Hollow porous monodisperse carboxyl polystyrene microsphere, preparation method and application of hollow porous monodisperse carboxyl polystyrene microsphere in kit for quantitatively detecting MxA

Abstract

The invention relates to the technical field of in-vitro diagnostic kits, in particular to hollow porous monodisperse carboxyl polystyrene microspheres, a preparation method and application of the hollow porous monodisperse carboxyl polystyrene microspheres in a kit for quantitatively detecting MxA. The preparation method of the microspheres comprises the steps that firstly, hydrophilic polystyrene microspheres are prepared to serve as stable particles; then fully mixing the Pickering particles with a first oil phase to form Pickering stable particles; then a surfactant and a second oil phase are added, the mixed system is stirred at a high speed, and the second oil phase permeates into the surfaces or pores of Pickering stable particles through interface interaction and molecular diffusion; under the action of an initiator, the polymeric monomers are subjected to a polymerization reaction, the low-boiling-point organic solvent is evaporated out along with temperature rise, and a polymer with a core part of a hollow structure and a shell layer of a porous structure is formed; and adding a carboxyl-containing functional monomer to form a hydrophilic polymer chain on the surface of the polymer, thereby finally obtaining the hollow porous monodisperse carboxyl polystyrene microspheres.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of in vitro diagnostic kits, and in particular to a kind of hollow porous monodisperse carboxyl polystyrene microspheres and preparation method and its application in quantitative detection MxA kit. BACKGROUND

[0002] Recent studies have shown that MxA protein can be used as a biomarker for viral infection, to diagnose and distinguish between bacterial and viral infections, improve the efficiency of disease diagnosis, and reduce the misuse of antibiotics. According to a study published in the journal Infectious Diseases and Therapy, researchers evaluated the performance of blood tests that combined MxA and CRP measurements to distinguish between viral and bacterial respiratory tract infections. The study developed an equation that used the absolute values of MxA and CRP to calculate the probability of bacterial infection, and the rule provided a 91.6% sensitivity and 90.4% negative predictive value for the diagnosis of bacterial infection. Currently, the CRP quantitative detection kit on the market is very mature, but the MxA quantitative detection field still needs to be improved.

[0003] MxA protein, Myxovirus resistance protein A, is a 78-kd antiviral protein produced by human cells induced by type I interferon (IFN-α / β), which has the advantages of biological activity stability, direct antiviral effect, long half-life, good specificity, and easy detection. Human MxA protein is distributed in the cytoplasm of cells, and a large amount of research data shows that MxA protein is closely related to viral infection and is very sensitive to viruses, even a small amount of virus can induce cell expression of MxA protein, so MxA protein can be used for early diagnosis of viral infection, and can also be used for differential diagnosis of clinical viral infection and bacterial or other microbial infection.

[0004] Although there are currently many MxA detection kits on the market, they are mostly based on enzyme-linked immunoassay, colloidal gold, fluorescence chromatography, and the most suitable application mode for popularization is to use latex-enhanced immunoturbidimetry reagents with a full-automatic analyzer, but no such products have been released. The root cause is that MxA requires very high detection sensitivity, and the particle size of the conventional microspheres on the market is too low, and the dispersibility is not good.

[0005] The detection principle of the latex-enhanced immunoturbidimetry reagent is that carboxyl polystyrene microspheres are coupled with antibodies through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and then are immunoreacted with antigens, so that the signal of the to-be-detected substance is amplified, thereby precise detection is realized. As a key raw material for the latex-enhanced immunoturbidimetry, the carboxyl polystyrene microspheres on the market generally have a particle size range of 60-500 nm, and are generally prepared by emulsion polymerization or seed polymerization. The microspheres with small particle size generally have low sensitivity. In order to improve the sensitivity of antigen detection, microspheres with large particle size are generally selected. Although the microspheres with large particle size can improve the sensitivity, the microspheres with large particle size are very aggregated and settled during the storage process due to the effects of density and surface charge, and have poor monodispersity, which seriously affects the accuracy of experimental results.

[0006] The patent CN110790853B provides a preparation process of carboxyl polystyrene microspheres without cleaning, which comprises the following steps: pouring a functional monomer solution into an external aqueous phase solution at one time, the functional monomer being one or more of acrylic acid, methacrylic acid and ethyl acrylate, stirring, and then adding an initiator to initiate a polymerization reaction; (2) after 5 min-2 h of initiation of polymerization, a mixture of styrene and divinylbenzene is added at one time, and then the mixture is reacted for a period of time, and then the mixture is cooled and filtered to obtain carboxyl polystyrene microspheres. However, the carboxylated polystyrene prepared by this method has a small particle size, about 100-300 nm, which is difficult to meet the requirements of some high-sensitivity detection items of the latex immunoturbidimetry.

