JPS5945302A - Manufacture of reactive hydrophilic latex particles marked with coloring matter or fluorescent compound - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a process for making reactive hydrophilic latex particles marked with five dyes or fluorescent compounds that is easily carried out with few difficult requirements. Particles containing incorporated dyes or fluorescent compounds are of great importance, for example in immunology, since they are suitable for detecting antigens or antibodies in human or veterinary tissues. . This detection method is particularly advantageous for making tissue sections, especially in histology. Another application is the selective marking of cells bearing uptakers identified on their surfaces.

Latex particles are produced by radical emulsion polymerization in a known manner. In this case, a milk-like emulsion is formed when a poorly water-soluble monomer is stirred together with an aqueous solution of an emulsifier. By adding a water-soluble radical initiator, polymerization is initiated at an appropriate temperature. The resulting latex is extremely stable in the presence of emulsifiers, since each particle is surrounded by a layer of ionized soap molecules and thus protected from agglomeration. However, the enclosing layer causes the latex particles to exhibit properties that are completely different from those of the original polymer.

The disadvantages of classical systems are partly avoided by so-called emulsifier-free emulsion polymerization. This polymerization results in a 7 tesox that contains no emulsifiers and thus is extremely pure. Such latexes include, for example, polytetrafluoroethylene I
4. Stable latexes produced from polystyrene, polyvinyl acetate, and polycarboxylic styrene are produced especially in the polymerization of ionogenic particles, in which case the ionogenic groups on the particle surface play the role of emulsifiers. 4, however, for immunological purposes the intermediates described above are particularly unsuitable. This is because the introduction of active groups that enable coupling with biologically and/or immunologically active substances is expensive and difficult. Purchase! Permit open wholesale 1s No. 54685
Hydrophilic latex particles have been developed based on the present invention, which can bind biologically and/or immunologically active proteins in the cylinder, and in doing so, the structure of the ζ substance and the stability of the latex have been developed. The hydrophilic latex particles are prepared by adding monomers containing epoxy groups, optionally in the presence of intermediates and water-soluble initiators that affect other particle properties in the desired direction, but without decomposition. Additional monomers which influence the 3° particle properties obtained by emulsion polymerization or by polymerization without the addition of stabilizers include, for example, polymerizable derivatives of intermediates of dyes or fluorescent compounds. can be used. In this case, hydrophilic IIA Nilatex particles marked with dyes or fluorescent compounds are obtained.

Another possibility for producing colored or fluorescent latex particles for use in immunology is to prepare ready-made latex particles, as described for example in U.S. Pat. No. 4,108,972. Reaction of dyes or fluorescent compounds +'11 Both methods described above have the following common drawbacks. It is absolutely necessary to synthesize reactive derivatives of dyes or fluorescent compounds in large numbers and by complicated methods. Furthermore,
In the method described in U.S. Pat. No. 4,108,972, the reaction of the reactive derivative of the dye or fluorescent compound takes place almost exclusively on the surface of the particles, i.e. biological and/or immunological in origin. In cases where a reaction occurs with the reactive groups of the loaded particles for cassillation with the active substance, the reactive and acid-accessible groups are first reacted with the dye or fluorescent compound. Anti(HH.

However, it is particularly disadvantageous that biologically and/or immunologically active proteins 71 are left with only low-reactivity and difficult-to-obtain groups for Cassilling's /ζ of 1υ1. 1 Furthermore, the higher the degree of substitution, the less free surface area is available for coupling. Therefore, the advantages of emulsion polymerization without emulsifiers are lost.

The above-mentioned drawback 1 of the known processes for producing reactive hydrophilic latex particles marked with dyes or fluorescent compounds is avoided by the Jj method of the present invention.

The present invention provides a method for producing reactive hydrophilic latex particles marked with a dye or a fluorescent compound. It is characterized in that a mixture of monocular bodies is polymerized or copolymerized in the presence of a dye or a fluorescent compound. The polymerization is carried out in an aqueous dispersion with exclusion of hydrogen using water-soluble radical-forming initiators, but on the basis of the emulsification method with the addition of emulsifiers or stabilizers.

