CN114031067A - Carbon dots with superoxide dismutase activity and preparation method thereof - Google Patents
Carbon dots with superoxide dismutase activity and preparation method thereof Download PDFInfo
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- 102000019197 Superoxide Dismutase Human genes 0.000 title claims abstract description 33
- 108010012715 Superoxide dismutase Proteins 0.000 title claims abstract description 33
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- 238000002360 preparation method Methods 0.000 title abstract description 24
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- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
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- BMMGVYCKOGBVEV-UHFFFAOYSA-N oxo(oxoceriooxy)cerium Chemical compound [Ce]=O.O=[Ce]=O BMMGVYCKOGBVEV-UHFFFAOYSA-N 0.000 description 1
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- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B32/00—Carbon; Compounds thereof
- C01B32/15—Nano-sized carbon materials
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
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Abstract
The invention provides a carbon dot with superoxide dismutase activity and a preparation method thereof, and the carbon dot comprises the following steps: mixing a carbon material and a nitric acid solution to obtain a mixture solution, wherein the concentration of the mixture solution is 10-20mg mL‑1The concentration of nitric acid is 15M,10M or 5M; stirring the mixture solution at 100-200 ℃, boiling and refluxing until the carbon material is completely etched to obtain clear liquid; sequentially neutralizing, removing impurities, concentrating and freeze-drying the clear solution to obtain carbon dots with the activity of the superoxide dismutase; the preparation method provided by the invention has the following stepsGreen, simple and convenient steps, small size, good biocompatibility, stable dispersibility and the like; has wide prospect in the practical application of oxidative damage protection, anti-aging, anti-inflammatory treatment and the like.
Description
Technical Field
The invention belongs to the research field of nano-enzyme, mimic enzyme and inorganic catalytic nano-material, and particularly relates to a carbon dot with superoxide dismutase activity and a preparation method thereof.
Background
As an extremely important biocatalyst, natural enzymes are widely applied to the fields of pharmacy, chemical industry, food processing, agriculture and the like due to the characteristics of high catalytic efficiency, strong substrate specificity, mild reaction conditions, good stereoselectivity and the like. However, the wide application of natural enzymes is greatly hindered due to the disadvantages of high preparation and purification costs, poor operational stability, sensitive catalytic activity to environmental conditions, and difficult recovery and reuse. In order to overcome the above problems, researchers have long been working on mimic enzymes. First reported Fe since 20073O4Since nanoparticles have peroxidase mimetic activity, various nanomaterials having mimetic activity, such as noble metals (gold, silver, palladium, platinum, etc.), metal oxides (iron oxide, copper oxide, cerium oxide, etc.), carbon nanomaterials, etc., have been widely developed and studied. Compared with natural enzymes, the nano-enzyme has the advantages of low cost, high stability, strong durability and the like, and is widely applied to biomedical applications such as biosensing, antibiosis, cell protection, disease diagnosis and treatment and the like.
However, the noble metal and metal oxide nanoenzymes reported at present are difficult to be applied in practical applications due to their disadvantages of insufficient stability, complex synthesis, difficult mass production, high cost, etc. Compared with the carbon-based nanoenzyme taking the carbon structure as the main body, the carbon-based nanoenzyme has good biocompatibility, and is widely applied to the field of mimic enzyme due to the definite electronic and geometric structures of the carbon-based nanoenzyme. The carbon dots serving as a zero-dimensional carbon nano material emerging in recent years can be used as an electron donor and an electron acceptor due to small particle size, large specific surface area and excellent conductivity, are ideal carbon catalysts for homogeneous catalysis, and have wide application prospects in biomedical applications such as biochemical analysis, disease diagnosis and treatment. However, the carbon dot nanoenzyme obtained by the current preparation method is mainly used for simulating the activity of natural Peroxidase (POD), and the application of the carbon dot nanoenzyme is greatly limited, especially in the aspects of biomedical applications such as cell protection, anti-aging, disease diagnosis and treatment and the like. Therefore, there is a need to develop a new preparation method and synthesize a carbon dot nanoenzyme having other mimic enzyme activities.
