JPH0548245B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial application field) Polymers that can be used for immunodiagnosis through antigen-antibody reactions or for enzyme reactions as immobilized enzymes by immobilizing antigens, antibodies, or enzymes that are biological proteins on the functional groups on the surface of microparticles. This invention relates to a method for producing acrolein fine particles. (Prior art) Acrolein has double bonds and aldehyde groups in its molecule, so its polymer is highly reactive and has attracted industrial interest as a component of reactive polymers. Research is being done on related uses. Conventionally, when acrolein is polymerized in an aqueous solvent, an anionic surfactant is generally used as a dispersant, and an anionic surfactant and a nonionic surfactant are also used to improve the polymerization stability of the resulting emulsion. Attempts are being made to use them together. A method using a sulfite adduct of polyacrolein as a dispersant has also been proposed. (Problems to be Solved by the Invention) When a surfactant is used as a dispersant when acrolein is polymerized in an aqueous solvent, the resulting polyacrolein emulsion has the disadvantage of lacking stability against electrolytes. There is. Furthermore, when a surfactant is used as a dispersant during polymerization, a part of the dispersant is adsorbed on the surface of the fine particles of the polyacrolein emulsion that is produced, and when the protein is attached to the surface of the polyacrolein fine particles, This dispersant has an adverse effect on Of course, the free dispersant contained in the emulsion can be processed by ion exchange method,
Although it is possible to remove the dispersant using techniques such as dialysis and washing, the stability of the polyacrolein fine particles becomes extremely poor when the dispersant is removed, causing deformation and aggregation of the particles, making them practically unusable. Become. Furthermore, when a polyacrolein sulfite adduct is used as a dispersant when acrolein is polymerized in an aqueous solvent, if the polyacrolein sulfite adduct is insufficiently purified, the particle size distribution of the resulting polyacrolein emulsion will be affected. is large, and particles with a bimodal distribution are often obtained. Therefore, there is a drawback that purification of the polyacrolein sulfite adduct requires a long time. (Means for solving the problem) Polymerization of acrolein is carried out by radical polymerization.

[Formula] 1, 2 addition and vinyl type polymerization occur preferentially, and the polymerization rate is low, making it impossible to obtain a sufficiently stable emulsion. In polymerization using alkali

