Preparation method of magnetic particles, preparation method of gold magnetic particles and application of gold magnetic particles

Abstract

The invention provides a preparation method of magnetic particles, a preparation method of gold magnetic particles and application of the gold magnetic particles, and belongs to the field of adsorption. The invention provides a preparation method of magnetic particles, which comprises the following steps: trivalent iron salt, a reducing agent, an anionic surfactant and a polyol solvent are mixed for solvothermal reaction to generate ferroferric oxide with anions adsorbed on the surface, and the ferroferric oxide with the anions adsorbed on the surface is the magnetic particles. According to the invention, negative ions adsorbed on the surface bring electronegativity to ferroferric oxide, so that certain charge shielding exists among magnetic particles, and therefore, effective dispersion can be carried out, and agglomeration is reduced.

Description

Technical Field

[0001] The present invention relates to the field of adsorption, and in particular to a method for preparing magnetic particles, a method for preparing gold magnetic particles and applications thereof. Background Art

[0002] Immunoassay is a method of quantitatively measuring the analyte through specific reactions between biological molecules, such as the reaction between antigens and antibodies, and using the signals of markers such as colloidal gold, fluorescence and radioactive isotopes. Colloidal gold is the most widely used tracer and can be observed as dark brown particles under a microscope. When colloidal gold aggregates in large quantities at the corresponding ligands, it forms red or pink spots visible to the naked eye. In addition, colloidal gold has a large surface plasmon resonance absorption and a large molar extinction coefficient, which facilitates quantitative optical detection.

[0003] Gold-modified nanomagnetic binary composite particles (gold magnetic particles) are a new type of nanomaterial. The structure of gold magnetic particles includes two parts: a core and a surface modification. Usually, magnetic particles are used as the core, and a gold shell or gold particles are used as the modification layer. Therefore, gold magnetic particles contain the dual characteristics and advantages of colloidal gold and magnetic particles. On the one hand, they have the optical and physicochemical properties of gold colloids and can interact with thiol groups. The surface of gold magnetic particles can easily assemble various thiol-derived biological probe molecules such as antibodies and nucleic acids. This non-covalent coupling method maintains the conformation and biological activity of the probe molecules to the greatest extent, which is convenient for optical detection. In addition, gold magnetic particles can control the directional movement of particles through magnetic response, achieve simple and rapid separation, and enrich the captured target molecules, reducing the time required for operation.

[0004] The co-precipitation method is a commonly used method for preparing magnetic particles. Although the process is simple, the prepared magnetic particles are not uniform in size and are prone to agglomeration. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing magnetic particles, a method for preparing gold magnetic particles and applications thereof. The magnetic particles prepared by the present invention have good dispersibility.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing magnetic particles, comprising the following steps:

[0008] The trivalent iron salt, the reducing agent, the anionic surfactant and the polyol solvent are mixed to carry out a solvothermal reaction to generate ferroferric oxide with anions adsorbed on the surface. The ferroferric oxide with anions adsorbed on the surface is the magnetic particle.

[0009] Preferably, the ferric iron salt includes one or more of ferric chloride, ferric sulfate, ferric nitrate and ferric acetate.

[0010] Preferably, the reducing agent includes one or more of sodium acetate, urea, sodium borohydride and oxalic acid.

[0011] Preferably, the ratio of the amount of the reducing agent to the amount of the iron ions in the ferric iron salt is 1:0.05-0.25.

[0012] Preferably, the anionic surfactant includes one or more of citrate, polyacrylamide and oleate.

[0013] Preferably, the ratio of the amount of iron ions in the ferric iron salt to the amount of anionic surfactant is 1:0.2-0.8.

[0014] Preferably, the temperature of the solvent thermal reaction is 220-300° C. and the time is 8-12 hours.

[0015] The present invention also provides a method for preparing gold magnetic particles, comprising the following steps:

[0016] The dispersion of magnetic particles prepared by the preparation method described in the above technical solution is mixed with a polymer solution for electrostatic adsorption to obtain positively charged magnetic particles; the polymer is positively charged and contains reducing groups;

[0017] The positively charged magnetic particles, water and a soluble gold salt solution are mixed to carry out a reduction reaction to obtain the gold magnetic particles.

[0018] Preferably, the reducing group includes one or more of an imine group, a quaternary ammonium group, an imidazole group and a pyridyl group.

