CS228215B1 - Method for the production of light wood soles for ladies summer walking shoes - Google Patents

Description

The invention relates to a process for the preparation of reactive hydrophilic latex particles with a fluorescent label by a simple and undemanding method, particles comprising a built-in fluorescent label are very important e.g. in immunology when used to detect antigens or antibodies in human or animal tissues: particularly suitable for studying tissue sections in histology. Another application is to selectively label cells bearing defined receptors on their surface.

Latex particles have traditionally been synthesized by emulsion radical polymerization, a widely used method in industry. The water-insoluble monomer is then mixed in an aqueous emulsifier solution to obtain milk-like emulsions. If a water-soluble radical initiator is now added to this emulsion, polymerization is started at a suitable temperature. The resulting latex is very stable in the presence of emulsifiers. Each of its particles is surrounded by an ionized layer of soap molecules that prevent coagulation. However, this layer causes the surface of the particles to have completely different chemical properties than the polymer itself.

The disadvantage of the classical system is partially eliminated by so-called emulsifier emulsion polymerization. This also leads to latexes which do not contain an undesirable e-emulsifier and therefore substantially cleaner. Thus, for example, latexes were prepared from polytetrafluoroethylene, polystyrene, polyvinyl acetate, polycarboxystyrene.

Stable latexes arise, in particular, from the polymerization of ionogenic monomers, with the ionogenic groups being situated on the surface of the particles in such a way that they assume the function of emulsifiers. For immunological purposes, all of the above-mentioned monomers are not well suited, since their activation allowing the biologically and / or immunologically active substance to bind in a subsequent sequence is difficult or at least difficult. Therefore, hydrophilic latex particles have been developed. They are capable of covalently binding biologically and / or immunologically active substances without compromising the structure of the bound substance on one side or breaking the stability of the emulsion on the other and are the subject of MS. AO No. 225 010. According to this application, hydrophilic latex particles are synthesized by emulsion polymerizing monomers containing epoxy groups in the molecule or in the polymer. in combination with other monomers causing desirable changes in particle properties, in the presence of a water-soluble radical-forming initiator, but without the addition of any emulsifier or emulsion stabilizer. Monomeric, radically polymerizable derivatives of colored or fluorescent compounds in which residues are methacrylated or ecrylolated can also be used as one of the other monomers used.

Another known method for obtaining fluorescently labeled latex particles for immunology applications described in US Pat. No. 4,108,972 consists in the reaction of ready-made latex particles with reactive derivatives of fluorescent or colored compounds. The latter two methods have the common disadvantage of having to synthesize the appropriate derivative, which is often long and laborious.

In the latter case, the fact that the reaction of the colored or fluorescent compound with the latex particle takes place almost exclusively on the particle surface and here with the ¹ groups originally intended for binding of the biologically and / or immunologically active substance. Since not all groups are equally reactive for a variety of reasons, the groups that are most accessible first react and the less reactive or less readily available groups remain for the binding of biologically and / or immunologically active substances which are more demanding.

In addition, at high substitution, the surface area of the particles is very different in composition from the original polymer, and the advantage of the emulsifier process is washed away.

These disadvantages are overcome by the process for preparing the reactive hydrophilic latex particles with a fluorescent label according to the invention. The present invention provides a process for the preparation of hydrophilic latex particles with a fluorescent label characterized by polymerizing a monomer containing a polymerizable double bond and a epoxy group selected from the group consisting of 3,4-epoxy-t-butene, glycidyl vinyl ether, glycidyl vinyl phthalate, glycidyl methacrylate, glycidyl methacrylate alone or in the presence of an additional monochrome divinyl monomer in an amount of less than 99 wt. selected from the group consisting of styrene, vinyl acetate, isoprene, divinylbenzene, ethylenedimethacrylate, and in the presence of 0.15 wt. fluorescent and / or coloring agents soluble in monomers selected from the group consisting of fluorescein, rosamine, rhodamine, coumarin, acridine and their derivatives after dispersion in 8 to 16 times the amount of water in which 0.5 to 1.5 g / l of initiator is dissolved free-radical polymerization at 0 to 80 ° C for 5 to 40 hours.

The process according to the invention is further characterized in that the partition coefficient of the fluorescent and / or colored substance between the aqueous and polymerizing phases is changed by means of a phase transfer catalyst.

The process for preparing the fluorescently labeled particles of the present invention is sensitive to the presence of atmospheric oxygen and must therefore be removed from all vessel components by boiling or distillation and manipulation under an inert atmosphere of nitrogen, argon or other inert gas.

The polymerization itself proceeds preferably at a volume ratio of water to monomer phase in the feed of from 8: 1 to 16: 1, at a temperature of 0 to 80 ° C, depending on the initiator used, for a period of 5 to 40 hours.

(A monomer containing a polymerizable double bond and at the same time an epoxy group can only be used in one or a mixture thereof. The epoxide content of the particles, as well as their other chemical and physical properties, is preferably adjusted by adding another monomer such as styrene, isoprene, vinyl acetate.

