CN110343218B - Immunomagnetic bead and preparation method thereof - Google Patents

Immunomagnetic bead and preparation method thereof Download PDF

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CN110343218B
CN110343218B CN201910495778.7A CN201910495778A CN110343218B CN 110343218 B CN110343218 B CN 110343218B CN 201910495778 A CN201910495778 A CN 201910495778A CN 110343218 B CN110343218 B CN 110343218B
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CN110343218A (en
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王鹏
侯立威
吴志能
谌庆春
李向东
张军
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Dongguan HEC Tech R&D Co Ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/14—Polymerisation; cross-linking
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F257/00—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00
    • C08F257/02—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00 on to polymers of styrene or alkyl-substituted styrenes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00—Use of inorganic substances as compounding ingredients
    • C08K3/18—Oxygen-containing compounds, e.g. metal carbonyls
    • C08K3/20—Oxides; Hydroxides
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/0018—Diamagnetic or paramagnetic materials, i.e. materials with low susceptibility and no hysteresis
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F41/00—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00—Use of inorganic substances as compounding ingredients
    • C08K3/18—Oxygen-containing compounds, e.g. metal carbonyls
    • C08K3/20—Oxides; Hydroxides
    • C08K3/22—Oxides; Hydroxides of metals
    • C08K2003/2265—Oxides; Hydroxides of metals of iron
    • C08K2003/2275—Ferroso-ferric oxide (Fe3O4)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K2201/00—Specific properties of additives
    • C08K2201/011—Nanostructured additives

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Abstract

The invention provides an immunomagnetic bead and a preparation method thereof, comprising the following steps: swelling the polystyrene microspheres; synthesizing magnetic composite microspheres at high temperature and high pressure; acid dissolution is carried out to remove the exposed magnetic particles on the surface of the magnetic composite microsphere and the surface cross-linking modification of the magnetic particles. The immunomagnetic beads prepared by the method have the advantages of uniform particle size, good magnetic responsiveness, stable structure and consistent magnetic content.

Description

Immunomagnetic bead and preparation method thereof
Technical Field
The invention relates to the technical field of composite material preparation, in particular to a preparation method of immunomagnetic beads.
Background
The magnetic bead is a composite functional microsphere with superparamagnetism, and can be coupled with protein, DNA and the like due to the modified functional groups such as amino, carboxyl and the like on the surface. Therefore, the method has wide application in cell separation, immobilized enzyme, immunodiagnosis, tumor targeted therapy, DNA extraction and the like.
The chemiluminescent immunomagnetic beads have high requirements in all magnetic beads, and the magnetic microspheres have the requirements of proper particle size, high particle size uniformity, magnetic response consistency and the like. The price of foreign commercialized magnetic beads is high, the technology is strict and confidential, the defects of non-uniform particle size, poor magnetic response and inconsistent magnetic response commonly occur in the domestic magnetic beads, and more seriously, the phenomenon that nano magnetic particles and microspheres fall off to pollute a sample occurs in the using process.
Therefore, it is required to provide a method for preparing immunomagnetic beads with uniform particle size, good magnetic responsiveness, stable structure and simple operation.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides an immunomagnetic bead and a preparation method thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preparation method of immunomagnetic beads comprises the following steps:
s1, swelling the polystyrene microspheres;
s2, synthesis of magnetic composite microspheres: dispersing the polystyrene microspheres swelled by S1 and an alkaline reagent in an organic solvent X to form emulsion A; mixing iron salt, water and an organic solvent X to form a solution B; mixing the emulsion A and the solution B, adding a surfactant to form uniform emulsion, and reacting in a high-pressure reaction kettle to obtain magnetic composite microspheres;
s3, removing exposed magnetic particles on the surface of the magnetic composite microsphere through acid dissolution;
and S4, carrying out surface cross-linking modification on the magnetic composite microspheres processed by the S3 to obtain the product.
In some embodiments, the polystyrene microspheres in S1 are uniform-particle-size microspheres of 1-5 μm.
In some embodiments, the swelling method of the polystyrene microspheres in S1 is: dispersing polystyrene microspheres into water containing a surfactant, adding a solvent C and a swelling agent, performing ultrasonic emulsification, and stirring at a certain temperature.
