Composite magnetic particles and a method for preparing the same

Abstract

The application relates to the technical field of composite magnetic particles, and discloses a composite magnetic particle and a preparation method thereof. The composite magnetic particle in the application is Fe3O4@ZIF-8 / CS nanoparticles, the nanoparticles are synthesized by coating magnetic Fe3O4 with ZIF-8 to form Fe3O4@ZIF-8, then chemical bonding reaction occurs between the amino groups on the surface of the Fe3O4@ZIF-8 and the grafted carboxyl groups in modified chitosan, and finally the modified chitosan is modified to the surface of the Fe3O4@ZIF-8.

Description

Technical Field

[0001] This invention relates to the field of composite magnetic particle technology, specifically to a composite magnetic particle and its preparation method. Background Technology

[0002] Nucleic acid isolation and purification are core steps in molecular biology, clinical diagnostics, forensic identification, and biotechnology. Traditional nucleic acid extraction methods, such as phenol-chloroform extraction and centrifugation, while widely used, generally suffer from drawbacks such as cumbersome operation, long processing time, the need for large amounts of toxic organic solvents, and difficulty in achieving automation and high throughput. In recent years, solid-phase extraction technology, especially separation methods based on magnetic nanoparticles, has become a research hotspot in the field of nucleic acid extraction due to its simplicity, lack of centrifugation requirements, and ease of achieving high throughput and automation. Fe3O4 magnetic nanoparticles possess superparamagnetism and a high specific surface area, enabling rapid separation under an external magnetic field, demonstrating significant advantages in the field of bioseparation. However, exposed Fe3O4 nanoparticles are prone to aggregation, have poor chemical stability, and lack specific binding sites on their surface, making them difficult to use directly and efficiently for nucleic acid extraction.

[0003] To improve the dispersibility and stability of magnetic nanoparticles, SiO2 is widely used as a protective shell for the Fe3O4 core. Coating Fe3O4 with SiO2 using the sol-gel method can form Fe3O4@SiO2 composite particles with a core-shell structure. Studies have shown that Fe3O4@SiO2 nanoparticles with appropriately ligand-modified surfaces can significantly improve the binding efficiency of nucleic acid molecules, exhibiting excellent separation performance in RNA extraction. However, the interaction between a simple SiO2 layer or simply amino-functionalized magnetic particles and DNA is still mainly electrostatic adsorption. The selective adsorption capacity and binding capacity for DNA in complex biological samples need further improvement, and their stability during extraction still requires optimization. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a composite magnetic particle and its preparation method.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A method for preparing composite magnetic particles specifically includes the following steps:

[0007] (1) Preparation of carboxylic acid-modified Fe3O4:

[0008] Sodium citrate dihydrate and anhydrous sodium acetate were stirred in ethylene glycol for 30 minutes. Then, ferric chloride hexahydrate was added while stirring and stirred until a mixture was formed. The mixture was then transferred to a reaction vessel for hydrothermal reaction. After the reaction was completed, the mixture was centrifuged, washed until the pH of the supernatant was neutral, dried, and ground to obtain carboxylic acid modified Fe3O4.

[0009] Furthermore, the hydrothermal reaction temperature is 190-200℃, and the reaction time is 8-10h;

[0010] Furthermore, the ratio of sodium citrate dihydrate, anhydrous sodium acetate, ethylene glycol, and ferric chloride hexahydrate is 0.005-0.02 mol: 0.375 mol: 40 mL: 0.1 mol.

[0011] (2) Preparation of Fe3O4@ZIF-8 nanoparticles:

[0012] 2-Methylimidazole and 2-aminobenzimidazole were stirred evenly in 20 mL of methanol to prepare an organic ligand solution. Carboxylic acid-modified Fe3O4 was ultrasonically dispersed in 40 mL of methanol, and then zinc nitrate hexahydrate was added and stirred at room temperature for 30 min. The organic ligand solution was then slowly added and stirred for 15 min. The mixture was then transferred to a water bath for reaction. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain Fe3O4@ZIF-8 nanoparticles.

[0013] Furthermore, the reaction temperature in the water bath is 60-70℃, and the reaction time is 6-12h;

[0014] Further, the ratio of the carboxylic acid-modified Fe3O4, zinc nitrate hexahydrate, 2-methylimidazole and 2-aminobenzimidazole is 0.2g:0.8-1.0g:2.2-3g:0.5-1.0g.

[0015] (3) Preparation of Fe3O4@ZIF-8 / CS nanoparticles:

[0016] Modified chitosan was stirred evenly in MES buffer (pH 5.0-6.0), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added, and the mixture was stirred at room temperature for 20-40 min. Fe3O4@ZIF-8 nanoparticles were then added, and the pH of the system was adjusted to neutral with Tris-HCl buffer (pH 7.4). The mixture was stirred at 30-40℃ for 12-24 h, magnetically separated, washed, and dried to obtain Fe3O4@ZIF-8 / CS nanoparticles.