[0007] In summary, (1) the MxA protein concentration of healthy people is about 40 ng / mL (different methods will be slightly different, but will not differ by orders of magnitude), and it exists in peripheral blood cells. The sample type is whole blood, and at least 10 times or more dilution is required during detection, so the detection sensitivity, linearity, stability and other aspects of the reagent are required to be very high. (2) From the perspective of microspheres, small particle size is difficult to meet the sensitivity requirements; large particle size is easy to settle, and has poor monodispersity, which affects the accuracy of experimental results. SUMMARY

[0008] The first object of the present application is to provide a hollow porous monodisperse carboxyl polystyrene microsphere and a preparation method thereof.

[0009] The second object of the present application is to provide an application of the hollow porous monodisperse carboxyl polystyrene microsphere in a quantitative detection MxA kit.

[0010] To achieve the above object, the present application provides the following technical solutions:

[0011] A preparation method of a hollow porous monodisperse carboxyl polystyrene microsphere, in particular:

[0012] First, hydrophilic polystyrene microspheres containing sodium sulfonate groups on the surface are prepared by emulsion polymerization, which are used as stable particles for subsequent reactions; then, the stable particles are mixed with the first oil phase as the stabilizer of the system, and the stable particles self-assemble at the oil-water interface to form particles with Pickering stabilization, i.e. Pickering stable particles, wherein the first oil phase is a low-boiling organic solvent;

[0013] Subsequently, a small amount of surfactant is added to the system to further stabilize the reaction system, and a polymerization monomer and a low-boiling organic solvent are added as the second oil phase, and the mixed system is treated by high-speed stirring, and the second oil phase penetrates into the surface or pores of the Pickering stable particles through interfacial interaction and molecular diffusion; then, under the action of the initiator, the polymerization monomer is polymerized, and as the reaction temperature rises, the low-boiling organic solvent in the system is continuously heated and evaporated, and finally a polymer with a hollow structure in the core and a porous structure in the shell is formed.

[0014] After the above reaction continues for a period of time and the system reaction state is stable, a functional monomer containing a carboxyl group is added to the system, and a hydrophilic polymer chain is formed on the surface of the polymer through subsequent reactions, and finally hollow porous monodisperse carboxyl polystyrene microspheres are obtained.

[0015] Further, the following steps are included:

[0016] (1) Preparation of stable particles: polystyrene stable particles are prepared by emulsion polymerization, nitrogen is passed to remove oxygen in the system, a surfactant is added, and after stirring and dissolving, styrene monomer and hydrophilic monomer are added, and under the action of the initiator, the monomers begin to polymerize, and the reaction is carried out at 70-85°C for 4-10h.

[0017] (2) Preparation of hollow porous monodisperse carboxyl polystyrene microspheres: in a pure water medium, first pass nitrogen to remove oxygen, then ultrasonically disperse the stable particles prepared in step (1), then add a low-boiling organic solvent as the first oil phase, and place it in a water bath, and stir at high speed; then add a surfactant to reduce the surface tension, and then add a mixture of a polymerization monomer and a low-boiling organic solvent as the second oil phase, and stir at high speed, and under the action of the initiator, the monomers begin to polymerize, and after the reaction is carried out at 85°C for 2.5h, a functional monomer is added, and the functional monomer continues to polymerize, and the reaction is continued for 4-6h.

[0018] In step (1), the surfactant is an anionic surfactant, including sodium dodecyl sulfate or sodium dodecyl benzene sulfonate, preferably sodium dodecyl sulfate.

[0019] In step (1), the hydrophilic monomer is sodium styrene sulfonate, and the volume-to-mass ratio of styrene to sodium styrene sulfonate is 1:0.04-2.

[0020] In step (1), the initiator is a water-soluble initiator, including one or more of potassium persulfate, sodium persulfate, and ammonium persulfate, preferably potassium persulfate.

[0021] In step (2), the first oil phase includes n-hexane or cyclohexane, wherein the mass ratio of the first oil phase to the stable particles is 1:0.1-2; the surfactant is an anionic surfactant, including at least one of sodium dodecyl sulfate and sodium dodecylbenzene sulfonate, preferably sodium dodecyl sulfate.

