CN112034164B - Chemiluminescence immune magnetic ball and preparation method thereof - Google Patents
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Abstract
The invention discloses a chemiluminescent immunomagnetic microsphere, which comprises a magnetic microsphere with a surface chemically modified with a p-toluenesulfonylated group, and is obtained by coupling with an antigen or an antibody. Also discloses a preparation method of the chemiluminescence immune magnetic microsphere, which comprises the following steps: polymerizing to obtain latex particles of polystyrene having a porous structure on the surface; mixing latex particles with a ferrous salt aqueous solution, and preparing a magnetic complex by an in-situ reduction method; carrying out hydroxylation modification on the magnetic complex to obtain a hydroxylated magnetic microsphere; and (3) reacting the hydroxylated magnetic microspheres with p-toluenesulfonyl chloride to obtain the p-toluenesulfonylated magnetic microspheres. The p-toluenesulfonylated magnetic microspheres prepared by the technology have high coupling rate to antibodies, and the coupled chemiluminescence immunoassay magnetic beads have uniform particle size, good magnetic responsiveness, high accuracy for luminescence detection and high thermal stability, and can meet the clinical use requirements.
Description
Technical Field
The invention belongs to the technical field of functional materials and analysis, and particularly relates to a chemiluminescent immune magnetic ball and a preparation method thereof.
Background
The chemiluminescence immune magnetic microsphere adopts the magnetic microsphere as a carrier, compared with other solid phase extraction materials, the magnetic material has the advantages of super paramagnetic property, small size, large surface area and the like, and can be fully contacted with an analyte in a solution to ensure high-efficiency adsorption. The magnetic material can be quickly separated from the mother liquor; the magnetic material can also be directly used for pretreatment of complex samples containing solid particles, microorganisms or high viscosity, and does not need steps such as filtration, centrifugation and the like in the middle. However, a simple magnetic material has a limited adsorption capacity, and it is necessary to modify the surface thereof appropriately to improve selectivity and adsorption performance. Common modifying groups comprise C18, phenyl, amino and polymer, however, modification of the groups often causes low efficiency of coupling antigen or antibody, low accuracy and poor thermal stability in subsequent detection and analysis processes.
Disclosure of Invention
The chemiluminescent immunomagnetic microspheres prepared by the invention and immunomagnetic beads coupled with antigen and antibody are used for chemiluminescent detection, and have high accuracy and good thermal stability.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the invention provides a chemiluminescence immune magnetic ball, which comprises a magnetic microsphere with a surface chemically modified with a p-toluenesulfonylation group, and is obtained by coupling with an antigen or an antibody.
Further, the magnetic microspheres are polystyrene magnetic microspheres.
In another aspect of the present invention, there is provided a method for preparing a chemiluminescent immunomagnetic sphere, comprising the steps of:
s1, polymerizing to obtain polystyrene latex particles with porous structures on the surfaces;
s2, mixing the latex particles obtained in the step S1 with an aqueous solution of ferrous salt, and preparing a magnetic complex by an in-situ reduction method;
s3, carrying out hydroxylation modification on the magnetic complex to obtain a hydroxylated magnetic microsphere;
s4, reacting the hydroxylated magnetic microspheres with p-toluenesulfonyl chloride to obtain p-toluenesulfonylated magnetic microspheres;
s5, mixing the p-toluenesulfonylated magnetic microspheres with an antigen or an antibody, and reacting for 6-8 hours to obtain the chemiluminescent immune magnetic spheres.
Specifically, the step S1 includes the following steps:
s11 preparation of seed emulsion
Polymerizing an emulsifier, distilled water, a monomer organic phase and an initiator to obtain a seed emulsion;
s12 Pre-emulsification of monomers
Mixing an emulsifier with a monomer organic phase, and fully stirring until a system presents a stable emulsion to obtain a monomer pre-emulsion;
s13 preparation of latex particles
Simultaneously dripping the initiator and the monomer pre-emulsion into the seed emulsion; heating to 84-86 ℃ for curing treatment; reducing the temperature to 40-45 ℃, and adjusting the pH value to be alkalescent to obtain an emulsion; and sieving the obtained emulsion to obtain the polystyrene latex particles.