Further, in the swelling method, the surfactant is sodium lauryl sulfate, sodium dodecyl sulfonate, sodium dodecyl benzene sulfonate, or sodium stearate; the solvent C is absolute ethyl alcohol; the swelling agent is organic solvent which can dissolve polystyrene such as dibutyl phthalate, ethyl acetate, butyl acetate, cyclohexane, toluene and the like; the temperature is 40-80 ℃; the ultrasonic time is 10 min; the stirring time for stirring is 5-20 h.
Further, in the swelling method, the temperature is 40 ℃, 50 ℃, 60 ℃, 70 ℃ or 80 ℃.
Further, in the swelling method, the stirring time of the stirring is 5h, 8h, 10h, 12h, 15h, 18h, or 20 h.
The swelling mainly has the function of increasing the porosity of the polystyrene microsphere and is beneficial to the diffusion of iron ions and reducing agent into the polystyrene during the synthesis process, so that more Fe is synthesized in the polystyrene microsphere3O4。
In some of the embodiments, the alkaline agent in S2 is sodium hydroxide, potassium hydroxide, or ammonia; the ferric salt in S2 is FeCl3、Fe(NO3)3、Fe2(SO4)3Or their crystalline hydrates; the organic solvent in the S2 is one of polyhydric alcohols such as ethylene glycol, glycerol, pentaerythritol, butanediol and neopentyl glycol; the surfactant in the S2 is one of carboxylate such as anhydrous sodium acetate, potassium acetate, sodium propionate, sodium oleate and the like.
Further, the emulsion A in S2 is obtained by dispersing the polystyrene microspheres obtained in S1 into a glycol solution of sodium hydroxide, violently stirring and then carrying out ultrasonic treatment.
Further, the solution B in the S2 is obtained by mixing ethylene glycol, ferric trichloride hexahydrate and water and then performing ultrasonic treatment.
In some embodiments, the pressure in the S2 is 0.3MPa to 3MPa, the reaction temperature is 150 ℃ to 250 ℃, and the reaction time is 5 to 20 hours.
In some of these embodiments, the pressure in S2 is between 0.3MPa and 2 MPa.
In some of these embodiments, the pressure in S2 is between 0.3MPa and 1.5 MPa.
In some of these embodiments, the pressure in S2 is between 0.3MPa and 1 MPa.
In some of these embodiments, the pressure in S2 is between 0.3MPa and 0.6 MPa.
In still other embodiments, the pressure in S2 is 0.3MPa, 0.4MPa, 0.5MPa, 0.6MPa, 0.9MPa, 1.0MPa, 1.5MPa, 2MPa, or 3 MPa.
In some of these embodiments, the reaction temperature in S2 is 160 ℃ to 220 ℃.
In some of these embodiments, the reaction temperature in S2 is 170 ℃ to 210 ℃.
In still other embodiments, the reaction temperature in S2 is 150 ℃, 170 ℃, 190 ℃, 200 ℃, 210 ℃, 230 ℃, or 250 ℃.
In some of these embodiments, the reaction time in S2 is 6 to 20 hours.
In still other embodiments, the reaction time in S2 is 6h, 8h, 9h, 10h, 12h, 14h, 15h, 16h, 18h, or 20 h.
In some embodiments, the molar weight ratio of the basic agent to the iron salt in S2 is (0.5-2): 1; the volume ratio of the water in the S2 to the total amount of the organic solvent X is (1-3): 10; the molar weight ratio of the surfactant to the iron salt in the S2 is (1-10): 1.
in some embodiments, the mass ratio of the basic agent to the iron salt in S2 is 1: 4.
in some embodiments, the volume ratio of water to the total amount of organic solvent X in S2 is (10-18): 60.
in some embodiments, the mass ratio of surfactant to iron salt in S2 is 4: 5.
specifically, the synthesis method of the magnetic composite microsphere in S2 includes: dispersing the polystyrene microspheres swelled by S1 into an organic solvent X solution of NaOH to form emulsion A; mixing ferric trichloride hexahydrate, water and an organic solvent X to form a solution B; mixing the emulsion A and the solution B, adding anhydrous sodium acetate to form a uniform emulsion, removing undispersed large particles, and reacting in a high-pressure reaction kettle at 150-250 ℃ under 0.3-3 MPa for 5-20 h; wherein the molar weight ratio of the sodium hydroxide to the ferric trichloride hexahydrate is (0.5-2): 1; the volume ratio of water to ethylene glycol is (1-3): 10; the molar weight ratio of the anhydrous sodium acetate to the ferric trichloride hexahydrate is (1-10): 1.