[0022] In step (2), the second oil phase is a mixture of styrene monomer and low-boiling-point organic solvent in a volume ratio of 1:0.01-0.1; the initiator is a water-soluble initiator, including one or more of potassium persulfate, sodium persulfate, and ammonium persulfate, preferably ammonium persulfate.

[0023] In step (2), the functional monomer is a carboxyl-containing monomer, including one or more of acrylic acid and methacrylic acid, preferably acrylic acid; wherein the volume ratio of styrene monomer to functional monomer is 1:0.04-0.2.

[0024] The hollow porous monodisperse carboxylated polystyrene microspheres prepared by this invention have a particle size range of 0.4-0.6 μm, good monodispersity, and are not easy to settle. They can achieve the goal of highly sensitive quantitative detection of analytes in the field of biochemical latex-enhanced immunoturbidimetry.

[0025] The hollow porous monodisperse carboxylated polystyrene microspheres of this invention can be used to prepare a latex immunoturbidimetric assay kit for MxA protein. The latex preparation method is as follows:

[0026] (1) Microsphere dilution: The prepared microspheres were diluted with 50 mM pH 6.0 MES buffer to a concentration of 2 mg / mL;

[0027] (2) Activation: Weigh 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and prepare a stock solution of 10 mg / mL with 50 mM pH 6.0 MES buffer; add the EDC stock solution to the above solution to make the final concentration 0.5 mg / mL, and incubate at 37℃ with shaking for 0.5 hours. After completion, centrifuge at 13000 r / min for 25 min to remove the supernatant and resuspend in PB buffer of pH 7.2.

[0028] (3) Coupling: Dilute the MxA antibody to 1 mg / mL with PB buffer at pH 7.2 and add it to the activated microspheres at a volume ratio of 1:5. Incubate at 37°C with shaking for 2 hours.

[0029] (4) Blocking: and after completion, add bovine serum albumin to a final concentration of 1%, incubate at 37°C constant temperature for 1 hour; centrifuge the above-mentioned reagent after blocking to remove the supernatant, resuspend and mix evenly with 10 mM Tris buffer containing 4-8% trehalose at pH 7.5, and then disperse by ultrasonic, which is the MxA antibody-microsphere complex, namely R2 solution.

[0030] The kit prepared by the present application needs to be used together with an instrument, including but not limited to a biochemical analyzer, a blood cell instrument, a specific protein instrument, etc., and the recommended parameters of the biochemical analyzer are as follows: sample amount: sample / R1 / R2 = 12 / 120 / 40 μL, wavelength: secondary wave / main wave = 0 / 660 nm, including but not limited to the above parameters.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] (1) The present application prepares a hollow porous monodisperse carboxyl polystyrene microsphere with a particle size range of 0.4-0.6 μm. The microsphere has a hollow structure and a shell porous structure, and the surface carboxyl group is a long-chain structure. The three structures synergize to reduce the density of the microsphere, improve the hydrophilicity of the microsphere, and make the microsphere not easy to aggregate and settle in pure water, and the microsphere has good monodispersity.

[0033] (2) The hollow porous monodisperse carboxyl polystyrene microsphere can realize the purpose of accurately detecting target proteins in the latex immunoturbidimetric method for detecting MxA protein, and meets the high sensitivity requirement for the to-be-detected substance.

[0034] (3) Through comparison of various performances, it is found that the kit prepared by the hollow porous monodisperse carboxyl polystyrene microsphere of the present application shows significant advantages in analysis sensitivity, dispersity, stability and other performances. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is a flow chart for preparing the hollow porous monodisperse polystyrene microsphere.

[0036] Figure 2 It is the R2 accelerated stability of the example. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0038] Example 1

[0039] AsFigure 1 As shown in the preparation method of a kind of hollow porous monodisperse carboxyl polystyrene microspheres, specifically:

[0040] (1) stable particles 1: in three-necked flask, 100mL pure water is added, nitrogen is passed for 20min to remove oxygen in the system, 150mg of surfactant sodium dodecyl sulfate (SDS) is added, and is dissolved by ultrasonic. Again, 8mL of styrene (St) and 0.4g of sodium styrene sulfonate are added, and are emulsified by ultrasonic for 30min. Then, it is placed in a constant temperature water bath, the temperature is set to 75℃, and when the temperature reaches 60℃, 100mg of initiator potassium persulfate (KPS) is added. The reaction is stopped after 6h to prepare seed microspheres, which are stored in pure water.

