The method of obtaining monodisperse synthetic polymer latex with carboxypropanoyl the surface of the particles

 

(57) Abstract:

Describes how to obtain monodisperse synthetic polymer latex with carboxypropanoyl the surface of the particles by bisimulations emulsion polymerization of the vinyl monomer in an aqueous-alkaline medium in the presence of a carboxyl-containing initiator when heated. It differs in that the vinyl monomer is used alone or a mixture of monomers of a number of: styrene, vinyl acetate, (meth)acrylates and in the original reaction mixture is further added salt with the ability to create buffer solutions in the field of alkaline pH or a mixture of such salts at a concentration of salt in the calculation of the aqueous phase of 0.005-0,150 M Invention may find application in colloid chemistry, biochemistry, biotechnology and medicine, mainly to obtain carriers immunoreagents in the immunoassay. The technical result - the creation of a method of obtaining a polymer latex, complying with the requirements for holders of immunoreagents.

The invention relates to the chemistry of macromolecular compounds, more specifically, to a method for producing monodisperse synthetic polymer latexes with a specified particle diameter.

Poluchenii, biotechnology and medicine. So, they can act as carriers of immunoreagents in immunoassay and model colloids, calibration standards in the analysis of variance.

The latexes used in the immunoassay must meet certain requirements:

1. The optimum diameter of the latex particles (microspheres) - 0.5 to 2.0 μm, as particles of such size, form in immunochemical large agglutinates, which facilitates the reaction accounting, which in most cases is visually [gull N. A. // Immunological diagnosis of viral infections. - M.: Medicine. 1985. S. 121].

2. The monodispersity of the microspheres (homogeneity for a given size).

3. The presence of surface functional groups (ionic and nonionic) [Prokopov N. And., Gritskova I. A., Cherkasov C. R., A. Chalykh E. // USP. 1996. So 65. S. 178.].

4. The particles must have a high capacity to attachable immunoreagents in the absence of loose surface layer of the polymer absorbing and shielding attached substances when carrying out the immunochemical reaction [Kondo A. // J. Immunol. Methods. 1990. V. 135. P. 111].

Bisimulation emulsion polymerization in the present time is the main zwesten way to obtain monodisperse synthetic copolymer latex with carboxypropanoyl surface by bisimulations emulsion copolymerization of polar monomer - styrene and the polar monomer is methacrylic acid in the presence of initiator [Eliseeva Century. And., Balazova, T. D. // USP. 1991. So 60. N 2. S. 308].

The latex obtained in a known manner, is used as a carrier of immunoreagents, as it allows you to produce as adsorption immobilization of proteins on the surface of the particles and their chemical binding to carboxyglutamic. However, upon receipt of the latex in the statistical distribution of the parts of methacrylic acid chain of the copolymer is enriched surface layer of these particles hydrophilic units and formed a loose surface layer [Muroi, S., Hosoi, K., Ishikawa T. // J. Appl. Polym. Sci. 1967. V. 11. P. 1963; Vizayendran B. R. // J. Appl. Polym. Sci. 1979. V. 23. P. 893]. The structure of such a surface layer depends on many conditions of synthesis of the particles and their subsequent processing [Okubo M, Kanaida K. , Matsumoto T. // J. Appl. Polym. Sci. 1987. V. 33. N 5. P. 1511] that leads, first, to poor reproducibility of the parameters of the latex (diameter, monodispersity, the uniformity of the surface charge of the particles), and secondly, to the shielding gidrogenizirovannye areas of polymer chains immunoreagents in the surface layer of the particles, which reduces the efficiency of immunochemical reactions [Kondo A. // J. Immunol. Methods. with carboxypropanoyl surface by bisimulations emulsion polymerization of styrene, initiated by persulfate [Kamel A. A. // J. Dispers. Sci. Technol. 1981. V. 2. P. 183].

The surface of the obtained latex polyfunctionally: along with sulphapyridine groups as a result of their partial hydrolysis are carboxy - and hydroxy-group, the number and location of which are difficult to control. In order to avoid this drawback and, thereby, to improve the quality and reproducibility of the properties of the resulting latex, solitaire group hydrolyzing boiling latex with subsequent oxidation of the hydroxy groups. However, this operation does not provide a complete translation of hydroxyl groups to carboxyl and multi-processing leads to significant loss of the final product.

