Preparation method of iron-cobalt-based magnetic matrix material and application of iron-cobalt-based magnetic matrix material in immobilized trypsin

Abstract

The invention discloses a preparation method of an iron-cobalt-based magnetic matrix material and application of the iron-cobalt-based magnetic matrix material in immobilized trypsin, the material structure is controlled by changing the proportion of iron and cobalt and preparation conditions, porous iron-cobalt matrixes with different structures and morphologies are prepared, then the surfaces of the porous iron-cobalt matrixes are coated with silicon dioxide, and the iron-cobalt-based magnetic matrix material is prepared. And forming a silicon dioxide coating, performing alkali etching on the silicon dioxide coating to form small dents on the surface, and further performing pore expansion by using salts with different concentrations to prepare the iron-cobalt-based magnetic matrix material. And combining the iron-cobalt-based magnetic matrix material with trypsin to obtain the immobilized trypsin. The preparation method has the beneficial effects that the material preparation process is simple, the magnetism of the matrix material is strong and stable, and the surface area is large, so that the modification capacity of trypsin is improved, and meanwhile, the prepared immobilized trypsin has different pore structures and material morphologies, can realize rapid and efficient enzymolysis of protein, and has a magnetic separation effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of magnetic material and enzyme immobilization, and particularly relates to a preparation method of a cobalt-iron-based magnetic matrix material and application thereof in immobilized trypsin. BACKGROUND

[0002] As an important functional material, magnetic matrix materials have wide application prospects in enzyme immobilization, catalysis, separation and other fields. Among them, cobalt-iron-based magnetic materials have attracted much attention due to their excellent magnetic properties and chemical stability.

[0003] An ideal magnetic matrix material should have high specific surface area, controllable pore structure, excellent magnetic response and good biocompatibility. At present, related researches mainly focus on single-component or simple binary magnetic nanomaterials (such as Fe3O4 nanoparticles), magnetic alloy materials (such as iron-cobalt alloy) and porous magnetic composite materials. For example, Chinese patent CN120866947A discloses a quaternary niobium-iron-cobalt sulfide (Nb3(FeCo)S6) crystal and a chemical vapor transport preparation method thereof. The material has significant magnetic anisotropy and is mainly used in the field of spin electronics, high-density magnetic storage and other physical devices. However, it is a dense inorganic crystal and does not have a porous structure suitable for loading biological macromolecules. Moreover, the synthesis conditions are harsh (high temperature and high vacuum), and the compatibility with biological systems and application scenarios are quite different. Chinese patent document CN108231309A discloses a preparation method of magnetic silica microspheres. The method prepares a core-shell structure through co-precipitation and sol-gel method, thereby improving the dispersibility and surface modifiability of the material. However, such materials usually have iron oxide as the magnetic core, and the saturation magnetization is limited, resulting in slow magnetic separation speed and low efficiency. In the application of biological sample separation or enzyme immobilization that requires fast and efficient processing, there is a bottleneck.

[0004] For iron-cobalt alloy matrix, the existing technology mainly focuses on preparation through high-temperature reduction or carbothermal reduction method. Researchers can obtain materials with different magnetic properties by adjusting the ratio of iron and cobalt. However, these methods often have difficulty in precisely controlling the micro-morphology, pore structure and specific surface area of the material at the same time. For example, traditional high-temperature solid-phase reaction is prone to cause particle agglomeration and pore collapse, and the specific surface area of the obtained material is generally low (usually less than 100 m 2 / g), which limits its application potential in enzyme immobilization requiring high loading capacity. In addition, the surface chemical properties of iron-cobalt alloy are single, and lack of active sites that can effectively interact with enzyme molecules, which often causes weak binding force and easy enzyme shedding when directly used for enzyme immobilization. SUMMARY

