Highly dispersed magnetic gold nanoparticles, method for preparing the same and use thereof

Abstract

The application relates to the field of immunochromatography, in particular to a high-dispersibility magnetic nanogold particle and a preparation method and application thereof, the preparation method comprises the following steps: 1) preparing Fe3O4 magnetic microparticles by using a hydrothermal synthesis method; 2) preparing polystyrene magnetic particles with controllable color and surface amination by using a dispersion polymerization method; and 3) preparing magnetic nanogold particles by combining nanogold with the polystyrene magnetic particles with controllable color and surface amination through electrostatic adsorption by using a citric acid reduction method. The magnetic nanogold particle prepared by the application has a particle size range of 300-500 nm, controllable color, high sensitivity, good dispersibility and good stability, the color polystyrene shell layer is introduced to realize color regulation of the microsphere particles, the magnetic mechanical property, the optical property and the immunochromatography technology are combined, the qualitative and quantitative double-detection functions of the to-be-detected objects can be realized, and the different colors can realize the purpose of multi-color and multi-link detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunochromatography, and in particular to a kind of high dispersity magnetic gold nanoparticles and its preparation method and application. BACKGROUND

[0002] Colloidal gold technology is a kind of antigen-antibody immune reaction combined with colloidal gold labeling tracer technology, which is used for qualitative and quantitative detection of antigen and antibody content. Due to its rapid, simple, low cost, good stability and other advantages, colloidal gold detection has been widely used in clinical detection field. However, colloidal gold particles are small, generally tens of nanometers, which has problems of low sensitivity, large batch difference and high cost.

[0003] Color microspheres, as a hot raw material in immunochromatography technology, have advantages of high sensitivity, rich color, good stability, easy scale production and small batch difference compared with colloidal gold, which is favored by many researchers. However, both methods need repeated centrifugation process, especially colloidal gold is more dependent on centrifuge. At present, the application of magnetic gold nanoparticles in immunochromatography field has been reported. Gold magnetic particle immunochromatography technology combines magnetic properties, optical properties and immunochromatography technology, which can not only realize qualitative and semi-quantitative analysis of the detected substance, but also realize quantitative detection of the detected substance by using its magnetic properties, which is not possessed by colloidal gold and color microspheres.

[0004] CN106370843 A discloses a rapid determination method of clenbuterol based on gold magnetic immunochromatography. The core-shell nanocomposite material is prepared by reducing the magnetic material with inner layer of γ-Fe2O3 and outer layer of nano-Au, so that the particle size range is 20-80 nm, and then combined with antigen to realize quantitative detection of the analyte. However, the gold magnetic particles prepared by this method have small particle size and low sensitivity, and pure iron oxide magnetic nanoparticles are easily oxidized in air, resulting in aggregation and poor dispersity.

[0005] CN107309436 A discloses Fe3O4 / SiO2 / Au gold magnetic nanoparticles, its preparation method and application. Gold nanoparticles with uniform particle size distribution are prepared by sodium citrate reduction method, Fe3O4 nanoparticles coated with SiO2 on the outer layer are used as magnetic core, gold nanoparticles are assembled on the surface of Fe3O4@SiO2, and Fe3O4 / SiO2 / Au gold magnetic nanoparticles with consistent morphology, good dispersity and core-shell structure are formed. The method uses sol-gel method to coat the magnetic material with silica, which effectively prevents the magnetic material from being oxidized by contacting with air, improves the dispersity of the particles, and realizes quantitative analysis and detection of the detected substance.

[0006] However, the gold magnetic particles prepared by the above two methods have the following disadvantages: (1) the silica-coated magnetic particles have small particle size and low sensitivity; (2) the magnetic particles are easily oxidized, have poor stability, are prone to aggregation, and have poor dispersibility; (3) the gold magnetic particles have single color, and the detection kit has single color; and (4) the detection function is single, and in the field of immunochromatography, the qualitative analysis detection of the analyte is mainly performed. SUMMARY

[0007] In view of the above problems, the application provides a highly dispersible magnetic gold nanoparticle, a preparation method and application thereof.

[0008] To achieve the above object, the application provides the following technical scheme.

[0009] A preparation method of a highly dispersible magnetic gold nanoparticle comprises the following steps.

[0010] (1) Fe3O4 magnetic microparticles are prepared by a hydrothermal synthesis method.

[0011] (2) color-controllable and amino-functionalized polystyrene magnetic particles are prepared by a dispersion polymerization method.

