Preparation method and application of magnetic nanoparticles based on triazolinone modification

Abstract

The invention relates to the technical field of magnetic nanoparticle preparation methods, and discloses a triazolinone modification-based magnetic nanoparticle preparation method, which comprises the following steps: 1, dissolving 3-chloromethyl-1, 2, 4-triazoline-5-ketone in DMF (Dimethyl Formamide); then adding Fe3O4-NH2 and potassium carbonate to obtain a mixture; 2, carrying out condensation reflux on the mixture; then naturally cooling to obtain a product; step 3, washing the product DMF with water until the pH value of the washing liquid is neutral; then drying; and step 4, obtaining the CMTO (at) Fe3O4-NH2 magnetic nano particles. The CMTO-at-Fe3O4-NH2 magnetic nanoparticle capturing material is very concerned due to the unique physical and chemical properties and the wide application prospect of the CMTO-at-Fe3O4-NH2 magnetic nanoparticle capturing material. The CMTO-at-Fe3O4-NH2 magnetic nanoparticles have larger specific surface area and higher adsorption capacity, can quickly adsorb target molecules, and can be quickly separated through an external magnetic field. Besides, the surfaces of the CMTO-at-Fe3O4-NH2 magnetic nanoparticles are easy to modify, and various functional groups can be introduced, so that selective capture of specific proteins or biomarkers is realized.

Description

Technical Field

[0001] The invention relates to the technical field of magnetic nanoparticle preparation methods, and in particular to a triazolinone-modified magnetic nanoparticle preparation method and application. Background Art

[0002] Solid phase extraction (SPE) technology facilitates the separation and concentration of analytes from liquid samples, or the cleaning of previously processed extracts. Compared with common methods such as isoelectric precipitation, organic solvent precipitation, dialysis, ultrafiltration, chromatography, electrophoresis, and crystallization, SPE has the advantages of less solvent use, simple operation, high selectivity, and less interference. This can save pretreatment time, improve efficiency, and prompt people to explore new adsorbents, using specific interactions with proteins to overcome the main shortcomings of traditional protein sample pretreatment. Separation and purification technology can significantly improve the purity of target substances and reduce impurity content. This is crucial for experiments, production or analysis that require high-purity raw materials, such as drug synthesis, enzyme-catalyzed reactions, and biosensor preparation. After obtaining a single substance through separation and purification, its physical properties (such as melting point, boiling point, solubility, etc.) and chemical properties (such as reactivity, stability, etc.) can be studied more accurately, and then the relationship between its structure and function can be deeply understood. The field of protein separation and purification is to design functional solid phase materials with special molecular modifications that can interact with protein domains while retaining the structure and activity of proteins. Isolating and purifying specific proteins lays the foundation for analyzing life processes, revealing disease mechanisms, and developing treatment strategies, and is crucial for subsequent detailed biochemical and structural studies.

[0003] Magnetic solid phase extraction (MSPE) aims to achieve a simple, low-cost and rapid SPE process. Magnetic nanoparticles (MNPs) are a new type of organic-inorganic functional material composed of magnetic materials and polymers. Because of their excellent superparamagnetism and unique physical and chemical properties, including good biocompatibility, high dispersion stability, high specific surface area, and the ability to manipulate external magnetic fields. MNPs have improved the ability to capture target compounds from complex matrices.

[0004] In the field of protein analysis, solid phase extraction technology can not only effectively separate and enrich target proteins, but also improve separation purity and detection sensitivity by selecting appropriate fillers. Although traditional solid phase extraction fillers such as silica gel, activated carbon, and gel can meet the analysis needs to a certain extent, their selectivity, reusability and stability still need to be improved. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing magnetic nanoparticles modified with triazolinone and its application, so as to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing magnetic nanoparticles modified with triazolinone, comprising the following specific steps:

[0007] Step 1, dissolving 3-chloromethyl-1,2,4-triazolin-5-one in DMF; then adding Fe3O4-NH2 and potassium carbonate to obtain a mixture;

[0008] Step 2, condensing and refluxing the mixture, and then cooling it naturally to obtain a product;

[0009] Step 3, washing the product with DMF water until the pH of the washing solution is neutral; then drying;

[0010] Step 4: Obtain CMTO@Fe3O4-NH2 magnetic nanoparticles.

