph with TMB as a substrate;
FIG. 6 shows the nano-enzyme Cu/g-C prepared in example 1 3 N 4 As peroxidase, H is used 2 O 2 A test pattern for a substrate; wherein the left graph is different H 2 O 2 Testing Cu/g-C at concentration 3 N 4 Catalytic oxidation H 2 O 2 The right image is H 2 O 2 An enzyme activity profile for a substrate;
FIG. 7 shows the nano-enzyme Cu/g-C prepared in example 1 3 N 4 As a graph of the bactericidal effect of peroxidase on staphylococcus aureus;
FIG. 8 shows the nano-enzyme Cu/g-C prepared in example 1 3 N 4 Is a result of the biocompatibility test of (2);
FIG. 9 is a nano-enzyme Fe/g-C prepared in comparative example 1 3 N 4 、Zn/g-C 3 N 4 、Cr/g-C 3 N 4 、K/g-C 3 N 4 、g-C 3 N 4 As a comparative graph of the glucose oxidase test.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The term "room temperature" as used herein refers to 15-35℃unless otherwise specified.
Example 1
Step 1, block g-C 3 N 4 Is prepared from the following steps: 6g dicyandiamide is put into a ceramic calcination boat, the temperature is uniformly increased from 20 ℃ to 550 ℃ in 4 hours under the air atmosphere, and the heat is preserved for 4 hours under the air atmosphere; naturally cooling to room temperature after heat preservation is finished, and grinding to obtain a block g-C 3 N 4 ；
Step 2, g-C 3 N 4 Preparation of nanosheets: 3g of block g-C 3 N 4 Dispersed in 30mL anhydrous H 2 SO 4 Stirring for 1 hr to obtain g-C 3 N 4 /H 2 SO 4 A mixture; 10mL of deionized water was gradually added to g-C 3 N 4 /H 2 SO 4 In the mixture, a clear solution was obtained. The clear solution was then added sequentially to 90mL of absolute ethanol and stirred for 18 hours, dialyzed against deionized water using a dialysis bag (membrane cutoff: 3500 kDa) to neutrality (to remove residual SO) 4 2- And ethanol), drying at 60deg.C, and collecting to obtain g-C 3 N 4 Nanometer sheet (g-C) 3 N 4 NS)；
Step 3, nanoenzyme (Cu/g-C 3 N 4 ) Is prepared from the following steps: 200mg of copper nitrate and 2.0g of g-C prepared in step 2 3 N 4 Mixing NS and deionized water, stirring for 2 hours, drying on a hot plate, putting the obtained mixture into a semi-closed porcelain boat with a cover, placing the semi-closed porcelain boat into a tube furnace, keeping the temperature for 9 hours at 130 ℃ at a speed of 10 ℃/min under argon gas flow, heating to 550 ℃ at a heating speed of 8.67 ℃/min, and keeping the temperature for 1 hour; cooling to room temperature after heat preservation, washing with 30mL deionized water for 5 times, and vacuum drying to obtain nano-enzyme (Cu/g-C) 3 N 4 )。
The nano-enzyme Cu/g-C prepared in the example 3 N 4 As can be seen from FIG. 1, the transmission electron microscopy of (C) is shown in FIG. 1, and the nano-enzyme Cu/g-C 3 N 4 The size of (2) is about 500nm, and Cu atoms are uniformly doped in g-C 3 N 4 The surface of the nanoplatelets.
The nano-enzyme Cu/g-C prepared in the example 3 N 4 As can be seen from FIG. 2, the introduction of Cu can effectively improve g-C 3 N 4 To the near infrared region.
The nano-enzyme Cu/g-C prepared in the example 3 N 4 As a graph for the glucose oxidase test, a graph is shown in FIG. 3 (specifically, cu/g-C 3 N 4 Mixing the nano enzyme with glucose with different concentrations, illuminating for 30 minutes, adding N, N diethyl-1, 4-phenylenediamine sulfate (DPD) and horseradish Peroxidase (POD) into the solution, and measuringTesting light absorption capacity to obtain enzyme activity curve), wherein the left graph shows the test of different hydrogen peroxide (H) under different glucose concentrations 2 O 2 ) The generated ultraviolet-visible graph, the right graph is the enzyme activity curve. As can be seen from FIG. 3, the nano-enzyme Cu/g-C 3 N 4 V relative to substrate glucose max Is 7.27X10 -8 M s -1 K is as follows m Is 1.915mM.
The nano-enzyme Cu/g-C prepared in the example 3 N 4 The graph for testing the bactericidal effect of glucose oxidase is shown in FIG. 4 (specifically, staphylococcus aureus S.aureus Xen36 was centrifuged and resuspended in sterile PBS to obtain bacterial liquid at a concentration of 2×10) 7 CFU/mL; cu/g-C 3 N 4 And mixing the nano enzyme with the bacterial liquid, adding glucose, irradiating under visible light for 30min, and evaluating the sterilization effect by a gradient dilution and coating method. As can be seen from FIG. 4, the nano-enzyme Cu/g-C 3 N 4 The glucose oxidase can effectively kill staphylococcus aureus, and the sterilization efficiency reaches 100% when the concentration is 12.5 ug/mL.
