Adsorption type polystyrene microsphere, preparation method and application of adsorption type polystyrene microsphere in detection of D-dimer

Abstract

The invention relates to the technical field of immunodetection, in particular to adsorption type polystyrene microspheres, a preparation method and application of the adsorption type polystyrene microspheres in detection of D-dimers, the preparation method comprises the following steps: 1) adding pure water into a flask, filling nitrogen to remove oxygen in a system, then adding a proper amount of anionic surfactant and a small amount of alkali to adjust the ionic strength of the system, and stirring to obtain a reaction solution; adding a styrene monomer and a halogenated hydrocarbon monomer, carrying out ultrasonic emulsification, transferring into a water bath kettle, and adding an initiator to react, so as to prepare polystyrene microspheres containing halogen groups on the surfaces; 2) dispersing polystyrene microspheres containing halogen groups on the surfaces into an acetonitrile solution, and adsorbing and removing water in a system by using a molecular sieve; under the action of K2CO3 and a catalyst KI, PVA and halogenated alkane are subjected to a nucleophilic substitution reaction, and the adsorption type polystyrene microspheres with the surfaces rich in hydroxyl groups are prepared. The polystyrene microspheres with non-functionalized surfaces are prepared, coated antibodies do not need to be activated, and the polystyrene microspheres are combined with D-dimer antibodies through electrostatic interaction.

Description

Technical Field

[0001] The present invention relates to the field of immunoassay technology, and specifically relates to an adsorption-type polystyrene microsphere, a preparation method and application thereof in detecting D-dimer. Background Art

[0002] D-dimer is a specific degradation product produced by the cross-linking of fibrin monomers by activated factor XIII and then hydrolyzed by plasmin. It is also a specific marker of the fibrinolytic process. D-dimer originates from the cross-linked fibrin clots dissolved by plasmin. Under pathological conditions, the dynamic balance between coagulation and fibrinolysis is disrupted, the coagulation tendency is enhanced, and thus the fibrin degradation products increase, leading to an increase in the D-dimer content. The increase in D-dimer levels indicates that fibrin thrombosis and fibrinolysis occur in the body, so it can be used clinically as a molecular marker of hypercoagulable state and hyperfibrinolysis in the body.

[0003] Common detection methods for D-dimer include latex agglutination, enzyme-linked immunosorbent assay and colloidal gold method. The principle of latex agglutination is that D-dimer in the plasma to be tested interacts with the monoclonal antibody coated on the latex particles to produce a flocculent precipitation reaction. Enzyme-linked immunosorbent assay uses two monoclonal antibodies against D-dimer as the center of the antigen, two antibody sandwich method, and adds a substrate of horseradish peroxidase for color development. Colloidal gold method uses the same antibody sandwich, that is, the coated antibody captures the antigen (D-dimer) in the plasma, and the same antibody coupled with colloidal gold is added for color development. Therefore, it is also centered on the antigen, and the antibodies are on both sides of the sandwich, but they are the same antibodies. Because the antibody has high specificity, it can bind to a variety of fragments containing D-dimer to increase the sensitivity of the test. Although it can occasionally bind to D fragments, no hooking phenomenon occurs.