Specifically, in the step of S11, the volume ratio of the monomer organic phase, water, the emulsifier and the initiator is 100 (500-1500) to (1-3) to (1-5).
Specifically, the step S3 includes the following steps:
s31, mixing the magnetic composite and epoxy polysiloxane, dripping glacial acetic acid at a constant speed under the conditions of ice bath and stirring, and stirring for reaction after dripping;
s32, suction filtering the reaction product, and sequentially adding deionized water, methanol, deionized water and 0.1M NaHCO3Washing with solution to obtain SiO on the surface2Drying the epoxy-coated magnetic microspheres, and diluting the epoxy-coated magnetic microspheres into suspension;
s33, adding mercaptoethanol into the suspension obtained in the step S32, oscillating for reaction, and washing to obtain the hydroxylated magnetic microspheres.
Specifically, the S4 includes the following steps:
s41, replacing a buffer system for storing the hydroxylated magnetic microspheres with an acetone solution, adjusting the pH value to be alkalescent, and cooling to-2-8 ℃ to obtain an acetone suspension of the hydroxylated magnetic microspheres;
s42, adding p-toluenesulfonyl chloride dropwise into the acetone suspension, stirring, after the reaction is finished, performing suction filtration, and washing with acetone and deionized water in sequence until the mixture is neutral to obtain the p-toluenesulfonylated magnetic microsphere.
Further, the reaction temperature in step S42 is controlled to be 20-30 ℃.
Further, the step S5 is carried out at a pH of 9-10, and the pH value is adjusted by ammonia water. Compared with the prior art, the invention has the advantages that:
the p-toluenesulfonylated magnetic microspheres prepared by the technology have high coupling rate to antibodies, and the coupled chemiluminescence immunoassay magnetic beads have uniform particle size, good magnetic responsiveness, high accuracy for luminescence detection and high thermal stability, and can meet the clinical use requirements.
Drawings
FIG. 1 is a SEM photograph provided in example 10 of the present invention
FIG. 2 is an SEM photograph provided in comparative example 1 of the present invention.
Fig. 3 is a particle size distribution diagram provided in example 10 of the present invention.
Fig. 4 is a graph showing the particle size distribution provided in comparative example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Preparation of chemiluminescence immune magnetic ball
1. Experimental Material
The main materials are as follows: styrene (St), chromatographically pure; 2-ethylhexyl acrylate (2-EHA), chromatographically pure; ethylene Glycol Dimethacrylate (EGDMA), chromatographically pure; toluene, dioctyl phthalate DOP, cyclohexanol, n-butanol, cetyl alcohol and lauryl alcohol are all chromatographically pure; NaHCO 23And analyzing the purity; triethylamine, analytically pure;
preparation of a monomer organic phase: uniformly mixing styrene, 2-ethylhexyl acrylate and ethylene glycol dimethacrylate, and adding a pore-foaming agent for mixing to prepare a monomer organic phase; wherein the pore-forming agent is selected from one of toluene, dioctyl phthalate DOP, cyclohexanol, n-butanol, cetyl alcohol or lauryl alcohol; the volume ratio of the styrene to the 2-ethylhexyl acrylate to the ethylene glycol dimethacrylate to the pore-foaming agent is (10-12) and 2:2 (0.1-0.5).
Preparing an emulsifier: 0.45g of CTAB cetyltrimethylammonium bromide was weighed out and dissolved in 9mL of redistilled water to prepare a 5% (w/v) aqueous solution.
Preparing an initiator: 0.05g of potassium persulfate (KPS) was dissolved in 1mL of redistilled water to prepare a 5% (w/v) aqueous solution.
Preparation of 0.1M aqueous sodium bicarbonate: 8.4g of sodium bicarbonate is weighed, dissolved in distilled water and made to volume of 1000 mL.