The nano-enzyme Cu/g-C prepared in the example 3 N 4 As a test chart of peroxidase using TMB as a substrate, a test chart is shown in FIG. 5 (specifically: nano-enzyme Cu/g-C 3 N 4 And H is 2 O 2 TMB mix, test light absorption capacity at 650 nm; by varying the TMB concentration, different absorption values were obtained, and an enzyme activity curve was obtained), wherein the left plot shows the Cu/g-C tested at different TMB concentrations 3 N 4 Catalytic oxidation H 2 O 2 The right panel shows the graph of enzyme activity using TMB as substrate. As can be seen from FIG. 5, the nano-enzyme Cu/g-C 3 N 4 V relative to the substrate TMB max Is 3.8X10 -8 Ms -1 K is as follows m Is 0.26mM.
The nano-enzyme Cu/g-C prepared in the example 3 N 4 As peroxidase, H is used 2 O 2 The test chart for the substrate is shown in FIG. 6 (specifically: nano-enzyme Cu/g-C 3 N 4 And H is 2 O 2 Mixing with TMB, testing light absorption energy at 650nmForce; by changing H 2 O 2 Different absorption values are obtained to obtain an enzyme activity curve), wherein the left graph shows different H 2 O 2 Testing Cu/g-C at concentration 3 N 4 Catalytic oxidation H 2 O 2 The right image is H 2 O 2 Is the enzyme activity curve graph of the substrate. As can be seen from FIG. 6, the nano-enzyme Cu/g-C 3 N 4 Relative to substrate H 2 O 2 V of concentration max Is 13.26X10 -8 M s -1 K is as follows m Is 0.25mM.
The nano-enzyme Cu/g-C prepared in the example 3 N 4 The graph of the sterilizing effect of peroxidase on Staphylococcus aureus is shown in FIG. 7 (specifically, staphylococcus aureus S.aureus Xen36 is centrifugally separated and resuspended in sterile PBS to obtain bacterial liquid with the concentration of 2×10) 7 CFU/mL; nano enzyme Cu/g-C 3 N 4 Mixing with fungus solution, adding H 2 O 2 The bactericidal effect was evaluated by gradient dilution and coating after 3 hours incubation at 37 ℃. As can be seen from FIG. 7, the nano-enzyme Cu/g-C 3 N 4 As peroxidase, the staphylococcus aureus can be completely killed at the concentration of 6.3 ug/mL.
The nano-enzyme Cu/g-C prepared in the example 3 N 4 The results of the biocompatibility test of (1) are shown in FIG. 8 (specifically: the number of surviving cells after 12 hours in which L929 cells were mixed with the material). As can be seen from FIG. 8, the survival rate of the cells was 75% or more after the addition of the material to the cells, indicating that the nano-enzyme Cu/g-C 3 N 4 Has the advantages of low toxicity and good biocompatibility.
Example 2
Step 1, block g-C 3 N 4 Is prepared from the following steps: 6g dicyandiamide is put into a ceramic calcination boat, the temperature is increased from 20 ℃ to 500 ℃ at a constant speed in 3 hours under the air atmosphere, and the temperature is kept for 5 hours under the air atmosphere; naturally cooling to room temperature after heat preservation is finished, and grinding to obtain a block g-C 3 N 4 ；
Step 2, g-C 3 N 4 Preparation of nanosheets: 3g of block g-C 3 N 4 Dispersed in 30mL anhydrous H 2 SO 4 Stirring for 1 hr to obtain g-C 3 N 4 /H 2 SO 4 A mixture; 10mL of deionized water was gradually added to g-C 3 N 4 /H 2 SO 4 In the mixture, a clear solution was obtained. The clear solution was then added sequentially to 120mL of absolute ethanol and stirred for 16 hours, dialyzed against deionized water using a dialysis bag (membrane cutoff: 3500 kDa) to neutrality (to remove residual SO) 4 2- And ethanol), drying at 50deg.C, and collecting to obtain g-C 3 N 4 Nanometer sheet (g-C) 3 N 4 NS)；
Step 3, nanoenzyme (Cu/g-C 3 N 4 ) Is prepared from the following steps: 200mg of copper nitrate and 2.0g of g-C prepared in step 2 3 N 4 Mixing NS and deionized water, stirring for 2 hours, drying on a hot plate, putting the obtained mixture into a semi-closed porcelain boat with a cover, placing the semi-closed porcelain boat into a tube furnace, keeping the temperature for 10 hours at 140 ℃ at a speed of 9 ℃/min under argon gas flow, heating to 600 ℃ at a heating speed of 9 ℃/min, and keeping the temperature for 1.3 hours; cooling to room temperature after heat preservation, washing with 30mL deionized water for 5 times, and vacuum drying to obtain nano-enzyme (Cu/g-C) 3 N 4 ). Results: the nano-enzyme Cu/g-C prepared in the example 3 N 4 Can effectively realize the dual-functional performance of glucose oxidase and peroxidase and kill staphylococcus aureus.
Results: the nano enzyme prepared by the embodiment has the activities of glucose oxidase and peroxidase, and the sterilization efficiency on staphylococcus aureus reaches 100%.
