CN115818695A - Preparation method of litchi-shaped cuprous oxide/copper oxide nano-microspheres with tetrapeptidase activity - Google Patents
Preparation method of litchi-shaped cuprous oxide/copper oxide nano-microspheres with tetrapeptidase activity Download PDFInfo
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Abstract
The invention discloses a litchi-shaped cuprous oxide/copper oxide nano microsphere (Cu) with tetrapeptidase activity 2 O/CuO NMSs), belonging to the technical field of bionic nanoenzymology. Cu 2 The preparation of O/CuO NMSs is completed by a one-step solvothermal method to prepare Cu 2 The O/CuO NMSs have the same enzymatic activity as 4 natural enzymes such as peroxidase, catalase, superoxide dismutase and laccase. Wherein, cu 2 The peroxidase, superoxide dismutase and laccase activities of O/CuO NMSs are fully proved by constructing a classical colorimetric system, while the activities of Cu 2 The catalase activity of O/CuO NMSs was evaluated by the degree of pressure change in the system. In summary, the Cu prepared by the present invention 2 The O/CuO NMSs are multi-enzyme active nano materials with wide application prospect.
Description
Technical Field
The invention belongs to the technical field of bionic nano composite materials, and relates to Cu 2 A preparation method of O/CuO nano-microspheres, in particular to Cu 2 A preparation method of a nanocomposite with O/CuO NMSs quadruple enzyme activity.
Background
Natural enzymes are important biocatalysts that mediate a variety of reactions in biological processes such as signal transduction, metabolism, and digestion. Because of high substrate specificity and catalytic efficiency, the catalyst is widely applied to the fields of industry, medical treatment, biology and the like. However, natural enzymes are limited in practical use due to their poor stability, sensitivity to environmental changes, and difficulty in purification and storage. Therefore, researchers have been working on nanomaterials having enzyme-like activity (nanoenzymes) to replace natural enzymes. Researchers reported as early as 2007 that a nano-material Fe with peroxidase-like activity 3 O 4 And (4) NPs. The nano enzyme as a replica of natural enzyme has the advantages of low cost, convenient preparation, good stability, good durability and the like, thereby being widely concerned.
In recent years, researchers at home and abroad report a plurality of nano materials with simulated enzyme activity. The nano material can singly show peroxidase, oxidase, catalase, laccase, superoxide dismutase, ascorbic acid oxidase mimic activity and the like. Moreover, some nanomaterials may even exhibit dual or multiple enzyme activities simultaneously. Although nanoenzyme science has achieved great success, some bottlenecks still exist, and further development and application of nanoenzymes are limited. The defects of the nano enzyme mainly exist in the following points: firstly, the preparation method of the nano enzyme has no major breakthrough. The synthesis route is complex and time-consuming, the synthesis conditions are harsh, and the large-scale production is difficult; secondly, the catalytic activity of the nano enzyme is still far lower than that of the corresponding natural counterpart; thirdly, the enzyme-like activity of the nano-enzyme is single, the bioavailability is low, and the multi-enzyme mimics have the problems of mutual interference and the like. Therefore, the research on the nano enzyme is still significant.
Disclosure of Invention
The invention aims to provide litchi-shaped cuprous oxide/cupric oxide (Cu) with tetraase activity 2 O/CuO) nano-microsphere, aiming at solving the problems of complex synthetic route of nano-enzyme, rigorous synthetic conditions, single enzyme-like activity and difficult large-scale production.
1. Cu (copper) 2 Preparation of O/CuO NMSs
Cu 2 The O/CuO NMSs are prepared by a one-step solvothermal method, specifically, copper nitrate trihydrate is used as a copper source, isonicotinic acid is used as a ligand, the copper nitrate trihydrate and the isonicotinic acid are dissolved in a mixed solution of N, N-dimethylacetamide, absolute ethyl alcohol and water, the mixture is subjected to uniform ultrasonic dispersion, the mixture is subjected to reaction for 18 to 24 hours at the temperature of 150 ℃, the mixture is naturally cooled to room temperature to obtain a black-brown solution, the black-brown solution is subjected to centrifugal washing to collect precipitates, and the precipitates are dried to prepare litchi-shaped Cu with the tetrazyme activity 2 O/CuO nano-microspheres.