[0012] (3) magnetic gold nanoparticles are prepared by electrostatic adsorption of citric acid reduction method.

[0013] In the step (1), Fe3O4 magnetic microparticles with uniform particle size are prepared by the hydrothermal synthesis method, FeCl3·6H2O and PEG 6000 are dispersed in ethylene glycol, after dissolution, anhydrous sodium acetate is added to the dispersion liquid, and the reaction is carried out overnight, and then the Fe3O4 magnetic microparticles are obtained by magnetic separation and washing, and the particle size is 100-200 nm. The mass ratio of FeCl3·6H2O, CH3COONa, PEG 6000 and ethylene glycol is 1:1-10:0.05-0.5:30-60.

[0014] The step (2) is specifically as follows: the magnetic microparticles and the oil-soluble dye prepared in the step (1) are dispersed in an ethanol aqueous solution by a dispersion polymerization method, oxygen is removed by nitrogen, then polystyrene magnetic particles with controllable color and surface amination are prepared by using PVP as a dispersant, styrene as a monomer, methacrylamide as a functional monomer, AIBN as an initiator, and reaction at 70-80 DEG C overnight, wherein the volume ratio of ethanol to water in the ethanol aqueous solution is 1:0-0.2; the mass ratio of Fe3O4 magnetic microparticles to styrene monomers is 0.08-0.2:1; the mass ratio of the dye to the styrene monomers is 0.1-0.5:1; the volume ratio of methacrylamide to the styrene monomers is 0.1-0.5:1; the mass ratio of the initiator to the styrene monomers is 0.01-0.03:1; and the mass ratio of the dispersant to the solvent is 0.008-0.03:1.

[0015] The step (3) is specifically as follows: the chloroauric acid is reduced by using a sodium citrate reduction method, and then combined with the polystyrene magnetic particles with controllable color and surface amination in the step (2), so as to prepare magnetic gold nanoparticles, wherein the particle size of the magnetic gold nanoparticles is 300-500 nm.

[0016] The high-dispersibility magnetic gold nanoparticles prepared by the method can be used for preparing a colorful multi-linked test kit and an immunochromatography.

[0017] The high-dispersibility magnetic gold nanoparticles prepared by the method are used for the immunochromatography, and the specific antibody labeling process is as follows:

[0018] (1) 20 muL of magnetic gold nanoparticles with a solid content of 5% are taken in a 2mL EP tube, 1mL of a labeling buffer is added, magnetic separation is performed, the supernatant is removed, 1mL of the labeling buffer is added, ultrasonic dispersion is performed, the above steps are repeated three times, and finally the magnetic gold nanoparticles are stored in 1mL of the labeling buffer;

[0019] (2) 0.5mg of an antibody is taken in the above 2mL EP tube, mixed quickly, and then incubated at room temperature for 2h;

[0020] (3) 100 muL of BSA blocking solution with a mass fraction of 10% is added to the above EP tube, mixed at room temperature, and then incubated for 30min;

[0021] (4) magnetic separation is performed, the supernatant is removed, 1mL of a storage solution is added for ultrasonic dispersion, the above steps are repeated three times, and finally the magnetic gold nanoparticles are stored in 1mL of the storage solution.

[0022] Compared with the prior art, the method has the following beneficial effects:

[0023] (1) The present application coats magnetic particles with color-controllable surface-aminoated polystyrene microspheres by dispersion polymerization, and then adsorbs nano-gold on the surface of the surface-aminoated magnetic particles by electrostatic adsorption, with a particle size range of 300-500 nm, which can effectively improve the detection sensitivity.

[0024] (2) The color-controllable color polystyrene layer can control the color of the particles, and can realize multi-color and multi-association detection, and has good stability and color controllability. The combination of magnetic properties, optical properties and immunochromatography technology can realize the qualitative and quantitative detection functions of the to-be-detected substances.

[0025] (3) The magnetic nano-gold particles can be used to prepare multi-association detection kits, such as respiratory tract triple detection, and different colors of the balls are used to combine different antibodies, and finally the color of the detection line is used to judge whether the virus is infected.

[0026] (4) The magnetic nano-gold particles can effectively prevent the oxidation and magnetic leakage of the magnetic core, and improve the dispersibility of the microspheres. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Particle size analysis diagram of the magnetic gold nano-particles prepared in Example 1.