Preparation of 40% (w/v) ferrous chloride aqueous solution: weighing 40g FeCl2Dissolved in 60mL of deionized water.
Preparation of 75% (w/v) sodium citrate aqueous solution: 75g of sodium citrate was weighed out and dissolved in 25mL of deionized water.
Preparation of 15% (w/v) sodium nitrite aqueous solution: 15g of sodium nitrite, NaNO2, was weighed into 85mL of deionized water.
2. Experimental methods
2.1 preparation of polystyrene latex particles
(1) Preparation of seed emulsion
Installing a thermometer, a condenser pipe and an inert gas inlet pipe on a four-neck flask, mixing an emulsifier and secondary distilled water in the flask, stirring to dissolve the emulsifier and the secondary distilled water, and adjusting the pH value of a reaction system to 7.0-8.0 by using 0.1M sodium bicarbonate; dripping a monomer organic phase, and after dripping, stirring and emulsifying at high speed for 15min to form white uniform non-layered emulsion; introducing nitrogen into the bottle for 5min (controlling the airflow to blow into the system at a flow rate not more than 5L/s, and reducing monomer loss because the air outlet does not aim at the liquid level); heating to 60-80 ℃, reducing the stirring speed to 200-220 r/min, taking the initiator, and slowly dripping the initiator into a four-neck flask at a constant speed within 30 min. And after the initiator is dripped, continuously preserving the heat for 15min to prepare the seed emulsion.
Wherein the emulsifier is 1-3% (v/v) of the monomer organic phase, the initiator accounts for 1-5% (v/v) of the monomer organic phase, and the volume ratio of the water phase to the monomer organic phase is (15-5) to 1. The volume ratio of the monomer organic phase, water, emulsifier and initiator is 100 (500-1500) (1-3) (1-5).
(2) Pre-emulsification of monomers
And adding an emulsifier and a monomer organic phase into a single-neck flask, fully stirring and emulsifying, wherein the emulsified system presents stable pre-emulsion (when the room temperature is low, the emulsification effect is poor under the condition, the layering is easy, the emulsification can be carried out under the condition of heating to 35-40 ℃, or the pre-emulsification is carried out under the condition of heating by ultrasound). Wherein the volume ratio of the emulsifier to the monomer organic phase is 1 (2-4).
(3) Preparation of latex particles
Continuously and uniformly dripping an initiator and the pre-emulsion (obtained in the step (2)) into the seed emulsion (obtained in the step (1)) through two feeding pipes at the temperature of between 60 and 80 ℃; heating to 85 +/-1 ℃, and curing for 1 h; reducing the temperature to 45 ℃, and adjusting the pH value to 7-8 by using triethylamine; and (3) passing the obtained emulsion through a copper mesh screen to obtain polystyrene latex particles with the particle size of about 200-500 nm.
2.2 Fe3O4Preparation of magnetic microparticles
Mixing a suspension (with a solid content of 30%) of polystyrene latex particles, a ferrous chloride solution and an ammonium acetate solution in a three-neck flask in a nitrogen atmosphere, stirring the mixed solution, heating to 70 ℃, and adjusting the pH value of the mixed solution to 7.0-7.2 by using ammonia water;
slowly and uniformly dripping 15mL of sodium nitrite solution into the reaction liquid through a constant-pressure dropping funnel, keeping the reaction temperature at 70 ℃ after dripping is finished for about 1 hour, keeping the whole reaction process in an oxygen-free atmosphere, maintaining the pH value of the reaction liquid at 7.0-7.2, cooling to room temperature after the reaction is finished, performing suction filtration, repeatedly washing the reaction product in an ultrasonic cleaning machine for 5 times with deionized water, 60s each time, and finally drying for 24 hours at 40 ℃ in a vacuum drying oven to obtain a magnetic complex.
2.3 surface hydroxyl modification of magnetic microparticles
Mixing the magnetic complex obtained in step 2.2 with epoxy-terminated polysiloxane, slowly and uniformly dripping glacial acetic acid under the condition of stirring ice bath, and continuously stirring and reacting at room temperature for 3 hours after dripping; wherein the mixing ratio of the magnetic composite and the epoxy-terminated polysiloxane is (3-6) g:50 ml.