[0005] In view of the above deficiencies, the present application aims to provide a preparation method of iron-cobalt-based magnetic matrix material and its application in immobilized trypsin, which has a porous structure, high specific surface area and strong magnetism, is suitable for efficient immobilization of trypsin, and can be used in protein enzymolysis, biosensing and other fields. A preparation method of iron-cobalt-based magnetic matrix material, comprising the following steps: (1) Dissolve polyvinylpyrrolidone in water, add cobalt salt and iron salt, and dry at 90-110 DEG C after stirring reaction to obtain a precursor; (2) Carbonize the precursor under nitrogen protection at 500-800 DEG C to obtain Co7Fe3 / Co material; (3) Disperse the material obtained in step (2) in a mixed solution of ethanol and water, add ammonia and silane material, stir, and then add tetraethyl orthosilicate dropwise for coating reaction to obtain Co7Fe3 / Co material coated with silica; (4) Disperse the coated material in ethanol, add ammonium nitrate for reflux pore expansion treatment, and then wash and dry to obtain iron-cobalt-based magnetic matrix material.

[0006] Further, the cobalt salt in step (1) is cobalt nitrate hexahydrate, the iron salt is iron nitrate nonahydrate, and the mass ratio of cobalt salt, iron salt and polyvinylpyrrolidone is (1-5):(0.5-1):1.

[0007] Further, the carbonization temperature in step (2) is 600-700 DEG C, the heating rate is 5 DEG C / min, and the carbonization time is 0.5-5 h.

[0008] Further, in step (3), the amount of ammonia is 0.1-3 g per 100 mg of magnetic matrix material, the amount of template agent is 0.05-1 g per 100 mg of magnetic matrix material, and the amount of tetraethyl orthosilicate is 0.01-0.7 g per 100 mg of magnetic matrix material; the template agent is one or more of cetyltrimethylammonium bromide, trioctylmethylammonium bromide and dodecyltrimethylammonium bromide.

[0009] Further, in step (4), the amount of ammonium nitrate is 0.5-2 g per 100 mg of coated material, and the reflux time is 1-5 h.

[0010] Further, the iron-cobalt-based magnetic matrix material has a porous structure, a specific surface area of 150-200 m 2 / g, and strong magnetism, and is suitable for enzyme immobilization carrier.

[0011] The application further provides application of the iron-cobalt-based magnetic matrix material in immobilized trypsin, specifically, the prepared iron-cobalt-based magnetic matrix material is combined with trypsin to prepare immobilized trypsin. The specific immobilization steps are as follows: The magnetic matrix material is mixed with water, and after freeze-drying, trypsin and ammonium bicarbonate solution are added, and after oscillation, filtration is performed, and the unabsorbed enzyme is washed with ammonium bicarbonate solution to obtain immobilized trypsin.

[0012] Further, the mass ratio of the trypsin to the magnetic matrix material is 1:10-1:50.

[0013] The application has the following beneficial effects: (1) the iron-cobalt-based magnetic matrix material prepared by the preparation method has a porous structure, and the specific surface area reaches 150-200 m 2 / g, which is significantly improved compared with less than 100 m 2 / g of the prior art, and can provide more enzyme binding sites; (2) the prepared iron-cobalt-based magnetic matrix material has strong magnetism, and the magnetic response is better than that of traditional Fe3O4 materials, and can realize rapid and efficient magnetic separation; (3) the prepared iron-cobalt-based magnetic matrix material is suitable for efficient immobilization of trypsin, and solves the problems of single chemical properties of traditional iron-cobalt alloy surfaces and easy falling off of enzymes; (4) the prepared iron-cobalt-based magnetic matrix material can be used in protein enzymolysis, biosensing and other fields, and improves the stability, reusability and operation convenience of the enzyme; (5) the application overcomes the defects of particle agglomeration and pore collapse in the traditional preparation method by precisely controlling the micro-morphology and pore structure of the material. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 BET graph of the iron-cobalt magnetic matrix material prepared in embodiment 1 of the application; Figure 2 SEM graph of the iron-cobalt magnetic matrix material prepared in embodiment 1 of the application; Figure 3 TEM graph of the porous silica-coated Co7Fe3 / Co material prepared by using (a) cetyltrimethylammonium bromide and (b) dodecyltrimethylammonium bromide in embodiment 2 of the application. DETAILED DESCRIPTION