[0028] Figure 2 The curve diagram of the magnetic signal intensity of the magnetic gold nano-particles combined with RSV antibodies for RSV virus antigen detection and the concentration of RSV virus antigen in Example 1.

[0029] Figure 3 The structure diagram of the multi-color and multi-association detection card in Example 5. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0031] Example 1

[0032] A preparation method of high-dispersibility magnetic nano-gold particles, comprising the following steps:

[0033] (1) Solvothermal preparation of Fe3O4 nano-magnetic core

[0034] FeCl3·6H2O 4.5g, PEG6000 400mg, dissolved in 200mL ethylene glycol, stirring to dissolve. Again weighed anhydrous sodium acetate 4.5g added to the above solution, stirring to completely dissolved; the dissolved uniform viscous liquid transfer to 500mL high temperature reactor, tighten the lid, placed in a 200℃ oven heating overnight; after the reactor temperature natural cooling, the black reaction liquid poured out, magnetic separation; first with anhydrous ethanol wash three times, then with pure water wash three times, save in pure water, determination of Fe3O4 content, nitrogen gas to save oxygen, avoid oxidation.

[0035] (2) Blue polymer shell coated by dispersion polymerization

[0036] The magnetic microspheres 0.5g and solvent blue 1g prepared in step (1) were poured into a reaction bottle, 10mL pure water and 90mL anhydrous ethanol were added, and ultrasonic dispersion was performed. Nitrogen was passed for 20min, 1g dispersant PVP was added and dissolved. 70mg initiator was dissolved in 5mL styrene and 2mL methyl acrylamide, and then added to the reaction bottle after the temperature reached 70℃, and reacted overnight. Anhydrous ethanol was washed for 3 times, pure water was washed for 3 times, and finally saved in 50mL pure water.

[0037] (3) Magnetic microspheres coated with nanometer gold

[0038] The magnetic microspheres coated with blue polymer shell prepared in step (3), 200mg dispersant PVP and 20mL 0.013mol / L sodium citrate were added to a reaction bottle, ultrasonic dispersion was performed, then the reaction bottle was added to a constant temperature water bath, and high speed stirring was performed at 80℃. After the temperature reached 80℃, 60mL of 1% chloroauric acid was added dropwise, and the reaction was terminated after 8h. Alcohol was washed for 3 times, water was washed for 3 times, and finally saved in pure water, the solid content was measured, and it was ready for use.

[0039] Example 2

[0040] A method for preparing a highly dispersed magnetic nanometer gold particle, comprising the following steps:

[0041] (1) Preparation of Fe3O4 nanometer magnetic core by solvothermal method

[0042] FeCl3·6H2O 4.5g, PEG6000 400mg, dissolved in 200mL ethylene glycol, stirring to dissolve. Again weighed anhydrous sodium acetate 4.5g added to the above solution, stirring to completely dissolved; the dissolved uniform viscous liquid transfer to 500mL high temperature reactor, tighten the lid, placed in a 200℃ oven heating overnight; after the reactor temperature natural cooling, the black reaction liquid poured out, magnetic separation; first with anhydrous ethanol wash three times, then with pure water wash three times, save in pure water, determination of Fe3O4 content, nitrogen gas to save oxygen, avoid oxidation.

[0043] (2) Coating red polymer shell by dispersion polymerization

[0044] The magnetic microparticles prepared in step (1) 0.5 g and solvent red 1 g were poured into a reaction bottle, 10 mL of pure water and 90 mL of anhydrous ethanol were added, and ultrasonic dispersion was performed. Nitrogen was passed for 20 min, 1 g of dispersant PVP was added and dissolved. 70 mg of initiator was dissolved in 5 mL of styrene and 2 mL of methyl methacrylamide, and after the temperature reached 70°C, it was added to the reaction bottle and reacted overnight. Anhydrous ethanol was washed for 3 times, pure water was washed for 3 times, and finally stored in 50 mL of pure water.

[0045] (3) Coating nanometer gold on the surface of magnetic microparticles

[0046] The magnetic microparticles coated with red polymer shell prepared in step (2), 200 mg of dispersant PVP and 20 mL of 0.013 mol / L sodium citrate were added to the reaction bottle, and ultrasonic dispersion was performed. After ultrasonic dispersion, the reaction bottle was placed in a constant temperature water bath, and high speed stirring was performed at 80°C. After the temperature reached 80°C, 60 mL of 1% mass fraction chloroauric acid was added dropwise, and the reaction was terminated after 8 h. Alcohol was washed for 3 times, water was washed for 3 times, and finally stored in pure water, and the solid content was measured.

