PS latex particle with high reactivity with pepsinogen antibody as well as preparation method and application of PS latex particle

Abstract

The invention belongs to the field of preparation of affinity polymer microspheres, and relates to PS latex particles with high reactivity with a pepsinogen antibody as well as a preparation method and application of the PS latex particles. Crosslinked polystyrene is adopted as an inner core, the structural stability and the refractive index property of the latex particle are guaranteed, the acrylate monomer and the acrylic copolymer are adopted as a shell layer, adjustable carboxyl reaction sites are provided on the surface of the latex particle, and the polyacrylate component with the low glass transition temperature has the rubbery state characteristic, so that the stability of the latex particle is improved, and the service life of the latex particle is prolonged. The carboxyl on the shell layer has higher reaction efficiency. The carboxylated PS latex nanoparticles can be used as a raw material of a kit for latex turbidimetry detection of gastric cancer markers, the reaction efficiency of latex particles and pepsinogens (PGI and PGII) is improved, the detection performance of immunoturbidimetry is enhanced, and the obtained latex kit has increased detection sensitivity and a linear range.

Description

Technical Field

[0001] The present invention belongs to the field of preparation of affinity polymer microspheres, and particularly relates to a carboxylated PS latex nanoparticle with high reactivity to pepsinogen antibody and a preparation method thereof, which is used to enhance the sensitivity of latex turbidimetric immunoassay for gastric cancer markers. Background Art

[0002] In latex enhanced turbidimetric immunoassay (LETIA), antibodies or antigens are modified on the surface of latex nanoparticles by non-covalent adsorption or chemical bond coupling to form a latex-antibody (antigen) complex. Through the immune reaction with antigens (or antibodies) in the sample, an antibody-antigen-latex particle complex flocculent is obtained. As the immune reaction continuously occurs, the aggregated flocculents continuously increase, thus changing the light transmittance or light scattering property of the dispersion at a specific wavelength (such as 600 nm). Therefore, by measuring the change in turbidity before and after the immune reaction, the concentration of antigens (antibodies) in the sample can be calculated, so as to achieve the purpose of detection. Compared with other immunoassay methods, such as chemiluminescence immunoassay, radioimmunoassay, enzyme-linked immunosorbent assay, etc., LETIA has the advantages of simple and convenient detection method, no radioactive pollution, accurate quantification, good stability, etc. More importantly, it can achieve high-throughput and multi-channel sample detection through an automatic biochemical analyzer, so the detection cost is greatly reduced, saving time and effort, and has been increasingly applied to clinical detection. So far, LETIA has been applied to detect various detection items such as C-reactive protein, serum amyloid A, rheumatoid factor, cystatin C, α1-microglobulin, β2-microglobulin, retinol-binding protein, neutrophil gelatinase-associated lipocalin, transferrin, α2-macroglobulin, lipoprotein a, etc. The diagnostic fields have covered multiple fields such as rheumatism, renal function, liver function, infection, blood lipid, myocardium, diabetes, etc., and have broad market prospects.

[0003] In LETIA, polystyrene (PS) latex nanoparticles are usually used. Antibodies can be adsorbed on the surface of the latex in a non-covalent bond form through hydrophobic interaction with PS. However, the stability of the antibody-latex nanoparticle complex obtained by this method is poor. Therefore, currently, methods such as surface modification or copolymerization of PS latex nanoparticles are often used to introduce reactive groups on their surfaces, and antibodies are fixed on the latex particles by chemical bonding to improve the stability of the reagent.

[0004] Carboxylated PS latex nanoparticles can be obtained by copolymerizing styrene and acrylic acid. In LETIA, it is often used to react with the amino group of antibodies, and antibodies are grafted onto the surface of latex particles through amidation reactions. Therefore, in order to improve the sensitivity of the LETIA method, in addition to considering improving the refractive index of the latex particles themselves, attention also needs to be paid to the surface carboxyl content of the latex and the surface chemical environment of the latex.

[0005] Copolymerizing polystyrene and acrylic acid monomers in an emulsion polymerization system is the main preparation process for latex nanoparticles used to enhance immunoturbidimetry. Although the carboxyl content can be adjusted within a certain range, the glass transition temperatures of polystyrene, polyacrylic acid, and their copolymers are relatively high (higher than 100 °C). Therefore, when antibody molecules adsorbed on the latex surface react with carboxyl groups, limited by the rigidity of the polymer chains, the reaction between carboxyl groups and amino groups on antibodies cannot be promoted by adjusting the chain conformation.