The reaction product is filtered, washed with deionized water, methanol and deionized water three times, and then washed with 0.1M NaHCO3Washing with the solution for 5 times to obtain SiO on the surface2Coating epoxy group magnetic microsphere, vacuum drying, and adding 0.1M NaHCO3Diluted to contain surface SiO25% (w/w) of epoxy-coated magnetic microspheres.
Mercaptoethanol was added to the suspension and after 10 minutes of shaking the reaction was carried out with 300ml of 0.1M NaHCO3Washing the solution for 3 times, and then washing the solution for 5 times by using deionized water to obtain the hydroxylated magnetic microspheres. Mercaptoethanol surface SiO2The proportion of the epoxy-coated magnetic microspheres is (2-3) g:5 mL.
2.4 magnetic microsphere para-toluenesulfonylation modification
Replacing a buffer system for storing the hydroxylated magnetic microspheres with an acetone solution, adjusting the pH to 8-9, stirring, uniformly mixing, and cooling to 0 ℃ to obtain an acetone suspension of the hydroxylated magnetic microspheres;
and dropwise adding p-toluenesulfonyl chloride into the acetone suspension, stirring, performing suction filtration after the reaction is finished, and washing the solution to be neutral by using acetone and deionized water in sequence to obtain the p-toluenesulfonylated magnetic microsphere.
Wherein, the p-toluenesulfonyl chloride is added in batches, the adding amount is not more than 0.1g/30ml of the hydroxylated magnetic microspheres each time, the total p-toluenesulfonyl chloride added is not less than 0.5g/30ml of the hydroxylated magnetic microspheres, the dropwise addition is finished at room temperature within 1.5h, and the mixture is stirred and mixed uniformly. After the reaction, the mixture is filtered, washed to be neutral by acetone and deionized water respectively, and stored at 4 ℃. In order to reduce side reactions, the reaction temperature is preferably controlled to be 20-30 ℃.
2.5 preparation of chemiluminescent immunomagnetic spheres
And (3) mixing the p-toluenesulfonylated magnetic microsphere obtained by the preparation method in the embodiment with an antigen or an antibody in an ammonia environment, and reacting for 6-8 hours to complete coupling to obtain the chemiluminescent immunomagnetic sphere. Compared with the traditional coupling process, the coupling process needs more than 24 hours, and the coupling time is greatly shortened.
Specifically, the present invention employs the above experimental method, and the following examples and comparative examples are performed as shown in table 1. In Table 1, the volume ratio of the monomer organic phase, water, emulsifier and initiator in the preparation process of the seed emulsion is represented as A, the volume ratio of the emulsifier in the monomer pre-emulsification process to the monomer organic phase is represented as B, the mixing ratio of the magnetic complex and the epoxy-terminated polysiloxane in the surface hydroxyl modification process of the magnetic particles is represented as C, and the ratio of the mercaptoethanol to the surface SiO in the surface hydroxyl modification process of the magnetic particles is represented as C2The proportion of the epoxy-coated magnetic microspheres is marked as D. The reaction conditions in step S5 are denoted as E, in particular the pH and the reagents used for adding the pH
TABLE 1
Performance characterization of p-toluenesulfonylated magnetic microspheres and chemiluminescent immunomagnetic spheres
The invention performs particle size and appearance characterization, magnetic characterization and functional group characterization on the p-toluenesulfonylated magnetic microspheres, and evaluates the coupling effect of the chemiluminescent immunomagnetic spheres prepared from the p-toluenesulfonylated magnetic microspheres.