Example 3
Step 1, block g-C 3 N 4 Is prepared from the following steps: 6g dicyandiamide is put into a ceramic calcination boat, the temperature is increased from 20 ℃ to 600 ℃ at a constant speed in 5 hours under the air atmosphere, and the temperature is kept for 3 hours under the air atmosphere; naturally cooling to room temperature after heat preservation is finished, and grinding to obtain a block g-C 3 N 4 ；
Step 2, g-C 3 N 4 Preparation of nanosheets: 3g of block g-C 3 N 4 Dispersed at 30mL anhydrous H 2 SO 4 Stirring for 1 hr to obtain g-C 3 N 4 /H 2 SO 4 A mixture; 10mL of deionized water was gradually added to g-C 3 N 4 /H 2 SO 4 In the mixture, a clear solution was obtained. The clear solution was then added sequentially to 105mL of absolute ethanol and stirred for 20 hours, dialyzed against deionized water using a dialysis bag (membrane cutoff: 3500 kDa) to neutrality (to remove residual SO) 4 2- And ethanol), drying at 55deg.C, and collecting to obtain g-C 3 N 4 Nanometer sheet (g-C) 3 N 4 NS)；
Step 3, nanoenzyme (Cu/g-C 3 N 4 ) Is prepared from the following steps: 200mg of copper nitrate and 2.0g of g-C prepared in step 2 3 N 4 Mixing NS and deionized water, stirring for 2 hours, drying on a hot plate, putting the obtained mixture into a semi-closed porcelain boat with a cover, placing the semi-closed porcelain boat into a tube furnace, keeping the temperature for 8 hours at the speed of 11 ℃/min under argon gas flow, heating to 500 ℃ at the heating speed of 8 ℃/min, and keeping the temperature for 2 hours; cooling to room temperature after heat preservation, washing with 30mL deionized water for 5 times, and vacuum drying to obtain nano-enzyme (Cu/g-C) 3 N 4 )。
Results: the nano enzyme prepared by the embodiment has the activities of glucose oxidase and peroxidase, and the sterilization efficiency on staphylococcus aureus reaches 100%.
Comparative example 1
The difference from example 1 is that Cu is changed to other metals (Fe, zn, cr, K respectively) to prepare nano-enzyme Fe/g-C respectively 3 N 4 Nano enzyme Zn/g-C 3 N 4 Nano enzyme Cr/g-C 3 N 4 Nano enzyme K/g-C 3 N 4 。
Test of nano enzyme Fe/g-C prepared in comparative example 1 3 N 4 Nano enzyme Zn/g-C 3 N 4 Nano enzyme Cr/g-C 3 N 4 Nano enzyme K/g-C 3 N 4 As a result of the function of glucose oxidase and comparison with example 1, the results are shown in FIG. 9 (specifically: g-C 3 N 4 And nano enzyme Cu/g-C 3 N 4 、Fe/g-C 3 N 4 、Zn/g-C 3 N 4 、Cr/g-C 3 N 4 、K/g-C 3 N 4 After 30 minutes of light irradiation, DPD and POD were added to the solution to test the light absorption capacity, respectively, by mixing with glucose. As can be seen from FIG. 9, cu/g-C was compared with other metal-modified nanoenzymes 3 N 4 Shows the strongest absorption peak, indicating the generation of H 2 O 2 The amount of (2) is the largest. Thus, in the present invention, the choice of metal is a critical parameter, while Cu has more excellent properties.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (2)
1. A nano-enzyme with multistage enzyme-linked reaction performance is characterized in that the nano-enzyme is Cu/g-C 3 N 4 By modifying Cu to g-C 3 N 4 Obtaining the nano-sheet;
the preparation method of the nano-enzyme with the multistage enzyme-linked reaction performance comprises the following steps:
combining a Cu metal precursor with g-C 3 N 4 Adding the nano-sheets into water, mixing and stirring uniformly, and drying to obtain a mixture;
calcining the mixture in an inert atmosphere, cooling, washing and drying to obtain the nano enzyme with the multi-stage enzyme-linked reaction performance;
the Cu metal precursor is copper nitrate;
the Cu metal precursor and the g-C 3 N 4 The mass ratio of the nano-sheets is 1:10;
the calcination is specifically as follows: heating to 130-150 ℃ at the speed of 9-11 ℃/min and preserving heat for 8-10h, and then continuously heating to 500-600 ℃ at the speed of 8-9 ℃/min and preserving heat for 1-2h;
the g-C 3 N 4 The preparation method of the nano-sheet comprises the following steps:
step 1, calcining dicyandiamide to obtain a block g-C 3 N 4 ；
Step 2, the block g-C 3 N 4 Dissolving in anhydrous sulfuric acid to obtain a mixture, diluting the mixture to obtain a clear solution, adding the clear solution into anhydrous ethanol, stirring, dialyzing to neutrality, and drying to obtain the g-C 3 N 4 A nano-sheet.
2. The use of a nanoenzyme with multi-stage enzyme-linked reaction performance according to claim 1 in multi-stage enzyme-linked reaction and sterilization.
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