[0011] According to the above technical scheme, in step 1, 3-chloromethyl-1,2,4-triazolin-5-one is 0.24 g, DMF is 50 mL, Fe3O4-NH2 is 10 mg, and potassium carbonate is 0.345 g.

[0012] According to the above technical scheme, the flask in step 2 is a 100 mL round-bottom flask; the temperature in the oil bath is 150° C., and the condensation reflux time is 10 h; it is naturally cooled to room temperature; the number of DMF washings in step 3 is 3 times; the drying temperature and time are 60° C. and 12 h, respectively.

[0013] According to the above technical solution, the preparation method of Fe3O4-NH2 includes

[0014] 1) firstly dissolving a mixture of FeCl3·6H2O and sodium acetate in ethylene glycol, and then dropwise adding hyperbranched polyethyleneimine with continuous stirring to form a yellow colloidal mixture;

[0015] 2) transferring the yellow colloidal mixture into an autoclave with a capacity of 400 g, placing the autoclave in an oven for reaction, and then cooling;

[0016] 3) Separating the final product obtained in 2) by magnetic separation, washing the magnetic solid product with anhydrous ethanol, then with distilled water, and finally drying to obtain Fe3O4-NH2.

[0017] According to the above technical scheme, in 1), FeCl3·6H2O is 0.135 g, sodium acetate is 1.8 g, ethylene glycol is 20 mL, and hyperbranched polyethyleneimine is 0.5 g, and continuous stirring is carried out at room temperature for 45 minutes.

[0018] According to the above technical scheme, 18.0 mL of the yellow colloidal mixture in 2) was transferred to a 50 mL polytetrafluoroethylene sealed stainless steel autoclave; the autoclave was reacted in an oven for 12 hours; the oven temperature was set to a constant temperature of 200° C.; and cooled to room temperature.

[0019] According to the above technical solution, in 3), the washing with anhydrous ethanol and the washing with distilled water are both performed twice; the drying temperature and time are 60° C. and 12 h respectively.

[0020] Application of CMTO@Fe3O4-NH2 magnetic nanoparticles in the separation and purification of hemoglobin.

[0021] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0022] CMTO@Fe3O4-NH2 magnetic nanoparticle capture materials have attracted much attention due to their unique physical and chemical properties and broad application prospects. CMTO@Fe3O4-NH2 magnetic nanoparticles have a large specific surface area and high adsorption capacity, can quickly adsorb target molecules, and achieve rapid separation through an external magnetic field. In addition, the surface of CMTO@Fe3O4-NH2 magnetic nanoparticles is easy to modify, and various functional groups can be introduced to achieve selective capture of specific proteins or biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0024] Figure 1 It is the scanning electron microscope image and the transmission electron microscope image in the present invention;

[0025] Figure 2 The Fourier infrared spectrum, Raman analysis result, EDS analysis result and VSM analysis result in the present invention;

[0026] Figure 3 It is the effect diagram of pH value and ionic strength on protein adsorption efficiency in the present invention;

[0027] Figure 4 It is the effect diagram of adsorption time and adsorption temperature on adsorption efficiency in the present invention;

[0028] Figure 5 It is a graph showing the reproducibility of the recovery and adsorption performance of Hb adsorbed on CMTO@Fe3O4-NH2 by different types of eluents in the present invention;

[0029] Figure 6 It is the SDS-PAGE result diagram in the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] See also Figure 1-5 The present invention provides a technical solution: a method for preparing magnetic nanoparticles modified with triazolinone, comprising the following specific steps:

[0032] Synthesis of Magnetic Nanoparticles Fe3O4-NH2

[0033] In this experiment, amino-coated magnetic nanoparticles (Fe3O4-NH2) were prepared by solvothermal synthesis using polyethyleneimine (PEI) as a protective agent.

[0034] 1) A mixture of 0.135 g of FeCl3·6H2O and 1.8 g of sodium acetate was dissolved in 20 mL of ethylene glycol. 0.5 g of hyperbranched polyethyleneimine (PEI, 25 kDa) was added dropwise and stirred continuously at room temperature for 45 minutes to form a yellow colloidal mixture.