The known method (prototype) obtain monodisperse synthetic polymer latex with carboxypropanoyl surface by bisimulations emulsion polymerization of styrene in an alkaline environment (pH of 7.2 and 11.6) when the mass ratio of the phases styrene : water = 1:(5-10) in the presence of a carboxyl-containing azoinitiator - 4,4'-azobis-(4-cianfarano acid) (PPM) concentration 0,010-0,007 M in the calculation of the aqueous phase at 801oC. In this way the carboxylation of the surface of the particles is due to the initiator [L,25-0,45 microns (min relative variance of 4.7% - the diameter of 0.36 μm), which is not enough to obtain high-quality immunodiagnostics (see above requirements latexes for immunoassay), so in the prototype, there is no indication to test known latexes as carriers for immunoreagents.

When seeking to obtain a known method of latexes with particle diameters, large, 0.45 µm, latexes lose monodispersity (relative variance exceeds 20%).

The analysis of the above analogues allows to assert that the problem of creation of latexes with carboxypropanoyl the surface of the particles - carriers for immunoreagents currently remains relevant.

Objective of the claimed invention is the creation of synthetic polymer latex, complying with the requirements for holders of immunoreagents.

This task is solved by the claimed method of obtaining monodisperse synthetic polymer latex with carboxypropanoyl the surface of the particles.

The invention has a combination of the following essential features:

1. Spend bisimulation emulsion polymerization of the vinyl monomer.

1.1. As the vinyl monomer COI when the mass ratio of the phases monomer : water=1: (5-25).

1.3. The process is carried out in an alkaline medium (pH initial mixture 7-14).

1.4. The polymerization is carried out in the presence of a carboxyl-containing initiator concentration calculated on the aqueous phase of 0.001-0,020 M

1.5. The polymerization is conducted by heating (75-95oC) in an atmosphere of inert gas.

1.6. In the original reaction mixture is further added salt with the ability to create buffer solutions in the field of alkaline pH or a mixture of such salts at a concentration of salt in the calculation of the aqueous phase of 0.005-0,150 M

This set of essential features of the claimed method provides the technical result - obtaining monodisperse polymer latex with optimal media immunoreagents properties by increasing particle diameter of 0.5-2.5 μm with a narrow size distribution of particles in comparison with the method of the prototype and achievements carboxypropanoyl surface with a wider interval specified and controlled surface concentration of carboxyl groups (1 to 6 mEq/m2and the higher the upper limit of this interval than the latex obtained by the method prototype (2.0 to 2.6 mg-EQ/m2).

Additional the national surface (for example, combination-carboxy - epoxy-groups - see example # 4).

Distinctive features of the proposed method from the prototype method are signs of 1.1, 1.6, i.e. the number of monomers used and their combination, as well as the introduction in the original reaction mixture an additional ingredient - salt with the ability to create buffer solutions in the field of alkaline pH or a mixture of such salts at a concentration of salt in the calculation of the aqueous phase of 0.005-0,150 M

Analysis of the known prior art did not allow to find a solution that exactly matches the set of essential characteristics of the claimed method. This confirms the novelty of the proposal.

Only the set of essential features of the proposed method allows to achieve the above effect. From the popularity of certain features of the invention not apparent from the evidence of the possibility of obtaining latex optimal for media immunoreagents properties. So, still failed to achieve the optimum, despite attempts by varying the known conditions for obtaining latex: concentration, pH of the environment, because the change of one parameter leads to improvement, such as latex, but the violation of monodispersity [Lishansky I. S. and others, leads to an increase of particle size, formed during bisimulations polymerization [Goodwin J. W., Colloid. Polym. Sci., 1974, V. 252, p. 464], in the present case has not justified itself. The authors claimed invention were conducted special experiments using NaCl as an additive in the original reaction mixture (according to the above-mentioned method Goodwin J. W.). Indeed, were able to obtain latexes with an average diameter of latex particles is about 0.5 μm, however, it was usasales distribution of latex particles by size, i.e. they have lost the property of monodispersity, and the whole effect of the claimed invention was not achieved. Used in the invention salts have known for them buffer properties, and initially it was assumed that their introduction into the reaction mixture will increase the ability to control the displacement of acid-base equilibrium in the synthesis process. Indeed, the authors of the claimed invention found a positive effect of these salts on maintaining an alkaline pH during the reaction, particularly if the specified initial pH 7-8. However, a completely unexpected and unpredictable in advance was the fact that the introduction of these salts, and only the claimed interval SNAC to 2.5 μm), the surface quality (surface concentration carboxypropyl in the absence of loose surface layer up to 6 mEq/m2), monodispersity. The achieved effect cannot be explained only buffer properties of these salts. Thus, in the present method introduces a new functional relationship "structure - property". This suggests accordance of the present invention, the eligibility condition of "inventive step" ("non-obviousness").