The molar mass ratio of the copper nitrate trihydrate to the isonicotinic acid is 1 to 2: 1. The volume ratio of the N, N-dimethylacetamide to the absolute ethyl alcohol to the water is 8 to 10 to 4 to 6 to 1. The centrifugation is carried out at 8000r/min for 1 to 3min. The precipitate was dried at 40 to 60 ℃ for 8 hours.
Cu as described above 2 Cu (NO) in the preparation of O/CuO NMSs 3 ) 2 ·3H 2 O provides a Cu source for chelation with isonicotinic acid, which contains a carboxylic acid-based organic ligand. The carboxyl can be bonded with the multi-valence metal ions in a changeable coordination mode to construct the nano material with a novel structure, so that more possibilities are provided for simulating multi-activity nano enzyme. Meanwhile, the use of the transition metal elements Cu and isonicotinic acid meets the requirement of low cost of the nano material.
2. Cu (copper) 2 Four-enzyme activity study of O/CuO NMSs
Exploration of peroxidase-like activity: cu constructed under acidic conditions 2 O/CuO NMSs-H 2 O 2 In the reaction system of-TMB, cu 2 The O/CuO NMSs exhibit catalytic properties equivalent to native peroxidase activity. In hydrogen peroxide (H) 2 O 2 ) In the presence of Cu 2 O/CuO NMSs effectively catalyze H 2 O 2 The chromogenic substrate 3,3', 5' -Tetramethylbenzidine (TMB) is oxidized to a blue product (TMB in the oxidized state). The enzyme activity of the nanoenzyme can be obtained by analyzing the change of the absorbance at 652 nm.
Exploration of catalase-like activity: based on H 2 O 2 Is unstableIs decomposed to produce O under the action of catalase 2 And H 2 And O. Thus, in a closed weak base environment, H is constructed 2 O 2 And Cu 2 O/CuO NMSs-H 2 O 2 The pressure values of the reaction system and the two systems were measured and used as evaluation H 2 O 2 The decomposition rate of (2) is strong or weak. And H 2 O 2 System phase ratio of Cu 2 O/CuO NMSs-H 2 O 2 Cu in reaction system 2 O/CuO NMSs effectively decompose H 2 O 2 Resulting in the generation of a large amount of gas that increases the gas pressure in the space.
Exploration of laccase-like activity: dopamine is used as a chromogenic substrate, and laccase can catalyze and oxidize the dopamine to generate a reddish brown substance. Based on the property of laccase activity, dopamine hydrochloride (HCl-DA) and Cu are constructed under the weak base condition because the hydrochloric acid group of the dopamine hydrochloride can be removed to form dopamine under the weak base condition 2 An O/CuO NMSs-HCl-DA reaction system. As a result, cu 2 The O/CuO NMSs catalyze the oxidation of dopamine and show a characteristic peak at 475 nm.
Exploration of superoxide dismutase-like: the research of the superoxide dismutase adopts a classical nitro tetrazole blue light reduction method. Firstly, a nitrotetrazolium blue chloride (NBT) -riboflavin-methionine-Ethylene Diamine Tetraacetic Acid (EDTA) reaction system is constructed under the condition of alkalescence, and the system can lead riboflavin to generate superoxide anion free radicals (O) when being irradiated by light 2- ) And reducing nitro-blue tetrazolium chloride into a blue-purple formazan product with the lambda max of 560 nm. Once superoxide dismutase is introduced into the system constructed above, the amount of blue-violet formazan product formed is greatly reduced, as superoxide dismutase eliminates the superoxide anion radicals produced. Constructed Cu 2 The O/CuO NMSs-NBT-riboflavin-methionine-EDTA reaction system showed weak absorption at 560nm, thus indicating Cu 2 The O/CuO NMSs have superoxide dismutase-like properties.