Disclosure of Invention
The invention aims to provide a carbon dot with superoxide dismutase activity and a preparation method thereof, and solves the defects in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a preparation method of carbon dots with superoxide dismutase activity, which comprises the following steps:
mixing a carbon material and a nitric acid solution to obtain a mixture solution, wherein the concentration of the mixture solution is 10-20mg mL < -1 >, and the concentration of nitric acid is 15M,10M or 5M;
stirring the mixture solution at 100-200 ℃, boiling and refluxing until the carbon material is completely etched to obtain clear liquid;
and sequentially neutralizing, removing impurities, concentrating and freeze-drying the clear solution to obtain the carbon dots with the activity of the superoxide dismutase.
Preferably, the carbon material comprises carbon fiber powder, coke, carbon black or graphite powder.
Preferably, the specific process of neutralizing the clear solution is:
neutralization was performed with NaOH until the pH of the clear solution was neutral.
Preferably, the specific process for removing impurities is as follows:
and (4) sequentially dialyzing, filtering and ultrafiltering the neutralized mixed solution to obtain a C-dots solution.
Preferably, the process of dialysis is: dialyzing with a dialysis bag having a molecular cut-off of 3500Dalton, and collecting the obtained liquid.
Preferably, the specific process of filtration is:
the filtration was carried out with a 0.22 μm filter to obtain a filtered liquid.
Preferably, the specific process for performing ultrafiltration is:
and sequentially carrying out ultrafiltration on the obtained filtrate by using ultrafiltration tubes with molecular cut-off of 100kDa, 50kDa, 30kDa, 10kDa and 3kDa to obtain a C-dots solution with the molecular cut-off of less than 3 kDa.
A carbon dot having superoxide dismutase activity, which is produced based on the method.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a carbon dot with superoxide dismutase activity and a preparation method thereof, aiming at solving the technical problems mentioned in the background technology, a large amount of cheap and easily obtained carbon materials are used as raw materials to prepare a batch of C-dots with small size, good biocompatibility and stable dispersibility, and the preparation method is simple, convenient and green and can be produced in large batch; compared with the reported C-dots with peroxidase activity (POD-like), the C-dots obtained by the preparation method has different catalytic performance from the prior art, and has superoxide dismutase activity (SOD-like) and hydroxyl radical scavenging property.
Meanwhile, the method is suitable for oxidizing various carbon materials, and can obtain C-dots with SOD-like, and mass production can be carried out; in addition, the preparation method can be used for screening C-dots with higher SOD-like activity in batches, and provides application values for the C-dot nanoenzyme in the fields of oxidative damage protection, anti-aging, anti-inflammatory treatment and the like.
Drawings
FIG. 1 shows the superoxide dismutase activity of the product obtained in example 2;
FIG. 2 shows the superoxide dismutase activity of the product obtained in example 3;
FIG. 3 is TEM and HRTEM pictures of different products of example 4;
FIG. 4 shows the superoxide dismutase activity of various products of example 4;
FIG. 5 shows hydroxyl radical scavenging properties of various products of example 4;
FIG. 6 is a graph of the total antioxidant capacity of the different products of example 4.
FIG. 7 shows intracellular ROS and O of different products of example 42 ·-The ability to purge.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
The invention aims to provide a carbon dot with superoxide dismutase activity and a preparation method thereof, and the application value of nano enzyme in the fields of oxidative damage protection, anti-aging, anti-inflammatory treatment and the like is improved by utilizing the SOD-like activity of the carbon dot.
The invention provides a preparation method of carbon dots with superoxide dismutase activity, which comprises the following steps:
c-dots preparation: weighing a proper amount of carbon material in a three-neck flask, and ultrasonically dispersing in a nitric acid (15M,10M,5M) solution to obtain a mixture solution, wherein the concentration of the mixture solution is 10-20mg mL-1(ii) a Stirring the mixed solution at 100-200 ℃, boiling and refluxing until the carbon material is completely etched to obtain brown clear liquid, stopping the reaction, naturally cooling to room temperature, and collecting to obtain a C-dots mixed solution; because the obtained C-dots mixed solution is in an acid environment, NaOH is used for neutralizing until the pH value is neutral for subsequent further treatment; then, in order to remove the salt in the neutralized mixed solution and byproducts generated in the reaction process, transferring the mixed solution into a dialysis bag with a molecular cut-off of 3500Dalton for dialysis for one week; collecting the liquid in the dialysis bag, and filtering with 0.22 μm filter membrane to remove impurities; for further purification, sequentially ultrafiltering the obtained filtrate by using ultrafiltration tubes with molecular cut-off of 100kDa, 50kDa, 30kDa, 10kDa and 3kDa, and separating to obtain C-dots solution with molecular cut-off less than 3 kDa; and finally, collecting the obtained filtrate, concentrating by using a rotary evaporator, and freeze-drying by using a vacuum freeze-drying machine to obtain a C-dots sample.