[0017] Furthermore, the ratio of the modified chitosan, MES buffer, EDC, NHS and Fe3O4@ZIF-8 nanoparticles is 0.5-1.0g:20mL:0.05-0.2g:0.05-0.2g:0.5g.

[0018] Furthermore, the modified chitosan is prepared by the following steps:

[0019] Step A1: Add 1-ethyl-5-aminobenzimidazole to a mixture of anhydrous dichloromethane and anhydrous chloroform and stir to dissolve. Then add triethylamine and stir for 5-10 min. Then cool to 0-5℃ in an ice-water bath. Under nitrogen protection, slowly add triphenylmethyl chloride anhydrous dichloromethane solution over 20-30 min. Then heat to room temperature and stir for 2-4 h. Then add anhydrous methanol and stir for 20-30 min. Finally, post-treat the reaction solution to obtain intermediate product 1.

[0020] Further, in step A1, the ratio of the amounts of 1-ethyl-5-aminobenzimidazole, anhydrous dichloromethane, anhydrous chloroform, triethylamine, triphenylmethylchloroanhydrous dichloromethane solution, and anhydrous methanol is 0.01 mol: 20-30 mL: 1-2 mL: 0.022-0.03 mol: 10 mL: 2-5 mL;

[0021] Further, the triphenylmethyl chloride anhydrous dichloromethane solution mentioned in step A1 is prepared by mixing and stirring triphenylmethyl chloride and anhydrous dichloromethane at a volume ratio of 0.01-0.025 mol: 10 mL;

[0022] Further, the post-processing step described in step A1 is as follows: the reaction solution is concentrated to 10 mL under reduced pressure, chloroform is added and stirred for 5 min, then washed successively with 1 mol / L dilute hydrochloric acid, distilled water and saturated saline, anhydrous sodium sulfate is added and dried for 30 min, filtered and concentrated under reduced pressure, and recrystallized.

[0023] Step A2: Mix intermediate product 1 in acetonitrile and stir until homogeneous. Heat to 50-55℃, then add bromobutane and heat to reflux. Monitor by TLC. Stop the reaction immediately after the starting material spot disappears. Remove acetonitrile by rotary evaporation. Cool to 30-50℃ and add 80wt% aqueous acetic acid solution. Heat to reflux for 1-2 hours. Cool to room temperature and then dilute in ice water. Extract with ether. Neutralize the aqueous phase to pH 8-9 with saturated sodium bicarbonate solution. Extract the aqueous phase with dichloromethane. Combine the organic phases, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the modifier.

[0024] Further, the ratio of intermediate product 1, acetonitrile, bromobutane and aqueous acetic acid in step A2 is 0.01 mol: 20 mL: 0.01-0.015 mol: 20-30 mL;

[0025] Step A3: Dissolve glyoxal in deionized water, add the modifier and heat to 40-50℃ and stir for 3-5 hours. Add chitosan solution and react at room temperature for 24 hours. Adjust the pH of the system to 9-10. Then slowly add 5wt% sodium borohydride aqueous solution under ice-water bath and stir for 24 hours. Adjust the pH to neutral, filter under reduced pressure, wash and dry. This is the benzimidazole quaternary ammonium salt modified chitosan.

[0026] Further, the ratio of glyoxal, deionized water, modifier, chitosan solution and sodium borohydride aqueous solution in step A3 is 0.01-0.015 mol: 20 mL: 0.01-0.015 mol: 10 mL: 10-15 mL;

[0027] Further, the chitosan solution in step A3 is prepared by dissolving 1g of chitosan in 10mL of 1wt% acetic acid solution;

[0028] Step A4: Stir the benzimidazole quaternary ammonium salt modified chitosan in isopropanol until homogeneous, add potassium hydroxide and stir to alkalize and swell for 1-2 hours, then slowly add chloroacetic acid isopropanol solution, heat to 60-70℃ and stir to react for 2-4 hours, remove the supernatant, add deionized water, adjust the pH to neutral, add ethanol to precipitate, filter, wash and dry to obtain modified chitosan.

[0029] Further, in step A4, the ratio of benzimidazole quaternary ammonium salt modified chitosan, isopropanol, potassium hydroxide, chloroacetic acid isopropanol solution, deionized water and ethanol is 2g:50mL:3-5g:10mL:100mL:200mL.

[0030] Further, the chloroacetic acid and isopropanol solution described in step A4 is prepared by mixing and stirring chloroacetic acid and isopropanol at a ratio of 4-5g:10mL.

[0031] On the other hand, the present invention also provides a composite magnetic particle prepared according to the above preparation method.

