polymer particles

Abstract

Monodisperse polymer particles useful in biological assays and other applications are provided. The present invention relates to monodisperse magnetic hydrogel polymer particles comprising a magnetic material and a polymer, wherein the polymer (a) has a log P of less than about 0.5. oct / wat The present invention also relates to monodisperse coated hydrogel polymer particles comprising a polymer and a coating, wherein the polymer is formed from (a) a hydrophilic vinyl monomer having a log P of less than about 0.5, and (b) a crosslinker comprising at least two vinyl groups. oct / wat (a) a hydrophilic vinyl monomer having a log P; and (b) a crosslinker comprising at least two vinyl groups. Methods for forming the monodisperse magnetic hydrogel polymer particles and the monodisperse coated polymer particles are also provided.

Description

[Technical Field]

[0001] The present invention relates to monodisperse polymer particles useful in biological assays and other applications. The monodisperse polymer particles are magnetic and / or coated. The present invention also relates to methods for preparing such particles and methods of using the particles, as well as other subject matter. [Background technology]

[0002] background Polymer particles are used in a wide variety of applications, such as biological assays or sequencing applications. In some of these applications, it is desirable to include a magnetic material in the polymer particles, for example, to enable magnetic separation of the polymer particles. In some of these applications, it is desirable to provide a coating on the polymer particles, for example, to modify the surface properties of the particles. Base polymer particles are typically made by emulsion polymerization or dispersion / precipitation polymerization.

[0003] Emulsion polymerization can be used to form relatively monodisperse polymer particles ranging from 50 to 1,000 nm. However, the formation of monodisperse polymer particles has several important limitations. First, all particles must be formed as part of the same batch within a short time period compared to the polymerization time. Second, growth conditions must be controlled to ensure that polymer particle growth is identical for all particles. Third, because this process requires the transfer of monomer from large reservoir droplets to swell into growing particles, the particles must either be uncrosslinked or have a very low level of crosslinking, as a higher level of crosslinking would prevent particle swelling.

[0004] Precipitation or dispersion polymerization can be used to create larger particles, ranging from 0.5 to 10 micrometers. Precipitation polymerization is carried out in a solvent (e.g., alcohol) that dissolves the monomer but not the polymer. Monodispersity can be achieved when the polymer precipitates from solution, resulting in rapid particle formation. Relatively large amounts of steric stabilizer are required to stabilize the particles after precipitation. The amount and type of stabilizer can vary the particle size, and porous particles can be achieved by using high levels of crosslinking agents. However, the conditions in precipitation polymerization are difficult to control. For example, it is very difficult to control porosity, which is a result of how the pore structure is increased as the polymer precipitates from solution. The difficulties of precipitation polymerization mean that in practice, it is nearly impossible to produce porous particles with the same monomer composition and porosity with more than very limited size variation.

[0005] One way to address some of these problems is to produce crosslinked porous or solid monodisperse polymer particles by a two-step process, named the Ugelstad process after the late Professor John Ugelstad, as described, for example, in EP-B-3905 and US-A-4530956. An improved Ugelstad process is described in WO 00 / 61647. In the Ugelstad process, seed particles, suitably prepared by emulsion polymerization, are converted into monodisperse particles in two steps by seed suspension polymerization. In the first step, the seed particles are swollen by preparing a fine (e.g., submicrometer) aqueous emulsion of a relatively low molecular weight water-insoluble substance, followed by the addition of a water-immiscible organic solvent (e.g., acetone), so that the water-insoluble substance diffuses into the seed particles. The water-insoluble substance is advantageously a heat-activated polymerization initiator. In the second step, the solvent is then removed, immobilizing the water-insoluble substance in the seed particles, which also incorporate large amounts of monomer and crosslinker. After initiation of polymerization, for example by heating to activate the initiator, larger polymer particles are produced. Thus, the Ugelstad process involves preparing seed particles by emulsion polymerization and expanding the seed particles by suspension polymerization. The smallest monodisperse particles described in the aforementioned prior art have an average diameter of 1 μm.

[0006] In a simplified version of the Ugelstad process, for example, when the oligomer weight-average molecular weight corresponds to a maximum of 50 monomer units (approximately 5000 molecular weight in the case of polystyrene), enhanced swelling capacity can be achieved simply by using oligomeric seed particles. This is described in US4530956 (Patent Document 4). In another version of the Ugelstad process, described in WO2010 / 125170 (Patent Document 5), oligomeric seed particles can be used to produce monodisperse particles with average diameters in the submicrometer range.

[0007] Particles produced according to the Ugelstad process or its variants can be made magnetic, for example, as described in WO 00 / 61647 (Patent Document 3) or WO 2010 / 125170 (Patent Document 5). However, particles produced by the Ugelstad process and its variants are typically produced from hydrophobic monomers such as styrene using an oil (discontinuous phase) in an aqueous (continuous phase) system. Therefore, the resulting polymer particles are hydrophobic. Hydrophobic particles suffer from nonspecific absorption problems when used in biological applications. This means that hydrophobic polymer particles are typically surface-modified, for example, with a coating, to increase the hydrophilicity of the surface before use in biological applications.

[0008] US2014 / 0073715 (Patent Document 6) describes a method for producing monodisperse hydrophilic particles. The method uses a monomer with a hydrophobic protecting group attached to the monomer, which is polymerized to form particles and then the protecting group is removed. However, the addition and removal of the protecting group increases the complexity of the process and can limit the number of different types of monomers that can be used in the process.

[0009] It is clear that known magnetic monodisperse polymer particles and methods for producing such particles suffer from many limitations, which also apply to coated monodisperse polymer particles. Therefore, there is a need for new monodisperse polymer particles and new production methods.

[0010] It is an object of the present invention to provide monodisperse magnetic polymer particles and methods for making monodisperse magnetic polymer particles having a low coefficient of variation (CV) and / or a low percentage of polydispersity. It is also an object of the present invention to provide coated monodisperse polymer particles and methods for making coated monodisperse polymer particles having a low coefficient of variation (CV) and / or a low percentage of polydispersity. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] EP-B-3905 [Patent Document 2] US-A-4530956 [Patent Document 3] WO00 / 61647 [Patent Document 4] US4530956 [Patent Document 5] WO2010 / 125170 [Patent Document 6] US2014 / 0073715 Summary of the Invention

[0012] Summary of the Disclosure The present invention is based in part on the recognition that seed particles comprising hydrophilic oligomers can be used in a novel method to form crosslinked monodisperse polymer particles. The present invention is also based on the recognition that the formed crosslinked monodisperse polymer particles can be made magnetic by depositing a magnetic material within the pores of the crosslinked monodisperse polymer particles. The present invention is also based on the recognition that the formed crosslinked monodisperse polymer particles can be coated with other substances.

[0013] According to a first aspect of the present invention, a magnetic material is provided, comprising a magnetic material and a polymer, and (a) having a log P of less than about 0.6. oct / wat Monodisperse magnetic hydrogel polymer particles are provided, which are formed from (a) a hydrophilic vinyl monomer having a (log P) and (b) a crosslinker comprising at least two vinyl groups.

[0014] In one embodiment, the hydrophilic vinyl monomer is (i) a monomer comprising -C(O)NH2, -C(O)NHCH3, -C(O)NHCH2CH3, or -C(O)N(CH3)2; (ii) Formula (Ib): Monomer of the compound TIFF0007765421000001.tif20128, (iii) Formula (Ic): Monomer of the compound of TIFF0007765421000002.tif20128, (iv) 4-acrylomorpholine or 1-vinylimidazole monomers, and (v) Monomers containing at least two vinyl groups, with no primary amine, no secondary amine, no hydroxyl, and no carboxylic acid. wherein R 1 and R 1a are each independently selected from —H, —CH 3 , —CH 2 CH 3 , or —CH 2 C(O)OH.

[0015] A second aspect of the invention is a polymer comprising: (a) a log P of less than about 0.6; oct / wat (a) a hydrophilic vinyl monomer having a log P; and (b) a crosslinker comprising at least two vinyl groups, wherein the particles further comprise a coating.

[0016] In one embodiment, the hydrophilic vinyl monomer is (i) a monomer comprising -C(O)NH2, -C(O)NHCH3, -C(O)NHCH2CH3, or -C(O)N(CH3)2; (ii) Formula (Ib): Monomer of the compound TIFF0007765421000003.tif20128, (iii) Formula (Ic): Monomer of the compound TIFF0007765421000004.tif20128, (iv) 4-acrylomorpholine or 1-vinylimidazole monomers, and (v) Monomers containing at least two vinyl groups, with no primary amine, no secondary amine, no hydroxyl, and no carboxylic acid. wherein R 1 and R 1a are each independently selected from —H, —CH 3 , —CH 2 CH 3 , or —CH 2 C(O)OH.

[0017] A third aspect of the present invention provides monodisperse hydrogel polymer particles formed from (a) a hydrophilic vinyl monomer comprising a monomer selected from 4-acryloylmorpholine and 1-vinylimidazole, and (b) a crosslinker comprising at least two vinyl groups.

[0018] A fourth aspect of the present invention provides a method for forming monodisperse hydrogel polymer particles, e.g., of the third aspect. In one embodiment, the method comprises: forming a solution (a) of at least 2% by weight of a hydrophilic vinyl monomer comprising a monomer selected from 4-acryloylmorpholine and 1-vinylimidazole in an aqueous solution, the solution also comprising a crosslinker comprising at least two vinyl groups; forming a solution (b) of a stabilizer in an organic solvent, mixing solutions (a) and (b) to form a water-in-oil emulsion (c), and adding monodisperse seed particles to the emulsion, wherein the organic solvent is immiscible in water, and at least one of solutions (a) and (b) comprises a radical initiator; forming swollen particles from monodisperse seed particles in an emulsion; and polymerizing the swollen particles to form monodisperse hydrogel polymer particles; Includes.

[0019] In one embodiment, the method comprises: forming a solution (a) of at least 2% by weight of hydrophilic vinyl monomers comprising monomers selected from 4-acryloylmorpholine and 1-vinylimidazole in an aqueous solution, the solution also comprising a chain transfer agent; forming a solution (b) of the stabilizer in an organic solvent, the organic solvent being immiscible in water, and at least one of solution (a) and solution (b) comprising a radical initiator; mixing solutions (a) and (b) to form a water-in-oil emulsion (c) and adding monodisperse seed particles to the emulsion; forming swollen grains from monodisperse seed grains in an emulsion; polymerizing the swollen particles to form monodisperse polymer particles; forming a solution (d) of the stabilizer in an organic solvent, the organic solvent being immiscible in water; forming a solution (e) of at least 2% by weight of a hydrophilic vinyl monomer comprising a monomer selected from 4-acryloylmorpholine and 1-vinylimidazole in an aqueous solution, the aqueous solution also comprising a crosslinker comprising at least two vinyl groups, and at least one of solution (d) and solution (e) comprising a radical initiator; mixing solutions (d) and (e) to form a water-in-oil emulsion (f) and adding monodisperse polymer particles to the emulsion; forming swollen polymer particles from monodisperse polymer particles in an emulsion; and polymerizing the swollen polymer particles to form monodisperse hydrogel polymer particles; Includes.

[0020] A fifth aspect provides a method for forming monodisperse magnetic hydrogel polymer particles. The method includes forming an aqueous suspension containing monodisperse hydrogel polymer particles and a magnetic material or a magnetic material precursor. When the aqueous suspension contains a magnetic material precursor, the magnetic material precursor is converted to a magnetic material. The magnetic material (whether or not the aqueous suspension initially contained a magnetic material precursor) is associated with the polymer particles. The monodisperse hydrogel polymer particles have (a) a log P of less than about 0.6. oct / wat In one embodiment, forming the aqueous suspension comprises (a) a hydrophilic vinyl monomer having a log P, and (b) a crosslinker comprising at least two vinyl groups. 2+ and / or Fe 3+ ions, and converting the magnetic material precursor to a magnetic material includes raising the pH to above 8 and precipitating the magnetic material.

[0021] A sixth aspect provides a method for forming coated, monodisperse hydrogel polymer particles. The method includes forming a suspension containing monodisperse hydrogel polymer particles and at least one epoxide, at least one silicate, or at least one silane, and reacting the epoxy groups of the at least one epoxide with surface functional groups of the particles or reacting the silicate to form a silica coating. The monodisperse hydrogel polymer particles have a log P less than (a) oct / wat (log P), and (b) a crosslinker containing at least two vinyl groups.

[0022] A seventh aspect comprises monodisperse magnetic hydrogel polymer particles or coated monodisperse hydrogel polymer particles obtainable by the method of the fifth or sixth aspect. The monodisperse magnetic hydrogel polymer particles or coated monodisperse hydrogel polymer particles may be obtainable by the method of the fifth or sixth aspect.

[0023] An eighth aspect includes the use of monodisperse magnetic hydrogel polymer particles of the first or seventh aspect in an assay method.

[0024] A ninth aspect includes the use of coated monodisperse hydrogel polymer particles of the second or seventh aspect in an assay method.

[0025] A tenth aspect includes the use of monodisperse hydrogel polymer particles of the third aspect, or monodisperse hydrogel polymer particles formed according to the method of the fourth aspect, in an assay method.

[0026] One embodiment of the present invention provides particles obtained by the process described herein.

[0027] One embodiment of the present invention provides particles having the characteristics of particles obtained by the methods disclosed herein; while such particles are obtained by the processes described herein, they are characterized only by their properties and not by their method of manufacture, and therefore the scope of protection of the claims relating to particles specified by their characteristics is determined solely by the characteristics of the particles, excluding their actual method of manufacture.

[0028] The products, processes, and uses of the present invention are not limited to the subject matter just described, and are more fully described in the following description and claims. [Brief explanation of the drawings]

[0029] Embodiments of the present invention are further described below with reference to the accompanying drawings. [Figure 1] 1 is a schematic representation of a one-step swellable particle formation process of the present disclosure, including a representation of exemplary monomers. [Figure 2] FIG. 1 is a schematic diagram of a single-stage particle formation process of the present disclosure. [Figure 3] FIG. 1 is a schematic diagram of the two-stage swellable particle formation process of the present disclosure, including representations of exemplary monomers and chain transfer agents. [Figure 4] FIG. 1 is a schematic diagram of the two-stage swellable particle formation process of the present disclosure. [Figure 5] The use of the particles of the present invention in the amplification and / or detection of nucleotides is demonstrated. [Figure 6] 1 shows the signal as a function of antigen concentration obtained in the assay of Example 67. Diamonds represent results for particles of Example 34, squares represent results for particles of Example 35, and triangles represent results for particles of Example 36. [Figure 7] 1 shows the signal as a function of antigen concentration obtained in the assay of Example 68. Diamonds represent results using particles from Example 34, squares represent results using particles from Example 35, and triangles represent results using particles from Example 36. [Figure 8]1 shows the signal as a function of antigen concentration obtained in the assay of Example 69. Diamonds represent results using particles from Example 34, squares represent results using particles from Example 37, and triangles represent results using particles from Example 38. [Figure 9] The non-specific binding obtained with the particles of Examples 34, 35, and 36 is shown in comparison to commercially available MyOne™ epoxy particles. DETAILED DESCRIPTION OF THE INVENTION

[0030] Detailed Description Throughout this description and the claims, the words "comprise," "contain," and variations thereof, mean, for example, "including, but not limited to," and they are not intended to (and do not) exclude other moieties, additives, components, integers, or steps. Throughout this description and the claims, the singular encompasses the plural unless the context requires otherwise. Specifically, where the indefinite article is used, the specification shall be understood to contemplate plural as well as singular, unless the context requires otherwise.

[0031] It is to be understood that features, integers, characteristics, compounds, chemical moieties, or groups described in connection with a particular aspect, embodiment, or example of the invention are applicable to other aspects, embodiments, or examples described herein, except where incompatible. All features disclosed herein (including any accompanying claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any embodiment disclosed herein. The invention extends to any novel, or any novel combination of features disclosed herein (including any accompanying claims, abstract, and drawings), or any novel, or any novel combination of steps of any method or process so disclosed.

[0032] The reader's attention is directed to all articles and documents related to this application, filed contemporaneously or prior to this specification, and made available for inspection herewith, the contents of all such articles and documents being incorporated herein by reference.

[0033] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0034] In one embodiment, the present invention provides novel magnetic polymer particles, in another embodiment, processes by which the novel particles can be prepared, and in other embodiments, methods of using the polymer particles.

[0035] The polymer particle formation process involves a water-in-oil emulsion, i.e., a discontinuous water phase suspended in a continuous oil phase. The formed polymer particles can then be made magnetic and / or coated.

[0036] Thus, the polymer particle formation process described herein involves the production of two distinct particles, namely, particles that undergo swelling and polymerization processes. polymer particles Form seed particles Accordingly, the terms "seed particles" and "polymer particles" are used herein as follows.

[0037] "Seed particles", unless the context requires otherwise, mean particles obtained by dispersion polymerization and used as intermediates in the polymer particle formation process.

[0038] "Polymer particles" refer to particles that can be made from seed particles by suspension polymerization in the processes described herein. After polymerization, a magnetic material can be added to the particles. For example, if the polymer particles contain pores, the magnetic material can be deposited in at least a portion of the pores. After polymerization (and optionally the addition of the magnetic material), the particles can be coated, for example, with an organic (e.g., polymer) or silica coating.

[0039] The polymer particles may be "formed" from specific hydrophilic vinyl monomers and crosslinkers. The specific hydrophilic monomers may include one or more monomers. The specific crosslinkers may include one or more crosslinkers. In this context, "formed from" may mean that the polymer consists of the reacted specific hydrophilic vinyl monomers and crosslinkers. In this context, "formed from" may mean that the polymer contains the specific hydrophilic vinyl monomers and crosslinkers along with small amounts of other vinyl monomers and / or crosslinkers. For example, the polymer may contain at least 90% by weight of the specific monomers and crosslinkers and no more than 10% by weight of other vinyl monomers and / or crosslinkers; for example, the polymer may contain at least 95% by weight of the specific monomers and crosslinkers and no more than 5% by weight of other vinyl monomers and / or crosslinkers. For example, the polymer may contain at least 98% by weight of the specific monomers and crosslinkers and no more than 2% by weight of other vinyl monomers and / or crosslinkers; for example, the polymer may contain at least 99% by weight of the specific monomers and crosslinkers and no more than 1% by weight of other vinyl monomers and / or crosslinkers.

[0040] "Magnetic material" refers to a material that responds to a magnetic field. When a magnetic material is paramagnetic, its magnetic properties turn off when the magnetic field is removed. When a magnetic material is superparamagnetic, it becomes saturated at a relatively low magnetic field, and the shutoff of its magnetic properties upon removal of the magnetic field is very fast / instantaneous. When a magnetic material is ferromagnetic, all of its magnetic atoms in each domain add a positive contribution to the net magnetization. When a magnetic material is ferrimagnetic, some magnetic atoms in each domain are opposed, but the material as a whole exhibits a net magnetization. Both ferromagnetic and ferrimagnetic materials retain their magnetic properties even after the external magnetic field is removed. Above the material's Curie temperature, ferromagnetic and ferromagnetic materials become paramagnetic. Magnetic properties can also be affected by the size of the magnetic particles in the magnetic material; some materials are ferromagnetic and ferrimagnetic at larger particle sizes, but superparamagnetic at appropriately small particle sizes (e.g., nm scale). For example, ferrimagnetic materials, such as iron oxide, form superparamagnetic crystals when the crystal size is small enough (eg, on the order of 15 nm scale or less for iron oxide).

[0041] "Magnetic material precursor" means a substance that can be converted to provide a magnetic material. The magnetic material precursor can include solvated transition metal ions (e.g., polyvalent cations of Fe, Ni, Co, or combinations thereof, optionally mixed with polyvalent cations of Al, Mn, Cu, Zn, Ca, Ge, Te, Ti, or Sn and / or rare earth elements). The solvated transition metal ions and / or rare earth ions can be converted to a magnetic material, for example, as oxides, by any process that precipitates the ions. Precipitation can be caused, for example, by a pH change, solvent removal, or temperature change. For example, the magnetic material precursor can include solvated transition metal ions (e.g., polyvalent cations of Fe, Ni, Co, or combinations thereof, optionally mixed with polyvalent cations of Al, Mn, Cu, Zn, Ca, Ge, Te, Ti, or Sn and / or rare earth elements). 2+ and / or Fe 3+ ions, and may be provided by an aqueous suspension of less than pH 6 containing Fe ions. 2+ and / or Fe 3+ The ions can be converted to magnetic materials by precipitation, for example by raising the pH to above 8.

[0042] "Coating" refers to a coating applied to the surface of a substrate. The coating may be a full coating that completely covers the substrate, or a partial coating that only covers a portion of the substrate's surface. The coating may be applied to the substrate by at least one of any suitable type of bond, such as a covalent bond, a metallic bond, an ionic bond, a hydrogen bond, a van der Waals interaction, a hydrophobic interaction, etc. A porous substrate may be coated such that at least a portion of the coating resides within the pores while maintaining the porous structure (e.g., so that the coating partially fills the pores). If the substrate comprises a polymer, the coating (or at least a portion thereof) may be provided by grafting. If the coating (or at least a portion thereof) is provided by grafting, the coating may be a partial coating. In the present disclosure, unless otherwise required by context, the substrate is a polymer particle. The polymer particle may be a magnetic polymer particle or a polymer particle that does not include a magnetic material.

[0043] "Grafting," in the context of polymer chemistry, includes a reaction in which one or more block species are attached to the backbone of a macromolecule as side chains having constitutional or configurational characteristics different from those of the backbone. When the substrate is a polymer particle or polymer particles and the coating (or at least a portion thereof) is provided by grafting, the polymer of the polymer particle provides the backbone and the coating provides the side chains (or side chains).

[0044] Reference to "hydrogel" with respect to a polymer, e.g., a polymer particle, refers to a polymer gel in which the swelling agent is water. The hydrogel polymer can absorb at least 20% of its weight in water. The hydrogel polymer can absorb at least 45%, at least 65%, at least 85%, at least 100%, or at least 300% of its weight in water. For example, the hydrogel polymer can absorb at least 1000%, at least 1500%, or even at least 2000% of its weight in water.

[0045] References to "transparent" with respect to polymer particles (e.g., hydrogel polymer particles) mean that the particles are porous and molecules or other reagents of interest can readily diffuse through aqueous solutions into the pores of the particles. For example, crosslinked hydrogel polymer particles of the present disclosure can be transparent to oligonucleotides and nucleic acid amplification and sequencing reagents even when polymerase is bound to the oligonucleotides, e.g., the oligonucleotides can be partially or entirely located within the pores.

[0046] References herein to "average" diameter, unless otherwise specified, refer to the modal diameter of the crosslinked polymer particles (e.g., crosslinked hydrogel polymer particles, magnetic hydrogel polymer particles, and / or coated hydrogel polymer particles) or to the z-average diameter of the seed particles. The modal diameter may be measured by a disc centrifuge, for example, by a CPS disc centrifuge. The z-average diameter may be the z-average diameter measured by dynamic light scattering (also known as photon correlation spectroscopy). Further disclosed are embodiments in which the mean diameter is the modal diameter, for example, the modal diameter determined by optical microscopy, throughout the entire range.

[0047] The term "monodisperse" means that for a plurality of particles (e.g., at least 100, more preferably at least 1000), the particles have a coefficient of variation (CV) or % polydispersity of their diameters of less than 20%, for example less than 15%, typically less than 10%, and optionally less than 8%, for example less than 5%.

[0048] The term "Mw" refers to the weight average molecular weight of a polymer. It has the following formula: TIFF0007765421000005.tif14128, where M i is the molecular weight of a particular chain and N iis the number of chains of that molecular weight. Mw can be measured using gel permeation chromatography (GPC) against a series of standard polymers with a particular eluent solvent system. For example, the Mw of the oligomer or polymer in the seed particles can be measured by GPC against polystyrene standards using DMF with 0.01 M LiBr as the eluent.

[0049] References herein to "polydispersity" or "% polydispersity" refer to a value for dynamic light scattering data derived from the "polydispersity index." The polydispersity index is a number calculated from a simple two-parameter fit to correlation data, e.g., dynamic light scattering data, as defined in ISO standard documents 13321:1996 E and ISO 22412:2008. The polydispersity index is dimensionless and is scaled so that values ​​less than 0.05 are rarely found except in highly monodisperse standards. A polydispersity index value greater than 0.7 for a particle sample indicates that the sample has a very broad particle size distribution, e.g., the particles are not monodisperse.

[0050] As used herein, "alkyl" and "C x ~C y The term "alkyl" (where x is at least 1 and less than 10, and y is greater than 10) includes reference to straight or branched chain alkyl moieties having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. This term includes reference to, for example, methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl, or tert-butyl), pentyl, hexyl, and the like. In particular, alkyl can be "C1-C6 alkyl," i.e., alkyl having 1, 2, 3, 4, 5, or 6 carbon atoms, or "C1-C4 alkyl," i.e., alkyl having 1, 2, 3, or 4 carbon atoms. The term "lower alkyl" includes reference to alkyl groups having 1, 2, 3, or 4 carbon atoms. Alkyl, where chemically possible, independently in each occurrence, includes oxo, =NR a , =NOR a , Halo, Nitro, Cyano, NRa R a , N.R. a S(O)2R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , S.R. a , S(O)R a , S(O)2OR a , S(O)2R a , S(O)NR a R a , CO2R a C(O)R a ,CONR a R a , C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, and R a is independently at each occurrence selected from H and C1-C4 alkyl.

[0051] As used herein, "alcohol" C x ~C y The term "alcohol" (where x is at least 1 and less than 10, and y is greater than 10) includes reference to a straight-chain or branched-chain alcohol moiety having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The term includes reference to the corresponding alkyl substituted with one or more hydroxyl (-OH) groups. Thus, the term includes reference to a monohydroxy-substituted alcohol or polyol (e.g., a diol or triol). Specifically, the alcohol can be a "C1-C6 alcohol," i.e., an alkyl having 1, 2, 3, 4, 5, or 6 carbon atoms and one or more hydroxyl groups, or a "C1-C4 alcohol," i.e., an alcohol having 1, 2, 3, or 4 carbon atoms and one or more hydroxyl groups.

[0052] The symbol TIFF0007765421000006.tif7128 represents the point of attachment of the moiety to the remainder of the compound.

[0053] As used herein, the term "log P" refers to the octanol-water partition coefficient (log P oct / wat ) The log P of a compound can be determined by any one of a variety of methods. In particular, for the compounds used in embodiments and other compounds disclosed herein, log P can be measured according to the shake flask method described in J. Sangster, "Octanol-water partition coefficients of simple organic compounds," J. Phys. Chem. Ref. Data, Vol. 18, No. 3, 1989, pp. 1111-1227, section 2.1.a, page 1116, which is incorporated herein by reference in its entirety. Log P can also be calculated from the structure of a compound using software; for example, log P can be calculated using ChemBioDraw® Ultra 14.0 from CambridgeSoft Corp.

[0054] Since log P is the organic phase-aqueous phase partition coefficient, this value for a given compound correlates with the proportion of monomer in the aqueous phase. In particular, the lower this value (including more negative values), the higher the proportion of the compound (e.g., monomer or crosslinker) in water or aqueous phase. The compound (e.g., monomer or crosslinker) used in the present invention should have a sufficiently low log P so that the compound is available in sufficient amounts for polymerization at equilibrium, for example, according to the method for preparing particles disclosed herein. Therefore, log P can be useful for characterizing the compounds used in the present invention.

[0055] Based on the molecular structure of a compound, it is possible to estimate the tendency of a molecule to reside in the organic or aqueous phase. For example, monomers containing more hydrophilic atoms such as nitrogen and oxygen and fewer carbon atoms are more likely to have more hydrophilic groups and are more likely to partition into the aqueous phase.

[0056] Because compounds of interest (e.g., monomers and / or crosslinkers) of the present invention are typically polymerizable vinyl monomers, a formula for calculating the ratio between the number of hydrophobic and hydrophilic atoms in a compound can be used to obtain an estimate of the compound's relative tendency to partition into the aqueous phase. For a given class of compounds, values ​​calculated from such a formula can be (approximately) correlated with log P. Such a formula can be used instead of, or in addition to, log P to determine monomers useful in the present invention.

[0057] For example, the hydrophilic vinyl monomers of the present invention (e.g., R 1 Suitable formulas for the compounds of formula I, such as compounds of formula I, where is -H, -CH are as follows: TIFF0007765421000007.tif10128, where N C is the number of carbon atoms in the monomer, and N N is the number of nitrogen atoms in the monomer, and N O is the number of oxygen atoms in the monomer. 原子 Where R is less than 2.75, acrylamide, vinylimidazole, acryloylmorpholine, vinylphosphonic acid, and vinylsulfonic acid are suitable, but 原子 Acrylates and other monomer classes are suitable where R is 2 or less and the monomer has a water solubility of 50 g / L or more (e.g., at 25°C). In general, acrylamides are more soluble in the aqueous phase than analogous acrylates, and R 原子 This reflects the difference in the passing criteria. 原子 The formulas and cutoff criteria can also be applied to crosslinkers used in the present invention, such as compounds of formula (IIa) or (IIb). R for exemplary monomers and crosslinkers 原子 The values ​​are shown in Table 1.

[0058] polymer particles The present invention includes embodiments in which the particles are polymeric and monodisperse. The present invention includes embodiments in which the particles are porous. In accordance with the present invention, the particles are magnetic and / or include a coating. Unless the context requires otherwise, references hereinafter to "particles" and "polymer particles" include references to monodisperse magnetic hydrogel polymer particles of the present invention and / or monodisperse coated hydrogel polymer particles of the present invention.

[0059] The particles may be in populations of at least 100, such as at least 1000. For example, for measurements, the particles may be in populations of at least 100, such as at least 1000. For example, in certain end uses, the particles may conveniently be in populations of at least 100, such as at least 1000.

[0060] "Monodisperse" means that for a plurality of particles (e.g., at least 100, more preferably at least 1000), the particles have a coefficient of variation (CV) of their diameters of less than 20%, e.g., less than 15%, typically less than 10%, and optionally less than 8%, e.g., less than 5%. Certain types of polymer particles have a CV of less than 10%. CV is defined as (standard deviation) / mean x 100, where "mean" is the average particle diameter and standard deviation is the standard deviation of particle sizes. The present invention also includes embodiments in which "mean" is either the z-average particle diameter or the modal particle diameter. CV is preferably calculated for the major mode. Thus, some particles below or above the modal size may not be considered in the calculation, which may be based, for example, on about 90% of the total particle count (i.e., detectable particles). Such CV determinations can be performed in a CPS disc centrifuge.

[0061] The polymer particles can be produced by the polymer particle formation process described hereinafter, for example, by using the monodisperse seed particles described herein.

[0062] Returning now to polymer particles, the present specification discloses crosslinked hydrogel polymer particles. A characteristic of crosslinked hydrogel polymer particles is that when placed in an aqueous solution that is a good solvent for the polymer, the particles swell instead of dissolving. For example, water is a good solvent for hydrogel particles containing acrylamide polymers. Hydrogel particles are hydrophilic, swell in water, and can be made with a variety of different porosities. The crosslinked hydrogel particles disclosed herein offer a combination of low nonspecific binding, monodispersity, and porosity, which provides advantages when the particles are used in biological assays. Furthermore, the nonspecific binding and porosity characteristics can be modified by including a magnetic material in the particles and / or coating the particles.

[0063] The level of crosslinking in the polymer particles produced by this process can be expressed as the weight percent (wt%) of crosslinker monomer contained in the total monomers used in the suspension polymerization. The wt% of crosslinker monomer can correspond to the weight percent of crosslinker in the matrix polymer (i.e., the weight percent of crosslinker in the dry weight of the crosslinked polymer particles). Thus, if the monomers used in the suspension polymerization are, for example, monofunctional acrylamide and difunctional acrylamide, the proportion of difunctional acrylamide (crosslinker monomer) is calculated as a weight percent based on the total weight of the difunctional acrylamide and monofunctional acrylamide. Typical levels of crosslinking include greater than 1 wt% crosslinker, for example, greater than 2 wt% crosslinker, for example, greater than 5 wt% crosslinker. For example, the level of crosslinking can be greater than 10 wt% crosslinker, or greater than 15 wt% crosslinker, for example, greater than 20 wt% crosslinker or greater than 25 wt% crosslinker. The level of crosslinking can be, for example, 5-60% by weight crosslinker, such as 10-50% by weight crosslinker, such as 20-40% by weight crosslinker, or even 20-30% by weight crosslinker. The level of crosslinking can be 1-30% by weight crosslinker, such as 1-20% by weight crosslinker, such as 1-10% by weight crosslinker, or the level of crosslinking can be 2-30% by weight crosslinker, such as 2-20% by weight crosslinker, such as 2-10% by weight crosslinker, suitable for example for porous hydrogel particles.

