JP2010500919A - Solid support - Google Patents

Abstract

  A solid support is described that includes a polymer-impregnated bead having beads in the beads and a polymer disposed in the pores. The support is particularly useful for a range of further applications, as well as solid phase synthesis of peptides, and immobilization of molecules, especially enzymes.

Description

  The present invention relates to a solid support comprising a polymer, a method for preparing a support and the use of the support in a solid phase method. Supports can be used in a wide range of physical and chemical methods that require interaction with the substrate, such as solid phase organic synthesis, solid phase extraction, solid phase reagents, species fixation, cell culture, catalysis, chromatography And useful for medical diagnosis.

Solid support materials useful for solid phase synthesis methods are known. A wide range of physical and chemical methods (including synthesis of organic molecules, especially peptides and oligonucleotides, species immobilization, supports in catalysis, ion exchange, species extraction from materials and chromatography) are solid Support material is used.
The synthesis of multi-stage organic molecules is typically produced in each stage, and many intermediates for separating the intermediate before proceeding to the subsequent stage (the intermediate is utilized as a feedstock). It involves a separation process. These methods may be time consuming and expensive, and may not be effective with respect to yield. Intermediates often require purification to remove excess reagents and reaction byproducts, and operations such as precipitation, filtration, two-phase solvent extraction, solid phase extraction, crystallization and chromatography may be used. .
Solid phase synthesis offers several advantages over liquid phase synthesis. For example, the isolation procedure used for liquid phase synthesis may be avoided to some extent by reversibly binding the target molecule to a solid support. Excess reagents and some of the by-products can be removed by filtration and washing the solid support. The target molecule can be recovered in several ways in high yield or even in substantially quantitative yield. High yield recovery in liquid phase synthesis is often difficult. In addition, the time required to perform operations on solid supports is typically much less than the time required to perform comparable steps in liquid phase synthesis.
Species fixation in a range of methods is also known. For example, polymer supports are commonly used for catalyst immobilization for use in traditional organic chemistry (including chemical catalysis and biocatalysis). The immobilized enzyme can be used in a wide range of methods (including organic chemistry) or chiral resolution, eg, the use of immobilized penicillin amidase for resolution of secondary alcohols (E. Baldaro et al., Tet. Asym. 4, 1031, (1993)) and immobilized penicillin G amidase has been used for hydrolysis of benzylpenicillin in the manufacture of amoxicillin (Carleysmith, SW and Lilly, MD, Biotechnol. Bioeng., 21, 1057-73, 1979).

Solid supports such as polymer beads may also be used to immobilize biological macromolecules for medical and diagnostic applications. Examples of this application include the immobilization of proteins, monoclonal antibodies and polyclonal antibodies. Cell culture is usually performed on solid supports having specific surface characteristics and morphology. The immobilized enzyme is a sensor for generating a signal, such as a glucose oxidase / peroxidase coupled enzyme system (which generates hydrogen peroxide in the presence of glucose, which in turn gives a colored, fluorescent or luminescent signal. It may be used as a detection of glucose by a peroxidase substrate for the oxidation of a wide variety of substrates.
Various phosphors, whose fluorescence is sensitive to specific cations or anions, may be immobilized on polymer beads to indicate the concentration of specific ions, including hydrogen ions for pH measurement.
Polymer particles are often used in chromatography where the solid support is referred to as the stationary phase. In some forms of chromatography, the stationary phase is expensive and may limit its use. The physical properties of the stationary phase may not be suitable for obtaining full effectiveness in certain applications. For example, soft polymers are often used for affinity chromatography, ion exchange chromatography and gel permeation chromatography, but may not be used effectively at high flow rates due to the deformable nature of the particles. Rigid macroporous polymers used in other modes of chromatography are often mechanically fragile and are subsequently problematic for short lifetimes.

Application of solid supports or stationary phases in chromatographic separations (eg used in complex high technology separation and mining industries used in the pharmaceutical and biotechnology industries for the purification of expensive products using preparative chromatography) (Including large-scale separations) are very extensive. Most of the world's palladium, an important component in catalytic converters and other industrial processes, can be purified using immobilized crown ethers (Traczyk, FP; Bruening, RL; Izatt, NE “metal ions from aqueous solutions. Application of molecular recognition technology (MRT) for removal and recovery of wastes ”; In Fortschritte in der Hydrometallurgie; 1998, Vortrage beim 34. Metallurgischen Seminar des Fachausschusses fuer Metallurgische Aus-und Weiterbildung der GDMB; 18-20 November 1998; Goslar) .
The use of polymer particles in solid phase extraction and preparation of solid phase reagents is also known in the chemical, pharmaceutical and biotechnology industries.
Known solid phase supports generally comprise polymer particles of special size and physical properties suitable for application. For ease of use, these polymer particles are often spherical and have a specific particle size distribution. The spherical nature of the particles improves the flow and filtration characteristics of the polymer. The use of a solid support has operational advantages but is disadvantageous for the solid phase approach. For example, commercially available supports commonly used for solid phase synthesis of peptides and oligonucleotides may be expensive. The polymer particles may typically be made by a dispersion or emulsion polymerization process in which a solution of monomers is dispersed in an immiscible solvent (continuous phase) prior to initiation of polymerization. The produced polymer particles are typically subsequently filtered, washed and classified. These methods are disadvantageous in several respects including monomer loss to the continuous phase, generation of a range of particle sizes and generation of fine particles during polymerization. Monomer loss to the continuous phase may not be effective in terms of both raw material costs and environmental costs. Classification of polymer particles to separate the particle sizes required for a particular application can be labor intensive and complex processes, typically sieving and / or air classification (these are With a loss of rate). “Fine” particles are usually produced. These fines may be problematic for the separation of the polymer beads and may require additional processing, such as sedimentation and decanting for their removal.
In addition to the undesirable cost of manufacture and the waste during preparation, certain disadvantages may arise due to the physical properties of known polymer particles. Microporous and macroporous polymers are commonly used and their production may be expensive and complex.
Microporous polymers have a relatively low level of cross-linking agent, which allows the polymer particles to solvate and thus swell in a suitable solvent. However, microporous polymer particles are generally soft and generally not suitable for use at high flow rates in packed column beds. In addition, soft polymers may be compressed undesirably, for example, causing fouling during filtration, often resulting in compression penetration into the used sinter or mesh.
Macroporous polymers have a high level of crosslinker in the polymer matrix and contain large pores. These polymer particles are generally hard and have good flow properties in packed columns. Hard macropore particles and macroreticular particles are more suitable for high flow rates in packed column beds. However, due to the hard nature, the particles are brittle and may structurally break under physical stress.

  Reducing the costs associated with manufacturing costs, waste, physical integrity of the support and poor product performance by providing a preformed solid bead with pores that can be used effectively as a container for polymer supports May be.

  In a first aspect, the present invention provides a polymer-impregnated bead characterized in that the bead has one or more holes in the bead, preferably penetrating the bead and a polymer disposed in the one or more holes. A solid support is provided.