[0047] Example 3

[0048] A preparation method of a highly dispersed magnetic nanometer gold particle, comprising the following steps:

[0049] (1) Preparation of Fe3O4 nanometer magnetic core by solvothermal method

[0050] FeCl3·6H2O 4.5 g, PEG6000 400 mg were weighed and dissolved in 200 mL of ethylene glycol, and stirred to dissolve. Anhydrous sodium acetate 4.5 g was weighed and added to the above solution, and stirred until completely dissolved; the uniformly dissolved viscous liquid was transferred to a 500 mL high temperature reaction kettle, the lid was tightly screwed, and it was placed in an oven at 200°C for heating overnight; after the reaction kettle temperature was naturally cooled, the black reaction liquid was poured out and magnetically separated; first washed with anhydrous ethanol for 3 times, then washed with pure water for 3 times, and stored in pure water, and the Fe3O4 content was measured.

[0051] (2) Coating black polymer shell by dispersion polymerization

[0052] The magnetic microparticles prepared in step (1) 0.5 g and black pigment (alcohol soluble) 1 g were poured into a reaction bottle, 10 mL of pure water and 90 mL of anhydrous ethanol were added, and ultrasonic dispersion was performed. Nitrogen was passed for 20 min, 1 g of dispersant PVP was added and dissolved. 70 mg of initiator was dissolved in 5 mL of styrene and 2 mL of methyl methacrylamide, and after the temperature reached 70°C, it was added to the reaction bottle and reacted overnight. Anhydrous ethanol was washed for 3 times, pure water was washed for 3 times, and finally stored in 50 mL of pure water.

[0053] (3) Magnetic micro-particle surface coated with nano-gold

[0054] The magnetic micro-particles coated with black polymer shell prepared in step (2), 200 mg dispersing agent PVP and 20 mL 0.013 mol / L sodium citrate were added into a reaction bottle, and after ultrasonic dispersion, the reaction bottle was added into a constant temperature water bath, and stirred at high speed at 80°C. After the temperature reached 80°C, 60 mL 1% chloroauric acid was added dropwise, and the reaction was terminated after 8 h. The product was washed with alcohol three times, washed with water three times, and finally stored in pure water, and the solid content was measured.

[0055] Example 4 Single detection - application examples of magnetic gold nanoparticles of examples 1-3 (taking respiratory syncytial virus (RSV) detection as an example)

[0056] (1) 20 μL magnetic gold nanoparticles with a solid content of 5% were taken into a 2 mL EP tube, 1 mL labeling buffer was added, and the supernatant was removed after magnetic separation. Then 1 mL labeling buffer was added, and the mixture was ultrasonically dispersed for three times, and finally stored in 1 mL labeling buffer;

[0057] (2) 0.5 mg antibody was taken into the above 2 mL EP tube, and after rapid mixing, the mixture was incubated at room temperature for 2 h;

[0058] (3) 100 μL 10% BSA blocking solution was added into the above EP tube, and the mixture was incubated at room temperature for 30 min;

[0059] (4) The supernatant was removed after magnetic separation, 1 mL storage solution was added and ultrasonically dispersed for three times, and finally stored in 1 mL storage solution.

[0060] Example 5 Multiple detection

[0061] The blue magnetic gold nanoparticles in example 1 were selected and coupled with influenza A virus (Flu A) antibody according to the process of example 4;

[0062] The red magnetic gold nanoparticles in example 2 were selected and coupled with influenza B virus (Flu B) antibody according to the process of example 4;

[0063] The black magnetic gold nanoparticles in example 3 were selected and coupled with mycoplasma pneumoniae virus (MP) antibody according to the process of example 4.

[0064] The experimental results are as follows:

[0065] Table 1 is the particle size results of Fe3O4 magnetic micro-particles and magnetic gold nanoparticles of examples 1-3. The prepared Fe3O4 particle size is 104 nm, and the particle size distribution of the prepared magnetic gold nanoparticles of examples 1-3 is 326 nm, 331 nm and 333 nm. Figure 1The particle size analysis chart of the magnetic gold nanoparticles in Example 1 shows that the particle size distribution is narrow, indicating that the gold magnetic particles have uniform particle size and good dispersibility.