[0064] The level of crosslinking can be, for example, greater than 30% crosslinker by weight or greater than 40% crosslinker by weight in highly crosslinked particles. The level of crosslinking can be 10-90% crosslinker by weight, 20-80% crosslinker by weight, or 25-75% crosslinker by weight, e.g., 25-60% crosslinker by weight or 30-50% crosslinker by weight. In highly crosslinked particles, the level of crosslinking can be up to 100% crosslinker by weight, e.g., a hydrophilic vinyl monomer can be the crosslinker.

[0065] When the particles are magnetic polymer particles (e.g., magnetic hydrogel polymer particles), the level of crosslinking is preferably greater than 10% by weight crosslinker, e.g., greater than 30% by weight crosslinker or greater than 40% by weight crosslinker. These crosslinking levels may provide particles with better retention of magnetic material compared to particles with lower levels of crosslinking. The level of crosslinking may be 10-90% by weight crosslinker, 20-80% by weight crosslinker, or 25-75% by weight crosslinker, e.g., 25-60% by weight crosslinker or 30-50% by weight crosslinker.

[0066] As mentioned above, crosslinked particles swell when placed in a good solvent for the polymer. The amount of swelling, measured, for example, as an increase in diameter, is related to the level of crosslinking. Particles with a higher degree of crosslinking typically swell less than particles made from similar polymers but with a lower degree of crosslinking. This property can be used to determine the relative level of crosslinking in a sample of polymer particles by comparing the sample to a series of standards with known different levels of crosslinking.

[0067] The particles suitably comprise addition polymers made by polymerizing one or more vinyl-unsaturated monomers. The unsaturated vinyl monomers may comprise an optionally substituted generalized vinyl group, -CR=CR'H, where R is H or alkyl (e.g., R is -CH3 or -CH2CH3), and R' is H, alkyl (e.g., -CH3 or -CH2CH3), or a functional group (e.g., carboxylic acid, phosphonic acid, primary amine, or secondary amine). The vinyl-unsaturated monomers may comprise a generalized vinyl group, -CR=CH2, where R is H or alkyl (e.g., R is -CH3 or -CH2CH3). The vinyl-unsaturated monomers may comprise a vinyl group, -CH=CH2. The monomers may be hydrophilic vinyl monomers, e.g., having a log P value of less than about 1, e.g., a log P value of less than about 0.5. The hydrophilic vinyl monomer can be a vinyl monomer having a log P value of less than about 0.5. The hydrophilic vinyl monomer can include an optionally substituted generalized vinyl group, -CR=CR'H, where R is H or alkyl (e.g., R is -CH3 or -CH2CH3), and R' is H, alkyl (e.g., -CH3 or -CH2CH3), or a functional group (e.g., a carboxylic acid, a phosphonic acid, a primary amine, or a secondary amine). The hydrophilic vinyl monomer can include a generalized vinyl group, -CR=CH2, where R is H or alkyl (e.g., R is -CH3 or -CH2CH3). The hydrophilic vinyl monomer can include a vinyl group, -CH=CH2. The monomer can be a vinyl monomer having a log P of less than about 0.6, for example, a log P of less than about 0.52. For example, the monomer can be a vinyl monomer having a log P of less than about 0.3 or less than about 0.2, for example, the monomer can be a vinyl monomer having a log P of less than about 0.1. The monomer may be a vinyl monomer having a log P of less than about 0, for example, less than about -0.2. The monomer may be a vinyl monomer having a log P of greater than about -3.8, for example, greater than about -3 or about -2. The monomer may be a vinyl monomer having a log P of 0.6 to -3.The monomer may be a vinyl monomer having a log P of 0.5 to −2, such as a log P of 0 to −2, for example a log P of −0.2 to −2. In particular, the monomer may be a vinyl monomer which also comprises a hydrophilic group, such as an acrylamide monomer or an acrylate monomer.

[0068] The monomer is R 原子 When the monomers are acrylamide, vinylimidazole, acryloylmorpholine, vinylphosphonic acid, and vinylsulfonic acid, specific values ​​for R 原子 is R 原子 If the monomer is an acrylate of another monomer class, the specific R 原子 is R 原子 may be 2 or less, and the monomer has a water solubility (eg, at 25° C.) of 50 g / L or more.

[0069] The monomer used in the particles has the formula (I): TIFF0007765421000008.tif15128, During the ceremony, R 1 is -H, -CH3, -CH2CH3, or -CH2C(O)OH; R 1a is -H, -CH3, -CH2CH3, -CH2C(O)OH, or -C(O)R 2 and R 1b is -C(O)R 2 is -P(O)(OH)2, is -S(O)2OH, or is a heterocycle having 3 to 8 ring atoms wherein the heteroatoms are selected from N, O, or S; or R 1a or R 1 R combined with 1b is -C(O)-OC(O)-, R 2 -OR 3 , -N(R 4 )R 5 or an -N-linked amino acid; R3 is selected from -H, -C1-C6 alkyl, or -C1-C6 alcohol; and R 4 and R 5 are each independently selected from -H, -C1-C6 alkyl, -C1-C6 haloalkyl, and -C1-C6 alcohol; or R 4 and R 5 together with the nitrogen to which they are attached form a heterocycle having 3 to 8 ring atoms, the heteroatoms being selected from N, O, or S.

[0070] In one embodiment, R 1 is -H, -CH3, -CH2CH3, or -CH2C(O)OH; R 1a is -H or -C(O)R 2 and R 1b is -C(O)R 2 , -P(O)(OH)2, or -S(O)2OH, or R 1a Or R 1 R combined with 1b is -C(O)-OC(O)-, R 2 -OR 3 or -N(R 4 )R 5 is selected from R 3 is selected from -H, -C1-C6 alkyl, or -C1-C6 alcohol; and R 4 and R 5 are each independently selected from -H, -C1-C6 alkyl, -C1-C6 haloalkyl, and -C1-C6 alcohol.

[0071] R 3 is -C1-C6 alkyl or -C1-C6 alcohol, the alkyl or alcohol may each independently be oxo, =NR a , =NOR a , Halo, Nitro, Cyano, NR a Ra , N.R. a S(O)2R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , S.R. a , S(O)R a , S(O)2OR a , S(O)2R a , S(O)NR a R a , CO2R a C(O)R a ,CONR a R a , C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4 haloalkyl, and R a is independently selected at each occurrence from H and C1-C4 alkyl. For example, R 3 is -C1-C6 alkyl or -C1-C6 alcohol, the alkyl or alcohol may each independently be, if chemically possible, OR a or CO2R a and optionally R a is H.

[0072] R 4 and / or R 5 When each of the groups is -C1-C6 alkyl, -C1-C6 haloalkyl, or -C1-C6 alcohol, each of the groups independently represents, if chemically possible, oxo, =NR a , =NOR a , Halo, Nitro, Cyano, NR a R a , N.R. a S(O)2R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , S.R.a , S(O)R a , S(O)2OR a , S(O)2R a , S(O)NR a R a , CO2R a C(O)R a ,CONR a R a , C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4 haloalkyl, and R a is independently selected at each occurrence from H and C1-C4 alkyl. For example, R 4 and / or R 5 When is -C1-C6 alkyl, -C1-C6 haloalkyl, or -C1-C6 alcohol, each independently represents, if chemically possible, OR at each occurrence. a or CO2R a and optionally R a is H.

[0073] R 1 R can be -H, -CH, or -CHCH. 1 can be -H or -CH3. For example, R 1 can be -H. 1 can be —CHC(O)OH.

[0074] R 1a R can be -H, -CH, or -CHCH. 1a can be -H. 1a is -C(O)R 2 It can be. R 1a can be —CHC(O)OH.

[0075] R 1b is -C(O)R 2 It can be. R 1b can be -P(O)(OH). R 1bcan be -S(O)2OH. 1b -C(O)R 2 When R 1a can be -H. R 1b -C(O)R 2 When R 1a -C(O)R 2 R 2 is optional -OR 3 It can be. R 1b When is -P(O)(OH)2, R 1a can be -H, R 1 can be -H, -CH, or -CHCH (e.g., -H). 1b When is -S(O)2OH, R 1a can be -H, R 1 can be -H, -CH3, or -CH2CH3 (e.g., -H).

[0076] R 1b R can be a heterocycle having 3 to 8 ring atoms, the heteroatoms being selected from N, O, or S. 1b can be imidazolyl.

[0077] R 1a or R 1 R combined with 1b can be —C(O)—OC(O)—.

[0078] R 2 HA-OR 3 It can be. R 2 -N(R 4 )R 5 It can be. R 2 can be -NH, -NHCH, -NHCHCH, or -NH(CH). 2 can be an N-linked amino acid. The N-linked amino acid can be selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, β-alanine, and methionine.

[0079] R3 can be -H. 3 can be -C1-C6 alkyl. For example, R 3 each independently represent, at each occurrence, oxo, halo, cyano, NR a R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , CO2R a C(O)R a ,CONR a R a , C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, and R a is independently at each occurrence selected from H and C1-C4 alkyl, e.g., R a is -H. R 3 can be a -C1 to C6 alcohol. For example, R 3 each independently represent, at each occurrence, oxo, halo, cyano, NR a R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , CO2R a C(O)R a ,CONR a R a , C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4 haloalkyl, and R a is independently selected at each occurrence from H and C1-C4 alkyl, e.g., R a is -H.

[0080] R 4 can be -H or -C1-C6 alkyl. 4 can be -C1-C6 alkyl. For example, R4 each independently represent, at each occurrence, oxo, halo, cyano, NR a R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , CO2R a C(O)R a ,CONR a R a , C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, and R a is independently selected at each occurrence from H and C1-C4 alkyl, e.g., R a is -H.

[0081] R 5 can be -H or -C1-C6 alkyl. 5 can be -C1-C6 alkyl. For example, R 5 each independently represent, at each occurrence, oxo, halo, cyano, NR a R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , CO2R a C(O)R a ,CONR a R a , C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, and R a is independently selected at each occurrence from H and C1-C4 alkyl, e.g., R a is -H.

[0082] R 4 and R 5R can be taken together with the nitrogen to which they are attached to form a heterocycle having 3 to 8 ring atoms (e.g., 4, 5, or 6 ring atoms) in which the heteroatoms are selected from N, O, or S. 4 and R 5 can be taken together with the nitrogen to which they are attached to form a morpholino heterocycle.

[0083] The monomer used in the particles has the formula (Ia): TIFF0007765421000009.tif20128, wherein R 1 , R 2 , and R 1a is as defined elsewhere.

[0084] The monomer used in the particles has the formula (Ib): TIFF0007765421000010.tif20128, wherein R 1 and R 1a is as defined elsewhere. In one embodiment, R 1 and R 1a are each independently selected from —H, —CH 3 , —CH 2 CH 3 , or —CH 2 C(O)OH.

[0085] The monomer used in the particles has the formula (Ic): TIFF0007765421000011.tif20128, wherein R 1 and R 1a is as defined elsewhere. In one embodiment, R 1 and R 1a are each independently selected from —H, —CH 3 , —CH 2 CH 3 , or —CH 2 C(O)OH.

[0086] The compound of Formula (I), (Ia), (Ib), or (Ic) may have a log P value of less than about 1, for example, a log P of less than about 0.5. The compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of less than about 0.6, for example, a log P of less than about 0.52. The compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of less than about 0.5. For example, the compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of less than about 0.3 or less than about 0.2, for example, the compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of less than about 0.1. The compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of less than about 0, for example, a log P of less than about -0.2. A compound of Formula (I), (Ia), (Ib), or (Ic) may have a log P of greater than about -3.8, for example, a log P of greater than about -3 or about -2. A compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of 0.6 to -3. A compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of 0.5 to -2, for example, a log P of 0 to -2, for example, a log P of -0.2 to -2.

[0087] The compounds of formula (I) or (Ia) have an R of less than 2.75. 原子 and the compound is an acrylamide, a vinylimidazole, an acryloylmorpholine, a vinylphosphonic acid, or a vinylsulfonic acid, or R 原子 The compound of formula (Ib) or (Ic) has an R of less than 2.75 and an aqueous solubility of 50 g / L or more, and the compound is an acrylate or other monomer class. 原子 may have:

[0088] The monomers may include at least one hydrophilic vinyl monomer containing an amide group selected from -C(O)NH, -C(O)NHCH, -C(O)NHCHCH, or -C(O)N(CH). The monomers may include at least one hydrophilic vinyl monomer containing a primary amide group (-C(O)NH).

[0089] Acrylamide monomers and / or acrylate monomers may be specifically mentioned. Suitable monomers include acrylamide (prop-2-enamide), N-(hydroxymethyl)acrylamide, N-hydroxyethylacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, 4-acrylomorpholine, 3-acrylamidopropanoic acid, methacrylamide, N-(2)-(hydroxyethyl)methacrylamide, N-(3-aminopropyl)methacrylamide, hydroxypropylacrylamide, N,N-dimethylacrylamide, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, acrylic acid; other acrylamide monomers, acrylic monomers, methacrylamide monomers, or methacrylic monomers having a log P value of less than about 1 (e.g., having a log P value of less than about 0.5); 原子 other acrylic or methacrylamide monomers having R 原子 Other acrylic or methacrylic acid monomers having a σ of 2 or less and a water solubility of 50 g / L or more (eg, at 25° C.) are included.

[0090] The monomers may comprise a mixture of monomers. For example, the monomers may comprise at least one monomer defined above and at least one compatible functional monomer. Exemplary functional monomers are hydrophilic vinyl monomers as defined herein, including carboxylic acids (-COOH), phosphonic acids (-P(O)(OH)), sulfonic acids (-S(O)OH), primary amines, or secondary amines. For example, the monomers can include at least one monomer of formula (I), (Ia), (Ib), or (Ic) that does not include a carboxylic acid (-COOH), phosphonic acid (-P(O)(OH)), sulfonic acid (-S(O)OH), primary amine, or secondary amine, and a monomer of formula (I), (Ia), or (Ib) that includes at least one carboxylic acid (-COOH), phosphonic acid (-P(O)(OH)), sulfonic acid (-S(O)OH), primary amine, or secondary amine.

[0091] In highly crosslinked particles, the monomer can be or include a crosslinker, such as those defined elsewhere herein, e.g., the monomer can be or include at least one compound of formula (IIa) or (IIb).

[0092] The functional monomer can be a vinyl monomer having a log P value of less than about 1, for example, less than about 0.5, including a carboxylic acid or a primary amine. The functional monomer can be a vinyl monomer having a log P of less than about 0.6, for example, less than about 0.52. The functional monomer can be a vinyl monomer having a log P of less than about 0.5. For example, the functional monomer can be a vinyl monomer having a log P of less than about 0.3 or less than about 0.2, for example, the functional monomer can be a vinyl monomer having a log P of less than about 0.1. The functional monomer can be a vinyl monomer having a log P of less than about 0, for example, less than about -0.2. The functional monomer can be a vinyl monomer having a log P of greater than about -3.8, for example, greater than about -3 or about -2. The functional monomer can be a vinyl monomer having a log P of 0.6 to -3. The functional monomer can be a vinyl monomer having a log P of 0.5 to -2, for example, a log P of 0 to -2, for example, a log P of -0.2 to -2. The functional monomer may be a compound of formula (I), (Ia), or (Ib) containing a carboxylic acid, a phosphonic acid, a sulfonic acid, or a primary amine. The functional monomer may contain a carboxylic acid, a phosphonic acid, or a sulfonic acid, for example, a carboxylic acid or a phosphonic acid. The functional monomer may be an acrylamide monomer containing a carboxylic acid or a primary amine.

[0093] Suitable functional monomers include 3-acrylamidopropionic acid, 4-acrylamidobutanoic acid, 5-acrylamidopentanoic acid, N-(3-aminopropyl)methacrylamide, acrylic acid, fumaric acid, itaconic acid, vinylphosphonic acid, vinylsulfonic acid, and 1-vinylimidazole. Further suitable functional monomers include anhydrides, such as maleic anhydride or itaconic anhydride, which can be hydrolyzed during polymerization to provide a carboxylic acid group.

[0094] When at least one functional monomer is present, the amount of functional monomer can be about 0.1 to about 100 mol%, for example, about 0.2 to about 50 mol%, for example, about 0.5 to about 40 mol%, or about 1 to about 30 mol% (e.g., about 2 to about 20 mol%). For example, the amount of functional monomer can be at least about 2 to about 100 mol%, for example, about 2 to about 50 mol%, for example, about 4 to about 40 mol%, or about 4 to about 30 mol% (e.g., about 2 to about 20 mol%). The amount of functional monomer can be about 5 to about 100 mol%, for example, about 5 to about 80 mol%. Molar % can refer to the amount of functional monomer as a mole % of the total hydrophilic vinyl monomer. The amount of functional monomer can be about 0.1 to about 60 wt%, for example, about 0.2 to about 50 wt%, for example, about 0.5 to about 40 wt%. The amount of functional monomer can be about 1 to about 60% by weight, for example, about 2 to about 50% by weight, for example, about 2 or 3 to about 40% by weight. The amount of functional monomer can be about 0.1 to about 10% by weight, for example, about 0.2 to about 5% by weight, for example, about 0.5 to about 2% by weight. The weight percentage can refer to the weight percentage of functional monomer contained in the total monomer used in the polymerization process. The total monomer can include, for example, at least one hydrophilic vinyl monomer that is not a functional monomer, a crosslinker, and a functional monomer.

[0095] Crosslinking can be achieved by incorporating a crosslinker containing at least two (e.g., two) vinyl groups (-CH=CH) as a comonomer. The crosslinker can be represented by formula (IIa) or (IIb): TIFF0007765421000012.tif51128, In the formula, R 6 is selected from -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 hydroxyalkyl-, -C1-C6 ether-, and a polyether, the polyether comprising 2 to 100 C2-C3 ether units; R 7 and R 8are each independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, -C3-C6 cycloalkyl, -C1-C6 hydroxyalkyl, or -C1-C6 ether; or R 7 and R 8 are linked together to form a -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 hydroxyalkyl-, -C1-C6 ether-, polyether, the polyether containing 2 to 100 C2-C3 ether units; R 9 is -N(R 11 )C(O)CH=CH2, R 10 is -H and -N(R 12 )C(O)CH=CH2, and R 11 and R 12 are each independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, -C3-C6 cycloalkyl, -C1-C6 hydroxyalkyl, or -C1-C6 ether.

[0096] The cross-linking agent may be at least one compound of formula (IIa): The cross-linking agent may be at least one compound of formula (IIb):

[0097] R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are independently, when chemically possible, each independently in each occurrence oxo, =NR a , =NOR a , Halo, Nitro, Cyano, NR a R a , N.R. a S(O)2R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , S.R.a , S(O)R a , S(O)2OR a , S(O)2R a , S(O)NR a R a , CO2R a C(O)R a ,CONR a R a , C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4 haloalkyl; R a is independently selected at each occurrence from H, C1-C4 alkyl, and C1-C4 alkenyl.

[0098] R 6 R may be selected from -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 hydroxyalkyl-, and -C1-C6 ether-. 6 R may be selected from -C1-C6 alkyl- and -C1-C6 hydroxyalkyl-. 6 R can be -C1-C6 alkyl-, for example, -CH2-, -(CH2)2-, -(CH2)3-, or -(CH2)4-, for example, -(CH2)2-. 6 may be —C1-C6 hydroxyalkyl-, for example, —C(OH)H—, —(C(OH)H)2-, —(C(OH)H)3-, or —(C(OH)H)4-, for example, —(C(OH)H)2-.

[0099] R 6 can be -C1-C6 heteroalkyl-, where optionally the heteroatom is an amine (e.g., a tertiary amine). For example, -C1-C6 heteroalkyl- can be -C(O)R on the heteroatom. a and optionally the heteroatom is an amine, e.g., R 6 can be —CH2CH2N(C(O)CH═CH2)CH2CH2—.

[0100] R 6 When R is a polyether, the polyether may be linear or branched.6 R may be a polyether containing 2 to 100 C2-C3 ether units, for example, a polyether containing 2 to 50 C2-C3 ether units. 6 can be a polyether containing 2 to 100 C2 ether units, for example, a polyether containing 2 to 50 C2 ether units. For example, R 6 is -(CH2) r (OCH2CH2) n O(CH2) s wherein r and s are each independently 2 or 3 (e.g., 2), and n is an integer from 1 to 100 (e.g., 1 to 50 or 1 to 25, e.g., 5 to 50 or 5 to 25). Without wishing to be bound by any theory, it is believed that crosslinkers comprising polyethers (e.g., R 6 is a polyether) is believed to have excellent solubility in the aqueous phase. This means that such crosslinkers can be used to provide particles with low levels of crosslinking (e.g., 1-20 wt. % crosslinker, or 1-10 wt. % crosslinker), and such polyethers containing crosslinkers are particularly suitable for providing particles with relatively high levels of crosslinking, e.g., greater than 20 wt. % crosslinker, greater than 25 wt. % crosslinker, or greater than 30 wt. % crosslinker. For example, the level of crosslinking can be 10-90 wt. % crosslinker, 20-80 wt. % crosslinker, or 25-75 wt. % crosslinker, e.g., 25-60 wt. % crosslinker, or 30-50 wt. % crosslinker.

[0101] R 7 and / or R 8 and / or R 11 and / or R 12 can be H. For example, R 7 and / or R 8 can be H. For example, R 11 and / or R 12 can be H.

[0102] R 7 and R 8may be linked together to form -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 hydroxyalkyl-, -C1-C6 ether-, polyethers, the polyethers containing 2 to 100 C2-C3 ether units. 7 and R 8 are bonded together to form -C1-C6 alkyl-, R 7 and R 8 The group defined by R 6 For example, R 6 can be -C1-C6 alkyl-, R 7 and R 8 can be -C1-C6 alkyl-.

[0103] R 6 may be selected from -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 ether-, or a polyether, the polyether comprising 2 to 100 C2-C3 ether units; R 7 and R 8 may each independently be selected from —H, —C1-C6 alkyl, —C1-C6 heteroalkyl, —C3-C6 cycloalkyl, or —C1-C6 ether; or R 7 and R 8 are linked together to form a -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 ether-, polyether, the polyether containing 2 to 100 C2-C3 ether units, and R 11 and R 12 are each independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, -C3-C6 cycloalkyl, or -C1-C6 ether.

[0104] The compound of Formula (IIa) or (IIb) may have a log P value of less than about 1, for example, less than about 0.5. The compound of Formula (IIa) or (IIb) may have a log P of less than about 0.6. The compound of Formula (IIa) or (IIb) may have a log P of less than about 0.5. For example, the compound of Formula (IIa) or (IIb) may have a log P of less than about 0.3 or less than about 0.2, for example, the compound of Formula (IIa) or (IIb) may have a log P of less than about 0.1. The compound of Formula (IIa) or (IIb) may have a log P of less than about 0, for example, less than about -0.2. The compound of Formula (IIa) or (IIb) may have a log P of greater than -3.8, for example, greater than about -3 or about -2. The compound of Formula (IIa) or (IIb) may have a log P of 0.6 to -3. The compound of formula (IIa) or (IIb) may have a log P of 0.5 to -2, such as a log P of 0 to -2, for example a log P of -0.2 to -2.

[0105] The compound of formula (IIa) or (IIb) has an R of less than 2.75. 原子 may have:

[0106] Exemplary crosslinkers for use in the particles of the present invention include N,N'-(1,2-dihydroxyethylene)bisacrylamide, N,N'-methylenebis(acrylamide), N,N'-ethylenebis(acrylamide), piperazine diacrylamide, glycerol 1,3-diglycerolate diacrylate, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW ≦2000), 4-arm PEG-acrylamide (MW ≦2000), N,N-bis(2-acrylamidoethyl)acrylamide. Exemplary crosslinkers N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW 2000 or less), and 4-arm PEG-acrylamide (MW 2000 or less), (particularly N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide) are particularly suitable for use in highly crosslinked particles and mechanically more stable particles, e.g., particles having a crosslinking level of at least 20% by weight of the crosslinker (e.g., a crosslinking level of at least 30% by weight of the crosslinker). One embodiment of the present invention includes the use of a combination of crosslinkers. A specific monomer may be mentioned, acrylamide (prop-2-ene-amide), for which N,N'-(1,2-dihydroxybisacrylamide) is a suitable crosslinker. A specific monomer may be referred to as hydroxymethyl acrylamide, for which N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is a suitable crosslinking agent. A specific monomer may be referred to as hydroxyethyl acrylamide, for which N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is a suitable crosslinking agent.

[0107] Crosslinkers may include N,N'-methylenebis(acrylamide), N,N'-ethylenebis(acrylamide), piperazine diacrylamide, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW ≦2000), 4-arm PEG-acrylamide (MW ≦2000), N,N'-bis(2-acrylamidoethyl)acrylamide.

[0108] The crosslinker can be a compound that does not contain a primary amine, a secondary amine, a hydroxyl, or a carboxylic acid. The crosslinker can be a compound of formula (IIa) or formula (IIb) that does not contain a primary amine, a secondary amine, a hydroxyl, or a carboxylic acid.

[0109] Some exemplary monomers and crosslinkers that can be used are listed in Table 1. Some other monomers are listed in Table 2. The log P values ​​for the monomers listed in Tables 1 and 2 were determined using ChemBioDraw® Ultra 14.0 from CambridgeSoft Corp. C , N N , and N O represents the number of carbon, nitrogen, and oxygen atoms in the monomer or crosslinker, respectively, 原子 =N C / (N N +N O ) according to R 原子 values ​​were calculated. In some particles that are embodiments of the present invention, the monomers in Table 2 are not used, for example, due to the log P value of the monomer. Additionally, the monomers methyl acrylate and methacrylic acid in Table 2 may be used in conventional Ugelstad procedures, and therefore these monomers and other monomers with higher log P values ​​may be less preferred for use in particles of the present invention and may be made using the polymer particle formation process described herein.

[0110] Table 1: Exemplary Monomers and Crosslinkers TIFF0007765421000013.tif244136 TIFF0007765421000014.tif24433 TIFF0007765421000015.tif244144 TIFF0007765421000016.tif246139 TIFF0007765421000017.tif246133 TIFF0007765421000018.tif246122

[0111] Table 2: Other monomers and crosslinkers TIFF0007765421000019.tif249119 TIFF0007765421000020.tif24994

[0112] The level of crosslinking in the crosslinked hydrogel polymer particles can be expressed as a weight percent (wt%) of crosslinker monomer contained in the total monomers used in the polymerization. Typical crosslinking levels include greater than 1 wt% crosslinker, e.g., greater than 2 wt% crosslinker, e.g., greater than 5 wt% crosslinker. For example, the level of crosslinking can be greater than 10 wt% crosslinker, or greater than 15 wt% crosslinker, e.g., greater than 20 wt% crosslinker (e.g., greater than 30 wt% crosslinker or greater than 40 wt% crosslinker). The level of crosslinking can also be 1-60 wt% crosslinker, e.g., 5-30 wt% crosslinker. For example, the level of crosslinking can be 5-60 wt% crosslinker, e.g., 10-50 wt% crosslinker. The level of crosslinking can also be, for example, 15-40 wt% crosslinker, e.g., 20-40 wt% crosslinker, e.g., 20-30 wt% crosslinker. The level of crosslinking can be, for example, 10-90% by weight crosslinker, 20-80% by weight crosslinker, or 25-75% by weight crosslinker, e.g., 25-60% by weight crosslinker or 30-50% by weight crosslinker, in highly crosslinked particles. The level of crosslinking can also be 1-30% by weight crosslinker, e.g., 1-20% by weight crosslinker, e.g., 1-10% by weight crosslinker. The level of crosslinking can also be 2-30% by weight crosslinker, e.g., 2-20% by weight crosslinker, e.g., 2-10% by weight crosslinker. In highly crosslinked particles, the level of crosslinking can be up to 100% by weight crosslinker, e.g., a hydrophilic vinyl monomer can be the crosslinker.

[0113] In one embodiment, the particles may have an average diameter of at least 500 nm, such as at least 600 nm, optionally at least 800 nm, such as in the case of particles having a diameter of at least 1 μm.

[0114] In one embodiment, the particles may have an average diameter of 10 μm or less, such as 5 μm or less, optionally 3 μm or less, such as in the case of particles having a diameter of 2 μm or less.

[0115] In one embodiment, the present invention comprises a class of polymer particles having an average diameter of from 0.5 μm to 10 μm, for example, from 0.8 μm to 5 μm.

[0116] The size and size distribution of the polymer particles may be determined as described below under the heading "Analytical Methods."

[0117] The present disclosure includes porous polymer particles having a porosity of at least 5%, e.g., at least 10% or at least 30%. The present disclosure includes porous particles having a porosity of 20% to 95%, particularly 30% to 95%, e.g., 40% to 95%, e.g., 50% to 80%.

[0118] Because acrylamide and acrylate polymers have a density of approximately 1.3 g / mL, while water has a density of approximately 1 g / mL, porosity can be calculated after determining the density of the hydrogel polymer particles. Porous polymer particles can be transparent to solvating molecules; for example, porous polymer particles can be transparent to oligonucleotides and nucleic acid amplification reagents as well as sequencing reagents (e.g., primers, nucleotides, and polymerase). While this method of determining porosity works well for intermediate hydrogel polymer particles, it is not effective for determining the porosity of magnetic polymer particles because magnetic materials are typically denser than polymers. Furthermore, this method only works well for coated particles whose coatings have a density similar to that of the acrylamide and acrylate polymers.

[0119] Porosity can be determined for magnetic or coated particles of known size (e.g., diameter) in a swollen (i.e., water-saturated hydrogel particle) state by comparing the mass of the swollen particle to the mass of the dry particle. The particles can be dried, for example, by placing them in a vacuum desiccator for at least 24 hours. Because the pores of swollen hydrogel particles contain primarily water, the volume of water present in the pores can be determined (using the density of water). This volume can be compared to the total volume of the swollen hydrogel particle (calculated from the particle diameter) to determine the porosity of the particle. This method is believed to be valid for magnetic polymer particles and coated polymer particles.

[0120] In one embodiment, the particles comprise functional groups. The functional groups may be selected from hydroxyl, carboxylic acid (—COOH), phosphonic acid (—P(O)(OH)), sulfonic acid (—S(O)(OH)), primary amine, and secondary amine. The functional groups may be selected from carboxylic acid, phosphonic acid, and sulfonic acid (—S(O)(OH)), e.g., the functional groups may be selected from carboxylic acid and phosphonic acid. The functional groups may include carboxylic acid and / or phosphonic acid and / or sulfonic acid (e.g., carboxylic acid and / or phosphonic acid). The functional groups (e.g., carboxylic acid and / or phosphonic acid) can enhance the deposition level of magnetic material in the polymer particles comprising the magnetic material. The functional groups may enhance bonding of the coating to the polymer of the coated polymer particle, e.g., the functional groups may provide chemical means for covalent, ionic, or hydrogen bonding.

[0121] In one embodiment, the functional groups are provided by the hydrophilic vinyl monomer (e.g., a compound of Formula (Ia) or (Ib)) and not by the crosslinker (e.g., a compound of Formula (IIa) or (IIb)). The functional groups can be enhanced to facilitate binding with target analytes (e.g., oligonucleotides) or target receptors. Exemplary methods for enhancing the functional groups of the particles of the present invention are described in the section "Uses of the Particles" herein.

[0122] In one embodiment, the polymer particles comprise a magnetic material (e.g., the particles are magnetic hydrogel polymer particles). The porous polymer particles may comprise a magnetic material, for example, one or more magnetic materials, within the pores. The present invention is not limited with respect to the identity of the magnetic material; for example, the magnetic material may comprise at least one of a paramagnetic material, a superparamagnetic material, a ferromagnetic material, or a ferrimagnetic material. The polymer particles may comprise a paramagnetic material. The polymer particles may comprise a superparamagnetic material. The polymer particles may comprise a ferromagnetic material, a ferrimagnetic material, or both.

[0123] In one embodiment, the polymer particles described herein comprise nanoparticle magnetic materials and / or superparamagnetic materials, particularly superparamagnetic crystals. The use of superparamagnetic materials in the particles of the present invention can be particularly advantageous because the lack of magnetic properties in the absence of an applied magnetic field reduces aggregation of the polymer particles and aids in dispersion of the polymer particles. This is beneficial when magnetic polymer particles are used in assays, as it facilitates uniform distribution of the magnetic polymer particles during the assay, and a relatively strong magnetic field is only applied when it is desired to isolate the polymer particles from the bulk of the assay solution.