Illustrative embodiments of the solid support of the present invention are shown in plan, side and cross-sectional views. 1 shows a support in which the polymer plug 3 is generally cylindrical. 1 shows a support in which the polymer plug 4 is generally cylindrical and the beads 1 are tubular. 2 shows a solid support of the present invention having a bulging shaped hole 2 in a bead 1. Etched glass beads are shown. Etched glass beads are shown. Figure 5 shows 15/0 etched glass beads with polymer plugs in the bead holes. A group of 15/0 glass beads with polymer plugs in the pores of the beads is shown. Figure 5 shows 15/0 etched glass beads with ninhydrin dyed polymer rings lining the pores of the beads.

The term “polymer” as used herein includes inorganic polymers (of which silica will be an example) and organic polymers (of which polystyrene will be an example).
Advantageously, the beads are rigid, mechanically strong and can be utilized at high flow rates in a packed column bed. The beads are also easily filtered in a batch mode operation to allow rapid processing.
Suitably the beads comprise an inert material. The inert material is preferably selected from glass, ceramics, polymers, metals such as steel, wood and other natural materials. In a preferred embodiment, the beads are preformed from glass. Glass seed beads commonly used in the jewelry industry are particularly beneficial for this application. They are manufactured on a large scale, are inexpensive and provide a useful dimensional and structural integrity support.
The beads are preferably spherical, approximately spherical or elliptical. The spherical nature of the beads is advantageous in many applications, such as facilitating packing in the column and improving flow characteristics across the bed during filtration. However, irregularly shaped, oval and other shaped beads may be used.
In another embodiment, a short piece of tubing may be used.
The present invention may use beads of any size, but the larger the polymer plug in the pores in the bead, the less effective the diffusion of the material into the polymer. The pores in the beads have a diameter perpendicular to the axis of the holes in the beads, preferably less than 2 mm, in particular less than 1 mm, more preferably from 0.01 to 0.75 mm, from 0.01 to 0.5 mm, in particular from 0.05 to 0.5 mm. The smaller the beads, the better the diffusion in the polymer to which the material is bound or absorbed, and the closer the packing between the beads.
The diameter of the beads is suitably 2 mm or less, preferably 1.5 mm or less, desirably 1.2 mm or less. The beads are preferably at least 0.01 mm in diameter and desirably at least 0.05 mm. In a preferred embodiment, the beads have a diameter of 0.1 to 1.2 mm, more preferably 0.1 to 1.0 mm, desirably 0.3 to 0.8 mm. In general, the beads are preferably as small as possible in terms of function, but this should be balanced with the economics of producing smaller beads. Optimally, the beads have a diameter of 0.4 to 0.7 mm.

The present invention is distinguished from a porous support in that the bead preferably comprises a single pore, and that the pore is typically of a size larger than that associated with the porous material. The solid support preferably comprises a single plug or mass of polymer within the pores. The beads may be porous, but it is also necessary to have pores in addition to the pores that may be present. The holes are preferably dumbbell-shaped or puffy (wider in the center), which helps physical retention of the polymer in the holes, or the holes may be cylindrical.
In a preferred embodiment, the pores of the beads are lined with a polymer so that the substrate can pass through the beads with the polymer lining. This can provide a more accessible surface area for contact and improve the flow of the substrate in the beads.
Suitable beads may be obtained commercially, for example from Miyuki beads and Toho beads, or by cutting the capillary tube material into short pieces and then heating them to a temperature just below the point at which the glass melts. It may be made. At this temperature, a small piece of capillary tube material shrinks to form beads.
Typically, glass seed beads of 11/0 and 15/0 sizes are readily available. For example, a 15/0 sized bead is approximately 1.15 mm in size in the direction of the hole and approximately 1.55 mm wide.
Suitably the polymer is produced in the pores of the beads. The polymer may be covalently bound to the beads directly or indirectly. The beads may be made from a material having an active site, for example if the wood provides hydroxyl groups in the cellulosic material, the polymer may be bound directly. If the beads are made from a more inert material, such as glass, it may be desirable to treat the beads to provide an active site to which the polymer can bind. If the bead comprises glass, it is preferably treated with an etchant, preferably a fluoride etch solution, such as a hydrogen fluoride solution and an ammonium difluoride solution, to provide a surface suitable for reaction with the derivative.

It is desirable for the beads to be derivatized to provide an active site for reaction with the polymer. Known materials and methods for derivatizing the glass surface may be used. In a particularly preferred embodiment, the derivative comprises a silane and an active site for attachment to the polymer. The active site is preferably a vinyl group.
Silane group has the formula _{- (O) n Si [(} CH 2) p [Z] q CR = CR 2] (4-n) ( wherein, n is from 1 to 3, p is 0 to 6 And R is independently H or alkyl, q is 0 or 1, and Z is a divalent linking group.
Z is preferably a group of the formula — (CH ₂ ) _r NRC (O) —, where r is from 1 to 6.
In a particularly preferred embodiment, the support comprises glass beads with bound silane groups thereto, the silane group is selected from -O ₃ SiCH = CH ₂ and _{-O 3 Si (CH 2) 3} NHCOCH = CH 2 . The —O ₃ SiCH═CH ₂ group is preferably derived from triethoxyvinylsilane. The —O ₃ Si (CH ₂ ) ₃ NHCOCH═CH ₂ group is preferably derived from aminopropyltrimethoxysilane, which is reacted with glass, and then its amine group is reacted with acryloyl chloride.
The polymer may be any suitable material according to the desired application. In a preferred embodiment, the polymer is an organic polymer and is a polymer resin, polyacrylamide, polystyrene, cellulose, polydimethylacrylamide, polymethyl methacrylate, polyurea, polyacryloylmorpholine and polybetahydroxyester, polyhype, polyalkylene glycol, such as Selected from polyethylene glycols and polypropylene glycols and polysaccharides such as agarose.
The polymer may be an inorganic polymer and is preferably selected from alumina, silica and other metal oxides.

The polymer may be further reacted to obtain special functionality for a given application. Preferably, the polymer is reacted with a compound having at least two functional groups, one functional group reacts with the polymer and the other functional group provides free functionality for use in the desired application. To give. In a preferred embodiment, polymers such as polydimethylacrylamide copolymer and polyacryloylmorpholine copolymer with N-acryloyl sarcosine methyl ester are reacted with a diamine compound such as ethylene diamine. For example, amine functionalized supports are suitable for use in peptide synthesis, oligonucleotide synthesis and solid phase organic chemistry.
The amine functionalized support may be further functionalized, for example, by conversion to a carboxylic acid, optionally using succinic acid. As an example, an amine functionalized support may be treated with N-hydroxysuccinimide and 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride in preparation for immobilizing proteins such as protein A.
In a further embodiment, the support comprises a bead and a polymer and additional material within the bead's pores. Examples of additional materials include inert materials, such as chemically inert materials with high absorbency. A particularly preferred inert material is polyhype. Polyhype is porous and highly absorbent. This material is particularly preferred for applications where the substance is absorbed by the support.
The solid support of the present invention may also comprise a functional material supported by a polymer. Examples of suitable functional materials include catalysts, initiators for organic synthesis, eg peptide synthesis, pharmaceutically active substances, agrochemical active substances, macromolecules, enzymes, nucleic acid sequences and proteins.
The present invention is particularly useful for supporting noble metal catalysts such as palladium catalysts. A particularly advantageous example is palladium acetate. In a preferred embodiment, palladium acetate is supported with polyurea.