[0015] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0016] A preparation method of a cobalt-iron-based magnetic matrix material, comprising the following steps: (1) Polyvinylpyrrolidone is completely dissolved in deionized water to form a uniform polymer solution. Cobalt nitrate hexahydrate and ferric nitrate nonahydrate are sequentially added to the solution, and the mass ratio of cobalt salt, iron salt and polyvinylpyrrolidone is (1-5):(0.5-1):1, preferably the molar ratio of cobalt to iron is 7:3. The reaction is continuously stirred at room temperature to fully complex the metal salt and polyvinylpyrrolidone. After the reaction is completed, the mixed solution is placed in an oven and dried at 90-110°C until completely dehydrated to obtain a polymer precursor containing metal ions. The metal ions in the precursor are uniformly wrapped by polyvinylpyrrolidone, providing a basis for forming uniformly dispersed metal nanoparticles in the subsequent carbonization process.

[0017] (2) The precursor is placed in a tube furnace, nitrogen is introduced to establish an inert protective atmosphere to prevent the metal from being oxidized at high temperature. The carbonization reaction is slowly heated to 500-800°C at a heating rate of 5°C / min. Preferably, the carbonization temperature is controlled at 600-700°C, the heating rate is 5°C / min, and the carbonization time is 1h. Under this condition, polyvinylpyrrolidone is pyrolyzed and carbonized, and the metal salt is reduced to metal nanoparticles to form Co7Fe3 / Co-600 material. The cobalt-iron alloy particles in the material are uniformly dispersed in the carbon matrix, and have good magnetic properties.

[0018] (3) The Co7Fe3 / Co-600 material obtained in step (2) is dispersed in a mixed solution of ethanol and water, and ultrasonic treatment is performed to ensure complete dispersion. Ammonia water is added to adjust the pH value, and a template agent is added. Under continuous stirring, tetraethyl orthosilicate is slowly added for sol-gel coating reaction. The amount of tetraethyl orthosilicate is 0.2-0.5g per 100mg of magnetic matrix material. During the reaction, tetraethyl orthosilicate is hydrolyzed to form silicic acid, which is then condensed to form a silica shell layer, encapsulating the magnetic particles to obtain Co7Fe3 / Co material coated with porous silica.

[0019] (4) The coated material is dispersed in anhydrous ethanol, and ammonium nitrate is added for reflux. The amount of ammonium nitrate is 0.5-1.5g per 100mg of coated material, and the reflux temperature is 60°C for 1h. Using ammonium nitrate solution for reflux can remove the cationic surfactant under mild conditions, avoiding oxidation of the material at high temperature caused by high-temperature calcination. During the reflux process, the ammonium ions dissociated from the ammonium nitrate solution can ion exchange with the cationic end of the template agent, so that it is completely removed in the form of a soluble salt. After the reflux is completed, the product is collected by magnetic separation, washed repeatedly with deionized water and ethanol to remove residual ammonium nitrate and other impurities, and finally dried at 60°C for 12 hours to obtain the final cobalt-iron-based magnetic matrix material.

[0020] in combination Figures 1 to 3 as shown: Example One A preparation method of iron-cobalt-based magnetic matrix material, through polyvinylpyrrolidone-mediated precursor preparation, high-temperature carbonization, silica coating and hole expansion treatment, an iron-cobalt-based magnetic matrix material with porous structure and strong magnetism is prepared.

[0021] Step 1: precursor preparation Dissolve 1 g of polyvinylpyrrolidone in 50 ml of ionized water to form a uniform polymer solution. Add 1.22 g of cobalt nitrate hexahydrate and 0.73 g of iron nitrate nonahydrate to the solution in turn, and strictly control the molar ratio of cobalt to iron to be 7:3. Stir the reaction continuously at room temperature to allow the metal salt to fully complex with the polyvinylpyrrolidone. After the reaction is complete, place the mixed solution in an oven and dry it at 100°C until it is completely dehydrated to obtain a polymer precursor containing metal ions. The metal ions in the precursor are uniformly coated with polyvinylpyrrolidone, providing a basis for the formation of uniformly dispersed metal nanoparticles in the subsequent carbonization process.