[0004] However, all of the above methods require functionalized polystyrene microspheres to be activated by EDC, NHS, etc., and then to be chemically bonded to antibodies before detecting the target antigen, which is a cumbersome process. Summary of the invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide an adsorption type polystyrene microsphere and a preparation method thereof and its application in detecting D-dimer. The adsorption type polystyrene microsphere is used for biochemical immunoturbidimetry, the microsphere surface is enriched with hydroxyl groups, and it binds to the D-dimer antibody through electrostatic interaction, and the antigen to be detected can be directly detected without activating the coated antibody.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention first prepares polystyrene microspheres rich in alkyl halogen on the surface by emulsion polymerization, and then wraps PVA on the surface of the microspheres by nucleophilic substitution reaction of halogenated alkane and alcohol hydroxyl group, so that the surface of the microspheres is rich in a large number of hydroxyl groups, and a large number of PVA side chains are also present on the surface of the microspheres and dispersed in the aqueous solution, so as to prevent the microspheres from aggregating and improve the monodispersity of the microspheres. Then, the R1 reagent is prepared: the adsorption type polystyrene microspheres are mixed with trehalose, the base NaOH is added to adjust the pH to 7-8, and finally a small amount of preservative Proclin 300 is added; the R2 reagent is prepared: the D-dimer antibody is mixed with 4-hydroxyethylpiperazineethanesulfonic acid buffer (MESbuffer), sodium chloride, PEG-6000 and bovine serum albumin, and finally a small amount of Proclin 300 is added. The sample to be tested is sequentially mixed with R1 and R2, the difference between the initial absorbance and the measured absorbance is obtained, and the concentration of the D-dimer is obtained according to the standard curve.

[0008] Specifically, a method for preparing adsorption-type polystyrene microspheres comprises the following steps:

[0009] (1) preparing polystyrene microspheres containing halogen groups on the surface by emulsion polymerization: adding pure water to a reaction container, filling with nitrogen to remove oxygen in the system, adding an appropriate amount of anionic surfactant and a small amount of alkali to adjust the ionic strength of the system, adding styrene monomer and halogenated alkane monomer, ultrasonic emulsification, transferring to a constant temperature water bath, adding an initiator to react, and preparing polystyrene microspheres containing halogen groups on the surface;

[0010] (2) The super-hydrophilic hydroxylated polystyrene microspheres were prepared by the nucleophilic substitution method: the polystyrene microspheres containing halogen groups on the surface were dispersed in an acetonitrile solution, and the water in the system was removed by molecular sieve adsorption; under the action of K2CO3 and catalyst KI, PVA and halogenated alkanes underwent nucleophilic substitution reaction to obtain adsorbed polystyrene microspheres with rich hydroxyl groups on the surface.

[0011] Furthermore, in step (1), the anionic surfactant is sodium dodecyl sulfate, the base is NaOH, the halogenated alkane monomer is ethyl 2-chloromethylacrylate, and the initiator is ammonium persulfate or potassium persulfate.

[0012] Furthermore, in step (1), the volume ratio of pure water to styrene is 1:0.05-0.2, the volume ratio of styrene to halogenated alkane monomer is 1:0.01-0.2, the mass ratio of pure water to surfactant is 1:0.0001-0.001, the mass ratio of pure water to NaOH is 1:0.0001-0.0005, and the mass ratio of styrene to initiator is 1:0.008-0.02.

[0013] Furthermore, in the step (1), after ultrasonic emulsification for 30 minutes, the mixture is transferred to a constant temperature water bath, an initiator is added at 60° C., and the mixture is reacted at 70-85° C. for 5-10 hours.

[0014] Furthermore, in the step (2), the mass ratio of the polystyrene microspheres containing halogen groups on the surface to the PVA is 1:0.2-0.5.

[0015] Furthermore, in the step (2), the reaction is carried out at 60-80° C. for 6-12 hours.

[0016] The application of the adsorption type polystyrene microspheres prepared by the present invention in detecting D-dimer comprises the following steps:

[0017] (1) Preparation of R1 reagent: 1 g / L trehalose, 0.08%-0.12% w / w adsorption polystyrene microspheres, 0.02%-

[0018] 0.04% v / v Proclin 300, 1 mol / L NaOH to adjust pH to 7.0-8.0;

[0019] (2) Preparation of R2 reagent: 50 mM MES buffer, pH 6.5, 0.5 g / L NaCl, 5 g / L PEG-6000, 2 g / L BSA, 0.05 mg / mL D-dimer, 0.02%-0.04% v / v Proclin 300;

[0020] (3) The sample to be tested is mixed with R1 reagent and R2 reagent in sequence, with the sampling volume being 9 μL, 225 μL, and 75 μL, respectively. The difference between the initial absorbance and the measured absorbance is obtained, and the concentration of D-dimer is obtained according to the standard curve.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The method of the present invention prepares adsorption-type polystyrene microspheres with a particle size range of 180-240 nm. The coated antibody does not need to be activated, and the microspheres are combined with the D-dimer antibody through electrostatic interaction. The steps are simple and the D-dimer antigen to be detected can be quantitatively detected by biochemical immunoturbidimetry.