[Formula] 1, 2 addition, Vinyl type polymerization and

[Formula] It is known that a mixture of 3, 4 addition and aldehyde type polymerization occurs,
It is known to obtain polymers with a significant presence of C═C double bonds in the polymer. (Polymer Processing 22, 725 (1973)) The present inventors have completed the present invention as a result of intensive studies on the polymerization method of acrolein. That is, the present invention involves adding 0.5 to 10% by weight of a compound that generates hydrogen sulfite ions to acrolein when polymerizing acrolein in an aqueous solvent, and polymerizing under alkaline conditions without the presence of a catalyst that generates free radicals. The present invention relates to a method for producing polyacrolein fine particles characterized by the following. According to the present invention, the particle size of the polyacrolein fine particles obtained can be easily controlled by adjusting the amount of the compound that generates hydrogen sulfite ions added within the above range, and the particle size can be very uniform. A polyacrolein emulsion is obtained. Furthermore, unlike the use of conventional surfactants, there is no undesirable effect on protein binding. In the present invention, polymerization is carried out in an aqueous solvent, but it is also possible to use a small amount of a hydrophilic organic solvent such as methanol, ethanol, acetone, dioxane, dimethylformamide, dimethylsulfoxide, etc. In this case, the amount of the hydrophilic organic solvent used is preferably 25% by weight or less based on water. Various compounds can be used as the compound that generates hydrogen sulfite ions. For example, sulfite salts such as alkali metal sulfite salts, alkaline earth metal sulfite salts, ammonium sulfite salts, and sulfurous acid can be used. More specifically, examples include sodium bisulfite, sodium sulfite, sodium metabisulfite, potassium bisulfite, potassium sulfite, potassium metabisulfite, lithium sulfite, calcium sulfite, calcium bisulfite, ammonium sulfite, ammonium bisulfite, sulfite, and the like. It will be done. In the present invention, when a compound that generates bisulfite ions is added to an aqueous solvent, bisulfite ions are generated, which react with acrolein to form an adduct, and this adduct is used as an excellent dispersant. Seem. The amount of the compound that generates bisulfite ions used is in the range of 0.5 to 10% by weight based on acrolein. By varying the amount of the compound that generates bisulfite ions added within this range, the particle size of the polyacrolein fine particles in the resulting foliacrolein emulsion can be freely controlled, for example, from 0.05 microns to 4 microns. In the present invention, polymerization is carried out under alkaline conditions. If the reaction system becomes alkaline by adding a compound that generates bisulfite ions,
Polymerization may be carried out as is. If the reaction system does not become alkaline even after adding a compound that generates hydrogen sulfite ions, the reaction system may be made alkaline by adding an alkali. In this case, the alkali is not particularly limited and various types can be used, such as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkaline earth metal hydroxides such as calcium hydroxide, sodium carbonate, potassium carbonate, etc. In addition to carbonates such as, tertiary amines such as pyridine and trimethylamine can also be used. In the present invention, when polymerization is carried out under alkaline conditions, it is preferable that the pH of the reaction system at the start of polymerization is 12 or less, and in particular, the pH of the reaction system at the start of polymerization is 9 to 9.
It is preferable to set it to 10.5. PH of reaction system
The higher the value, the faster the reaction, but the Canitzaro reaction is more likely to occur as a side reaction, and the resulting polyacrolein fine particles are more likely to be colored. In addition, the reaction system
Although the PH decreases as the reaction progresses, it is not necessary to keep the PH constant. In the present invention, the ratio of acrolein to the solvent used is preferably such that the amount of acrolein is 100% by weight or less of the amount of the solvent used. The polymerization reaction is preferably carried out at room temperature. According to the present invention, the irritating odor of acrolein is almost eliminated in a short polymerization time of 1 to 2 hours, and the polymerization can be completed in a short time. The polyacrolein fine particles obtained by the present invention can be used as a carrier as is and sensitized with antigens, antibodies, etc. to be used as an immunodiagnostic test agent.
In order to further stabilize the polyacrolein fine particles, it is also possible to copolymerize the polyacrolein fine particles obtained by the present invention with other copolymerizable monomers, and then sensitize them with antigens, antibodies, etc. Examples of other copolymerizable monomers include methyl (meth)acrylate, styrene, ethyl (meth)acrylate, butyl (meth)acrylate, 2-