[0006] Therefore, how to develop a latex particle that can enhance the sensitivity of the latex turbidimetry method for detecting gastric cancer markers is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0007] In view of the problems in the prior art that the reaction efficiency between the surface carboxyl groups of carboxylated PS latex nanoparticles and antibodies is often not high, the antibody grafting rate is low, and it is not easy to have an immune reaction with the target protein to form latex aggregates, resulting in low sensitivity and limited linear range in latex turbidimetry detection, the present invention provides a preparation method of carboxylated PS latex nanoparticles with high reactivity with antibodies and applies it to the latex turbidimetry detection of gastric cancer markers - pepsinogen (PGI, PGII).

[0008] It should be noted that by designing a core-shell structure latex nanoparticle based on carboxylated polystyrene, the present invention can not only ensure the light scattering properties of the latex particles, but also use polyacrylate with a low glass transition temperature as the shell layer, making the latex surface layer have the elastic characteristics of a rubber state, which is beneficial to improving the reaction efficiency between the latex particles and antibodies, thereby improving the detection sensitivity and application range of the latex-enhanced immunoturbidimetry method.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The first technical object of the present invention is to provide a preparation method of carboxylated PS latex nanoparticles with high reactivity with pepsinogen antibodies, using a semi-continuous emulsion polymerization method, and the steps include pre-emulsification of the reaction solution and a semi-continuous seed emulsion polymerization process.

[0011] Optionally, the pre-emulsified reaction solution includes the following three kinds, namely emulsion A, which is composed of styrene, water, emulsifier, and crosslinking agent; emulsion B, which is composed of acrylate, water, emulsifier, crosslinking agent, and initiator; emulsion C, which is composed of acrylate, acrylic acid, emulsifier, initiator, and water; wherein,

[0012] In emulsion A, the mass-volume ratio of styrene in emulsion A is 10g - 30g / 100mL, the mass-volume ratio of the emulsifier in emulsion A is 0.1g - 2g / 100mL, and the mass-volume ratio of the crosslinking agent in emulsion A is 0.1g - 1g / 100mL;

[0013] In emulsion B, the mass-volume ratio of acrylate in emulsion B is 10g - 50g / 100mL, the mass-volume ratio of the emulsifier in emulsion B is 1g - 10g / 100mL, the mass-volume ratio of the crosslinking agent in emulsion B is 1g - 20g / 100mL, and the mass-volume ratio of the initiator in emulsion B is 0.1g - 1g / 100mL;

[0014] In emulsion C, the mass-volume ratio of acrylate in emulsion C is 20g - 50g / 100mL, the mass-volume ratio of acrylic acid in emulsion C is 5g - 20g / 100mL, the mass-volume ratio of the emulsifier in emulsion C is 0.1g - 5g / 100mL, and the mass-volume ratio of the initiator in emulsion C is 0.1g - 1g / 100mL.

[0015] After configuring the above three reaction solutions, place them in an ultrasonic disperser and ultrasonicate for 10 - 30 minutes to obtain a stable emulsion.

[0016] Moreover, the volume ratio of emulsions A, B, and C is 6:1:1.2 - 2.

[0017] Furthermore, the emulsifiers used in the preparation of the pre-emulsion include one or a mixture of two of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sorbitan monooleate, polyoxyethylene sorbitan fatty acid ester, and polyethylene glycol octylphenyl ether, and the weight of the emulsifier accounts for 0.5% - 2% of the total monomer amount.

[0018] The initiators used in the preparation of the pre-emulsion include one of potassium persulfate, sodium persulfate, and ammonium persulfate, and the weight ratio of the initiator is 0.1% - 1% of the total monomer amount.

[0019] The crosslinking agents used in the preparation of the pre-emulsion include one or a mixture of two of 1,4-butanediol diacrylate, divinylbenzene, N,N'-methylenebisacrylamide, and allyl methacrylate, and the weight of the crosslinking agent accounts for 5% - 20% of the total monomer amount.