[0041] (2) hollow porous monodisperse polystyrene microspheres 1: in three-necked flask, 100mL pure water is added, nitrogen is passed to remove oxygen, 1g of the stable particles prepared above is added, and is dispersed by ultrasonic. Then, 3mL of cyclohexane is added, and is stirred at high speed in water bath for 30min. Then, 30mg of surfactant sodium dodecyl sulfate is added and dissolved by stirring. Then, 10mL of styrene and 0.8mL of cyclohexane are added, and are stirred at high speed. When the temperature rises to 60℃, 100mg of initiator ammonium persulfate (APS) is added, and the reaction is carried out at 85℃ for 2.5h. Then, 1mL of acrylic acid is added dropwise, and the reaction is continued for 4h.

[0042] Example 2

[0043] As shown in the preparation method of a kind of hollow porous monodisperse carboxyl polystyrene microspheres, specifically: Figure 1

[0044] (1) stable particles 2: in three-necked flask, 100mL pure water is added, nitrogen is passed for 20min to remove oxygen in the system, 150mg of surfactant sodium dodecyl sulfate (SDS) is added, and is dissolved by ultrasonic. Again, 8mL of styrene (St) and 0.8g of sodium styrene sulfonate are added, and are emulsified by ultrasonic for 30min. Then, it is placed in a constant temperature water bath, the temperature is set to 75℃, and when the temperature reaches 60℃, 100mg of initiator potassium persulfate (KPS) is added. The reaction is stopped after 6h to prepare seed microspheres, which are stored in pure water.

[0045] (2) hollow porous monodisperse polystyrene microspheres 2: in three-necked flask, 100mL pure water is added, nitrogen is passed to remove oxygen, 1g of the stable particles prepared above is added, and is dispersed by ultrasonic. Then, 3mL of cyclohexane is added, and is stirred at high speed in water bath for 30min. Then, 25mg of surfactant sodium dodecyl sulfate is added and dissolved by stirring. Then, 15mL of styrene and 0.8mL of cyclohexane are added, and are stirred at high speed. When the temperature rises to 60℃, 100mg of initiator APS is added, and the reaction is carried out at 85℃ for 2.5h. Then, 1mL of acrylic acid is added dropwise, and the reaction is continued for 4h.

[0046] Example 3​

[0047] As shown in Figure 1 A method for preparing hollow porous monodisperse carboxyl polystyrene microspheres, specifically:

[0048] (1) Stable particles 3: 100 mL of pure water was added to a three-necked flask, and nitrogen was passed for 20 min to remove oxygen in the system. 150 mg of surfactant SDS was added and ultrasonically dissolved. Then 8 mL of styrene (St) and 1.2 g of sodium styrene sulfonate were added, and ultrasonic emulsification was performed for 30 min. Then it was placed in a constant temperature water bath, the temperature was set to 75℃, and when the temperature reached 60℃, 100 mg of initiator potassium persulfate (KPS) was added. The reaction was stopped after 6 h to prepare seed microspheres, which were stored in pure water.

[0049] (2) Hollow porous monodisperse polystyrene microspheres 3: 100 mL of pure water was added to a three-necked flask, and nitrogen was passed to remove oxygen. 1 g of the stable particles prepared above was added and ultrasonically dispersed. Then 3 mL of cyclohexane was added and placed in a water bath for high-speed stirring for 30 min. 10 mg of surfactant sodium dodecyl sulfate was added and stirred to dissolve. Then 20 mL of styrene and 0.8 mL of cyclohexane were added, and high-speed stirring was performed. When the temperature rose to 60℃, 100 mg of initiator APS was added, and the reaction was carried out at 85℃ for 2.5 h. 1 mL of acrylic acid was added dropwise, and the reaction was continued for 4 h.

[0050] Comparative Example 1

[0051] In a three-necked flask, 90 mL of ethanol and 5 mL of pure water were added, and nitrogen was passed for 20 min to remove oxygen in the system. 2 g of PVP K30 was added and stirred to dissolve. 100 mg of initiator AIBN was dissolved in 10 mL of St and 1 mL of acrylic acid, and then added to the flask. The reaction was carried out at 75℃ for 10 h.

