High-dispersity magnetic gold nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immunochromatography, in particular to high-dispersity magnetic nano-gold particles and a preparation method and application thereof, and the preparation method comprises the following steps: 1) preparing Fe3O4 magnetic particles by adopting a hydrothermal synthesis method; (2) preparing polystyrene magnetic particles with controllable colors and aminated surfaces by adopting a dispersion polymerization method; and (3) a citric acid reduction method is adopted, nanogold is combined with color-controllable and surface-aminated polystyrene magnetic particles through the electrostatic adsorption effect, and the magnetic gold nanoparticles are prepared. The prepared magnetic nanogold particles have the particle size range of 300-500 nm, are controllable in color, high in sensitivity, good in dispersity and good in stability, the color of the microsphere particles is regulated and controlled by introducing a colored polystyrene shell layer, the magnetic mechanical property, the optical property and the immunochromatography technology are combined, the purpose of qualitative and quantitative double detection functions of an object to be detected can be achieved, and the method is suitable for large-scale popularization and application. And the purpose of multi-color multi-connection detection can be achieved through different colors.

Description

Technical Field

[0001] The invention relates to the technical field of immunochromatography, and specifically relates to highly dispersed magnetic nano-gold particles and a preparation method and application thereof. Background Art

[0002] Colloidal gold technology is a technology that combines antigen-antibody immune reaction with colloidal gold labeling and tracing technology for qualitative and quantitative detection of antigen and antibody content. Due to its advantages such as rapidity, simplicity, low cost and good stability, colloidal gold detection has been widely used in the field of clinical testing. However, the particle size of colloidal gold is small, generally tens of nanometers, and there are problems such as low sensitivity, large batch-to-batch variation and high cost.

[0003] Colored microspheres are a hot raw material in immunochromatography technology. Compared with colloidal gold, they have the advantages of high sensitivity, rich colors, good stability, easy large-scale production and small batch differences, and are favored by many researchers. However, both methods require repeated centrifugation processes, especially colloidal gold is more dependent on centrifuges. At present, there have been reports on the application of magnetic nanogold particles in the field of immunochromatography. Gold magnetic particle immunochromatography technology combines magnetomechanical properties, optical properties and immunochromatography technology. It can not only realize qualitative and semi-quantitative analysis of the test object, but also use its magnetomechanical properties to realize quantitative detection of the test object, which is a feature that colloidal gold and colored microspheres do not have.

[0004] CN106370843 A discloses a method for rapid determination of clenbuterol based on gold magnetic immunochromatography, wherein the inner layer of γ-Fe 2 O 3 The outer layer of the magnetic material is coated with nano-Au to prepare a core-shell nanocomposite material with a particle size range of 20-80nm, and then combined with an antigen to achieve quantitative detection of the analyte. However, the gold magnetic particles prepared by this method have a small particle size and low sensitivity, and the pure iron oxide magnetic nanoparticles are easily oxidized in the air, resulting in agglomeration and poor dispersion.

[0005] CN107309436A discloses a Fe 3 O 4 / SiO 2 / Au gold magnetic nanoparticles and their preparation method and application, gold nanoparticles with uniform particle size distribution are prepared by sodium citrate reduction method, and then coated with SiO 2 Fe 3 O 4 Nanoparticles act as magnetic cores and gold nanoparticles are assembled on Fe 3 O 4 @SiO 2 The surface forms a Fe with consistent morphology, good dispersion and core-shell structure.3 O 4 / SiO 2 / Au gold magnetic nanoparticles. This method uses the sol-gel method to coat the magnetic material with silicon dioxide, effectively preventing the magnetic material from being oxidized by contact with air, improving the dispersibility of the particles, and realizing quantitative analysis and detection of the object to be detected.

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

[0007] In view of the above problems, the present invention provides a highly dispersible magnetic nano-gold particle and a preparation method and application thereof. The magnetic nano-gold particles prepared by the method have a particle size range of 300-500nm, polystyrene-coated magnetic particles, controllable color, high sensitivity, good dispersibility and good stability. A colored polystyrene shell layer is introduced to achieve the control of the color of the microsphere particles. The magnetic properties, optical properties and immunochromatography technology are combined to achieve the purpose of qualitative and quantitative dual detection functions of the object to be detected, and the different colors can achieve the purpose of colorful multi-link detection.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A method for preparing highly dispersible magnetic nano-gold particles comprises the following steps:

[0010] (1) Preparation of Fe by hydrothermal synthesis 3 O 4 Magnetic particles;

[0011] (2) Using dispersion polymerization, color-controllable and surface-amino-modified polystyrene magnetic particles were prepared;

[0012] (3) Using the citric acid reduction method, gold nanoparticles were combined with color-controllable and surface-amino-modified polystyrene magnetic particles through electrostatic adsorption to prepare magnetic gold nanoparticles.