[0035] 2) Transfer 18.0 mL of the mixture into a 50 mL polytetrafluoroethylene-sealed stainless steel autoclave. Set the constant temperature oven to 200°C, place the autoclave in the oven and react for 12 hours. Then cool to room temperature.

[0036] 3) The obtained product was separated by magnetic separation, washed twice with anhydrous ethanol and twice with distilled water, and dried at 60°C for 12 h to obtain Fe3O4-NH2.

[0037] Synthesis of triazolinone-modified CMTO@Fe3O4-NH2

[0038] Using the method of organic synthesis, step 4, dissolve 0.24g 3-chloromethyl-1,2,4-triazolin-5-one in 50mL DMF. Add 10mg Fe3O4-NH2 and 0.345g potassium carbonate to obtain a mixture. Step 5, transfer the mixture to a 100mL round-bottom flask, add a stirrer, condense and reflux at 150°C in an oil bath for 10h, transfer the mixture to a condensation reflux device consisting of a round-bottom flask, a spherical condenser and an oil bath, add a stirrer, condense and reflux in an oil bath, wherein the stirrer rotates automatically when the oil bath is heated, and cool naturally to room temperature. The obtained product is washed three times with DMF and three times with water until the pH of the washing liquid is neutral. Dry at 60°C for 12h to obtain CMTO@Fe3O4-NH2.

[0039] Synthesis of CMTO@Fe3O4-NH2:

[0040] Experimental materials:

[0041] Hemoglobin (Hb), serum albumin; cytochrome C (Cyt-C); transferrin (Trf); protein molecular weight standards (myosin, Mr 200.0 kDa; β-galactosidase, Mr 116.0 kDa; phosphatase b, Mr 97.2 kDa; bovine serum albumin, Mr 66.4 kDa; ovalbumin, Mr 44.3 kDa; carbonyl glycosides, Mr 29.0 kDa; trypsin inhibitor, Mr 20.1 kDa; lysozyme, Mr 14.3 kDa ; aprotinin, Mr 6.5 kDa); polyethyleneimine (PEI); 3-chloromethyl-1,2,4-triazolin-5-one (CMTO); sodium acetate, ethylene glycol, anhydrous ethanol, methanol, imidazole, N,N-dimethylformamide, tris(hydroxymethyl)aminomethane (Tris), sodium dodecyl sulfate (SDS), sodium chloride, boric acid, phosphoric acid, glacial acetic acid, sodium hydroxide, ammonium persulfate, Coomassie Brilliant Blue G250, Coomassie Brilliant Blue R250, glycine, methylenebisacrylamide, ammonium persulfate. All reagents were analytically pure unless otherwise stated, and no pretreatment was performed before use. The experimental water was secondary deionized water (ddH2O, 18 MΩcm).

[0042] Example 1 Preparation of CMTO@Fe3O4-NH2:

[0043] Using the method of organic synthesis, 0.24g 3-chloromethyl-1,2,4-triazolin-5-one was dissolved in 50mL DMF. 10mg Fe3O4-NH2 and 0.345g potassium carbonate were added. The mixture was transferred to a 100mL round-bottom flask, a stirrer was added, and the mixture was condensed and refluxed at 150°C in an oil bath for 10h, cooled naturally to room temperature, and the obtained product was washed three times with DMF and three times with water until the pH of the washing liquid was neutral. CMTO@Fe3O4-NH2 was obtained by drying at 60°C for 12h.

[0044] result:

[0045] Scanning electron microscopy (SEM), transmission electron microscopy (TEM), infrared spectroscopy (FT-IR), Raman spectroscopy (Raman), EDS energy spectrum, VSM magnetic performance tests of CMTO@Fe3O4-NH2 Figure 1 , 2 As shown, Figure 1 Scanning electron micrographs of Fe3O4-NH2 (A) and CMTO@Fe3O4-NH2 (B); Transmission electron micrographs of Fe3O4-NH2 (C) and CMTO@Fe3O4-NH2 (D). Figure 2 Fourier transform infrared spectra (A) and Raman analysis results (B) of Fe3O4-NH2 and CMTO@Fe3O4-NH2; EDS analysis results of CMTO@Fe3O4-NH2 (C); VSM analysis results of Fe3O4-NH2 and CMTO@Fe3O4-NH2 (D), which are relatively evenly dispersed nanoparticles of about 250nm, forming spherical particles with larger particle size and smoother surface, and proving that 3-chloromethyl-1,2,4-triazolin-5-one is successfully bound to the surface of magnetic nanomaterials.