[0124] The superparamagnetic crystals of the polymer particles can be of any material that can be deposited in superparamagnetic crystalline form on the polymer particles or in their pores, if the particles are porous. The magnetic material can include or be a metal oxide or alloy. The magnetic material can include or be an iron oxide, a ferrite such as magnetite or maghemite, or a combination thereof. A portion of the iron in the iron oxide, e.g., magnetite or maghemite, can be substituted (i.e., replaced) with Al, Mn, Ni, Cu, Co, Zn, Ca, Ge, Te, Ti, or Sn, or a combination thereof. In particular, the magnetite or maghemite can have a portion of their iron replaced by Mn. As mentioned, some particles contain a combination of magnetite and maghemite, in which case either the magnetite or maghemite, or both, can have such partial substitution of their iron content. Additionally or alternatively, the particles of the present disclosure may include iron-based metal nanoparticles and FeNi alloy nanoparticles, in either case to increase the saturation magnetization of the particles. When a portion of the iron is substituted with one or more other elements, the total amount substituted may be in the range of up to 5 mol%, for example, 0.1 to 5 mol%, for example, 0.5 to 4 mol%, for example, 1 to 3 mol%.

[0125] In one embodiment, the polymer particles are ferrimagnetic, and in particular, the magnetic material can include or be ferrimagnetic iron oxide crystals. Accordingly, the present disclosure includes polymer particles as described herein that include a magnetic material selected from ferrimagnetic iron oxide crystals, superparamagnetic iron oxide crystals, and combinations thereof.

[0126] The total amount of magnetic material present is generally greater than 20%, preferably greater than 25%, and desirably 30% or more, e.g., up to 85% or at least 50% by weight, e.g., 30-80% by weight. The percentages are weight percentages calculated relative to the weight of magnetic material (e.g., metal oxide) based on the total dry weight of the particles. Thus, when the magnetic material consists of superparamagnetic material, the total amount of superparamagnetic material present is generally greater than 20%, preferably greater than 25%, and desirably 30% or more, e.g., up to 85% or at least 50% by weight, e.g., 30-80% by weight, each percentage calculated relative to the weight of magnetic material (e.g., metal oxide) based on the total dry weight of the particles.

[0127] In one embodiment, the polymer particles include a coating. The polymer particles including the coating may include a magnetic material, or the polymer particles including the coating may not include a magnetic material. The coating is typically provided on the outer surface of the particle and on porous particles, and the coating may also be provided on the solvent-accessible surfaces of the pores. The coating may be a polymer coating. The coating may be a silica coating. The coating may completely cover the surface of the polymer particle. The coating may partially cover the surface of the polymer particle. The use of a coating may provide several advantages. For example, where appropriate, the coating may be used to modify the surface chemistry of the polymer particles in different applications. When the polymer particles include a magnetic material, the coating may serve to separate the magnetic material from the bulk of the solution in which the particles are suspended, for example, to prevent the magnetic material from leaching out of the particle and / or to prevent the magnetic material from reacting with other components present in the solution.

[0128] In one embodiment, the coating is a polymer coating. When the coating is a polymer coating, the coating (or at least a portion thereof) may be provided by grafting. In one embodiment, the coating is an organic coating formed from at least one epoxide monomer. The at least one epoxide monomer may comprise an epoxide monomer, for example, a mixture of at least two or at least three epoxide monomers.The at least one epoxide monomer (or mixture of epoxide monomers) may be selected from the group consisting of epichlorohydrin, epibromohydrin, isopropyl glycidyl ether, butyl glycidyl ether, allyl glycidyl ether, 1,4-butanediol diglycidyl ether (1,4-bis(2,3-epoxypropoxy)butane), neopentyl glycol diglycidyl ether, ethylene glycol diglycidyl ether, glycerin diglycidyl ether, glycidol, and glycidyl methacrylate, ethylhexyl glycidyl ether, methyl glycidyl ether, glycerol propoxylate triglycidyl ether, poly(propylene glycol) diglycidyl ether, 1,3 butylene diol diglycidyl ether, tert butyl glycidyl ether, 1,4 cyclohexanedimethanol diglycidyl ether, diethylene glycol diglycidyl ether, dodecyl glycidyl ether, O-(2,3 epoxypropyl)-O'-methyl polyethylene glycol glycidyl ether, glycidyl tetrafluoroethyl ether, 1,6 hexanediol diglycidyl ether, octyl glycidyl ether, decyl glycidyl ether, poly(epichlorohydrin-co-ethylene oxide-co-allyl glycidyl ether), polyethylene glycol diglycidyl ether, trimethylolethane triglycidyl ether, trimethylolpropane triglycidyl ether, tert-butyldimethylsilyl glycidyl ether, 1,2-epoxybutane, 1,2-epoxypentane, 1,2-epoxy-5-hexene, 1,2-epoxy-hexane, 1,2-epoxy-7-octene, 1,2-epoxyoctane, 1,2,7,8-diepoxyoctane, 1,2-epoxy-9-decene, 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, or a combination thereof. The at least one epoxide monomer (or mixture of epoxide monomers) may be selected from 1,4-butanediol diglycidyl ether, glycidol, glycidyl methacrylate, or combinations thereof.

[0129] In one embodiment, the coating comprises silica. The silica coating can be formed by reacting a silicate (e.g., Na2SiO3) in the presence of polymer particles at a pH of less than about 11. The silica coating can be formed by reacting an orthosilicate (e.g., tetraethylorthosilicate) in the presence of polymer particles at a temperature of at least 80°C (e.g., a temperature of at least 90°C or 100°C).

[0130] In one embodiment, the magnetic and / or coated particles can be coupled to affinity ligands, the nature of which will be selected based on their affinity for the specific analyte whose presence or absence in the sample is to be confirmed. Affinity ligands can include any molecule that can be linked to a particle and that is also capable of specific recognition of a particular analyte. Affinity ligands include monoclonal antibodies, polyclonal antibodies, antibody fragments, nucleic acids, oligonucleotides, proteins, oligopeptides, polysaccharides, sugars, peptides, nucleic acid molecules encoding peptides, antigens, drugs, and other ligands. Examples of suitable affinity ligands are available and well known in the published literature. The use of additional binding partners, secondary affinity ligands, and linking groups, which are routine in the art, will not be discussed further herein, although such species can be used with the particles of the present invention if desired.

[0131] The particles of the present invention can be stable in aqueous solution (i.e., can be resistant to polymer degradation) over the temperature range typical of aqueous solutions. Monodisperse crosslinked hydrogel polymer particles can be stable at temperatures up to about 100°C. For example, monodisperse crosslinked hydrogel polymer particles can be stable within a temperature range of about 0°C to about 100°C.

[0132] seed particles Polymer particles can be prepared by a polymer particle forming process that starts with specific seed particles.The applicant has found that the seed particles typically used in other processes, such as polystyrene seed particles, are not compatible with the present invention and the process of the present disclosure.Therefore, the present disclosure provides seed particles that are suitable for use in the present invention and the polymer particle forming process of the present disclosure.

[0133] The seed particles can be (and preferably are) monodisperse. The seed particles comprise multiple non-crosslinked oligomers of poly N,N-dimethylacrylamide and a z-average diameter of 100 nm to 1,500 nm. Each seed particle contains 1 x 10 6 mers of poly N,N-dimethylacrylamide. 5 Super (e.g. 1×10 6 For example, each seed particle may contain 5×10 of poly N,N-dimethylacrylamide. 6 The polymer may contain more than one non-crosslinked oligomer.

[0134] The seed particles may have a z-average diameter of 150 nm to 1,300 nm. For example, the seed particles may have a z-average diameter of 300 nm to 1,100 nm.

[0135] The oligomers have a weight average molecular weight (Mw) of about 2,000 Da to about 100,000 Da as measured by GPC against polystyrene standards. In addition to or alternative to the specified Mw ranges, each oligomeric polymer may contain about 30 to 2,000 (e.g., about 60 to about 1,000, or about 80 to about 500) monomer units.

[0136] The Mw of the seed particle oligomer can be less than 100,000 Da, optionally less than 50,000 Da, and even optionally less than 40,000 Da, for example, less than 30,000 Da. Mw can be greater than 4,000 Da, optionally greater than 5,000 Da, and even optionally greater than 6,000 Da, such as greater than 8,000 Da, for example, greater than 10,000 Da. For example, Mw can be between 5,000 Da and 70,000 Da, for example, between 6,000 Da and 60,000 Da, for example, between 7,000 Da and 50,000 Da, or between 8,000 Da and 40,000 Da.

[0137] Without being bound by any theory, the Mw of the oligomer of seed particle is believed to be an important feature of seed particle.For example, in order to provide the seed particle that forms monodisperse polymer particles during the particle formation process described herein, it may be necessary to have the Mw of the oligomer of seed particle within a certain range.If the seed particle polymer has a higher molecular weight, for example, because no chain transfer agent is used during seed particle formation (for example, the monodisperse microspheres of M.Babic and D.Horak, Macromolecular Reaction Engineering, 2007,1,86-94), the particles obtained from this process may be monodisperse, for example, CV may be greater than 20%, and therefore the particles are not suitable for use in this process.

[0138] The seed particles can have the characteristics of seed particles formed according to the methods of forming seed particles disclosed herein, for example, the seed particles can have the characteristics of seed particles formed by radical-initiated polymerization of N,N-dimethylacrylamide in an organic solvent in the presence of a stabilizer and a chain transfer agent.

[0139] Preparation of seed particles The polymer particles can be prepared by the processes described herein starting from specific seed particles. Thus, the present disclosure provides a method for preparing monodisperse seed particles.

[0140] Monodisperse seed particles can be made by a method including dissolving N,N-dimethylacrylamide, a stabilizer, a radical initiator, and a chain transfer agent in an organic solvent to form a reaction mixture, and heating the reaction mixture to activate the initiator, thereby forming monodisperse seed particles.

[0141] In this method, once the reaction mixture is formed in a suitable vessel, the reaction mixture is typically mixed, for example with a stirrer, during the heating step.

[0142] Preferably, the polymerization reaction will occur in a reaction mixture containing a minimum amount of oxygen. Therefore, before heating the reaction mixture, the reaction mixture can be purged from oxygen. Purging can include sparging with a chemically inert gas. The inert gas can be nitrogen or a noble gas (e.g., helium, neon, argon, krypton, or xenon). For example, the inert gas can be argon, helium, or nitrogen, such as argon or nitrogen.

[0143] This process can be considered a reverse precipitation polymerization in which hydrophilic seed particles precipitate from an organic solvent. The stabilizer can prevent seed particle aggregation and aid in the formation of monodisperse seed particles. The selection of a suitable stabilizer depends on the stabilizer's ability to dissolve in the organic solvent. The stabilizer can be a block copolymer of styrene and a polyolefin, such as a triblock copolymer based on styrene and ethylene / butylene, such as a linear triblock copolymer of styrene and ethylene / butylene. The stabilizer can be a triblock copolymer consisting of a poly(ethylene-co-butylene) midblock and polystyrene outer blocks. Exemplary stabilizers include Kraton A1535H, Kraton G1650M, Kraton G1652M, or Kraton G1657M, or a combination thereof.

[0144] The radical initiator can be a peroxide initiator or an azo initiator, for example, one that decomposes at elevated temperatures. Exemplary radical initiators include 2,2'-azobis(2-methylpropionitrile), 2,2'-azodi(2-methylbutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), dibenzoyl peroxide, and the like. The radical initiator can be 2,2'-azobis(2-methylpropionitrile). The radical initiator can be 2,2'-azodi(2-methylbutyronitrile). The temperature to which the reaction mixture can be heated will depend on the temperature to which the radical initiator is activated. Heating the reaction mixture to activate the initiator can include heating the reaction mixture to a temperature of at least 40°C, for example, to a temperature of at least 50°C, for example, to a temperature of at least 60°C, or for example, to a temperature of at least 70°C. For example, when the radical initiator is 2,2'-azobis(2-methylpropionitrile), the reaction mixture can be heated to a temperature of at least 50°C or at least 60°C.

[0145] The addition of a chain transfer agent reduces the molecular weight of the seed particle polymer by reacting with the free radicals in the growing polymer chains, terminating the chains and transferring lone electrons to radical species derived from the chain transfer agent. The radical species derived from the chain transfer agent then reacts with the monomer to form a radical from the monomer, which can then react with another monomer to initiate the formation of a new polymer chain. The chain transfer agent can be a thiol or a haloalkane. For example, the chain transfer agent can be selected from thiols (e.g., 1-octanethiol, hexanethiol, 6-mercapto-1-hexanol, benzylthiol), alkylthiols (e.g., 1-octanethiol, hexanethiol), carbon tetrachloride, and bromotrichloromethane. For example, the chain transfer agent can be 1-octanethiol. The total amount of chain transfer agent added can be in the range of 1 mole per 10 moles of monomer to 1 mole per 300 moles of monomer, for example, 1 mole per 20 moles of monomer to 1 mole per 100 moles of monomer, for example, about 1 mole of chain transfer agent per 30 moles of monomer. The addition time of the chain transfer agent is important to obtain monodisperse seed particles: the chain transfer agent should be present in the reaction mixture before the start of polymerization (i.e., before activation of the radical initiator). This finding is surprising, as for seed particles (e.g., polystyrene seed particles) used in the conventional Ugelstad process, if a chain transfer agent is added, it should be added after the start of particle formation, as taught in WO 2010 / 125170, the contents of which are incorporated herein by reference.

[0146] The organic solvent may comprise a mixture of an alkane component and an aromatic component. The organic solvent may comprise a single component, such as an alkane component or an aromatic component. The alkane component may be or may comprise hexane, heptane, or octane. The aromatic component may be C1-C 10 It may be or include an alkyl-substituted phenyl or a C1-C8 dialkyl-substituted phenyl, such as a C1-C4 alkyl-substituted phenyl, such as toluene. When the organic solvent includes a single component and that component is an aromatic component, the aromatic component may be a C4-C12 The alkane component may be an alkyl-substituted phenyl or a C2-C8 dialkyl-substituted phenyl. The alkane component may be heptane, and the aromatic component may be toluene. The alkane component and the aromatic component may be present in a weight ratio of alkane component:aromatic component of between about 0.5:1 and about 20:1, for example, the weight ratio of alkane component:aromatic component may be between about 1:1 and about 15:1, for example, between about 1:1 and about 10:1.

[0147] The reaction mixture can include N,N-dimethylacrylamide in an amount of about 2% to about 5% by weight, a stabilizer in an amount of about 1% to about 5% by weight, a radical initiator in an amount of about 0.01% to about 4% by weight (e.g., about 0.05% to about 0.25% by weight), and a chain transfer agent in an amount of about 0.05% to about 0.25% by weight.

[0148] The monodisperse seed particles may then be subjected to a particle formation process, for example as outlined below.

[0149] Particle preparation The present disclosure provides methods for forming monodisperse crosslinked hydrogel polymer particles. For example, the Ugelstad process requires that particles be formed in the oil phase of an oil-in-water emulsion, while the hydrogel polymer particles and corresponding hydrophilic monomers are (log P and / or R 原子 The Ugelstad process cannot be used to directly form such particles because the polymer may be preferentially soluble in water, as provided by (Illegible Text). Applicant has addressed this by providing the method of the present disclosure, which provides a particle formation process using a water (discontinuous phase) system in an oil (continuous phase) system. This is believed to represent the first such process that has been used to form monodisperse crosslinked polymer particles.

[0150] The present disclosure provides a method for forming monodisperse crosslinked hydrogel polymer particles. The method includes the steps of: forming a solution (a) of at least 2% by weight of a hydrophilic vinyl monomer in an aqueous solution, the aqueous solution also containing a crosslinker containing at least two vinyl groups; forming a solution (b) of a stabilizer in an organic solvent, the organic solvent being immiscible in water and at least one of solutions (a) and (b) containing a radical initiator; mixing solutions (a) and (b) to form a water-in-oil emulsion (c); adding monodisperse seed particles to the emulsion; allowing the monodisperse seed particles to form swollen particles in the emulsion; and polymerizing the swollen particles to form monodisperse crosslinked hydrogel polymer particles. As described below, this method can be considered a single-stage method because it includes a single swelling step and a single polymerization step.

[0151] Solution (a) can be formed by first forming a solution of at least 2% by weight of the hydrophilic vinyl monomer in an aqueous solution and then adding the crosslinking agent, which can be added before the hydrophilic vinyl monomer, or the hydrophilic vinyl monomer and crosslinking agent can be added to the solution at about the same time. Similarly, the stabilizer and radical initiator can be added to solution (b) at about the same time or sequentially.

[0152] In the context of this disclosure, an organic solvent is considered to be immiscible with water if, when water is mixed with the organic solvent in an amount of at least 5% by weight, the organic solvent and water separate into two separate phases.

[0153] The method involves swelling seed particles having an aqueous discontinuous phase in an oil continuous phase. Schematic diagrams of this process, which can be considered a one-step process because it involves a single swelling step and a single polymerization step, are provided in Figure 1 (shown with specific monomers) and Figure 2 (shown more generally). The method shown in Figures 1 and 2 is conveniently divided into two steps: swelling monodisperse seed particles 100 to form swollen seed particles 110, and polymerizing the monomers in the swollen seed particles to form crosslinked hydrogel polymer particles 120. Prior to the first step, a water-in-oil emulsion is formed. The water-in-oil emulsion is formed by mixing an aqueous solution containing the exemplary monomer acrylamide 101 and the exemplary crosslinker 1,2-dihydroxybisacrylamide 102 with an oil phase containing a steric stabilizer, and the emulsion is formed when the aqueous and oil phases are agitated (e.g., by stirring). If highly crosslinked particles are desired, the monomer can be replaced with a crosslinker, so that the only monomer present in the water-in-oil emulsion is the crosslinking monomer (i.e., the crosslinker, e.g., 1,2-dihydroxybisacrylamide 102). The water-in-oil emulsion also typically contains an initiator, which may be added to the oil phase. The initiator is a compound that, upon activation, initiates polymerization of the monomer and crosslinker. The monodisperse seed particles 100 comprise uncrosslinked oligomers of poly N,N-dimethylacrylamide 131 and are considered "activated seed particles." The seed particles 100 can be prepared as described elsewhere in this application. In a first step, the monodisperse seed particles 100 are added to the water-in-oil emulsion, and the emulsion is stirred for a period of time (e.g., at least 30 minutes or at least 1 hour, typically 4 to 48 hours). During this period, the monomer 101 and crosslinker 102 diffuse into the activated seed particles 100 to form swollen seed particles 110. The swollen seed particle 110 comprises a mixture of at least the monomer 101, the crosslinker 102, and the polymer from the activated seed particle 100. The swollen seed particle 110 may also contain other components, such as one or more porogens, which may become incorporated into the particle when contained in a water-in-oil emulsion.For example, in the illustrated method, water is also present in the swollen seed particles 110, and this water can be considered a porogen. The second step involves polymerizing the monomers 101 and crosslinkers 102 to form crosslinked hydrogel polymer particles 120. In the second step, polymerization is initiated by activating an initiator, for example, by heating the emulsion.

[0154] The provision of activated seed particles is a key feature of a successful Ugelstad process. The activation of the seed particles is typically provided by adding an organic compound with very low solubility as an emulsion to produce entropically activated seed particles. However, the applicant has surprisingly determined that this additional activation step is not necessary in this process, for example, when the seed particles comprise oligomers of poly(N,N-dimethylacrylamide).

[0155] Swelling is important to the particle formation process. This process requires that the monomer and (if present) porogen have limited solubility in the continuous phase. If the monomer and porogen have too high a solubility in the continuous phase, the solubility will not be sufficient to facilitate the monomer (and any porogen) entering the seed and forming swollen particles. On the other hand, if the monomer (and any porogen) has insufficient solubility in the continuous phase, there will be negligible diffusion through the continuous phase, and therefore, mass transport of the monomer to the seed will be impossible, preventing the formation of swollen particles. Solubility can be conveniently measured using log P. The time to complete swelling, i.e., the time until all the monomer (and any porogen) is colocalized on the seed particle, is highly dependent on factors such as temperature, solubility, and viscosity, and will therefore vary from system to system. Therefore, typical timescales for the step of causing monodisperse seed particles to form swollen particles will vary from 30 minutes to 48 hours. For example, the step of swelling monodisperse seed particles may be carried out for at least 30 minutes, e.g., at least 1 hour. The step of swelling the monodisperse seed particles may be carried out for at least 4 hours, at least 8 hours, or at least 12 hours. The upper limit of the swelling time is not believed to be critical, and it is believed that swelling times of several days, e.g., 3 days or 2 days, may be used.

[0156] The step of forming swollen particles from the monodisperse seed particles can include mixing the emulsion, for example, mixing the emulsion for substantially the entire swelling time. The step of forming swollen particles from the monodisperse seed particles can be carried out at a temperature of about 10°C to about 60°C, for example, about 10°C to about 40°C, for example, about 15°C to about 30°C. For example, the mixing can be carried out at a temperature of about 10°C to about 60°C, for example, about 10°C to about 40°C, for example, about 15°C to about 30°C.

[0157] Solution (a) comprises at least 2 wt. % of a hydrophilic vinyl monomer in aqueous solution. The aqueous solution may be water. The aqueous solution may comprise water and up to 50 wt. % (e.g., up to 30 wt. % or up to 25 wt. %, e.g., up to 20 wt. %, optionally up to 10 wt. %) of a water-miscible organic solvent. The water-miscible organic solvent may be a C1-C4 alcohol, e.g., ethanol or methanol, e.g., methanol. The water-miscible organic solvent may be a C2-C4 nitrile, e.g., acetonitrile.

[0158] The hydrophilic vinyl monomer may include an optionally substituted generalized vinyl group, -CR=CR'H, where R is H or alkyl (e.g., R is -CH3 or -CH2CH3), and R' is H, alkyl (e.g., -CH3 or -CH2CH3), or a functional group (e.g., a carboxylic acid, a phosphonic acid, a primary amine, or a secondary amine). The hydrophilic vinyl monomer may include a generalized vinyl group, -CR=CH2, where R is alkyl (e.g., R is -CH3 or -CH2CH3). The hydrophilic vinyl monomer may include a vinyl group, -CH=CH2. The hydrophilic vinyl monomer may be a vinyl monomer having a log P value of less than about 1, for example, a log P of less than about 0.5. The monomer may be a vinyl monomer having a log P of less than about 0.6 (e.g., less than about 0.52). The monomer may be a vinyl monomer having a log P of less than about 0.5. For example, the monomer can be a vinyl monomer having a log P of less than about 0.3 or less than about 0.2, e.g., the monomer can be a vinyl monomer having a log P of less than about 0.1. The monomer can be a vinyl monomer having a log P of less than about 0, e.g., less than about -0.2. The monomer can be a vinyl monomer having a log P of greater than about -3.8, e.g., greater than about -3 or about -2. The monomer can be a vinyl monomer having a log P of 0.6 to -3. The monomer can be a vinyl monomer having a log P of 0.5 to -2, e.g., a log P of 0 to -2, e.g., a log P of -0.2 to -2. In particular, the monomer can be a vinyl monomer that also contains a hydrophilic group, e.g., an acrylamide monomer or an acrylate monomer.

[0159] The monomer is R 原子 When the monomers are acrylamide, vinylimidazole, acryloylmorpholine, vinylphosphonic acid, and vinylsulfonic acid, specific values ​​for R 原子 is R 原子 If the monomer is an acrylate of another monomer class, the specific R 原子 is R 原子may be 2 or less, and the monomer has a water solubility (eg, at 25° C.) of 50 g / L or more.

[0160] The monomer used in the method may be at least one compound of formula (I), wherein R 1 is -H, -CH3, -CH2CH3, or -CH2C(O)OH; R 1a is -H, -CH3, -CH2CH3, -CH2C(O)OH, or -C(O)R 2 and R 1b is -C(O)R 2 is -P(O)(OH)2, is -S(O)2OH, or is a heterocycle having 3 to 8 ring atoms wherein the heteroatoms are selected from N, O, or S; or R 1a or R 1 R combined with 1b is -C(O)-OC(O)-, R 2 -OR 3 , -N(R 4 )R 5 or an -N-linked amino acid; R 3 is selected from -H, -C1-C6 alkyl, or -C1-C6 alcohol; and R 4 and R 5 are each independently selected from -H, -C1-C6 alkyl, -C1-C6 haloalkyl, and -C1-C6 alcohol; or R 4 and R 5 together with the nitrogen to which they are attached form a heterocycle having 3 to 8 ring atoms, the heteroatoms being selected from N, O, or S.

[0161] The monomer used in the method can be at least one compound of formula (I), wherein R 1 is -H, -CH3, -CH2CH3, or -CH2C(O)OH, and R 1a is -H or -C(O)R 2 and R1b is -C(O)R 2 , -P(O)(OH)2, or -S(O)2OH; or R 1a Or R 1 R combined with 1b is -C(O)-OC(O)- and R 2 -OR 3 or -N(R 4 )R 5 and R 3 is -H, -C1-C6 alkyl, or -C1-C6 alcohol, and R 4 and R 5 are each independently selected from -H, -C1-C6 alkyl, -C1-C6 haloalkyl, and -C1-C6 alcohol.

[0162] R 3 is -C1-C6 alkyl or -C1-C6 alcohol, the alkyl or alcohol may each independently be oxo, =NR a , =NOR a , Halo, Nitro, Cyano, NR a R a , N.R. a S(O)2R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , S.R. a , S(O)R a , S(O)2OR a , S(O)2R a , S(O)NR a R a , CO2R a C(O)R a ,CONR a R a , C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4 haloalkyl, and R a is independently selected at each occurrence from H and C1-C4 alkyl. For example, R3 is -C1-C6 alkyl or -C1-C6 alcohol, the alkyl or alcohol may each independently be, if chemically possible, OR a or CO2R a and optionally R a is H.

[0163] R 4 and / or R 5 When each of the groups is -C1-C6 alkyl, -C1-C6 haloalkyl, or -C1-C6 alcohol, each of the groups independently represents, if chemically possible, oxo, =NR a , =NOR a , Halo, Nitro, Cyano, NR a R a , N.R. a S(O)2R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , S.R. a , S(O)R a , S(O)2OR a , S(O)2R a , S(O)NR a R a , CO2R a C(O)R a ,CONR a R a , C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4 haloalkyl, and R a is independently selected at each occurrence from H and C1-C4 alkyl. For example, R 4 and / or R 5 When is -C1-C6 alkyl, -C1-C6 haloalkyl, or -C1-C6 alcohol, each independently represents, if chemically possible, OR at each occurrence. a or CO2R aand optionally R a is H.

[0164] R 1 R can be -H, -CH, or -CHCH. 1 can be -H or -CH3. For example, R 1 can be -H. 1 can be —CHC(O)OH.

[0165] R 1a R can be -H, -CH, or -CHCH. 1a can be -H. 1a is -C(O)R 2 It can be. R 1a can be —CHC(O)OH.

[0166] R 1b -C(O)R 2 It can be. R 1b can be -P(O)(OH). R 1b can be -S(O)2OH. 1b -C(O)R 2 When R 1a can be -H. 1b -C(O)R 2 When R 1a -C(O)R 2 R 2 is optional -OR 3 It can be. R 1b When is -P(O)(OH)2, R 1a can be -H, R 1 can be -H, -CH, or -CHCH (e.g., -H). 1b When is -S(O)2OH, R 1a can be -H, R 1 can be -H, -CH3, or -CH2CH3 (e.g., -H).

[0167] R 1bR can be a heterocycle having 3 to 8 ring atoms, the heteroatoms being selected from N, O, or S. 1b can be imidazolyl.

[0168] R 1a or R 1 R combined with 1b can be —C(O)—OC(O)—.

[0169] R 2 HA-OR 3 It can be. R 2 -N(R 4 )R 5 It can be. R 2 can be -NH, -NHCH, -NHCHCH, or -NH(CH). 2 can be an N-linked amino acid. The N-linked amino acid can be selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, β-alanine, and methionine.

[0170] R 3 can be -H. R 3 can be -C1-C6 alkyl. For example, R 3 each independently, at each occurrence, is selected from oxo, halo, cyano, NR a R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , CO2R a C(O)R a ,CONR a R a , C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl; R a is independently, at each occurrence, selected from H and C1-C4 alkyl, e.g., R a R can be -H, -C1-C6 alkyl substituted with one or two substituents.3 can be a -C1 to C6 alcohol. For example, R 3 can be a —C1-C6 alcohol substituted with one or two substituents, each of which independently at each occurrence is selected from oxo, halo, cyano, NR a R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , CO2R a C(O)R a ,CONR a R a , C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl; R a is independently selected at each occurrence from H and C1-C4 alkyl, e.g., R a is -H.

[0171] R 4 can be -H or -C1-C6 alkyl. 4 can be -C1-C6 alkyl. For example, R 4 each independently represent, at each occurrence, oxo, halo, cyano, NR a R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , CO2R a C(O)R a ,CONR a R a , C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, and R a is independently selected at each occurrence from H and C1-C4 alkyl, e.g., R a is -H.

[0172] R 5can be -H or -C1-C6 alkyl. 5 can be -C1-C6 alkyl. For example, R 5 each independently represent, at each occurrence, oxo, halo, cyano, NR a R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , CO2R a C(O)R a ,CONR a R a , C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, and R a is independently selected at each occurrence from H and C1-C4 alkyl, e.g., R a is -H.

[0173] R 4 and R 5 R can, together with the nitrogen to which they are attached, form a heterocycle having 3 to 8 ring atoms (e.g., 4, 5, or 6 ring atoms) in which the heteroatoms are selected from N, O, or S. 4 and R 5 can be taken together with the nitrogen to which they are attached to form a morpholino heterocycle.

[0174] The monomer used in the method can be at least one compound of formula (I), (Ia), (Ib), or (1c).

[0175] A compound of Formula (I), (Ia), (Ib), or (Ic) may have a log P value of less than about 1, for example, a log P of less than about 0.5. A compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of less than about 0.6 (e.g., less than about 0.52). A compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of less than about 0.5. For example, a compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of less than about 0.3 or less than about 0.2, for example, a compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of less than about 0.1. A compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of less than about 0, for example, a log P of less than about -0.2. A compound of Formula (I), (Ia), (Ib), or (Ic) may have a log P of greater than about -3.8, for example, a log P of greater than about -3 or about -2. A compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of 0.6 to -3. A compound of Formula (I), (Ia), (Ib), or (1c) may have a log P of 0.5 to -2, for example, a log P of 0 to -2, for example, a log P of -0.2 to -2.

[0176] When the compound of formula (I) or (Ia) is acrylamide, vinylimidazole, acryloylmorpholine, vinylphosphonic acid, or vinylsulfonic acid, the compound has an R 原子 or when the compound is an acrylate or other monomer class, may have 2 or less R 原子 and a water solubility of 50 g / L or greater. The compounds of formula (Ib) or (Ic) may have an R 原子 may have:

[0177] The monomers may include at least one hydrophilic vinyl monomer containing an amide group selected from -C(O)NH, -C(O)NHCH, -C(O)NHCHCH, or -C(O)N(CH). The monomers may include at least one hydrophilic vinyl monomer containing a primary amide group (-C(O)NH).

[0178] Acrylamide monomers and / or acrylic monomers may be specifically mentioned. Suitable monomers include acrylamide (prop-2-enamide), N-(hydroxymethyl)acrylamide, N-hydroxyethylacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, 4-acrylomorpholine, 3-acrylamidopropanoic acid, methacrylamide, N-(2-hydroxyethyl)methacrylamide, N-(3-aminopropyl)methacrylamide, hydroxypropylacrylamide, N,N-dimethylacrylamide, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, acrylic acid; other acrylamide monomers, acrylic monomers, methacrylamide monomers, or methacrylic monomers having a log P value of less than about 1 (e.g., having a log P value of less than about 0.5); and other acrylamide monomers, acrylic monomers, methacrylamide monomers, or methacrylic monomers having an R of less than 2.75. 原子 other acrylic or methacrylamide monomers having R 原子 and other acrylic or methacrylic acid monomers having a water solubility (eg, at 25° C.) of 50 g / L or greater.