preferably, the synthesis method of the magnetic composite microspheres in S2 is as follows: dispersing the polystyrene microspheres swelled by S1 into a glycol solution of NaOH to form emulsion A; mixing ferric trichloride hexahydrate, water and glycol to form a solution B; mixing the emulsion A and the solution B, adding anhydrous sodium acetate to form a uniform emulsion, removing undispersed large particles, and reacting in a high-pressure reaction kettle at 0.3-2 MPa and 160-210 ℃ for 5-10 h; wherein the molar weight ratio of the sodium hydroxide to the ferric trichloride hexahydrate is (1-2): 1; the volume ratio of water to ethylene glycol is (1-2): 10; the molar weight ratio of the anhydrous sodium acetate to the ferric trichloride hexahydrate is (1-5): 1.
more preferably, the synthesis method of the magnetic composite microspheres in S2 is as follows: dispersing the polystyrene microspheres swelled by S1 into a glycol solution of NaOH to form emulsion A; mixing ferric trichloride hexahydrate, water and glycol to form a solution B; mixing the emulsion A and the solution B, adding anhydrous sodium acetate to form a uniform emulsion, removing undispersed large particles, and reacting in a high-pressure reaction kettle at the temperature of 170-200 ℃ under the pressure of 0.3-1.5 MPa for 6-10 h; wherein the molar weight ratio of the sodium hydroxide to the ferric trichloride hexahydrate is (1-2): 1; the volume ratio of water to ethylene glycol is (1-2): 10; the molar weight ratio of the anhydrous sodium acetate to the ferric trichloride hexahydrate is (2-4): 1.
more preferably, the synthesis method of the magnetic composite microspheres in S2 is as follows: dispersing the polystyrene microspheres swelled by S1 into a glycol solution of NaOH to form emulsion A; mixing ferric trichloride hexahydrate, water and glycol to form a solution B; mixing the emulsion A and the solution B, adding anhydrous sodium acetate to form a uniform emulsion, removing undispersed large particles, and reacting in a high-pressure reaction kettle at the temperature of 170-210 ℃ under the pressure of 0.3-0.6 MPa for 6-20 hours; wherein the molar weight ratio of the sodium hydroxide to the ferric trichloride hexahydrate is (1-2): 1; the volume ratio of water to ethylene glycol is (1-3): 10; the molar weight ratio of the anhydrous sodium acetate to the ferric trichloride hexahydrate is (2-4): 1.
more preferably, the synthesis method of the magnetic composite microspheres in S2 is as follows: dispersing the polystyrene microspheres swelled by S1 into a glycol solution of NaOH to form emulsion A; mixing ferric trichloride hexahydrate, water and glycol to form a solution B; mixing the emulsion A and the solution B, adding anhydrous sodium acetate to form a uniform emulsion, removing undispersed large particles, and reacting in a high-pressure reaction kettle at the temperature of 170-210 ℃ under the pressure of 0.3-0.6 MPa for 6-20 hours; wherein the mass ratio of the sodium hydroxide to the ferric trichloride hexahydrate is 1: 4; the volume ratio of water to glycol is (10-18): 60, adding a solvent to the mixture; the mass ratio of the anhydrous sodium acetate to the ferric trichloride hexahydrate is 4: 5.
dispersing polystyrene microspheres into an iron salt solution containing a reducing agent to enable iron salt and the reducing agent to enter the polystyrene microspheres, and then carrying out reduction reaction to generate nano Fe under the conditions of high temperature and high pressure3O4Tightly combined with polystyrene microspheres; the other part of the ferric salt generates nano Fe on the surface of the polystyrene microsphere3O4The agglomeration of the polystyrene microspheres is prevented, and finally the magnetic composite microspheres are generated.
In some embodiments, the S3 dissolving the magnetic composite microspheres in an acidic solution, wherein the acidic solution is 0.1mol/L to 0.5mol/L hydrochloric acid solution, the dissolving temperature is 45 ℃ to 75 ℃, and the dissolving is performed at a stirring speed of 150r/min to 300 r/min.