[0023] (2) The present invention prepares polystyrene microspheres with a surface rich in halogen groups by emulsion polymerization, and then wraps PVA on the surface of the microspheres through a nucleophilic substitution reaction between halogenated alkanes and alcohol hydroxyl groups, so that the surface of the microspheres is rich in a large number of hydroxyl groups. A large number of PVA side chains exist on the surface of the microspheres and are dispersed in the aqueous solution, which prevents the microspheres from aggregating and improves the monodispersity of the microspheres. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is a structural diagram of adsorption-type polystyrene microspheres.

[0025] Figure 2 For comparison and detection with the calibration samples of Examples 1-3.

[0026] Figure 3 This is the correlation curve of Example 1.

[0027] Figure 4 This is the correlation curve of Example 2.

[0028] Figure 5 This is the correlation curve of Example 3. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] (1) Preparation of adsorption-type polystyrene microspheres (PS180)

[0032] Add 200mL of pure water to a three-necked flask, pass nitrogen for 20 minutes to remove oxygen in the system, add 50mg of ionic strength regulator NaOH and 50mg of anionic surfactant SDS, and dissolve by ultrasonic. Then add 20mL of styrene (St) and 0.2mL of ethyl 2-chloromethylacrylate, ultrasonic emulsify for 30 minutes, then place in a constant temperature water bath, set the temperature to 75°C, add 200mg of initiator ammonium persulfate (APS) when the temperature reaches 60°C, stop the reaction for 6 hours to obtain microspheres PS180. Wash once with water and store in pure water.

[0033] Take 500 mg of the above microspheres and disperse them in 50 mL of acetonitrile solution. Use molecular sieve adsorption method to remove water in the system. Place them in a three-necked flask, add 50 mg of K2CO3 and a small amount of catalyst KI, and finally add 100 mg of polyvinyl alcohol PVA. React at 60°C for 6 h to prepare PS180. Wash with water twice, adjust the solid content to 10%, and store in pure water.

[0034] (2) Prepare R1 and R2

[0035] Preparation R1: 1 g / L trehalose, 0.08% (w / w) PS180, 0.04% Proclin 300, 1 mol / L NaOH to adjust pH to 7.5, the solvent is pure water.

[0036] Preparation R2: 50 mM MES buffer, pH 6.5, 0.5 g / L NaCl, 5 g / L PEG-6000, 2 g / L BSA, 0.05 mg / mL D-dimer, 0.04% (v / v) Proclin 300, the solvent is pure water.

[0037] (3) D-dimer antigen detection

[0038] Calibrator test: The volumes of the calibrator solution, R1, and R2 were 9 μL, 225 μL, and 75 μL, respectively, and the absorbance (△Abs) corresponding to the six calibrator concentrations was measured;

[0039] Sample testing: The volumes of sample, R1, and R2 were 9 μL, 225 μL, and 75 μL, respectively. The absorbance (△Abs) corresponding to the concentrations of the 12 samples was measured.

[0040] Example 2

[0041] (1) Preparation of adsorption-type polystyrene microspheres (PS240)

[0042] Add 200mL of pure water to a three-necked flask, pass nitrogen for 20 minutes to remove oxygen in the system, add 50mg of ionic strength regulator NaOH and 40mg of anionic surfactant SDS, and dissolve by ultrasonic. Then add 20mL of styrene (St) and 0.2mL of ethyl 2-chloromethylacrylate, ultrasonic emulsify for 30 minutes, then place in a constant temperature water bath, set the temperature to 75℃, add 200mg of initiator ammonium persulfate (APS) when the temperature reaches 60℃, stop the reaction for 6 hours to obtain microspheres PS240. Wash once with water and store in pure water.