In order to avoid solubility of the resulting latex particles in water or organic solvents, which may be very acute in some, especially copolymeric feedstocks with strongly hydrophilic hoaonomers, conventional divinyl-type-type thickeners such as divisylbenzene, ethylenedimethacrylate and others are added to the polymerization feed.

The essential input into the process for the preparation of reactive nydrophilic latex particles is the presence of colored and / or fluorescent compounds dissolved in the monomer or monomer mixture prior to the commencement of emulsion polymerization.

Any water-soluble initiator commonly used in emulsion polymerization can be used as initiator. These are preferably peiOxodisulfáty, perborate, hydrogen peroxide and ionic derivatives ^2.2, -azodiisobutyronitrilu.

The reactive, hydrophilic, latex particles with a fluorescent label prepared by the process of the invention are perfectly spherical, all of virtually the same size (monodisperse) and having a diameter of from 0.2 to 1%.

The synthesized latex upon completion of the polymerization may contain residues of unreacted monomers and a colored or fluorescent substance, which is preferably removed by steam distillation, dialysis, or decantation using a centrifuge. A suitable method for controlling latex purification is by monitoring the fluorescence of the supernetant or dialysate.

The principle of incorporating a colored or fluorescent substance consists in transferring a radical from the polymerizing chain to a colored or fluorescent substance molecule, which itself or a part of it in the next stage serves as a starter for the next chain or is deactivated by recombination. The transfer reaction can take place both in the water phase to form fluorescent label terminated surface-active oligomers and in the aicellar organic phase.

The proportion of both types of reactions is dependent on the partition coefficient of the colored or fluorescent substance between the aqueous and organic phases and the diffusion rate between the phases. Increasing the solubility of the fluorescent substance in the organic micellar phase can be achieved by simple chemical modification.

The high efficiency of the transfer reactions taking place in the polymerization mixture containing the fluorescent substance can be attributed to their chemical structure. Most of these are phenolic-type aromatic compounds which are generally characterized by high transfer constants.

Increase of the interfacial transport can then be achieved also by means of phase carriers.

The terminal epoxy groups of the side chains in the latexes are highly reactive and can be readily modified by chemical reaction to form other groups more suitable for the intended use. As a rule, the fluorescence of the latex is not lost and its utility properties are preserved.

He did

10 mg of fluoresoein was dissolved in 20 g of glycidyl methacrylate and the dissolved oxygen was removed by a stream of nitrogen. 0.4 g of 3,3'-azobis-O-cyanobutanesulfonene sodium was dissolved in 200 ml of boiled redistilled water cooled in a stream of nitrogen; both components were transferred to a glass reactor and nitrogen was bubbled for 10 min. The reactor was then sealed and the mixture was allowed to react at 75 ° C for 24 hours with stirring (500 rpm). After this time, 95% conversion was achieved, the product being a latex formed by monodisperse spherical particles of yellow color with a diameter of 500 nm and fluorescing in UV light (yellow-green fluorescence). The polymer contains 0.05 mg of fluorescein (1 g of polyglycidyl methacrylate).

Example 2

As described in Example 1, a solution of 0.4 g of 3.3 ^z -azobis (sodium 3-cyanobutanesulfonate) and 200 ml of redistilled water and 20 g of a mixture containing 85 wt. styrene and 15% by weight. glycidyl methacrylate in which 10 mg of fluorescein and 20 mg of cetyltrimethylammonium chloride were dissolved. After mixing, the reaction mixture was heated with stirring at 500 rpm at 75 ° C for 24 hours. Unreacted monomer was removed by steam distillation. The resulting monodisperse copolymer particles have a diameter of 250 nm and exhibit yellow-green fluorescence in UV light. The polymer contains 0.03 mg of fluorescein / 1 kg of copolymers.

Example 3

The mixture prepared by mixing 0.3 g of 3,3'-azobis- (3-cyanobutanesulfonic acid) in 200 ml of redistilled water and 20 g of a mixture containing 85 wt. % vinyl acetate and 15 wt. glycidyl methacrylate. Under the polymerization conditions of Example 1, the conversion reached 80%, the latex formed consisting of monodisperse spherical particles with a diameter of 200 nm. The polymer contains 0.02 mg fluorescein / 1 g polymer.

Example

A batch consisting of a solution of 0.4 g of 3,3'-azobis- (3-cyanobutanesulfonate) in 200 ml of redistilled water and 20 g of a mixture of 15 wt. % glycidyl methacrylate and 85 wt. isoprene and containing 10 mg of fluorescein and 10 mg of cetyltrimethylammonium chloride per hour at 75 ° C. The conversion achieved was 75%, unreacted monomers were removed by steam distillation. The resulting stable latex contained monodisperse spherical particles and a diameter of 250 nm, showing yellow-green fluorescence.