Ethylhexyl (meth)acrylate, (meth)
Examples include acrylonitrile. Other copolymerizable monomers can be used in any proportion to polyacrolein, but it is preferably used in an amount of 100% by weight or less. When copolymerizing other copolymerizable monomers with the polyacrolein fine particles, the copolymerizable monomer may be added to the reaction system after the polymerization of acrolein of the present invention is almost completed, but it may be added during the polymerization of acrolein or during the polymerization of acrolein. A copolymerizable monomer may be added before starting the process. Persulfates, azo compounds, benzoyl peroxide, etc. are suitable as initiators used when copolymerizing copolymerizable monomers with polyacrolein fine particles, and redox catalysts are used by combining these with reducing agents. and more effective. The amount of initiator used is based on the copolymerizable monomer.
0.01 to 3% by weight, especially 0.05 to 1% by weight are preferred. Since many aldehyde groups exist on the surface of the polyacrolein fine particles obtained by the present invention, they are particularly useful as carriers for immunodiagnostic tests, and can be used with other copolymerizable polyacrolein fine particles or the like. Simply by contacting microparticles obtained by copolymerizing monomers with antigens or antibodies, the antigen or antibody can be firmly immobilized on the surface of the microparticles. The polyacrolein fine particles obtained by the present invention have a high specific gravity, and when the particles obtained by sensitizing them with an antigen or antibody are used as a diagnostic test agent in place of red blood cells in a test using the microtiter method, they are particularly excellent. Effects can be obtained. That is, when the polyacrolein fine particles obtained in the present invention are used as a carrier, a fixed amount of serum or urine containing an antibody or antigen corresponding to the sensitized antigen or antibody is serially diluted on a microplate. When added and left to stand after mixing, a clear agglomerated image appears in 1 to 3 hours. (Example) Example 1 Aqueous solution containing 0.2g of sodium bisulfite
Add 10g of acrolein to 90g and add 1 while stirring.
% sodium hydroxide was added until the pH reached 10, stirring was continued, and the polymerization was completed in 3 hours. At this time
PH drops to 7.8. In order to remove a small amount of unreacted acrolein and soluble polymers from the obtained polyacrolein emulsion, it was centrifuged.
Only the emulsion particles were precipitated by rotating at 2500 rpm for 10 minutes, and a polyacrolein emulsion was obtained by replacing the dispersion medium with water. When the particle size was observed and measured using a scanning electron microscope, it was found to be 1.96μ, with almost no particle size distribution. Example 2 Under the same conditions as Example 1, sodium bisulfite
When 0.1 g was used for polymerization, an emulsion with a particle size of 3.2 μm and almost no particle size distribution was obtained. Example 3 Sodium bisulfite under the same conditions as Example 1
When 0.5 g was used for polymerization, an emulsion with almost no particle size distribution and a particle size of 1.17 μm was obtained. Example 4 Sodium bisulfite under the same conditions as Example 1
When 1.0 g was used for polymerization, an emulsion with almost no particle size distribution and a particle size of 0.26 μm was obtained. Example 5 The reaction was carried out in the same manner as in Example 1 except that sodium hydroxide was used instead of sodium hydroxide in Example 1, and the same results as in Example 1 were obtained. Example 6 In Example 1, 0.2 g of sodium bisulfite
Instead of 0.24 g of sodium sulfite, polymerization was carried out as it was without adding 1% sodium hydroxide, and the reaction was otherwise carried out in the same manner as in Example 1.
Results similar to those in Example 1 were obtained. Example 7 In Example 1, 0.2 g of sodium bisulfite
The reaction was carried out in the same manner as in Example 1, except that 0.18 g of sodium pyrosulfite was used instead of , and the same results as in Example 1 were obtained. Example 8 In Example 1, 0.2 g of sodium bisulfite
The reaction was carried out in the same manner as in Example 1, except that 0.23 g of potassium hydrogen sulfite was used instead of, and the same results as in Example 1 were obtained. Example 9 In Example 1, 0.2 g of sodium bisulfite
When the reaction was carried out in the same manner as in Example 1 except that 0.3 g of potassium sulfite was used instead of 1% sodium hydroxide and the reaction was carried out as it was without adding 1% sodium hydroxide, the same results as in Example 1 were obtained. Example 10 In Example 1, sodium bisulfite 0.2