[0032] The beneficial effects of this invention are:

[0033] In this invention, Fe3O4@ZIF-8 / CS nanoparticles are synthesized by coating magnetic Fe3O4 with ZIF-8. Then, the amino groups on the surface of Fe3O4@ZIF-8 and the carboxyl groups grafted in modified chitosan undergo a chemical bonding reaction, thereby modifying the surface of Fe3O4@ZIF-8 with modified chitosan.

[0034] In this invention, Fe3O4 magnetic nanoparticles serve as the core of the entire composite structure, providing superparamagnetism. This allows the material to rapidly accumulate and separate under an external magnetic field, and then disperse uniformly again after the magnetic field is removed. This enables rapid solid-liquid phase separation during DNA extraction, replacing traditional centrifugation steps, simplifying the operation process, and making it suitable for automated high-throughput processing. The ZIF-8 shell has a regular microporous structure and a large specific surface area, providing a large number of physical adsorption sites for DNA, significantly improving the DNA capture capacity per unit mass of particles. When DNA fragments enter the nanopores of ZIF-8, they are spatially confined and "locked in," enhancing binding stability and reducing non-specific losses during washing. The ZIF-8 shell acts as a physical barrier, isolating the Fe3O4 core from direct contact with the external environment and inhibiting the dissolution and oxidation of iron ions. Simultaneously, ZIF-8 slowly degrades in an acidic environment (pH < 6), a characteristic that can be utilized during the DNA elution stage to release the DNA fragments captured by the pores.

[0035] The benzimidazole quaternary ammonium salt introduced into the modified chitosan carries a permanent positive charge, generating a strong electrostatic attraction with the DNA phosphate backbone, providing a powerful initial driving force for DNA capture and achieving rapid binding. Benzimidazole is formed by the fusion of an imidazole ring and a benzene ring, creating a larger π-conjugated system. Its aromatic ring and the aromatic ring of the DNA base undergo face-to-face π-π stacking, enhancing binding specificity and achieving "double locking" of DNA. The nitrogen atom on the imidazole ring has protonation / deprotonation properties. At low pH, it carries a positive charge to enhance adsorption, while at high pH, ​​deprotonation leads to surface charge reversal, generating electrostatic repulsion with DNA and actively promoting DNA release. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1: Modified chitosan was prepared by the following steps:

[0038] Step A1: Add 0.01 mol of 1-ethyl-5-aminobenzimidazole to a mixture of 20 mL of anhydrous dichloromethane and 1 mL of anhydrous chloroform and stir to dissolve. Then add 0.022 mol of triethylamine and stir for 5 min. Then cool the mixture to 0-5℃ in an ice-water bath. Under nitrogen protection, slowly add 10 mL of triphenylmethyl chloride anhydrous dichloromethane solution over 20 min. Then heat the mixture to room temperature and stir for 2 h. Then add 2 mL of anhydrous methanol and stir for 20 min. Finally, post-treat the reaction solution to obtain intermediate product 1.

[0039] Preferably, the triphenylmethyl chloride anhydrous dichloromethane solution is prepared by mixing and stirring triphenylmethyl chloride and anhydrous dichloromethane at a ratio of 0.01 mol: 10 mL.

[0040] Preferably, the post-processing step A1 is as follows: the reaction solution is concentrated to 10 mL under reduced pressure, chloroform is added and stirred for 5 min, then washed successively with 1 mol / L dilute hydrochloric acid, distilled water and saturated saline, anhydrous sodium sulfate is added and dried for 30 min, filtered and concentrated under reduced pressure, and recrystallized.

[0041] Step A2: Mix 0.01 mol of intermediate product 1 in 20 mL of acetonitrile and stir until homogeneous. Heat to 50 °C, then add 0.01 mol of bromobutane and heat to reflux. Monitor by TLC. Stop the reaction immediately after the starting material spot disappears. Remove acetonitrile by rotary evaporation. Cool to 30 °C and add 20 mL of 80 wt% acetic acid aqueous solution. Heat to reflux for 1 h and cool to room temperature. Then dilute in 100 mL of ice water and extract with 50 mL of diethyl ether. Neutralize the aqueous phase to pH 8 with saturated sodium bicarbonate solution and extract the aqueous phase with 30 mL of dichloromethane. Combine the organic phases, dry with 15 g of anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the modifier.

[0042] Step A3: Dissolve 0.01 mol glyoxal in 20 mL of deionized water, add 0.01 mol modifier and heat to 40 °C and stir for 3 h. Add 10 mL of chitosan solution and react at room temperature for 24 h. Adjust the pH of the system to 9. Then slowly add 10 mL of 5 wt% sodium borohydride aqueous solution under an ice-water bath and stir for 24 h. Adjust the pH to neutral, filter under reduced pressure, wash and dry to obtain benzimidazole quaternary ammonium salt modified chitosan.