Example 5

As described in Example 1, a solution of 0.4 g of 3,3'-azobis (3-cyanobutanesulfonic acid) in 200 ml of redistilled water and 20 g of glycidyl methacrylate, in which 10 mg of coumarin was dissolved and then mixed and polymerized, was separately deoxygenated. for 24 hours at 75 ° C with stirring. Unreacted monomer and low molecular weight substances were removed by dialysis. The conversion was 96%,

The resulting latex contained monodisperse spherical particles with a diameter of 520 nm, showing blue fluorescence in UV light. The polymer contained 0.04 mg coumarin / 1 g polymer.

100 ml of latex was added to the reaction with 100 ml of aqueous ammonia solution (25%) and allowed to react at room temperature for 24 hours, resulting in the formation of amino groups by aminolysis of the oxirane groups. / G polymer.

Example

As described in Example 1, separately deoxygenated solutions of 0.4 g of 3,3'-azobis (3-cyanobutanesulfonene sodium) in 200 ml of water and 20 g of glycidyl methacrylate with 1 g of ethylenedimethacrylate; 10 mg of rhodamine B and 20 mg of dodecyltrimethylammonium chloride were dissolved in the monomer mixture. After mixing, the reaction mixture was polymerized at 75 ° C for 12 hours with stirring. Unreacted monomer and low molecular weight substances were removed by pressure filtration.

The conversion was 96%, the resulting latex contained monodisperse spherical particles of 500 nm diameter, exhibiting UV fluorescence corresponding to 0.03 mg rhodamine B / 1 g. polymer. The reaction was charged with 100 mL of latex with 100 mL of 0.05 M H 2 SO 4 with heating at 0 ° C for 3 hours. Oxirane groups are hydrolyzed to form vialel diols. The size of the latex particles is maintained, and after latex treatment by dialysis against a 1% soda solution, the particles exhibit fluorescence corresponding to 0.03 mg of rhodamine B / 1 g of polymer. The hydrolyzed latex (100 mL) was oxidized with a solution of 0.1 N H ₄ O ₄ (200 mL) at room temperature for 24 hours with stirring. By dialysis of the latex against a 1% soda solution, unreacted periodic acid was removed and the total volume of the latex was adjusted to 100 ml by pressure filtration; latex particles contain 4 wt. The aldehyde groups and their fluorescence correspond to 0.01 mg of rhodamine B / 1 g of polymer.

Example 7

As described in Example 1, oxygen was removed from solutions of 0.4 g of 3,3'-azobis (3-cyanobutanesulfonic acid) in 300 ml of water and 20 g of glycidyl methacrylate with 0.5 g of divinylbenzene in which 15 mg of the coumarin condensation product were dissolved. with formaldehyde and dodecylamine. After mixing, the reaction mixture was polymerized at 75 ° C for 24 hours. Unreacted monomers and its molecular substances were removed by pressure filtration? the resulting latex contained 500 nm monodisperse spherical particles exhibiting blue fluorescence.

Example 8

KluoresCi-latex-OCHg-CH-pHg 0

NH.OH — Fluoresc-latex-OCH-CH-CH ^{1 2} 1 I

OH NH fluoresc ₂ + CH_0 -fluoresc, -latex-OGHn -GH-GH ' ² i I ²

OH NH-CH ₂ -fluoresc ₂

The latex prepared by the procedure of Example 1 and containing 0.05 mg of fluorescein / 1 g of polymer was diluted with an equal volume of 15% ammonium hydroxide solution and allowed to react for 24 hours at room temperature. Ammonolysis results in the formation of initial amino and hydroxyl groups from oxirane groups. After dialysis and adjusting the pH of the latex to 8, 10 mg of fluorescein and 0.5 ml of a 37% formaldehyde solution were added and the reaction mixture stirred for an additional 24 hours at room temperature and then dialyzed until the dialysate lost fluorescence with 1% soda solution. The modified latex contained 500 nm diameter monodisperse spherical particles and contained 0.10 mg fluorescein / 1 g polymer.

Claims (2)

SUBJECT OF THE INVENTION 1. A process for the preparation of reactive hydrophilic latex particles with a fluorescent label comprising polymerizing a monomer containing a polymerizable double bond and a epoxy group selected from the group consisting of 3,4-epoxy-1-butene, glycidyl vinyl ether, glycidyl vinyl phthalate, glycidyl methacrylate, glycidyl acrylate alone or in the presence of another mono- or divinyl monomer in an amount of less than 99 wt. % selected from the group consisting of styrene, vinyl acetate, isoprene, dlvinylbenzene, ethylenedimethacrylate and in the presence of 0.15 to 1 wt. fluorescent and / or coloring agent soluble monomers selected from the group consisting of fluorescein, rosamine, rhodamine, coumarin, acridine and their derivatives after dispersion in 8 to 10 times the amount of water in which 0.5 to 1.5 g / l of initiator is dissolved radical polymerization at 0 to 80 ° C for 5 to 40 hours. 2. A process according to claim 1, characterized in that the partition coefficient of the fluorescent and / or colored substance is changed by means of a phase transfer catalyst between the aqueous and polymerizing phases.