As the water-soluble monomer, an epoxy compound containing at least one C═C double bond in the molecule is advantageously used. In particular, epoquine alkylene compounds, bridyl esters or glycidyl ethers, such as glycidyl acrylate, glycidyl methacrylate, glycidyl vinyl ether, glycidyl vinyl phthalate, 3.4-
Preference is given to epoxy compound-1. A particular epoxy monomer or also a mixture of different epoxy monomers can be utilized in the process of the present invention. The concentration of epoxy compound in the monomer phase can be from 1 to 100 parts by weight.

The content of epoxy groups, as well as other chemical and physical properties, is advantageously determined by other monocular substances such as styrenes, dienes, acrylamides, methacrylamides, alkyl-, hydroxyargyl- and aminoalkyl acrylates or -
methacrylate, vinyl ether, vinyl ester, N
It can be controlled by adding vinylpyrrolidone, methacrylic or acrylaldehyde.

In most cases, especially when strongly hydrophilic comonomers are present, it is necessary to reduce the solubility of the resulting latex particles in water or organic solvents.

This is achieved by adding to the polymerization mixture customary divinyl crosslinking agents such as alkylene- or hydroxyalkylene acrylate-1 or -methacrylate, alkylene bisacrylic- or -methacrylamide, divinylbenzene. be able to.

The dye and the fluorescent compound are preferably present in the polymerization mixture in an amount of 0.05
Add in an amount of ~1 part by weight. Particularly advantageous according to the invention are fluorescein, rhosamine, rhodamine, coumarin, acridine and similar compounds.

As initiators it is possible to use all the water-soluble initiators customary for emulsion polymerizations. According to the invention, it is advantageous to use 2,2'-azodiisobutyronitrile derivatives of beroxodisulfate, oxoborate, hydrogen peroxide or ionogens in a concentration of 0.5 to 1.5.9 / l. be.

The present infusion method for producing reactive hydrophilic latex particles marked with dyes or fluorescent compounds is extremely sensitive to oxygen. Therefore, oxygen can be very carefully removed from the entire composition and the container by thorough boiling, distillation under an inert atmosphere, or introduction of nitrogen, argon or other inert gas.

The polymerization is preferably carried out according to the invention at a float to volume ratio between water and monomer phases of 8:1 to 16:1. Temperature is 0~
80 DEG C., preferably in the range from 60 to 80'G, and the reaction time in the range from 5 to 40 hours, the surface parameters depending on the selected initiator.

The reactive hydrophilic Latinx particles produced in the present invention are
They are spherical, monodisperse, subdivided particles of approximately the same affinity to each other in the strict sense, with a diameter of about 02 to 1.511 m. , colored or fluorescent latex particles may still contain residual amounts of unpolymerized monomers, dyes or fluorescent compounds after the end of emulsion polymerization, which can be removed by steam distillation, dialysis or several rounds of It can be removed by centrifugation and decantation. The pure IW of latex particles can advantageously be tracked by dialysis or by fluorescein testing of the waste water.

The introduction of dyes or fluorescent compounds can take place in the aqueous phase with the release of oligomers terminated with fluorescent molecules, similar to soaps, as well as in organic micelles.

The ratio of the two steps depends on the partition coefficient of the dye or fluorescent compound between the water and the organic phase and the rate of diffusion between the two phases.

The solubility of a dye or fluorescent compound in one or the other stream is primarily determined by its chemical structure.

The dyes or fluorescent compounds used in the invention are generally aromatic substances, such as phenols, which generally have a high transfer constant.

Solubility properties can be adjusted by simple chemical transformations. Finally, mesophase transport can also be enhanced with phase-transfer catalysts.

Free evoquine groups at the terminal positions of the latex are highly reactive. These can easily be converted by reactions known per se into other reactive groups, ie more suitable for coupling with biologically and/or immunologically active proteins. . In these reactions, the color and fluorescent properties of the latex particles are generally not lost.

Next, the method of the present invention will be explained in detail in Example 3. Example 1 Glycidyl methacrylate-1.
Atmospheric oxygen is removed by melting 0.071 g and passing through nitrogen. In 200 m6 of distilled water that has been boiled and cooled by passing nitrogen through it.