[0046] Application of CMTO@Fe3O4-NH2 magnetic nanoparticles in separation and purification of hemoglobin;

[0047] Example 2 Protein adsorption and elution:

[0048] Experimental steps:

[0049] In order to investigate the adsorption performance of CMTO@Fe3O4-NH2 magnetic nanoparticles on proteins, hemoglobin (Hb), serum albumin (HSA), transferrin (Trf) and cytochrome C (Cyt-C) were selected as model proteins. 1 mg CMTO@Fe3O4-NH2 was added to a 1 mL centrifuge tube and mixed with 500 μL 100 μg mL -1The protein solution was mixed and shaken for 45 minutes. The CMTO@Fe3O4-NH2 after protein adsorption was separated from the solution by magnetic separation. 300 μL of the supernatant was collected for hemoglobin, and the residual protein concentration in the solution was quantified by the absorption peak intensity of the dyed protein solution at 405 nm. 60 μL of the supernatant was collected for other proteins, and 300 μL of Coomassie Brilliant Blue G250 solution was added. The residual protein concentration in the solution was quantified by the absorption peak intensity of the dyed protein solution at 595 nm. The protein concentration before and after adsorption was calculated using the standard working curve. The adsorption efficiency of protein in the experiment was calculated by formula (1):

[0050]

[0051] Where: E1 is the adsorption efficiency of protein (%); C0 is the original concentration of protein prepared in the experiment (μgmL -1 ); C1 is the concentration of protein in the solution after adsorption (μgmL -1 ).

[0052] Collect the CMTO@Fe3O4-NH2 after magnetic separation and add 500μL 200μgmL -1 The imidazole solution was used as the eluent and the mixture was shaken for 15 minutes to elute the hemoglobin adsorbed on the surface of CMTO@Fe3O4-NH2. The absorption peak intensity of the supernatant was measured at 405 nm by magnetic separation. The elution efficiency was calculated based on the protein content in the eluent and the amount of protein adsorbed by the adsorbent. The protein elution rate in the experiment can be calculated by formula (2).

[0053]

[0054] Where E2 is the elution rate of the protein, C0 is the original concentration of the protein in the experiment, C1 is the concentration of white in the solution after adsorption, and C2 is the concentration of protein in the solution after elution.

[0055] Separation and purification of actual sample hemoglobin (Hb):

[0056] Take a certain volume of whole blood sample after red blood cell disruption and dilute it 1000 times with BR solution of pH=7. Take 1mgCMTO@Fe3O4-NH2, add 500μL diluted whole blood sample, shake for 45 minutes, and remove the supernatant. Add 500μL secondary water, shake for 15min, and then magnetically separate, remove the supernatant, and then add 500μL 200μgmL -1 The purified hemoglobin was eluted with imidazole solution for 30 minutes.

[0057] result:

[0058] Effects of pH and ionic strength on protein adsorption efficiency Figure 3 , The effect of pH value on the adsorption efficiency of Hb, HSA, Trf, and Cyt-C (A). Protein solution: 100 μg mL -1 ,500μL; CMTO@Fe3O4-NH2: 1mg (A); Effect of Nacl concentration on the adsorption efficiency of Hb and HSA under pH7.0 conditions (B), Effect of adsorption time and adsorption temperature on the adsorption efficiency Figure 4 , the figure shows the effect of adsorption temperature on the adsorption of Hb by CMTO@Fe3O4-NH2 (A); the effect of adsorption time on the adsorption of Hb by CMTO@Fe3O4-NH2 at 20°C (B); the adsorption isotherm of Hb adsorption by CMTO@Fe3O4-NH2 (C) the relationship between 1 / qeq and 1 / Ceq (D), protein solution: 100-900μgmL -1 ,0.1mL; CMTO@Fe3O4-NH2: 1mg; adsorption time: 45min, by Figure 4 It can be seen that with the increase of hemoglobin concentration, the unit adsorption amount of CMTO@Fe3O4-NH2 on Hb gradually increases, indicating that the adsorption behavior of CMTO@Fe3O4-NH2 on Hb has a good correlation with the Langmuir isotherm. Based on this, it can be judged that the adsorption of hemoglobin by the material is a monolayer adsorption. The recovery of hemoglobin adsorbed on CMTO@Fe3O4-NH2 by different types of eluents can be seen. Figure 5 , hemoglobin solution: 100 μg mL -1 , 500μL, pH7.0; CMTO@Fe3O4-NH2: 1.0mg; adsorption time: 45min; eluent volume: 500μL. SDS-PAGE analysis results are shown in Figure 6 Lane 1: protein extract of hemoglobin in blood; Lane 2: supernatant after CMTO@Fe3O4-NH2 adsorption; Lane 3: supernatant after secondary water pre-washing; Lane 4: supernatant after elution with 0.2molL-1 imidazole solution; Lane 5: 100μgmL -1 In the hemoglobin solution, the protein band appeared at 10.0 kDa, which was consistent with the band of hemoglobin, indicating that hemoglobin was separated and purified in the blood sample using CMTO@Fe3O4-NH2 as an adsorbent.

[0059] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing triazolinone-modified magnetic nanoparticles, characterized in that: The specific steps include: Step 1, dissolving 3-chloromethyl-1,2,4-triazolin-5-one in DMF; then adding Fe3O4-NH2 and potassium carbonate to obtain a mixture; Step 2, condensing and refluxing the mixture, and then cooling it naturally to obtain a product; Step 3, washing the product with DMF water until the pH of the washing solution is neutral; then drying; Step 4: Obtain CMTO@Fe3O4-NH2 magnetic nanoparticles.

2. The method for preparing triazolinone-modified magnetic nanoparticles according to claim 1, characterized in that: In step 1, 3-chloromethyl-1,2,4-triazolin-5-one is 0.24 g, DMF is 50 mL, Fe3O4-NH2 is 10 mg, and potassium carbonate is 0.345 g.

3. The method for preparing triazolinone-modified magnetic nanoparticles according to claim 2, characterized in that: In step 2, the flask is a 100 mL round-bottom flask; the temperature in the oil bath is 150° C., and the condensation reflux time is 10 h; the mixture is naturally cooled to room temperature; in step 3, the DMF washing is performed 3 times; the drying temperature and time are 60° C. and 12 h, respectively.

4. The method for preparing triazolinone-modified magnetic nanoparticles according to claim 1, characterized in that: The preparation method of Fe3O4-NH2 comprises 1) firstly dissolving a mixture of FeCl3·6H2O and sodium acetate in ethylene glycol, and then dropwise adding hyperbranched polyethyleneimine with continuous stirring to form a yellow colloidal mixture; 2) transferring the yellow colloidal mixture into an autoclave with a capacity of 1000 g, placing the autoclave in an oven for reaction, and then cooling; 3) Separating the final product obtained in 2) by magnetic separation, washing the magnetic solid product with anhydrous ethanol, then with distilled water, and finally drying to obtain Fe3O4-NH2.

5. The method for preparing triazolinone-modified magnetic nanoparticles according to claim 4, characterized in that: 1) The amount of FeCl3·6H2O is 0.135 g, the amount of sodium acetate is 1.8 g, the amount of ethylene glycol is 20 mL, and the amount of hyperbranched polyethyleneimine is 0.5 g. The mixture is stirred continuously at room temperature for 45 minutes.

6. The method for preparing triazolinone-modified magnetic nanoparticles according to claim 5, characterized in that: 2) 18.0 mL of the yellow colloidal mixture was transferred to a 50 mL polytetrafluoroethylene-sealed stainless steel autoclave; the autoclave was reacted in an oven for 12 hours; the oven temperature was set to a constant temperature of 200° C.; and cooled to room temperature.

7. The method for preparing triazolinone-modified magnetic nanoparticles according to claim 6, characterized in that: 3) Washing with anhydrous ethanol and distilled water were performed twice; the drying temperature and time were 60°C and 12h respectively.

8. Use of the CMTO@Fe3O4-NH2 magnetic nanoparticles as claimed in any one of claims 1 to 7 in the separation and purification of hemoglobin.

Images