[0179] Solution (a) may contain 60% by weight or less of hydrophilic vinyl monomer. For example, solution (a) may contain 55% by weight or less or 50% by weight or less of hydrophilic vinyl monomer. For example, solution (a) may contain 45% by weight or less or 40% by weight or less of hydrophilic vinyl monomer, for example, solution (a) may contain 30% by weight or less of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight of hydrophilic vinyl monomer. Solution (a) may contain at least 5% by weight of hydrophilic vinyl monomer. Solution (a) may contain at least 8% by weight of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 60% by weight or less of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% by weight of hydrophilic vinyl monomer and 60% by weight or less of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 50% by weight or less of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% by weight of hydrophilic vinyl monomer and 50% by weight or less of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 45% by weight or less of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% by weight of hydrophilic vinyl monomer and 45% by weight or less of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 30% by weight or less of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% by weight of hydrophilic vinyl monomer and 30% by weight or less of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and no more than 15% of hydrophilic vinyl monomer, for example, solution (a) may contain about 10% by weight of hydrophilic vinyl monomer.

[0180] The hydrophilic vinyl monomer may comprise a mixture of monomers. For example, the monomer may comprise at least one monomer defined above and at least one compatible functional monomer. Exemplary functional monomers are hydrophilic vinyl monomers as defined herein, including carboxylic acid (-COOH), phosphonic acid (-P(O)(OH)), sulfonic acid (-S(O)OH), primary amine, or secondary amine. For example, the monomers can include at least one monomer of formula (I), (Ia), or (Ib) that does not include a carboxylic acid (-COOH), phosphonic acid (-P(O)(OH)), sulfonic acid (-S(O)OH), primary amine, or secondary amine, and a monomer of formula (I), (Ia), (Ib), or (Ic) that includes at least one carboxylic acid (-COOH), phosphonic acid (-P(O)(OH)), sulfonic acid (-S(O)OH), primary amine, or secondary amine (e.g., includes at least one carboxylic acid (-COOH) or phosphonic acid (-P(O)(OH))).

[0181] The functional monomer can be a vinyl monomer having a log P value of less than about 1 (e.g., less than about 0.5), which includes a carboxylic acid or a primary amine. The functional monomer can be a vinyl monomer having a log P of less than about 0.6. The functional monomer can be a vinyl monomer having a log P of less than about 0.52. The functional monomer can be a vinyl monomer having a log P of less than about 0.5. For example, the functional monomer can be a vinyl monomer having a log P of less than about 0.3 or less than about 0.2, e.g., the functional monomer can be a vinyl monomer having a log P of less than about 0.1. The functional monomer can be a vinyl monomer having a log P of less than about 0, e.g., less than about -0.2. The functional monomer can be a vinyl monomer having a log P of greater than about -3.8, e.g., greater than about -3 or about -2. The functional monomer can be a vinyl monomer having a log P of 0.6 to -3. The functional monomer can be a vinyl monomer having a log P of 0.5 to -2, e.g., a log P of 0 to -2, e.g., a log P of -0.2 to -2. The functional monomer can be a compound of formula (I), (Ia), or (Ib) containing a carboxylic acid or a primary amine. The functional monomer can be an acrylamide monomer containing a carboxylic acid, a phosphonic acid, a sulfonic acid, or a primary amine. The functional monomer can contain a carboxylic acid, a phosphonic acid, or a sulfonic acid, e.g., a carboxylic acid or a phosphonic acid. Suitable functional monomers include 3-acrylamidopropionic acid, 4-acrylamidobutanoic acid, 5-acrylamidopentanoic acid, N-(3-aminopropyl)methacrylamide, acrylic acid, fumaric acid, itaconic acid, vinylphosphonic acid, vinylsulfonic acid, and 1-vinylimidazole. Additional suitable functional monomers include anhydrides, e.g., maleic anhydride or itaconic anhydride, which can be hydrolyzed to provide a carboxylic acid group after polymerization.

[0182] When at least one functional monomer is present, the amount of functional monomer can be about 0.1 to about 100 mol%, for example, about 0.2 to about 50 mol%, for example, about 0.5 to about 40 mol%, or about 1 to about 30 mol% (e.g., about 2 to about 20 mol%). For example, the amount of functional monomer can be at least about 2 to about 100 mol%, for example, about 2 to about 50 mol%, for example, about 4 to about 40 mol%, or about 4 to about 30 mol% (e.g., about 2 to about 20 mol%). The amount of functional monomer can be about 5 to about 100 mol%, for example, about 5 to about 80 mol%. Molar % can refer to the amount of functional monomer as a mole % of the total hydrophilic vinyl monomer. The amount of functional monomer can be about 0.1 to about 60 mol%, for example, about 0.2 to about 50 mol%, for example, about 0.5 to about 40 mol%. The amount of functional monomer can be about 1 to about 60% by weight, for example, about 2 to about 50% by weight, for example, about 2 or 3 to about 40% by weight. The amount of functional monomer can be about 0.1 to about 10% by weight, for example, about 0.2 to about 5% by weight, for example, about 0.5 to about 2% by weight. The mole percent can refer to the mole percent of functional monomer contained in the total hydrophilic vinyl monomer in solution (a) (i.e., a solution containing at least 2% by weight of hydrophilic vinyl monomer in water).

[0183] In highly crosslinked particles, the monomer may be or include a crosslinker, for example, the monomer may be or include at least one compound of formula (IIa) or (IIb).

[0184] The crosslinker used in this method can include at least two (e.g., two) vinyl groups (-CH=CH2). The crosslinker used in this method can be at least one of a compound of formula (IIa) or formula (IIb), where R 6 is selected from -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 hydroxyalkyl-, -C1-C6 ether-, or a polyether, the polyether containing 2 to 100 C2-C3 ether units, and R 7 and R 8are each independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, -C3-C6 cycloalkyl, -C1-C6 hydroxyalkyl, or -C1-C6 ether; or R 7 and R 8 are linked together to form -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 hydroxyalkyl-, -C1-C6 ether-, a polyether containing 2 to 100 C2-C3 ether units; R 9 is -N(R 11 )C(O)CH=CH2, and R 10 is -H and -N(R 12 )C(O)CH=CH2, and R 11 and R 12 are each independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, -C3-C6 cycloalkyl, -C1-C6 hydroxyalkyl, or -C1-C6 ether.

[0185] The cross-linking agent may be at least one compound of formula (IIa): The cross-linking agent may be at least one compound of formula (IIb):

[0186] R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are each independently, when chemically possible, oxo, =NR a , =NOR a , Halo, Nitro, Cyano, NR a R a , N.R. a S(O)2R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , S.R. a , S(O)R a , S(O)2ORa , S(O)2R a , S(O)NR a R a , CO2R a C(O)R a ,CONR a R a , C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4 haloalkyl; R a is independently selected at each occurrence from H, C1-C4 alkyl, and C1-C4 alkenyl.

[0187] R 6 R may be selected from -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 hydroxyalkyl-, and -C1-C6 ether-. 6 R may be selected from -C1-C6 alkyl- and -C1-C6 hydroxyalkyl-. 6 R can be -C1-C6 alkyl-, for example, -CH2-, -(CH2)2-, -(CH2)3-, or -(CH2)4-, for example, -(CH2)2-. 6 may be —C1-C6 hydroxyalkyl-, for example, —C(OH)H—, —(C(OH)H)2-, —(C(OH)H)3-, or —(C(OH)H)4-, for example, —(C(OH)H)2-.

[0188] R 6 can be -C1-C6 heteroalkyl-, where optionally the heteroatom is an amine (e.g., a tertiary amine). For example, -C1-C6 heteroalkyl- can be -C(O)R on the heteroatom. a and optionally the heteroatom is an amine, e.g., R 6 can be —CH2CH2N(C(O)CH═CH2)CH2CH2—.

[0189] R 6 When R is a polyether, the polyether may be linear or branched. 6R may be a polyether containing 2 to 100 C2-C3 ether units, for example, a polyether containing 2 to 50 C2-C3 ether units. 6 can be a polyether containing 2 to 100 C2 ether units, for example, a polyether containing 2 to 50 C2 ether units. For example, R 6 is -(CH2) r (OCH2CH2) n O(CH2) s where r and s are each independently 2 or 3 (e.g., 2), and n is an integer from 1 to 100 (e.g., 5 to 50 or 5 to 25). Without wishing to be bound by any theory, it is believed that crosslinkers comprising polyethers (e.g., R 6 is a polyether) is believed to have excellent solubility in the aqueous phase. This means that such crosslinkers can be used to provide particles with low levels of crosslinking (e.g., 1-20 wt. % crosslinker, or 1-10 wt. % crosslinker), and such polyethers containing crosslinkers are particularly suitable for providing particles with relatively high levels of crosslinking, e.g., greater than 20 wt. % crosslinker, greater than 25 wt. % crosslinker, or greater than 30 wt. % crosslinker. For example, the level of crosslinking can be 10-90 wt. % crosslinker, 20-80 wt. % crosslinker, or 25-75 wt. % crosslinker, e.g., 25-60 wt. % crosslinker, or 30-50 wt. % crosslinker.

[0190] R 7 and / or R 8 and / or R 11 and / or R 12 can be H. For example, R 7 and / or R 8 can be H. For example, R 11 and / or R 12 can be H.

[0191] R 7 and R 8may be linked together to form -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 hydroxyalkyl-, -C1-C6 ether-, polyethers, the polyethers containing 2 to 100 C2-C3 ether units. 7 and R 8 are bonded together to form -C1-C6 alkyl-, R 7 and R 8 The group defined by R 6 For example, R 6 can be -C1-C6 alkyl-, and R 7 and R 8 can be -C1-C6 alkyl-.

[0192] R 6 may be selected from -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 ether-, polyethers, where the polyethers contain from 2 to 100 C2-C3 ether units; R 7 and R 8 may each independently be selected from —H, —C1-C6 alkyl, —C1-C6 heteroalkyl, —C3-C6 cycloalkyl, or —C1-C6 ether; or R 7 and R 8 are linked together to form a -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 ether-, polyether, the polyether containing 2 to 100 C2-C3 ether units, and R 11 and R 12 are each independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, -C3-C6 cycloalkyl, or -C1-C6 ether.

[0193] The compound of Formula (IIa) or (IIb) may have a log P value of less than about 1, for example, less than about 0.5. The compound of Formula (IIa) or (IIb) may have a log P of less than about 0.6. The compound of Formula (IIa) or (IIb) may have a log P of less than about 0.5. For example, the compound of Formula (IIa) or (IIb) may have a log P of less than about 0.3 or less than about 0.2, for example, the compound of Formula (IIa) or (IIb) may have a log P of less than about 0.1. The compound of Formula (IIa) or (IIb) may have a log P of less than about 0, for example, less than about -0.2. The compound of Formula (IIa) or (IIb) may have a log P of greater than -3.8, for example, greater than about -3 or about -2. The compound of Formula (IIa) or (IIb) may have a log P of 0.6 to -3. The compound of formula (IIa) or (IIb) may have a log P of 0.5 to -2, such as a log P of 0 to -2, for example a log P of -0.2 to -2.

[0194] The compound of formula (IIa) or (IIb) has an R of less than 2.75. 原子 may have:

[0195] Exemplary crosslinkers for use in embodiments of the present invention include N,N'-(1,2-dihydroxyethylene)bisacrylamide, N,N'-methylenebis(acrylamide), N,N'-ethylenebis(acrylamide), piperazine diacrylamide, glycerol 1,3-diglycerolate diacrylate, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW ≦2000), 4-arm PEG-acrylamide (MW ≦2000), and N,N-bis(2-acrylamidoethyl)acrylamide. Exemplary crosslinkers N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW 2000 or less), and 4-arm PEG-acrylamide (MW 2000 or less) (e.g., N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide) are particularly suitable for use in highly crosslinked particles and mechanically more stable particles, i.e., particles having a crosslinking level of at least 20% by weight of the crosslinker (e.g., a crosslinking level of at least 30% by weight of the crosslinker). Embodiments can also include the use of combinations of crosslinkers.

[0196] Crosslinkers may include N,N'-methylenebis(acrylamide), N,N'-ethylenebis(acrylamide), piperazine diacrylamide, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW ≦2000), 4-arm PEG-acrylamide (MW ≦2000), N,N-bis(2-acrylamidoethyl)acrylamide.

[0197] The crosslinker can be a compound that does not contain a primary amine, a secondary amine, a hydroxyl, or a carboxylic acid. The crosslinker can be a compound of formula (IIa) or formula (IIb) that does not contain a primary amine, a secondary amine, a hydroxyl, or a carboxylic acid.

[0198] The level of crosslinking in the crosslinked hydrogel polymer particles formed by this method can be expressed as a weight percent (wt%) of crosslinker monomer contained in the total monomers used in the polymerization. Typical levels of crosslinking include greater than 5 wt% crosslinker, e.g., greater than 10 wt% crosslinker, or greater than 15 wt% crosslinker, e.g., greater than 20 wt% crosslinker. The level of crosslinking can also be, for example, 5-60 wt% crosslinker, e.g., 10-50 wt% crosslinker. The level of crosslinking can also be, for example, 15-40 wt% crosslinker, e.g., 20-40 wt% crosslinker, e.g., 20-30 wt% crosslinker. Thus, solution (a) can contain a crosslinker amount in a wt% equivalent to 5-60% of the amount of hydrophilic vinyl monomer, e.g., 10-50% of the amount of hydrophilic vinyl monomer. Thus, solution (a) may contain, for example, an amount of crosslinker in a weight % corresponding to 15 to 40% of the amount of hydrophilic vinyl monomer, for example 20 to 40% of the amount of hydrophilic vinyl monomer, for example 20 to 30% of the amount of hydrophilic vinyl monomer.

[0199] The level of crosslinking can be, for example, greater than 30% by weight crosslinker or greater than 40% by weight crosslinker (e.g., greater than 50% by weight crosslinker) in highly crosslinked particles. The level of crosslinking can be 10-90% by weight crosslinker, 20-80% by weight crosslinker, or 25-75% by weight crosslinker, for example, 25-60% by weight crosslinker, or 30-50% by weight crosslinker. In highly crosslinked particles, the level of crosslinking can be up to 100% by weight crosslinker; for example, the hydrophilic vinyl monomer can be the crosslinker; for example, the hydrophilic vinyl monomer and the crosslinker can be the same compound. Thus, solution (a) can contain an amount of crosslinker in a weight percent equivalent to 20-80% of the amount of hydrophilic vinyl monomer, for example, 25-60% of the amount of hydrophilic vinyl monomer.

[0200] A specific hydrophilic vinyl monomer may be referred to as acrylamide (prop-2-enamide), for which 1,2-dihydroxybisacrylamide is a suitable crosslinking agent. A specific hydrophilic vinyl monomer may be referred to as hydroxymethylacrylamide, for which N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is a suitable crosslinking agent. A specific hydrophilic vinyl monomer may be referred to as hydroxyethylacrylamide, for which N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is a suitable crosslinking agent. Further specific hydrophilic vinyl monomers and crosslinking agents are listed in Table 1.

[0201] The stabilizer may be or include a nonionic surfactant, such as a nonionic polymer surfactant. The nonionic surfactant may include at least one oligomeric surfactant. For example, the nonionic surfactant may include an oligomeric surfactant. The nonionic polymer or oligomeric surfactant may include at least one polyethyleneoxy group or at least one polypropyleneoxy group. The nonionic polymer surfactant may include at least one polyethyleneoxy group. The nonionic oligomeric surfactant may include at least one polyethyleneoxy group or at least one polypropyleneoxy group. The stabilizer may be selected from or include Hypermer 2296, Abil WE09, Abil EM90, and sorbitan monooleate (Span 80).

[0202] The polymerization may include activating a radical initiator. The radical initiator is typically activated by heating to form radicals that start the polymerization reaction, although other activation methods, such as radiation, may also be used. Activating the radical initiator may include heating the emulsion containing the swollen particles. Heating typically involves heating the emulsion to a temperature above the temperature at which the step of forming swollen particles from the monodisperse seed particles is performed. Heating may include heating to a temperature of at least 40°C, such as at least 50°C, for example at least 60°C or at least 70°C.

[0203] The radical initiator may be or may include a peroxide initiator or an azo initiator. For example, the radical initiator may be a peroxide initiator. For example, the radical initiator may be an azo initiator. An exemplary radical initiator is 2,2'-azobis-2,4-dimethylvaleronitrile. An exemplary radical initiator is 2,2'-azodi(2-methylbutyronitrile). The radical initiator may be present in solution (a). The radical initiator may be present in solution (b). The radical initiator may be present in the emulsion in an amount of about 0.1% to about 1.5% by weight. For example, the radical initiator may be present in the emulsion in an amount of about 0.6% to about 1.2% by weight, e.g., about 0.8% by weight.

[0204] The organic solvent used in the method for forming monodisperse crosslinked hydrogel polymer particles may comprise (or consist of) at least one of an aliphatic hydrocarbon, an aliphatic carbonate, an aliphatic ester, an aliphatic ether, an aromatic hydrocarbon, or a silicone, or a combination thereof. For example, the organic solvent may comprise (or consist of) at least two of an aliphatic hydrocarbon, an aliphatic carbonate, an aliphatic ester, an aliphatic ether, an aromatic hydrocarbon, and a silicone, or the organic solvent may comprise (or consist of) at least three of an aliphatic hydrocarbon, an aliphatic carbonate, an aliphatic ester, an aliphatic ether, an aromatic hydrocarbon, and a silicone. The organic solvent may comprise (or consist of) a mixture of heptane and toluene. The organic solvent may comprise (or consist of) a mixture of aliphatic hydrocarbons. The organic solvent may comprise (or consist of) bis(2-ethylhexyl)carbonate. The organic solvent may comprise (or consist of) bis(2-ethylhexyl)carbonate, an aliphatic hydrocarbon, and an aromatic hydrocarbon. The organic solvent may comprise (or consist of) bis(2-ethylhexyl) adipate.

[0205] The monodisperse seed particles added to the water-in-oil emulsion (c) may have the characteristics of monodisperse seed particles formed according to the method for preparing monodisperse seed particles disclosed herein. The monodisperse seed particles added to the water-in-oil emulsion (c) may be monodisperse seed particles formed according to the method for preparing monodisperse seed particles disclosed herein. The monodisperse seed particles may be monodisperse seed particles of the present disclosure, such as monodisperse seed particles of the present invention.

[0206] In one embodiment, the present invention provides a method for forming monodisperse crosslinked hydrogel polymer particles, comprising the steps of: forming a solution (a) of at least 2% by weight of a hydrophilic vinyl monomer in an aqueous solution, the aqueous solution also containing a chain transfer agent; forming a solution (b) of a stabilizer in an organic solvent, the organic solvent being immiscible in water and at least one of solutions (a) and (b) containing a radical initiator; mixing solutions (a) and (b) to form a water-in-oil emulsion (c) and adding monodisperse seed particles to the emulsion; allowing the monodisperse seed particles to form swollen particles in the emulsion; polymerizing the swollen particles to form monodisperse polymer particles; and mixing the solution (d) of a stabilizer in an organic solvent with the stabilizer in the organic solvent. The method includes the steps of forming a water-in-oil emulsion (e) of at least 2% by weight of a hydrophilic vinyl monomer in an aqueous solution, the aqueous solution also containing a crosslinker containing at least two vinyl groups, and at least one of the solutions (d) and (e) containing a radical initiator; mixing the solutions (d) and (e) to form a water-in-oil emulsion (f) and adding monodisperse polymer particles to the emulsion; allowing the monodisperse polymer particles to form swollen polymer particles in the emulsion; and polymerizing the swollen polymer particles to form monodisperse crosslinked hydrogel polymer particles. As described below, this method can be considered a two-step method because it includes a first swelling step and a first polymerization step, followed by a second swelling step and a second polymerization step. Compared to the one-step method, the two-step method can provide large, monodisperse crosslinked hydrogel polymer particles.

[0207] Similar to the one-step process, the two-step process involves swelling seed particles with an aqueous discontinuous phase in an oil continuous phase. Schematic diagrams of this process, which can be considered a two-step process because it involves two stages of swelling and two stages of polymerization, are provided in Figure 3 (shown with specific monomers) and Figure 4 (shown more generally). The process shown in Figures 3 and 4 is conveniently divided into four steps: swelling monodisperse seed particles 100 with monomer 103 and a chain transfer agent 105 to form swollen (monodisperse) seed particles 110; polymerizing the monomers in the swollen seed particles to form (uncrosslinked) hydrophilic polymer particles 130; swelling the hydrophilic polymer particles with monomer 103 and a crosslinker 102 to form swollen hydrophilic polymer particles 140; and polymerizing the monomers in the hydrogel polymer particles to form crosslinked hydrogel polymer particles 150. Prior to the first step, a water-in-oil emulsion is formed. The water-in-oil emulsion is formed by mixing an aqueous solution containing a monomer, e.g., hydroxymethylacrylamide 103, and a chain transfer agent, e.g., 1-thioglycerol 105, with an oil phase containing a steric stabilizer, and the emulsion is formed when the aqueous and oil phases are agitated (e.g., by stirring). The water-in-oil emulsion also typically contains an initiator, which may be added to the oil and / or aqueous phases. An initiator is a compound that, upon activation, initiates polymerization of the monomers. The monodisperse seed particles 100 comprise non-crosslinked oligomers of poly N,N-dimethylacrylamide 131 and are considered "activated seed particles." The seed particles 100 may be prepared as described elsewhere in this application.

[0208] In the first step, monodisperse seed particles 100 are added to a water-in-oil emulsion, and the emulsion is stirred for a period of time (e.g., at least 30 minutes or at least 1 hour, typically 4 to 48 hours). During this period, the monomer 103 and chain transfer agent 105 diffuse into the activated seed particles 100 to form swollen seed particles 110. The swollen seed particles 110 contain a mixture of at least the monomer 103, the chain transfer agent 105, and the polymer from the activated seed particles 100. The swollen seed particles 110 may also contain other components, such as one or more porogens, which may become incorporated into the particles when contained in a water-in-oil emulsion. For example, in the illustrated method, water is also present in the swollen seed particles 110, and this water may be considered a porogen. The second step involves polymerizing the monomer 103 to form (non-crosslinked) hydrophilic polymer particles 130. In the second step, polymerization is initiated by activating an initiator, for example, by heating the emulsion. The presence of the chain transfer agent 105 results in relatively short polymers (e.g., oligomers) of polyhydroxymethylacrylamide 132 in the hydrogel polymer particles. These relatively short polymers, combined with the lack of crosslinking, mean that the hydrophilic polymer particles 130 can act as larger "activated seed particles" suitable for further rounds of swelling and polymerization. The hydrophilic polymer particles contain relatively short, uncrosslinked polymers of polyhydroxymethylacrylamide 132 as well as uncrosslinked (typically fewer) oligomers 131 of poly N,N-dimethylacrylamide 131.

[0209] Before the third step, a water-in-oil emulsion is formed. This emulsion is formed by mixing an aqueous solution containing a monomer, e.g., hydroxymethylacrylamide 103, and a chain transfer agent, e.g., 1,2-dihydroxybisacrylamide 102, with an oil phase containing a steric stabilizer. If highly crosslinked particles are desired, the monomer can be replaced with a crosslinker, so that the only monomer present in the water-in-oil emulsion is the crosslinking monomer (i.e., the crosslinker). The water-in-oil emulsion also typically contains an initiator, which may be added to either the oil or aqueous phase. The initiator is a compound that, upon activation, initiates the polymerization of the monomer and crosslinker. In the third step (second swelling), hydrophilic polymer particles 130 are added to the water-in-oil emulsion, and the emulsion is stirred for a period of time (e.g., at least 30 minutes or at least 1 hour, typically 4 to 48 hours). During this period, the monomer 103 and crosslinker 102 diffuse into the hydrophilic polymer particles 130 to form swollen hydrogel polymer particles 140. The swollen hydrophilic polymer particles 140 include a mixture of at least the monomers 103, the crosslinker 102, and the polymers from the hydrophilic polymer particles 130. The swollen hydrogel polymer particles 140 may also include other components, such as one or more porogens, which can enter the particles when included in a water-in-oil emulsion. For example, in the illustrated method, water is also present in the swollen seed particles 110, and this water can be considered a porogen. The fourth step involves polymerizing the monomers 103 and the crosslinker 102 to form the crosslinked hydrogel polymer particles 104. In the fourth step, polymerization is initiated by activating an initiator, for example, by heating the emulsion.

[0210] The monomer, chain transfer agent, radical initiator, stabilizer, crosslinker, organic solvent, and optional porogen may each be as defined elsewhere herein. The monomer may be or include hydroxymethylacrylamide or hydroxyethylacrylamide. The chain transfer agent may be or include 1-thioglycerol. The radical initiator may be or include azobisdimethylvaleronitrile, such as 2,2'-azodi(2-methylbutyronitrile) (AMBN). The stabilizer may be or include Hypermer 2296, Abil WE09, and Abil EM90. The crosslinker may be 1,2-dihydroxybisacrylamide or N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide.

[0211] Solution (a) may contain 60% by weight or less of hydrophilic vinyl monomer. For example, solution (a) may contain 55% by weight or less or 50% by weight or less of hydrophilic vinyl monomer. For example, solution (a) may contain 45% by weight or less or 40% by weight or less of hydrophilic vinyl monomer, for example, solution (a) may contain 30% by weight or less of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight of hydrophilic vinyl monomer. Solution (a) may contain at least 5% by weight of hydrophilic vinyl monomer. Solution (a) may contain at least 8% by weight of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 60% by weight or less of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% by weight of hydrophilic vinyl monomer and 60% by weight or less of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 50% by weight or less of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% by weight of hydrophilic vinyl monomer and 50% by weight or less of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 45% by weight or less of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% by weight of hydrophilic vinyl monomer and 45% by weight or less of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 30% by weight or less of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% by weight of hydrophilic vinyl monomer and 30% by weight or less of hydrophilic vinyl monomer. Solution (a) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and no more than 15% of hydrophilic vinyl monomer, for example, solution (a) may contain about 10% by weight of hydrophilic vinyl monomer.

[0212] Solution (e) may contain 60% by weight or less of hydrophilic vinyl monomer. For example, solution (e) may contain 55% by weight or less or 50% by weight or less of hydrophilic vinyl monomer. For example, solution (e) may contain 45% by weight or less or 40% by weight or less of hydrophilic vinyl monomer, for example, solution (e) may contain 30% by weight or less of hydrophilic vinyl monomer. Solution (e) may contain at least 2% by weight of hydrophilic vinyl monomer. Solution (e) may contain at least 5% by weight of hydrophilic vinyl monomer. Solution (e) may contain at least 8% by weight of hydrophilic vinyl monomer. Solution (e) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 60% by weight or less of hydrophilic vinyl monomer, for example, solution (e) may contain at least 8% by weight of hydrophilic vinyl monomer and 60% by weight or less of hydrophilic vinyl monomer. Solution (e) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 50% by weight or less of hydrophilic vinyl monomer, for example, solution (e) may contain at least 8% by weight of hydrophilic vinyl monomer and 50% by weight or less of hydrophilic vinyl monomer. Solution (e) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 45% by weight or less of hydrophilic vinyl monomer, for example, solution (e) may contain at least 8% by weight of hydrophilic vinyl monomer and 45% by weight or less of hydrophilic vinyl monomer. Solution (e) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and 30% by weight or less of hydrophilic vinyl monomer, for example, solution (e) may contain at least 8% by weight of hydrophilic vinyl monomer and 30% by weight or less of hydrophilic vinyl monomer. Solution (e) may contain at least 2% by weight (e.g., 5% by weight) of hydrophilic vinyl monomer and no more than 15% of hydrophilic vinyl monomer, for example, solution (e) may contain about 10% by weight of hydrophilic vinyl monomer.

[0213] In highly crosslinked particles, the hydrophilic vinyl monomer of solution (e) can be or can include a crosslinker, for example, the monomer can be or can include at least one compound of formula (IIa) or (IIb).

[0214] The level of crosslinking in the crosslinked hydrogel polymer particles formed by this method can be expressed as the weight percent (wt%) of crosslinker monomer contained in the total monomers used in the polymerization (i.e., in the second polymerization step). The wt% of crosslinker monomer can correspond to the wt% of crosslinker in the matrix polymer (i.e., the wt% of crosslinker in the dry weight of the crosslinked polymer particle). Typical levels of crosslinking include greater than 5 wt% crosslinker, e.g., greater than 10 wt% crosslinker, or greater than 15 wt% crosslinker, e.g., greater than 20 wt% crosslinker (e.g., greater than 30 wt% crosslinker). The level of crosslinking can also be, for example, 5-60 wt% crosslinker, e.g., 10-50 wt% crosslinker. The level of crosslinking can also be, for example, 15-40 wt% crosslinker, e.g., 20-40 wt% crosslinker, e.g., 20-30 wt% crosslinker. The level of crosslinking can be 20-80% by weight crosslinker or 25-75% by weight crosslinker, for example, 25-60% by weight crosslinker or 30-50% by weight crosslinker, e.g., high-density particles. Thus, solution (e) can contain a crosslinker amount in a weight percentage corresponding to 5-60% of the amount of hydrophilic vinyl monomer, e.g., 10-50% of the amount of hydrophilic vinyl monomer. Thus, solution (e) can contain a crosslinker amount in a weight percentage corresponding to, e.g., 15-40% of the amount of hydrophilic vinyl monomer, e.g., 20-40% of the amount of hydrophilic vinyl monomer, e.g., 20-30% of the amount of hydrophilic vinyl monomer.

[0215] The level of crosslinking can be, for example, greater than 30% by weight crosslinker or greater than 40% by weight crosslinker (e.g., greater than 50% by weight crosslinker) in highly crosslinked particles. The level of crosslinking can be 10-90% by weight crosslinker, 20-80% by weight crosslinker, or 25-75% by weight crosslinker, e.g., 25-60% by weight crosslinker, or 30-50% by weight crosslinker. In highly crosslinked particles, the level of crosslinking can be up to 100% by weight crosslinker; for example, the hydrophilic vinyl monomer in solution (e) can be the crosslinker; e.g., the hydrophilic vinyl monomer and the crosslinker can be the same compound. Solution (e) can contain an amount of crosslinker in a weight percent equivalent to 20-80% of the amount of hydrophilic vinyl monomer, e.g., 25-60% of the amount of hydrophilic vinyl monomer.

[0216] The radical initiator may be present in each or at least one emulsion in an amount of about 0.1% to about 1.5% by weight, for example, the radical initiator may be present in each or at least one emulsion in an amount of about 0.6% to about 1.2% by weight, for example, the radical initiator may be present in each or at least one emulsion in an amount of about 0.8% by weight.

[0217] The addition of a chain transfer agent reduces the molecular weight of the polymer in the monodisperse polymer particles by reacting with the free radicals in the growing polymer chains to terminate them and transferring the lone electron to a radical species derived from the chain transfer agent. The radical species derived from the chain transfer agent then reacts with the monomer to form a radical from the monomer, which can then react with another monomer to initiate the formation of a new polymer chain. The chain transfer agent can be a thiol or a haloalkane. For example, the chain transfer agent can be selected from thiols (e.g., 1-thioglycerol, 1-octanethiol, hexanethiol, 6-mercapto-1-hexanol, and benzylthiol), alkylthiols (e.g., 1-octanethiol, hexanethiol), and thiol polyols (e.g., 1-thioglycerol). The chain transfer agent can be or include 1-thioglycerol. The total amount of chain transfer agent added can be in the range of 1 mole per 10 moles of monomer to 1 mole per 300 moles of monomer, such as 1 mole per 20 moles of monomer to 1 mole per 100 moles of monomer, for example, about 1 mole of chain transfer agent per 30 moles of monomer.

[0218] The monodisperse crosslinked hydrogel polymer particles formed according to the methods of the present invention can be porous. For example, the monodisperse crosslinked hydrogel polymer particles formed can have a porosity of at least 5%, e.g., at least 10%. The present disclosure includes porous monodisperse crosslinked hydrogel polymer particles having a porosity of 20% to 95%, particularly 30% to 90%, e.g., 40% to 90%, e.g., 50% to 80%. The porous monodisperse crosslinked hydrogel polymer particles formed can be transparent to solvating molecules, e.g., the porous monodisperse crosslinked hydrogel polymer particles can be transparent to oligonucleotides and nucleic acid amplification reagents and sequencing reagents (e.g., primers, nucleotides, and polymerase).

[0219] Monodisperse crosslinked hydrogel polymer particles formed according to the methods of the present invention may have a mode diameter of 0.5 μm to 10 μm, e.g., 0.5 μm to 5 μm. Monodisperse crosslinked hydrogel polymer particles formed may have a mode diameter of at least 500 nm, e.g., at least 600 nm, optionally at least 800 nm, such as for particles having a diameter of at least 1 μm. Monodisperse crosslinked hydrogel polymer particles formed according to the methods of the present invention may have a mode diameter of 10 μm or less, e.g., 5 μm or less, optionally 3 μm or less, such as for particles having a diameter of 2 μm or less. The size and size distribution of monodisperse crosslinked hydrogel polymer particles formed according to the methods of the present invention may be determined as described below under the heading "Analytical Methods."