The solid support of the present invention may be produced by an effective and relatively simple method. The present invention provides, in the second aspect, a step of preparing a bead having pores therein, a step of contacting the bead with a monomer or a solution of the monomer, a step of polymerizing the monomer to form a polymer, and optionally a polymer A method for producing a solid support material is provided, comprising the step of subjecting the beads comprising a further treatment to remove the polymer from the surface of the beads.
The polymerization is preferably initiated by methods known to those skilled in the art. For example, beads containing monomers or monomer solutions are added to a solvent that is immiscible with the monomer solvent and heated to effect polymerization. When the monomer solution is aqueous, the solvent is, for example, kerosene.
The polymer may be dried or cured by conventional means such as heating and UV irradiation.
Preferably, once the polymer is produced, the bead containing polymer is physically removed, for example, in a roll mill, so as to remove the polymer from the outer surface of the bead and leave the polymer disposed in one or more pores of the bead. Subject to mechanical wear.
Preferably, when the beads are exposed to a component monomer or component monomer solution of the intended polymer support, capillary action holds the solution in one or more pores and the polymer is It is generated by a known starting method that is known.
If desired, the method of manufacturing the solid support material may include a step of treating the surface of the bead to provide an active site prior to contacting the bead with the monomer or monomer solution.
Glass beads used in jewelry and textile trade are commonly known as seed beads. The use of seed beads has a particular advantage in that the polymer plug is preferably dumbbell shaped or blistered, and as such, the polymer is physically constrained due to its shape. Let's go. The polymer plug itself is preferably secured within the hole of the bead due to its shape.

In preferred cases, the polymer plug may be covalently attached to the rigid beads during or subsequent to polymerization. Also, one or more component monomers can be covalently bonded to the bead surface prior to initiation of polymerization.
The polymer-bead combination is particularly robust when the polymer is constrained in the pores due to physical constraints and is covalently bonded to the surface of the bead.
The solid support of the present invention may be used in any chemical or physical method in which the solid support is used.
In another embodiment, the present invention provides a solid support comprising a polymer-impregnated bead, wherein the bead has pores in the bead and the pore walls include a layer of polymer such that a polymer ring is provided in the pores of the bead. I will provide a.
Supports with polymer linings or rings rather than plugs can be made by the same method as with polymer plugs, except by using a dilute polymer solution. By adapting the concentration of the polymer solution, the thickness of the lining can be adjusted. In a preferred embodiment, the lining is at least 1 micron and desirably at least 5 microns thick to ensure that it is as thick as the size of the holes in the beads and still has a polymer ring rather than a solid plug. It may be possible to do. Suitably, the lining is up to 100 microns, preferably up to 50 microns, more preferably up to 20 microns thick. Particularly preferred ranges are thicknesses of 1-100 microns, 5-100 microns, and 5-50 microns.
For example, a support having a polymer lining of thickness from 1 micron to 20 microns is particularly advantageous for cell culture and medical diagnostic applications.

Solid supports are particularly useful for solid phase synthesis of organic species, especially macromolecules. In a preferred embodiment, the solid support may be used for the synthesis of peptides, oligonucleotides or oligosaccharides. Polydimethylacrylamide as the polymer support is particularly advantageous for peptide synthesis.
The solid supports of the present invention are also useful for solid phase extraction, such as ion extraction and ion exchange, to remove species from the liquid contacted with the support (batch form or as a stream on the support).
The solid support of the present invention is particularly useful for immobilizing solid phase reagents, metal catalysts and other catalysts, biocatalysts, enzymes, proteins, antibodies including polyclonal and monoclonal antibodies, whole cells and species including polymers. . The present invention is particularly advantageous for supporting enzymes such as horseradish peroxidase and glucose oxidase, particularly in combination with polydimethylacrylamide and other similar hydrophilic polymers.
The present invention is particularly useful for affinity chromatography, eg, immobilization of protein A affinity ligands. Affinity chromatography is mainly used for the separation of biological products such as biopharmaceuticals. Suitably the affinity ligand is immobilized in the stationary phase. This ligand has a particular affinity for the component of the biological mixture that is contacted with the support. Affinity may be based on any form of interaction, such as specific biological interactions such as found with enzymes and substrates, receptors and ligands and antigens and antibodies.
In a preferred embodiment, the affinity ligand comprises protein A and is used to interact with immunoglobulin. Protein A binds to the Fc region of some immunoglobulin antibodies, and many biopharmaceuticals are based on immunoglobulins.
In a further embodiment, the present invention provides a solid support comprising beads impregnated with beads in the beads and a polymer disposed in the pores, the polymer comprising polymer-impregnated beads comprising immobilized protein A.
In the art, stationary phases with large molecules, such as protein A, are available in two forms where the support is a macroporous resin or a softer support with low levels of cross-linking. Macroporous resins have a low surface area followed by low loading. Softer supports are made with sufficient crosslinker to provide sufficient rigidity for use in low to intermediate pressure chromatography. However, they are still relatively highly cross-linked and cannot be easily penetrated by biological macromolecules. As a result, the bands observed with chromatographic separation are relatively wide and not all of the immobilized ligands are accessible.

Advantageously, the present invention allows for the immobilization of suitable polymers containing very low levels of cross-linking, thus providing improved diffusion and access to all active sites in the polymer.
In a further preferred embodiment, the present invention includes a layer of polymer such that the bead has pores in the bead and the walls of the pore provide a polymer ring in the pores of the bead, the polymer comprising immobilized protein A. A solid support comprising polymer-impregnated beads is provided.
In a further embodiment, the solid support may be used as a support in the field of cell culture, particularly stem cell culture. Polyhydroxyesters as blocks or coated plates are often used for stem cell culture. In these systems, cells are often difficult to recover and are often detached from the polymer surface by physical stress. In this system, the physical stress on the cell-polymer interaction is mitigated due to the cage environment, i.e. the polymer is in the bead, not the outer surface of the bead, and the polymer is more used compared to the block polymer or coated plate. Provided in a physical form that can.
Solid supports may be used in applications involving electrically conducting polymers and light emitting polymers. A solid support comprising a light emitting polymer may be disposed on the display panel.
The supports of the present invention may be used to immobilize species including antibodies, oligonucleotides, enzymes or fluorophores and may be located in an array, each support assaying a different component of the solution. . Beads with ligands covalently bound to the polymer bound to the surface may be used as “wells”. The specific binding of the target ligand, such as an antigen or complementary DNA or RNA sequence, can then be detected using established methods.
The solid support of the present invention is useful for the preparation of stationary phases for chromatographic separations such as affinity chromatography, ion exchange chromatography, reverse phase chromatography, conventional phase chromatography, chiral chromatography and gel permeation chromatography. It is.