[0020] In the preparation of the pre-emulsion, the acrylate monomers used include one or two of methyl acrylate, ethyl acrylate, 2-hydroxyethyl acrylate, butyl acrylate, 2-ethylhexyl acrylate, methoxyethyl acrylate, ethoxyethyl acrylate, 4-hydroxybutyl acrylate, 2-hydroxyethyl acrylate, and 2-hydroxypropyl acrylate. The weight of the acrylate monomers accounts for 30%-50% of the total monomers.

[0021] Moreover, the weight of the acrylic acid monomer accounts for 2%-10% of the total monomers.

[0022] It should be noted that the total monomers refer to the total amount of styrene, acrylate, and acrylic acid.

[0023] Optionally, the specific method for preparing carboxylated PS latex nanoparticles by the semi-continuous seed emulsion polymerization method includes: adding emulsion A to a three-neck reaction flask, mechanically stirring at 100-300 revolutions per minute, raising the temperature to 70-85 °C, adding an initiator, and reacting for 2-4 h; then, adding emulsion B dropwise within 0.5-2 h and reacting for 0.5-2 h; finally, adding emulsion C dropwise within 0.5-2 h and reacting for 1-3 h, and finally cooling to room temperature to end the reaction.

[0024] It should be noted that the dropping order of the pre-emulsion reaction liquids A, B, and C cannot be reversed to avoid affecting the synthesis of the particles with carboxyl groups on the surface.

[0025] The second technical object of the present invention is to provide carboxylated PS latex nanoparticles prepared by the method as described above. The latex nanoparticles have a core-shell structure, with a cross-linked polystyrene inner core and a copolymer of acrylate monomers and acrylic acid as the outer shell, having a low glass transition temperature and exhibiting a rubber state at room temperature.

[0026] Specifically, the hydrodynamic diameter of the carboxylated PS latex nanoparticles is 80 nm - 300 nm, the particle size distribution index is less than 0.05, and the carboxyl density is 0.100 mmol / g - 0.400 mmol / g based on the dry sphere.

[0027] The third technical object of the present invention is to claim the application of the above core-shell structured carboxylated PS latex nanoparticles in the detection of pepsinogen by latex immunoturbidimetry.

[0028] Compared with the prior art, the advantages of the present invention are as follows:

[0029] 1. By adopting the semi-continuous seed emulsion polymerization method, the present invention first obtains cross-linked polystyrene seeds, and then gradually increases the size of the latex particles in batches by monomer dropping, and finally obtains monodisperse carboxylated PS latex nanoparticles, with the outermost layer being a copolymer of ethyl acrylate and acrylic acid.

[0030] 2. In the present invention, the core is crosslinked polystyrene, and the shell is a copolymer of acrylate monomers and acrylic acid. The acrylate monomers include one or a mixture of two of methyl acrylate, ethyl acrylate, 2-hydroxyethyl acrylate, butyl acrylate, 2-ethylhexyl acrylate, methoxyethyl acrylate, and ethoxyethyl acrylate. The shell has a low glass transition temperature and characteristics similar to those of polyacrylate elastomers; and finally, ethyl acrylate and acrylic acid copolymer are preferably used.

[0031] 3. In the preparation of the shell material of the present invention, acrylic acid copolymer monomers are added. On the one hand, it can adjust the glass transition temperature of the shell layer and increase the hardness of the soft layer. On the other hand, it can increase the surface carboxyl group content of the latex particles.

[0032] 4. The latex particles prepared by the method of the present invention can improve the coupling efficiency with the amino groups on the antibody molecules through the high-content carboxyl groups on the surface and the elastic surface environment of the shell, and can be used as the latex raw material for LETIA reagents.

[0033] 5. In the latex turbidimetric assay of pepsinogen, the latex particles prepared by the present invention are compared with the P0323 latex particles of JSR Corporation of Japan: the correlation coefficient R 2 is 0.99, the CV value is less than 5%, and at the same time, the low-end signal sensitivity is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0035] Figure 1 It is a schematic diagram of the preparation of core-shell structured carboxylated PS latex nanoparticles by semi-continuous seed emulsion polymerization.

[0036] Figure 2 It is a transmission electron microscope photograph of PS seeds.

[0037] Figure 3 It is a transmission electron microscope photograph of core-shell structured PS seeds (not carboxylated).

[0038] Figure 4 It is a transmission electron microscope photograph of core-shell structured carboxylated PS latex nanoparticles.

