HU196613B - Process for producing polyacrylamide - particularly coloured - gel beads by way of microsuspension polymerization - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to a novel microsuspension polymerization process, preferably a color polyacrylamide copolymer for the production of gel beads, in an aqueous solution of one or more initiators, one or more monomers, a crosslinking molecule, and one or more initiators of an emulsifier in a non-polar, water-immiscible inorganic solvent or solvent mixture. in the case of an aqueous solution of a substance to be polymerized containing an aqueous colloidal solution of a colorant, and dispersing a buffer solution under an inert gas atmosphere, the resulting organic solvent gel bead dispersion is poured into water at 80-100 ° C, allowed to cool, the phases are separated, and the aqueous medium is separated. The gel bead dispersion is sedimented, washed with water, subjected to fractional annealing in a known manner, and fractions of the desired particle size are collected and, if desired, within the colorless gel beads obtained. dyeing with in situ dye. -1-

Description

BACKGROUND OF THE INVENTION The present invention relates to a novel micro-slurry polymerization process, preferably to the production of colored polyacrylamide copolymer gel beads.

As is known, one of the most critical steps in radioinununoassay (R1A) is the separation of antibody-bound and free ligand. Therefore, some years after the appearance of radioimmunosay, the first so-called solid phase procedures were introduced. According to this method, the antiserum was coupled to Sephadex G-25 dextran gel, polystyrene, cellulose or Teflon (Biochem. J. 100, 31C / 1966 / Biochim. Biophys Acta 130, 257/1966). No. 4,455,411. U.S. Pat. No. 4,123,195 discloses the preparation of high molecular weight acrylamide polymers using persulphate, formaldehyde sodium sulfoxylate and 2,2'-azobis 2-amidopropane initiators. Similarly, the preparation of a high porosity bead polymer (20-800 μηι) is disclosed in DE 34 04 021 A1. These large particle size immunocarriers have a. the downside was that. In the incubation mixture, they settle rapidly, and to prevent this, the plastic tube containing the reaction mixture had to be rotated during the incubation period. DE 32 24 484 A1. In U.S. Patent No. 5,600,600, microspheres which fluoresce or magnetize by suspension polymerization of acrolein-type compounds in the presence of suitable surfactants are disclosed. are produced. The microspheres cannot be used under radioimnumoassay conditions because of their too small particle size (0.0380 pm) that cannot be sedimented by centrifuges used in the isotope laboratory. DE 31 06 456 A1. German Patent Specification No. 4,198,125 describes the preparation of a solid support for bead polymerization, particularly for immobilizing enzymes. DE 31 24 454 A1. describes the preparation of polymers for absorption in aqueous media, particularly in physiological fluids. A particle size polyacrylamide bead, which was practically non-sedimenting during the incubation period, was prepared which avoided rotation and allowed the antibody-coated gel particles to be pelleted after centrifugation (GIT-Labor Medizin 2/81).

The process, although having many advantages over the above, has the major drawback that after centrifugation, the small amount of gel is barely visible at the bottom of the test tube, so that a smaller proportion of the gel and the associated immune material are lost when aspirate is aspirated. This disadvantage was addressed by U.S. Patent No. 4,108,974. A process described in U.S. Patent No. 4,600,123, which in some cases attempted to dye a polyacrylamide by post-dyeing the already prepared acrylamide polymer with a certain ink called "Alcian" or "Alcian" yellow. This paint is physically bound to the polyacrylamide and adsorbed on the surface, with the disadvantage that it can be easily removed from the surface for certain external effects. Acrylamide polymer discolouration may also occur during application. The same drawback is with DE 26 52 646. which uses phthalocyanine or cadmium compounds to color the beads,

Although the methods described above have more or less failed to achieve their intended purpose, you may not have all the benefits that they were designed to achieve. so e.g. subsequent coloring can significantly alter the interfacial properties of the gel bead, thereby significantly altering its binding capacity. Certain - not very polar - dyes may alter the colloidal stability of the system, thereby losing the very benefit of using a microsphere, e.g. that a system containing microspheres in a long-dispersed state, due to the high specific surface area of the support and the good accessibility conditions, provides near homogeneous phase reaction conditions, such that the solid support reaction partner can be becomes removable.