Further, when the magnetic composite microspheres in the S3 are dissolved in an acidic solution, the amount and time of the acid are controlled, and the molar ratio of the hydrochloric acid to the iron salt is (3-4.5): and 1, controlling the acid dissolution time to be 12-24 hours according to the concentration of hydrochloric acid and the acid dissolution temperature so as to avoid insufficient dissolution or excessive dissolution. Magnetic composite microsphere treated with acid, smooth surface and free of Fe3O4And the residue is beneficial to the kinetic consistency of the coupling reaction.
In some embodiments, the crosslinking method in S4 is: dispersing the magnetic composite microspheres processed by S3 and a surfactant in water, adding a cross-linking agent, a functional monomer and an initiator, and heating to 60-100 ℃ for reaction for 4-20 h; separating and washing to obtain a product; wherein the surfactant is sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecyl benzene sulfonate or sodium stearate; the crosslinking agent is divinylbenzene; the functional monomer is acrylic acid such as methacrylic acid, acrylic acid or undecylenic acid; the initiator is azobisisobutyronitrile, azobisisoheptonitrile, benzoyl peroxide, lauroyl peroxide, dicumyl peroxide or potassium persulfate.
Through the modification of Divinylbenzene (DVB) and methacrylic acid (MAA), a compact crosslinking layer is formed on the surface of the microsphere, wherein carboxyl functional groups are distributed on the surface of the compact crosslinking layer, and the compact crosslinking layer is combined with the polystyrene microsphere base material in a chemical bond form, so that the structural stability of the microsphere is improved, and the microsphere is not easily damaged in the using process.
On the other hand, the invention provides an immunomagnetic bead which is structurally characterized by a magnetic composite microsphere core, a compact crosslinking layer interlayer and surface carboxyl functional groups, wherein the magnetic composite microsphere core is composed of PS and nano magnetic particles, the nano magnetic particles are dispersed and distributed in the PS microsphere, the compact crosslinking layer is coated on the outer layer of the magnetic composite microsphere core, and the carboxyl functional groups are distributed on the surface of the compact crosslinking layer.
Further, the immunomagnetic beads comprise a core, an interlayer and a surface, wherein the core is a magnetic composite microsphere 3, the interlayer is a compact crosslinking layer 4, and the surface is a carboxyl functional group 5; the inner core of the magnetic composite microsphere 3 consists of PS microspheres 2 and nano ferroferric oxide 1, wherein the nano ferroferric oxide 1 is dispersedly distributed in the PS microspheres 2, the outer layer of the inner core of the magnetic composite microsphere 3 is coated with a compact crosslinking layer 4, and carboxyl functional groups 5 are distributed on the surface of the compact crosslinking layer 4; as shown in fig. 2, 4 and 6.
On the other hand, the invention also provides the immunomagnetic beads obtained by the preparation method, which have stable structures and uniform surface appearances.
The invention has the beneficial effects that: (1) the magnetic composite microspheres are synthesized under the conditions of high temperature and high pressure, the reaction system is close to a homogeneous system, the consistency of the magnetic content percentage of the microspheres is ensured, and the nano Fe is ensured under the conditions of high temperature and high pressure3O4The particles are more tightly combined with the PS microspheres; (2) the acid dissolves the exposed magnetic particles on the surface, so that the coating effect of the microsphere is good, and the surface of the microsphere has no exposed Fe3O4(ii) a (3) After the acid dissolution is finished, the surface of the microsphere is modified by DVB, so that a net structure is formed on the surface of the microsphere, the structural stability of the microsphere is improved, the microsphere is not easy to damage in the using process, and the Fe is solved3O4The particles are easy to fall off to pollute the sample; (4) the magnetic beads prepared by the method have stable structure and uniform surface appearance.
Definition of terms
All ranges cited herein are inclusive, unless expressly stated to the contrary.
The terms "a" or "an" are used herein to describe elements and components described herein. This is done merely for convenience and to provide a general sense of the scope of the invention. Such description should be understood to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise. "plural" means two or more.
The numbers in this disclosure are approximate, regardless of whether the word "about" or "approximately" is used. The numerical value of the number may have differences of 1%, 2%, 5%, 7%, 8%, 10%, etc. Whenever a number with a value of N is disclosed, any number with a value of N +/-1%, N +/-2%, N +/-3%, N +/-5%, N +/-7%, N +/-8% or N +/-10% is explicitly disclosed, wherein "+/-" means plus or minus, and a range between N-10% and N + 10% is also disclosed.