[0220] Monodisperse crosslinked hydrogel polymer particles formed according to the methods of the present invention may have a CV of less than 20%, such as less than 15%. For example, the particles may have a CV of less than 10%, less than 8%, such as less than 5%.

[0221] The monodisperse crosslinked hydrogel polymer particles formed according to the method of the present invention may contain functional groups. The functional groups may be selected from hydroxyl, carboxylic acid (-COOH), primary amine, or secondary amine. In one embodiment, the functional groups are provided by the hydrophilic vinyl monomer (e.g., a compound of Formula (I)) and not by the crosslinker (e.g., a compound of Formula (IIa) or Formula (IIb)). The functional groups may be enhanced to facilitate binding with target analytes (e.g., oligonucleotides) or target receptors. Exemplary methods for enhancing particle functionality are described in the "Uses of the Particles" section herein.

[0222] Monodisperse crosslinked hydrogel polymer particles formed according to the methods of the present invention may contain oligonucleotides bound to the particles. The oligonucleotides may be bound to the monodisperse crosslinked hydrogel polymer particles via a linker. For example, each of the monodisperse hydrogel polymer particles (or a portion thereof) may contain multiple oligonucleotides bound to the particle. The multiple oligonucleotides may be the same for each individual crosslinked hydrogel polymer particle. For example, a first crosslinked hydrogel polymer particle may contain multiple oligonucleotides having a first sequence bound to the first particle, and a second crosslinked hydrogel polymer particle may contain multiple oligonucleotides having a second sequence bound to the second crosslinked hydrogel polymer particle. If the monodisperse crosslinked hydrogel polymer particles are porous, the oligonucleotides may be bound to the outer surface of the particle or to the inside of the pores. The pores may be large enough to make the particles transparent to oligonucleotides so that the oligonucleotides can be located partially or entirely within the pores, even when polymerase is bound to the oligonucleotides.

[0223] The monodisperse crosslinked hydrogel polymer particles formed according to the methods of the present invention may be stable in aqueous solution (i.e., may be resistant to polymer degradation) over the temperature range typical of aqueous solutions. The monodisperse crosslinked hydrogel polymer particles formed may be stable at temperatures up to about 100°C. For example, the monodisperse crosslinked hydrogel polymer particles formed may be stable within a temperature range of about 0°C to about 100°C.

[0224] One embodiment of the present invention provides particles obtained by, or having the characteristics of, the particles obtained by the preparation process described herein.

[0225] The polymer particle formation process described herein can be performed in a highly reproducible and scalable manner. Thus, the present invention can enable batch-to-batch and intra-batch consistency, which is essential for industrial applications. The present invention can also enable the production of pilot-scale batches of at least 300 g and industrial batches on a kilogram scale.

[0226] The polymer particle formation process described herein can be performed consistently without the problems that can occur with practical emulsion polymerization, such as particle agglomeration and product variability.

[0227] Preparation of magnetic particles In one embodiment, a method for forming monodisperse magnetic hydrogel polymer particles is provided, comprising forming an aqueous suspension comprising monodisperse hydrogel polymer particles and a magnetic material or a magnetic material precursor. The magnetic material precursor, if present, is converted to a magnetic material. The magnetic material is associated with the polymer particles. The magnetic material precursor can be, for example, a mixture of monodisperse hydrogel polymer particles and Fe 2+ and / or Fe 3+ ions, which can be converted to a magnetic material by raising the pH to above 8 and precipitating the magnetic material. The monodisperse hydrogel polymer particles can be made (e.g., made) according to the methods of forming monodisperse hydrogel polymer particles disclosed herein.

[0228] Aqueous suspensions with a pH below 6 contain Fe 2+ and Fe 3+ The aqueous suspension with a pH of less than 6 may contain FeCl2 and FeCl3. The aqueous suspension with a pH of less than 6 may contain Fe 2+ and Fe 3+ions, which may also include multivalent cations of metals selected from Al, Mn, Ni, Cu, Co, Zn, Ca, Ge, Te, Ti, or Sn, or combinations thereof. When the aqueous suspension includes multivalent cations of metals selected from Al, Mn, Ni, Cu, Co, Zn, Ca, Ge, Te, Ti, or Sn, or combinations thereof, the amount of multivalent cations is preferably Fe. 2+ and Fe 3+ It may comprise up to 5 mol % of the amount of ions.

[0229] Raising the pH to above 8 may involve adding ammonia (e.g., aqueous ammonia) to the aqueous suspension.

[0230] The magnetic material may be precipitated as a nanoparticle magnetic material and / or a superparamagnetic material. The magnetic material may be precipitated as a paramagnetic material. The magnetic material may be precipitated as a superparamagnetic material. The magnetic material may be precipitated as a ferromagnetic material, a ferrimagnetic material, or both. If the monodisperse hydrogel polymer particles are porous, the magnetic material may be precipitated within the pores, for example, the magnetic material may be precipitated within the pores as a superparamagnetic material.

[0231] The total amount of magnetic material present in the monodisperse magnetic hydrogel polymer particles formed by the process is generally greater than 20%, preferably greater than 25%, and desirably 30% or more, e.g., up to 85% or at least 50% by weight, e.g., 30-80% by weight. Percentages are weight percentages calculated relative to the weight of magnetic material (e.g., metal oxide) based on the total dry weight of the particles. When the magnetic material consists of a superparamagnetic material, the total amount of superparamagnetic material present can be greater than 20%, preferably greater than 25%, and desirably 30% or more, e.g., up to 85% or at least 50% by weight, e.g., 30-80% by weight, each percentage calculated relative to the weight of magnetic material (e.g., metal oxide) based on the total dry weight of the particles.

[0232] Preparation of coated particles In one embodiment, a method for forming coated monodisperse hydrogel polymer particles is provided, the method comprising: forming a suspension containing monodisperse hydrogel polymer particles and at least one epoxide, at least one silicate, or at least one orthosilicate; and reacting the epoxy groups of the at least one epoxide with surface functional groups of the particles, or reacting a silicate or silane to form a silica coating. The monodisperse hydrogel polymer particles can be produced according to the method for forming monodisperse hydrogel polymer particles disclosed herein. The monodisperse hydrogel polymer particles can be produced according to the method for forming monodisperse magnetic hydrogel polymer particles disclosed herein.

[0233] In one embodiment, the method includes reacting the epoxy groups of at least one epoxide with the surface functional groups of the particles. The suspension may include an organic solvent (e.g., an organic nitrile, such as acetonitrile, or an alcohol, such as isopropanol). The reaction may include a grafting reaction. The at least one epoxide monomer may include an epoxide monomer, for example, a mixture of at least two or at least three epoxide monomers.The at least one epoxide monomer (or mixture of epoxide monomers) may be selected from the group consisting of epichlorohydrin, epibromohydrin, isopropyl glycidyl ether, butyl glycidyl ether, allyl glycidyl ether, 1,4-butanediol diglycidyl ether (1,4-bis(2,3-epoxypropoxy)butane), neopentyl glycol diglycidyl ether, ethylene glycol diglycidyl ether, glycerin diglycidyl ether, glycidol, and glycidyl methacrylate, ethylhexyl glycidyl ether, methyl glycidyl ether, glycerol propoxylate triglycidyl ether, poly(propylene glycol) diglycidyl ether, 1,3 butylene diol diglycidyl ether, tert butyl glycidyl ether, 1,4 cyclohexanedimethanol diglycidyl ether, diethylene glycol diglycidyl ether, dodecyl glycidyl ether, O-(2,3 epoxypropyl)-O'-methyl polyethylene glycol glycidyl ether, glycidyl tetrafluoroethyl ether, 1,6 hexanediol diglycidyl ether, octyl glycidyl ether, decyl glycidyl ether, poly(epichlorohydrin-co-ethylene oxide-co-allyl glycidyl ether), polyethylene glycol diglycidyl ether, trimethylolethane triglycidyl ether, trimethylolpropane triglycidyl ether, tert-butyldimethylsilyl glycidyl ether, 1,2-epoxybutane, 1,2-epoxypentane, 1,2-epoxy-5-hexene, 1,2-epoxy-hexane, 1,2-epoxy-7-octene, 1,2-epoxyoctane, 1,2,7,8-diepoxyoctane, 1,2-epoxy-9-decene, 1,2-epoxydecane, 1,2-epoxydodecane, 1,2-epoxytetradecane, or a combination thereof. The at least one epoxide monomer (or mixture of epoxide monomers) can be selected from 1,4-butanediol diglycidyl ether, glycidol, glycidyl methacrylate, or a combination thereof. The weight ratio of the monodisperse hydrogel polymer particles to the at least one epoxide monomer can range from 1:10 to 10:1.

[0234] The surface functional groups can include amines. The amines can be derived from functional monomers used in the process for forming the monodisperse hydrogel polymer particles, or the amines can be introduced using standard chemistry. For example, the (non-amine) functional groups of the polymer particles can be modified with reagents that can convert the functional groups into reactive moieties that can undergo nucleophilic or electrophilic substitution, and the amines are introduced by selecting a suitable reagent, including a nucleophilic or electrophilic reagent.

[0235] In one embodiment, the method includes reacting a silicate or silane to form a silica coating. The hydrogel polymer particles may include iron oxide crystals (e.g., the hydrogel polymer particles may include a magnetic material that includes iron oxide crystals) and / or hydroxy groups. Without wishing to be bound by any theory, it is believed that the presence of iron oxide crystals and / or hydroxy groups in the hydrogel polymer particles promotes the formation of the silica coating.

[0236] In one embodiment, the method includes reacting a silicate to form a silica coating. The silicate can be a metasilicate, such as Na2SiO3. The suspension can include an aqueous solvent (e.g., the solvent can be water). Reacting the silicate to form the silica coating can include lowering the pH of the suspension to less than about 11 (e.g., less than 11, 10, or 7). The mass ratio of monodisperse hydrogel polymer particles to silicate can range from 1:10 to 10:1.

[0237] In one embodiment, the method includes reacting a silane to form a silica coating. The silane can be an organosilane, such as tetraethyl orthosilicate. The suspension can include an organic solvent, such as dipropylene glycol dimethyl ether. Reacting the orthosilicate to form the silica coating can include raising the temperature of the suspension to at least 80°C (e.g., a temperature of at least 90°C or 100°C). Reacting the orthosilicate to form the silica coating can include raising the temperature of the suspension to a temperature within a range of 80°C to 155°C, such as a temperature of 100°C to 150°C, for example, a temperature of 110°C to 150°C (e.g., a temperature of about 130°C). The mass ratio of monodisperse hydrogel polymer particles to orthosilicate can be in the range of 1:10 to 10:1.

[0238] Using Particles The particles can be used in many applications, such as polynucleotide sequencing, bioassays, information storage, color imaging, in vitro diagnostics, bioprocessing, diagnostic microbiology, biosensors, and drug delivery. Polymer particles containing magnetic materials are particularly useful in assays (e.g., when the polymer particles are conjugated with a target analyte, such as an affinity ligand), because the magnetic material allows the particles (and anything bound to them) to be separated from the bulk solution by application of a magnetic field.

[0239] Polymer particles can be activated to facilitate conjugation with target analytes, such as polynucleotides or other affinity ligands. Affinity ligands include monoclonal antibodies, polyclonal antibodies, antibody fragments, nucleic acids, oligonucleotides, proteins, oligopeptides, polysaccharides, sugars, peptides, nucleic acid molecules encoding peptides, immobilized metal affinity chromatography (IMAC) agents, antigens, drugs, and other ligands. If the affinity ligand is of clinical interest, polymer particles conjugated to the affinity ligand can be useful for in vitro diagnosis. In addition to the methods described below, suitable methods of activation and bioconjugation are described in G.T. Hermanson, Bioconjugate Techniques, 2013 (3 rd Edition), Academic Press, the contents of which are incorporated herein by reference in their entirety.

[0240] For example, functional groups on polymer particles can be enhanced to enable binding with target analytes or analyte receptors. In certain examples, the functional groups of polymer particles can be modified with reagents that can convert the functional groups into reactive moieties capable of undergoing nucleophilic or electrophilic substitution. For example, hydroxyl groups on polymer particles can be activated by replacing at least a portion of the hydroxyl groups with sulfonate groups or chlorines. Exemplary sulfonate groups can be derived from tresyl, mesyl, tosyl, or tosyl chloride, or any combination thereof. The sulfonate can act to replace the nucleophile with the sulfonate. The sulfonate can further react with the liberated chlorine to provide a chlorinated group that can be used in the process of binding particles. In another example, if the polymer particles contain amine or carboxylic acid groups (e.g., from a functional monomer), the amine or carboxylic acid groups can be activated.

[0241] For example, a target analyte or analyte receptor can be attached to a polymer particle via nucleophilic substitution with a sulfonate group or other activating group. In certain instances, a target analyte receptor terminated with a nucleophile, such as an amine or thiol, can undergo nucleophilic substitution to replace a sulfonate group on the surface of the polymer particle. As a result of activation, a conjugated particle can be formed.

[0242] An example of such a target analyte receptor terminated with a nucleophile is streptavidin, which is ideally suited for many different assay methods. For example, biotinylated single-stranded oligonucleotide probes bound to streptavidin-conjugated particles can be used to isolate sequence-specific DNA. The biotinylated probe is bound to the conjugated particles by mixing an appropriate amount of polymer particles with an excess of biotinylated probe. The particles / probe are then incubated with a DNA sample in a hybridization buffer, such as SSPE or SSC, under conditions appropriate for the length and sequence of the probe and DNA. When magnetic polymer particles are used, the magnetic properties of the polymer particles are utilized to wash away excess, unwanted DNA. The captured DNA can be detected / quantified by PCR or any other suitable technique. For example, biotinylated double-stranded DNA fragments bound to streptavidin-conjugated polymer particles can be used to isolate DNA sequence-specific binding proteins. Biotinylated DNA is conjugated to polymer particles by mixing an appropriate amount of beads with an excess of biotinylated DNA fragments. The particles / DNA are then incubated with a protein sample in a hybridization buffer under conditions appropriate for the protein under investigation. When magnetic polymer particles are used, the magnetic properties of the polymer particles are utilized to wash away excess, undesired proteins. The captured proteins can be eluted from the probe (by high salt, low salt, heat, low pH, etc.) for downstream applications and detection.

[0243] In another example, the sulfonated particles can be further reacted with mono- or polyfunctional mono- or poly-nucleophilic reagents that can form bonds to the particles while maintaining nucleophilic activity for oligonucleotides containing electrophilic groups, such as maleimides. Furthermore, the residual nucleophilic activity can be converted to electrophilic activity by binding to a reagent containing multiple electrophilic groups that will subsequently be bound to the oligonucleotides containing electrophilic groups.

[0244] In another example, a monomer containing a functional group (functional monomer) can be included in the monomer mixture during polymerization. The functional monomer can include, for example, an acrylamide containing a carboxylic acid, a phosphonic acid, a sulfonic acid, an ester, a halogen, or another amine-reactive group. The ester group can be hydrolyzed before reaction with an amine oligonucleotide or other amine-containing affinity ligand.

[0245] Exemplary amine-containing affinity ligands have the formula: TIFF0007765421000021.tif24128, wherein each R is independently hydrogen or a protecting group, and X is a C2-C 20 Represents an alkylene linker (e.g., C5 or C6 alkylene). This affinity ligand provides a carboxymethylated aspartate (Cm-Asp) group that can be attached to a particle (MPP), deprotected (when R is a protecting group), and complexed with a metal ion to form an IMAC agent. TIFF0007765421000022.tif30128 Thus, this ligand provides the ability to bind to His, Cys, Met, Gln, Asn, Lys, or Tyr residues present in histidine tags in recombinant proteins or metalloprotein active sites in natural proteins or peptides, and to bind to phosphorylated proteins or peptides. The generation, conjugation, and use of this affinity ligand in assays (e.g., using magnetic polymer particles) are described in US2010 / 0222508A1, the contents of which are incorporated herein by reference in their entirety.

[0246] Other activation chemistries incorporate multiple steps to convert specific functional groups to accommodate specific desired linkages. For example, sulfonate-modified hydroxyl groups can be converted to nucleophilic groups by several methods. In one example, the reaction of sulfonates with azide anions yields azide-substituted hydrophilic polymers. Azides can be used for direct conjugation to acetylene-substituted biomolecules via "CLICK" chemistry, which can be performed with or without copper catalysis. Optionally, azides can be converted to amines by, for example, catalytic reduction with hydrogen or reduction with organophosphines. The resulting amines can then be converted to electrophilic groups using various reagents, such as diisocyanates, bis-NHS esters, cyanuric chloride, or combinations thereof. In one example, the use of diisocyanates creates a urea linkage between the polymer and the linker, leaving residual isocyanate groups that can react with amino-substituted biomolecules to create a urea linkage between the linker and the biomolecule. In another example, the use of a bis-NHS ester results in an amide linkage between the polymer and the linker, leaving a residual NHS ester group that can react with an amino-substituted biomolecule to form an amide linkage between the linker and the biomolecule. In a further example, the use of cyanuric chloride results in an amino-triazine linkage between the polymer and the linker and two residual chlorotriazine groups, one of which can react with an amino-substituted biomolecule to form an amino-triazine linkage between the linker and the biomolecule. Other nucleophilic groups can be incorporated into particles via sulfonate activation. For example, reaction of sulfonated particles with thiobenzoate anion and the resulting hydrolysis of the thiobenzoate incorporates thiols into the particles, which can then react with maleimide-substituted biomolecules to form thiosuccinimide linkages to the biomolecule. Thiols can also be reacted with bromoacetyl groups.

[0247] Covalent attachment of biomolecules to non-volatile or polymeric substrates can be achieved using electrophilic moieties on the substrate coupled to nucleophilic moieties on the biomolecule, or nucleophilic linkages on the substrate coupled to electrophilic linkages on the biomolecule. Due to the hydrophilic nature of the most common biomolecules of interest, the solvent of choice for these couplings is water, or water containing some water-soluble organic solvent to disperse the biomolecule on the substrate. In particular, polynucleotides are generally coupled to substrates in aqueous systems due to their polyanionic nature. Because water competes with the electrophile and nucleophile by hydrolyzing the electrophile to an inactive moiety for conjugation, aqueous systems generally result in low yields of coupled product, the yield being based on the electrophilic moiety of the pair. When a high yield of the electrophilic moiety of the reactive pair is desired, a high concentration of nucleophile is required to drive the reaction and mitigate hydrolysis, resulting in inefficient use of the nucleophile. In the case of polynucleic acids, the metal phosphate counterion can be replaced with a lipophilic counterion to facilitate solubilization of the biomolecule in polar, non-reactive, non-aqueous solvents. These solvents can include amides or ureas, such as formamide, N,N-dimethylformamide, acetamide, N,N-dimethylacetamide, hexamethylphosphoramide, pyrrolidone, N-methylpyrrolidone, N,N,N'N'-tetramethylurea, N,N'-dimethyl-N,N'-trimethyleneurea, or combinations thereof; carbonates, such as dimethyl carbonate, propylene carbonate, or combinations thereof; ethers, such as tetrahydrofuran; sulfoxides and sulfones, such as dimethyl sulfoxide, dimethyl sulfone, or combinations thereof; hindered alcohols, such as tert-butyl alcohol; or combinations thereof.Lipophilic cations can include tetraalkylammonium or tetraarylammonium cations, such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, tetrapentylammonium, tetrahexylammonium, tetraheptylammonium, tetraoctylammonium, and alkyl and aryl mixtures thereof, tetraarylphosphonium cations, such as tetraphenylphosphonium, tetraalkylarsonium, or tetraarylarsonium, such as tetraphenylarsonium, and trialkylsulfonium cations, such as trimethylsulfonium, or combinations thereof. Conversion of polynucleic acids to organic solvent-soluble materials by exchanging metal cations for lipophilic cations can be carried out by a variety of standard cation exchange techniques.

[0248] The polymer particles can be activated to facilitate conjugation with target analytes, such as polynucleotides or other affinity ligands. For example, functional groups on crosslinked hydrogel polymer particles can be enhanced to allow binding with target analytes or analyte receptors. In certain examples, the functional groups of the polymer can be modified with a reagent that can convert the hydrophilic polymer functional groups into reactive moieties that can undergo nucleophilic or electrophilic substitution. In particular, if the polymer particles have carboxyl functional groups, these can be activated to facilitate conjugation with biomolecules, such as nucleic acids.

[0249] In embodiments in which particles are formed from comonomers containing hydroxyl groups, the hydroxyl groups on the hydrophilic particles can be activated by replacing at least a portion of the hydroxyl groups with sulfonate groups or chlorine. Exemplary sulfonate groups can be derived from tresyl, mesyl, tosyl, or tosyl chloride, or any combination thereof. The sulfonate can act to replace a nucleophile with the sulfonate. The sulfonate can further react with the liberated chlorine to provide a chlorinated group that can be used in the process of binding particles. In another example, amine groups on a hydrophilic polymer can be activated.

[0250] For example, a target analyte or analyte receptor can be attached to a hydrophilic polymer via nucleophilic substitution with a sulfonate group. In certain instances, a target analyte receptor terminated with a nucleophile, such as an amine or thiol, can undergo nucleophilic substitution to replace a sulfonate group on the surface of the hydrophilic polymer. As a result of activation, a conjugated particle can be formed.

[0251] In another example, if the particle contains an amine (e.g., if the particle is formed from a monomer that contains an amine or contains a coating that contains an amine), the nucleophilic amino group can be modified with a difunctional bis-electrophilic moiety, such as a diisocyanate or a bis-NHS ester, to provide a hydrophilic particle that is reactive to nucleophiles.

[0252] When conjugated to a polynucleotide, the polymer particles are 1 μm 3 At least 7 x 10 per 4 For example, the nucleotide density can be 1 μm 3 At least 10 per 5 , e.g., 1 μm 3 At least 10 per 6 , 1 μm 3 At least 5 x 10 per 6 , 1 μm 3 At least 8 x 10 per6 , 1 μm 3 At least 1 x 10 per 7 , or even 1 μm 3 At least 3 x 10 per 7 In a further example, the nucleotide density can be 1 μm 3 10 per 15 It can be the following:

[0253] Such polymer particles can be used in a variety of separation and analytical techniques. In particular, polymer particles can be useful for binding polynucleotides. Such bound polynucleotides can be useful for separating polynucleotides from solution or can be used in analytical techniques such as sequencing. In a specific example shown in Figure 5, such polymer particles can be used as supports for polynucleotides during sequencing techniques. For example, the particles can immobilize polynucleotides for sequencing using fluorescent sequencing techniques.

[0254] Generally, polymer particles can be engineered to contain biomolecules, including nucleosides, nucleotides, nucleic acids (oligonucleotides and polynucleotides), polypeptides, sugars, polysaccharides, lipids, or derivatives or analogs thereof. For example, polymer particles can be bound or attached to biomolecules. The termini or any internal portion of the biomolecule can be bound or attached to the polymer particle. The polymer particles can be bound or attached to biomolecules using linking chemistries. Linking chemistries include covalent or non-covalent bonds, including ionic bonds, hydrogen bonds, affinity bonds, dipole-dipole bonds, van der Waals bonds, and hydrophobic bonds. Linking chemistries include affinities between binding partners, e.g., an avidin moiety and a biotin moiety; an antigenic epitope and an antibody or immunologically reactive fragment thereof; an antibody and a hapten; a digoxigen moiety and an anti-digoxigen antibody; a fluorescein moiety and an anti-fluorescein antibody; an operator and a repressor; a nuclease and a nucleotide; a lectin and a polysaccharide; a steroid and a steroid-binding protein; an active compound and an active compound receptor; a hormone and a hormone receptor; an enzyme and a substrate; an immunoglobulin and protein A; or an oligonucleotide or polynucleotide and its corresponding complement.

[0255] 5, a plurality of polymer particles 204 (e.g., monodisperse crosslinked hydrogel polymer particles of the present disclosure) can be placed in solution with a plurality of polynucleotides 202. The plurality of particles 204 can be activated or otherwise prepared to bind with the polynucleotides 202. For example, the particle 204 can include an oligonucleotide that is complementary to a portion of one of the plurality of polynucleotides 202.

[0256] In certain embodiments, the polymer particles and polynucleotides are subjected to polymerase chain reaction (PCR) amplification, such as emulsion PCR. For example, dispersed phase droplets 206 or 208 can be formed as part of an emulsion and can contain particles or polynucleotides. In one example, polynucleotides 202 and hydrophilic particles 204 are provided at low concentrations and ratios relative to each other, such that a single polynucleotide 202 can be present in the same dispersed phase droplet as a single polymer particle 204. Other droplets, such as droplet 208, can contain a single polymer particle and no polynucleotides. Each droplet 206 or 208 can contain sufficient enzymes, nucleotides, salts, or other components to facilitate replication of the polynucleotide. Alternatively, amplification techniques such as recombinase polymerase amplification (RPA), with or without an emulsion, can be used.

[0257] In one aspect, the present invention provides the use of the hydrogel polymer particles of the present invention in nucleic acid amplification, which may be emulsion PCR.

[0258] In some embodiments, a method for nucleic acid amplification includes performing a primer extension reaction on a polynucleotide hybridized to an oligonucleotide bound to a polymer particle. In some embodiments, the polymer particle is a monodisperse crosslinked hydrogel polymer particle of the present disclosure. In some embodiments, the method for nucleic acid amplification includes (a) providing a polymer particle having a single-stranded oligonucleotide (e.g., a primer oligonucleotide) attached thereto; (b) providing a single-stranded template polynucleotide; (c) hybridizing the single-stranded oligonucleotide to the single-stranded template polynucleotide; and (d) contacting the single-stranded template polynucleotide with a polymerase and at least one nucleotide under conditions suitable for the polymerase to catalyze the polymerization of at least one nucleotide into a single-stranded oligonucleotide, thereby generating an extended single-stranded oligonucleotide. In some embodiments, the method further includes (e) removing (e.g., denaturing) the single-stranded template polynucleotide from the extended single-stranded oligonucleotide so that the single-stranded oligonucleotide remains attached to the polymer particle; (f) hybridizing the remaining single-stranded oligonucleotide to a second single-stranded template polynucleotide; and (g) contacting the second single-stranded template polynucleotide with a second polymerase and at least one second nucleotide under conditions suitable for the second polymerase to catalyze the polymerization of the second at least one nucleotide into the single-stranded oligonucleotide to produce the subsequent extended single-stranded oligonucleotide. In some embodiments, steps (e), (f), and (g) can be repeated at least once. In some embodiments, the polymerase and the second polymerase comprise a thermostable polymerase. In some embodiments, the conditions suitable for nucleotide polymerization include performing the nucleotide polymerization step (e.g., step (d) or (g)) at an elevated temperature. In some embodiments, the conditions suitable for nucleotide polymerization include performing the nucleotide polymerization step (e.g., step (d) or (g)) at alternating temperatures (e.g., high and relatively low temperatures). In some embodiments, the alternating temperatures range from 60 to 95°C.In some embodiments, the temperature cycle can be from about 10 seconds to about 5 minutes, or about 10 minutes, or about 15 minutes or more. In some embodiments, the nucleic acid amplification method can generate one or more polymer particles each attached to a plurality of template polynucleotides, each comprising a sequence complementary to the single-stranded template polynucleotide or the second single-stranded template polynucleotide. In some embodiments, each of the polymer particles can be bound to a plurality of single-stranded oligonucleotides (e.g., capture oligonucleotides). In some embodiments, steps (b), (c), (d), (e), (f), or (g) can be performed using a plurality of single-stranded polynucleotides. In some embodiments, at least a portion of the single-stranded oligonucleotides comprise a nucleotide sequence complementary (or partially complementary) to at least a portion of the single-stranded polynucleotide. In some embodiments, the method for nucleic acid amplification (as described above) can be performed in an aqueous phase solution (e.g., dispersed phase droplets) in an oil phase.

[0259] Following PCR, a particle, e.g., particle 210, is formed, which may contain polymer particles 212 and multiple copies 214 of a polynucleotide. For clarity, most of the polynucleotide 214 is shown on the exterior surface of particle 210. However, as also shown, the polynucleotide can extend (or be located) within particle 210. For example, hydrogels and hydrophilic particles can have a low concentration of polymer relative to water and thus be relatively porous. They can contain polynucleotide segments within and throughout particle 210, and polynucleotides can reside in pores and other openings. In particular, particle 210 allows diffusion of enzymes, nucleotides, primers, and reaction products used to monitor the reaction. A larger number of polynucleotides per particle generates a better signal.

[0260] In some embodiments, the polymer particles from the emulsion-breaking procedure can be collected and washed in preparation for sequencing. Collection can be performed by contacting biotin moieties (e.g., linked to amplified polynucleotide templates bound to the polymer particles) with avidin moieties and separating them from polymer particles lacking biotinylated templates. Collected polymer particles carrying double-stranded template polynucleotides can be denatured to obtain single-stranded template polynucleotides for sequencing. The denaturation step can include treatment with a base (e.g., NaOH), formamide, or pyrrolidone.

[0261] Another sequencing system is the Applied Biosystems SOLiD™ Sequencing System (Sequencing by Oligonucleotide Ligation and Detection), which uses stepwise cycle ligation reactions for high-throughput DNA sequencing. In this bead-based system, beads (i.e., polymer particles) loaded with DNA templates undergo sequential ligation and cleavage reactions using four-color fluorescently labeled octamer probes. These probes are delivered sequentially and serve to interrogate dinucleotide positions on the DNA strand. It would be desirable to support higher bead densities, which would facilitate an increased number of bead events per instrument run and improved probe chemistry, allowing for increased sequencing fidelity.

[0262] Sequencing by oligonucleotide ligation and detection involves attaching a nucleic acid target to a cross-linked polymer particle (bead), followed by immobilizing multiple particles on a surface. Each nucleic acid-bead conjugate contains a unique DNA sequence, and this type of sequencing technology is disclosed in International Publication No. WO2006 / 084132A2 (incorporated herein by reference).

[0263] The method for attaching beads to a support utilizes flat glass microscope slides irreversibly coated with streptavidin. The beads containing nucleic acids are contacted with biotinylated nucleotides (e.g., obtained by the action of biotinylated dNTPs and terminal deoxytransferase on the DNA target after attachment to the beads). Incubation of the biotinylated beads with the streptavidin-coated slide results in immobilization of the beads on the slide via biotin-streptavidin interaction. Kinetics-wise, this is a very effective attachment scheme, although movement of the beads on the slide has sometimes been observed under conditions required by DNA sequencing assays. When beads are present on the slide at high density (e.g., up to 100,000 beads / mm), the beads are not readily transported to the slide. 2 ), when interrogated multiple times (e.g., up to 25 times), any significant bead movement can prevent robust identification of a specific bead in subsequent scans within the dense population of beads.

[0264] Thus, US2009 / 0099027 (equivalent to WO2009 / 026546, both of which are incorporated herein by reference) describes a covalent bonding system for bead immobilization that reduces bead migration during sequencing and other forms of genetic analysis. The method includes reacting nucleophilic groups on the surface of a substrate with a molecule containing multiple electrophilic groups, thereby providing one or more free electrophilic groups on the surface of the substrate, and reacting nucleophilic groups on the surface of a particulate material with one or more free electrophilic groups on the surface of the substrate to covalently bond the particulate material to the substrate.

[0265] US2009 / 0099027 describes the modification of nucleophilic (more specifically amino-functional) surfaces with multifunctional electrophiles. For example, the electrophilic surface of a silicate glass microscope slide can be easily converted to a nucleophilic surface by reacting the surface groups with (aminopropyl)trialkoxysilane.

[0266] DNA target nucleic acids covalently attached to crosslinked polymer beads can be modified by the action of aminoalkyl dNTPs and terminal deoxytransferase on the DNA target after attachment to the beads. The nucleophilic amino groups on the DNA target can then react with residual electrophilic groups on the support surface to form multiple stable covalent bonds between the beads and the glass surface.

[0267] It has been found that stable covalent bonds can be formed between surfaces containing electrophilic groups and particles containing nucleophilic groups. Furthermore, beads containing nucleophilic amino groups from the action of amino-dNTPs and terminal deoxytransferase on DNA targets can be immobilized on modified surfaces under aqueous basic conditions. For example, a surface containing amino groups activated with benzene 1,4-diisothiocyanate can be used to immobilize beads with nucleophilic groups. Furthermore, the covalent bond appears to be very stable, and no bead movement is observed.