[0022] Step 2: high-temperature carbonization treatment Place the precursor in a tube furnace and introduce nitrogen to establish an inert protective atmosphere to prevent the metal from being oxidized at high temperatures. Slowly heat to 500-800°C at a rate of 5°C / min for carbonization. The carbonization temperature is preferably controlled at 600°C, the heating rate is 5°C / min, and the carbonization time is 1h. Under these conditions, the polyvinylpyrrolidone is pyrolyzed and carbonized, and the metal salt is reduced to metal nanoparticles to form a Co7Fe3 / Co-600 material. The cobalt-iron alloy particles in the material are uniformly dispersed in the carbon matrix, with good magnetic properties.

[0023] Step 3: porous silica coating Disperse 100 mg of the Co7Fe3 / Co-600 material obtained in step two in 150 mL of a 25% ethanol and water mixture, and ensure complete dispersion by ultrasonic treatment. Add 1 mL of pH adjuster to the dispersion, and then add 0.5 g of cetyltrimethylammonium bromide as a template agent. Under continuous stirring, slowly add 0.5 g of tetraethyl orthosilicate for sol-gel coating reaction. The amount of tetraethyl orthosilicate used is 0.5 g per 100 mg of magnetic matrix material. During the reaction, the tetraethyl orthosilicate hydrolyzes to form silicic acid, which then condenses to form a silica shell layer, encapsulating the magnetic particles to obtain a Co7Fe3 / Co material coated with porous silica.

[0024] Step 4: removal of template agent The 100 mg coated material was dispersed in 50 mL anhydrous ethanol, and 1 g ammonium nitrate was added for reflux. The amount of ammonium nitrate was 1 g per 100 mg of coated material, the reflux temperature was 60°C, and the time was 1 h. Refluxing with ammonium nitrate solution can remove the cationic surfactant under mild conditions, avoiding oxidation of the material at high temperature caused by high-temperature calcination. During the reflux process, the ammonium ions dissociated in the ammonium nitrate solution can exchange with the cationic end of the cetyltrimethylammonium bromide, so that the cetyltrimethylammonium bromide molecules are completely removed in the form of soluble salt. After the reflux was completed, the product was collected by magnetic separation, washed repeatedly with deionized water and ethanol to remove residual ammonium nitrate and other impurities, and finally dried at 60°C for 12 hours to obtain the final iron-cobalt-based magnetic matrix material.

[0025] The iron-cobalt-based magnetic matrix material prepared by the preparation method of the above embodiment one has a porous structure, a specific surface area of 150-200 m 2 / g, and strong magnetic characteristics, which can quickly respond under the action of an external magnetic field. The porous structure of the material provides a large number of active sites, making it suitable for use as an enzyme immobilization carrier, which can effectively improve the efficiency and stability of enzyme immobilization. The cobalt-iron alloy particles in the material endow it with excellent magnetic separation performance, facilitating rapid separation and recycling in biological catalysis processes.

[0026] Embodiment two Different from the above embodiment one, this embodiment aims to compare the pore sizes of the porous silica coating layers formed by using different template agents, analyze the influence of template agents on the pore sizes of the porous silica coating layers during material synthesis, and select appropriate template agents to obtain materials with appropriate pore sizes in different use situations.

[0027] The preparation methods of Co7Fe3 / Co-600 in steps 1 and 2 are the same as those in embodiment one.

[0028] Step 3: porous silica coating The 100 mg Co7Fe3 / Co-600 material obtained in the above steps was dispersed in 150 mL of a 25% ethanol and water mixture, and ultrasonic treatment was performed to ensure complete dispersion. 1 mL of ammonia water was added to the dispersion to adjust the pH value, and 0.5 g of cetyltrimethylammonium bromide and 0.7 g of dodecyltrimethylammonium bromide were added as template agents. Under continuous stirring, 0.5 g of tetraethyl orthosilicate was slowly added for sol-gel coating reaction. The amount of tetraethyl orthosilicate was 0.5 g per 100 mg of magnetic matrix material. During the reaction, the tetraethyl orthosilicate hydrolyzes to form silicic acid, which then condenses to form a silica shell layer, encapsulating the magnetic particles to obtain Co7Fe3 / Co material coated with porous silica.