It is an object of the present invention to provide a process that is quite generally applicable, easy and inexpensive, in which other comonomers such as acrylic and mesh may be used. acrylic acid is used to produce colored gel beads in one or two steps, preferably in the size range of 3 to 50 µm. The colored gel beads thus produced retain the adjusted color intensity during use for a long time and the coloring agent does not appreciably affect the surface bonding ore and the colloidal stability of the beads. A further requirement is that the stained gel should have a noticeable color, and that the pigment used for staining should not undergo a change of color or dissolve under the conditions of R 1A, - it should not be affected by staining. antiserum to gel beads, - the dye used should not impair the gel-bound antibody and the antigen binding properties of the liquid phase.

It has been found that many pigments are suitable for meeting the above requirements, but Berlin-blue is the most suitable of the possible pigments because of its color, solubility and chemical stability.

The present invention is based on the discovery that during the polymerization of acrylamide, acrylic acid and Ν, Ν'-bisacrylamide in aqueous solution, a Berlin-blue salt containing particles having a diameter of 8 to 10 μΜ can be added to the aqueous monomer solution. colloidal particles of the colorant remain immobilized in the crosslinking system (one step procedure).

Alternatively, a microsuspension process known per se for these monomers can be prepared by colorless gel beads and subsequently colored by preparation of a Berlin-blue colloidal gel bead (two step procedure).

In particular, it has been found that if the dyeing is carried out before the polymerization is carried out by mixing the monomers, and if the dyeing is carried out afterwards, a suitable colloidal dye which is physically but irreversibly bound is incorporated into the polymer. (immobilize), no longer diffuses out of it, does not interfere with the binding of the immune substance to the surface of the immunosorbent,

196 613 requirements. As a colloidal colorant for the above purposes, it is preferable to use Berlin-blue Fe ₄ (Fe / CN ₆ ) ·, especially when used in the form of a purified sol with a concentration of 0.5 to 1.0 g / 100 cin ^{3 in} aqueous phase. The color of the polyacrylamide bead polymer produced by the process of the invention varies from pale blue to dark blue, is a pleasant, noticeable color, and the coilidated dye particles are trapped in the polymer and can no longer diffuse therefrom. The gel beads thus colored have a stable pH in their preparation and application in a medium, i.e. pH 4-8, which is stable even after several washes. In the process of the invention, a suitable pH is obtained with a suitable buffer solution, for example tris (hydroxyncyl). The amount of buffer used is 0.075 mol / 100 cm ^{3 of} aqueous phase The buffer used and the pH range used depend on the colorant chosen.

Depending on the particle size fractions collected in the process of the invention, the colored polyacrylamide bead polymer can be used in different areas. However, radioimmunoassay kits are still the most widely used application. Blue fractions in the range of 3 to 50 µm are most suitable for this purpose.

Emulsifier for the stability of the process of the invention the w / o emulsions with non-polar, water immiscible inert organic solvent or solvent mixture -3X10 ^-1 2x10 ^-3 mol / dni ³ aqueous solution of the initiator is 0.5 to 2.0 pmole / 100 cm ³ aqueous phase Ν, Ν, Ν ', Ν'-tetranylethylenediamine (TEMED) solution, 0.3-2.0 nmol / 100 cm ³ aqueous phase ammonium persulfate (NH ₄ ) ₂ S ₂ O ₈ solution, 1 , 0-10.0 mol / 100 cm ³ aqueous phase N, N-methylene bisacrylamide (BISZ) crosslinker and 0.2-0.4 mol / 100 cm ³ aqueous phase acrylamide in an acrylic acid and optionally, depending on the desired color strength, a mixture of purified aqueous colorants containing 0.005-0.0500 g / 100 cm ^{3 of} aqueous phase, preferably a beryllium blue salt, and an aqueous buffer solution having a pH of 7-9, ratio of inert organic solvent immiscible with apolar water to water: 5: 1-10: 1 It is dispersed at 0-25 ° C in an inert gas atmosphere at 1000-1800 rpm, then polymerized at 0 50 ° C for 5-10 minutes at 1000-1800 rpm and then 1560 minutes at 600-1,400 rpm. The resulting organic solvent gel bead dispersion is poured into water at 80-100 ° C, optionally containing electrolyte, the resulting aqueous gel bead dispersion is separated, washed with water and then fractionated in a known manner. sedimentation is carried out and the crude fraction in the range of 3 to 50 µm is separated off, optionally concentrated and optionally stained inside the resulting colorless gel beads with a colorant produced in situ, preferably Berlin blue.