[0043] Take 500 mg of the above microspheres and disperse them in 50 mL of acetonitrile solution. Use molecular sieve adsorption method to remove water in the system. Place them in a three-necked flask, add 50 mg of K2CO3 and a small amount of catalyst KI, and finally add 100 mg of polyvinyl alcohol PVA. React at 60°C for 6 h to prepare PS240. Wash with water twice, adjust the solid content to 10%, and store in pure water.

[0044] (2) Prepare R1 and R2

[0045] Preparation R1: 1 g / L trehalose, 0.08% (w / w) PS240, 0.04% Proclin 300, 1 mol / L NaOH to adjust pH to 7.5, the solvent is pure water.

[0046] Preparation R2: 50 mM MES buffer, pH 6.5, 0.5 g / L NaCl, 5 g / L PEG-6000, 2 g / L BSA, 0.05 mg / mL D-dimer, 0.04% (v / v) Proclin 300, the solvent is pure water.

[0047] (3) D-dimer antigen detection

[0048] Calibrator test: The volumes of the calibrator solution, R1, and R2 were 9 μL, 225 μL, and 75 μL, respectively, and the absorbance (△Abs) corresponding to the six calibrator concentrations was measured;

[0049] Sample testing: The volumes of sample, R1, and R2 were 9 μL, 225 μL, and 75 μL, respectively. The absorbance (△Abs) corresponding to the concentrations of the 12 samples was measured.

[0050] Example 3

[0051] (1) Preparation of adsorption-type polystyrene microspheres (PS300)

[0052] Add 200mL of pure water to a three-necked flask, pass nitrogen for 20 minutes to remove oxygen in the system, add 50mg of ionic strength regulator NaOH and 25mg of anionic surfactant SDS, and dissolve by ultrasonic. Then add 20mL of styrene (St) and 0.2mL of ethyl 2-chloromethylacrylate, ultrasonic emulsify for 30 minutes, then place in a constant temperature water bath, set the temperature to 75℃, add 200mg of initiator ammonium persulfate (APS) when the temperature reaches 60℃, stop the reaction for 6 hours to obtain microspheres PS300. Wash once with water and store in pure water.

[0053] Take 500 mg of the above microspheres and disperse them in 50 mL of acetonitrile solution. Use molecular sieve adsorption method to remove water in the system. Place them in a three-necked flask, add 50 mg of K2CO3 and a small amount of catalyst KI, and finally add 100 mg of polyvinyl alcohol PVA. React at 60°C for 6 h to prepare PS300. Wash with water twice, adjust the solid content to 10%, and store in pure water.

[0054] (2) Prepare R1 and R2

[0055] Preparation R1: 1 g / L trehalose, 0.08% (w / w) PS300, 0.04% Proclin 300, 1 mol / L NaOH to adjust pH to 7.5, the solvent is pure water.

[0056] Preparation R2: 50 mM MES buffer, pH 6.5, 0.5 g / L NaCl, 5 g / L PEG-6000, 2 g / L BSA, 0.05 mg / mL D-dimer, 0.04% (v / v) Proclin 300, the solvent is pure water.

[0057] (3) D-dimer antigen detection

[0058] Calibrator test: The volumes of calibrator solution, R1, and R2 were 9 μL, 225 μL, and 75 μL, respectively, and the absorbance (△Abs) corresponding to the six calibrator concentrations was measured;

[0059] Sample testing: The volumes of sample, R1, and R2 were 9 μL, 225 μL, and 75 μL, respectively. The absorbance (△Abs) corresponding to the concentrations of the 12 samples was measured.

[0060] The experimental results are as follows:

[0061] Table 1 △Abs test results of calibrators

[0062]

[0063] a: Commercially available DD kit.

[0064] Table 2 △Abs test results of samples

[0065]

[0066] a: Commercially available DD kit.