In further applications, the solid support may be used as an absorbent. In this application, it is particularly advantageous for the support to comprise an inert absorbent material bound to beads (to which a polymer is bound). Polyhype is a particularly preferred inert material. The solid support may be used to absorb household spills such as tea, coffee and wine, or may be used in large scale applications to absorb oil from spills, for example. . The absorbent support may be used to absorb spills and then physically removed or, in the case of spilled oil in the water body, effectively trap the oil and then the bottom of the water body Sink in.
The solid support of the present invention may be used as a carrier to carry compounds that are released over a period of time, such as pharmaceutical or agrochemical compounds or compositions. This use provides a means to adjust the dose regime of the compound according to the loading of the compound in the support. In the case of pharmaceuticals, this is advantageous in that it does not require the patient to take large doses, e.g., with chemotherapy, rather than helping the precise dose of the active substance, e.g., continuous gradual release. might exist.
In a preferred embodiment, the present invention provides a solid support comprising polymer-impregnated beads, wherein the beads are disposed within and within the pores of the beads and further comprise a polymer comprising a pharmaceutical compound or composition.
The solid support of the present invention may be applied to any chemical, biological or physical solid process in which the polymer support is currently used.
The present invention is particularly useful for medical diagnostic tests such as immunoassays. Thus, the present invention further provides a solid support comprising polymer-impregnated beads (the beads in a support for selectively reacting or binding to the pores in the beads and the polymer disposed in the pores and the compounds in the sample. A medical diagnostic for detecting the presence of a compound in a sample, comprising a functional material supported by a polymer, for example an enzyme, for example with horseradish peroxidase, and contacting the sample with a solid support Provide a method.

The beads may also be loaded or packed into a polymer packed column and pores and interstitial spaces to form a monolith. The present invention further provides a solid support monolith comprising a plurality of solid support material beads of the present invention that may be packed in a mass, eg, a column arrangement, and optionally include a polymer in the interstitial space.
As desired, the interstitial space between the beads in the monolith can be filled with a polymer that is different from the polymer retained in the pores of the beads. In another embodiment, the interstitial spaces between the beads in the monolith may be filled with different components, such as cell culture nutrients. In this example, the cells may be cultured on a polymer matrix in the pores.
The polymer in the pores of the beads may be colored using, for example, a dye to provide aesthetic or functional properties.
The invention also includes embodiments in which the beads with polymer plugs or linings further comprise smaller beads with polymer plugs or linings in the pores of the larger beads. The polymer in the smaller beads may be the same as or different from the polymer in the larger beads.
1-4 show exemplary embodiments of the solid support of the present invention in plan, side and sectional views, respectively. 5 and 6 show etched glass beads for use in the present invention. 7-9 show photographs of the solid support of the present invention.
In FIG. 1, a hole 2 in a bead 1 has a polymer 3 disposed therein. From the cross-sectional view, the polymer end 4 is larger in diameter than the polymer center 5 to provide a dumbbell-shaped cross section, and the polymer 3 is physically retained within the pore 2 to provide increased strength to the support.
FIG. 2 shows a support in which the polymer plug 3 is generally cylindrical.
In FIG. 3, the polymer plug 4 is generally cylindrical and the bead 1 is tubular.
FIG. 4 shows the solid support of the present invention having a bulge-shaped hole 2 in the bead 1 and the polymer 3 is more in the hole than at the end so as to provide a means to physically hold the polymer plug in the hole 2. It ’s thick.
The etched glass beads in FIGS. 5 and 6 are shown before receiving the polymer plug or lining. In FIG. 5, etched 15/0 beads are shown next to smaller 0.25 mm glass beads and in FIG. 6 next to smaller 0.65 mm glass beads.
FIG. 7 shows 15/0 etched glass beads with polymer plugs in the pores of the beads.
FIG. 8 shows a group of 15/0 etched glass beads with polymer plugs in the pores of the beads, the polymer being colored with ninhydrin to more clearly show the polymer.
FIG. 9 shows a 15/0 etched glass bead with a polymer ring dyed with ninhydrin lining the pores of the bead.
The invention is illustrated by the following non-limiting examples.

Example 1-Preparation of beads with active surface
1. Glass beads (144 g) with a bead etching size of 15/0 were placed in a 250 cm ³ polypropylene bottle and covered with Dip'n Etch (100 cm ³ ), a solution of ammonium difluoride. The bottle was placed in an ultrasonic bath for 6 hours and left for 16 hours.
Wash the beads with water (10x50 cm ³ ), aqueous sodium hydroxide (15% w / v, 10x50 cm ³ ), water (10x50 cm ³ ), aqueous hydrochloric acid (1 mol / dm ³ , 10x50 cm ³ ), then water (10x50 cm ³ ) did. The beads were then dried at 100 ° C. for 1 hour (yield 138 g, BI).
Using the same procedure, a batch of size 11/0 beads (100 g) was etched to give 97 g of etched beads (BII).
2. Reaction of etched beads with vinyltriethoxysilane 30g of etched 15/0 size beads are placed in a polypropylene bottle and covered with a solution of vinyltriethoxysilane in methanol (40cm ³ , 1: 1v / v) Water (1 cm ³ ) was added. The mixture was placed in an ultrasonic bath for 1 hour, then washed with acetone and dried under a stream of nitrogen to give vinyl functional beads (BVI).
3. Insert etched beads aminopropyltrimethoxysilane reaction etched bead size 15/0 of (30g, BI) and 30g of polypropylene bottle, a solution of aminopropyltrimethoxysilane in methanol (40 cm ^3, 1 : covered with 1v / v), was added water (1cm ^3). The mixture was placed in an ultrasonic bath for 1 hour, then washed with acetone and dried under a stream of nitrogen to give aminopropyl functional beads (BAmI).
4. Reaction of aminopropyl-functionalized beads with acryloyl chloride The aminopropyl-functionalized beads (30 g, BAmI) prepared previously were covered in dichloromethane (30 cm ³ ), then acryloyl chloride (5 cm ³ ) followed by 4-methylmorpholine ( 5 cm ³ ) was added. The mixture was agitated and left for 1 hour, then washed with dichloromethane ( ³ × ³⁰ cm ³ ) and then dried in a stream of nitrogen to give acrylamide functional beads (BAcI).

Example 2-Preparation of a solid support
1.Polydimethylacrylamide polymerization
N, N-dimethylacrylamide (100 mmol, 9.9 g), N-acryloylsarcosine methyl ester (13 mmol, 2.0 g) and bis-acryloylethylenediamine (5 mmol, 0.82 g) and water (3 cm ³ ) in a round bottom flask. I put it in. Aqueous ammonium persulfate solution (0.75 g in 2 cm ³ ) was added. The acrylamide beads (30 g, BAcI) prepared in section 4 of Example 1 above were immediately added to the monomer solution and a slight vacuum was applied to remove bubbles from the pores of the beads.
Excess monomer solution was drained using a stainless steel sieve. The sieve containing the beads was then placed in an 80 ° C. kerosene bath to ensure that all of the beads were immersed in the kerosene.
After 2 hours, the sieve containing the beads was placed in a cold water bath and left for 1 hour. Now, the beads containing the polymer enclosed in the holes were transferred to an Erlenmeyer flask with a magnetic stir bar. Water (50 cm ³ ) was added and the mixture was stirred with a magnetic stirrer for 5 minutes. The supernatant containing small irregular particles of polymer eroded from the surface of the beads was removed by decanting. This washing process was repeated until the supernatant did not contain any particles of polymer observed. A bead-polymer composite solid support (BPI) was stored under water.

