RU2751881C1 - Method for producing monodisperse polymer microspheres with aldehyde groups - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: present invention relates to a method for producing monodisperse polymer microspheres with aldehyde groups, which can be used in the creation of diagnostic preparations for the latex-agglutination reaction in clinical diagnostic and immunological studies. The method involves a performance of a polymerization reaction using acrolein, while precipitation dispersed polymerization is carried out under conditions of simultaneous anionic and radical polymerization by preliminary preparation of reagents with washing and distillation of acrolein in the presence of hydroquinone. Then, the synthesis of polymer microspheres is carried out, for which distilled water is loaded into the reactor, into which polyvinylpyrrolidone, potassium persulfate and freshly distilled acrolein are added on an electromagnetic stirrer while constant stirring. Further, while stirring, a 5% solution of tetramethylethylenediamine (TMED) is added to the reaction mixture drop by drop to produce microspheres.EFFECT: claimed method makes it possible to obtain in a short time high-quality monodisperse polymer microspheres with aldehyde groups of 1.3-2.0 mcm in size, the content of aldehyde groups in which is 1.3 mmole/1g of the polymer.6 cl, 5 ex

Description

The alleged invention relates to the chemistry of macromolecular compounds, in particular to methods for producing synthetic polymer latex with aldehyde groups on the surface, which can be used in the creation of diagnostic preparations for the reaction of latex agglutination in clinical diagnostic and immunological studies.

Currently, the latex agglutination reaction (LAD) has become widespread, both in clinical diagnostic tests and in biochemical and immunological studies. A particularly widespread use of RLA is observed in the diagnosis of infectious diseases [1-4]. These tests use monodisperse suspensions of sensitized polymer microspheres. With the help of latex test systems, it is possible to determine more than a hundred causative agents of infectious diseases, including bacterial, viral, fungal and others. In addition, there are more than sixty different latex test systems that allow you to determine the Rh factor, blood group, the presence of drugs, poisons and drugs in the samples [2-6]

Known methods for producing polymer microspheres by methods of emulsifier-free, emulsion, seed, suspension, precipitation, dispersion polymerization. The choice of the synthesis method is determined by the need to obtain suspensions with desired properties.

In addition, for particles used in immunodiagnostic tests, the result of which is assessed by the sedimentation rate of agglutinates, special requirements may be imposed on the density of the polymer. Polymeric dispersions can contain groups on the surface that can directly react with biomolecules or form a chemical bond with them after the activation reaction of the microspheres. When choosing microspheres, a method for their synthesis and sensitization of biomolecules on them, it is necessary to take into account that many reactive groups are lost due to hydrolysis both during their synthesis and during sensitization. Polyacrolein carriers occupy a special place in biotechnology. Their important advantage is the presence of aldehyde groups on the surface of microparticles. These groups are stable in a wide range of pH of the medium and, under mild conditions, easily form a chemical bond with biological molecules, which is fundamental in the manufacture of diagnostic preparations.

Several methods are known for preparing polyacrolein latexes with aldehyde groups on the surface (7 - 10).

There is a known method of producing polyacrolein latexes by anionic precipitation polymerization of acrolein in an alkaline medium (8), where the pH is more than 11 with the gradual introduction of an alkali solution into the acrolein solution and only in the presence of a specially synthesized emulsifier-product of the interaction of polyglutaraldehyde oligomers with sodium bisulfite. Polymerization lasts 10 hours at room temperature.

However, the method is characterized by poor reproducibility, which is associated with the peculiarity of the polymerization of acrolein in an aqueous alkaline medium, the difficulty of controlling the rate of introduction of an alkaline solution and the non-standardness of the emulsifier used, in addition, this method is too time-consuming.

A known method of introducing monodisperse polymer particles of aldehyde groups into the surface layer by emulsifier-free emulsion copolymerization of styrene or methyl methacrylate with acrolein (11).

The disadvantage of this method is that it does not allow the introduction of particles of the amino group into the surface layers, since the presence of unprotected amino groups in the composition of the reaction mixture causes the termination of the free radical process of chain growth.