3.3'-Azobis(3-cyanobutane sodium-sulfonane)0. 'Dissolve 4g. Both components are placed in a glass reactor and treated with nitrogen for a further 10 minutes. Then,
The reactor was sealed and vigorously stirred (500r).
pm) 75°C (7) Chang'f f 2 hours The reaction is carried out 4, After this time, the reaction rate is 95% 31 The reaction product is colored yellow and t, + V red white These are the 1-dimensional r(+ dispersion beams) that emit light at , and these have a diameter of 500 [+111].

The polymer contains 0.05 fluorescein in polygrindyl methacrylate-1,1,q! 7 containing tJ, e.g.
2. In the same manner as described in Example 1, 3. 200 ml of distilled water in which 0.4.9 % of δ'-azobis (δ-anobutan sodium, surpona) was dissolved and 110 liters of fluorescein
Oxygen was removed separately from 20 g of a mixture consisting of 15% purinenormethacrylate and 85% methylene methacrylate in which 11 and cetyltrimethylammonium chloride were dissolved. After the ,/IL reaction, the reaction mixture is allowed to react for 24 hours at a temperature of 75° C. without vigorous mixing. 3 The unpolymerized residual monomers are removed by steam distillation.

The monodisperse copolymer particles produced have a diameter of 250 nm. These emit yellow-green fluorescence under UV red and white. The concentration of cruorescein is 0.03m! l//3° where 1 g of polymer is used Example 3 Distilled water 200.71/! middle 3
．． 3'-Anobis (3'-Anobutanna IS Li-Sulfonane) 0.3.9 M solution! :, glycidyl methacrylate-1 dissolved in yl A recene l Q +719
- Removal of oxygen from a mixture of 20 & 15 Chongshuu and Vinyl Acede 1-85ii candy and allowing them to react with each other. , under the polymerization conditions described in Example 1, the reaction rate is 80%. This latex consists of monodisperse spherical particles with a diameter of 200 nm.

The fluorine content is 0.02 my/polymer]-,
Example 4 In the same manner as described in Example 1, 3.9 in 2 OQ me of distilled water was prepared.
A solution of 0.4 g of 3'-anobis(3-cyanobutane-nathosulfonane) and glycidyl methacrylate-1
・20g mixture of 15 polymerization parts and 1 soprene 85 parts by weight
and fluorescein]-o:n';t and cetyl trimedelammonium zolomi'10m! ? and temperature 7
React at 5°C for 24 hours 1, the reaction rate is 75%. The residual monomer is removed by steam distillation. The stable latex is yellow-green fluorescent and contains dispersible, finely divided spherical particles with a diameter of 250 nm.

Example 5 3.3' in 20 ord of distilled water as described in Example 1.
-Azobis(3-cyanbutane-sodium-sulfonane) 0.4 g, glycidyl methacrylate-1-20 with coumarin IQ +719,! Oxygen is removed separately from 7. After mixing, the reaction mixture is polymerized for 25 hours at a temperature of 75° C. with vigorous stirring. Residual impurities and low molecular weight substances in the pond are removed by dialysis. This reaction rate is 96%. The latex obtained fluoresces in the U-red, white, and blue colors and contains monodisperse, finely divided spherical particles with a diameter of 520 nm. Coumarin content is 0
04 mg/g of polymer.

Subsequently, ammonia solution (25 ml) was added to 100 ml of this emulsion.
%) 100 m/ and left at room temperature for 24 hours. At this time, the epoxy group is converted into an amine group by the ammonolith. All other properties of the latex remain the same. This modified latex contains coumarin 0.03 m
9/Contains 1 g of polymer.

Example 6 In the same manner as described in Example 1, 3.3' of distilled water
Air oxygen is removed separately from 0.4 g of azobis(3-cyanobutane-sodium-sulfonane) and 20 g of glycidyl methacrylate with 1 g of ethylene dimethacrylate, 1 g of rhodamine B1omy and 20 m9 of F-decyltrimethylammonium chloride. After mixing, the reaction mixture is reacted for 12 hours at a temperature of 75° C. with vigorous stirring. Unreacted monomers and other low molecular weight substances are removed by pressure filtration. This reaction rate is 96%.