To confirm compliance with the decision of the eligibility condition "industrial applicability" and for a better understanding of the invention examples of specific performance.

Materials:

Technical initiators was purified twice by recrystallization from ethanol, dried in vacuum.

Purification of the monomers was carried out standard vacuum distillation. [Goodwin J. W. Brit. Polymer J., 1973, V. 5, N 2, p. 347].

Salt was used domestic production marks "H. H. and H. D. A.".

The diameters of the latex particles was measured using electron microscopy (Electron microscope JEM 100 S firm "JEON" Japan), the coefficient of polydispersity latex and relative dispersion of diameters .G. // USP. 1965, T. 34, No. 2, S. 276].

The surface concentration of carboxyl groups of the latex was determined after three times washing from water-soluble impurities sequential centrifugation and redispersion conductometric titration according to the method [Labib, M. E., Robertson, A. A. // J. Colloid Polym. Sci. 1980. V. 15. P. 2193].

Suspensions of bacterial cells of Neisseria meningitidis And obtained from the collection of the State scientific center of applied Microbiology.

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide - "Sigma", USA.

Bovine serum albumin -"Sigma", USA.

Polyclonal antibodies: for them to get used serum of animals (rabbits) immunized with a suspension of bacterial cells of Neisseria meningitidis A, with titles in reaction diffusion precipitation on Ouchterlony not lower than 1:8 [Primal X., Brock Th. // Basics of immunology. - M.: Mir. 1986. C. 100]. Selection was performed on protein A - sepharose. The analysis was carried out in the analytical electrophoresis on polyacrylamide gel [New methods of analysis of amino acids, peptides and proteins. - M.: Mir. 1974. S. 277]. Determination of the concentration of the antibodies was performed spectrophotometrically determining the optical density of a solution of antibody at a wavelength of 280 nm (A2802HPO4(concentration 0,040 M calculated on the aqueous phase), and 7.7 ml of fresh styrene copolymer (mass ratio of styrene/water = 1 : 14), pH = 11,3, polymerization was carried out at 80oC for 12 hours. Drove residual monomer and water vapor. The conversion of monomer was 98%. The particle size of the latex of 0.82 μm, coefficient of dispersion 1,008, the relative dispersion 5,12%. The surface concentration of carboxyl groups of the latex of 2.6 mEq/m2.

Example N 2

Conducted under the conditions of example No. 1 when the mass ratio of the styrene/water = 1 : 5, concentration CEC 0,010 M, Na2B4O70.005 M added a solution of NaOH concentration of 1.55 mol/l to pH of 10.7. The particle size of the latex of 1.06 μm, a coefficient of dispersion of 1,015, the relative variance of 7.2%. The surface concentration of carboxyl groups of the latex of 3.1 mEq/m2.

Example N 3

Conducted under the conditions of example No. 1 in mass ratio butylmethacrylate/water = 1 : 10, the concentration of 5,5'-azobis-(5-canitano acid) 0,010 M, Na2HPO40,150 the relative variance of 2.8%. The surface concentration of carboxyl groups of the latex - 1.0 mg - EQ/m2.

Example N 4

Conducted under the conditions of example No. 1 when the mass ratio (styrene + glycidylmethacrylate) / water = (0,8+0,2) : 10, the concentration of the IEC 0,010 M, KHC8H4O4(hydroptila potassium) 0,020 M Added a solution of NaOH concentration of 1.55 mol/l to pH of 9.5. The particle size of the latex of 0.62 μm, coefficient of dispersion 1,006, the relative variance of 3.4%. The surface concentration of carboxyl groups of the latex of 4.3 mEq/m2.