For the prototype, a method for producing polyacrolein particles with aldehyde groups on the surface with increased chemical stability by two-stage polymerization was chosen (9). At the first stage, traditional anionic precipitation polymerization of acrolein is carried out in an aqueous-alkaline medium with the addition of a 0.2N KOH solution to a pH of 10.5-11.0. The reaction mixture is stirred at a gradually increasing temperature to 35 ° C for 1.5-2.5 hours until the pH drops to 7.5-8.0. Further, in the second stage, radical prepolymerization is carried out. To do this, radical initiators (potassium persulfate, disodium isobisisomasyanic acid, benzoyl peroxide) are added to the reaction mixture in an amount of 3-6% by weight of acrolein and the process is continued for another 2-3 hours at a temperature of 40 to 90 ° C in an argon atmosphere. To obtain colored latexes, the first stage of the process is carried out in the presence of 0.02-1.5% by weight of acrolein of a water-soluble organic dye.

The disadvantage of this method is the need to control the optical density of the reaction mixture under the conditions of a rapidly flowing process and the moment the emulsifier is introduced into it.

In addition, the method is rather complicated, time-consuming, two-stage and, in fact, is a surface modification of acrolein latexes obtained by anionic polymerization in an aqueous alkaline medium.

A common disadvantage of polyacrolein latexes obtained by anionic precipitation polymerization is low chemical stability and significant contamination with oligomers.

The technical objective of the proposed invention is to develop a new method for creating a synthetic polymer latex that meets the requirements for carriers of immunoreagents, which allows in a short time (no more than 3 hours) to obtain high-quality monodisperse polymer microspheres with aldehyde groups on the surface for the design of immunodiagnostic drugs.

The task is achieved by the fact that, in the method of obtaining monodisperse polymer microspheres with aldehyde groups, including setting up a polymerization reaction using acrolein, precipitation dispersed polymerization is carried out under conditions of simultaneous anionic and radical polymerization by preliminary preparation of reagents with washing and distillation of acrolein in the presence of hydroquinone based on 0.2 g of the latter per 100 ml of acrolein at a temperature of (52-54) ° C, then the synthesis of polymer microspheres is carried out, for this, 20 ml of distilled water is loaded into a 50 ml reactor, into which 0.1 g of polyvinylpyrrolidone (obtaining a 0.5% aqueous solution), 0.05 g of potassium persulfate (1% by weight of acrolein), 6 ml (5 g) of freshly distilled acrolein, then 1 ml of a 5% solution of tetramethylethylenediamine is added dropwise to the reaction mixture with stirring (TEMED), to obtain colorless microspheres or 0.5 ml for floor teachings of colored microspheres, after the introduction of which the polymerization process begins, and the whole process is carried out with stirring for 2.5 - 3 hours at room temperature, obtaining colorless or colored microspheres with a size of 1.3 - 2 microns with an aldehyde group content of 1.3 mmol \ 1g of polymer.

At the same time, to prepare the TEMED reagent, the latter in an amount of 0.05 ml is dissolved in 1 ml of distilled water, obtaining a 5% solution.

In addition, aldehyde groups are determined by incubating the polymer dispersion with n-nitrophenylhydrazine in 0.05M phosphate buffer with pH 5.8 using a KFK-3 photometer, while the analysis is carried out by mixing 1 ml of a solution of n-nitrophenylhydrazine and 100 μl of a 5% polymer suspension , the samples are incubated for 6 hours with constant stirring and centrifuged at 6 thousand rpm for 15 minutes, and using the calibration curve, the final concentration of n-nitrophenylhydrazine in the supernatant is determined, then the difference between the initial and final concentration obtained after incubation , calculate the number of aldehyde groups.

Moreover, to prepare a 0.05M phosphate buffer solution (pH 5.8), 4 ml of 0.2M disubstituted sodium phosphate is mixed with 46 ml of 0.2M monosodium phosphate and brought to 200 ml with distilled water.

In addition, to obtain colored microspheres, a 1% aqueous solution of one of the safranin or thionine dyes is preliminarily prepared, and then, before adding tetramethylethylenediamine to the reaction mixture, 2 ml of safranin solution or 1 ml of thionine solution are added to this mixture.

In this case, the diameter of the polymer microspheres is determined by the method of electron transmission microscopy, for this, the material of the polymer dispersion is applied in a loop to the mesh of the substrate, the excess is removed with filter paper and dried, then the samples are viewed at various magnifications on a transmission electron microscope using the program for calculating the particle diameter.