The following definitions, as used herein, should be applied unless otherwise indicated. For the purposes of the present invention, the chemical elements are in accordance with the CAS version of the periodic Table of elements, and the 75 th version of the handbook of chemistry and Physics, 1994. In addition, general principles of Organic Chemistry can be referred to as described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausaltito: 1999, and "March's Advanced Organic Chemistry" by Michael B.Smith and Jerry March, John Wiley & Sons, New York:2007, the entire contents of which are incorporated herein by reference.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, unless a specific section is cited. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Drawings
Fig. 1 is an SEM image of the magnetic composite microsphere prepared in step 2 of example 1.
Fig. 2 is a schematic structural view of the magnetic composite microsphere prepared in step 2 of example 1.
FIG. 3 is an SEM image of acid-washed magnetic composite microspheres obtained in step 3 of example 1.
FIG. 4 is a schematic structural diagram of the magnetic composite microsphere after acid washing treatment in step 3 of example 1.
FIG. 5 is a SEM photograph of immunomagnetic beads prepared in step 4 of example 1.
FIG. 6 is a schematic diagram of the structure of immunomagnetic beads prepared in step 4 of example 1.
FIG. 7 is a Raman spectrum of immunomagnetic beads prepared in step 4 of example 1.
Fig. 8 is an SEM image of the magnetic composite microsphere prepared in comparative example 4.
Wherein, in fig. 2, 4 and 6: 1-nano Fe3O4(ii) a 2-PS microspheres; 3-magnetic composite microspheres; 4-dense crosslinked layer, 5-carboxyl functional group.
Detailed Description
The following abbreviations are used throughout the present invention:
st-styrene; SDS-sodium dodecyl sulfate; KPS-potassium persulfate; FeCl3-ferric chloride;
NaOH-sodium hydroxide; NaAc-sodium acetate; PS-polystyrene; MAA-methacrylic acid;
DVB-divinylbenzene; PBS solution-phosphate buffer.
Example 1
1. Swelling of polystyrene microspheres
Dispersing 2g of polystyrene microspheres (1 micron) into 100mL of an aqueous solution containing 0.1% SDS, adding 20mL of absolute ethyl alcohol and 5g of dibutyl phthalate, fully performing ultrasonic emulsification, adding the mixture into a four-neck flask with a mechanical stirrer, stirring for 10 hours at the water bath temperature of 50 ℃, centrifugally separating white solid particles after swelling, and respectively washing with pure water and absolute ethyl alcohol for 3-5 times for later use.
2. Synthesis of magnetic composite microspheres
Dissolving 1.25g of NaOH in 50mL of EG, adding the swollen polystyrene microspheres into the EG, violently stirring the mixture to disperse the polystyrene microspheres, and performing ultrasonic treatment to form uniform emulsion to obtain emulsion A; another 10mL EG and 5g FeCl were taken3·6H2O and 10mLH2And O, mixing and performing ultrasonic treatment to obtain a solution B, slowly dropwise adding the solution B into the emulsion A while vigorously stirring, continuously stirring for 10min after dropwise adding is finished, adding 4g of NaAc into the system, and forming uniform emulsion by vigorously stirring and ultrasonically treating.
And (3) sieving the obtained uniform emulsion with a 400-mesh standard sieve, adding the uniform emulsion into a 200mL high-pressure reaction kettle with a polytetrafluoroethylene inner container, keeping the temperature at 0.3MPa and 170 ℃ for 6h, taking out the uniform emulsion, cooling, separating a generated black precipitate by using a magnet, and washing the black precipitate for three times by using absolute ethyl alcohol to obtain the magnetic composite microsphere.
The SEM image of the magnetic composite microsphere is shown in FIG. 1; the structure schematic diagram is shown in fig. 2. In the structural schematic diagram, gray represents polystyrene microsphere base material, and black spots represent nano Fe3O4(ii) a The morphology shown in the SEM picture is consistent with the structure of the structural schematic diagram.