1. Particle size and morphology characterization
SEM scanning electron microscope, 1HMR nuclear magnetic spectrum and thermogravimetric analysis TGA are adopted to evaluate the performance of the p-toluenesulfonylated magnetic microsphere. The p-toluenesulfonylated magnetic microsphere provided by the invention is in a sphere-like shape (as shown in figure 1) through the observation of a scanning electron microscope, and the surface pore-forming effect is obvious. The particle size distribution is 1-12 μm (as shown in figure 3), and the surface of the benzene sulfonyl group is coated with modified particle size. As shown in FIGS. 2 and 4, the particle size distribution and magnetic sphere effect of the p-toluenesulfonylated magnetic microsphere prepared by the present invention are significantly better than those of the comparative examples.
2. Functional group characterization
The changes of the functional groups of the magnetic material were characterized by a Nicolet6700 type Fourier Infrared spectrometer (ThermoFisher Co., USA), and the infrared pattern of the magnetic beads was 1375-1And 1200cm-1The absorption peaks are respectively the absorption peaks of a benzene ring and S ═ O, and the method provided by the invention shows that the p-toluenesulfonylated magnetic microsphere is successfully obtained.
3. Coupling effect of chemiluminescent immunomagnetic spheres
3.1 coupling Process
1) Adding (60ug/15 muL AFP) antibody, (3mg/30 muL) magnetic beads, 200 muL 1M ammonia water and 355 muL coating solution into a 2mL round-bottom centrifuge tube in sequence to make the final concentration of the magnetic beads be 5 mg/mL; finally, the coupling volume is 600 mu L, and the mixture is stirred and mixed for 6-8 h at 37 ℃;
2) adsorbing the magnetic beads for 2min by a magnetic frame, and adsorbing the coating liquid;
3) washing with one volume of magnetic bead diluent for 4 times, and rotating at 37 deg.C for 5min each time;
4) sealing with magnetic bead sealing solution for 4h, and rotating at 37 deg.C for 4 h;
5) washing with one-time volume of magnetic bead diluent for 4 times, and rotating at 37 deg.C for 5min each time;
6) the storage method comprises the following steps: the coupled magnetic beads were diluted to 50mL of a magnetic bead dilution (magnetic bead concentration: 120. mu.g/mL) and stored at 4 ℃ until use.
And (3) carrying out protein quantitative analysis on the supernatant before and after coupling by using a Bradford method, and calculating the coupling efficiency.
3.2 reagent configuration
1. Protein standard solution: (Note: the dilution is preferably a sample-dissolving solution, but for convenience, 0.9% NaCl or PBS may be used)
(ii) 1 tube protein standard (30mg BSA) +1.2ml protein standard preparation-fully dissolved → 25mg/ml protein standard. (-20 ℃ C. storage)
② 20 mul 25mg/ml protein standard solution plus 980 mul diluent-fully mixed → 0.5mg/ml protein standard solution. (-20 ℃ C. storage)
2. BCA working solution: and (4) fully mixing the 50V reagent A +1V reagent B- → the BCA working solution. The temperature is stable for 24 h.
3.3 magnetic bead supernatant protein concentration determination (96-well plate)
1. And (3) standard substance: adding 0.5mg/ml protein standard: 0.1, 2, 4, 8, 12, 16, 20 μ l; add standard dilutions to 20 μ l: 20. 19, 18, 16, 12, 8, 4, 0 μ l (i.e. concentration gradient: 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/ml).
2. Testing and sample adding: the appropriate volume of sample is added, and if less than 20. mu.l, the volume is made up to 20. mu.l with standard dilution.
3. And (3) reagent reaction: mu.l of BCA working solution was added to each well and left at 37 ℃ for 20-30 minutes (room temperature 2 hours; 60 ℃ for 30 min).
4. And (3) detection: assay A562 with microplate reader. (540-595 nm). A standard curve was drawn and the protein concentration was calculated. Coupling ratio (pre-coupling protein concentration-post-coupling protein concentration) x supernatant volume/magnetosphere volume. The p-toluenesulfonylated magnetic microspheres prepared in examples 1 to 13 and comparative examples 1 to 10 were coupled to give chemiluminescent microspheres, the coupling efficiency and coupling time of which are shown in Table 2.