[0013] The step (1) is specifically to prepare Fe with uniform particle size by hydrothermal synthesis. 3 O 4 Magnetic particles, FeCl 3 6H 2 O and PEG 6000 were dispersed in ethylene glycol. After dissolution, anhydrous sodium acetate was added to the above dispersion and reacted overnight. Fe 3 O4 Magnetic particles, particle size 100-200nm. Among them, FeCl 3 6H 2 O、CH 3 The mass ratio of COONa, PEG6000 and ethylene glycol is 1:1-10:0.05-0.5:30-60.

[0014] Specifically, step (2) comprises dispersing the magnetic particles and oil-soluble dye prepared in step (1) in an ethanol aqueous solution by a dispersion polymerization method, deoxygenating with nitrogen, and then reacting at 70-80° C. overnight with PVP as a dispersant, styrene as a monomer, methacrylamide as a functional monomer, and AIBN as an initiator to obtain color-controllable and surface-amino polystyrene magnetic particles. The volume ratio of ethanol to water in the ethanol aqueous solution is 1:0-0.2; Fe 3 O 4 The mass ratio of magnetic particles to styrene monomer is 0.08-0.2:1, the mass ratio of dye to styrene monomer is 0.1-0.5:1; the volume ratio of methacrylamide to styrene monomer is 0.1-0.5:1, the mass ratio of initiator to styrene monomer is 0.01-0.03:1, and the mass ratio of dispersant to solvent is 0.008-0.03:1.

[0015] Specifically, the step (3) is to reduce chloroauric acid by sodium citrate reduction method, and then combine it with the color-controllable and surface-amino-polystyrene magnetic particles in step (2) to prepare magnetic nano-gold particles. The particle size of the magnetic nano-gold particles is 300-500nm.

[0016] The highly dispersible magnetic nano-gold particles prepared by the invention can be used to prepare colorful multi-link detection kits and immunochromatography.

[0017] The highly dispersible magnetic nano-gold particles prepared by the present invention are used for immunochromatography, and the specific antibody labeling process is as follows:

[0018] (1) Take 20 μL of magnetic gold nanoparticles with a solid content of 5% in a 2 mL EP tube, add 1 mL of labeling buffer, perform magnetic separation, remove the supernatant, add 1 mL of labeling buffer, and perform ultrasonic dispersion. Repeat three times and finally store in 1 mL of labeling buffer.

[0019] (2) Take 0.5 mg of antibody into the above 2 mL EP tube, mix quickly, and incubate at room temperature for 2 h;

[0020] (3) Add 100 μL of 10% BSA blocking solution to the above EP tube, mix and incubate at room temperature for 30 min;

[0021] (4) Magnetic separation: remove the supernatant, add 1 mL of preservation solution and perform ultrasonic dispersion. Repeat this process three times and finally store the cells in 1 mL of preservation solution.

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

[0023] (1) The present invention uses a dispersion polymerization method to coat the color-controllable surface amino-modified polystyrene microspheres with magnetic particles, and then adsorbs nano-gold on the surface of the surface amino-modified magnetic particles through electrostatic adsorption. The particle size ranges from 300 to 500 nm, which can effectively improve the detection sensitivity.

[0024] (2) The magnetic nano-gold particles are introduced into a colored polystyrene layer, which can adjust the color of the particles, realize colorful multi-link detection, and have good stability and controllable color. The magnetic properties, optical properties and immunochromatography technology are combined to realize the qualitative and quantitative dual detection functions of the object to be detected.

[0025] (3) The magnetic gold nanoparticles can be used to prepare multi-detection kits with their rich colors, such as respiratory triple detection, where different colored balls are used to bind different antibodies, and finally the color of the test line is used to determine whether a person is infected with a certain virus.