[0019] The present invention also provides the use of gold magnetic particles prepared by the preparation method described in the above technical scheme in the adsorption and separation of protein ligands.

[0020] The present invention provides a method for preparing magnetic particles, comprising the following steps:

[0021] The trivalent iron salt, the reducing agent, the anionic surfactant and the polyol solvent are mixed to carry out a solvothermal reaction to generate ferroferric oxide with anions adsorbed on the surface, and the ferroferric oxide with anions adsorbed on the surface is the magnetic particle.

[0022] In the present invention, the anions adsorbed on the surface bring negative charge to the ferroferric oxide, so that there is a certain charge shielding between the magnetic particles, thereby enabling effective dispersion and reducing agglomeration.

[0023] In the solvent thermal reaction, the trivalent iron ions are partially reduced to divalent iron ions under the action of the reducing agent, and precipitated as corresponding hydroxides under weak alkaline conditions formed by the ionization of the anionic surfactant, and condensed into ferroferric oxide particles at high temperature. When the concentration of the ferroferric oxide particles exceeds the saturation concentration, the crystal nuclei of ferroferric oxide begin to precipitate and eventually grow into crystals. The present invention can control the number of crystal nuclei in the solution and ultimately adjust the size of the magnetic particles by adjusting the amount of the anionic surfactant.

[0024] In the prior art, gold magnetic particles are often prepared by co-adsorbing magnetic particles with colloidal gold particles, the main disadvantage of which is that the degree of adsorption is not uniform and the morphology of the original magnetic particles is destroyed. However, based on the negatively charged magnetic particles, the present invention adsorbs a polymer with a reducing group and a positive charge on the surface of the magnetic particles through electrostatic adsorption, and uses the reducing group of the polymer to perform in-situ reduction of gold ions, thereby realizing the in-situ preparation of colloidal gold, improving the dispersion of colloidal gold, and not destroying the morphology of the original magnetic particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the result graph of the adsorption of SH-RGD by gold magnetic particles. DETAILED DESCRIPTION

[0026] The present invention provides a method for preparing magnetic particles, comprising the following steps:

[0027] The trivalent iron salt, the reducing agent, the anionic surfactant and the polyol solvent are mixed to carry out a solvothermal reaction to generate ferroferric oxide with anions adsorbed on the surface, and the ferroferric oxide with anions adsorbed on the surface is the magnetic particle.

[0028] In the present invention, the ratio of the amount of the reducing agent to the amount of the iron ion in the ferric iron salt is preferably 1:0.05-0.25, more preferably 1:0.1-0.2, and further preferably 1:0.12-0.15; the ferric iron salt preferably includes one or more of ferric chloride, ferric sulfate, ferric nitrate and ferric acetate; the reducing agent includes one or more of sodium acetate, urea, sodium borohydride and oxalic acid. Sodium acetate can also be a complexing agent.

[0029] In the present invention, the ratio of the amount of iron ions in the ferric iron salt to the amount of anionic surfactant is preferably 1:0.2-0.8, more preferably 1:0.3-0.6, and further preferably 1:0.4-0.5; the anionic surfactant preferably includes one or more of citrate, polyacrylamide and oleate; the citrate preferably includes sodium citrate; the oleate preferably includes sodium oleate.

[0030] In the present invention, the mass ratio of the ferric iron salt to the polyol solvent is preferably 1:10-30, more preferably 1:15-25, and further preferably 1:18-20; the polyol solvent preferably includes ethylene glycol and / or glycerol. Ethylene glycol can also be used as a reducing agent to reduce the ferric iron salt.

[0031] In the present invention, the temperature of the solvothermal reaction is preferably 220 to 300° C., more preferably 250 to 280° C., and the time is preferably 8 to 12 hours, more preferably 9 to 10 hours.

[0032] The present invention also provides a method for preparing gold magnetic particles, comprising the following steps:

[0033] The dispersion of magnetic particles prepared by the preparation method described in the above technical solution is mixed with a polymer solution for electrostatic adsorption to obtain positively charged magnetic particles; the polymer is positively charged and contains reducing groups;

[0034] The positively charged magnetic particles are mixed with a soluble gold salt solution to perform a reduction reaction to obtain the gold magnetic particles.

[0035] The present invention mixes the dispersion of the magnetic particles described in the above technical solution with a polymer solution for electrostatic adsorption to obtain positively charged magnetic particles.