5. Evaluation of luminescent Properties in chemiluminescent immunoassays
1) The chemiluminescence microspheres obtained in the above steps are prepared into 150ug/mL working solution.
2) AFP enzyme marker working solution concentration: 0.5 ug/mL;
3) 50 mul of working solution and 50 mul of AFP enzyme marker working solution are added into a reaction reagent tube and placed for 30min at 4 ℃. Wherein, the AFP calibrator concentration is 1ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 800ng/mL, and the linear correlation coefficient is obtained by machine measurement and linear fitting of measured values.
4) The AFP calibrants of 1ng/mL, 10ng/mL, 50ng/mL, 100ng/mL and 800ng/mL are used as upper samples, the magnetic beads provided by the embodiments 1-13 of the invention and the comparative examples 1-10 are respectively adopted for detection, the machine is used for detection, and the detection average deviation (as initial deviation) of the standards with different concentrations corresponding to the various magnetic beads is respectively calculated.
5) Further, the benzenesulfonyl magnetic beads prepared in examples 1 to 13 and comparative examples 1 to 10 provided by the invention were stored in a 5mL centrifuge tube containing a working solution, 4mL of each tube was subjected to labeling and sealing, the centrifuge tube was placed at 37 ℃ and 4 ℃ respectively, and after being placed for 6 days, the step of 5) was performed again, the detection was performed on the machine, and the average detection deviation of the standards with different concentrations corresponding to the various magnetic beads was calculated. The results are shown in Table 2
TABLE 2
As can be seen from Table 2:
1. comparative examples 1 to 6 show that, compared with example 10, the volume ratio of the monomer organic phase, water, emulsifier and initiator in the preparation process of the seed emulsion is not in the range of 100 (500-1500) to (1-3) to (1-5), so that the coupling rate and the linear dependence are remarkably reduced, and the initial average deviation, the 4 ℃ average deviation and the 37 ℃ average deviation are remarkably increased. The method shows that in the preparation process of the seed emulsion, the proper proportion of the monomer organic phase, the water, the emulsifier and the initiator is added, so that the coupling rate of the finally-worthy p-toluenesulfonylated magnetic microspheres can be improved, the detection accuracy of the p-toluenesulfonylated magnetic microspheres is improved, and the stability of the p-toluenesulfonylated magnetic microspheres can be improved.
2. Compared with the embodiment 10, the volume ratio of the emulsifier to the monomer organic phase in the monomer pre-emulsification process of the comparative examples 7 and 8 is not in the range of 1 (2-4), the coupling rate and the linear correlation are improved compared with the comparative examples 1-6, and the volume ratio is still obviously lower than that of the embodiment 10; the initial mean deviation, the 4 ℃ mean deviation, and the 37 ℃ mean deviation for each of comparative examples 7, 8 were reduced relative to comparative examples 1-6, but were still significantly higher than example 10. This shows that the monomer pre-emulsification process needs to be controlled on the premise of controlling the volume ratio of the monomer organic phase, water, emulsifier and initiator in the preparation process of the seed emulsion.
3. Comparative examples 9, 10, 11 and 12 are examples 10, respectively, in which the magnetic composite body and the terminal epoxy group were reacted during the modification of the surface hydroxyl group of the magnetic fine particlesThe mixing ratio of polysiloxane is not controlled to be (3-6) g:50ml, mercaptoethanol: surface SiO2The proportion of the epoxy-coated magnetic microspheres is not controlled to be (2-3) g:5 ml. Comparative examples 9, 10, 11 and 12 each had an improved coupling ratio and linear dependence relative to comparative examples 7-8, but were still significantly lower than example 10; the initial mean deviation, the 4 ℃ mean deviation, and the 37 ℃ mean deviation for each of comparative examples 9, 10, 11, and 12 were reduced relative to comparative examples 7-8, but were still significantly higher than example 10. This shows that, on the premise of controlling the volume ratio of the monomer organic phase, water, emulsifier and initiator in the preparation process of the seed emulsion, the surface hydroxylation modification of the magnetic microspheres is controlled, and further the further benzene sulfonylation modification process is indirectly controlled (only the p-toluenesulfonyl chloride is not less than 0.5g/30ml of the hydroxylated magnetic microspheres), so that the initial average deviation, the 4 ℃ average deviation and the 37 ℃ average deviation of the obtained p-toluenesulfonylated magnetic microspheres are further reduced, and the higher coupling rate is ensured.