2. Reaction of bead-polymer composite and ethylenediamine The water on the bead-polymer composite (about 33 g, BPI) prepared earlier was drained and the beads were covered with ethylenediamine. The mixture was left overnight (about 16 hours) and then washed with water (10 × 50 cm ³ ).
The amine functional beads (BPAmI) were stored under water. The solid support is suitable for use in peptide synthesis.
Polyhype polymerized styrene (3 cm ³ ), divinylbenzene (7 cm ³ ) and sorbitan monooleate (span 80) (2 cm ³ ) in 3.11 / 0 beads in a beaker and stirred at 200 rpm to produce a smooth emulsion Until then, water (300 cm ³ ) containing ammonium persulfate (0.75 g) was gradually added.
Size 11/0 etched beads (30 g, BII) were mixed with about 50 cm ³ of this emulsion in a round bottom quick fit flask. A slight vacuum was applied to expel air from the pores of the beads and the mixture was heated at 60 ° C. for 2 hours.
The resulting solid block was broken with a spatula and water (about 50 cm ³ ) was added. Water (50 cm ³ ) was added and the mixture was stirred with a magnetic stirrer for 5 minutes. The supernatant containing small irregular particles of polymer eroded from the surface of the beads was removed by decanting. This washing process was repeated until the supernatant did not contain any particles of polymer observed. The bead-polymer composite was dried at 100 ° C. overnight and stored dry (BPII).

Polymerization of polyhype in 4.15 / 0 beads 15/0 etched beads (34 g) were mixed with about 50 cm ^{3 of} the emulsion previously prepared in Example 2-3 and subjected to the same operation. The resulting bead-polymer composite was dried overnight at 100 ° C. and stored dry (yield 34.5 g, BPIII).
5.Polyacryloylmorpholine polymerization in BPIII
4-acryloylmorpholine (90 mmol, 12.65 g), N-acryloyl sarcosine methyl ester (15 mmol, 2.36 g) and bis-acryloylethylenediamine (2 mmol, 0.35 g) and water (1 cm ³ ) were placed in a round bottom flask. . Aqueous ammonium persulfate solution (0.5 g in 1 cm ³ ) was added. The polyhype beads prepared in Example 2-4 above (34.55 g, BPIII) were immediately added to the monomer solution, and a slight vacuum was applied to exclude remaining bubbles from the bead pores.
Excess monomer solution was drained using a stainless steel sieve. The sieve containing the beads was then placed in an 80 ° C. kerosene bath to ensure that all of the beads were immersed in the kerosene. As an alternative to heating under immersion in kerosene, the beads could be subjected to UV irradiation at room temperature.
After 2 hours, the sieve containing the beads was placed in a cold water bath and left for 1 hour. Now, the beads containing the polymer enclosed in the holes were transferred to an Erlenmeyer flask with a magnetic stir bar. Water (50 cm ³ ) was added and the mixture was stirred with a magnetic stirrer for 5 minutes. The supernatant containing small irregular particles of polymer eroded from the surface of the beads was removed by decanting. This washing process was repeated until the supernatant did not contain any particles of polymer observed. Bead-polymer composite (BPIV) was stored under water.
50 beads were removed and dried at 100 ° C. overnight (181 mg). The final weight corresponded to the addition of 1 g polymer per 10 g glass beads.
6. Reaction of bead-polymer composite and ethylenediamine The water on the bead-polymer composite (BPIV) prepared earlier was drained and the beads were covered with ethylenediamine. The mixture was left overnight (about 16 hours) and then washed with water (10 × 50 cm ³ ). The amine functional beads (BPAmII) were stored under water and suitable for use in peptide synthesis.

Example 3-Synthesis of Leu-enkephalinamide
1. Coupling of internal reference amino acid Amino-functional beads (11.8 cm ³ , BPAmII produced in Example 2-6) were placed in a glass chromatography column (diameter 17 mm) and N, N-dimethylformamide ( DMF) aliquots (10 × 10 cm ³ ).
Fmoc-Ala-OH (1.25 g, 4 mmol) (Fmoc = 9-fluorenylmethyloxycarbonyl) and 2- (1H-benzotriazol-1-yl) -N, N, N ′, N′-tetramethyl Uronium tetrafluoroborate (TBTU) (1.21 g, 3.8 mmol) was dissolved in DMF (3 cm ³ ). 4-Methylmorpholine (NMM) (0.528 cm ³ , 4.8 mmol) was added and the mixture was preactivated for 2-3 minutes before being added to the column and drained to the beads under gravity.
The coupling reaction was completed by ninhydrin assay within 3 hours. The beads were washed under gravity with an aliquot of DMF (10 × 10 cm ³ ).
Piperidine / DMF (10 cm ³ , 20% v / v) was added to the column and discharged onto the beads under gravity. The reaction was left for 10 minutes. Performs second treatment with piperidine ^{/ DMF (10cm 3, 20%} v / v) over 20 minutes, the beads were washed with DMF (10x10cm ^3).
2. Binder coupling
Assay Fmoc-Am-Rink-OH (2.05 g, 3.8 mmol) using the procedure shown in Example 3-1 except that Fmoc-Am-Rink-OH was used instead of Fmoc-Ala-OH Coupling for 5 hours, followed by treatment with piperidine / DMF.

3. Fmoc -Leu-OH coupling Fmoc-Leu-OH (1.32) using the procedure shown in Example 3-1 except that Fmoc-Leu-OH was used instead of Fmoc-Ala-OH. g, 4 mmol) were coupled as assayed for 4 hours and treated with piperidine / DMF.
4. Fmoc -Phe-OH coupling Fmoc-Phe-OH (1.55) using the procedure shown in Example 3-1 except that Fmoc-Phe-OH was used instead of Fmoc-Ala-OH. g, 4 mmol) were coupled as assayed for 16 hours and treated with piperidine / DMF.
5. Fmoc -Gly-OH coupling Fmoc-Gly-OH (1.19) using the procedure shown in Example 3-1 except that Fmoc-Gly-OH was used instead of Fmoc-Ala-OH. g, 4 mmol) were coupled as assayed for 16 hours and treated with piperidine / DMF.
6. Fmoc -Gly-OH coupling Fmoc-Gly-OH (1.19) using the procedure shown in Example 3-1 except that Fmoc-Gly-OH was used instead of Fmoc-Ala-OH. g, 4 mmol) were coupled as assayed for 16 hours and treated with piperidine / DMF.
7.Coupling of Fmoc-Tyr (tBu) -OH and then using the procedure shown in Example 3-1 except that Fmoc-Tyr (tBu) -OH was used instead of Fmoc-Ala-OH. -Tyr (tBu) -OH (1.84 g, 4 mmol) was coupled as assayed for 16 hours and treated with piperidine / DMF.
8. The peptide cleavage beads were washed with dichloromethane (5x10cm ^3), was added trifluoroacetic acid containing water ^{(TFA) (10cm 3, 5} % v / v), was discharged to the beads under gravity. The polymer in the beads turned red, indicating that the cleavage was progressing. After 10 minutes, a further aliquot of TFA (10 cm ³ ) was added and the mixture was cleaved for 1 hour.
The beads were washed with TFA (5 × 10 cm ³ ). The combined TFA cleavage solution and washings were reduced to oil on a rotary evaporator. The oil was triturated with diethyl ether to produce a white solid. The ether was removed by decanting and the peptide was air dried for 2 hours (yield 308 mg).
The peptide was shown to contain a single major component by reverse phase HPLC and had the expected molecular weight as measured by MALDI-TOF mass spectrometry.