[0043] Preferably, the chitosan solution in step A3 is prepared by dissolving 1g of chitosan in 10mL of 1wt% acetic acid solution;

[0044] Step A4: Stir 2g of benzimidazole quaternary ammonium salt modified chitosan in 50mL of isopropanol until homogeneous. Add 3g of potassium hydroxide and stir to alkalize and swell for 1h. Then slowly add 10mL of chloroacetic acid isopropanol solution. Heat to 60℃ and stir to react for 2h. Remove the supernatant, add 100mL of deionized water, adjust the pH to neutral, add 200mL of ethanol to precipitate, filter, wash, and dry to obtain modified chitosan.

[0045] Preferably, the chloroacetic acid and isopropanol solution in step A4 is prepared by mixing and stirring chloroacetic acid and isopropanol at a ratio of 4g:10mL.

[0046] Example 2: Modified chitosan was prepared by the following steps:

[0047] Step A1: 0.01 mol of 1-ethyl-5-aminobenzimidazole was added to a mixture of 25 mL of anhydrous dichloromethane and 1.5 mL of anhydrous chloroform and stirred to dissolve. Then, 0.026 mol of triethylamine was added and stirred for 8 min. The mixture was then placed in an ice-water bath and cooled to 0-5 °C. Under nitrogen protection, 10 mL of triphenylmethyl chloride anhydrous dichloromethane solution was slowly added over a period of 25 min. The mixture was then heated to room temperature and stirred for 3 h. Finally, 3.5 mL of anhydrous methanol was added and stirred for 25 min. The reaction solution was then post-treated to obtain intermediate product 1.

[0048] Preferably, the triphenylmethyl chloride anhydrous dichloromethane solution is prepared by mixing and stirring triphenylmethyl chloride and anhydrous dichloromethane at a ratio of 0.02 mol: 10 mL.

[0049] Preferably, the post-processing step A1 is as follows: the reaction solution is concentrated to 10 mL under reduced pressure, chloroform is added and stirred for 5 min, then washed successively with 1 mol / L dilute hydrochloric acid, distilled water and saturated saline, anhydrous sodium sulfate is added and dried for 30 min, filtered and concentrated under reduced pressure, and recrystallized.

[0050] Step A2: Mix 0.01 mol of intermediate product 1 in 20 mL of acetonitrile and stir until homogeneous. Heat to 55 °C, then add 0.013 mol of bromobutane and heat to reflux. Monitor by TLC. Stop the reaction immediately after the starting material spot disappears. Remove acetonitrile by rotary evaporation. Cool to 40 °C and add 25 mL of 80 wt% acetic acid aqueous solution. Heat to reflux for 1.5 h and cool to room temperature. Then dilute in 100 mL of ice water and extract with 50 mL of diethyl ether. Neutralize the aqueous phase to pH 8.5 with saturated sodium bicarbonate solution and extract the aqueous phase with 30 mL of dichloromethane. Combine the organic phases, dry with 15 g of anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the modifier.

[0051] Step A3: Dissolve 0.013 mol glyoxal in 20 mL of deionized water, add 0.013 mol modifier and heat to 45 °C and stir for 4 h. Add 10 mL of chitosan solution and react at room temperature for 24 h. Adjust the pH of the system to 9.5. Then slowly add 12 mL of 5 wt% sodium borohydride aqueous solution under an ice-water bath and stir for 24 h. Adjust the pH to neutral, filter under reduced pressure, wash and dry to obtain benzimidazole quaternary ammonium salt modified chitosan.

[0052] Preferably, the chitosan solution in step A3 is prepared by dissolving 1g of chitosan in 10mL of 1wt% acetic acid solution;

[0053] Step A4: Stir 2g of benzimidazole quaternary ammonium salt modified chitosan in 50mL of isopropanol until homogeneous. Add 4g of potassium hydroxide and stir to alkalize and swell for 1.5h. Then slowly add 10mL of chloroacetic acid isopropanol solution. Heat to 65℃ and stir to react for 3h. Remove the supernatant, add 100mL of deionized water, adjust the pH to neutral, add 200mL of ethanol to precipitate, filter, wash, and dry to obtain modified chitosan.

[0054] Preferably, the chloroacetic acid and isopropanol solution in step A4 is prepared by mixing and stirring chloroacetic acid and isopropanol at a ratio of 4.5g:10mL.

[0055] Example 3: Modified chitosan was prepared by the following steps:

[0056] Step A1: Add 0.01 mol of 1-ethyl-5-aminobenzimidazole to a mixture of 30 mL of anhydrous dichloromethane and 2 mL of anhydrous chloroform and stir to dissolve. Then add 0.03 mol of triethylamine and stir for 10 min. Then cool the mixture to 0-5℃ in an ice-water bath. Under nitrogen protection, slowly add 10 mL of triphenylmethyl chloride anhydrous dichloromethane solution over 30 min. Then heat the mixture to room temperature and stir for 4 h. Then add 5 mL of anhydrous methanol and stir for 30 min. Finally, post-treat the reaction solution to obtain intermediate product 1.