[0036] In the present invention, the mass ratio of the magnetic particles to the polymer is preferably 1:0.3-0.5, more preferably 1:0.4-0.45; the mass concentration of the magnetic particles in the dispersion of the magnetic particles is preferably 0.05%; the mass content of the polymer in the polymer solution is preferably 5%; the volume ratio of the dispersion of the magnetic particles to the polymer solution is preferably 1.2:1; the polymer is positively charged and contains reducing groups; the reducing groups preferably include one or more of imine groups, quaternary ammonium groups, imidazole groups and pyridine groups; the polymer preferably includes one or more of polyethylene imine, polybetaine, chitosan and polylysine.

[0037] After electrostatic adsorption, the present invention preferably performs magnetic separation and washing on the obtained product to remove free polymers, thereby obtaining the positively charged magnetic particles.

[0038] After obtaining the positively charged magnetic particles, the present invention mixes the positively charged magnetic particles, water and a soluble gold salt solution to perform a reduction reaction to obtain the gold magnetic particles.

[0039] In the present invention, the mixing preferably includes dispersing the magnetic particles in water and then mixing with a soluble gold salt solution.

[0040] In the present invention, the method for preparing the soluble gold salt solution preferably comprises the following steps:

[0041] The soluble gold salt, the pH adjuster and water are mixed and dissolved to obtain the soluble gold salt solution.

[0042] In the present invention, the soluble gold salt preferably includes one or more of chloroauric acid, bromoauric acid and cyanauric acid; the pH regulator preferably includes Na 2 CO 3 ; The pH value of the soluble gold salt solution is preferably 8-10.

[0043] In the present invention, the reduction reaction is preferably carried out in a boiling state of the mixed solution, the temperature of the reduction reaction is preferably 80-100°C, more preferably 85-90°C; the time is preferably 0.5-3h, more preferably 1-2.5h, and further preferably 1.5-2h. The magnetic particles with reducing groups have reducing properties, and gold ions are reduced on the surface of the magnetic particles to generate single gold particles, which aggregate into nano-sized gold particles in the solution.

[0044] After the reduction reaction, the present invention preferably performs magnetic separation on the obtained reduction reaction product, and then washes and dries the obtained solid to obtain the gold magnetic particles.

[0045] In the present invention, the mass ratio of the positively charged magnetic particles to gold in the gold magnetic particles is preferably 1:0.01-0.2.

[0046] The present invention also provides the use of gold magnetic particles prepared by the preparation method described in the above technical scheme in the adsorption and separation of protein ligands.

[0047] The preparation method of the magnetic particles, the preparation method of the gold magnetic particles and their applications provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] Preparation of magnetic particles by solvothermal method 1

[0050] Weigh 1.35 g of ferric chloride hexahydrate and dissolve it in 20 mL of ethylene glycol. After stirring to dissolve, add 2.7 g of anhydrous sodium acetate and 1.0 g of sodium citrate to the reaction system. After magnetic stirring to dissolve, transfer it into a reactor and heat it to 250 ° C for 8 hours. After cooling to room temperature, transfer the reaction product out of the reactor, wash it repeatedly with distilled water and ethanol, and then vacuum dry it.

[0051] The generated particles are spherical and have good dispersibility. The particle size is about 200nm, and the edge of each particle is clear and complete spherical without agglomeration.

[0052] Example 2

[0053] Preparation of magnetic particles by solvothermal method 2

[0054] Weigh 1.35 g of ferric chloride hexahydrate and dissolve it in 20 mL of ethylene glycol. After stirring to dissolve, add 2.7 g of anhydrous sodium acetate and 0.75 g of sodium citrate to the reaction system. After magnetic stirring to dissolve, transfer it into a reactor and heat it to 250 ° C for 8 hours. After cooling to room temperature, transfer the reaction product out of the reactor, wash it repeatedly with distilled water and ethanol, and then vacuum dry it.

[0055] The generated particles are spherical and have good dispersibility. The particle size is about 400nm, and the edge of each particle is clear and complete spherical without agglomeration.