[0026] (4) The magnetic gold nanoparticles can effectively prevent the magnetic core from being oxidized and magnetic leakage, and improve the dispersibility of the microspheres. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a particle size analysis diagram of the magnetic gold nanoparticles prepared in Example 1.

[0028] Figure 2 This is a curve diagram of the magnetic signal intensity and RSV virus antigen concentration of the RSV virus antigen detected after the magnetic gold nanoparticles are combined with RSV antibodies in Example 1.

[0029] Figure 3 This is a schematic diagram of the structure of the multi-color multi-link detection card of Example 5. 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] Example 1

[0032] A method for preparing highly dispersible magnetic nano-gold particles comprises the following steps:

[0033] (1) Solvothermal preparation of Fe 3 O 4 Nano magnetic core

[0034] Weigh FeCl 3 6H 2 4.5g O and 400mg PEG6000 were dissolved in 200mL ethylene glycol and stirred to dissolve. Then 4.5g anhydrous sodium acetate was weighed and added to the above solution and stirred until completely dissolved; the uniform viscous liquid after dissolution was transferred to a 500mL high-temperature reactor, the lid was tightened, and it was placed in a 200℃ oven and heated overnight; after the reactor temperature cooled naturally, the black reaction liquid was poured out and magnetically separated; it was first washed three times with anhydrous ethanol, then washed three times with pure water, and stored in pure water for determination of Fe 3 O 4 Content, deoxygenated and stored in nitrogen to avoid oxidation.

[0035] (2) Dispersion polymerization method to coat the blue polymer shell

[0036] Pour 0.5g of the magnetic particles obtained in step (1) and 1g of solvent blue into a reaction bottle, add 10mL of pure water and 90mL of anhydrous ethanol, and disperse by ultrasonic. Pass nitrogen for 20min, add 1g of dispersant PVP, and dissolve. Dissolve 70mg of initiator in 5mL of styrene and 2mL of methacrylamide, add to the reaction bottle at 70℃, and react overnight. Wash with anhydrous ethanol 3 times, wash with pure water 3 times, and finally store in 50mL of pure water.

[0037] (3) Magnetic particles coated with gold nanoparticles

[0038] Add the blue polymer shell-coated magnetic microparticles obtained in step (3), 200 mg of dispersant PVP and 20 mL of 0.013 mol / L sodium citrate into a reaction bottle. After ultrasonic dispersion, place the reaction bottle in a constant temperature water bath and stir at 80°C. After the temperature reaches 80°C, add 60 mL of 1% chloroauric acid dropwise. After 8 hours, terminate the reaction. Wash with alcohol three times, wash with water three times, and finally store in pure water. Measure the solid content and set aside.

[0039] Example 2

[0040] A method for preparing highly dispersible magnetic nano-gold particles comprises the following steps:

[0041] (1) Solvothermal preparation of Fe 3 O 4 Nano magnetic core

[0042] Weigh FeCl 3 6H 24.5g O and 400mg PEG6000 were dissolved in 200mL ethylene glycol and stirred to dissolve. Then 4.5g anhydrous sodium acetate was weighed and added to the above solution and stirred until completely dissolved; the uniform viscous liquid after dissolution was transferred to a 500mL high-temperature reactor, the lid was tightened, and it was placed in a 200℃ oven and heated overnight; after the reactor temperature cooled naturally, the black reaction liquid was poured out and magnetically separated; it was first washed three times with anhydrous ethanol, then washed three times with pure water, and stored in pure water for determination of Fe 3 O 4 Content, deoxygenated and stored in nitrogen to avoid oxidation.

[0043] (2) Dispersion polymerization method to coat the red polymer shell

[0044] Pour 0.5g of the magnetic particles obtained in step (1) and 1g of solvent red into a reaction bottle, add 10mL of pure water and 90mL of anhydrous ethanol, and disperse by ultrasonic. Pass nitrogen for 20min, add 1g of dispersant PVP, and dissolve. Dissolve 70mg of initiator in 5mL of styrene and 2mL of methacrylamide, add to the reaction bottle at 70℃, and react overnight. Wash with anhydrous ethanol 3 times, wash with pure water 3 times, and finally store in 50mL of pure water.