[0268] Surface-immobilized beads can be used in the method of analyzing nucleic acid sequences based on the repeated cycle of double-stranded extension along single-stranded templates by ligation.This type of sequencing method is disclosed in U.S. Patent No. 5,750,341, U.S. Patent No. 5,969,119 and U.S. Patent No. 6,306,597B1 and International Publication No. WO2006 / 084132A2.Each of these publications is incorporated herein by reference in its entirety.Furthermore, the techniques described in the above-mentioned publications can be used to analyze (for example, sequence) the nucleic acid templates attached to the support-bound particles described herein.Immobilized beads can be used in sequencing methods that do not necessarily use ligation steps, for example, sequencing using labeled nucleotides with removable protecting groups that prevent polynucleotide chain elongation (for example, U.S. Patent No. 6,664,079, U.S. Patent No. 6,232,465 and U.S. Patent No. 7,057,026, each of which is incorporated herein by reference in its entirety). The immobilized beads can be used in a variety of techniques in which the signal on the beads is repeatedly detected through multiple cycles.

[0269] The beads used in SOLiD sequencing can be monodisperse magnetic and / or coated hydrogel particles of the present disclosure. Accordingly, one embodiment includes the use of monodisperse particles in the methods and products disclosed in the publications mentioned in the above paragraph, and the present applicant considers all such uses, methods, and products to be within the scope of the present invention and reserves the right to claim them. The use of submicron particles in SOLiD sequencing allows for the attachment of higher density particles to glass surfaces (e.g., glass panels or microscope slides). Also included in one embodiment is a method of performing SOLiD sequencing using monodisperse particles of the present disclosure, in which the monodisperse submicron particles of the present disclosure are bound to a nucleic acid target and immobilized on a surface, such as a glass surface. The method of immobilization is not critical and can be covalent or non-covalent; an example of non-covalent binding is via streptavidin / avidin-biotin binding. Covalent bonding can be as described in, for example, US2009 / 0099027 and WO2009 / 026546, but any other suitable technique for covalent bonding can be used.Therefore, one embodiment includes the method of forming an article of manufacture, which further comprises the monodisperse submicrometer particles of the present disclosure being bonded to nucleic acid, and optionally, the nucleic acid-containing particles obtained are immobilized on a surface, for example, a glass surface.Nucleic acid can be used as a target in sequencing, for example, by using SOLiD sequencing.

[0270] for example, (a) hybridizing a first initializing oligonucleotide probe to a target polynucleotide to form a probe-target duplex, wherein the oligonucleotide probe has an extendable probe end, the target polynucleotide is bound to a polymer particle that is a member of a population of polymer particles disclosed herein, and the particle is covalently attached to the surface of a solid support; (b) ligating a first end of an extender oligonucleotide probe to the extendable probe end, thereby forming an extended duplex comprising the extender oligonucleotide probe, wherein the extender oligonucleotide probe comprises a cleavage site and a detectable label; (c) identifying one or more nucleotides in the target polynucleotide by detecting a label attached to the newly ligated extender oligonucleotide probe; (d) cleaving the newly ligated extender oligonucleotide probe at the cleavage site to generate an extendable probe end, wherein the cleavage removes a portion of the newly ligated extender oligonucleotide probe that includes the label from the probe-target duplex; and (e) repeating steps (b), (c), and (d) until the sequence of the nucleotides in the target polynucleotide is determined. A method is provided, comprising:

[0271] (a) hybridizing a primer to a target polynucleotide to form a primer-target duplex, wherein the target polynucleotide is attached at its 5' end to a polymer particle that is a member of a population of polymer particles disclosed herein, and the polymer particle is covalently attached to a surface of a support; (b) contacting the primer-target duplex with a polymerase and one or more different nucleotide analogs to incorporate the nucleotide analogs onto the 3' end of the primer, thereby forming an extended primer strand, wherein the incorporated nucleotide analog terminates the polymerase reaction and each of the one or more nucleotide analogs comprises: (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil and analogs thereof; (ii) a unique label attached to the base or its analog via a cleavable linker; (iii) a deoxyribose; and (iv) a cleavable chemical group that caps the -OH group at the 3' position of the deoxyribose; (c) washing the surface of the support to remove any unincorporated nucleotide analogues; (d) detecting the unique label attached to the newly incorporated nucleotide analog, thereby identifying the newly incorporated nucleotide analog; (e) optionally, permanently capping any unreacted -OH groups on the extended primer strand; (f) cleaving the cleavable linker between the newly incorporated nucleotide analog and the unique label; (g) cleaving the chemical group capping the -OH group at the 3' position of the deoxyribose of the newly incorporated nucleotide analogue to uncap the -OH group; (h) washing the surface of the support to remove the cleaved compound; (i) Repeating steps (b) to (h) Also provided is a method for sequencing a nucleic acid, comprising:

[0272] The polymer particles of the present disclosure can be used in any method of nucleic acid sequencing that includes polymer particles.One embodiment includes the particles of the present disclosure that are bound to nucleic acid, and the method of sequencing nucleic acid that includes the step of binding nucleic acid to a population of particles of the present disclosure.Nucleic acid can be DNA or RNA.

[0273] The present disclosure includes articles of manufacture (e.g., articles of manufacture) comprising a plurality of monodisperse particles of the present disclosure attached to a substrate, such as a glass surface, via, for example, streptavidin-biotin linkages, avidin-biotin linkages, or covalent linkages, as described, for example, in US2009 / 0099027 and WO2009 / 026546. The particles may be attached to the substrate via nucleic acids. The present disclosure includes the use of monodisperse particles of the present disclosure to produce such articles. One embodiment includes the use of the attachment chemistry described in US2009 / 0099027 and WO2009 / 026546 to attach monodisperse particles of the present disclosure to a substrate, and the applicant reserves the right to claim methods using such chemistry and the products thereof. Accordingly, the present specification incorporates by reference the disclosures of US2009 / 0099027 and WO2009026546.

[0274] Thus, embodiments include methods of subjecting the functionalized monodisperse polymer particles of the present disclosure to one or more further reactions to obtain desired products. Other embodiments include the use of these products in the present application.

[0275] Considering the consistency of quality and characteristics that the particles of the present disclosure can have, they can be used in methods that involve carrying out processes with conjugated substances selected from, for example, labels, biological molecules, and biological structures, such as biological molecules such as amino acids, sugars, nucleotides, and nucleosides, and multimers made by condensing two or more such monomers together, such as polypeptides, proteins, polysaccharides, oligonucleotides, and nucleic acids. Labels can include dyes, such as fluorescent dyes, quenchers, enzymes, and semiconductor nanocrystals. Embodiments of the present invention are suitable for such applications, as well as for the production of conjugated substances. (i) a conjugate comprising a population of particles of the present disclosure, at least some of which are bound to a conjugated substance, e.g., those just described; (ii) a method comprising the step of attaching at least a portion of the population of particles of the present disclosure to a substance, such as those just described; (iii) attaching at least a portion of the population of particles of the present disclosure to a substrate. Includes.

[0276] Analysis method Molecular weight measurement The weight-average molecular weight (Mw) of the oligomer in the seed particles can be determined from measurements performed using gel permeation chromatography (GPC). In GPC, a series of polymer particle standards are run and used to generate a calibration curve. The Mw of the oligomer can be measured by GPC relative to polystyrene standards using DMF containing 0.01 M LiBr as the eluent. Because these Mw values ​​are calculated relative to standards of a different polymer (polystyrene) than that of the seed particles, the calculated Mw represents a relative value, not an absolute value. Therefore, although the measurements are reproducible, they do not provide the actual Mw.

[0277] A summary of the GPC methods used in the examples provided herein is as follows: The following experimental conditions were used: Eluent: DMF containing 0.01M LiBr Precolumn: PSS GRAM, 10 μm, Guard ID 8.0 mm x 50 mm Column: PSS GRAM, 10 μm, Linear M ID 8.0 mm x 300 mm PSS GRAM, 10 μm, Linear M ID 8.0 mm x 300 mm Temperature: 70°C Pump: PSS SECcurity 1260 HPLC pump Flow rate: 1.0mL / min Injection system: PSS SECcurity 1260 autosampler Injection volume: 50μL Sample concentration: 3.0g / L Detector: PSS SECcurity 1260 Refractive Index Detector (RID) Chromatography data system: PSS WinGPC UniChrom version 8.2 Polystyrene standards with different molecular weights were measured under the above experimental conditions to obtain a calibration curve.The samples were then analyzed.The Mw of the samples was then calculated based on the PS calibration curve.

[0278] Size and size distribution The size distribution of a sample can be measured using a disc centrifuge, such as the CPS Disc Centrifugation™ on a Disc Centrifuge Model DC20000, using the protocol provided by the instrument manufacturer. Accurate results require calibration with standards of similar density to the sample being analyzed; therefore, suitable polymer standards are only used if available, for example, a set of small polystyrene particle standards for the particles of the present disclosure, which primarily comprise polystyrene. If the sample being measured has an unknown density, for example, for porous particles, measurements obtained by CPS disc centrifugation will be reproducible but will not provide the actual diameter.

[0279] A summary of the CPS Disc Centrifuge™ used in the examples provided herein is as follows: Those skilled in the art can readily adapt this method as appropriate for other samples, for example, by selecting suitable gradients, disc speeds, and standard particles based on the size and porosity of the particles being analyzed. Disc centrifugation analysis was performed on a CPS instruments CPS DC20000 with a disc speed of 10,000 rpm and a gradient of 8–24 wt% sucrose in 1.5 g / L SDS (aqueous solution). The gradient was generated using a CPS instruments automated gradient pump, and the volume of the injected gradient was 16–17 mL. Prior to injection, samples were diluted to approximately 0.01 wt% in MilliQ HO. The method used for the analysis had the following settings: maximum diameter 4.0 μm, minimum diameter 0.1 μm, particle density 1.6 g / mL, particle refractive index 1.592, particle absorption 0.2, particle asphericity 1, calibration standard diameter 1.098, calibration standard density 1.6, standard half maximum 0.2 μm, liquid density 1.06 g / mL, liquid refractive index 1.355. The reported size is the absorption peak diameter, and the CV is determined by setting boundaries around the main peak.

[0280] Photon correlation spectroscopy (PCS) can be used to obtain the hydrodynamic diameter of particles in the form of z-average. The measurement is independent of particle density and is based on the Brownian motion of small particles. PCS measurements of nano-sized particles can be obtained, for example, using a Malvern Zetasizer Nano-ZS, Model ZEN3600. Further details and methods can be found in the Malvern Zetasizer Nano series instruction manual (incorporated herein in its entirety by reference).

[0281] Another technique that can be used to determine particle size and size distribution is optical microscopy. A population of beads can be prepared by placing an aqueous solution containing beads on a microscope slide, then capturing images of the beads at a suitable level of magnification, for example, 100x or greater, and analyzing the size of the beads using image analysis software.

[0282] The optical microscopy method used in the examples provided herein is outlined below. Microscopy samples were prepared by sandwiching a 0.12 mm diameter, 9 mm Secure-Seal™ spacer between a cover glass and a Secure-Seal™ hybridization chamber gasket. The formed cavity was filled with 8 μL of beads diluted in 150 mM NaCl, followed by brief centrifugation and mounting on a microscope slide. Phase-contrast optical microscopy was then performed on an Olympus IX81 inverted microscope using a 100x lens. Multiple images were automatically collected using a Matlab-based macro for μManager. An out-of-focus image was collected as background and subtracted from the phase-contrast image. The background-subtracted image was then thresholded using Otsu's method to identify individual beads for size analysis. Standard-sized green fluorescent polystyrene beads were used to calibrate the bead size measured by fluorescence microscopy. A series of Alexa 488-labeled hydrogel beads was then used to calibrate the phase-contrast size to the fluorescence size.

[0283] Optical microscopy is the preferred method for measuring particle size because it provides measurements independent of particle density. Disk centrifuge analysis is the preferred method for measuring particle size distribution (CV) because optical microscopy is based on image analysis, and the presence of artifacts in the image can result in artificially high CV.

[0284] magnetic susceptibility Magnetic susceptibility was determined using a Bartington Instruments MS3 meter, magnetic susceptibility system, equipped with an MS2B dual frequency sensor. Samples for measurement were prepared by weighing 100.0 ± 20.0 mg of particles diluted in 7.0000 ± 0.1000 g of aluminum oxide. Each batch was analyzed in duplicate with mass-specific correction using the following settings: measurement duration 1 second, number of repetitions 1, and number of cycles 100.

[0285] Vibrating Sample Magnetometry (VSM) VSM was performed on a LakeShore 7400. 3–10 mg of sample was weighed and placed in a locking ring gelatin capsule (Torpac). To prevent movement during measurement, the capsule was secured with adhesive and allowed to dry overnight. Hysteresis curves were collected by first setting the magnetic field to 10,000 Oe, then gradually decreasing it to -10,000 Oe, and finally gradually increasing it to 10,000 Oe.

[0286] Iron content Quantitative measurement of the iron content of magnetic particles was performed by Alcontrol Laboratories Norway by dissolving 0.11 g of particles in 5 mL of aqua regia followed by microwave heating. The sample was diluted to 50 mL with H2O and finally diluted 250 times. The iron concentration was then determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis performed on a Varian 720-ES instrument. [Example]

[0287] Seed particle synthesis Example 1 A 2 L jacketed round-bottom flask equipped with a mechanical anchor stirrer and condenser was charged with 34.6 g of distilled N,N-dimethylacrylamide, 27.6 g of Kraton G1650, 1.5 g of 2,2'-azobis(2-methylpropionitrile), 747.9 g of heptane, 187.0 g of toluene, and 1.2 g of 1-octanethiol. Once everything was dissolved, the reaction mixture was purged with Ar(g) for 60 minutes. The reaction was then heated to 71 °C using a temperature-controlled water bath connected to the reactor and stirred at 400 rpm for 16 hours. The resulting dispersion was analyzed by dynamic light scattering, which gave a z-average diameter of 360 nm.

[0288] Example 2 A 2 L jacketed round-bottom flask equipped with a mechanical anchor stirrer and condenser was charged with 34.6 g of distilled N,N-dimethylacrylamide, 27.7 g of Kraton G1650, 1.4 g of 2,2'-azobis(2-methylpropionitrile), 747 g of heptane, 187 g of toluene, and 2.5 g of 1-octanethiol. Once everything was dissolved, the reaction mixture was purged with Ar(g) for 60 minutes. The reaction was then heated to 71 °C using a temperature-controlled water bath connected to the reactor and stirred at 400 rpm for 16 hours. The resulting dispersion was analyzed by dynamic light scattering, which gave a z-average diameter of 970 nm.

[0289] Synthesis of polymer particles from low molecular weight seed particles Example 3 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 7.4 g of acrylamide (AAm), 2.5 g of N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBAAm), and 14.9 g of HO. The mixture was shaken at 61 °C until everything was dissolved.

[0290] Solution 2 (stabilizer): 33 g of Abil WE09, 36 g of mineral oil, and 145 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stir bar until everything was dissolved.

[0291] Solution 3 (emulsion): 66 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. Further emulsification was performed by sonication using a Hielscher Ultrasound Technology UP 200s with a frequency setting of 0.9 and an amplitude setting of 40%. In the next step, Solution 3 and 9.5 g of PDMAAm seeds (Example 1) were charged into a 250 mL three-neck round-bottom flask equipped with a condenser, mechanical stirrer, and gas inlet, and stirred at 100 rpm at 60 °C for 1 hour. Finally, the flask was purged with Ar(g) for 15 minutes, after which 2.6 g of 2,2'-azodi(2-methylbutyronitrile) (AMBN) dissolved in 2.6 g of toluene was added and heated to 70 °C for 7 hours with stirring at 50 rpm.

[0292] Redispersion: 30 g of crude product was added to a 250 mL centrifuge flask and charged with 1 g of N-methyl-2-pyrrolidone (NMP). The flask was shaken for 2 hours and then centrifuged at 2000 rpm for 5 minutes. The supernatant was removed and the product was further washed twice with NMP and three times with HO before finally being redispersed in HO.

[0293] Example 4 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 7.4 g of N-[tris(hydroxymethyl)methyl]acrylamide (THMAAm), 2.5 g of N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBAAm), and 14.9 g of HO. The mixture was shaken at 61 °C until everything was dissolved.

[0294] Solution 2 (stabilizer), solution 3 (emulsion), and redispersant: same as in Example 3.

[0295] Example 5 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 7.4 g of hydroxyethylacrylamide (HEAAm), 2.5 g of N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBAAm), and 14.9 g of HO. The mixture was shaken at 61 °C until everything was dissolved.

[0296] Solution 2 (stabilizer), solution 3 (emulsion), and redispersant: same as in Example 3.

[0297] Example 6 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 7.5 g of hydroxymethylacrylamide (HMAAm), 5.0 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 12.4 g of HO. The mixture was stirred until everything was dissolved.

[0298] Solution 2 (stabilizer): 35 g of Abil WE09, 38 g of mineral oil, and 152 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stir bar until everything was dissolved.

[0299] Solution 3 (emulsion): 71 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. Further emulsification was performed by sonication using a Hielscher Ultrasound Technology UP 200s with a frequency setting of 0.9 and an amplitude setting of 40%. In the next step, Solution 3 and 4.2 g of PDMAAm seeds (Example 1) were charged into a 250 mL three-necked round-bottom flask equipped with a condenser, a mechanical stirrer, and a gas inlet, and stirred at 100 rpm at 19 °C for 1 hour. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70 °C with stirring at 50 rpm for 7 hours.

[0300] Redispersion: 50 g of crude product was added to a 250 mL centrifuge flask and charged with 1 g of N-methyl-2-pyrrolidone (NMP). The flask was shaken for 1 hour and then centrifuged at 4000 rpm for 5 minutes. The supernatant was removed and the product was further washed twice with NMP and five times with HO before finally being redispersed in HO.

[0301] Example 7 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 7.5 g of hydroxymethylacrylamide (HMAAm), 2.5 g of piperazine diacrylamide, and 15.0 g of HO. The mixture was stirred until everything was dissolved.

[0302] Solution 2 (stabilizer), solution 3 (emulsion), and redispersant: same as in Example 6.

[0303] Example 8 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 7.5 g of hydroxymethylacrylamide (HMAAm), 5.0 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 12.4 g of HO. The mixture was stirred until everything was dissolved.

[0304] Solution 2 (stabilizer): 24 g of Abil WE09, 26 g of mineral oil, and 104 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 250 mL beaker and mixed with a magnetic stir bar until everything was dissolved.

[0305] Solution 3 (emulsion): Same as in Example 6.

[0306] Redispersion: 25 g of crude product was added to a 250 mL centrifuge flask and charged with 1 g of N-methyl-2-pyrrolidone (NMP). The flask was shaken for 3 days and then centrifuged at 5000 rpm for 10 minutes. The supernatant was removed and the product was further washed twice with NMP and four times with HO before finally being redispersed in HO.

[0307] Example 9 Solution 1 (monomer phase): A 500 mL Duran flask was charged with 36.8 g of hydroxymethylacrylamide (HMAAm), 2.6 g of acrylic acid, 39.4 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 118.3 g of HO. The mixture was shaken until everything was dissolved.

[0308] Solution 2 (stabilizer): 7.6 g of 2,2'-azodi(2-methylbutyronitrile) (AMBN), 95.6 g of Abil WE09, 104.3 g of mineral oil, and 417.2 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 1 L beaker and mixed with a magnetic stir bar until all was dissolved.

[0309] Solution 3 (emulsion): 520 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. Further emulsification was performed by sonication using a Hielscher Ultrasound Technology UP 200s with a frequency setting of 0.9 and an amplitude setting of 40%. In the next step, Solution 3 and 40 g of PDMAAm seeds (Example 2) were charged into a 1 L jacketed cylindrical reactor equipped with a condenser, mechanical stirrer, and gas inlet and stirred at 100 rpm at 22 °C for 80 minutes. Finally, the flask was purged with Ar(g) for 30 minutes and then heated to 70 °C for 20 hours with stirring at 55 rpm.

[0310] Redispersion and analysis: The crude product was divided into 2 x 1 L centrifuge flasks and centrifuged at 3600 rpm for 15 minutes. The supernatant was removed and the particles were redispersed in isopropanol. The redispersed particles were then divided into a total of 4 x 1 L centrifuge bottles and washed four times with isopropanol and eight times with HO before finally being redispersed in HO. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter to a standard was determined to be 1.62 μm with a CV of 16.1.

[0311] Example 10 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 3.7 g of acrylic acid, 3.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 17.3 g of HO. The mixture was shaken until everything was dissolved.

[0312] Solution 2 (stabilizer): 2.6 g of 2,2'-azodi(2-methylbutyronitrile) (AMBN), 33 g of Abil WE09, 36 g of mineral oil, and 144 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stir bar until all was dissolved.

[0313] Solution 3 (emulsion): 66 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. Further emulsification was performed by sonication using a Hielscher Ultrasound Technology UP 200s with a frequency setting of 0.9 and an amplitude setting of 40%. In the next step, Solution 3 and 9.9 g of PDMAAm seeds (Example 1) were charged into a 250 mL three-necked round-bottom flask equipped with a condenser, mechanical stirrer, and gas inlet and stirred at 100 rpm at 20°C for 1 hour. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C for 7 hours with stirring at 62 rpm.

[0314] Redispersion and analysis: 77 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 minutes. The supernatant was removed and the particles were redispersed in isopropanol followed by a total of three washes in isopropanol. The particles were redispersed in HO, and the redispersed particles were transferred to a 1 L centrifuge flask and washed twice with HO. The batch was then transferred to 3 x 250 mL centrifuge flasks and washed once more before finally being redispersed in HO. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter to a standard was determined to be 0.37 μm with a CV=5.4%.

[0315] Example 11 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 1.9 g of hydroxymethylacrylamide (HMAAm), 1.9 g of acrylic acid, 3.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 17.3 g of HO. The mixture was shaken until everything was dissolved.

[0316] Solution 2 (stabilizer), Solution 3 (emulsion): Same as in Example 10.

[0317] Redispersion and analysis: As in Example 10, but using 78 g of crude oil. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter to a standard was determined to be 0.43 μm with a CV=5.6%.

[0318] Example 12 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 2.8 g of hydroxymethylacrylamide (HMAAm), 0.9 g of acrylic acid, 3.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 17.3 g of HO. The mixture was shaken until everything was dissolved.

[0319] Solution 2 (stabilizer), Solution 3 (emulsion): Same as in Example 10.

[0320] Redispersion and analysis: As in Example 10, but using 78 g of crude oil. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter to a standard was determined to be 0.43 μm with a CV=5.2%.

[0321] Example 13 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 3.7 g of hydroxymethylacrylamide (HMAAm), 3.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 17.3 g of HO. The mixture was shaken until everything was dissolved.

[0322] Solution 2 (stabilizer): 2.6 g of 2,2'-azodi(2-methylbutyronitrile) (AMBN), 33 g of Abil WE09, 36 g of mineral oil, and 144 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stir bar until all was dissolved.

[0323] Solution 3 (emulsion): 66 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. Further emulsification was performed by sonication using a Hielscher Ultrasound Technology UP 200s with a frequency setting of 0.9 and an amplitude setting of 40%. In the next step, Solution 3 and 9.9 g of PDMAAm seeds (Example 1) were charged into a 250 mL three-necked round-bottom flask equipped with a condenser, mechanical stirrer, and gas inlet and stirred at 100 rpm at 20°C for 1 hour. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C for 7 hours with stirring at 62 rpm.

[0324] Redispersion and analysis: 78 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 minutes. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 minutes. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H2O and divided into a total of three 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed three times with H2O before finally being redispersed in H2O. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter to a standard was determined to be 0.43 μm with a CV of 5.4%.

[0325] Example 14 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 3.4 g of hydroxyethylacrylamide (HEAAm), 0.3 g of acrylic acid (AA), 3.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 17.3 g of HO. The mixture was shaken until everything was dissolved.

[0326] Solution 2 (stabilizer), Solution 3 (emulsion): Same as in Example 13.

[0327] Redispersion and analysis: 77 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 minutes. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 minutes. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H2O and divided into a total of three 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed three times with H2O before finally being redispersed in H2O. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter to a standard was determined to be 0.41 μm with a CV of 6.1%.

[0328] Example 15 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 3.4 g of N-[tris(hydroxymethyl)methyl]acrylamide (THMAAm), 0.3 g of acrylic acid (AA), 3.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 17.3 g of HO. The mixture was shaken until everything was dissolved.

[0329] Solution 2 (stabilizer), Solution 3 (emulsion): Same as in Example 13.

[0330] Redispersion and analysis: 78 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 minutes. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 minutes. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H2O and divided into a total of three 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed three times with H2O before finally being redispersed in H2O. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter to a standard was determined to be 0.42 μm with a CV=5.7%.

[0331] Example 16 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 3.2 g of hydroxymethylacrylamide (HMAAm), 0.5 g of fumaric acid, 3.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 17.3 g of HO. The mixture was heated to 40 °C and mixed with a magnetic stir bar until everything was dissolved.

[0332] Solution 2 (stabilizer): 2.6 g of 2,2'-azodi(2-methylbutyronitrile) (AMBN), 33 g of Abil WE09, 35 g of mineral oil, and 140 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 250 mL beaker and mixed with a magnetic stir bar until all was dissolved.

[0333] Solution 3 (emulsion): 66 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. Further emulsification was performed by sonication using a Hielscher Ultrasound Technology UP 200s with a frequency setting of 0.9 and an amplitude setting of 40%. In the next step, Solution 3 and 9.9 g of PDMAAm seeds (Example 1) were charged into a 250 mL three-necked round-bottom flask equipped with a condenser, mechanical stirrer, and gas inlet and stirred at 100 rpm at 20°C for 1 hour. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C with stirring at 65 rpm for 7 hours.

[0334] Redispersion and analysis: 77 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 minutes. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 minutes. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H2O and divided into a total of three 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed three times with H2O before finally being redispersed in H2O. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter to a standard was determined to be 0.44 μm with a CV=5.5%.

[0335] Example 17 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 3.1 g of hydroxymethylacrylamide (HMAAm), 0.6 g of itaconic acid, 3.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 17.3 g of HO. The mixture was shaken until everything was dissolved.

[0336] Solution 2 (stabilizer), Solution 3 (emulsion): Same as in Example 16.

[0337] Redispersion and analysis: 77 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 minutes. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 minutes. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H2O and divided into a total of three 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed three times with H2O before finally being redispersed in H2O. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter to a standard was determined to be 0.47 μm with a CV=5.4%.

[0338] Example 18 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 3.2 g of hydroxymethylacrylamide (HMAAm), 0.5 g of vinylphosphonic acid, 3.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 17.3 g of HO. The mixture was shaken until everything was dissolved.

[0339] Solution 2 (stabilizer), Solution 3 (emulsion): Same as in Example 16.

[0340] Redispersion and analysis: 77 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 minutes. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 minutes. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H2O and divided into a total of three 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed three times with H2O before finally being redispersed in H2O. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter to a standard was determined to be 0.42 μm with a CV=5.3%.

[0341] Example 19 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 0.6 g of hydroxymethylacrylamide (HMAAm), 0.7 g of acrylamidopropionic acid (AAPA), 1.2 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 22.2 g of HO. The mixture was shaken until everything was dissolved.

[0342] Solution 2 (stabilizer): 2.6 g of 2,2'-azodi(2-methylbutyronitrile) (AMBN), 32 g of Abil WE09, 35 g of mineral oil, and 140 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stir bar until all was dissolved.

[0343] Solution 3 (emulsion): 65 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. Further emulsification was performed by sonication using a Hielscher Ultrasound Technology UP 200s with a frequency setting of 0.9 and an amplitude setting of 40%. In the next step, Solution 3 and 9.9 g of PDMAAm seeds (Example 1) were charged into a 250 mL three-necked round-bottom flask equipped with a condenser, mechanical stirrer, and gas inlet and stirred at 100 rpm at 20°C for 1 hour. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C for 7 hours with stirring at 57 rpm.

[0344] Redispersion and analysis: To a 1 L centrifuge flask, 71 g of crude product, 500 mL of HO, and 300 mL of 2-BuOH were added. The flask was placed on a shaker table and then centrifuged. The 2-BuOH was removed and 150 mL of 2-BuOH was added. The particles were redispersed in HO and divided into a total of four 250 mL centrifuge bottles and centrifuged. The washed redispersed particles were washed three more times with HO before finally being redispersed in HO. Bright-field microscopy was performed by diluting the redispersed product to a concentration of 0.01 wt% in 150 mM NaCl solution, and image statistics were collected from 23 images to obtain a mode diameter of 0.85 μm.

[0345] Example 20 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 1.8 g of hydroxymethylacrylamide (HMAAm), 0.7 g of acrylamidopropionic acid (AAPA), 2.5 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 19.7 g of HO. The mixture was shaken until everything was dissolved.

[0346] Solution 2 (stabilizer), Solution 3 (emulsion): Same as in Example 19.

[0347] Redispersion and analysis: 70 g of crude product, 500 mL of HO, and 300 mL of 2-BuOH were added to a 1 L centrifuge flask. The flask was placed on a shaker table and then centrifuged. The 2-BuOH was removed and 150 mL of 2-BuOH was added. This was repeated two more times, after which the particle suspension was divided into a total of 6 x 250 mL centrifuge bottles and centrifuged. The particles were redispersed in HO and centrifuged. The redispersed particles were washed five times with HO before finally being redispersed in HO. Bright-field microscopy was performed by diluting the redispersed product to a concentration of 0.01 wt% in 150 mM NaCl solution, and image statistics were collected from 25 images to obtain a mode diameter of 0.83 μm.

[0348] Example 21 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 4.2 g of hydroxymethylacrylamide (HMAAm), 0.7 g of acrylamidopropionic acid (AAPA), 4.9 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 14.8 g of HO. The mixture was shaken until everything was dissolved.

[0349] Solution 2 (stabilizer): 2.6 g of 2,2'-azodi(2-methylbutyronitrile) (AMBN), 33 g of Abil WE09, 35 g of mineral oil, and 140 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stir bar until all was dissolved.

[0350] Solution 3 (emulsion): 66 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. Further emulsification was performed by sonication using a Hielscher Ultrasound Technology UP 200s with a frequency setting of 0.9 and an amplitude setting of 40%. In the next step, Solution 3 and 9.9 g of PDMAAm seeds (Example 1) were charged into a 250 mL three-necked round-bottom flask equipped with a condenser, mechanical stirrer, and gas inlet and stirred at 100 rpm at 20°C for 1 hour. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C with stirring at 58 rpm for 7 hours.

[0351] Redispersion and analysis: To a 1 L centrifuge flask, 71 g of crude product, 500 mL of HO, and 300 mL of 2-BuOH were added. The flask was placed on a shaker table and then centrifuged. The 2-BuOH was removed and 150 mL of 2-BuOH was added. This was repeated two more times, after which the particle suspension was divided into a total of 6 x 250 mL centrifuge bottles and centrifuged. The particles were redispersed in HO and centrifuged. The redispersed particles were washed five times with HO before finally being redispersed in HO. Bright-field microscopy was performed by diluting the redispersed product to a concentration of 0.01 wt% in 150 mM NaCl solution, and image statistics were collected from 18 images to obtain a mode diameter of 0.82 μm.

[0352] Example 22 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 5.5 g of hydroxymethylacrylamide (HMAAm), 0.7 g of acrylamidopropionic acid (AAPA), 6.2 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 12.3 g of HO. The mixture was shaken until everything was dissolved.

[0353] Solution 2 (stabilizer), Solution 3 (emulsion): Same as in Example 21.

[0354] Redispersion and analysis: To a 1 L centrifuge flask, 68 g of crude product, 500 mL of HO, and 300 mL of 2-BuOH were added. The flask was placed on a shaker table and then centrifuged. The 2-BuOH was removed and 150 mL of 2-BuOH was added. This was repeated two more times, after which the particle suspension was divided into a total of 6 x 250 mL centrifuge bottles and centrifuged. The particles were redispersed in HO and centrifuged. The redispersed particles were washed five times with HO before finally being redispersed in HO. Bright-field microscopy was performed by diluting the redispersed product to a concentration of 0.01 wt% in 150 mM NaCl solution, and image statistics were collected from 22 images to obtain a mode diameter of 0.86 μm.

[0355] Example 23 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 3.1 g of hydroxymethylacrylamide (HMAAm), 0.7 g of acrylamidopropionic acid (AAPA), 3.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 17.3 g of HO. The mixture was shaken until everything was dissolved.