Example 4-Immobilization of enzyme
1. Coupling of glucose oxidase and horseradish peroxidase to a solid support Glucose oxidase (black Aspergillus, 27.6 mg) was dissolved in sodium acetate (5.5 cm ³ , 0.1 mol / dm ³ pH 5.5). Horseradish peroxidase (25.7 mg) was dissolved in sodium acetate (5.14 cm ³ , 0.1 mol / dm ³ pH 5.5).
Aliquots (3 cm ³ ) of these solutions were dialyzed separately for 1 hour against water (500 cm ³ ) and then against sodium acetate (500 cm ³ , 0.1 mol / dm ³ pH 5.5). Each formulation was further dialyzed against sodium acetate (200 cm ³ , 0.1 mol / dm ³ pH 5.5) over 3 hours.
Cold m- sodium periodate Aliquots (0.27 cm ^3, 88 mmol / dm ³⁾ of each of the enzyme by adding to the dialyzed enzyme solution of each of the volume of 2.7 cm ³ of polypropylene tubes in 5 cm ³ Periodate oxidation of carbohydrates was performed. The tube was wrapped in aluminum foil to protect the contents from light and mixed with a bottle roll for 20 minutes. The reaction was stopped by addition of a 10-fold dilution of glycerol in water (20 mm ³ ) and rapid mixing.
0.1 mol / dm ³ MES of 300 cm ³ volume of the reaction byproducts over 1 hour in the fridge, 0.15 mol / dm ³ NaCl, was removed by exhaustive dialysis of the oxidized enzyme solution respectively for pH 5.0. Further dialysis of each bead formulation was carried out against 300 cm ³ in 300 ml ³ volume of this buffer over the course of 1 hour and then overnight in the fridge. Final dialysis of each formulation against 400 cm ³ buffer was performed. These oxidized enzyme preparations were stored frozen until needed.

The bead batch BPAmI produced in Example 2-2 was added to a double 10 cm ³ polypropylene tube in water to obtain a packed volume of about 1 cm ³ . The supernatant was decanted and left in the water (10 cm ³ ) in the fridge overnight. Further water washing (10 cm ³ ) and washing with 0.1 mol / dm ³ sodium phosphate, 0.15 mol / dm ³ NaCl, pH 7.4 ( ³ × 10 cm ³ ) was performed with a bottle roll each time for 10 minutes of mixing. The last extended 30 minute wash of each bead formulation was performed and the supernatant volume was reduced to about 1.5 cm ³ .
The beads were treated with succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH) to convert the primary amines of the beads to protected hydrazine. In practice, SANH (25 mg) was dissolved in dimethyl sulfoxide (DMSO) (1.2 cm ³ ) and an aliquot (0.3 cm ³ ) was added to each of the bead formulations and stirred. 0.1M sodium phosphate, 0.15M NaCl, pH 7.4 buffer (1.5 cm ³ ) was added to each tube and mixed with a bottle roll at room temperature for 4 hours. The supernatant was aspirated and washed ( ³ × 10 cm ³ ) with buffer followed by water ( ³ × 10 cm ³ ). The beads were then washed with 0.1 mol / dm ³ MES, 0.15 mol / dm ³ NaCl, pH 5.0 (4 × 10 cm ³ ), leaving the bead formulation as a pellet.
Appropriately add 1 cm ³ of oxidized glucose oxidase or oxidized horseradish peroxidase to each bead pellet and mix for 3 hours in the dark at room temperature via the aldehyde of each of the oxidized enzymes. Coupling to all SANH-modified beads was performed.
The supernatant was aspirated and each bead formulation was washed with pH 5.0 buffer (5 × 10 cm ³ ) followed by water (5 × 10 cm ³ ), each time the bead suspension was shaken quickly and then the supernatant was carefully aspirated. The bead formulation was stored in 10 cm ³ volumes of water in a fridge (BPAmII-horseradish peroxidase and BPAmII-glucose peroxidase).

Example 5-Test of immobilized enzyme
Immobilized horseradish peroxidase The immobilized horseradish peroxidase beads produced in Example 4 were washed 3 times in saline phosphate buffer (PBS) for 2 minutes per wash. PBS was removed by pouring from aqueous sodium phosphate solution (0.1 mol / dm ³ ) containing hydrogen peroxide (0.35 mmol / dm ³ ) and diaminobenzidine (0.5 mg / cm ³ ) adjusted to pH 6.4 with citric acid. The dyeing medium is added. After 5 minutes, the beads stained completely dark brown, indicating the presence of the enzyme and confirming that the enzyme was active.
Staining was performed at 37 ° C. The beads were washed to remove the staining media and photographed at 50x with a Nikon inverted microscope.
Immobilized glucose oxidase Immobilized glucose oxidase beads were washed 3 times in saline phosphate buffered saline (PBS) for 2 minutes per wash. Except pouring PBS, all PBS pH 6.9 in D- glucose (42 mmol / dm ^3), phenazine meso sulfate (0.1 mg / cm ³⁾ and nitroblue consisting tetrazolium (0.5 mg / cm ³⁾ staining medium Was added. After 5 minutes, the beads stained completely dark blue, indicating the presence of the enzyme and confirming that the enzyme was active.
Staining was performed at 37 ° C. The beads were washed to remove the staining media and photographed at 50x with a Nikon inverted microscope.