[0045] (3) Magnetic particles coated with gold nanoparticles

[0046] Add the magnetic microparticles coated with the red polymer shell prepared in step (2), 200 mg of the dispersant PVP and 20 mL of 0.013 mol / L sodium citrate into the reaction bottle. After ultrasonic dispersion, add the reaction bottle to a constant temperature water bath and stir at 80°C. After the temperature reaches 80°C, add 60 mL of 1% chloroauric acid dropwise. After 8 hours, terminate the reaction. Wash with alcohol three times, wash with water three times, and finally store in pure water. Measure the solid content and set aside.

[0047] Example 3

[0048] A method for preparing highly dispersible magnetic nano-gold particles comprises the following steps:

[0049] (1) Solvothermal preparation of Fe 3 O 4 Nano magnetic core

[0050] Weigh FeCl 3 6H 24.5g O and 400mg PEG6000 were dissolved in 200mL ethylene glycol and stirred to dissolve. Then 4.5g anhydrous sodium acetate was weighed and added to the above solution and stirred until completely dissolved; the uniform viscous liquid after dissolution was transferred to a 500mL high-temperature reactor, the lid was tightened, and it was placed in a 200℃ oven and heated overnight; after the reactor temperature cooled naturally, the black reaction liquid was poured out and magnetically separated; it was first washed three times with anhydrous ethanol, then washed three times with pure water, and stored in pure water for determination of Fe 3 O 4 Content, deoxygenated and stored in nitrogen to avoid oxidation.

[0051] (2) Dispersion polymerization method to coat the black polymer shell

[0052] Pour 0.5g of magnetic particles prepared in step (1) and 1g of melanin (alcohol soluble) into a reaction bottle, add 10mL of pure water and 90mL of anhydrous ethanol, and disperse by ultrasonic. Pass nitrogen for 20min, add 1g of dispersant PVP, and dissolve. Dissolve 70mg of initiator in 5mL of styrene and 2mL of methacrylamide, add to the reaction bottle at 70℃, and react overnight. Wash with anhydrous ethanol 3 times, wash with pure water 3 times, and finally store in 50mL of pure water.

[0053] (3) Magnetic particles coated with gold nanoparticles

[0054] Add the magnetic microparticles coated with black polymer shell prepared in step (2), 200 mg of dispersant PVP and 20 mL of 0.013 mol / L sodium citrate into a reaction bottle. After ultrasonic dispersion, place the reaction bottle in a constant temperature water bath and stir at 80°C. After the temperature reaches 80°C, add 60 mL of 1% chloroauric acid dropwise. After 8 hours, terminate the reaction. Wash with alcohol three times, wash with water three times, and finally store in pure water. Measure the solid content and set aside.

[0055] Example 4 Single Detection - Application Examples of Magnetic Gold Nanoparticles in Examples 1-3 (Taking Respiratory Syncytial Virus (RSV) Detection as an Example)

[0056] (1) Take 20 μL of magnetic gold nanoparticles with a solid content of 5% in a 2 mL EP tube, add 1 mL of labeling buffer, perform magnetic separation, remove the supernatant, add 1 mL of labeling buffer, and perform ultrasonic dispersion. Repeat three times and finally store in 1 mL of labeling buffer.

[0057] (2) Take 0.5 mg of antibody into the above 2 mL EP tube, mix quickly, and incubate at room temperature for 2 h;

[0058] (3) Add 100 μL of 10% BSA blocking solution to the above EP tube, mix and incubate at room temperature for 30 min;

[0059] (4) Magnetic separation: remove the supernatant, add 1 mL of preservation solution and perform ultrasonic dispersion. Repeat this process three times and finally store the cells in 1 mL of preservation solution.

[0060] Example 5 Multiplex Detection

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

[0064] The experimental results are as follows:

[0065] Table 1 is Fe 3 O 4 The particle size results of the magnetic particles and the magnetic gold nanoparticles of Examples 1-3 are as follows: 3 O 4 The particle size is 104 nm, and the particle size distribution of the magnetic nano-gold particles prepared in Examples 1-3 is 326 nm, 331 nm, and 333 nm. Figure 1 This is a particle size analysis diagram of the magnetic gold nanoparticles in Example 1. It can be seen from the figure that the particle size distribution is narrow, indicating that the particle size of the gold magnetic particles is uniform and the dispersion is good.