[0057] Preferably, the triphenylmethyl chloride anhydrous dichloromethane solution is prepared by mixing and stirring triphenylmethyl chloride and anhydrous dichloromethane at a ratio of 0.025 mol: 10 mL.

[0058] Preferably, the post-processing step A1 is as follows: the reaction solution is concentrated to 10 mL under reduced pressure, chloroform is added and stirred for 5 min, then washed successively with 1 mol / L dilute hydrochloric acid, distilled water and saturated saline, anhydrous sodium sulfate is added and dried for 30 min, filtered and concentrated under reduced pressure, and recrystallized.

[0059] Step A2: Mix 0.01 mol of intermediate product 1 in 20 mL of acetonitrile and stir until homogeneous. Heat to 55 °C, then add 0.015 mol of bromobutane and heat to reflux. Monitor by TLC. Stop the reaction immediately after the starting material spot disappears. Remove acetonitrile by rotary evaporation. Cool to 50 °C and add 30 mL of 80 wt% acetic acid aqueous solution. Heat to reflux for 2 h and cool to room temperature. Then dilute in 100 mL of ice water and extract with 50 mL of diethyl ether. Neutralize the aqueous phase to pH 9 with saturated sodium bicarbonate solution and extract the aqueous phase with 30 mL of dichloromethane. Combine the organic phases, dry with 15 g of anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the modifier.

[0060] Step A3: Dissolve 0.015 mol glyoxal in 20 mL of deionized water, add 0.015 mol modifier and heat to 50 °C and stir for 5 h. Add 10 mL of chitosan solution and react at room temperature for 24 h. Adjust the pH of the system to 10. Then slowly add 15 mL of 5 wt% sodium borohydride aqueous solution under an ice-water bath and stir for 24 h. Adjust the pH to neutral, filter under reduced pressure, wash and dry to obtain benzimidazole quaternary ammonium salt modified chitosan.

[0061] Preferably, the chitosan solution in step A3 is prepared by dissolving 1g of chitosan in 10mL of 1wt% acetic acid solution;

[0062] Step A4: Stir 2g of benzimidazole quaternary ammonium salt modified chitosan in 50mL of isopropanol until homogeneous. Add 5g of potassium hydroxide and stir to alkalize and swell for 2 hours. Then slowly add 10mL of chloroacetic acid isopropanol solution. Heat to 70℃ and stir to react for 4 hours. Remove the supernatant, add 100mL of deionized water, adjust the pH to neutral, add 200mL of ethanol to precipitate, filter, wash, and dry to obtain modified chitosan.

[0063] Preferably, the chloroacetic acid and isopropanol solution in step A4 is prepared by mixing and stirring chloroacetic acid and isopropanol at a ratio of 5g:10mL.

[0064] Example 4: A method for preparing composite magnetic particles, specifically including the following steps:

[0065] (1) Preparation of carboxylic acid-modified Fe3O4:

[0066] 0.005 mol sodium citrate dihydrate and 0.375 mol anhydrous sodium acetate were stirred in 40 mL of ethylene glycol for 30 min. Then, 0.1 mol ferric chloride hexahydrate was added while stirring and stirred until homogeneous to form a mixture. The mixture was then transferred to a reaction vessel for hydrothermal reaction. After the reaction was completed, the mixture was centrifuged, washed until the pH of the supernatant was neutral, dried, and ground to obtain carboxylic acid modified Fe3O4.

[0067] Preferably, the hydrothermal reaction temperature is 190°C and the reaction time is 8 hours.

[0068] (2) Preparation of Fe3O4@ZIF-8 nanoparticles:

[0069] 2.2 g of 2-methylimidazole and 0.5 g of 2-aminobenzimidazole were stirred evenly in 20 mL of methanol to prepare an organic ligand solution. 0.2 g of carboxylic acid-modified Fe3O4 was ultrasonically dispersed in 40 mL of methanol, followed by the addition of 0.8 g of zinc nitrate hexahydrate, and stirred at room temperature for 30 min. Then, the organic ligand solution was slowly added and stirred for 15 min. The mixture was then transferred to a water bath for reaction. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain Fe3O4@ZIF-8 nanoparticles.

[0070] Preferably, the reaction temperature in the water bath is 60°C and the reaction time is 6 hours.