Example N 5

Conducted under the conditions of example No. 1 when the mass ratio of the styrene/water = 1 : 25, concentration CEC 0,020 M NaHCO30,010 M, was added a solution of NaOH concentration of 1.55 M to pH 7.0. The particle size of the latex of 0.52 μm, coefficient of dispersion 1,007, the relative dispersion of 4.9%. The surface concentration of carboxyl groups of the latex to 5.7 mEq/m2.

Example N 6

Conducted under the conditions of example No. 1 in mass ratio methylmethacrylat/water= 1: 7, the concentration of the IEC 0,010 M, NaH2C6H5O7(sodium citrate) 0.005 M Na2HPO40.005 M added a solution of NaOH concentration of 1.55 M to a pH of 10.2. The particle size of the latex of 1.52 μm, coefficient of dispersion 1,012, the relative dispersion of 8.2%. Surface is the second example No. 1 when the mass ratio of the styrene/water = 1 : 10, concentration CEC 0.001 M, Na2PO40.005 M added a solution of NaOH concentration of 1.55 M to pH of 14.0. The particle size of the latex of 2.50 μm, coefficient of dispersion 1,019, the relative dispersion of 9.6%. The surface concentration of carboxyl groups of the latex to 1.4 mEq/m2.

Example N 8

Sensitization of latex with carboxypropanoyl surface, obtained in the conditions of example No. 1: a 1% suspension of latex particles in deionized water was treated with a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide 5-10 mg/ml, was mixed in a ratio of 1:1 by volume with a solution of polyclonal antibodies to Neisseria meningitidis A (100 µg/ml) were incubated for 2-6 hours at 37oC. Sensitized latexes were transferred into 0.1 M glycine buffer (0.9% NaCl, pH 8,3) with 1% bovine serum albumin. The final concentration of latex in diagnostic kits ever dilution buffer to 0.25%.

Example N 9

Sensitization of latex with carboxypropanoyl surface, obtained in the conditions of example No. 2 were made in the conditions of example No. 8.

Example N 10

Tests obtained based assays obtained in the examples NN 8 and 9 were produced by the following method:

1. Preparing a suspension of bacterial cells is Tekle with 20 µl of bacterial suspension and incubated at circular rocking.

3. For control (the definition of spontaneous agglutination) 20 μl of the latex was mixed on glass with 20 ml of phosphate-saline buffer.

4. Records of the results of the reaction were made visually on a scale from (++++) to (-), agglutination (++++) meant the formation of large aggregates of latex particles within 1-2 min, ( + + + ) the formation of large aggregates of latex particles for 5 min, ( ++ ) - education units of medium size, (+) - formation of aggregates of small size and (-) - absence of aggregation (see. table).

Analysis of examples of specific performance shows that the inventive method has allowed to solve the problem. The resulting latexes are characterized by a particle diameter in the range of 0.5 - 2.5 μm, while the relative dispersion does not exceed 10%; achieved surface concentration of carboxyl groups in the range of 1 to 6 mEq/m2. Using the obtained latexes when designing immunodiagnostics allows to obtain products with a high sensitivity at the level of the best foreign analogues [Bangs L. B. Commercial latex diagnostic tests. The latex course. 1994. V. 12/1].

The invention is not limited to the above examples.

Outside the interval declared the end of the Sri salt concentrations 0.004 M in the calculation of the aqueous phase the size of the latexes of not greater than 0.45 µm.

2. By increasing the concentration of salts to 0.16 M observed broadening of the distribution of the latex particles by size and increasing the amount of coagulum, which reduces the yield of the target product.

The method of obtaining monodisperse synthetic polymer latex with carboxypropanoyl the surface of the particles by bisimulations emulsion polymerization of the vinyl monomer in an aqueous-alkaline medium in the presence of a carboxyl-containing initiator by heating, characterized in that the vinyl monomer is used alone or a mixture of monomers of a number of styrene, vinyl acetate, (meth)acrylates and in the original reaction mixture is further added salt with the ability to create buffer solutions in the field of alkaline pH or a mixture of such salts at a concentration of salt in the calculation of the aqueous phase of 0.005 - 0,150 M

 

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