[0066] Table 1 Particle size of Fe3O4 and magnetic gold nanoparticles in Examples 1-3

[0067] Name Fe3O4 Example 1 Example 2 Example 3 Particle size 104 nm 326 nm 331 nm 333 nm

[0068] Table 2 shows the detection results of the magnetic gold nanoparticles prepared in Examples 1-3 after binding with RSV antibodies on RSV virus antigens, which can be detected after dilution by 10 times, 20 times, 50 times and 100 times.

[0069] Figure 2 The curve chart of the magnetic signal intensity of the magnetic gold nanoparticles in Example 1 after binding with RSV antibodies on RSV virus antigens and the concentration of RSV virus antigens shows that the magnetic signal intensity has good correlation with the concentration, and quantitative detection can be realized.

[0070] Table 2 Detection of magnetic gold nanoparticles in Examples 1-3 after binding with RSV antibodies

[0071] Dilution factor 10 times 20 times 50 times 100 times 1000 times Example 1 Positive Positive Positive Positive Negative Example 2 Positive Positive Positive Positive Negative Example 3 Positive Positive Positive Positive Negative

[0072] Table 3 shows that the color microspheres prepared in Examples 1-3 after binding with respiratory virus type A influenza virus antibody, type B influenza virus antibody and mycoplasma pneumonia virus antibody can detect the respiratory virus quality control product diluted by 50 times, and all show positive, indicating that the three viruses can be detected.

[0073] Table 3 Multiple color respiratory virus triple detection

[0074] Virus type Flu A Flu B MP Detection result Positive Positive Positive

[0075] Figure 3 The structure schematic diagram of the multiple color multiple detection card in Example 5 is shown, in which the blue gold magnetic particles are coupled with Flu A antibody, the red gold magnetic particles are coupled with Flu B antibody, and the black gold magnetic particles are coupled with MP antibody, and the results can be judged by color, which can be judged very intuitively, and the purpose of multiple color multiple detection is realized.

[0076] Although the embodiments of the present application have been shown and described, it can be understood by those skilled in the art that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing highly dispersed magnetic gold nanoparticles, characterized in that, Includes the following steps: (1) Fe3O4 magnetic microparticles with uniform particle size were prepared by hydrothermal synthesis. FeCl3·6H2O and PEG 6000 were dispersed in ethylene glycol and dissolved. Anhydrous sodium acetate was added to the above dispersion and reacted overnight. Fe3O4 magnetic microparticles with a particle size of 100-200 nm were obtained by magnetic separation and washing. (2) The magnetic microparticles and oil-soluble dye prepared in step (1) were dispersed in an ethanol-water solution by dispersion polymerization. Nitrogen gas was passed through to remove oxygen. Then, PVP was used as the dispersant, styrene as the monomer, methacrylamide as the functional monomer, and AIBN as the initiator. The reaction was carried out overnight at 70-80℃ to obtain polystyrene magnetic particles with controllable color and surface aminated. The volume ratio of ethanol to water in the ethanol-water solution was 1:0-0.2; the mass ratio of Fe3O4 magnetic microparticles to styrene monomer was 0.08~0.2:1; the mass ratio of dye to styrene monomer was 0.1~0.5:1; the volume ratio of methacrylamide to styrene monomer was 0.1~0.5:1; the mass ratio of initiator to styrene monomer was 0.01~0.03:1; and the mass ratio of dispersant to solvent was 0.008~0.03:

1. (3) Using the citric acid reduction method, gold nanoparticles are combined with polystyrene magnetic particles with controllable color and surface aminoation through electrostatic adsorption to prepare magnetic gold nanoparticles with a particle size of 300-500 nm.

2. The method for preparing highly dispersed magnetic gold nanoparticles according to claim 1, characterized in that: In step (1), the mass ratio of FeCl3·6H2O, CH3COONa, PEG6000 and ethylene glycol is 1:1-10:0.05-0.5:30-60.

3. The method for preparing highly dispersed magnetic gold nanoparticles according to claim 1, characterized in that: Specifically, step (3) involves reducing chloroauric acid using sodium citrate reduction and then combining it with the color-controllable and surface-aminated polystyrene magnetic particles from step (2) to prepare magnetic gold nanoparticles.

4. Highly dispersed magnetic gold nanoparticles prepared by any of the preparation methods described in claims 1-3.

5. The application of the highly dispersed magnetic gold nanoparticles according to claim 4 in the preparation of a multicolor multi-detection kit.

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