The method is carried out as follows.

Preliminary preparation of the reagents necessary for the synthesis is carried out. For polymerization, a vinyl monomer, acrolein, is used, which is initially purified; for this, an aqueous solution is prepared at a mass ratio of the monomer: water phases = 1: 4. Then the resulting mixture is distilled in the presence of hydroquinone at the rate of 0.2 g of hydroquinone per 100 ml of acrolein. The distillation is carried out in a heated fume hood. A fraction boiling at 52-54 ° C is used in this work.

Next, 0.05 ml of tetramethylethylenediamine (TEMED) is dissolved in 1 ml of distilled water, obtaining a 5% solution. Weighed portions of potassium persulfate 0.10 g and polyvinylpyrrolidone 0.10 g are prepared on a laboratory balance with an accuracy of 0.01 g. Aqueous solutions of dyes - safranin and thionine are prepared.

In addition, a 0.005M solution of monosubstituted potassium phosphate is prepared in advance for the subsequent study of the physicochemical properties.

After that, the process of polymerization of vinyl monomer - acrylamide is carried out directly. Polymerization is carried out in the presence of a redox system (potassium persulfate - polyvinylpyrrolidone - TEMED).

To do this, 20 ml of distilled water is loaded into a 50 ml reactor, into which, with constant stirring on an electromagnetic stirrer, 0.1 g of polyvinylpyrrolidone (obtaining a 0.5% aqueous solution), 0.05 g of potassium persulfate (1% by weight of acrolein ), 6 ml (5 g) of freshly distilled acrolein, then, while stirring, 1 ml of a 5% TEMED solution (to obtain colorless microspheres) or 0.5 ml to obtain colored microspheres is added dropwise to the reaction mixture, after the introduction of which the polymerization process begins. The whole process is carried out with stirring for 2.5 to 3 hours at room temperature. The final stage of the proposed technology consists of determining the physicochemical characteristics of polymer microspheres, namely:

- The diameter of polymer microspheres is determined by electron transmission microscopy. For this, the native material of the polymer dispersion is applied in a loop onto a mesh-substrate, the excess is removed with filter paper and dried. The samples are viewed at various magnifications on a transmission electron microscope using a program to calculate the particle diameter.

- To determine the content of active adehyde groups, the polymer dispersion is incubated with n-nitrophenylhydrazine in 0.05M phosphate buffer with pH 5.8. The calibration dependence of absorption at a wavelength of 392 nm on the concentration of n-nitrophenylhydrazine in the presence of 0.05M phosphate buffer pH 5.8 is obtained using a KFK-3 photometer (ZOMZ, RUSSIA). Analysis of the test samples by mixing 1 ml of a solution of n-nitrophenylhydrazine and 100 μl of a 5% polymer suspension. The samples are incubated for 6 hours with constant stirring and centrifuged at 6 thousand rpm. within 15 minutes. The calibration curve is used to determine the final concentration of p-nitrophenylhydrazine in the supernatant. From the difference between the initial and final concentration obtained after incubation, the number of aldehyde groups is calculated.

The proposed technology, the proposed method, as a set of essential features, ensures the achievement of a technical result, namely:

- obtaining monodisperse polymer latex with a particle size of 1.3-2.0 microns, optimal for the carrier of immunoreagents;

- chemical and mechanical stability due to the presence of reactive aldehyde groups on the surface of the particles (1.3 mmol \ 1 g of polymer);

- in a one-stage polymerization process for 2.5-3 hours at room temperature.

Distinctive features of the proposed method from the prototype are: - the technology is carried out in one stage, polymerization is initiated by the redox system potassium persulfate-polyvinylpyrrolidone-tetramethylethylenediamine. This system generates the formation of radicals, and the organic base TEMED (promoter) is the source of hydroxyl ions. Thus, radical and anionic polymerization of acrolein with crosslinking of the polymer in the entire volume of the formed latex particles is carried out simultaneously.

Example 1.

Confirming the practical preparation of polyacrolein latex.