Example 6-Immobilization of protein A
Coupling of rProtein A to beads The packed bed volume (5 cm ³ ) of bead batch BPAmII produced in Examples 2-6 was distributed into 50 cm ³ polypropylene tubes. Excess water was decanted and the bead formulation was washed with water (5 × 40 cm ³ ), each time the beads were resuspended and they were allowed to settle under gravity. Washing with sodium borate (0.1 mol / dm ³ , pH 8.3, 4 × 40 cm ³ ) was carried out in the same way, and the beads were further frozen overnight in 50 cm ³ of this buffer and left to stand.
In order to convert the amines of the beads to carboxylic acid groups, they were treated with succinic anhydride. Succinic anhydride (1.2 g) was dissolved in DMSO (60 cm ³ ). The borate buffer was decanted from the beads and an aliquot (30 cm ³ ) of this succinic anhydride solution was added to the bead formulation, shaken rapidly and mixed in a bottle roll for 6 hours at room temperature. The bead formulation was washed with water (8 × 40 cm ³ ), quickly resuspending the beads each time, and the supernatant was aspirated. An aliquot of morpholinoethanesulfonic acid (MES) buffer (25 mmol / dm ³ , pH 5.0, 40 cm ³ ) was added, and the beads were then left overnight in the fridge. Further washing of the beads with this buffer (2 × 40 cm ³ ) was performed, leaving the beads as a pellet.
N- hydroxysuccinimide (1.6 g) of cold MES buffer (25 mmol / dm ^3, pH5.0,32cm ³⁾ was dissolved in an aliquot of this solution (15cm ³⁾ was added to the bead pellet was quickly mixed. 1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, 1.6 g) was dissolved in MES buffer (25 mmol / dm ³ , pH 5.0, 32 cm ³ ) and an aliquot (15 cm ³ ) was added to the bead formulation. The bead formulation was activated by mixing with a bottle roll for 30 minutes. The supernatant was decanted and each formulation was quickly washed with MES buffer (25 mmol / dm ³ , pH 5.0, 5 × 40 cm ³ ), leaving the beads as pellets.
Immediately, the r Protein A solution (of 15cm ^3, 25 mmol / dm ³ MES, in pH5.0 4mg / cm ³⁾ aliquots of a (10 cm ³⁾ was added to the beads formulation, a coupling with mixing in a bottle roll Proceed for 2 hours at room temperature. The supernatant was decanted and an aliquot (30 cm ³ ) of Trizma-HCl, pH 7.4 was added and mixed for 2 hours to block the remaining N-hydroxysuccinimide ester. The supernatant was decanted, water washed (8x40cm ³⁾ performed in bead formulations, leaving the beads in water (10cm ³⁾ (BPAmII- Protein A).

Example 7-Use as Absorbent The solid support produced in Example 2-3 above was used to absorb red oil spills in amounts many times the mass of the support. When the solid support was applied to the spill, the red oil was completely absorbed by the solid support, illustrating its use as an absorbent.
Example 8 Encapsulation of Palladium Acetate Beads prepared according to Example 1, Part 3, amino-functionalized beads, BAmI (1 g), palladium acetate (0.1 g) and poly (phenyl isocyanate) in chloroform (0.86 g) Coformaldehyde) (0.64 g) was added and left for 5 minutes. A vacuum was applied instantaneously, the beads were transferred to a sieve and the excess palladium acetate / poly (phenylisocyanate coformaldehyde) solution was drained. The sieve containing the beads was immersed in a water bath and allowed to stand overnight for complete polymerization. The beads were washed with DMF and left to stir in DMF for an additional 24 hours to ensure that all polymer debris was removed from the exterior of the beads. The beads were washed with water and air dried.

Example 9
Etching of the glass surface of a new bead Small glass beads with a hollow center (new bead, Fig. 4, 3.86 g) are placed in a 50 cm ³ polypropylene bottle and Dip'n Etch [ammonium difluoride] solution (10 cm ³ ). The bottle was placed in an ultrasonic bath for 2 hours and then left for 16 hours.
The beads were washed with water (10 × ⁵ cm ³ ), aqueous sodium hydroxide (15% w / v, 10 × ⁵ cm ³ ), water (10 × ⁵ cm ³ ), aqueous hydrochloric acid (1 mol / dm ³ , 10 × ⁵ cm ³ ) and then water (10 × ⁵ cm ³ ). . The beads were then dried for 1 hour at 100 ° C. (yield 2.8 g).
Reaction of etched beads with aminopropyltrimethoxysilane A solution of aminopropyltrimethoxysilane (0.1 cm ³ ) in ethanol: water (5 cm ³ , 95: 5 v / v) was prepared and left for 10 minutes. The previously prepared etched beads (2 g) were placed in a polypropylene bottle and covered with this preactivation solution. The mixture was placed in an ultrasonic bath for 1 hour, then washed with acetone and dried under a stream of nitrogen to give aminopropyl functional beads. The beads were then cured at 110 ° C. for 2 hours.

Reaction of aminopropyl-functionalized beads with acryloyl chloride The aminopropyl-functionalized beads (2 g) prepared previously were covered in dichloromethane (2 cm ³ ) containing 4-methylmorpholine (0.5 cm ³ ) and in dichloromethane (2 cm ³ ). A solution of acryloyl chloride (0.5 cm ³ ) was added slowly over 5 minutes. The mixture was agitated and left for 1 hour, then washed with dichloromethane ( ³ × ⁵ cm ³ ) and then dried in a stream of nitrogen to give acrylamide functional beads.
Polymerization of polydimethylacrylamide
N, N-dimethylacrylamide (10 mmol, 1 g), N-acryloylsarcosine methyl ester (1.3 mmol, 0.2 g) and bis-acryloylethylenediamine (0.25 mmol, 0.04 g) and water (0.3 cm ³ ) in a round bottom flask. I put it in. Aqueous ammonium persulfate (0.08 g in 0.2 cm ³ ) was added. The previously prepared acrylamide beads (2 g) were immediately added to the monomer solution and a slight vacuum was applied to remove bubbles from the bead pores.
Excess monomer solution was drained using a stainless steel sieve. The sieve containing the beads was then left overnight under a UV lamp.
The sieve containing the beads was placed in a cold water bath and left for 1 hour. Now, the beads containing the polymer enclosed in the holes were transferred to an Erlenmeyer flask with a magnetic stir bar. Water (5 cm ³ ) was added and the mixture was stirred with a magnetic stirrer for 5 minutes. The supernatant containing small irregular particles of polymer eroded from the surface of the beads was removed by decanting. This washing process was repeated until the supernatant did not contain any particles of polymer observed. The bead-polymer composite solid support was stored under water.

Example 10
Reaction of etched beads with aminopropyltrimethoxysilane A solution of aminopropyltrimethoxysilane (1 cm ³ ) in ethanol: water (50 cm ³ , 95: 5 v / v) was prepared and left for 10 minutes. Etched size 15/0 beads (30 g, BI) were placed in a polypropylene bottle and covered with this preactivation solution. The mixture was placed in an ultrasonic bath for 1 hour, then washed with acetone and dried under a stream of nitrogen to give aminopropyl functional beads. The beads were then cured at 110 ° C. for 2 hours.
Cover in dichloromethane (15cm ³⁾ containing an amino-functionalized bead and acryloyl chloride reaction destination prepared amino-functionalized beads (30 g) 4-methylmorpholine (5 cm ^3), acryloyl in dichloromethane (15 cm @ 3) A solution of chloride (5 cm ³ ) was added slowly over 5 minutes. The mixture was agitated and left for 1 hour, then washed with dichloromethane ( ³ × ³⁰ cm ³ ) and then dried in a stream of nitrogen to give acrylamide functional beads.
Polymerization of polydimethylacrylamide
N, N-dimethylacrylamide (100 mmol, 9.9 g), N-acryloylsarcosine methyl ester (13 mmol, 2.0 g) and bis-acryloylethylenediamine (5 mmol, 0.82 g) and water (3 cm ³ ) in a round bottom flask. I put it in. Aqueous ammonium persulfate solution (0.75 g in 2 cm ³ ) was added. The previously prepared acrylamide beads (30 g) were immediately added to the monomer solution and a slight vacuum was applied to remove air bubbles from the bead pores.
Excess monomer solution was drained using a stainless steel sieve. The sieve containing the beads was then left overnight under a UV lamp.
The sieve containing the beads was placed in a cold water bath and left for 1 hour. Now, the beads containing the polymer enclosed in the holes were transferred to an Erlenmeyer flask with a magnetic stir bar. Water was added (50 cm ^3), stirred for 5 min the mixture with a magnetic stirrer. The supernatant containing small irregular particles of polymer eroded from the surface of the beads was removed by decanting. This washing process was repeated until the supernatant did not contain any particles of polymer observed. The bead-polymer composite solid support was stored under water.
Example 11
A solid support with a lining was produced. The procedure of Example 2 was followed except that the water and ammonium persulfate levels were doubled to 1.5 g in 6 cm ³ water and 4 cm ³ ammonium persulfate solution, respectively. Beads with a polymer lining of about 50 microns thickness were produced.