[0056] Example 3

[0057] Preparation of magnetic particles by solvothermal method 3

[0058] Weigh 1.35 g of ferric chloride hexahydrate and dissolve it in 20 mL of ethylene glycol. After stirring to dissolve, add 2.7 g of anhydrous sodium acetate and 0.5 g of sodium citrate to the reaction system. After magnetic stirring to dissolve, transfer it into a reactor and heat it to 250 ° C for 8 hours. After cooling to room temperature, transfer the reaction product out of the reactor, wash it repeatedly with distilled water and ethanol, and then vacuum dry it.

[0059] The generated particles are spherical and have good dispersibility. The particle size is about 800nm, and the edge of each particle is clear and complete spherical without agglomeration.

[0060] Application Example 1

[0061] Magnetic particle surface positive charge modification

[0062] 6 mL of a dispersion of citric acid-modified magnetic particles (Example 1) with a mass concentration of 0.05% was added to a 25 mL glass bottle, and 5 mL of a polyethyleneimine solution (with a PEI content of 5 wt%) was added thereto, and the mixture was stirred for reaction.

[0063] The free PEI was removed by magnetic separation and washing, and the PEI-modified magnetic particles were collected and redispersed in 6 mL of deionized water to obtain a 0.05% MP-PEI dispersion. When these negatively charged magnetic particles were mixed with the PEI solution, the positively charged molecular chains of PEI were automatically attached to the particle surface through electrostatic adsorption, and the surface was transformed from negatively charged to strongly positively charged.

[0064] Colloidal gold modification of positively charged magnetic particles

[0065] 1 mL of 1 wt% chloroauric acid (AuCl 4 ·3H 2 O) solution, 1.5 mL 0.2 M Na 2 CO 3The solution and 87.5 mL of deionized water were added to a three-necked flask in sequence to obtain a soluble gold salt solution with a pH value of 10. The obtained soluble gold salt solution was then heated to boiling. 10 mL of MP-PEI dispersion with a mass concentration of 0.05% was quickly added to the flask and kept boiling for 30 minutes. After the reaction was completed, the particles were retained by magnetic separation, the product was washed with deionized water 3 times, and finally dispersed in 10 mL of water to obtain gold magnetic particles (the mass ratio of positively charged magnetic particles to gold was 1:0.2).

[0066] Immobilization of thiol compounds by gold-modified magnetic particles

[0067] The SH-RGD polypeptide (molecular weight 10K) containing a thiol group was dissolved in 10 mL of 0.1 mol / L NaOH solution, and the resulting solutions were fixed to 10 mL, 20 mL, 50 mL and 100 mL, respectively. The polypeptide solution had a maximum absorption peak at 327 nm, and a concentration-absorbance standard curve of SH-RGD was drawn.

[0068] Add 0.2 mg to 1.6 mg of the above-mentioned gold magnetic particles into 2 mL of SH-RGD solution containing 25 ug SH-RGD polypeptide, shake in the dark at room temperature for 4 hours, and separate the adsorbed gold magnetic particles under a magnetic field to obtain gold magnetic particle-SH-RGD polypeptide.

[0069] The concentration of SH-RGD in the supernatant and the original solution was detected, and the adsorption amount of gold magnetic particles was calculated by subtraction method. The results are as follows: Figure 1 shown.

[0070] Depend on Figure 1 It can be seen that the adsorption of gold magnetic particles to SH-RGD is about 5ug / mg. Thiol groups are widely present in the biological molecular structure in nature, which lays a good foundation for the application of this nanoparticle in the biological field.

[0071] Application of gold magnetic particles-SH-RGD peptide in SPR detection

[0072] 0.01g of the above-mentioned gold magnetic particle-SH-RGD polypeptide and 10mL of water were ultrasonically dispersed for 5 minutes using an ultrasonic disperser to obtain a dispersion with a mass concentration of 0.1%. Then, the gold magnetic particle-SH-RGD polypeptide in the dispersion was adsorbed to the bottom of the test tube using a magnetic stand, and the clean supernatant was absorbed; then 10mL of a mixed solution of 50mg / mL EDC (activator, the activated polypeptide can bind to the protein) and NHS (the mass ratio of EDC to NHS is 1:1, and the mass ratio of EDC to gold magnetic particle-SH-RGD polypeptide is 0.1:1) was added, and the reaction was oscillated for 10 minutes; the gold magnetic particle-SH-RGD polypeptide was adsorbed to the bottom of the test tube again using a magnetic stand, the supernatant was extracted, 10mL of 0.01M PBS buffer was added, and the mixture was resuspended. Then slowly drop 100uL of goat anti-rabbit antibody solution, with an antibody dosage of 0.10mg; stir for 1-2h at 2-8℃, add 10% BSA (final concentration of 0.5%) of the obtained solution mass for blocking (BSA quenches the unreacted EDC and NHS, that is, the BSA protein terminates the activation reaction of EDC and NHS) for 0.5h; adsorb the blocked gold magnetic particles-SH-RGD polypeptide-antibody to the bottom of the test tube, extract the supernatant, and then add the preservation solution, seal and store at 4℃ in the dark for later use.