3. Removal of exposed magnetic particles on surface of magnetic composite microsphere
Adding the magnetic composite microspheres into a 250mL four-mouth flask with mechanical stirring, adding 200mL hydrochloric acid solution with the concentration of 0.5mol/L, dissolving at 60 ℃ for 12h, keeping the stirring speed at 250r/min, changing the particles into light gray after the completion, separating the magnetic particles by using a magnet, and washing 3 times by using absolute ethyl alcohol and pure water respectively.
An SEM image of the magnetic composite microspheres subjected to acid washing treatment is shown in FIG. 3; the schematic structure is shown in fig. 4. After acid dissolution, the main component of the surface is polystyrene, and only a small amount of Fe3O4And (4) residue, which is consistent with the structure shown in the structural schematic diagram.
4. Surface crosslinking of magnetic particles
Dispersing light gray magnetic particles into 100mL of aqueous solution containing 0.1% SDS, adding the aqueous solution into a four-neck flask with a mechanical stirrer, adding 0.1g of DVB, 0.5g of MAA, 10mL of absolute ethyl alcohol and 0.1g of KPS, introducing nitrogen and condensed water, keeping the stirring speed of 250r/min, reacting for 10 hours in a water bath kettle at 80 ℃, separating out magnetic microspheres by using a magnet after the reaction is finished, and washing for 3 times by using the absolute ethyl alcohol and pure water respectively to obtain a target sample; the target sample was dispersed in PBS solution and stored at 4 ℃.
The SEM image of the immunomagnetic beads prepared by the invention is shown in FIG. 5; the schematic structure is shown in fig. 6. The surface of the magnetic bead is mainly a spherical cross-linked network structure formed by divinylbenzene and methacrylic acid, and Fe can not be observed3O4Presence of (a); further by Raman detection, as shown in FIG. 7, Fe is not shown3O4The characteristic peak of (a) is consistent with a structural schematic diagram.
The magnetic beads prepared by the method of the invention (1) have uniform particle size (1-5 μm); (2) the magnetic responsiveness of the magnetic beads is good, 1.5mL of magnetic bead pure water dispersion (20mg/mL) can finish adsorption within 20-30 seconds on a magnetic frame, and the solution becomes clear; (3) the structure is stable, and the magnetic bead structure is not damaged after 300W ultrasound for 5 min; (4) and (3) washing the magnetic beads with absolute ethyl alcohol for three times, drying at 80 ℃, and performing thermogravimetric analysis for 5 times, wherein the deviation of the 5 times thermogravimetric results is within 3%.
Example 2
The water consumption of the solution B in the second step of the example 1 is adjusted to 18mL, the pressure is adjusted to 0.6MPa, the temperature is increased to 210 ℃, the reaction time is prolonged to 20h, other process steps, parameters and operations are unchanged, and finally the performance and the structure of the magnetic beads obtained by the experiment are similar to those of the example 1.
Comparative example 1
The water consumption of the solution B in the second step of the embodiment 1 is increased to 25mL, and other process steps and raw material proportions are unchanged, so that the magnetic composite microspheres cannot be synthesized; the reason why the synthesis is not possible is that the water content is too high, and the reaction rapidly generates Fe (OH)3Large particle precipitation, which can not be dehydrated and can convert crystal form to generate magnetic Fe under the condition of existence of a large amount of water3O4。
Comparative example 2
The temperature of the second step of high-temperature synthesis in example 1 is reduced to 145 ℃, and other process conditions and material ratios are unchanged, so that the magnetic microspheres cannot be synthesized. The main reason for the failure of synthesis is that the temperature is too lowMethod for generating magnetic Fe by completing crystal transformation3O4。
Comparative example 3
The second step of example 1 reduces the synthesis time to 4h, and other process condition steps and material ratios are unchanged, so that the magnetic microspheres cannot be synthesized. The main reason for the failure of synthesis is that the crystal transformation to produce magnetic Fe cannot be completed in a short time3O4。
Comparative example 4
The water consumption of the second step solution B of example 1 was reduced to 5mL, and the other process steps and raw material consumption were unchanged.