[0039] Figure 5 It is a particle size distribution diagram of core-shell structured carboxylated PS latex nanoparticles.

[0040] Figure 6Calibration curves for Example 5 and Comparative Example 1.

[0041] Figure 7 Correlation analysis for Example 5 and Comparative Example 1. Detailed implementation manners

[0042] Next, in combination with the embodiments of the present invention and the accompanying drawings of the specification, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0043] The embodiments of the present invention disclose a preparation method of core-shell structured carboxylated PS latex nanoparticles.

[0044] To better understand the present invention, the following embodiments are used to further specifically illustrate the present invention, but it should not be construed as a limitation of the present invention. For those skilled in the art, some non-essential improvements and adjustments made according to the above invention content are also considered to fall within the protection scope of the present invention.

[0045] Next, in combination with specific embodiments, the technical solutions of the present invention will be further described.

[0046] Example 1

[0047] Using a semi-continuous seeded emulsion polymerization method, core-shell structured (ethyl acrylate and acrylic acid copolymer shell) carboxylated PS latex nanoparticles with a hydrodynamic diameter of 300 nm are prepared ( Figure 1 )

[0048] Prepare emulsion A, including 0.32 g of sodium dodecyl sulfate, 40.60 g of styrene, 0.41 g of divinylbenzene and 200 mL of water; prepare emulsion B, including 0.01 g of sodium dodecyl sulfate, 1.05 g of sorbitan monooleate, 10.25 g of ethyl acrylate, 2.08 g of N,N-methylenebisacrylamide, 0.15 g of sodium persulfate and 30 mL of water; prepare emulsion C, containing 0.29 g of sodium dodecyl sulfate, 20.15 g of ethyl acrylate, 4.08 g of acrylic acid, 0.25 g of sodium persulfate and 30.0 mL of water; pre-emulsify the prepared solutions and place them in an ultrasonic disperser for ultrasonic treatment for 30 minutes to obtain stable emulsions A, B, and C.

[0049] Add emulsion A into a 500 mL three-necked reaction flask, heat it in an oil bath to 80 °C and mechanically stir at a speed of 200 revolutions per minute. Add 0.2 g of sodium persulfate to initiate the polymerization process and react for 2 h. Then, add emulsion B dropwise within 1 h and react for 2 h. Finally, add emulsion C dropwise within 1 h and react for 1 h. Finally, cool it to room temperature to end the reaction.

[0050] After emulsion A reacts for 2 h, take a small amount of PS seeds and observe the particle size of the PS seeds by transmission electron microscopy, which is about 220 nm ( Figure 2 ). After adding emulsion B and reacting for 2 h, take a sample and observe the particle size by transmission electron microscopy, which is about 250 nm ( Figure 3 ). After adding emulsion C and reacting for 1 h, core-shell structured carboxylated PS latex nanoparticles are prepared. Observed by transmission electron microscopy, they have a good spherical shape, uniform particle size distribution, and the particle size is 290 nm ( Figure 4 ), which is 70 nm larger than the particle size of the PS seeds. The increased part of the particle size is contributed by the carboxylated acrylate shell layer. Measured by a particle size analyzer, the core-shell structured carboxylated PS latex nanoparticles are dispersed in PBS solution, and the hydrodynamic diameter is 300 nm ( Figure 5 ), the particle size dispersion index PDI is 0.04, and the carboxyl density is 0.150 mmol / g based on the dry sphere.

[0051] Example 2

[0052] The polymerization method is the same as that in Example 1. Adopt a semi-continuous seed emulsion polymerization method to prepare core-shell structured carboxylated PS latex nanoparticles with a hydrodynamic diameter of 210 nm.

[0053] Prepare emulsion A, including 0.67 g of sodium dodecyl sulfate, 41.62 g of styrene, 0.58 g of divinylbenzene and 200 mL of water; prepare emulsion B, including 0.02 g of sodium dodecyl sulfate, 1.26 g of sorbitan monooleate, 12.75 g of ethyl acrylate, 3.18 g of N,N-methylenebisacrylamide, 0.15 g of sodium persulfate and 30 mL of water; prepare emulsion C, containing 0.45 g of sodium dodecyl sulfate, 22.15 g of ethyl acrylate, 5.66 g of acrylic acid, 0.26 g of sodium persulfate and 30.0 mL of water. Pre-emulsify the prepared solutions and place them in an ultrasonic disperser for ultrasonic treatment for 30 minutes to obtain stable emulsions A, B, and C.