[0073] Place the packaged SPR microfluidic chip into a spotter, dilute the gold magnetic particle-SH-RGD polypeptide-antibody to a working concentration (100 μg / mL); turn on the spotter, spot the gold magnetic particle-SH-RGD polypeptide-antibody solution into the reaction area of ​​the chip, and add a sample volume (5 μL); then use a freeze dryer to freeze-dry to obtain a gold magnetic particle antibody chip, and then cover the gold magnetic particle antibody chip with a surface coating for standby use.

[0074] Turn on the instrument, load the gold magnetic particle antibody chip and the conventional antibody chip respectively, and detect 100uL solution with concentrations of 0, 5, 50, and 500ng / kg according to the set program. Read the reaction signal values ​​of the gold magnetic particle antibody chip and the conventional antibody chip respectively. The results are shown in Table 1:

[0075] Table 1 Reaction signal values ​​of gold magnetic particle antibody chip and conventional antibody chip

[0076]

[0077] Gold magnetic particle-SH-RGD peptide can form local field resonance with the SPR chip to further enhance the signal value of SPR.

[0078] in conclusion:

[0079] The present invention uses a solvothermal method to synthesize nano-magnetic ferroferric oxide particles with uniform size and surface modification of citric acid, and the size of the magnetic particles can be adjusted by the amount of sodium citrate used. On this basis, positively charged polyethyleneimine (PEI)-coated nano-magnetic particles are prepared by electrostatic adsorption. Due to its reducing property, the PEI-coated magnetic particles can reduce chloroauric acid in situ to synthesize binary modified gold magnetic particle complexes. Nano-gold is distributed on the surface of the magnetic particles. Research and development has found that this material has a specific absorption capacity for thiol-containing polypeptide substances and can be used as a potential carrier material for biological detection. It can resonate with the SPR chip on the surface plasmon resonance detector to further enhance the signal and improve the sensitivity of detection.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing magnetic particles, characterized in that: The following steps are involved: The trivalent iron salt, the reducing agent, the anionic surfactant and the polyol solvent are mixed to carry out a solvothermal reaction to generate ferroferric oxide with anions adsorbed on the surface. The ferroferric oxide with anions adsorbed on the surface is the magnetic particle.

2. The preparation method according to claim 1, characterized in that: The ferric iron salt includes one or more of ferric chloride, ferric sulfate, ferric nitrate and ferric acetate.

3. The preparation method according to claim 1, characterized in that: The reducing agent includes one or more of sodium acetate, urea, sodium borohydride and oxalic acid.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The ratio of the amount of the reducing agent to the amount of the iron ions in the trivalent iron salt is 1:0.05-0.

25.

5. The preparation method according to claim 1, characterized in that: The anionic surfactant includes one or more of citrate, polyacrylamide and oleate.

6. The preparation method according to claim 1 or 5, characterized in that: The ratio of the amount of iron ions in the trivalent iron salt to the amount of anionic surfactant is 1:0.2-0.

8.

7. The preparation method according to claim 1, characterized in that: The temperature of the solvent thermal reaction is 220-300° C., and the time is 8-12 hours.

8. A method for preparing gold magnetic particles, characterized in that: The following steps are involved: The dispersion of magnetic particles prepared by the preparation method according to any one of claims 1 to 7 is mixed with a polymer solution for electrostatic adsorption to obtain positively charged magnetic particles; the polymer is positively charged and contains reducing groups; The positively charged magnetic particles, water and a soluble gold salt solution are mixed to carry out a reduction reaction to obtain the gold magnetic particles.

9. The preparation method according to claim 8, characterized in that: The reducing group includes one or more of an imine group, a quaternary ammonium group, an imidazole group and a pyridyl group.

10. Use of the gold magnetic particles prepared by the preparation method according to claim 8 or 9 in the adsorption and separation of protein ligands.

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