[0071] (3) Preparation of Fe3O4@ZIF-8 / CS nanoparticles:

[0072] 0.5 g of the modified chitosan prepared in Example 1 was stirred evenly in 20 mL of MES buffer (pH 5.0), 0.05 g of EDC and 0.05 g of NHS were added and stirred at room temperature for 20 min, then 0.5 g of Fe3O4@ZIF-8 nanoparticles were added, and the pH of the system was adjusted to neutral with Tris-HCl buffer (pH 7.4). The mixture was stirred at 30 °C for 12 h, magnetically separated, washed, and dried to obtain Fe3O4@ZIF-8 / CS nanoparticles.

[0073] Example 5: A method for preparing composite magnetic particles, specifically including the following steps:

[0074] (1) Preparation of carboxylic acid-modified Fe3O4:

[0075] 0.01 mol sodium citrate dihydrate and 0.375 mol anhydrous sodium acetate were stirred in 40 mL of ethylene glycol for 30 min. Then, 0.1 mol ferric chloride hexahydrate was added while stirring and stirred until homogeneous to form a mixture. The mixture was then transferred to a reaction vessel for hydrothermal reaction. After the reaction was completed, the mixture was centrifuged, washed until the pH of the supernatant was neutral, dried, and ground to obtain carboxylic acid modified Fe3O4.

[0076] Preferably, the hydrothermal reaction temperature is 195°C and the reaction time is 9 hours.

[0077] (2) Preparation of Fe3O4@ZIF-8 nanoparticles:

[0078] 2.6 g of 2-methylimidazole and 0.75 g of 2-aminobenzimidazole were stirred evenly in 20 mL of methanol to prepare an organic ligand solution. 0.2 g of carboxylic acid-modified Fe3O4 was ultrasonically dispersed in 40 mL of methanol, followed by the addition of 0.9 g of zinc nitrate hexahydrate, and stirred at room temperature for 30 min. Then, the organic ligand solution was slowly added and stirred for 15 min. The mixture was then transferred to a water bath for reaction. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain Fe3O4@ZIF-8 nanoparticles.

[0079] Preferably, the reaction temperature in the water bath is 65°C and the reaction time is 9 hours.

[0080] (3) Preparation of Fe3O4@ZIF-8 / CS nanoparticles:

[0081] 0.75 g of the modified chitosan prepared in Example 2 was stirred evenly in 20 mL of MES buffer (pH 5.5), 0.1 g of EDC and 0.1 g of NHS were added and stirred at room temperature for 30 min, then 0.5 g of Fe3O4@ZIF-8 nanoparticles were added, and the pH of the system was adjusted to neutral with Tris-HCl buffer (pH 7.4). The mixture was stirred at 35 °C for 16 h, magnetically separated, washed, and dried to obtain Fe3O4@ZIF-8 / CS nanoparticles.

[0082] Example 6: A method for preparing composite magnetic particles, specifically including the following steps:

[0083] (1) Preparation of carboxylic acid-modified Fe3O4:

[0084] 0.02 mol sodium citrate dihydrate and 0.375 mol anhydrous sodium acetate were stirred in 40 mL of ethylene glycol for 30 min. Then, 0.1 mol ferric chloride hexahydrate was added while stirring and stirred until a mixture was formed. The mixture was then transferred to a reaction vessel for hydrothermal reaction. After the reaction was completed, the mixture was centrifuged, washed until the pH of the supernatant was neutral, dried, and ground to obtain carboxylic acid modified Fe3O4.

[0085] Preferably, the hydrothermal reaction is carried out at a temperature of 200°C for 10 hours.

[0086] (2) Preparation of Fe3O4@ZIF-8 nanoparticles:

[0087] 3 g of 2-methylimidazole and 1.0 g of 2-aminobenzimidazole were stirred evenly in 20 mL of methanol to prepare an organic ligand solution. 0.2 g of carboxylic acid-modified Fe3O4 was ultrasonically dispersed in 40 mL of methanol, followed by the addition of 1.0 g of zinc nitrate hexahydrate, and stirred at room temperature for 30 min. Then, the organic ligand solution was slowly added and stirred for 15 min. The mixture was then transferred to a water bath for reaction. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain Fe3O4@ZIF-8 nanoparticles.

[0088] Preferably, the reaction temperature in the water bath is 70°C and the reaction time is 12 hours.

[0089] (3) Preparation of Fe3O4@ZIF-8 / CS nanoparticles:

[0090] 1.0 g of the modified chitosan prepared in Example 3 was stirred evenly in 20 mL of MES buffer (pH 6.0), 0.2 g of EDC and 0.2 g of NHS were added and stirred at room temperature for 40 min, then 0.5 g of Fe3O4@ZIF-8 nanoparticles were added, and the pH of the system was adjusted to neutral with Tris-HCl buffer (pH 7.4). The mixture was stirred at 40 °C for 24 h, magnetically separated, washed, and dried to obtain Fe3O4@ZIF-8 / CS nanoparticles.