[0029] Step 4: Removal of the template agent The coated 100 mg material was dispersed in 50 mL of anhydrous ethanol, and 1 g of ammonium nitrate was added for reflux. The amount of ammonium nitrate used was 1 g per 100 mg of coated material, the reflux temperature was 60°C, and the time was 1 h. Refluxing with ammonium nitrate solution can remove the cationic surfactant under mild conditions, avoiding oxidation of the material at high temperature caused by high-temperature calcination. After refluxing, the product was collected by magnetic separation, washed repeatedly with deionized water and ethanol to remove residual ammonium nitrate and other impurities, and finally dried at 60°C for 12 hours to obtain the final iron-cobalt-based magnetic matrix material.

[0030] The pore size of the silica coating layer on the surface of the iron-cobalt-based magnetic matrix material prepared by the above-mentioned method of Example Two was 3-5 nm when hexadecyltrimethylammonium bromide was used as the template agent, and the pore size of the silica coating layer on the surface of the iron-cobalt-based magnetic matrix material prepared by the above-mentioned method of Example Two was 1-2 nm when dodecyltrimethylammonium bromide was used as the template agent. Thus, different template agents can be selected to control the pore size of the material according to different substrates.

[0031] An application method of an iron-cobalt-based magnetic matrix material is as follows: Example Three Step 1: Preparation of an iron-cobalt-based magnetic matrix material The preparation of the iron-cobalt-based magnetic material was the same as the preparation method of the material in Example One.

[0032] Step 2: Preparation of immobilized trypsin: The prepared iron-cobalt-based magnetic matrix material was prepared into a 5 mg / mL solution, 100 μL of which was taken into a 1.5 mL centrifuge tube, freeze-dried, and then 100 μL of 1 mg / mL trypsin and 100 μL of 50 mmol / L ammonium bicarbonate were added, shaken at 25°C for 1 h, filtered, and immobilized trypsin was obtained.

[0033] Accurately weigh 1 mg of standard protein (bovine serum albumin), add 100 μL of 8 mol / L urea in 50 mmol / L NH4HCO3 buffer, wait for the protein to completely dissolve, then add 5 μL of 200 mmol / L DTT, and react at 56°C for 45 min to open the disulfide bond. Then add 20 μL of 200 mmol / L IAA, avoid light for 30 min to alkylate, and dilute to the desired protein concentration with 50 mmol / L ammonium bicarbonate (pH 8.0) solution.

[0034] Immobilized trypsin was added to the processed protein sample and incubated at 37 °C for 16 h. After the reaction was completed, the sample was filtered, and the immobilized trypsin was recycled. The filtrate was the enzymatic hydrolysis solution.

[0035] Step 3: Protein Enzymatic Digestion Experiment Immobilized trypsin hydrolysis process: Iron-cobalt based magnetic matrix material immobilized with trypsin is added to the processed protein sample and incubated at 37 ℃ for 16 h. After the reaction is completed, magnetic separation is used to separate the material from the solution to terminate the reaction. The recovered material can be recycled, and the filtrate is the enzymatic hydrolysis reaction solution.

[0036] Free solution enzymatic hydrolysis process: Trypsin was added to the treated protein sample at an enzyme:protein ratio of 1:40 and incubated at 37 °C for 24 h. The enzymatic hydrolysis reaction was terminated with formic acid after the reaction was completed.

[0037] After detection using MALDI-TOF-MS, the enzymatic digestion efficiency was evaluated by comparing the results with those of the free solution with the amino acid coverage and the number of matched peptides obtained from the search. The experimental results are shown in Table 1.