0.05 g of potassium persulfate and 6 ml of freshly distilled acrolein are dissolved in 20 ml of 0.5% polyvinylpyrrolidone. To the resulting solution with constant stirring on an electromagnetic stirrer add 1 ml of 5% aqueous solution of tetramethylethylenediamine. The reaction mixture is stirred for 3 hours at room temperature. Colorless particles with an average diameter of 1.4 μm are obtained. The polymer latex yield is 80%.

Example 2.

Demonstrating the preparation of colored polymer microspheres.

The polymerization process is carried out according to the technology described in example 1, but 2 ml of a 1% aqueous solution of the dye - safranin-T is introduced into the acrolein solution before polymerization and the polymerization is initiated by adding 0.5 ml of a 5% aqueous solution of the promoter-tetramethylethylenediamine to the reaction mixture. Particles of red color with a size of 2 μm are obtained.

Example 3.

Demonstrating the preparation of colored polymer microspheres.

The polymerization process is carried out according to the technology described in example 1, but 1 ml of a 1% aqueous solution of thionine is introduced into the acrolein solution before polymerization and the polymerization is initiated by adding 0.5 ml of a 5% aqueous solution of a promoter-tetramethylethylenediamine to the reaction mixture. Violet particles with a particle size of 1.3 μm are obtained.

Example 4.

Confirming the practical use of polymer latex in immunodiagnostics. Preparation of polymeric antigenic pseudotuberculosis diagnosticum.

To obtain a pseudotuberculosis polymeric antigenic diagnosticum, the plasmid-free strain H14 / 84 Y. pseudotuberculosis was used (from the collection of the Museum of Living Cultures of the FKUZ of the Rostov-on-Don Anti-Plague Institute). Sensitin is prepared using Y. pseudotuberculosis outer membrane proteins. As sensitin, a pool of 10 protein antigens with molecular weights in the range of 28-80 kDa, with a total protein concentration of 0.9-1.3 mg / ml, is used, which is dissolved in 1 ml of 0.1 M bicarbonate buffer solution (pH 9, 2).

Then, sensitin is immobilized on a polymer carrier, which is a suspension of microspheres at the rate of 1 mg of protein per 100 mg of carrier. Spherical polymer particles colored with safranin-T (red), 2 μm in size, containing 1.3 mM / g of aldehyde groups are used as a carrier. The incubation of sensitin on the carrier is carried out for 2 hours at room temperature with stirring on an electromagnetic stirrer, after which the suspension is left for 16-18 hours at a temperature of 4-6 ° C, then unbound adehyde groups are blocked, washed three times with buffered saline (pH 7, 2) and centrifuged at 3000 rpm for 10 minutes. at room temperature, then suspended in a gelatinous sucrose medium, creating a dilution of 1: 1 and poured into insulin vials of 1 ml, and then lyophilized. The resulting diagnosticum makes it possible to carry out studies to identify antibodies to the causative agent of pseudotuberculosis in one stage with visual consideration of the color reaction. The results of the study are obtained within 2 hours, which makes it possible to classify the preparation obtained on polyacrolein microspheres as a means of express diagnostics.

Example 5.

Confirming the practical use of polymer latex in immunodiagnostics. Preparation of polymeric immunoglobulin antitoxic diagnosticum for quantitative determination of toxin production of Vibrio cholerae.

To obtain a diagnosticum for the determination of toxin production by Vibrio cholerae, experimental antitoxic immune rabbit sera are initially obtained. For the preparation of sensitin, immunoglobulins are isolated from rabbit sera. Then, sensitin is immobilized on a polymer carrier, which is a violet suspension of microspheres with a size of 1.3 µm stained with the thionine dye.

The polymeric carrier is sensitized with the obtained immunoglobulins. To immobilize sensitin, the precipitate of the washed carrier is suspended in a borate buffer (pH8.4), immunoglobulins to cholera toxin are added to it and left for contact with stirring for 2 hours at 20 ° C. As a result, aldehyde reactive groups on the surface of particles interact specifically with amino groups of protein molecules of immunoglobulins. After that, the suspension is cooled to 4-5 ° C. and kept at this temperature for 16-18 hours.