The resulting gel bead dispersion is preferably washed with water until the additive and monomer free.

In the one-step process, it is expected that the colloidal additive will interfere with, render poorly the product, or possibly prevent the polymerization of the acrylamide. Aqueous polymerization of acrylamide is highly sensitive to oxygen and other additives present in the polymerization system. The microspheres polymer is produced in a system in which the average particle size is close to the upper limit of the colloidal size range, so that the specific surface area of the system formed is large. This makes the highly mixed system more sensitive to external and internal influences. A further difficulty may be that the emulsifier used may interact with the microphase-type coloring colloid, thereby transferring the coloring agent to the organic phase through its altered interface properties. This phenomenon would render the adjustment of the color intensity irreversible.

It is known that colloidal particles, mainly due to their large specific surface area, are capable of binding to a variety of materials at their interface, or that the particles bind to structures of sufficient charge and polarity.

Thus, in the two-step process, it would be expected that the Berlin-blue sol particles formed in the system would uncontrollably bind (immobilize) not only inside the gel bead, but also on its surface, thereby decisively affecting the polymer and solids. interfacial groups of the copolymer with respect to in vitro material (s), enzyme (s) binding, and colloidal stability of the gel bead dispersion.

In both processes, it would be expected that, due to the relatively wide pore size distribution of the gel, some of the colloidal particles would be removed from the gel bead by diffusion, thereby attenuating the intensity of the adherent color.

Polyacrylamide gel beads having a diameter of 3-50 µm and stained with a Berlin-blue salt produced by the microsuspension polymerization process of the present invention can be used as radioimmunoassay kits.

The invention is further illustrated by the following non-limiting examples, without thereby limiting the scope of the invention.

Example 1

In a three-necked round-bottom flask equipped with a stirrer, a nitrogen inlet and a dispenser, a solution of toluene-sorbitol-inono-startate (Sjan 60), 200 cm ³ 0.96 g / 100 cnr ³ 2.2X10 ³ mol / dm ³ , Nitrogen gas was bubbled through the solution at 1000 rpm while the temperature of the solution was adjusted to between 0 and 5 ° C. The mixer was then ramped to 1500 rpm and 5 cm ^{3 of} Tris (hydroxymethyl) aminomethane (hereinafter TRIS) buffer solution (36.6 g of TRIS and 48 cm ^{3 of} aqueous 1 N hydrochloric acid) was added to the solution. solution/

100 cm ³ ) 37.7 μΐ N, N, N ', Nhetiamethyl ethylenediamine (TEMED), 5 cm ³ aqueous ammonium persulphate solution (2 g ammonium persulphate / 100 cm ³ ) 10 cm ³ colored aqueous monomer solution (28 g acrylamide, 2.8 g acrylic acid, 2.21 g N, N'-methylene bisacrylamide / 100 cm ³ and 1 cm ³ (0.5 g / 100 cin ³ ) beryllium blue sol and dispersed until the toluene layer, until the desired particle size (of the checks carried out microscopically). Subsequently, the temperature was raised to 40 ^e C 2 minutes after 5 minutes of polymerization time, the stirrer speed is reduced to 800 revolutions / minute and the polymerization was carried out for another 30 minutes under these conditions.