Due to litchi like Cu 2 The multi-enzyme activity of the O/CuO nano-microspheres is realized, and the nano-enzyme has potentialThe biological application of (1). Based on Cu 2 The nanometer enzyme (catalase-like enzyme and superoxide dismutase-like enzyme) with the antioxidant activity of the O/CuO nanometer microspheres can effectively remove active oxygen species through a cascade reaction, and treat various diseases caused by oxidative stress. On the other hand, based on Cu 2 The nanometer enzyme (peroxidase-like enzyme) with the oxidation promoting activity of the O/CuO nanometer microsphere can catalyze the generation of a large number of active oxygen species in vivo through a cascade reaction and a synergistic effect, and is widely applied to biosensing, anticancer and antibacterial treatment. However, individual antioxidant or pro-oxidative nanoenzymes are also limited in biomedical applications. For example, in anti-tumor applications, the role of pro-oxidative nanoenzymes is limited due to hypoxia of the tumor microenvironment. The synergistic effect of antioxidant enzyme and pro-oxidase can provide oxygen and promote the generation of active oxygen species to kill tumor cells. In the treatment of acute kidney injury, the synergistic effect of antioxidant enzymes and pro-oxidase enzymes can effectively control the increase in reactive oxygen species production caused by disease. Thus, litchi like Cu 2 The synergistic effect of the antioxidation-like and the pro-oxidation-like nanoenzymes of the O/CuO nanospheres can greatly expand the application range of the nanoenzymes.
In conclusion, the invention discloses a method for preparing a multienzyme active nano material by a one-step solvothermal method, and prepared Cu 2 The O/CuO NMSs have the same enzyme activity as 4 natural enzymes such as peroxidase, catalase, superoxide dismutase and laccase. Wherein, cu 2 The peroxidase, superoxide dismutase and laccase activities of O/CuO NMSs are fully proved by constructing a classical colorimetric system, while the activities of Cu 2 The catalase activity of O/CuO NMSs was evaluated by the degree of pressure change in the system. In summary, the Cu prepared by the present invention 2 The O/CuO NMSs are multi-enzyme active nano materials with wide application prospect.
Drawings
FIG. 1 is Cu prepared according to the present invention 2 A four-enzyme activity schematic diagram of O/CuO NMSs;
FIG. 2A is Cu 2 The scanning electron microscope image of the O/CuO NMSs is magnified to 1 μm, and the scanning electron microscope image of the boxed part of the FIG. 2A is magnified to 200 nm;
FIG. 2C is Cu 2 Energy dispersive X-ray spectrograms of O/CuO NMSs,
FIG. 2D is Cu 2 The scanning electron microscope image of the O/CuO NMSs is magnified to 0.5 μm, and FIG. 2E is the scanning electron microscope image of the boxed portion of FIG. 2D magnified to 100 nm;
FIG. 2F is a scanning electron micrograph, enlarged to 5nm, of a selected portion of the box of FIG. 2D, wherein the inset is a diffraction pattern of electrons in selected areas of the figure;
FIGS. 2G, 2H, 2I, 2J and 2K are Cu 2 An elemental map image of O/CuO NMSs;
FIG. 3 is Cu 2 X-ray powder diffraction spectra of O/CuO NMSs;
FIG. 4 is TMB, TMB-H 2 O 2 ，Cu 2 O/CuO NMSs- H 2 O 2 ，Cu 2 O/CuO NMSs-TMB and Cu 2 O/CuO NMSs-TMB-H 2 O 2 Ultraviolet-visible absorption spectrograms of the four reaction systems;
FIG. 5 is H 2 O 2 And Cu 2 O/CuO NMSs-H 2 O 2 A gas pressure contrast diagram of two reaction systems;
FIG. 6 is DA, H 2 O 2 And Cu 2 A graph of ultraviolet-visible absorption spectra of an O/CuO NMSs-DA reaction system;
FIG. 7 shows NBT-riboflavin-methionine-EDTA and Cu 2 Two reaction systems of O/CuO NMSs-NBT-riboflavin-methionine-EDTA have ultraviolet-visible absorption spectrograms.
Detailed Description
The invention is further illustrated by the following examples.
Example 1
Cu 2 Preparation of O/CuO NMSs
First, 48.5mg of copper nitrate trihydrate and 24.6mg of isonicotinic acid were accurately weighed and added in this order to a mixed solution containing 18mL of N, N-dimethylacetamide, 10mL of anhydrous ethanol and 2mL of distilled water. Next, the mixture was dispersed by ultrasonic waves for 10min to be completely dissolved. Next, the mixed solution was charged into a 50mL reaction vessel containing polytetrafluoroethylene and reacted at 150 ℃ for 24 hours. After cooling to room temperature, the solid was collected by centrifugation at 8000 r/min. Tighten upThen respectively washing the solid in the last step three times by using N, N-dimethylacetamide, ethanol and deionized water, and collecting precipitate, wherein the precipitate is Cu 2 O/CuO NMSs. Finally, adding Cu 2 Drying O/CuO NMSs in a vacuum drying oven at 60 ℃ for 8h to obtain Cu 2 Powder samples of O/CuO NMSs.