Free aldehyde groups are blocked by introducing a 0.5% gelatose solution into a suspension in buffered saline (pH 7.0-7.1) and incubating for 2 hours at room temperature with constant stirring. As a result of the stabilization of gelatosis, it allows blocking the reactive groups on the surface of the particles that did not bind to immunoglobulins during the sensitization process.

The suspension of the diagnosticum is centrifuged and washed three times with buffered saline (pH 7.0-7.1) from excess gelatose, and the final precipitate is suspended in 0.1% gelatose solution in buffered saline (pH 7.0-7.1) ...

Diagnosticum is designed to detect cholera toxin and determine the level of toxin production of Vibrio cholerae serogroup O1 in broth cultures when isolated from environmental objects and from humans using the latex agglutination reaction. The results of the study are obtained within 2 hours, which makes it possible to classify the drug obtained on polyacrolein microspheres as a means of express diagnostics.

The use of the proposed invention allows, due to the new method, in a short time to obtain high-quality monodisperse polymer microspheres with aldehyde groups on the surface of the microspheres in a one-stage precipitation-dispersed polymerization process.

The microspheres obtained by the proposed method can be used to obtain immobilized enzymes, antibodies, toxins, drugs and other compounds with the aim of using them in scientific and practical activities, in particular, as affinity sorbents. In addition, suspension immunodiagnostic preparations can be developed on their basis.

Sources of information

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Claims (6)

1. A method of obtaining monodisperse polymer microspheres with aldehyde groups, including setting up a polymerization reaction using acrolein, characterized in that precipitation dispersed polymerization is carried out under conditions of simultaneous anionic and radical polymerization by preliminary preparation of reagents with washing and distillation of acrolein in the presence of hydroquinone at the rate of 0 , 2 g of the latter per 100 ml of acrolein at a temperature of 52-54 ° C, then the synthesis of polymer microspheres is carried out, for this, 20 ml of distilled water is loaded into a reactor with a capacity of 50 ml, into which 0.1 g of polyvinylpyrrolidone ( obtaining a 0.5% aqueous solution), 0.05 g of potassium persulfate (1% by weight of acrolein), 6 ml (5 g) of freshly distilled acrolein, then with stirring, 1 ml of a 5% solution is added dropwise to the reaction mixture tetramethylethylenediamine (TEMED), to obtain colorless microspheres or 0.5 ml to obtain colored microspheres, after the introduction of which the polymerization process begins, and the whole process is carried out with stirring for 2.5 - 3 hours at room temperature, obtaining colorless or colored microspheres with a size of 1.3 - 2 microns with an aldehyde group content of 1.3 mmol / 1 g polymer. 2. The method according to claim 1, characterized in that for the preparation of the TEMED reagent, the latter in an amount of 0.05 ml is dissolved in 1 ml of distilled water, obtaining a 5% solution. 3. The method according to claim 1, characterized in that the aldehyde groups are determined by incubating the polymer dispersion with n-nitrophenylhydrazine in 0.05M phosphate buffer with pH 5.8 using a KFK-3 photometer, wherein the analysis is carried out by mixing 1 ml of solution n-nitrophenylhydrazine and 100 μl of a 5% polymer suspension, the samples are incubated for 6 hours with constant stirring and centrifuged at 6 thousand rpm for 15 minutes, and using the calibration curve, the final concentration of n-nitrophenylhydrazine in the supernatant is determined, then, from the difference between the initial and final concentrations obtained after incubation, the number of aldehyde groups is calculated. 4. The method according to claim 3, characterized in that for the preparation of 0.05M phosphate buffer solution (pH 5.8), 4 ml of 0.2M disubstituted sodium phosphate is mixed with 46 ml of 0.2M monosodium phosphate and adjusted with distilled water to 200 ml. 5. The method according to claim 1, characterized in that to obtain colored microspheres, a 1% aqueous solution of one of the safranin or thionine dyes is preliminarily prepared, and then, before adding tetramethylethylenediamine to the reaction mixture, 2 ml of safranin solution or 1 ml thionine solution. 6. The method according to claim 1, characterized in that the diameter of the polymer microspheres is determined by the method of electron transmission microscopy, for this, the polymer dispersion material is applied in a loop on a mesh-substrate, the excess is removed with filter paper and dried, then the samples are viewed at different magnifications on a transmission electron microscope using a program to calculate the particle diameter.