After stirring, the toluene gel bead dispersion was poured into boiling distilled water (4 x 5 vol.) In a round bottom flask with stirring and reflux, and the mixture was allowed to cool while stirring. The toluene phase which is substantially free of gel particles is separated from the aqueous phase by decantation and the gel bead dispersion in the aqueous phase is sedimented.

The gel beads are resuspended in 4-5 times distilled water and sedimented to wash the cmulgcator and unreacted components. This operation is repeated 3-4 times.

Approximately 4-5 portions of the parallel gel bead dispersion were combined and the fraction was pelleted. The largest (φ 40 μηι) and smallest (φ 5 μηι) fractions were removed and repeated two to three times, and the resulting diluted gel bead dispersion was concentrated by settling.

Thus, a maximum concentration of 50 g of swollen gel / 100 cm ³ gel bead dispersion is obtained.

Degree of swelling: 8 g water / 1 g dry gel (microscopic method).

The particle size and particle size distribution were determined microscopically by a phase contrast method, and it was determined that almost 80% of the particles fall within the range of 5-10 μιη.

The acrylic acid content of the gel beads was determined by conductive titration with 0.1 N sodium hydroxide. The acrylic acid content is 10 mol%, i.e. every tenth monomer unit contains a carboxyl group.

Sedimentation rate: the gel bead dispersion does not settle in pure water after 3 hours.

Degree of crosslinking: 27, i.e. molar ratio of crosslinker to monomer: 1:27.

sodium persulfate solution and 2.2 cm <^{3> of} distilled water are dispersed in 200 cm < ³ > of 2X10 ² mol / dm ³ of toluene Span 60 at 1000 rpm at a temperature of 10: 1 at 25 [deg.] C. The polymerization was carried out at 35 ° C and 800 rpm for 60 minutes. The emulsion was quenched with 15 mol% aqueous sodium chloride. The washing and fractionation were carried out as in Example 1.

The color polyacrylamide gel beads thus obtained had an acrylic acid content of 5 mol% (by conductive titration with 0.1 N sodium hydroxide).

Degree of cross-linking: 40, i.e. ratio of cross-linker to monomer: 1:40.

Example 3

Each was prepared as in Example 1 with the following differences: 28 g acrylamide, 0.735 g BISZ, 0.7 g acrylic acid and 1 cm ³ (0.75 g / 100 cm ³ ) Berlin blue in 100 cm ³ aqueous 10 CIN ³ monomer mixture containing a solution of 5 cm ³ of Tris solution, 20 μΐ TEMED tube 5 cin ^3, 1 g / 100 cm ³ concentrated aqueous ainmónium-peiszulfát solution of 100 cm ³ 3, ΟΧΙΟ ² mol / cm ³ koncetnrációjú toluene in Span 60 at 0 ° C at 1200 rpm until the desired droplet size is reached. The polymerization was carried out at 40 ° C and 800 rpm for 30 minutes. Elimination of the emulsion is 600 cm ³ of hot distilled water pouring, stirring is carried out. After cooling, phase separation and fractionation were performed as in Example 1.

The acrylic acid content of the colored polyacrylamide gel beads thus obtained was 2.5 mol% (conductivity titration, 0.1 N aqueous sodium hydroxide solution).

Example 4

For 5 ml of the 1% gel bead dispersion prepared in Example 1, 0.3 ml of 1% ferric chloride (FeCl ₃ ) solution was added with gentle stirring at room temperature, followed by 0.3 ml of 1% potassium ferrocyanate (K ₄ Fe) after 5 minutes. (CN) ₆ ) After a reaction time of 10 minutes, the suspension is suctioned or centrifuged and the beads are washed with distilled water. The bead wash is repeated at least three times. The resulting dye is well detectable in the aqueous phase in the immunosorbent and does not appreciably discolour at pH 4 to 8 for at least 1 month.