Structural characterization: FIGS. 2A, B, D, E and F are Cu 2 Scanning electron micrographs of O/CuO NMSs, transmission Electron microscopy imaging of Cu 2 The morphology of O/CuO NMSs is characterized, and Cu can be seen 2 The O/CuO NMSs have a litchi-like morphology; while Cu can be seen in FIG. 2C 2 The O/CuO NMSs contain C, N, O and Cu elements, and the elements mapping imaging (FIGS. 2G-K) shows that the C, N, O and Cu elements are uniformly distributed in Cu 2 O/CuO NMSs. FIG. 3 is Cu 2 X-ray powder diffraction spectrum diagram of O/CuO NMSs (atomic Power systems), and Cu in the spectrum diagram 2 Characteristic peaks of O and CuO and Cu 2 The characteristic diffraction peaks of O (JCPDS No. 48-0667) and CuO (JCPDS No. 48-1548) are consistent, which indicates that Cu 2 Formation of O/CuO NMS from Cu 2 O and CuO.
Cu 2 Four-enzyme simulation activity exploration of O/CuO NMSs
Cu 2 Peroxidase activity of O/CuO NMSs by oxidation in H 2 O 2 Under conditions that catalyze the oxidation of the chromogenic substrate TMB to form oxidation state TMB. First, cu is added 2 O/CuO NMSs(13 μg/mL，10μL)、H 2 O 2 (10M, 60. Mu.L) and TMB (50mM, 100. Mu.L) were sequentially added to a buffer solution of acetic acid-sodium acetate (pH =4.4, 0.2M), and the total volume of the reaction system was 2mL. Then, after reacting at room temperature for 2min, the absorbance at 655nm was recorded by an ultraviolet-visible spectrophotometer. TMB, TMB-H 2 O、Cu 2 O/CuO NMSs-H 2 O 2 And Cu 2 Construction of O/CuONMSs-TMB reaction system and Cu 2 O/CuO NMSs-TMB-H 2 O 2 Essentially the same, the only difference being that the TMB reaction system is deficient in Cu 2 O/CuO NMSs and H 2 O 2 ；Cu 2 O/CuO NMSs-H 2 O 2 TMB is lacked in the reaction system; cu 2 O/CuO NMSs-TMB reactionThe system is short of H 2 O 2 . Cu in FIG. 4 2 O/CuO NMSs-TMB-H 2 O 2 The absorbance of the reaction system at 655nm is obviously higher than that of TMB, TMB-H 2 O 2 ，Cu 2 O/CuO NMSs-H 2 O 2 ，Cu 2 O/CuO NMSs-TMB and Cu 2 O/CuO NMSs-TMB-H 2 O 2 Prove that Cu 2 The O/CuO NMS catalytically oxidizes TMB to Ox TMB, which is blue in color.
Cu 2 The catalase activity of O/CuO NMSs is based on catalase-catalyzed H 2 O 2 The decomposition product gas of (2) was confirmed. First, a 1.5mL injection vial containing 940 μ L of a buffer solution of Tris (hydroxymethyl) aminomethane (Tris) -hydrochloric acid (HCl) (pH =8,0.05m) was charged with Cu 2 O/CuO NMSs (40. Mu.L, 13. Mu.g/mL) and H 2 O 2 (10. Mu.L, 10M). Then screwing a cover, reacting at room temperature for 20min, measuring the pressure value of the mixed system by using a portable barometer, and reacting with the single H 2 O 2 The pressure values of the systems were compared. FIG. 5 is the measured H 2 O 2 ，Cu 2 O/CuO NMSs-H 2 O 2 Pressure value of reaction system due to Cu 2 O/CuO NMSs catalyze H 2 O 2 Causes a rise in pressure in the enclosed space.