[0052] Example 4

[0053] The MxA test kit (latex immunoturbidimetry) prepared by using the hollow porous monodisperse polystyrene microspheres includes:

[0054] Reagent R1:

[0055] MES buffer solution with pH 6.0 50 mM;

[0056] Protective agent BSA 20 mM;

[0057] Coagulation accelerator (PEG 20000) 1%;

[0058] Preservative (sodium azide) 0.1 g / L;

[0059] Reagent R2 (latex preparation method):

[0060] (1) Microsphere dilution: The microspheres prepared in Examples 1-3 and Comparative Example 1 were diluted with 50 mM MES buffer at pH 6.0 to a concentration of 2 mg / mL;

[0061] (2) Activation: EDC was weighed and prepared into a stock solution of 10 mg / mL with 50 mM MES buffer at pH 6.0; EDC stock solution was pipetted into the above solution to a final concentration of 0.5 mg / mL, and incubated at 37°C for 0.5 hours with constant shaking, after which the supernatant was removed by centrifugation at 13,000 r / min for 25 min, and resuspended with PB buffer at pH 7.2;

[0062] (3) Coupling: two MxA antibodies (Nanjing Liding Medical Technology Co., Ltd., Catalog No.: mAb03, mAb04) were each diluted to 1 mg / mL with PB buffer at pH 7.2, and added to the above activated microspheres at a volume ratio of 1:5, and incubated at 37°C for 2 hours with constant shaking;

[0063] (4) Blocking: After completion, 1% bovine serum albumin was added to a final concentration, and incubated at 37°C for 1 hour with constant shaking; the above blocked reagent was centrifuged to remove the supernatant, and resuspended with 10 mM Tris buffer containing 4-8% trehalose at pH 7.5, mixed well, and then dispersed by ultrasonic, to obtain the MxA antibody-microsphere complex, i.e. R2 solution;

[0064] Testing: R1 was used with R2 to test the calibrated sample on a specific protein analyzer. First, load sample diluent R3 (Nanjing Liding Medical Technology Co., Ltd., Catalog No.: LD-YX-013), reagent R1 and reagent R2 on the instrument. After the instrument sucks the whole blood sample, it will first be diluted 1:10 with the sample diluent, and after 300 s of reaction, reagent R1 will be added, and after 60 s of incubation at 37°C, reagent R2 will be added for incubation for 240 s. The instrument starts collecting data 10 s after adding R1, reads the first point A1, and after 240 s of incubation after adding R2, reads the second point A2. The absorbance of the sample A = A2-A1. The loading parameters are: main wavelength 660 nm, sample 12 μL, R1 120 μL, R2 40 μL.

[0065] The particle size and dispersion index of the hollow porous carboxyl polystyrene microspheres prepared in Examples 1-3 and the carboxyl polystyrene microspheres prepared by dispersion polymerization in Comparative Example 1 are shown in Table 1. The particle size of the microspheres in Comparative Example 1 was 517 nm, and the dispersion index was 0.048, indicating that the particle size of the microspheres in Comparative Example 1 was relatively uniform. The particle sizes of the microspheres in Examples 1-3 were 408 nm, 536 nm and 612 nm, respectively, and the dispersion indexes were all less than 0.05, indicating that the particle sizes of the microspheres had good uniformity.

[0066] Table 1 Particle size and dispersion index of microspheres

[0067]

[0068] Table 2 is the light absorbance result of latex R2 prepared in Example 4, which is detected by the specific protein analyzer with reagent Rl. The light absorbance value of latex R2 prepared by the microspheres of Comparative Example 1 is very low, and it has no correlation with the sample concentration value. The core reason for this phenomenon may be that during the test, after the latex is combined with reagent Rl, the microspheres rapidly aggregate and settle due to the insufficient "molecular arm length" on the surface of the microspheres and weak steric hindrance. The light absorbance of latex R2 prepared by the microspheres of Examples 1-3 has good correlation with the sample concentration value, and the analysis sensitivity gradually increases with the increase of particle size.

[0069] Table 2 Light absorbance of latex R2

[0070]

[0071]

[0072] Figure 2 The latex R2 prepared in Examples 1-3 is detected with Rl to test the accelerated stability of two samples after being accelerated at 37℃ for 0, 1, 3, 5, and 7 days. Since the analysis sensitivity of the microspheres of Comparative Example 1 is too low, it does not participate in the evaluation of accelerated stability. From the results, it can be seen that the accelerated stability of the latex prepared in the microspheres of Examples 1 and 2 is within 10% at 37℃ for 7 days, and the accelerated stability of the latex prepared in the microspheres of Example 3 is about 20% at 37℃ for 7 days, which may be related to the particle size.