[0054] Add emulsion A into a 500 mL three-necked reaction flask, heat it in an oil bath to 80 °C and mechanically stir at a speed of 300 revolutions per minute. Add 0.25 g of sodium persulfate to initiate the polymerization process and react for 3 h. Then, add emulsion B dropwise within 0.5 h and react for 2 h. Finally, add emulsion C dropwise within 0.5 h and react for 1 h. Finally, cool it to room temperature to end the reaction.

[0055] As determined by a particle size analyzer, the core-shell structured carboxylated PS latex nanoparticles are dispersed in a PBS solution, with a hydrodynamic diameter of 210 nm, a particle size dispersion index PDI of 0.05, and a carboxyl density of 0.270 mmol / g based on the dry sphere.

[0056] Example 3

[0057] The polymerization method is the same as that in Example 1. A semi-continuous seed emulsion polymerization method is adopted to prepare core-shell structured carboxylated PS latex nanoparticles with a hydrodynamic diameter of about 120 nm.

[0058] Prepare emulsion A, including 1.08 g of sodium dodecyl sulfate, 41.56 g of styrene, 0.61 g of divinylbenzene and 200 mL of water; prepare emulsion B, including 0.05 g of sodium dodecyl sulfate, 2.28 g of sorbitan monooleate, 11.08 g of ethyl acrylate, 5.01 g of N,N-methylenebisacrylamide, 0.20 g of sodium persulfate and 30 mL of water; prepare emulsion C, containing 0.58 g of sodium dodecyl sulfate, 25.10 g of ethyl acrylate, 7.56 g of acrylic acid, 0.35 g of sodium persulfate and 30.0 mL of water. Pre-emulsify the prepared solutions and place them in an ultrasonic disperser for ultrasonic treatment for 30 minutes to obtain stable emulsions A, B, and C.

[0059] Add emulsion A to a 500 mL three-necked reaction flask, heat it in an oil bath to 75 °C and mechanically stir at a speed of 300 revolutions per minute. Add 0.36 g of sodium persulfate to initiate the polymerization process and react for 3 h; then, gradually add emulsion B dropwise within 1 h and react for 2 h; finally, gradually add emulsion C dropwise within 1 h and react for 2 h, and finally cool to room temperature to end the reaction.

[0060] As determined by a particle size analyzer, the core-shell structured carboxylated PS latex nanoparticles are dispersed in a PBS solution, with a hydrodynamic diameter of 120 nm, a particle size dispersion index PDI of 0.04, and a carboxyl density of 0.350 mmol / g based on the dry sphere.

[0061] Example 4

[0062] The polymerization method is the same as that in Example 1. A semi-continuous seed emulsion polymerization method is adopted to prepare core-shell structured carboxylated PS latex nanoparticles with a hydrodynamic diameter of about 80 nm.

[0063] Prepare Emulsion A, which includes 1.56 g of sodium dodecyl sulfate, 42.38 g of styrene, 0.48 g of divinylbenzene and 200 mL of water; prepare Emulsion B, which includes 0.17 g of sodium dodecyl sulfate, 3.06 g of sorbitan monooleate, 12.04 g of ethyl acrylate, 1.85 g of N,N-methylenebisacrylamide, 0.20 g of sodium persulfate and 30 mL of water; prepare Emulsion C, which contains 0.85 g of sodium dodecyl sulfate, 21.05 g of ethyl acrylate, 6.07 g of acrylic acid, 0.34 g of sodium persulfate and 30.0 mL of water. Pre-emulsify the prepared solutions, place them in an ultrasonic disperser and ultrasonicate for 30 minutes to obtain stable Emulsions A, B and C.

[0064] Add Emulsion A into a 500 mL three-necked reaction flask, heat it in an oil bath to 80 °C and mechanically stir at a speed of 200 revolutions per minute, add 0.41 g of sodium persulfate to initiate the polymerization process, and react for 3 h; then, dropwise add Emulsion B within 1 h and react for 2 h; finally, dropwise add Emulsion C within 1 h and react for 2 h, and finally cool to room temperature to end the reaction.