[0091] Comparative Example 1: This comparative example is a composite magnetic particle. The difference from Example 6 is that Fe3O4@ZIF-8 prepared in Example 3 is used instead of Fe3O4@ZIF-8 / CS nanoparticles prepared in Example 3. All other aspects are the same.

[0092] Comparative Example 2: This comparative example is a composite magnetic particle. The difference from Example 6 is that Fe3O4@SiO2 is used instead of the Fe3O4@ZIF-8 / CS nanoparticles prepared in Example 3. All other aspects are the same.

[0093] The performance of the composite magnetic particles prepared in Examples 4-6 and Comparative Examples 1-2 was tested:

[0094] (1) DNA extraction and elution: The ground sample powder (100 mg shrimp sample) was transferred to a centrifuge tube pre-filled with 400 μL of lysis buffer and RNase. The mixture was quickly mixed and the centrifuge tube was heated in a 70°C water bath for 10 min. After heating, the sample was centrifuged and 300 μL of supernatant was transferred to a new centrifuge tube. A mixed solution (including 300 μL of binding buffer, 300 μL of isopropanol, and 30 μL of composite magnetic particles (hereinafter referred to as magnetic beads)) was added to the centrifuge tube, vortexed to mix, and the centrifuge tube was placed in a magnetic rack and the liquid was aspirated. The magnetic beads were then washed twice with 500 μL of protein removal buffer and 600 μL of rinsing buffer. After washing, 100 μL of elution buffer was added to the system, vortexed to mix, and incubated at 65°C for 3 min. Centrifuge tubes were placed in a magnetic rack. After the magnetic beads were completely adsorbed, the supernatant was the DNA solution. The pH of the mixed solution was 5, and the composite magnetic particles used were the magnetic beads from Examples 1-3 and Comparative Examples 1-2.

[0095] (2) Calculation of DNA extraction rate: The DNA concentration was determined using a Thermo Fisher Qubit4 fluorescence analyzer. First, a concentration test system was prepared, with a Qubit buffer to dye ratio of 200:1 to prepare the mix solution. Then, the DNA to be tested was added to the concentration test system, with a mix solution to DNA ratio of 199:1. The concentration test system was then placed in the Qubit analyzer to read the DNA concentration. The extraction rate was calculated as: extraction rate = [(A1-A2) / A1] × 100, where A1 is the concentration of the DNA to be extracted; A2 is the concentration of the extracted DNA.

[0096] Lysis buffer preparation: Weigh 19.15 g guanidine hydrochloride (Gu-HCl), 0.185 g EDTA, 0.03 g NaCl, and 2 mL Triton X-100, and bring the volume to 100 mL with ddH2O. Mix well. Binding buffer: Weigh 20 g PEG-6000 and 17.5 g NaCl, and bring the volume to 100 mL with ddH2O. Wash buffer: Add 70 mL anhydrous ethanol and bring the volume to 100 mL with ddH2O. Eluent: Weigh 0.121 g Tris-HCl and 0.04 g EDTA, adjust the pH to 8.0, and bring the volume to 100 mL with ddH2O.

[0097] The test results are shown in Table 1:

[0098] Table 1: Performance Test Results

[0099]

[0100] As can be seen from Table 1, the composite magnetic particles prepared by this invention have a high DNA extraction rate and have good application prospects in DNA extraction technology.

[0101] The above content is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the scope defined by the inventive concept, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing composite magnetic particles, characterized in that, The composite magnetic particles are Fe3O4@ZIF-8 / CS nanoparticles; The Fe3O4@ZIF-8 / CS nanoparticles specifically include the following steps: (1) Preparation of carboxylic acid-modified Fe3O4: Sodium citrate dihydrate and anhydrous sodium acetate were stirred in ethylene glycol, and then ferric chloride hexahydrate was added and stirred to form a mixture. The mixture was then transferred to a reaction vessel for hydrothermal reaction. After the reaction was completed, the mixture was centrifuged, washed until the pH of the supernatant was neutral, dried, and ground to obtain carboxylic acid modified Fe3O4. (2) Preparation of Fe3O4@ZIF-8 nanoparticles: 2-Methylimidazole and 2-aminobenzimidazole were stirred evenly in methanol to prepare an organic ligand solution; carboxylic acid-modified Fe3O4 was dispersed in methanol, then zinc nitrate hexahydrate was added and stirred, and then the organic ligand solution was slowly added and stirred. The mixture was then transferred to a water bath for reaction. After the reaction was completed, the mixture was centrifuged, washed and dried to obtain Fe3O4@ZIF-8 nanoparticles. (3) Preparation of Fe3O4@ZIF-8 / CS nanoparticles: Modified chitosan was stirred evenly in MES buffer, EDC and NHS were added and stirred at room temperature, then Fe3O4@ZIF-8 nanoparticles were added, and the pH of the system was adjusted to neutral with Tris-HCl buffer. The mixture was stirred, magnetically separated, washed, and dried to obtain Fe3O4@ZIF-8 / CS nanoparticles.