Cu 2 The laccase activity of O/CuO NMSs was demonstrated based on the laccase catalysis of dopamine oxidation to reddish brown amino complexes. Firstly, cu is mixed 2 O/CuO NMSs (50. Mu.L, 13. Mu.g/mL) and HCl-DA (10 mM, 100. Mu.L) were added sequentially to 1.85 mL of tris (hydroxymethyl) aminomethane-hydrochloric acid (pH =7.7, 0.05M) buffer solution. After 20min of reaction at room temperature, the change in absorbance at a wavelength of 475nm was recorded by an ultraviolet-visible spectrophotometer, since the color of the reaction system changed from colorless to reddish brown. FIG. 6 shows Cu 2 The O/CuO NMSs effectively catalyze the oxidation of dopamine to generate a reddish brown substance, and the absorbance at 475nm is obviously increased.
Cu 2 Superoxide dismutase derived from O/CuO NMSs was confirmed by reducing nitrotetrazolium chloride blue using active oxygen generated by photoreaction. Firstly, the methodNitro-tetrazolium chloride blue (10 mM, 20. Mu.L), riboflavin (1M, 10. Mu.L), methionine (1M, 30. Mu.L) and ethylenediaminetetraacetic acid (1 mM, 20. Mu.L) were added to a tris (hydroxymethyl) aminomethane-hydrochloric acid (pH =7.5, 0.05M) buffer solution, and the total volume of the reaction system was always kept at 2mL. After the reaction was carried out for 5 minutes in the dark and for 5 minutes in the light (5W), the absorbance at 560nm was recorded by an ultraviolet-visible spectrophotometer. Then, cu was added to a tris (hydroxymethyl) aminomethane-hydrochloric acid (PH =7.5,0.05m) buffer solution 2 O/CuO NMSs (20. Mu.L, 13. Mu.g/mL), nitrotetrazolium chloride (10 mM, 20. Mu.L), riboflavin (1M, 10. Mu.L), methionine (1M, 30. Mu.L), ethylenediaminetetraacetic acid (10mM, 20. Mu.L) were recorded as absorbance at 560nm by an ultraviolet-visible spectrophotometer. Finally, the absorbance at 560nm of the two systems was compared, and NBT-riboflavin-methionine-EDTA and Cu are shown in FIG. 7 2 Absorbance change of O/CuO NMSs-NBT-riboflavin-methionine-EDTA reaction system, cu 2 O/CuO NMS eliminates superoxide anion radicals generated by the presence of riboflavin under light, resulting in a significant decrease in absorbance at 560 nm.
Claims (6)
1. The preparation method of the litchi-shaped cuprous oxide/copper oxide nano-microsphere with the tetrase activity is characterized by comprising the following steps of: copper nitrate trihydrate is used as a copper source, isonicotinic acid is used as a ligand, the copper nitrate trihydrate and the isonicotinic acid are dissolved in a mixed solution of N, N-dimethylacetamide, absolute ethyl alcohol and water, the mixture is subjected to ultrasonic dispersion uniformly, the mixture reacts for 18 to 24 hours at the temperature of 150 to 180 ℃, the mixture is naturally cooled to room temperature, precipitates are collected through centrifugal washing and dried, and litchi-shaped cuprous oxide/copper oxide nano microspheres with the tetrazyme activity are prepared.
2. The method for preparing litchi-shaped cuprous oxide/cupric oxide nano-microspheres with tetrase activity as claimed in claim 1, wherein the method comprises the following steps: the molar mass ratio of the copper nitrate trihydrate to the isonicotinic acid is 1 to 2: 1.
3. The method for preparing litchi-shaped cuprous oxide/cupric oxide nano-microspheres with tetrase activity as claimed in claim 1, wherein the method comprises the following steps: the volume ratio of the N, N-dimethylacetamide to the absolute ethyl alcohol to the water is 8 to 10: 4 to 6: 1.
4. The method for preparing litchi-shaped cuprous oxide/copper oxide nano-microspheres with tetrase activity as claimed in claim 1, wherein the method comprises the following steps: the centrifugation is carried out for 1 to 3min at 8000 r/min.
5. The method for preparing litchi-shaped cuprous oxide/cupric oxide nano-microspheres with tetrase activity as claimed in claim 1, wherein the method comprises the following steps: the precipitate was dried at 40 to 60 ℃ for 8 hours.
6. The method for preparing litchi-shaped cuprous oxide/cupric oxide nano-microspheres with tetrase activity as claimed in claim 1, wherein the method comprises the following steps: cu 2 The O/CuO nano microsphere has the following four enzyme activities: peroxidase, catalase, laccase, and superoxide dismutase.
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