[0065] Measured by a particle size analyzer, the core-shell structured carboxylated PS latex nanoparticles are dispersed in PBS solution, the hydrodynamic diameter is 80 nm, the particle size dispersion index PDI is 0.05, and the carboxyl density is 0.400 mmol / g based on the dry weight.

[0066] Example 5

[0067] Use the 300 nm core-shell structured carboxylated PS latex nanoparticles prepared in Example 1 for latex enhanced immunoturbidimetric assay of pepsinogen (II), and select the latex product of Japan's JSR Corporation as a comparison (product number P0323) to illustrate the difference in sensitivity between the two.

[0068] Method for binding antibody to latex particles:

[0069] Wash the 300 nm core-shell structured carboxylated PS latex nanoparticles 2-3 times with 10 mL of MES buffer by centrifugation; then add 10 mL of MES buffer to prepare a concentration of 0.1% w / v and ultrasonically disperse the particles; then add 20 mg / mL of EDC to a final concentration of 5 mg / mL and react at room temperature for 30 minutes, continuously mix to activate the carboxyl groups on the particle surface; wash the activated particles 2-3 times with boric acid buffer by centrifugation and then ultrasonically disperse them in 5 mL of boric acid buffer; mix the particle dispersion and pepsinogen (II) antibody solution and react at room temperature for 3 hours; then centrifuge and wash, and redisperse in glycine buffer (0.05 M glycine + 5% trehalose + 2% BSA + 0.1% EDTA, pH 8.0), ultrasonically disperse; finally store at 4 °C for use.

[0070] Comparative Example 1

[0071] Wash P03232-3 three times by centrifugation with 10 mL of MES buffer; then add 10 mL of MES buffer to prepare a concentration of 0.1% w / v, and ultrasonically disperse the particles; then add 20 mg / mL EDC to a final concentration of 5 mg / mL, react at room temperature for 30 minutes, continuously mix, and perform carboxyl activation on the particle surface; wash the activated particles 2-3 times by centrifugation with boric acid buffer, and then ultrasonically disperse them in 5 mL of boric acid buffer; mix the particle dispersion and pepsinogen (II) antibody solution, and react at room temperature for 3 hours; then centrifuge and wash, and redisperse in glycine buffer (0.05 M glycine + 5% trehalose + 2% BSA + 0.1% EDTA, pH 8.0), and ultrasonically disperse; finally store at 4 °C for use.

[0072] Test Example

[0073] Test the kits prepared in Example 5 and Comparative Example 1 on Hitachi 7180 using PGII clinical sample standards with concentrations of 0 mg / L, 5 mg / mL, 15 mg / L, 30 mg / mL, 60 mg / L, 100 mg / mL (S1-S6) respectively. After the reaction, use the concentration as the abscissa and the reaction signal value to make a standard curve, and the results are shown in Table 1.

[0074] Table 1

[0075] PGII (Example 5) PGII (Comparative Example) Blank Absorbance 8616 8641 Concentration 1 0 67 55 Concentration 2 5 218 161 Concentration 3 15 581 493 Concentration 4 30 1355 1251 Concentration 5 60 2496 2315 Concentration 6 100 4312 4205

[0076] Use 50 clinical samples and test them with two groups of reagents respectively. The results are shown in Table 2. Figure 7 The correlation coefficient is R 2 = 0.99.

[0077] Table 2

[0078]

[0079]

[0080] Table 3 shows the stability test (CV < 5%).

[0081]

[0082] The reagents prepared from the P0323 latex particles of JSR Corporation in Japan and the reagents prepared from the latex particles of Example 1 had blank absorbances of 8616 and 8641 respectively, both of which were less than 16000 required by the pepsinogen (II) antibody, and the deviation between the two was only 0.29%. When the antigen concentration was 5 ng / mL, the analytical absorbances were 161 and 218 respectively. The reagents prepared from both latex particles met the technical requirements of 200 ± 20%, and the latex particles prepared in Example 1 had stronger absorbance and thus higher sensitivity.

[0083] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing carboxylated PS latex nanoparticles having high reactivity with pepsinogen antibodies, characterized in that: The semi-continuous emulsion polymerization method is adopted, and the steps include pre-emulsification of the reaction solution and semi-continuous seed emulsion polymerization process; the details are as follows: 1) ultrasonically treating the pre-emulsified reaction solution for 10-30 minutes to obtain stable emulsions, namely emulsion A, emulsion B and emulsion C; 2) stirring emulsion A at 100-300 r / min, raising the temperature to 70-85° C., adding the initiator, and reacting for 2-4 hours; then, adding emulsion B dropwise within 0.5-2 hours, and reacting for 0.5-2 hours; finally, adding emulsion C dropwise within 0.5-2 hours, reacting for 1-3 hours, and cooling to room temperature to obtain core-shell structured carboxylated PS latex nanoparticles.