[0073] Although the embodiments of the present application have been shown and described, it can be understood by those skilled in the art that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing hollow porous monodisperse carboxyl polystyrene microspheres, characterized by: First, hydrophilic polystyrene microspheres containing sodium sulfonate groups on the surface are prepared by emulsion polymerization, which are used as stable particles for subsequent reactions; then, the stable particles are mixed with the first oil phase as the stabilizer of the system, and self-assembly of the stable particles occurs at the oil-water interface to form particles with Pickering stability, namely Pickering stable particles, wherein the first oil phase is a low-boiling organic solvent; Subsequently, a small amount of surfactant is added to the system to further stabilize the reaction system, and polymerizable monomers and a low-boiling organic solvent are added as the second oil phase, and the mixed system is treated by high-speed stirring, and the second oil phase penetrates into the surface or pores of the Pickering stable particles through interfacial interaction and molecular diffusion; then, under the action of the initiator, the polymerizable monomers undergo polymerization reaction, and as the reaction temperature rises, the low-boiling organic solvent in the system is continuously heated and evaporated, and finally a polymer with a hollow structure in the core and a porous structure in the shell is formed; After the above reaction continues for a period of time and the system reaction state is stable, a functional monomer containing carboxyl groups is added to form hydrophilic polymer chains on the surface of the polymer through subsequent reactions, and finally hollow porous monodisperse carboxyl polystyrene microspheres are obtained.

2. The method for preparing hollow porous monodisperse carboxylated polystyrene microspheres according to claim 1, characterized in that, The method comprises the following steps: (1) Preparation of stable particles: polystyrene stable particles are prepared by emulsion polymerization, nitrogen is passed to remove oxygen in the system, a surfactant is added, and after stirring and dissolving, styrene monomers and hydrophilic monomers are added, and under the action of an initiator, the monomers begin to polymerize, and the reaction is carried out at 70-85°C for 4-10h; (2) Preparation of hollow porous monodisperse carboxyl polystyrene microspheres: in a pure water medium, first pass nitrogen to remove oxygen, then add the stable particles prepared in step (1) and ultrasonic dispersion, then add a low-boiling organic solvent as the first oil phase, place it in a water bath, and stir at high speed, then add a surfactant to reduce the surface tension, then add a mixture of polymerizable monomers and a low-boiling organic solvent as the second oil phase, and stir at high speed, then under the action of an initiator, the monomers begin to polymerize, then after 2.5h of reaction at 85°C, a functional monomer is added, and the functional monomer continues to polymerize, and the reaction is continued for 4-6h.

3. The method for preparing hollow porous monodisperse carboxylated polystyrene microspheres according to claim 2, characterized in that: In step (1), the surfactant is an anionic surfactant, including sodium dodecyl sulfate or sodium dodecyl benzene sulfonate.

4. The method for preparing hollow porous monodisperse carboxylated polystyrene microspheres according to claim 2, characterized in that: In step (1), the hydrophilic monomer is sodium styrene sulfonate, and the volume-to-mass ratio of styrene to sodium styrene sulfonate is 1:0.04-2.

5. The method for preparing hollow porous monodisperse carboxylated polystyrene microspheres according to claim 2, characterized in that: In step (1), the initiator is a water-soluble initiator, including one or more of potassium persulfate, sodium persulfate, and ammonium persulfate.

6. The method for preparing hollow porous monodisperse carboxylated polystyrene microspheres according to claim 2, characterized in that: In step (2), the first oil phase includes n-hexane or cyclohexane, and the mass ratio of the first oil phase to the stable particles is 1:0.1-2; the surfactant is an anionic surfactant, including at least one of sodium dodecyl sulfate and sodium dodecyl benzene sulfonate.

7. The method for preparing hollow porous monodisperse carboxylated polystyrene microspheres according to claim 2, characterized in that: In the step (2), the second oil phase is a mixture of styrene monomer and low boiling point organic solvent, the volume ratio is 1:0.01-0.1; the initiator is water-soluble initiator, including one or more of potassium persulfate, sodium persulfate, ammonium persulfate.

8. The method for preparing hollow porous monodisperse carboxylated polystyrene microspheres according to claim 7, characterized in that: In the step (2), the functional monomer is a carboxyl-containing monomer, including one or more of acrylic acid, methacrylic acid, wherein the volume ratio of styrene monomer to functional monomer is 1:0.04-0.

2.

9. The hollow porous monodisperse carboxyl polystyrene microspheres prepared by the process of any one of claims 1 to 8, characterized in that: The particle size range is 0.4-0.6 μm.

10. Use of the hollow porous monodisperse carboxyl polystyrene microspheres of claim 9 in the preparation of a latex immunoturbidimetry test kit for MxA protein.

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