4. Example 10 with respect to examples 1-9, the volume ratio of the monomer organic phase to water to the emulsifier to the initiator during the preparation of the seed emulsion, the volume ratio of the emulsifier to the monomer organic phase during the monomer pre-emulsification, the mixing ratio of the magnetic complex to the epoxy-terminated polysiloxane during the modification of the surface hydroxyl groups of the magnetic particles, and the ratio of mercaptoethanol to surface SiO2The proportion of the epoxy-coated magnetic microspheres is optimized, so that the coupling effect is better, and the stability of immunoassay is better. In examples 11 and 12, on the basis of example 10, the antibody coupling process is optimized, the coupling effect is better, and the immunoassay stability is better. In addition, in comparative examples 13 and 14, the surface benzenesulfonyl and sulfonylation modified immune microspheres are synthesized by the existing method, the antibody coupling process needs a coupling agent, the coupling time is longer, and the stability of immunoassay is poor.
In summary, the tosylated magnetic microsphere prepared by the technology has high coupling rate to the antibody, the chemiluminiscence immunoassay magnetic bead obtained after coupling has uniform particle size and good magnetic responsiveness, and the chemiluminiscence immunoassay magnetic bead has high accuracy and high thermal stability and can meet the clinical use requirement.
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
Claims (3)
1. A preparation method of a chemiluminescence immune magnetic ball is characterized in that the chemiluminescence immune magnetic ball is obtained by coupling a magnetic microsphere with a surface chemically modified by a p-benzenesulfonyl group and an antigen or an antibody; the preparation method comprises the following steps:
s1, polymerizing to obtain polystyrene latex particles with porous structures on the surfaces;
s2, mixing the latex particles obtained in the step S1 with an aqueous solution of ferrous salt, and preparing a magnetic complex by an in-situ reduction method;
s3, carrying out hydroxylation modification on the magnetic complex to obtain a hydroxylated magnetic microsphere;
s4, reacting the hydroxylated magnetic microsphere with p-toluenesulfonyl chloride to obtain a p-toluenesulfonylated magnetic microsphere;
s5, mixing the p-toluenesulfonylated magnetic microspheres with an antigen or an antibody in an ammonia water environment, and reacting for 6-8 hours to obtain the chemiluminescent immunomagnetic spheres;
the step of S1 includes the steps of:
s11 preparation of seed emulsion
Polymerizing an emulsifier, distilled water, a monomer organic phase and an initiator to obtain a seed emulsion; mixing an emulsifier and secondary distilled water in a flask, stirring to dissolve the emulsifier, and adjusting the pH value of a reaction system to 7.0-8.0 by using 0.1M sodium bicarbonate; dripping a monomer organic phase, and after dripping, stirring and emulsifying at high speed for 15min to form white uniform non-layered emulsion; introducing nitrogen into the bottle for 5 min; heating to 60-80 ℃, reducing the stirring speed to 200-220 r/min, taking an initiator, slowly and uniformly dripping the initiator into a flask bottle within 30min, and continuing to keep the temperature for 15min after finishing dripping the initiator to prepare a seed emulsion; the volume ratio of the monomer organic phase, water, emulsifier and initiator is 100 (500-1500) to (1-3) to (1-5); wherein, the preparation of the emulsifier: weighing 0.45g of cetyl trimethyl ammonium bromide CTAB, and dissolving in 9mL of secondary distilled water to prepare 5% (w/v) aqueous solution; preparing an initiator: 0.05g of potassium persulfate (KPS) is weighed and dissolved in 1mL of secondary distilled water to prepare 5% (w/v) aqueous solution; preparation of a monomer organic phase: uniformly mixing styrene, 2-ethylhexyl acrylate and ethylene glycol dimethacrylate, and adding a pore-foaming agent to prepare a monomer organic phase; wherein the pore-foaming agent is selected from one of toluene, dioctyl phthalate DOP, cyclohexanol, n-butyl alcohol, hexadecanol or dodecanol; the volume ratio of the styrene to the 2-ethylhexyl acrylate to the ethylene glycol dimethacrylate to the pore-foaming agent is (10-12) and 2:2 (0.1-0.5);