2. The method for preparing composite magnetic particles according to claim 1, characterized in that, The ratio of sodium citrate dihydrate, anhydrous sodium acetate, ethylene glycol, and ferric chloride hexahydrate is 0.005-0.02 mol: 0.375 mol: 40 mL: 0.1 mol.

3. The method for preparing composite magnetic particles according to claim 1, characterized in that, The ratio of the carboxylic acid-modified Fe3O4, zinc nitrate hexahydrate, 2-methylimidazole, and 2-aminobenzimidazole is 0.2g:0.8-1.0g:2.2-3g:0.5-1.0g.

4. The method for preparing composite magnetic particles according to claim 1, characterized in that, The ratio of modified chitosan, MES buffer, EDC, NHS and Fe3O4@ZIF-8 nanoparticles is 0.5-1.0g:20mL:0.05-0.2g:0.05-0.2g:0.5g.

5. The method for preparing composite magnetic particles according to claim 1, characterized in that, The modified chitosan is prepared by the following steps: Step A1: 1-Ethyl-5-aminobenzimidazole was added to a mixture of anhydrous dichloromethane and anhydrous chloroform and stirred until dissolved. Triethylamine was then added and stirred. The mixture was then cooled to 0-5°C in an ice-water bath. Under nitrogen protection, a solution of triphenylmethyl chloride in anhydrous dichloromethane was slowly added. The mixture was then heated to room temperature and stirred. Anhydrous methanol was then added and stirred. The reaction solution was then post-treated to obtain intermediate product 1. Step A2: Mix intermediate product 1 in acetonitrile and stir until homogeneous. Then add bromobutane and heat to reflux. Monitor by TLC. Stop the reaction immediately after the starting material spot disappears. Remove acetonitrile by rotary evaporation. Cool to 30-50℃ and add 80wt% aqueous acetic acid solution. Heat to reflux and cool to room temperature. Then dilute in ice water and extract with ether. Neutralize the aqueous phase to pH 8-9 with saturated sodium bicarbonate solution. Extract the aqueous phase with dichloromethane. Combine the organic phases, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the modifier. Step A3: Dissolve glyoxal in deionized water, add the modifier and heat and stir, add chitosan solution, react at room temperature, then adjust the pH of the system to 9-10, then slowly add 5wt% sodium borohydride aqueous solution under ice water bath and stir, then adjust the pH to neutral, filter under reduced pressure, wash and dry, benzimidazole quaternary ammonium salt modified chitosan. Step A4: Stir the benzimidazole quaternary ammonium salt modified chitosan in isopropanol until homogeneous, add potassium hydroxide and stir to alkalize and swell, then slowly add chloroacetic acid isopropanol solution, heat and stir to react, remove the supernatant, add deionized water, adjust the pH to neutral, add ethanol to precipitate, filter, wash and dry to obtain modified chitosan.

6. The method for preparing composite magnetic particles according to claim 5, characterized in that, In step A1, the ratio of 1-ethyl-5-aminobenzimidazole, anhydrous dichloromethane, anhydrous chloroform, triethylamine, triphenylmethyl chloride anhydrous dichloromethane solution, and anhydrous methanol is 0.01 mol: 20-30 mL: 1-2 mL: 0.022-0.03 mol: 10 mL: 2-5 mL. The triphenylmethyl chloride anhydrous dichloromethane solution is prepared by mixing and stirring triphenylmethyl chloride and anhydrous dichloromethane at a ratio of 0.01-0.025 mol: 10 mL.

7. The method for preparing composite magnetic particles according to claim 5, characterized in that, The ratio of intermediate product 1, acetonitrile, bromobutane and acetic acid aqueous solution used in step A2 is 0.01mol:20mL:0.01-0.015mol:20-30mL.

8. The method for preparing composite magnetic particles according to claim 5, characterized in that, The ratio of glyoxal, deionized water, modifier, chitosan solution and sodium borohydride aqueous solution in step A3 is 0.01-0.015 mol: 20 mL: 0.01-0.015 mol: 10 mL: 10-15 mL. The chitosan solution is prepared by dissolving 1 g of chitosan in 10 mL of 1 wt% acetic acid solution.

9. The method for preparing composite magnetic particles according to claim 5, characterized in that, In step A4, the ratio of benzimidazole quaternary ammonium salt modified chitosan, isopropanol, potassium hydroxide, chloroacetic acid isopropanol solution, deionized water, and ethanol is 2g:50mL:3-5g:10mL:100mL:200mL. The chloroacetic acid isopropanol solution is prepared by mixing and stirring chloroacetic acid and isopropanol at a ratio of 4-5g:10mL.

10. A composite magnetic particle, characterized in that, It is prepared according to any one of claims 1-9.

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