Example 2

Each was prepared as in Example 1 with the following differences: 28 g acrylamide, 1.47 g BISZ, 1.4 g acrylic acid and 2 cm ³ (0.5 / 100 cm ³ ) Berlin-blue sol per 100 cm ^Of a colored monomer mixture containing ³ aqueous solutions of 10 cm ^{3 of} 5 cm ^{3 of} TRlSZ buffer, 12.5 μΐ TEMED, 2.8 cm ³ (1 g / 100 cin ³ ) of aqueous ammonia4

Example 5

Use of Berlin-blue colored polyacrylamide gel in RIA kits.

The blue acrylic acid acrylamide N, N'-methylene bisacrylamide bead polymer prepared in Example 1 was used as immunosorbent in a thyroxine (T4) radioimmunoassay as follows: The wet gel was suspended in 0.003 mol of pH 6.5 phosphate buffer (1 g).

196,613 gels + 5 ml buffer), anti-T4 immune serum is added and incubated for 1 hour at + 4 ° C after mixing.

(The amount of immune serum depends on the liter.) An appropriate amount of 1- 3- (3-dimethylaminopropyl) carbodiimide HCl is added to the reaction mixture and incubated overnight at + 4 ° C. After centrifugation the next morning, the gel was washed extensively with physiological saline (5 x 10 x 10 vol). Finally, the gel is washed with 0.5% human serum albumin-containing barbitol buffer and resuspended in barbitol buffer. (1 g wet gel at 10).

Measure the standard series and the serum samples to be tested as follows:

Weigh out 2020 μΙ of the standard solutions (1-6) and samples, 200 μ tr of the tracer and 20–20 μΐ of the prepared immunosorbent prepared as above. After vortexing, homogenize and incubate for 1 hour at 37 ° C. After incubation for 20 minutes at ₂ θ centrifuged at 2000-3000 g for the tubes. The supernatant is aspirated from the precipitate and the radioactivity of each tube is measured.

The results are evaluated by plotting the counts of the 6 standard solutions versus the T4 concentration (pg / dl) of the standards, and. read off the T4 concentration for the cpin value for each sample.

Claims (1)

A patent claim Procedure - preferably colored - 20-200 crosslinking degree polyacrylamide copolymer gel bead in the production of microsuspension polymerization means, characterized in that the emulsifier for the stability of a W / O type emulsions 2X ΙΟ ^-3 -3X10 in nonpolar, AEM water-miscible inert organic solvent or solvent mixture ^{- In an} aqueous solution of ¹ mol / dm ^{3, the} initiator is 0.5 2.0 μιηοΙ / 100 cm ³ aqueous phase Ν, Ν, Ν ', Ν'-tetraethyl ethylene diuun (TEMED) solution, 0.3-2.0 nmol / 100 cm 3 aqueous phase of ammonium persulfate (NH ₄ ) ₂ S ₂ O ₈ , 1.0 to 10.0 mol / 100 cm ^{3 of} aqueous phase Ν, Ν-methylene bisacrylamide (BISZ) crosslinker and With 2 to 0.4 mol / 100 cm ^{3 of} aqueous phase acrylamide, 2 to 15 mole% b: m acrylic acid, and optionally 0.005 to 0.0500 g / 100 cm ^{3 of} aqueous phase purified aqueous dyes, preferably beryllium. blue sol containing po limiting mixture, and an aqueous buffer solution to provide a pH of 7-9, the ratio of inert organic solvent immiscible with apolar water to water at 5: 1 to 10: 1 to 0 at 25 ° C in an inert gas sphere of 1000 to 1800 dispersed at 0-50 ° C for 5-10 minutes at 1000-1800 rpm and then for 15-16 minutes at 600-1,400 rpm, resulting in an organic solvent medium gel bead dispersion of 80-100 ° C. The resulting aqueous medium gel bead dispersion is separated, washed with water, fractionated by settling in a known manner and the crude fraction in the range of 3 to 50 gm is separated, optionally concentrated and optionally inside the gel beads, the coloring is carried out with a colorant produced in situ, preferably Berlin-blue. Zuk.