1-bead 2-hole 3-polymer 4-end of polymer 5-center of polymer

Claims (43)

  A solid support comprising polymer-impregnated beads, wherein the beads have pores in the beads and a polymer disposed in the pores.   The solid support of claim 1, wherein the beads comprise an inert material.   3. A solid support according to claim 2, wherein the inert material is selected from glass, ceramic, polymer, wood (or other natural material) and metal.   4. A solid support according to any one of claims 1 to 3, wherein the beads are generally spherical or elliptical.   The solid support according to any one of claims 1 to 3, wherein the beads are tubular.   The solid support according to any one of claims 1 to 3, wherein the beads have an irregular shape.   7. A solid support according to any one of the preceding claims, wherein the pores in the beads have a dumbbell-shaped length method cross section.   7. A solid support according to any one of claims 1 to 6, wherein the pores in the beads have a longitudinal cross-section that is generally tubular.   7. A solid support according to any one of the preceding claims, wherein the pores in the beads have a longitudinal cross-section that is generally blistered (wider at the center).   10. A solid support according to any one of claims 1 to 9, wherein the polymer in the beads has a particle size of less than 2 mm.   The solid support according to any one of claims 1 to 10, wherein the polymer in the beads has a particle size of 0.01 to 0.5 mm.   12. A solid support according to any one of claims 1 to 11, wherein the polymer is produced in the pores of the beads.   13. A solid support according to any one of claims 1 to 12, wherein the polymer is covalently bonded directly or indirectly to the beads.   14. A solid support according to any one of claims 1 to 13, wherein the beads comprise glass and have been treated with an etchant to obtain an active site suitable for reaction with a derivative for binding to the polymer. .   15. A solid support according to any one of claims 1 to 14, wherein the beads are derivatized with silane to obtain active sites for reaction with the polymer.   The solid support according to claim 15, wherein the active site is a vinyl group. Silane group is represented by the formula:-(O) _n Si [(CH ₂ ) _p [Z] _q CR = CR ₂ ] _(4-n) (where n is 1 to 3 and p is 0 to 6) The solid support according to claim 15 or 16, wherein R is independently H or alkyl, q is 0 or 1, and Z is a divalent linking group. Z has the formula _{- (CH 2) r NRC (} O) - ( wherein, r is a is from 1 to 6) group of a solid support according to claim 17. Silane groups are selected from -O ₃ SiCH = CH ₂ and _{-O 3 Si (CH 2) 3} NHCOCH = CH 2, the solid support of any one of claims 15 18.   20. A solid support according to any one of claims 1 to 19, wherein the active site comprises a functional group other than silane, which functional group can form a covalent bond between the bead and the polymer.   21. Any one of claims 1 to 20, wherein the polymer is selected from polyacrylamide, polystyrene, cellulose, polydimethylacrylamide, polydimethylmethacrylate, polyurea, polyacryloylmorpholine, polybetahydroxyester, polyhype, polyalkylene glycol, and polysaccharide. The solid support according to Item.   The support contains an inert material within the pores of the bead and the polymer is bound to the inert material or, if the inert material is porous, retained within the pores of the inert material The solid support according to any one of claims 1 to 21.   The solid support of claim 21, wherein the inert material comprises a polyhype or a porous inorganic polymer, such as porous silica.   24. The solid support according to any one of claims 1 to 23, further comprising a functional material supported by a polymer.   Functional materials are catalysts, initiator species for peptide synthesis, initiator species for oligonucleotide synthesis, initiator species for solid phase organic synthesis, pharmaceutically active substances, agrochemical active substances, proteins or other organisms 25. A solid support according to claim 24, selected from biological macromolecules.   26. A solid support according to any one of claims 1 to 25, wherein the polymer is in the form of a solid plug in the pores in the beads.   26. A polymer-impregnated bead, wherein the bead comprises a polymer-impregnated bead, wherein the bead has a hole in the bead and the wall of the hole includes a layer of polymer so as to provide a ring of polymer in the hole of the bead. Solid support.   28. The solid support of claim 27, wherein the polymer layer has a thickness of 1 to 100 microns.   26. A functional material comprising and supported by or supported by a solid support according to any one of claims 1 to 25 (the functional material is for interaction with a compound in a biological sample) Medical diagnostic product for detecting the presence of a compound in a biological sample for analysis, characterized in that it comprises a specific binding site).   30. The medical diagnostic product of claim 29, wherein the functional material comprises an enzyme supported by a polymer.   29. A monolith comprising a solid support according to any one of claims 1 to 28 contained in a column.   Solid support comprising polymer-impregnated beads (the beads are supported by a polymer in the support for selectively reacting or binding to the pores in the beads and the polymer disposed in the pores and the compound in the biological sample. Medical diagnostic method for detecting the presence of a compound in a biological sample, characterized in that the biological sample is contacted with a solid support.   Providing a bead having pores therein, contacting the bead with a monomer or monomer solution, polymerizing the monomer to form a polymer and optionally subjecting the bead containing the polymer to further processing. A method for producing a solid support material, comprising the step of removing the polymer from the surface of the beads.   34. The method of claim 33, wherein the monomer or monomer solution is added to the beads and the polymerization is carried out in the presence of a solvent (which is immiscible with the monomer or monomer solvent).   35. A method according to claim 33 or 34, wherein the beads comprising the polymer are subjected to physical wear so as to remove the polymer from the outer surface of the beads and leave the polymer in the pores of the beads.   32. Use of a solid support according to any one of claims 1 to 31 in a chemical, biological or physical method.   37. Use according to claim 36 of a solid support for solid phase synthesis of a species selected from peptides, oligonucleotides, oligosaccharides.   37. Use of a solid support for solid phase extraction.   38. Use according to claim 36 of a solid support for solid phase organic chemistry.   37. A solid support for immobilization of a species selected from solid phase reagents, metal catalysts and other catalysts, biocatalysts, enzymes, proteins, antibodies including polyclonal and monoclonal antibodies, whole cells and polymers. use.   37. Use according to claim 36 of a solid support for cell culture.   37. Use of a solid support in the preparation of a stationary phase for chromatographic separation.   Use of a solid support as an absorbent according to claim 36.

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