2. The method for preparing carboxylated PS latex nanoparticles according to claim 1, characterized in that: The volume ratio of the emulsion A, emulsion B and emulsion C is 6:1:1.2-2; Moreover, the emulsion A is composed of styrene, water, emulsifier, and crosslinking agent; the emulsion B is composed of acrylate, water, emulsifier, crosslinking agent, and initiator; and the emulsion C is composed of acrylate, acrylic acid, emulsifier, initiator, and water; wherein, In emulsion A, the mass volume ratio of styrene to emulsion A is 10g-30g / 100mL, the mass volume ratio of emulsifier to emulsion A is 0.1g-2g / 100mL, and the mass volume ratio of cross-linking agent to emulsion A is 0.1g-1g / 100mL; In emulsion B, the mass volume ratio of acrylate to emulsion B is 10g-50g / 100mL, the mass volume ratio of emulsion B is 1g-10g / 100mL, the mass volume ratio of crosslinking agent to emulsion B is 1g-20g / 100mL, and the mass volume ratio of initiator to emulsion B is 0.1g-1g / 100mL; In emulsion C, the mass volume ratio of acrylate to emulsion C is 20g-50g / 100mL, the mass volume ratio of acrylic acid to emulsion C is 5g-20g / 100mL, the mass volume ratio of emulsifier to emulsion C is 0.1g-5g / 100mL, and the mass volume ratio of initiator to emulsion C is 0.1g-1g / 100mL.

3. The method for preparing carboxylated PS latex nanoparticles according to claim 2, characterized in that: The emulsifier comprises one or two of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sorbitan monooleate, polyoxyethylene sorbitan fatty acid ester and polyethylene glycol octylphenyl ether, and the weight of the emulsifier accounts for 0.5%-2% of the total monomer amount.

4. The method for preparing carboxylated PS latex nanoparticles according to claim 2, characterized in that: The initiator includes one of potassium persulfate, sodium persulfate and ammonium persulfate, and the weight of the initiator is 0.1%-1% of the total amount of the monomers.

5. The method for preparing carboxylated PS latex nanoparticles according to claim 2, characterized in that: The crosslinking agent includes one or two of diacrylate-1,4-butylene glycol, divinyl benzene, N,N-methylenebisacrylamide and allyl methacrylate, and the weight of the crosslinking agent accounts for 5%-20% of the total monomer amount.

6. The method for preparing carboxylated PS latex nanoparticles according to claim 2, characterized in that: The acrylate monomer includes one or two of methyl acrylate, ethyl acrylate, hydroxyethyl acrylate, butyl acrylate, 2-ethylhexyl acrylate, methoxyethyl acrylate, ethoxyethyl acrylate, 4-hydroxybutyl acrylate, 2-hydroxyethyl acrylate, and 2-hydroxypropyl acrylate, and the weight of the acrylate monomer accounts for 30%-50% of the total monomer amount.

7. The method for preparing carboxylated PS latex nanoparticles according to claim 2, characterized in that: The weight of acrylic acid monomer accounts for 2% to 10% of the total monomers.

8. A carboxylated PS latex nanoparticle prepared by the method according to claim 1, characterized in that: The latex nanoparticles have a core-shell structure, the inner core is cross-linked polystyrene, the outer shell is acrylate monomer and acrylic copolymer, have a low glass transition temperature, and exhibit a rubber state at room temperature.

9. The carboxylated PS latex nanoparticles according to claim 8, characterized in that The hydrodynamic diameter of the PS latex nanoparticles is 80nm-300nm, the particle size dispersion index is less than 0.05, and the carboxyl density is 0.100mmol / g-0.400mmol / g in terms of dry bulb.

10. Use of the carboxylated PS latex nanoparticles prepared by the method of claim 1 or the carboxylated PS latex nanoparticles as claimed in claim 8 in latex turbidimetric detection of gastric cancer markers - pepsinogen (PGI, PGII).

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