Wherein the carbon material is carbon fiber powder, coke, carbon black or graphite powder.
Superoxide dismutase Activity of C-dots: the superoxide dismutase activity of C-dots is detected by using a superoxide dismutase detection kit (Biyun, S0101M) and ESR.
Hydroxyl radical scavenging properties of C-dots: terephthalic Acid (TA) can capture OH to generate high-fluorescence 2-hydroxy terephthalic acid, the excitation wavelength is 320nm, and the emission peak is 425 nm. The hydroxyl radical scavenging property of C-dots is verified by the change of characteristic peak at 425 nm. In addition, the elimination of hydroxyl radicals can also be detected by means of ESR.
Total antioxidant capacity of C-dots: and (3) detecting the total antioxidant capacity level of the C-dots by using a total antioxidant capacity detection kit (Biyuntian, S0119).
Intracellular ROS and O of C-dots2 ·-Clearing capacity: using ROS and O2 ·-Specific fluorescent probes, DCFH-DA and DHE, assess ROS and O of C-dots at the cellular level2 ·-Very high scavenging capacity.
The invention has the advantages of novelty and remarkable characteristics
(1) The preparation method provided by the invention is suitable for the oxidation of various carbon materials, and all the available C-dots with superoxide dismutase activity have strong universality.
(2) The preparation method provided by the invention can be used for screening raw materials of C-dots with higher superoxide dismutase activity in batches, and provides application values for nano-enzyme in the fields of oxidative damage protection, ageing resistance, anti-inflammatory treatment and the like.
(3) The preparation method provided by the invention has the characteristics of green synthesis, simple and convenient steps, small size, good biocompatibility, stable dispersibility and the like, is beneficial to improving the combination of the catalyst and the substrate, and can obtain a good catalytic effect by reaching a small amount.
Example 1
C-dots preparation: weighing a proper amount of coke in a three-neck flask, and ultrasonically dispersing the coke in a 15M nitric acid (15M,10M,5M) solution to obtain a mixture solution, wherein the concentration of the mixture solution is 10mg mL-1(ii) a Stirring the mixed solution at 110 ℃, boiling and refluxing until the carbon material is completely etched to obtain clear liquid, stopping the reaction, naturally cooling to room temperature, and collecting to obtain a C-dots mixed solution; since the obtained C-dots mixed solution is in an acid environment, NaOH is used for neutralization for subsequent further treatment; followed by removing the salt from the neutralized mixed solutionAnd byproducts generated in the reaction process, transferring the mixed solution into a dialysis bag with the molecular interception amount of 3500Dalton for dialysis for one week; collecting the liquid in the dialysis bag, and filtering with 0.22 μm filter membrane to remove impurities; for further purification, sequentially ultrafiltering the obtained filtrate by using ultrafiltration tubes with molecular cut-off of 100kDa, 50kDa, 30kDa, 10kDa and 3kDa, and separating to obtain C-dots solution with molecular cut-off less than 3 kDa; and finally, collecting the obtained filtrate, concentrating by using a rotary evaporator, and freeze-drying by using a vacuum freeze-drying machine to obtain a C-dots sample.
Example 2
C-dots preparation: weighing a proper amount of carbon materials (carbon fiber powder, carbon black, graphite powder and the like) in a three-neck flask, and ultrasonically dispersing the carbon materials in a 15M nitric acid solution to obtain a mixture solution, wherein the concentration of the mixture solution is 16.7mg mL-1(ii) a Stirring and boiling the mixed solution at 100, 110, 130 or 200 ℃ respectively, refluxing until the carbon material is completely etched to obtain clear liquid, stopping the reaction, naturally cooling to room temperature, and collecting to obtain a C-dots mixed solution; since the obtained C-dots mixed solution is in an acid environment, NaOH is used for neutralization for subsequent further treatment; then, in order to remove the salt in the neutralized mixed solution and byproducts generated in the reaction process, transferring the mixed solution into a dialysis bag with a molecular cut-off of 3500Dalton for dialysis for one week; collecting the liquid in the dialysis bag, and filtering with 0.22 μm filter membrane to remove impurities; for further purification, sequentially ultrafiltering the obtained filtrate by using ultrafiltration tubes with molecular cut-off of 100kDa, 50kDa, 30kDa, 10kDa and 3kDa, and separating to obtain C-dots solution with molecular cut-off less than 3 kDa; and finally, collecting the obtained filtrate, concentrating by using a rotary evaporator, and freeze-drying by using a vacuum freeze-drying machine to obtain a C-dots sample.