s12 Pre-emulsification of monomers
Mixing an emulsifier with a monomer organic phase, and fully stirring until a system presents a stable emulsion to obtain a monomer pre-emulsion; the volume ratio of the emulsifier to the monomer organic phase is 1 (2-4);
s13 preparation of latex particles
Dropping the initiator and the monomer pre-emulsion into the seed emulsion at the temperature of between 60 and 80 ℃; heating to 84-86 ℃ for curing treatment for 1 h; reducing the temperature to 40-45 ℃, and adjusting the pH value to be alkalescent to obtain an emulsion; sieving the obtained emulsion to obtain polystyrene latex particles with the particle size of 200-500 nm;
the step of S2 includes:
mixing a suspension of polystyrene latex particles, a ferrous chloride solution and an ammonium acetate solution in a three-neck flask in a nitrogen atmosphere, stirring the mixed solution, heating to 70 ℃, and adjusting the pH value of the mixed solution to 7.0-7.2 by using ammonia water; slowly and uniformly dripping 15mL of sodium nitrite solution into the reaction solution through a constant-pressure dropping funnel, keeping the reaction temperature at 70 ℃ after dripping is finished for about 1 hour, keeping the whole reaction process in an oxygen-free atmosphere, maintaining the pH value of the reaction solution between 7.0 and 7.2, cooling to room temperature after the reaction is finished, performing suction filtration, repeatedly washing the reaction product for 5 times in an ultrasonic cleaning machine by using deionized water for 60s each time, and finally drying in a vacuum drying oven at 40 ℃ for 24 hours to obtain a magnetic complex;
the step of S3 includes the steps of:
s31, mixing the magnetic composite and epoxy-terminated polysiloxane, dropwise adding glacial acetic acid at a constant speed under the conditions of ice bath and stirring, and stirring to react for 3 hours after dropwise adding; wherein the mixing ratio of the magnetic composite and the epoxy-terminated polysiloxane is (3-6) g:50 ml;
s32, suction filtering the reaction product, washing with deionized water, methanol and deionized water for three times, and then 0.1M NaHCO3Washing with the solution for 5 times to obtain SiO on the surface2Coating epoxy group magnetic microsphere, drying and using 0.1M NaHCO3Diluted to contain surface SiO25% (w/w) of suspension of epoxy-coated magnetic microspheres;
s33, mercaptoethanol is added into the suspension obtained in the step S32, after shaking reaction for 10 minutes, 300mL0.1M NaHCO is added3Washing the solution for 3 times, and then washing the solution for 5 times by using deionized water to obtain hydroxylated magnetic microspheres; mercaptoethanol surface SiO2The proportion of the epoxy-coated magnetic microspheres is (2-3) g:5 mL;
the S4 includes the following steps:
s41, replacing a buffer system for storing the hydroxylated magnetic microspheres with an acetone solution, adjusting the pH value to be alkalescent, and cooling to-2-8 ℃ to obtain an acetone suspension of the hydroxylated magnetic microspheres;
s42, adding p-toluenesulfonyl chloride dropwise into the acetone suspension, stirring, after the reaction is finished, performing suction filtration, and washing with acetone and deionized water in sequence until the mixture is neutral to obtain the p-toluenesulfonylated magnetic microsphere.
2. The method according to claim 1, wherein the reaction temperature in step S42 is controlled to 20 to 30 ℃.
3. The method of claim 1, wherein the step S5 is performed at a pH of 9-10, and the pH is adjusted by ammonia water.
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