[0038] Table 1. Evaluation of the effect of iron-cobalt based magnetic matrix material on BSA enzymatic hydrolysis after immobilization of trypsin. Immobilized trypsin Free solution Number of matched peptides 71.5% 49.5% Amino acid coverage 87 33 As shown in Table 1, the immobilized trypsin has a significantly improved enzymatic hydrolysis efficiency and a significantly shortened hydrolysis time compared to free solution. The magnetism imparted by the iron-cobalt based magnetic matrix material enables rapid and efficient recovery and separation.

[0039] It should be noted that the various parameters, ratios, and steps shown in the embodiments and accompanying drawings of this invention are merely preferred examples for clearly illustrating the technical solution of this invention, and are not intended to limit the scope of implementation of this invention. Specific mass ratios such as 1.22g:0.73g:1g and specific temperatures of 600℃ in the embodiments are exemplary and not exclusive. Any other combination of parameters falling within the scope defined in the specification, as long as it can prepare the same "iron-cobalt based magnetic matrix material," falls within the protection scope of this patent's technical solution.

[0040] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0041] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing an iron-cobalt-based magnetic matrix material, characterized in that, Includes the following steps: (1) Dissolve polyvinylpyrrolidone in water, add cobalt salt and iron salt, stir the reaction and dry at 90℃~110℃ to obtain the precursor; (2) The precursor was carbonized at 500℃~800℃ under nitrogen protection to obtain Co7Fe3 / Co material; (3) Disperse the material obtained in step (2) in a mixed solution of ethanol and water, add ammonia and template agent, stir and then add tetraethyl orthosilicate dropwise to carry out the coating reaction, and obtain Co7Fe3 / Co material coated with porous silica. (4) The coated material is dispersed in ethanol, ammonium nitrate is added for reflux to remove the template agent, and after washing and drying, the iron-cobalt based magnetic matrix material is obtained.

2. The method for preparing an iron-cobalt based magnetic matrix material according to claim 1, characterized in that: In step (1), the cobalt salt is cobalt nitrate hexahydrate, the iron salt is iron nitrate nonahydrate, and the mass ratio of cobalt salt, iron salt and polyvinylpyrrolidone is (1-5):(0.5-1):

1.

3. The method for preparing an iron-cobalt-based magnetic matrix material according to claim 1, characterized in that: In step (2), the carbonization temperature is 600℃~700℃, the heating rate is 5℃ / min, and the carbonization time is 0.5~5h.

4. The method for preparing an iron-cobalt-based magnetic matrix material according to claim 1, characterized in that: In step (3), the amount of ammonia water used is 0.1 to 3g per 100mg of magnetic matrix material, the amount of template agent used is 0.05 to 1g per 100mg of magnetic matrix material, and the amount of tetraethyl orthosilicate used is 0.01 to 0.7g per 100mg of magnetic matrix material.

5. The method for preparing an iron-cobalt-based magnetic matrix material according to claim 1, characterized in that: In step (3), the template agent is one or more of hexadecyltrimethylammonium bromide, trioctylmethylammonium bromide, and dodecyltrimethylammonium bromide.

6. The method for preparing an iron-cobalt-based magnetic matrix material according to claim 1, characterized in that: In step (4), the amount of ammonium nitrate used is 0.5 to 2 g per 100 mg of coating material, and the reflux time is 1 to 5 h.

7. An iron-cobalt-based magnetic matrix material prepared by the method according to any one of claims 1 to 6, characterized in that: The material has a porous structure and a specific surface area of ​​150–200 m². 2 / g, with strong magnetism, is suitable for enzyme immobilization carriers.

8. The application of an iron-cobalt based magnetic matrix material in immobilized trypsin, characterized in that: Immobilized trypsin is prepared by combining the magnetic matrix material of claim 7 with trypsin.

9. The application according to claim 8, characterized in that, The immobilization steps are as follows: The magnetic matrix material was mixed with water, freeze-dried, and then trypsin and ammonium bicarbonate solution were added. After shaking and filtration, the unadsorbed enzyme was washed with ammonium bicarbonate solution to obtain immobilized trypsin.

10. The application according to claim 9, characterized in that: The mass ratio of the trypsin to the magnetic matrix material is 1:10 to 1:50.

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