FIG. 1 SOD-like activity assay of C-dots in example 2. The results show that C-dots with SOD-like activity can be obtained by using various carbon materials (carbon fiber powder, coke, carbon black, graphite powder and the like) as carbon sources and adopting a method of etching by using 15M nitric acid, and the SOD-like activity of the C-dots depends on the strengthThe kind of carbon source and the oxidation time. The superoxide dismutase detection kit is Biyuntian S0101M; the final concentration of the sample used was 50ug mL-1。
Example 3
C-dots preparation: weighing a proper amount of coke in a three-neck flask, and respectively ultrasonically dispersing the coke in a 15M,10M or 5M nitric acid solution to obtain a mixture solution, wherein the concentration of the mixture solution is 16.7mg mL-1(ii) a Stirring and boiling the mixed solution at 110, 160 or 200 ℃ respectively, refluxing until the carbon material is completely etched to obtain clear liquid, stopping the reaction, naturally cooling to room temperature, and collecting to obtain a C-dots mixed solution; since the obtained C-dots mixed solution is in an acid environment, NaOH is used for neutralization for subsequent further treatment; then, in order to remove the salt in the neutralized mixed solution and byproducts generated in the reaction process, transferring the mixed solution into a dialysis bag with a molecular cut-off of 3500Dalton for dialysis for one week; collecting the liquid in the dialysis bag, and filtering with 0.22 μm filter membrane to remove impurities; for further purification, sequentially ultrafiltering the obtained filtrate by using ultrafiltration tubes with molecular cut-off of 100kDa, 50kDa, 30kDa, 10kDa and 3kDa, and separating to obtain C-dots solution with molecular cut-off less than 3 kDa; and finally, collecting the obtained filtrate, concentrating by using a rotary evaporator, and freeze-drying by using a vacuum freeze-drying machine to obtain a C-dots sample.
FIG. 2 SOD-like activity assay of C-dots in example 3. The results show that nitric acid of different concentrations etches the carbon material, and has little influence on the SOD-like activity thereof, and the SOD-like activity which depends on the oxidation time as described above exists. The superoxide dismutase detection kit is Biyuntian S0101M; the final concentration of the sample used was 50ug mL-1。
FIG. 3 TEM and HR-TEM of C-dots in example 3. Indicating a C-dots particle size of about 1.3nm and the presence of C-dots (100) by measuring its lattice size, d 0.21nm, consistent with reported results, indicating successful preparation of C-dots.
FIG. 4 SOD-like activity assay of C-dots in example 3. A, picture A: indicating the presence of C-dotsThe SOD-like activity with concentration dependence, the enzyme activity of which reaches 4000-. The superoxide dismutase detection kit is Dojindo S311-10; the final concentration of the sample is 0-100ug mL-1. And B, drawing: the SOD-like activity of C-dots was indirectly judged by capturing the signal of DMPO/. OOH adduct using ESR. In detail, L-met and riboflavin react under LED irradiation to generate superoxide (O)2 ·-) The DMPO can capture O2 ·-Forming DMPO/. OOH adducts. All samples were taken after 5 minutes of light exposure in PBS buffer solution (25mM, pH7.4) containing 13mM L-met, 20. mu.M riboflavin, and 25mM DMPO. ESR results showed that C-dots are on O2 ·-There was a concentration-dependent scavenging effect, indicating that C-dots have excellent SOD-like activity. The final concentration of the sample is 0-50ug mL-1
FIG. 5 measurement of hydroxyl radical (. OH) scavenging property of C-dots in example 3. A, picture A: the hydroxyl radical scavenging properties of C-dots were indirectly judged by trapping the signal of DMPO/. OH adduct using ESR. OH is produced by using the Fenton reaction system. Specifically, all samples were at 1mM Fe2+And 4mM H2O2After incubation in PBS buffer (25mM, pH7.4) at 37 ℃ for 15 minutes, DMPO/. OH adduct was formed by capturing. OH with DMPO. ESR results showed that C-dots had a concentration-dependent OH-scavenging effect, indicating that C-dots had a concentration-dependent OH-scavenging property. And B, drawing: TA is a non-fluorescent compound that reacts with OH to produce a fluorescent aromatic hydroxylated product (2-hydroxy terephthalic acid) with excitation and emission peaks of 320 and 425nm, respectively. In detail, the samples used contained 0.5mM TA and 10mM H2O2Was subjected to ultraviolet irradiation for 15min with 25mM PBS buffer (pH 7.4), and then analyzed by a fluorescence spectrometer. The results show concentration dependent OH scavenging properties.