The monodisperse and UV red-white fluorescent particles have a diameter of 50
It has 0 nm.

Rhodamine B @ amount is 003! n9/1 g of polymer.

100 ml of 0.05 mol/l sulfuric acid are added to 13 ml of this emulsion HI OQ and subsequently heated to 80° C. for 3 hours.

At this time, the epoxy group is converted into the adjacent hydroxy group. The particle size remains the same. After dialysis against 1% soda solution, the particles were rhodamine B0.03r.
It emits a fluorescence equivalent to ng/g of polymer to 1 lj.

Next, 100 ml of the hydrolyzed latex was
Treat with 200 ml of mol/l periodic acid solution for 24 hours at room temperature with stirring. Dry filtration reduces capacity to 100
Reduce to i711. The latex particles thus obtained contain quaternary aldehyde groups. This fluorescence corresponds to a concentration of 0.01 m9 of rhodamine B and 1 g of combined rhodamine.

Example 7 As described in Example 1, 3,3'-azobis( Atmospheric oxygen is removed from the solution of 3-Nanobutan sulpon.

After mixing, the reaction mixture is polymerized for 24 hours at a temperature of 75°C. Unpolymerized monomer components and other low molecular weight substances are removed by pressure filtration. The latex contains dispersed spherical particles with a diameter of 500 nm and emits blue fluorescence.

Example 8 HNH2 prepared according to Example 1 and fluorescein 0.05 m
9/polymer l, 1 in the same volume to the latex containing 9
Add 5% ammonia solution and leave at room temperature for 24 hours. Then dialyze and adjust the pt value to PI+8. Add 0.5 m6 of fluorescein 1o;rv and'o:formaldehyde (37%).-1 Under constant stirring, the mixture is kept at room temperature for a further 24 hours. hold 3 then 1
% soda solution. The modified latex obtained consists of dispersible, finely divided spherical particles with a diameter of 500 I]171. This is full 1st/Sein 10m
g/g of polymer.

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Claims (1)

[Claims] (1) A method for producing reactive hydrophilic latex particles marked with 8 dyes or fluorescent compounds, in which one type of intermediater that is poorly soluble in water or various types of sparsely soluble in water are used. One-component polymerization by emulsion polymerization in which the corpus luteum is dispersed in water together with a dye or a fluorescent compound and in the presence of a water-soluble initiator forming radicals in the exclusion of oxygen, but without the addition of emulsifiers or stabilizers. or copolymerization of reactive hydrophilic latex particles marked with a dye or a fluorescent compound. 2. The method according to claim 1, characterized in that the water-dissolvable il-i form is at least one epoxy compound containing a polymerizable C==C double bond. , the method according to claim 2, characterized in that the epoxy compound is 3,4-epoxyzotene-1, glycidyl vinyl ether, glycidyl vinyl phthalate, glycidyl acrylate or glycidyl methacrylate. The method according to any one of claims 1 to 3, which is carried out by adding an agent. 5. As the dye or fluorescent compound, fluorescein, rhodamine, rhodamine, coumarin or haacridine or the r The method according to any one of claims 1 to 6, characterized in that the derivative is a derivative of a compound. 6. The distribution coefficient of the dye or fluorescent compound between the aqueous phase and the organic phase; 7. The method of claim 1, wherein the diffusion rate is varied by a phase-1 transfer catalyst. 7. The method of claim 1, wherein the concentration of the dye or fluorescent compound is from 0.05 to 1% by weight. The method according to any one of Items 1 to 6. 8. During emulsion polymerization, the float ratio is 8:1 to volume.
l6: ], is! The method according to any one of claims 1 to 7. 39. Process according to any one of claims 1 to 8, wherein the initiator concentration is between 0.5 and 1.5 g/'l. 10 The concentration of the epoxy compound in the monomer phase is between 1 and 10
0 weight person. The method according to any one of claims 1 to 9. 11. The method according to any one of claims 1 to 10, wherein the emulsion polymerization is carried out at a temperature range of 0 to 80°C for 5 to 40 hours.