[0066] Table 1Fe 3 O 4 and the particle size of the magnetic nano-gold particles in Examples 1-3

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

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

[0069] Figure 2 This is a curve diagram of the magnetic signal intensity and RSV virus antigen concentration for RSV virus antigen detection after the magnetic gold nanoparticles are combined with RSV antibodies in Example 1. It can be seen that the magnetic signal intensity has a good correlation with the concentration, and quantitative detection can be achieved.

[0070] Table 2 Example 1-3 Magnetic nanogold particles coupled to RSV antibody detection

[0071] Dilution multiple 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 the colored microspheres prepared in Examples 1-3 were coupled with respiratory virus antibodies, influenza A virus antibodies, and Mycoplasma pneumoniae virus antibodies, and then the triple test was diluted 50 times of the respiratory virus quality control product, all of which showed positive results, indicating that all three viruses can be detected.

[0073] Table 3 Multi-color respiratory virus triple test

[0074] Virus Type Flu A Flu B MP Test results Positive Positive Positive

[0075] Figure 3 This is a schematic diagram of the structure of the colorful multi-link detection card of Example 5, in which blue gold magnetic particles are coupled to Flu A antibodies, red gold magnetic particles are coupled to Flu B antibodies, and black gold magnetic particles are coupled to MP antibodies. The results can be judged by color, and judgments can be made very intuitively, thereby achieving the purpose of colorful multi-link detection.

[0076] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing highly dispersible magnetic nano-gold particles, characterized in that: The following steps are involved: (1) Fe3O4 magnetic particles were prepared by hydrothermal synthesis; (2) Using dispersion polymerization, color-controllable and surface-amino-modified polystyrene magnetic particles were prepared; (3) Using the citric acid reduction method, gold nanoparticles were combined with color-controllable and surface-amino-modified polystyrene magnetic particles through electrostatic adsorption to prepare magnetic gold nanoparticles.

2. The method for preparing highly dispersible magnetic nano-gold particles according to claim 1, characterized in that: The step (1) specifically comprises the following steps: preparing Fe3O4 magnetic particles with uniform particle size by hydrothermal synthesis method, dispersing FeCl3·6H2O and PEG 6000 in ethylene glycol, adding anhydrous sodium acetate to the above dispersion after dissolution, reacting overnight, and obtaining Fe3O4 magnetic particles with a particle size of 100-200 nm by magnetic separation and washing.

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

4. The method for preparing highly dispersible magnetic nano-gold particles according to claim 1, characterized in that: The step (2) is specifically as follows: the magnetic particles and oil-soluble dye prepared in step (1) are dispersed in an ethanol aqueous solution by a dispersion polymerization method, nitrogen is passed through to deoxygenate, and then PVP is used as a dispersant, styrene is used as a monomer, methacrylamide is used as a functional monomer, and AIBN is used as an initiator, and the reaction is carried out at 70-80° C. overnight to prepare color-controllable and surface-amino polystyrene magnetic particles.

5. The method for preparing highly dispersible magnetic nano-gold particles according to claim 4, characterized in that: In the step (2), the volume ratio of ethanol to water in the ethanol aqueous solution is 1:0-0.2; the mass ratio of Fe3O4 magnetic particles to styrene monomer is 0.08-0.2:1, and the mass ratio of dye to styrene monomer is 0.1-0.5:

1.

6. The method for preparing highly dispersible magnetic nano-gold particles according to claim 5, characterized in that: In the step (2), the volume ratio of methacrylamide to styrene monomer is 0.1-0.5:1, the mass ratio of initiator to styrene monomer is 0.01-0.03:1, and the mass ratio of dispersant to solvent is 0.008-0.03:

1.

7. The method for preparing highly dispersible magnetic nano-gold particles according to claim 6, characterized in that: The step (3) specifically comprises reducing chloroauric acid by sodium citrate reduction method, and then combining the chloroauric acid with the color-controllable and surface-amino-modified polystyrene magnetic particles in step (2) to prepare magnetic nano-gold particles.

8. The method for preparing highly dispersible magnetic nano-gold particles according to claim 7, characterized in that: The particle size of magnetic gold nanoparticles is 300-500nm.

9. Highly dispersible magnetic nano-gold particles obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the highly dispersible magnetic nanogold particles according to claim 8 in the preparation of a multi-color multi-link detection kit and immunochromatography.

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