[0356] Solution 2 (stabilizer): 2.6 g of 2,2'-azodi(2-methylbutyronitrile) (AMBN), 32 g of Abil WE09, 35 g of mineral oil, and 140 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stir bar until all was dissolved.

[0357] Solution 3 (emulsion): 66 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. Further emulsification was performed by sonication using a Hielscher Ultrasound Technology UP 200s with a frequency setting of 0.9 and an amplitude setting of 40%. In the next step, Solution 3 and 9.9 g of PDMAAm seeds (Example 1) were charged into a 250 mL three-necked round-bottom flask equipped with a condenser, mechanical stirrer, and gas inlet and stirred at 100 rpm at 20°C for 1 hour. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C with stirring at 58 rpm for 7 hours.

[0358] Redispersion and analysis: 70 g of crude product, 500 mL of HO, and 300 mL of 2-BuOH were added to a 1 L centrifuge flask. The flask was placed on a shaker table and then centrifuged. The 2-BuOH was removed and 150 mL of 2-BuOH was added. This was repeated two more times, after which the particle suspension was divided into a total of 6 x 250 mL centrifuge bottles and centrifuged. The particles were redispersed in HO and centrifuged. The redispersed particles were washed five times with HO before finally being redispersed in HO. Bright-field microscopy was performed by diluting the redispersed product to a concentration of 0.01 wt% in 150 mM NaCl solution, and image statistics were collected from 22 images to obtain a mode diameter of 0.84 μm.

[0359] Example 24 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 3.1 g of hydroxymethylacrylamide (HMAAm), 0.7 g of acrylamidopropionic acid (AAPA), 3.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 17.3 g of HO. The mixture was shaken until everything was dissolved.

[0360] Solution 2 (stabilizer), Solution 3 (emulsion): Same as in Example 23.

[0361] Redispersion and analysis: 71 g of crude product, 500 mL of HO, and 300 mL of 2-BuOH were added to a 1 L centrifuge flask. The flask was placed on a shaker table and then centrifuged. The 2-BuOH was removed and 150 mL of 2-BuOH was added. This was repeated two more times, after which the particle suspension was divided into a total of 6 x 250 mL centrifuge bottles and centrifuged. The particles were redispersed in HO and centrifuged. The redispersed particles were washed five times with HO before finally being redispersed in HO. Bright-field microscopy was performed by diluting the redispersed product to a concentration of 0.01 wt% in 150 mM NaCl solution, and image statistics were collected from 25 images to obtain a mode diameter of 0.85 μm.

[0362] Example 25 Solution 1 (monomer phase): A 500 mL Duran flask was charged with 28.6 g of hydroxymethylacrylamide (HMAAm), 0.95 g of acrylamidopropionic acid (AAPA), 19.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 29.4 g of HO. The mixture was shaken until everything was dissolved.

[0363] Solution 2 (stabilizer): 3.8 g of 2,2'-azodi(2-methylbutyronitrile) (AMBN), 48 g of Abil WE09, 53 g of mineral oil, and 210 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 600 mL beaker and mixed with a magnetic stir bar until everything was dissolved.

[0364] Solution 3 (emulsion): 262 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. Further emulsification was performed by sonication using a Hielscher Ultrasound Technology UP 200s with a frequency setting of 0.9 and an amplitude setting of 40%. In the next step, Solution 3 and 40 g of PDMAAm seeds (Example 1) were charged into a 1 L jacketed round-bottom flask equipped with a condenser, mechanical stirrer, and gas inlet and stirred at 100 rpm at 22 °C overnight. Finally, the flask was purged with Ar(g) for 30 minutes and then heated to 70 °C with stirring at 64 rpm for 7 hours.

[0365] Redispersion and Analysis: The crude product was divided into two 250 mL centrifuge flasks and centrifuged at 5000 rpm for 15 minutes and 7000 rpm for 10 minutes. The supernatant was removed, and the particles were redispersed in isopropanol. The redispersed particles were then divided into a total of six 250 mL centrifuge bottles and washed four times with isopropanol and six times with HO before finally being redispersed in HO. The particles were then filtered through three different filtration mesh fabrics: Sefar Nitex 03-64 / 32, Sefar Nitex 03-30 / 18, and Sefar Nitex 03-1 / 1. The product was analyzed using an optical microscope and a disc centrifuge. Bright-field microscopy was performed by diluting the redispersed product to a concentration of 0.01 wt% in 150 mM NaCl solution. Image statistics were collected from 18 images, yielding a mode diameter of 0.91 μm. The relative disc centrifuge diameter using CPS Method 3 was determined to be 0.54 μm with a CV=6.2%.

[0366] Example 26 Solution 1 (monomer phase): A 500 mL Duran flask was charged with 27.0 g of hydroxymethylacrylamide (HMAAm), 2.6 g of acrylamidopropionic acid (AAPA), 19.7 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 49.3 g of HO. The mixture was shaken until everything was dissolved.

[0367] Solution 2 (stabilizer), Solution 3 (emulsion): Same as in Example 25.

[0368] Redispersion and Analysis: The crude product was divided into two 250 mL centrifuge flasks and centrifuged at 5000 rpm for 25 minutes. The supernatant was removed, and the particles were redispersed in isopropanol. The redispersed particles were then divided into a total of six 250 mL centrifuge bottles and washed four times with isopropanol and five times with HO before finally being redispersed in HO. The particles were then filtered through two different filtration mesh cloths: Sefar Nitex 03-30 / 18 and Sefar Nitex 03-1 / 1. The product was analyzed using optical microscopy and a disc centrifuge. Bright-field microscopy was performed by diluting the redispersed product to a concentration of 0.01 wt% in 150 mM NaCl solution, and image statistics were collected from 21 images to obtain a mode diameter of 0.90 μm. The relative disc centrifuge diameter using CPS Method 3 was determined to be 0.53 μm with a CV of 5.2%.

[0369] Example 27 Solution 1 (monomer phase): A 500 mL Duran flask was charged with 24.8 g of hydroxymethylacrylamide (HMAAm), 5.0 g of acrylamidopropionic acid (AAPA), 19.9 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 49.7 g of HO. The mixture was shaken until everything was dissolved.

[0370] Solution 2 (stabilizer): 3.8 g of 2,2'-azodi(2-methylbutyronitrile) (AMBN), 52 g of Abil WE09, 57 g of mineral oil, and 228 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 1000 mL beaker and mixed with a magnetic stir bar until everything was dissolved.

[0371] Solution 3 (emulsion): 284 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. Further emulsification was performed by sonication using a Hielscher Ultrasound Technology UP 200s with a frequency setting of 0.9 and an amplitude setting of 40%. In the next step, Solution 3 and 17 g of PDMAAm seeds (Example 1) were charged into a 1 L jacketed round-bottom flask equipped with a condenser, mechanical stirrer, and gas inlet and stirred at 100 rpm at 25 °C overnight. Finally, the flask was purged with Ar(g) for 30 minutes and then heated to 70 °C with stirring at 62 rpm for 7 hours.

[0372] Redispersion and analysis: The crude product was divided into two 250 mL centrifuge flasks and centrifuged at 3000 rpm for 20 minutes and 5000 rpm for 10 minutes. The supernatant was removed, and the particles were redispersed in isopropanol. The redispersed particles were then divided into a total of six 250 mL centrifuge bottles and washed four times with isopropanol and six times with HO before finally being redispersed in HO. The particles were then filtered through two different filtration mesh cloths: Sefar Nitex 03-30 / 18 and Sefar Nitex 03-1 / 1. The product was analyzed using an optical microscope and a disc centrifuge. Bright-field microscopy was performed by diluting the redispersed product to a concentration of 0.01 wt% in 150 mM NaCl solution, and image statistics were collected from 30 images to obtain a mode diameter of 1.0 μm. The relative disc centrifuge diameter using CPS Method 3 was determined to be 0.57 μm with a CV=6.3%.

[0373] Magnetization of polymer particles Example 28 Magnetization: A 50 mL three-necked round-bottom flask equipped with a mechanical stirrer was charged with 9.8 g of particle suspension in HO (Example 3, 3.1% dry content) and purged with Ar(g) for 20 min, after which 0.18 g of FeClx4HO and 0.20 g of FeClx6HO were added. The mixture was stirred at 250 rpm for 10 min, 1.24 g of NH (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0374] Work-up and analysis: The batch was added to a 250 mL centrifuge flask and washed 12 times with HO. A 1 mL sample of the batch was added to a 2 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0375] Example 29 Magnetization: A 50 mL three-necked round-bottom flask equipped with a mechanical stirrer was charged with 7.5 g of particle suspension in HO (Example 4, 4.0% dry content) and 2.4 mL of HO and purged with Ar(g) for 20 min, after which 0.18 g of FeCl2 x 4 HO and 0.20 g of FeCl3 x 6 HO were added. The mixture was stirred at 250 rpm for 10 min, 1.24 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0376] Work-up and analysis: The batch was added to a 250 mL centrifuge flask and washed 12 times with HO. A 1 mL sample of the batch was added to a 2 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0377] Example 30 Magnetization: A 50 mL three-necked round-bottom flask equipped with a mechanical stirrer was charged with 6.0 g of particle suspension in HO (Example 5, 5.0% dry content) and 3.8 mL of HO and purged with Ar(g) for 20 min, after which 0.18 g of FeCl2 x 4 HO and 0.20 g of FeCl3 x 6 HO were added. The mixture was stirred at 250 rpm for 10 min, 1.24 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0378] Work-up and analysis: The batch was added to a 250 mL centrifuge flask and washed 12 times with HO. A 1 mL sample of the batch was added to a 2 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0379] Example 31 Magnetization: A 50 mL three-necked round-bottom flask equipped with a mechanical stirrer was charged with 9.7 g of particle suspension in HO (Example 6, 3.1% dry content), 0.18 g of FeCl2 x 4HO, and 0.20 g of FeCl3 x 6HO. The mixture was stirred at 250 rpm for 5 min and then purged with Ar(g) for 25 min. Finally, 1.24 mL of NH3 (25%) was added and the reaction was allowed to proceed under Ar(g) for 1 h.

[0380] Work-up and analysis: The batch was added to a 250 mL centrifuge flask and washed 7 times with HO. A 1.5 mL sample of the batch was added to a 1.5 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0381] Example 32 Magnetization: A 50 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 9.5 g of particle suspension in HO (Example 7, 3.1% dry content), 0.18 g of FeCl2 x 4HO, and 0.20 g of FeCl3 x 6HO. The mixture was stirred at 250 rpm for 5 min and then purged with Ar(g) for 25 min. Finally, 1.24 mL of NH3 (25%) was added and the reaction was allowed to proceed under Ar(g) for 1 h.

[0382] Work-up and analysis: The batch was added to a 250 mL centrifuge flask and washed 7 times with HO. A 1.5 mL sample of the batch was added to a 1.5 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0383] Example 33 Magnetization: A 50 mL three-necked round-bottom flask equipped with a mechanical stirrer was charged with 9.1 g of particle suspension in HO (Example 8, 3.1% dry content), 0.18 g of FeCl2 x 4HO, and 0.20 g of FeCl3 x 6HO. The mixture was stirred at 250 rpm for 5 min and then purged with Ar(g) for 25 min. Finally, 1.24 mL of NH3 (25%) was added and the reaction was allowed to proceed under Ar(g) for 1 h.

[0384] Work-up and analysis: The batch was added to a 250 mL centrifuge flask and washed 7 times with HO. A 1.5 mL sample of the batch was added to a 1.5 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0385] Example 34 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 72 g of particle suspension in HO (Example 25, 5.6% dry content), 2.4 g of FeCl2 x 4HO, and 2.7 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 15 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0386] Work-up and analysis: The batch was divided into 2 x 250 mL centrifuge flasks and washed 15 times with HO. The Fe content in the dried particles was determined to be 190 mg / g, and the magnetic susceptibility was 64 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 16.0 Am 2 / kg.

[0387] Example 35 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 72 g of particle suspension in HO (Example 25, 5.6% dry content), 4.8 g of FeCl2 x 4HO, and 5.4 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 30 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0388] Work-up and analysis: The batch was divided into 2 x 250 mL centrifuge flasks and washed 30 times with HO. The Fe content in the dried particles was determined to be 255 mg / g, and the magnetic susceptibility was 72 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 21.5 Am 2 / kg.

[0389] Example 36 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 72 g of particle suspension in HO (Example 25, 5.6% dry content), 7.3 g of FeCl2 x 4HO, and 8.2 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 45 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0390] Work-up and analysis: The batch was divided into 2 x 250 mL centrifuge flasks and washed 45 times with HO. The Fe content in the dried particles was determined to be 336 mg / g, and the magnetic susceptibility was 81 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 30.5 Am 2 / kg.

[0391] Example 37 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 71 g of particle suspension in HO (Example 26, 5.6% dry content), 7.3 g of FeCl2 x 4HO, and 8.2 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 45 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0392] Workup and analysis: The batch was divided into two 250 mL centrifuge flasks and washed a total of 65 times (5 times with HO, followed by 1 time in 1 M acetic acid and 59 times with HO). The Fe content in the dried particles was determined to be 248 mg / g, and the magnetic susceptibility was 62 10 -5 m 3 / kg, saturation magnetization at 10,000 Oe is 20.6 Am 2 / kg.

[0393] Example 38 Magnetization: A 100 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 39 g of particle suspension in HO (Example 27, 5.1% dry content), 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0394] Workup and analysis: The batch was divided into 2 x 250 mL centrifuge flasks and washed 65 times (5 times with HO, followed by 1 time in 1 M acetic acid and 59 times with HO). The magnetic susceptibility was 72 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 24.7 Am 2 / kg.

[0395] Example 39 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 35 g of particle suspension in HO (Example 19, 1.7% dry content), 33 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0396] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 9 times with HO, 2 times with 0.01 M citrate buffer (pH 5.3), and 28 times with HO. The Fe content in the dried particles was determined to be 478 mg / g, and the magnetic susceptibility was 72 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 37.8 Am 2 / kg.

[0397] Example 40 Magnetization: A 100 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 42 g of particle suspension in HO (Example 20, 1.8% dry content), 1.5 g of HO, 2.3 g of FeCl2 x 4HO, and 2.6 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 14 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0398] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 9 times with HO, 2 times with 0.01 M citrate buffer (pH 5.3), and 27 times with HO. The Fe content in the dried particles was determined to be 363 mg / g, and the magnetic susceptibility was 67 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 31.0 Am 2 / kg.

[0399] Example 41 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 58 g of particle suspension in HO (Example 23, 4.1% dry content), 9.4 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0400] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 9 times with HO, 2 times with 0.01 M citrate buffer (pH 5.3), and 27 times with HO. The Fe content in the dried particles was determined to be 233 mg / g, and the magnetic susceptibility was 53 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 20.2 Am 2 / kg.

[0401] Example 42 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 68 g of particle suspension in HO (Example 21, 3.5% dry content), 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0402] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 9 times with HO, 2 times with 0.01 M citrate buffer (pH 5.3), and 38 times with HO. The Fe content in the dried particles was determined to be 177 mg / g, and the magnetic susceptibility was 42 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 15.0 Am 2 / kg.

[0403] Example 43 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 58 g of particle suspension in HO (Example 22, 5.1% dry content), 9.0 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0404] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 9 times with HO, 2 times with 0.01 M citrate buffer (pH 5.3), and 38 times with HO. The Fe content in the dried particles was determined to be 205 mg / g, and the magnetic susceptibility was 52 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 16.6 Am 2 / kg.

[0405] Example 44 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 51 g of particle suspension in HO (Example 24, 3.5% dry content), 17 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0406] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 9 times with HO, 2 times with 0.01 M citrate buffer (pH 5.3), and 27 times with HO. The Fe content in the dried particles was determined to be 288 mg / g, and the magnetic susceptibility was 68 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 23.8 Am 2 / kg.

[0407] Example 45 Magnetization: A 1 L cylindrical reactor equipped with a mechanical stirrer was charged with 252 g of particle suspension in HO (Example 9, 5.6% dry content), 18.4 g of FeCl2 x 4HO, and 20.5 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 112 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0408] Work-up and analysis: The batch was divided into 2 x 1 L centrifuge flasks and washed 33 times with H2O.

[0409] Example 46 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 44 g of particle suspension in HO (Example 10, 4.5% dry content), 31 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0410] Work-up and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 21 times with HO. A 1.5 mL sample of the batch was added to a 1.5 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0411] Example 47 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 37 g of particle suspension in HO (Example 11, 5.6% dry content), 39 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0412] Work-up and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 30 times with H2O.

[0413] Example 48 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 34 g of particle suspension in HO (Example 12, 5.6% dry content), 41 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0414] Work-up and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 30 times with HO. A 1.5 mL sample of the batch was added to a 1.5 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0415] Example 49 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 42 g of particle suspension in HO (Example 13, 4.7% dry content), 33 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0416] Work-up and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 34 times with HO. A 1.5 mL sample of the batch was added to a 1.5 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0417] Example 50 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 44 g of particle suspension in HO (Example 14, 4.5% dry content), 31 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0418] Work-up and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 40 times with HO. A 1.5 mL sample of the batch was added to a 1.5 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0419] Example 51 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 44 g of particle suspension in HO (Example 15, 4.5% dry content), 31 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0420] Work-up and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 40 times with HO. A 1.5 mL sample of the batch was added to a 1.5 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0421] Example 52 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 35 g of particle suspension in HO (Example 16, 5.65% dry content), 40 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0422] Work-up and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 27 times with HO. A 1.5 mL sample of the batch was added to a 1.5 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0423] Example 53 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 45 g of particle suspension in HO (Example 17, 4.47% dry content), 30 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0424] Work-up and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 27 times with HO. A 1.5 mL sample of the batch was added to a 1.5 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0425] Example 54 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 49 g of particle suspension in HO (Example 18, 4.12% dry content), 26 g of HO, 3.7 g of FeCl2 x 4HO, and 4.1 g of FeCl3 x 6HO. The mixture was stirred at 150 rpm for 10 min and then purged with Ar(g) for 30 min. Finally, the stirring speed was set to 250 rpm, 22 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0426] Work-up and analysis: The batch was transferred to a 250 mL centrifuge flask and washed 27 times with HO. A 1.5 mL sample of the batch was added to a 1.5 mL Eppendorf tube and placed on a DynaMag™-2 magnet; within 1 minute the sample had completely migrated towards the magnet.

[0427] Epoxy-coated polymer particles Example 55 - Tosyl activation of polyacrylamide particles To a 13 wt% solution of the particles of Example 9 in acetone (46.7 mL), 0.7 g of pyridine and 1.4 g of p-toluenesulfonyl chloride were added with stirring. The reaction was then further stirred at room temperature overnight. Upon completion, the supernatant was removed and worked up by washing with 4 x 250 mL of acetone.

[0428] Example 56 - Amination of Tosylated Polyacrylamide Particles Five grams of the tosylated particles of Example 55 in acetone were stripped of solvent and then resuspended in a total of 75 grams of 2,2'-(ethylenedioxy)bis(ethylamine) (EDEA). The mixture was stirred at 72°C for 4 hours. The reaction product was washed with 3 x 300 mL of HO, 3 x 300 mL of 10 mM acetic acid (aqueous), 3 x 300 mL of HO, and finally 4 x 300 mL of N-methylpyrrolidinone.

[0429] Example 57 - Epoxy Coating of Aminated Polyacrylamide Particles From 4.1 g of aminated particles of Example 56 in 265.5 g of NMP, 235 g of solvent was removed, and the remaining 8.5 g of 1,4-butanediol diglycidyl ether, 3.3 g of glycidol, and 6.1 g of glycidyl methacrylate were added with stirring. After 5 minutes, the temperature was raised to 87°C, and the mixture was stirred for 20 hours. The reaction product was then cooled for 30 minutes and then washed with 200 mL of acetone, 2 x 200 mL of methanol, 2 x 200 mL of 30:70 methanol:isopropanol, and 4 x 200 mL of isopropanol.

[0430] Example 58 - Grafting of acrylic acid onto polyacrylamide particles 52 mg of 2,2'-azobis(2-methylpropionitrile) (AIBN) was dissolved in 20 g of isopropanol by stirring, and this was added to 4.3 g of the particles of Example 57 in 47 g of isopropanol. 1.1 mL of acrylic acid was added, and the mixture was heated to 72 °C with stirring. After reaching the desired temperature, the reaction was continued for 20 hours. Subsequently, it was cooled for 20 minutes and washed with 2 × 300 mL of isopropanol and 3 × 200 mL of methanol.

[0431] Example 59 - One-Step Epoxy Coating of Polyacrylamide Particles To a 10 wt% solution of the particles of Example 35 in acetonitrile (1.42 mL) was added 0.12 g of 1,4-butanediol diglycidyl ether, 0.05 g of glycidol, and 0.09 g of glycidyl methacrylate. Then, 0.03 g of lutidine and 0.05 g of p-toluenesulfonic acid monohydrate were also added, and the reaction was shaken at 70°C for 19 hours. After cooling, the reaction was worked up by washing with 4 x 5 mL of acetone.

[0432] Silica-coated polymer particles Example 60 A 50 mL three-necked round-bottom flask equipped with a mechanical stirrer was charged with 1.5 g of Na2SiO3 and 10 mL of H2O. The pH of the solution was adjusted to pH 12-13 with 1 M HCl, after which 7.8 g of the particles of Example 35 (6.4 wt. % dry content) was added. The mixture was stirred at 200 rpm for 10 minutes, after which the flask was lowered into an oil bath preheated to 60 °C. 1 M HCl was then added dropwise to adjust the pH to pH 6. The mixture was then stirred for 15 minutes, after which the reaction mixture was transferred to a 100 mL Duran flask and subjected to five magnetic shifts in H2O. The SiO2 coating was observed at 1094 cm in FTIR spectroscopy performed on a dry sample. -1 This was confirmed by the appearance of a peak at

[0433] Example 61 A 50 mL three-necked round-bottom flask equipped with a mechanical stirrer was charged with 0.5 g of Na2SiO3 and 10 mL of H2O. The pH of the solution was adjusted to pH 12-13 with 1 M HCl, after which 7.8 g of the particles of Example 35 (dry content 6.4 wt%) was added. The mixture was stirred at 200 rpm for 5 minutes, after which the flask was lowered into an oil bath preheated to 60 °C. 1 M HCl was then added dropwise to adjust the pH to pH 2.5. The mixture was then stirred for 5 minutes, after which the reaction mixture was transferred to a 250 mL Duran flask and subjected to seven magnetic shifts in H2O. The SiO2 coating was observed at 1094 cm in FTIR spectroscopy performed on a dry sample. -1 This was confirmed by the appearance of a peak at

[0434] Example 62 A 50 mL three-necked round-bottom flask equipped with a mechanical stirrer was charged with 0.5 g of Na2SiO3 and 10 mL of H2O. The pH of the solution was adjusted to pH 12-13 with 1 M HCl, after which 8.9 g of the particles of Example 26 (dry content 5.6 wt%) was added. The mixture was stirred at 200 rpm for 5 minutes, after which the flask was lowered into an oil bath preheated to 60 °C. 1 M HCl was then added dropwise to adjust the pH to pH 2.5. The mixture was then stirred for 5 minutes, after which the reaction mixture was transferred to a 250 mL centrifuge flask and subjected to five centrifugal shifts in H2O. The SiO2 coating was confirmed by FTIR spectroscopy performed on a dried sample at 1094 cm -1 This was confirmed by the appearance of a peak at

[0435] Example 63 - Silane Route A 20 mL screw-top vial was filled with 7.8 g of the particles of Example 35 (dry content 6.4 wt%), and three magnetic shifts were performed in methanol and five magnetic shifts in dipropylene glycol dimethyl ether. The suspension was then transferred to a 50 mL three-necked round-bottom flask equipped with a mechanical stirrer, followed by the addition of 4.7 g of tetraethyl orthosilicate and 80 μL of H2O. The reaction mixture was then heated to 130 °C and stirred overnight. The reaction mixture was then transferred to a 20 mL screw-top vial, and five magnetic shifts were performed in dipropylene glycol dimethyl ether and five magnetic shifts in H2O. Finally, the particle suspension was stirred for three days. The SiO2 coating was observed at 1094 cm in FTIR spectroscopy performed on a dried sample. -1 This was confirmed by the appearance of a peak at

[0436] Antibody Coupling and Functional Assays Example 64 Anti-TnI mAb was conjugated to the particles of Examples 34, 35, and 36 according to the following procedure. 5 mg of beads (500 μL of 1% stock solution) was loaded into a 2 mL Sarstedt tube. The tube was placed on a magnet, the supernatant was removed, and 1 mL of 15 mM MES buffer (pH 6.0) was added. The MES buffer washing step was repeated three times, followed by resuspension in 205 μL of MES buffer. Subsequently, 45 μL of EDC (20 mg / mL in H2O) was added, the tube was vortexed, and then incubated on a roller at room temperature for 30 minutes. The tube was placed on a magnet, the supernatant was removed, and then resuspension in 200 μL of MES buffer was added. 52 μL of anti-TnI mAb SDIX (3.8 mg / mL) was added to the tube, the tube was vortexed, and then incubated on a roller at room temperature for 2 hours. The particles were then washed twice with 1 mL of TBST buffer (50 mM Tris-HCl pH 7.4 containing 140 mM NaCl, and 0.1% Tween-20) and subsequently resuspended in TBST buffer overnight on a roller at 37° C. Finally, the particles were washed a final time with 1 mL of TBST buffer and then resuspended in 985 μL of TBST buffer to obtain a 5 mg / mL particle suspension.

[0437] Example 65 The anti-hGH mAb GhG2 was conjugated to the particles of Examples 34, 35, and 36 according to the following procedure. 5 mg of particles (500 μL of 1% stock solution) was loaded into a 2 mL Sarstedt tube. The tube was placed on a magnet, the supernatant was removed, and 1 mL of 15 mM MES buffer (pH 6.0) was added. The MES buffer washing step was repeated three times, followed by resuspension in 205 μL of MES buffer. 45 μL of EDC (20 mg / mL in H2O) was then added, the tube was vortexed, and then incubated on a roller at room temperature for 30 minutes. The tube was placed on a magnet, the supernatant was removed, and then resuspension in 190 μL of MES buffer was performed. 60 μL of anti-hGH mAb GhG2 (3.5 mg / mL) was added to the tube, the tube was vortexed, and then incubated on a roller at room temperature for 2 hours. The particles were then washed twice with 1 mL of TBST buffer (50 mM Tris-HCl pH 7.4 containing 140 mM NaCl, and 0.1% Tween-20) and subsequently resuspended in TBST buffer overnight on a roller at 37° C. Finally, the particles were washed a final time with 1 mL of TBST buffer and then resuspended in 985 μL of TBST buffer to obtain a 5 mg / mL particle suspension.

[0438] Example 66 Anti-TnI mAb was conjugated to the particles of Examples 34, 37, and 38 according to the following procedure. A 2 mL Sarstedt tube was loaded with 5 mg of particles (500 μL of 1% stock solution). The tube was placed on a magnet, the supernatant was removed, and 1 mL of 15 mM MES buffer (pH 6.0) was added. The MES buffer washing step was repeated three times, followed by resuspension in 205 μL of MES buffer. Subsequently, 45 μL of EDC (20 mg / mL in H2O) was added, the tube was vortexed, and then incubated on a roller at room temperature for 30 minutes. The tube was placed on a magnet, the supernatant was removed, and then resuspension in 205 μL of MES buffer was added. 42.5 μL of anti-TnI mAb SDIX (4.7 mg / mL) was added to the tube, the tube was vortexed, and then incubated on a roller at room temperature for 2 hours. The particles were then washed twice with 1 mL of TBST buffer (50 mM Tris-HCl pH 7.4 containing 140 mM NaCl, and 0.1% Tween-20) and subsequently resuspended in TBST buffer overnight on a roller at 37° C. Finally, the particles were washed a final time with 1 mL of TBST buffer and then resuspended in 985 μL of TBST buffer to obtain a 5 mg / mL bead suspension.

[0439] Example 67 A 96-well microplate (Greiner-bio-one) was used for the experiment, and four different antigen concentrations (0, 5 ng / mL, 50 ng / mL, and 100 ng / mL recombinant human growth hormone in bovine serum) were screened in triplicate for each batch of particles. 50 μL of antigen solution and 50 μL of detection antibody (mAb GhB9-AP) were added to each well, and the plate was sealed with tape and incubated on a shaker at 37°C for 15 minutes. Subsequently, 100 μL of suspended particles coupled with anti-hGH mAb GhG2 prepared according to Example 65 were added, and the plate was sealed with tape and incubated on a shaker at 37°C for 30 minutes. The microplate was placed on a magnet, the supernatant was removed, and the beads were washed 3 times with 200 μL of TBST buffer before finally being resuspended in 20 μL of TBST buffer. Finally, substrate (DynaLight Substrate w / RapidGlow) was added and the plate was read on a BioTek Synergy™ 4 plate reader. The results obtained are shown in Figure 6.

[0440] Example 68 A 96-well microplate (Greiner-bio-one) was used for the experiment, and four different antigen concentrations (0, 0.5 ng / mL, 5 ng / mL, and 50 ng / mL of human cardiac troponin ITC complex in bovine serum) were screened in triplicate for each particle batch. 50 μL of antigen solution and 50 μL of detection antibody (mAb 560) were added to each well, the plate was sealed with tape, and the mixture was incubated on a shaker at 37°C for 10 minutes. Subsequently, 100 μL of suspended particles coupled with anti-TnI mAb SDIX prepared according to Example 6564 were added, the plate was sealed with tape, and the mixture was incubated on a shaker at 37°C for 30 minutes. The microplate was placed on a magnet, the supernatant was removed, and the beads were washed 3 times with 200 μL of TBST buffer before finally being resuspended in 20 μL of TBST buffer. Finally, substrate (DynaLight Substrate w / RapidGlow) was added and the plate was read on a BioTek Synergy™ 4 plate reader. The results obtained are shown in Figure 7.

[0441] Example 69 A 96-well microplate (Greiner-bio-one) was used for the experiment, and four different antigen concentrations (0, 0.5 ng / mL, 5 ng / mL, and 50 ng / mL of human cardiac troponin ITC complex in bovine serum) were screened in triplicate for each particle batch. 50 μL of antigen solution and 50 μL of detection antibody (mAb 560) were added to each well, and the plate was sealed with tape and incubated on a shaker at 37°C for 10 minutes. Subsequently, 100 μL of suspended particles coupled with anti-TnI mAb SDIX prepared according to Example 6566 were added, sealed with tape, and incubated on a shaker at 37°C for 30 minutes. The microplate was placed on a magnet, the supernatant was removed, and the beads were washed 3 times with 200 μL of TBST buffer before finally being resuspended in 20 μL of TBST buffer. Finally, substrate (DynaLight Substrate w / RapidGlow) was added and the plate was read on a BioTek Synergy™ 4 plate reader. The results obtained are shown in Figure 8.

[0442] Example 70 A 96-well microplate (Greiner-bio-one) was used for the experiment to evaluate the nonspecific binding of the particles of Examples 34, 35, and 36 and compare them with commercially available Dynabeads® MyOne™ epoxy. First, 10 μL of bead suspension (10 mg / mL) was diluted with 990 μL of TBST buffer, and each particle batch was evaluated in triplicate up to the microwell. Next, 100 μL of AE conjugate (mouse IgG conjugated to acridinium ester (AE) diluted in 30 mM MES buffer) was added, the plate was sealed with tape, and incubated at 37°C for 30 minutes. The plate was placed on a magnet, the supernatant removed, and then washed 3 times with 300 μL of TBST buffer. Finally, the particles were resuspended in 20 μL of TBST using a shaker, followed by the addition of a trigger solution of 75 μL of 1.32% H 2 O 2 and 75 μL of 0.35 N NaOH, and the plate was read in a Berthold Centro LB 960 microplate luminometer.

[0443] The results are shown in Figure 9, which demonstrate that the particles of each of Examples 34, 35, and 36 have lower levels of nonspecific binding than the MyOne™ epoxy particles. These results, combined with the results shown in Figures 6-8, demonstrate that the particles of the present invention are useful in assays and related bead-based separation applications.

[0444] Seed particle synthesis Example 71 A 2 L jacketed round-bottom flask equipped with a mechanical anchor stirrer and condenser was charged with 34.6 g of distilled N,N-dimethylacrylamide, 27.7 g of Kraton G1650, 1.4 g of 2,2'-azobis(2-methylpropionitrile), 747 g of heptane, 187 g of toluene, and 1.2 g of 1-octanethiol. Once everything was dissolved, the reaction mixture was purged with Ar(g) for 60 minutes. The reaction was then heated to 71 °C using a temperature-controlled water bath connected to the reactor and stirred at 350 rpm for 20 hours. The resulting dispersion was analyzed by dynamic light scattering, which gave a z-average diameter of 382 nm.