FIG. 6 Total antioxidant capacity of C-dots in example 3 was measured. The total antioxidant capacity detecting reagent kit (Biyuntian, S0119) is one kind of total antioxidant capacity detecting reagent kit for various antioxidant solutions with ABTS as color developing agentA kit for detecting the chemical ability. Dynamic detection of A by addition of C-dots solutions of different concentrations734And (3) absorbing, and utilizing Trolox as an antioxidant to prepare a standard curve, and calculating to obtain the total antioxidant capacity of the C-dots to reach 8-10 mmol/g.
FIG. 7 intracellular ROS and O of C-dots in example 32 ·-And clearing and detecting. A, picture A: DCFH is an ROS specific fluorescent probe, when the Rosup induces, strong green fluorescence appears, and after cells are treated with 100 mu g/mL C-dots for 8h in advance, the green fluorescence is obviously reduced, which indicates that the C-dots have good ROS scavenging capacity, while the blank group and the C-dots treated group do not appear green fluorescence, which indicates that the C-dots have good biological safety. And B, drawing: DHE is an O2 ·-Specific fluorescent probes, when subjected to the same treatment as described above, also showed good O2 ·-The ability to purge.
Claims (8)
1. A method for preparing carbon dots with superoxide dismutase activity is characterized by comprising the following steps:
mixing a carbon material and a nitric acid solution to obtain a mixture solution, wherein the concentration of the mixture solution is 10-20mg mL-1The concentration of nitric acid is 15M,10M or 5M;
stirring the mixture solution at 100-200 ℃, boiling and refluxing until the carbon material is completely etched to obtain clear liquid;
and sequentially neutralizing, removing impurities, concentrating and freeze-drying the clear solution to obtain the carbon dots with the activity of the superoxide dismutase.
2. The method for preparing carbon dots having superoxide dismutase activity as claimed in claim 1, wherein the carbon material comprises carbon fiber powder, coke, carbon black or graphite powder.
3. The method for preparing carbon dots with superoxide dismutase activity as claimed in claim 1, wherein the specific process for neutralizing the clear solution is as follows:
neutralization was performed with NaOH until the pH of the clear solution was neutral.
4. The method for preparing carbon dots with superoxide dismutase activity according to claim 1, wherein the specific process for removing impurities is as follows:
and (4) sequentially dialyzing, filtering and ultrafiltering the neutralized mixed solution to obtain a C-dots solution.
5. The method for preparing carbon dots with superoxide dismutase activity as claimed in claim 4, wherein the dialysis process comprises: dialyzing with a dialysis bag having a molecular cut-off of 3500Dalton, and collecting the obtained liquid.
6. The method for preparing carbon dots with superoxide dismutase activity as claimed in claim 4, wherein the filtration process comprises:
the filtration was carried out with a 0.22 μm filter to obtain a filtered liquid.
7. The method for preparing carbon dots with superoxide dismutase activity according to claim 4, wherein the specific process for carrying out ultrafiltration is as follows:
and sequentially carrying out ultrafiltration on the obtained filtrate by using ultrafiltration tubes with molecular cut-off of 100kDa, 50kDa, 30kDa, 10kDa and 3kDa to obtain a C-dots solution with the molecular cut-off of less than 3 kDa.
8. A carbon dot having superoxide dismutase activity, which is produced by the method according to any one of claims 1 to 7.
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