90 g of an aqueous ethanol solution containing 0.2 g of sodium bisulfite instead of 90 g of an aqueous solution containing g
The reaction was carried out in the same manner as in Example 1 except that [water:ethanol=90:10 (weight ratio)] was used, and the same results as in Example 1 were obtained. Example 11 The reaction was carried out in the same manner as in Example 10, except that acetone was used instead of ethanol, and the same results as in Example 1 were obtained. Example 12 The reaction was carried out in the same manner as in Example 1 except that calcium hydroxide was used instead of sodium hydroxide, and the same results as in Example 1 were obtained. Example 13 In Example 1, sodium bisulfite 0.2
The reaction was carried out in the same manner as in Example 1 except that 0.26 g of ammonium sulfite was used in place of 0.2 g of ammonium sulfite, and the same results as in Example 1 were obtained. Example 14 In Example 1, sodium bisulfite 0.2
The reaction was carried out in the same manner as in Example 1, except that 1.23 g of a sulfite aqueous solution containing 10% by weight of sulfur dioxide was used instead of 1.2 g of sulfur dioxide, and the same results as in Example 1 were obtained. Comparative Example 1 A polyacrolein agglomerate polymerized in water using ammonium persulfate was washed with water to give a concentration of 2.5 per gram.
Add g of sodium bisulfite and stir to dissolve. After complete dissolution, the solution was poured into a cellophane tube and dialyzed against distilled water for one day to remove excess sodium bisulfite. Using the thus obtained sulfite adduct of polyacrolein as a dispersant, an aqueous solution containing 0.2 g of this adduct
Acrolein was polymerized in the same manner as in Example 1 using 90 parts. The particle diameters of the fine particles of the obtained polyacrolein emulsion had a bimodal distribution centered around 2μ and 0.5μ. Example 15 Aqueous solution containing 0.2g of sodium bisulfite
Add 10 parts of acrolein to 90 parts, add 1% aqueous sodium hydroxide solution with stirring until pH = 9.5, continue the reaction for 3 hours, then introduce nitrogen gas into the reaction vessel to drive out oxygen. Add 2 parts of methyl methacrylate and 1 part of 2% ammonium persulfate aqueous solution, and react at a reaction temperature of 50°C for 5 hours.
Polymerization was carried out at 65°C for 2 hours to complete the copolymerization of methyl methacrylate. When washed in the same manner as in Example 1 and the particle size was measured, the average particle size was 2.18μ, and the particles had almost no particle size distribution. Reference Example 1 Measurement of Rheumatoid Factor The polyacrolein emulsion obtained in Example 3 was dispersed to a solid content concentration of 0.5%.
1 part of 0.05M phosphate buffered saline (PBS) solution and 5 mg/ml of heat-denatured human gamma globulin (Sigma human gamma globulin soluble by heat treatment at 63°C for 10 minutes)
Mix with 1 part of PBS solution dissolved in 37
Shake slowly for 1 hour at ℃, then add 1 part of bovine serum albumin (BSA) 1% PBS solution,
Shake at ℃×1 hour. The precipitate obtained by centrifugation (1500 rpm x 5 min) was washed three times with PBS and finally dispersed in a PBS solution containing 0.1% BSA to give a sensitized latex concentration of 0.5% to obtain a sensitized latex. . Separately, 25μ of PBS containing 0.1% BSA was added to each well of a 96-well V-type microplate, and normal human serum and RA (+) serum diluted 10 times with the same PBS (a mixture of 3 people) was added to the first well. ) was added and serially diluted 2 times using a diluter. Add 25μ of sensitizing latex to each well,
The mixture was shaken with a mixer and left at room temperature for 2 hours. The aggregation image was as follows.

【table】

[Table] (Effects of the invention) According to the present invention, by adjusting the amount of the compound that generates hydrogen sulfite ions added, the particle size of the obtained polyacrolein fine particles can be easily controlled. A highly stable polyacrolein emulsion with a very uniform particle size with almost no distribution can be obtained in a relatively short time, and acrolein, which has a strong odor, can be almost completely removed in a short time.
100% polymerization can be completed. Furthermore, when sensitizing the surface of the polyacrolein fine particles obtained according to the present invention with an antigen or antibody,
No untoward effects arise from the use of compounds that generate bisulfite ions.

Claims (1)

[Claims] 1. Polymerization characterized in that when acrolein is polymerized in an aqueous solvent, 0.5 to 10% by weight of a compound that generates hydrogen sulfite ions is added to the acrolein, and the polymerization is carried out under alkaline conditions without the presence of a catalyst that generates free radicals. A method for producing acrolein fine particles.