[0445] Synthesis of polymer particles from low molecular weight seed particles Example 72 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 5.0 g of N-(hydroxymethyl)-acrylamide (HMAAm), 2.4 g of vinylimidazole (VIm), 7.4 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 34 g of HO. The mixture was shaken until everything was dissolved.

[0446] Solution 2 (stabilizer): 60 g of Abil WE09, 66 g of mineral oil, 5.0 g of 2,2'-azobis(2-methylbutyronitrile) (AMBN), and 263 g of diethylhexyl carbonate (Tegosoft DEC) were added to a 500 mL beaker and mixed with a magnetic stir bar until all was dissolved.

[0447] Solution 3 (emulsion): 127 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. In the next step, Solution 3 and 24 g of PDMAAm seeds (Example 71) were charged into a 500 mL jacketed reaction vessel equipped with a condenser, mechanical stirrer, and gas inlet, stirred at 100 rpm at room temperature for 1 hour, and purged with Ar(g) for 25 minutes. Finally, the heating was set to 70° C. with stirring at 60 rpm for 7 hours.

[0448] Redispersion: The crude product was added to a 250 mL centrifuge and centrifuged at 3000 rpm for 15 minutes. The supernatant was removed and the product was washed three times with isopropanol and four times with HO before finally being redispersed in HO.

[0449] Example 73 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 6.9 g of N-(hydroxymethyl)-acrylamide (HMAAm), 0.48 g of vinylimidazole (VIm), 7.4 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 34 g of HO. The mixture was shaken until everything was dissolved.

[0450] Solution 2 (stabilizer): Same as in Example 72.

[0451] Solution 3 (emulsion): 127 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. In the next step, Solution 3 and 24 g of PDMAAm seeds (Example 71) were charged into a 500 mL jacketed reaction vessel equipped with a condenser, mechanical stirrer, and gas inlet, stirred at 100 rpm at room temperature for 1 hour, and purged with Ar(g) for 25 minutes. Finally, the heating was set to 70° C. with stirring at 60 rpm for 7 hours.

[0452] Redispersion: The crude product was added to a 250 mL centrifuge and centrifuged at 3000 rpm for 15 minutes. The supernatant was removed and the product was washed three times with isopropanol and four times with HO before finally being redispersed in HO.

[0453] Example 74 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 5.2 g of N-(hydroxymethyl)-acrylamide (HMAAm), 1.2 g of vinylimidazole (VIm), 0.93 g of acrylic acid, 7.4 g of N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm), and 34 g of HO. The mixture was shaken until everything was dissolved.

[0454] Solution 2 (stabilizer): Same as in Example 72.

[0455] Solution 3 (emulsion): 127 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. In the next step, Solution 3 and 24 g of PDMAAm seeds (Example 71) were charged into a 500 mL jacketed reaction vessel equipped with a condenser, mechanical stirrer, and gas inlet, stirred at 100 rpm at room temperature for 1 hour, and purged with Ar(g) for 25 minutes. Finally, the heating was set to 70° C. with stirring at 60 rpm for 7 hours.

[0456] Redispersion: The crude product was added to a 250 mL centrifuge and centrifuged at 3000 rpm for 15 minutes. The supernatant was removed and the product was washed three times with isopropanol and four times with HO before finally being redispersed in HO.

[0457] Example 75 Solution 1 (monomer phase): A 250 mL Duran flask was charged with 2.875 g acrylic acid (AA), 8.651 g N-acryloylmorpholine, 3.820 g DHEBA, and 61.19 g HO. The mixture was shaken at 61° C. until everything was dissolved.

[0458] Solution 2 (stabilizer): 96.62 g of Abil WE09, 105.09 g of mineral oil, 420.32 g of diethylhexyl carbonate (Tegosoft DEC), and 7.938 g of AMBN were added to a 1 L beaker and mixed with a magnetic stir bar until everything was dissolved.

[0459] Solution 3 (emulsion): 198.20 g of Solution 2 was added to Solution 1, followed by mixing with an Ultraturrax for 1 minute. In the next step, Solution 3 and 36.70 g of PDMAAm seeds (Example 71) were charged into a 300 mL reactor equipped with a condenser, mechanical stirrer, and gas inlet and stirred at 100 rpm for 1 hour and 15 minutes at room temperature. Finally, the flask was purged with Ar(g) for 30 minutes and then heated to 70°C with stirring at 50 rpm for 7 hours.

[0460] Redispersion: The crude product was added to a 1 L centrifuge flask and centrifuged at 5000 rpm for 40 minutes. The supernatant was removed and the product was further washed three times with isopropanol and three times with HO before finally being redispersed in HO.

[0461] Magnetization of polymer particles Example 76 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 89 g of particle suspension in HO (Example 72, 3.4% dry content), 26 g of HO, 7.3 g of FeCl2 x 4HO, and 8.2 g of FeCl3 x 6HO. The mixture was stirred at 100 rpm for 1 h and then purged with Ar(g) for 45 min. Finally, the stirring speed was set to 320 rpm, 45 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0462] Work-up and analysis: The batch was transferred to a 1 L centrifuge flask and washed 35 times with HO. The Fe content in the dried particles was determined to be 412 mg / g, and the magnetic susceptibility was 72 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 33 Am 2 / kg.

[0463] Example 77 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 72 g of particle suspension in HO (Example 73, 4.2% dry content), 43 g of HO, 7.3 g of FeCl2 x 4HO, and 8.2 g of FeCl3 x 6HO. The mixture was stirred at 100 rpm for 1 h and then purged with Ar(g) for 45 min. Finally, the stirring speed was set to 320 rpm, 45 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0464] Work-up and analysis: The batch was transferred to a 1 L centrifuge flask and washed 35 times with HO. The Fe content in the dried particles was determined to be 404 mg / g, and the magnetic susceptibility was 76 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 33 Am 2 / kg.

[0465] Example 78 Magnetization: A 250 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 91 g of particle suspension in HO (Example 74, 3.3% dry content), 24 g of HO, 7.3 g of FeCl2 x 4HO, and 8.2 g of FeCl3 x 6HO. The mixture was stirred at 100 rpm for 1 h and then purged with Ar(g) for 45 min. Finally, the stirring speed was set to 320 rpm, 45 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 h.

[0466] Work-up and analysis: The batch was transferred to a 1 L centrifuge flask and washed 35 times with HO. The Fe content in the dried particles was determined to be 399 mg / g, and the magnetic susceptibility was 70 10 -5 m 3 / kg, and the saturation magnetization at 10,000 Oe is 31 Am 2 / kg.

[0467] Example 79 Magnetization: A 100 mL three-neck round-bottom flask equipped with a mechanical stirrer was charged with 36.10 g of particle suspension in HO (Example 75, 2.81% dry content), 33.02 g of HO, 2.478 g of FeCl2 x 4HO, and 2.791 g of FeCl3 x 6HO. The mixture was stirred at 100 rpm for 50 minutes and then purged with Ar(g) for 40 minutes. Finally, the stirring speed was set to 300 rpm, 15.18 g of NH3 (25%) was added, and the reaction was allowed to proceed under Ar(g) for 1 hour and 20 minutes.

[0468] Work-up and analysis: The batch was transferred to a 1 L centrifuge flask and washed 30 times with H2O.

[0469] Further embodiments The present disclosure also includes embodiments described in the following numbered paragraphs: 1. A magnetic material; (a) log P less than about 0.6 oct / wat (log P), and (b) a crosslinker containing at least two vinyl groups a polymer formed from 1. Monodisperse magnetic hydrogel polymer particles comprising: 2. The polymer particles of paragraph 1, wherein the hydrophilic vinyl monomer has a log P of less than about 0.3, and optionally the log P is less than about 0. 3. The polymer particles according to item 1 or 2, wherein the hydrophilic vinyl monomer has a log P of 0.5 to -2, and optionally the log P is 0 to -2. 4. Polymer particles according to any of the preceding paragraphs, wherein the hydrophilic vinyl monomer is an acrylamide monomer and / or an acrylate monomer. 5. The hydrophilic vinyl monomer is represented by formula (I): TIFF0007765421000023.tif15128, During the ceremony, R 1 is -H, -CH3, -CH2CH3, or -CH2C(O)OH; R 1a is -H or -C(O)R 2 and R 1b But -C(O)R 2 , -P(O)(OH)2, or -S(O)2OH, or R 1a Or R 1 R combined with 1b is -C(O)-OC(O)-, R 2 But, -OR 3 or -N(R 4 )R 5 and R 3 is -H, -C1-C6 alkyl, or -C1-C6 alcohol, and R 4 and R 5are each independently selected from -H, -C1-C6 alkyl, -C1-C6 haloalkyl, and -C1-C6 alcohol; R 3 and / or R 4 and / or R 5 may independently represent, when chemically possible, oxo, =NR a , =NOR a , halo, nitro, cyano, NR a R a , N.R. a S(O)2R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , S.R. a , S(O)R a , S(O)2OR a , S(O)2R a , S(O)NR a R a , CO2R a C(O)R a ,CONR a R a , C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, and R a is independently selected at each occurrence from H and C1-C4 alkyl. 6.R 1a is -H and R 1b -C(O)R 2 and optionally R 2 -OR 3 Item 5. The polymer particles according to item 4, 7.R 1b Item 5. The polymer particles according to item 4, wherein is —P(O)(OH) 2 . 8.R 3 Item 8. The polymer particles according to any one of Items 5 to 7, wherein is —H. 9.R 4 is -H or -C1-C6 alkyl, and optionally R 4Item 9. The polymer particles according to any one of items 5 to 8, wherein is —H. 10.R 5 can be -H or -C1-C6 alkyl, and optionally R 5 Item 10. The polymer particles according to any one of items 5 to 9, wherein is —H. 11. Polymer particles according to any preceding paragraph, wherein the hydrophilic vinyl monomer is selected from or comprises acrylamide, hydroxymethylacrylamide, [tris(hydroxymethyl)methyl]acrylamide, 3-acrylamidopropanoic acid, methacrylamide, 2-hydroxyethylmethacrylamide, (3-aminopropyl)methacrylamide, or 2-hydroxyethylacrylate, and optionally the hydrophilic vinyl monomer is selected from or comprises one of acrylamide(prop-2-enamide), hydroxymethylacrylamide, trishydroxymethylmethacrylamide, 2-hydroxyethylmethacrylate, acrylic acid, itaconic acid, fumaric acid, vinylphosphonic acid, vinylsulfonic acid, itaconic anhydride, and maleic anhydride. 12. Polymer particles according to any preceding paragraph, wherein the hydrophilic vinyl monomer contains a primary amide group (-C(O)NH2). 13. The crosslinker has formula (IIa) or (IIb): TIFF0007765421000024.tif51128, In the formula, R 6 is selected from -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 hydroxyalkyl-, -C1-C6 ether-, or a polyether, the polyether comprising 2 to 100 C2-C3 ether units; R 7 and R 8 are each independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, -C3-C6 cycloalkyl, -C1-C6 hydroxyalkyl, or -C1-C6 ether; or R 7 and R 8are bonded together to form a -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl-, -C1-C6 hydroxyalkyl-, -C1-C6 ether-, polyether, the polyether containing 2 to 100 C2-C3 ether units; R 9 But -N(R 11 )C(O)CH=CH2, R 10 -H and -N(R 12 )C(O)CH=CH2, and R 11 and R 12 are each independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, -C3-C6 cycloalkyl, -C1-C6 hydroxyalkyl, or -C1-C6 ether; Optionally, R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 and each independently, when chemically possible, be oxo, ═NR a , =NOR a , halo, nitro, cyano, NR a R a , N.R. a S(O)2R a , N.R. a CONR a R a , N.R. a CO2R a , OR a , S.R. a , S(O)R a , S(O)2OR a , S(O)2R a , S(O)NR a R a , CO2R a C(O)R a ,CONR a R a, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl; R a is independently selected at each occurrence from H, C1-C4 alkyl, and C1-C4 alkenyl. 14.R 6 But (CH2) r (OCH2CH2) n O(CH2) s and r and s are each independently 2 or 3; and n is an integer from 1 to 100, Item 14. The compound according to item 13, wherein r and s are each 2 and n is an integer of 1 to 50. 15.R 7 and / or R 8 Item 15. The polymer particles according to item 13 or 14, wherein 16. The polymer particles of any preceding paragraph, wherein the crosslinker has a log P value of less than about 1, optionally less than about 0.5, and further optionally less than about 0. 17. The crosslinker is or is not selected from the group consisting of N,N'-(1,2-dihydroxyethylene)bisacrylamide, N,N'-methylenebis(acrylamide), N,N'-ethylenebis(acrylamide), glycerol 1,3-diglycerolate diacrylate, piperazine diacrylamide, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW 2000 or less), 4-arm PEG-acrylamide (MW 2000 or less), and / or N,N-bis(2-acrylamidoethyl)acrylamide. or the crosslinker is or comprises 1,2-dihydroxybis-acrylamide, or the crosslinker is or comprises N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW 2000 or less), and 4-arm PEG-acrylamide (MW 2000 or less), optionally wherein the crosslinker is or comprises N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide. 18. The polymer particles of any preceding paragraph, wherein the crosslinking agent does not contain a primary amine, a secondary amine, a hydroxyl, or a carboxylic acid. 19. Polymer particles according to any preceding paragraph, wherein the level of crosslinking is from 1 to 60% by weight crosslinker, optionally from 1 to 30% by weight crosslinker, or the level of crosslinking is from 10 to 90% by weight crosslinker, optionally from 25 to 60% by weight crosslinker. 20. The polymer particles according to any one of items 1 to 3, wherein the hydrophilic vinyl monomer is a crosslinking agent defined in any one of items 13 to 19. 21. A polymer particle according to any preceding paragraph, wherein the polymer comprises pores and the pores contain at least a portion of a magnetic material, optionally the magnetic material being substantially located within the pores. 22. The polymer particles according to item 21, wherein the pores provide the polymer with a porosity level of 40% to 95%. 23. Polymer particles according to any of the preceding paragraphs, wherein the magnetic material is a nanoparticle magnetic material and / or the magnetic material is a superparamagnetic material. 24. Polymer particles according to any preceding paragraph, wherein the magnetic material is a metal oxide or alloy and / or the magnetic material comprises ferromagnetic iron oxide crystals and / or superparamagnetic iron oxide crystals. 25. The polymer particles of any preceding paragraph, further comprising a coating. 26. The polymer particles according to paragraph 25, wherein the coating is a polymer coating formed from at least one epoxide monomer. 27. The polymer particles of claim 26, wherein the coating comprises silica. 28. (a) log P less than about 0.6 oct / wat (log P), and (b) a crosslinker containing at least two vinyl groups a polymer formed from and a coating. 29. The polymer particles according to item 28, wherein the coating is or comprises an organic coating or a silica coating. 30. The polymer particles according to item 28 or 29, wherein the coating is an organic coating formed from at least one epoxide monomer. 31. The polymer particles according to paragraph 28 or 29, wherein the coating comprises silica. 32. The polymer particles according to any one of paragraphs 28 to 31, wherein the hydrophilic vinyl monomer is as further defined in any one of paragraphs 2 to 12 or 20. 33. The polymer particles according to any one of items 28 to 32, wherein the crosslinking agent is as further defined in any one of items 13 to 19. 34. The polymer particles according to any one of items 28 to 33, wherein the polymer contains pores. 35. Polymer particles according to any preceding paragraph, having an average diameter of from 0.5 μm to 10 μm, optionally from 0.5 μm to 5 μm. 36. Polymer particles according to any preceding paragraph, having an average diameter of at least 0.5 μm, optionally at least 0.6 μm, further optionally at least 0.7 μm or at least 0.8 μm. 37. Polymer particles according to any preceding paragraph, having a coefficient of variation (CV) of less than 20%, optionally said CV is less than 15%, and further optionally said CV is less than 10%. 38. The polymer particle of any preceding paragraph, wherein the polymer particle comprises a functional group, optionally the functional group being selected from a carboxylic acid (—COOH), a primary amine, or a secondary amine. 39. Monodisperse Hydrogel Polymer Particles and Fe 2+ and / or Fe 3+ forming an aqueous suspension having a pH of less than 6, the suspension comprising: increasing the pH to greater than 8; and Precipitating the magnetic material 1. A method of forming the monodisperse magnetic hydrogel polymer particles, comprising: the monodisperse hydrogel polymer particles (a) log P less than about 0.6 oct / wat (log P), and (b) a crosslinker containing at least two vinyl groups The method of claim 1, wherein 40. The aqueous suspension having a pH of less than 6 contains Fe 2+ and Fe 3+ 40. The method of claim 39, wherein the suspension comprises FeCl2 and FeCl3. 41. The method of claim 39 or 40, further comprising forming a polymer coating and / or a silica coating on the magnetic hydrogel polymer particles. 42. The method of claim 41, wherein the step of forming the polymer coating comprises forming a suspension comprising monodisperse magnetic hydrogel polymer particles and at least one epoxide, and reacting epoxy groups of the at least one epoxide with surface functional groups of the magnetic hydrogel polymer particles, optionally wherein the surface functional groups are amines. 43. The method of claim 41, wherein the step of forming the silica coating comprises forming a suspension containing monodisperse magnetic hydrogel polymer particles and a silicate or orthosilicate, and reacting the silicate or silane to form the silica coating. 44. The method of claim 43, wherein reacting the silicate to form a silica coating comprises lowering the pH of the suspension to less than 11. 45. The method of claim 43, wherein reacting the orthosilicate to form a silica coating comprises increasing the temperature of the suspension to at least 80°C. 46. ​​Forming a suspension comprising monodisperse hydrogel polymer particles and at least one epoxide or silicate or orthosilicate; reacting the epoxy groups of at least one epoxide with the surface functional groups of said particles; or reacting the silicate or the orthosilicate to form a silica coating. 1. A method of forming coated monodisperse hydrogel polymer particles, comprising: the monodisperse hydrogel polymer particles (a) log P less than about 0.6 oct / wat (log P), and (b) a crosslinker containing at least two vinyl groups The method of claim 1, wherein 47. The method of claim 46, wherein the surface functional group is an amine. 48. The method of claim 46, wherein the step of reacting the silicate to form a silica coating comprises lowering the pH of the suspension to less than 11. 49. The method of claim 46, wherein the step of reacting the orthosilicate to form a silica coating comprises increasing the temperature of the suspension to above 90°C. 50. Forming a solution (a) of at least 2% by weight of a hydrophilic vinyl monomer in an aqueous solution, the solution also containing a crosslinker containing at least two vinyl groups; forming a solution (b) of a stabilizer in an organic solvent, mixing solutions (a) and (b) to form a water-in-oil emulsion (c), and adding monodisperse seed particles to the emulsion, wherein the organic solvent is immiscible in water, and at least one of solutions (a) and (b) comprises a radical initiator; allowing said monodisperse seed particles to form swollen particles in said emulsion; and polymerizing the swollen particles to form monodisperse hydrogel polymer particles. Item 50. The method according to any one of Items 39 to 49, wherein the monodisperse hydrogel polymer particles are formed according to a method comprising: 51. The method of claim 50, wherein the hydrophilic vinyl monomer is as further defined in any of claims 2 to 12 or 20. 52. The method of paragraph 50 or 51, wherein the cross-linking agent is as further defined in any of paragraphs 13 to 19. 53. The stabilizer is a non-ionic surfactant, and optionally the non-ionic surfactant is a non-ionic polymeric surfactant; Further optionally, the nonionic polymeric surfactant comprises at least one polyethyleneoxy group or at least one polypropyleneoxy group, and / or 53. The method according to any one of Items 50 to 52, wherein the nonionic polymer surfactant is an oligomeric surfactant. 54. The method of any one of items 50 to 53, wherein the stabilizer is selected from Hypermer 2296, Abil EM90, and sorbitan monooleate. 55. The method of any one of paragraphs 50 to 53, wherein the radical initiator is a peroxide initiator or an azo initiator, and optionally the radical initiator is 2,2'-azobis-2,4-dimethylvaleronitrile. 56. The organic solvent comprises at least one of an aliphatic hydrocarbon, an aliphatic carbonate, an aliphatic ester, an aliphatic ether, an aromatic hydrocarbon, or a silicone, or a combination thereof; and / or 56. The method of any one of items 50 to 55, wherein the organic solvent comprises a mixture of heptane and toluene, a mixture of aliphatic hydrocarbons, bis(2-ethylhexyl) carbonate, or bis(2-ethylhexyl) adipate, optionally mixed with aliphatic and aromatic hydrocarbons. 57. The method according to any one of items 50 to 56, wherein the solution (a) contains 60% by weight or less of a hydrophilic vinyl monomer. 58. The method of any of paragraphs 50 to 57, wherein solution (a) comprises at least 2% by weight of a hydrophilic vinyl monomer and not more than 60% by weight of a hydrophilic vinyl monomer, and optionally, solution (a) comprises at least 5% by weight of a hydrophilic vinyl monomer and not more than 45% by weight of a hydrophilic vinyl monomer. 59. The method of any one of items 50 to 58, wherein solution (a) contains about 10% by weight of a hydrophilic vinyl monomer. 60. The method of any of paragraphs 50 to 59, wherein the initiator is present in the emulsion in an amount of about 0.1% to about 1.5% by weight, and optionally, the initiator is present in the emulsion in an amount of about 0.6% to about 1.2% by weight, and further optionally, the initiator is present in the emulsion in an amount of about 0.8% by weight. 61. The method of any one of items 50 to 60, wherein the polymerizing step includes activating the radical initiator. 62. Forming a solution (a) of at least 2% by weight of a hydrophilic vinyl monomer in an aqueous solution, the aqueous solution also containing a chain transfer agent; forming a solution (b) of the stabilizer in an organic solvent, the organic solvent being immiscible in water, and at least one of solution (a) and solution (b) comprising a radical initiator; mixing solutions (a) and (b) to form a water-in-oil emulsion (c) and adding monodisperse seed particles to said emulsion; allowing said monodisperse seed particles to form swollen grains in said emulsion; polymerizing the swollen particles to form monodisperse polymer particles; forming a solution (d) of the stabilizer in an organic solvent, wherein the organic solvent is immiscible in water; forming a solution (e) of at least 2% by weight of a hydrophilic vinyl monomer in an aqueous solution, the aqueous solution also containing a crosslinker containing at least two vinyl groups, and at least one of solution (d) and solution (e) containing a radical initiator; mixing solutions (d) and (e) to form a water-in-oil emulsion (f), and adding the monodisperse polymer particles to the emulsion; allowing said monodisperse polymer particles to form swollen polymer particles in said emulsion; and polymerizing the swollen polymer particles to form the monodisperse hydrogel polymer particles. Item 50. The method according to any one of Items 39 to 49, wherein the monodisperse hydrogel polymer particles are formed according to a method comprising: 63. The method of any of paragraphs 50 to 62, wherein the monodisperse seed particles comprise a Z-average diameter of 100 nm to 1500 nm, and each seed particle comprises a plurality of non-crosslinked oligomers of poly N,N-dimethylacrylamide. 64. The method of any of paragraphs 50 to 63, wherein the monodisperse seed particles are formed by radical-initiated polymerization of N,N-dimethylacrylamide ...

Claims

1. (a) a log P of less than 0.6 oct / wat (log P), and (b) a crosslinker containing at least two vinyl groups a polymer formed from Coating and 1. A monodisperse coated hydrogel polymer particle comprising: The hydrophilic vinyl monomer is selected from the group consisting of the following monomers: (i) Monomers including hydroxymethyl acrylamide, [tris(hydroxymethyl)methyl]acrylamide, 4-acryloylmorpholine, 3-acrylamidopropanoic acid, methacrylamide, 2-hydroxyethyl methacrylamide, (3-aminopropyl)methacrylamide, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, itaconic acid, fumaric acid, vinylphosphonic acid, vinylsulfonic acid, itaconic anhydride, 1-vinylimidazole, maleic anhydride, N-hydroxyethyl acrylamide, or N-(2-hydroxyethyl)methacrylamide; (ii) Formula (Ib): The compound (iii) Formula (Ic): The compound In the formula, R 1 and R 1a are each independently —H, —CH 3 , -CH 2 CH 3 , or -CH 2 C(O)OH, and (v) a monomer containing at least two vinyl groups, which does not contain a primary amine, a secondary amine, a hydroxyl, or a carboxylic acid; and at least one hydrophilic vinyl monomer selected from The monodisperse coated hydrogel polymer particles, wherein the monodisperse coated hydrogel polymer particles have a coefficient of variation (CV) of their diameter of less than 20%.

2. the coating is or comprises an organic coating or a silica coating; Optionally, the coating comprises an organic coating formed from at least one epoxide monomer; or Optionally, the coating comprises silica.

3. 3. The polymer particle of claim 1, wherein the hydrophilic vinyl monomer is or comprises an acrylate monomer.

4. log P is calculated from the structure of the monomer using CambridgeSoft Corp's ChemBioDraw® Ultra 14.0; and / or The hydrophilic vinyl monomer has the following formula: by R 原子 and a monomer having the formula: In the formula, N C is the number of carbon atoms in the hydrophilic vinyl monomer, and N N is the number of nitrogen atoms in the hydrophilic vinyl monomer, and N O is the number of oxygen atoms in the hydrophilic vinyl monomer, When the hydrophilic vinyl monomer is vinylimidazole, acryloylmorpholine, vinylphosphonic acid, or vinylsulfonic acid, R 原子 is less than 2.75, and When the hydrophilic vinyl monomer is an acrylate or other monomer class, R 原子 4. The polymer particles according to claim 1, wherein the solubility of the hydrophilic vinyl monomer in water at 25° C. is 50 g / L or more.

5. 5. The polymer particle of claim 1, wherein the hydrophilic vinyl monomer is selected from or comprises 4-acryloylmorpholine, methacrylamide, and vinylphosphonic acid.

6. The crosslinker is represented by formula (IIa) or (IIb): and In the formula, R 6 But, -C 1 ~C 6 Alkylene-, -C 1 ~C 6 Heteroalkylene-, -C to C 6 Cycloalkylene-, -C 1 ~C 6 Hydroxyalkylene-, -C 1 ~C 6 ether-, or polyether, wherein the polyether has 2 to 100 C 2 ~C 3 containing ether units, R 7 and R 8 are each independently —H, —C 1 ~C 6 Alkyl, —C 1 ~C 6 Heteroalkyl, —C 3 ~C 6 cycloalkyl, —C 1 ~C 6 Hydroxyalkyl, or -C 1 ~C 6 ethers, or R 7 and R 8 are bonded together to form -C 1 ~C 6 Alkylene-, -C 1 ~C 6 Heteroalkylene-, -C to C 6 Cycloalkylene-, -C 1 ~C 6 Hydroxyalkylene-, -C 1 ~C 6 ether- or polyether, wherein the polyether has 2 to 100 C 2 ~C 3 containing ether units, R 9 But -N(R 11 )C(O)CH=CH 2 and R 10 is -H and -N(R 12 )C(O)CH=CH 2 is selected from R 11 and R 12 are each independently —H, —C 1 ~C 6 Alkyl, —C 1 ~C 6 Heteroalkyl, —C 3 ~C 6 cycloalkyl, —C 1 ~C 6 hydroxyalkyl, or —C 1 ~C 6 6. The polymer particles according to claim 1, wherein the polymer particles are selected from the group consisting of ethers.

7. R 6 But, -C 1 ~C 6 Alkylene-, -C 1 ~C 6 Heteroalkylene-, -C to C 6 Cycloalkylene-, -C 1 ~C 6 ether-, or polyether, wherein the polyether has 2 to 100 C 2 ~C 3 containing ether units, R 7 and R 8 are each independently —H, —C 1 ~C 6 Alkyl, —C 1 ~C 6 Heteroalkyl, —C 3 ~C 6 cycloalkyl, or -C 1 ~C 6 ethers, or R 7 and R 8 are bonded together to form -C 1 ~C 6 Alkylene-, -C 1 ~C 6 Heteroalkylene-, -C to C 6 Cycloalkylene-, -C 1 ~C 6 ether- or polyether, wherein the polyether has 2 to 100 C 2 ~C 3 containing ether units, and R 11 and R 12 are each independently —H, —C 1 ~C 6 Alkyl, —C 1 ~C 6 Heteroalkyl, —C 3 ~C 6 cycloalkyl, or —C 1 ~C 6 ethers, and / or R 6 However, (CH 2 ) r (OCH 2 CH 2 ) n O (CH 2 ) s and where: r and s are each independently 2 or 3; and n is an integer from 1 to 99, Optionally, 7. The polymer particles of claim 6, wherein r and s are each 2 and n is an integer from 1 to 50.

8. the crosslinker has a log P value of less than 1, optionally less than 0.5, and further optionally less than 0; Optionally, 8. The polymer particle of claim 1, wherein log P is calculated from the structure of the crosslinker using ChemBioDraw® Ultra 14.0 from CambridgeSoft Corp.

9. the crosslinker is or comprises N,N'-(1,2-dihydroxyethylene)bisacrylamide, N,N'-methylenebis(acrylamide), N,N'-ethylenebis(acrylamide), glycerol 1,3-diglycerolate diacrylate, piperazine diacrylamide, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW ≦2000), 4-arm PEG-acrylamide (MW ≦2000), and / or N,N-bis(2-acrylamidoethyl)acrylamide; or the crosslinker is or comprises 1,2-dihydroxybis-acrylamide, or the crosslinker is or comprises N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW 2000 or less), and 4-arm PEG-acrylamide (MW 2000 or less); optionally, the cross-linking agent is or comprises N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide; and / or 9. The polymer particles of claim 1, wherein the crosslinking agent does not contain a primary amine, a secondary amine, a hydroxyl, or a carboxylic acid.

10. the polymer comprises pores; providing a polymer in which the pores have a porosity level of between 40% and 95%; 10. The polymer particle according to any one of claims 1 to 9, wherein the porosity is determined from the volume of water present in the pores compared to the total volume of the polymer particle saturated with water.

11. the polymer particles have an average diameter of from 0.5 μm to 10 μm, optionally from 0.5 μm to 5 μm; and / or 11. The polymer particles of any one of claims 1 to 10, having a coefficient of variation (CV) of less than 15%, optionally said CV being less than 10%.

12. forming a suspension comprising monodisperse hydrogel polymer particles and at least one epoxide or silicate or orthosilicate; and reacting the epoxy groups of the at least one epoxide with the surface functional groups of the particles; or reacting the silicate or the orthosilicate to form a silica coating.

1. A method of forming coated monodisperse hydrogel polymer particles, comprising: the monodisperse hydrogel polymer particles (a) a log P of less than 0.6 oct / wat (log P), and (b) a crosslinker containing at least two vinyl groups is formed from The hydrophilic vinyl monomer is selected from the group consisting of the following monomers: (i) Monomers including hydroxymethyl acrylamide, [tris(hydroxymethyl)methyl]acrylamide, 4-acryloylmorpholine, 3-acrylamidopropanoic acid, methacrylamide, 2-hydroxyethyl methacrylamide, (3-aminopropyl)methacrylamide, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, itaconic acid, fumaric acid, vinylphosphonic acid, vinylsulfonic acid, itaconic anhydride, 1-vinylimidazole, maleic anhydride, N-hydroxyethyl acrylamide, or N-(2-hydroxyethyl)methacrylamide; (ii) Formula (Ib): The compound (iii) Formula (Ic): The compound In the formula, R 1 and R 1a are each independently —H, —CH 3 , -CH 2 CH 3 , or -CH 2 C(O)OH, and (v) a monomer containing at least two vinyl groups, which does not contain a primary amine, a secondary amine, a hydroxyl, or a carboxylic acid; and at least one hydrophilic vinyl monomer selected from The method, wherein the monodisperse coated hydrogel polymer particles have a coefficient of variation (CV) of their diameter of less than 20%.

13. the surface functional groups are amines, or the step of reacting the silicate to form a silica coating comprises lowering the pH of the suspension to less than 11; or 13. The method of claim 12, wherein the step of reacting the orthosilicate to form a silica coating comprises raising the temperature of the suspension to above 90°C.

14. Use of polymer particles according to any one of claims 1 to 11 in an assay method.

15. Use of polymer particles according to any one of claims 1 to 11 in nucleic acid amplification, comprising: Optionally, The use, wherein the nucleic acid amplification is emulsion PCR.

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