The SEM image of the resulting magnetic composite microspheres is shown in FIG. 8, but the magnetic microspheres were produced, but the structure shown in FIG. 2 could not be obtained, and Fe was produced3O4Self-assembled into spherical particles of about 200nm and adhered to the surface of the PS microsphere, and the interior of the PS microsphere is free from Fe3O4。
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (11)

1. A preparation method of immunomagnetic beads is characterized by comprising the following steps:
s1, swelling the polystyrene microspheres;
s2, synthesis of magnetic composite microspheres: dispersing the polystyrene microspheres swelled by S1 and an alkaline reagent in an organic solvent X to form emulsion A; mixing iron salt, water and an organic solvent X to form a solution B; mixing the emulsion A and the solution B, adding a surfactant to form uniform emulsion, and reacting in a high-pressure reaction kettle to obtain magnetic composite microspheres;
s3, removing exposed magnetic particles on the surface of the magnetic composite microsphere through acid dissolution;
s4, performing surface cross-linking modification on the magnetic composite microspheres processed by the S3 to obtain products;
wherein the reaction temperature in the S2 is 150-250 ℃, the pressure of a reaction system is 0.3-3 MPa, the reaction time is 5-20h, and the volume ratio of the volume of the water in the S2 to the total volume of the organic solvent X used by the emulsion A and the solution B is (1-3): 10.
2. the method of claim 1, wherein the polystyrene microspheres in S1 are uniform microspheres with a particle size of 1-5 μm.
3. The method of claim 1, wherein the swelling of the polystyrene microspheres in S1 is performed by: dispersing polystyrene microspheres into water containing a surfactant, adding a solvent C and a swelling agent, performing ultrasonic emulsification, and stirring at a certain temperature.
4. The method of claim 3, wherein the surfactant is sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecyl benzene sulfonate, or sodium stearate; the solvent C is absolute ethyl alcohol; the swelling agent is dibutyl phthalate, ethyl acetate, butyl acetate, cyclohexane or toluene; the temperature is 40-80 ℃; the stirring time for stirring is 5-20 h.
5. The method of claim 1, wherein the basic reagent in S2 is sodium hydroxide, potassium hydroxide or ammonia water; the ferric salt in S2 is FeCl3、Fe(NO3)3、Fe2(SO4)3Or their crystalline hydrates; the organic solvent X in the S2 is ethylene glycol, glycerol, pentaerythritol, butanediol or neopentyl glycol; the surfactant in the S2 is anhydrous sodium acetate, potassium acetate, sodium propionate or sodium oleate.
6. The method of claim 1, wherein the emulsion A of S2 is prepared by dispersing the polystyrene microspheres obtained from S1 in NaOH solution of ethylene glycol, vigorously stirring, and subjecting to ultrasound.
7. The method of claim 1, wherein the solution B in S2 is obtained by mixing ethylene glycol, ferric chloride hexahydrate and water, and then subjecting the mixture to ultrasound.
8. The method of claim 1, wherein the molar weight ratio of the basic reagent to the iron salt in S2 is (0.5-2): 1; the molar weight ratio of the surfactant to the iron salt in the S2 is (1-10): 1.
9. the method of claim 1, wherein the magnetic composite microspheres in S3 are dissolved in an acidic solution, wherein the acidic solution is 0.1mol/L to 0.5mol/L hydrochloric acid solution, the dissolution temperature is 45 ℃ to 75 ℃, and the dissolution is performed at a stirring speed of 150r/min to 300 r/min.
10. The method of claim 1, wherein the cross-linking step in S4 comprises: dispersing the magnetic composite microspheres processed by S3 into SDS solution, adding a cross-linking agent, a functional monomer and an initiator, heating to 60-100 ℃ and reacting for 4-20 h; separating and washing to obtain a product; wherein the crosslinking agent is divinylbenzene; the functional monomer is methacrylic acid, acrylic acid or undecylenic acid; the initiator is azobisisobutyronitrile, azobisisoheptonitrile, benzoyl peroxide, lauroyl peroxide, dicumyl peroxide or potassium persulfate.
11. The immunomagnetic bead obtained by the preparation method according to any one of claims 1 to 10, wherein the immunomagnetic bead is structurally characterized by a magnetic composite microsphere core, a compact cross-linking layer interlayer and surface carboxyl functional groups, wherein the magnetic composite microsphere core is composed of PS and nano magnetic particles, the nano magnetic particles are dispersedly distributed in the PS microsphere, the compact cross-linking layer is coated on the outer layer of the magnetic composite microsphere core, and the carboxyl functional groups are distributed on the surface of the compact cross-linking layer.
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