[0067] The structure of the prepared adsorption polystyrene microspheres is shown in Figure 1 As shown. Combined with Table 1, it can be seen that the test results of Example 1 are lower than those of the control, and the test results of Examples 2 and 3 are higher than those of the control, among which the test results of the calibrators of Examples 1 and 2 are relatively close to the control. Figure 2-4 It can be seen that the correlations of Example 1 and Example 2 are R 2 =0.992 and R 2 =0.9922, which has a good correlation, while Example 3 is presumably due to the large particle size, and the linearity and correlation are poor.

[0068] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing adsorption-type polystyrene microspheres, characterized in that: The following steps are involved: (1) preparing polystyrene microspheres containing halogen groups on the surface by emulsion polymerization: adding pure water to a reaction container, filling with nitrogen to remove oxygen in the system, adding an appropriate amount of anionic surfactant and a small amount of alkali to adjust the ionic strength of the system, adding styrene monomer and halogenated alkane monomer, ultrasonic emulsification, transferring to a constant temperature water bath, adding an initiator to react, and preparing polystyrene microspheres containing halogen groups on the surface; (2) The super-hydrophilic hydroxylated polystyrene microspheres were prepared by the nucleophilic substitution method: the polystyrene microspheres containing halogen groups on the surface were dispersed in an acetonitrile solution, and the water in the system was removed by molecular sieve adsorption; under the action of K2CO3 and catalyst KI, PVA and halogenated alkanes underwent nucleophilic substitution reaction to obtain adsorbed polystyrene microspheres with rich hydroxyl groups on the surface.

2. The method for preparing adsorption-type polystyrene microspheres according to claim 1, characterized in that: In the step (1), the anionic surfactant is sodium dodecyl sulfate, the base is NaOH, the halogenated alkane monomer is ethyl 2-chloromethylacrylate, and the initiator is ammonium persulfate or potassium persulfate.

3. The method for preparing adsorption-type polystyrene microspheres according to claim 2, characterized in that: In the step (1), the volume ratio of pure water to styrene is 1:0.05-0.2, the volume ratio of styrene to halogenated alkane monomer is 1:0.01-0.2, the mass ratio of pure water to surfactant is 1:0.0001-0.001, the mass ratio of pure water to NaOH is 1:0.0001-0.0005, and the mass ratio of styrene to initiator is 1:0.008-0.

02.

4. The method for preparing adsorption-type polystyrene microspheres according to claim 3, characterized in that: In the step (1), after ultrasonic emulsification for 30 minutes, the mixture is transferred to a constant temperature water bath, an initiator is added at 60° C., and the mixture is reacted at 70-85° C. for 5-10 hours.

5. The method for preparing adsorption-type polystyrene microspheres according to claim 4, characterized in that: In the step (2), the mass ratio of the polystyrene microspheres containing halogen groups on the surface to PVA is 1:0.2-0.

5.

6. The method for preparing adsorption-type polystyrene microspheres according to claim 5, characterized in that: In the step (2), the reaction is carried out at 60-80°C for 6-12 hours.

7. Adsorption-type polystyrene microspheres obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the adsorption-type polystyrene microspheres according to claim 7 in detecting D-dimer.

9. The use of the adsorption-type polystyrene microspheres in detecting D-dimer according to claim 8, characterized in that: The following steps are involved: (1) Prepare R1 reagent: 1 g / L trehalose, 0.08%-0.12% w / w adsorption polystyrene microspheres, 0.02%-0.04% v / v Proclin 300, and adjust the pH to 7.0-8.0 with 1 mol / L NaOH; (2) Preparation of R2 reagent: 50 mM MES buffer, pH 6.5, 0.5 g / L NaCl, 5 g / L PEG-6000, 2 g / L BSA, 0.05 mg / mL D-dimer, 0.02%-0.04% v / v Proclin 300; (3) The sample to be tested is mixed with R1 reagent and R2 reagent in sequence, with the sampling volume being 9 μL, 225 μL, and 75 μL, respectively. The difference between the initial absorbance and the measured absorbance is obtained, and the concentration of D-dimer is obtained according to the standard curve.

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