Polymer particles

Abstract

The present invention relates to monodisperse magnetic hydrogel polymer particles comprising a magnetic material and a polymer formed from: (a) a hydrophilic vinyl monomer having a log P oct / wat (log P) is less than about 0.5; and (b) a crosslinker comprising at least two vinyl groups. The present invention also relates to monodisperse coated hydrogel polymer particles comprising a polymer 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 (log P) is less than about 0.5; and (b) a crosslinker comprising at least two vinyl groups; and a coating. Also provided are methods of forming monodisperse magnetic hydrogel polymer particles and monodisperse coated polymer particles.

Description

[0001] This application is a divisional application of a patent application with application number 201880012834.0, application date January 19, 2018, and invention name “Polymer Particles”.

[0002] The present invention relates to monodisperse polymer particles that can be used in bioassays and other applications. The monodisperse polymer particles are magnetic and / or coated. The present invention also relates to methods of making such particles and methods of using the particles, as well as other subjects. Background Art

[0003] Polymer particles are used in various applications, such as bioassays or sequencing applications. In some of these applications, it is desirable to include a magnetic material in the polymer particles, such as to allow magnetic isolation of the polymer particles. In some of these applications, it is desirable to provide a coating on the polymer particles, such as to change the surface properties of the particles. The base polymer particles are typically prepared by emulsion polymerization or dispersion / precipitation polymerization.

[0004] Emulsion polymerization can be used to form relatively monodisperse polymer particles of 50-1,000 nm. However, the formation of monodisperse polymer particles does have a number of important limitations. First, all particles must be formed as part of the same batch and in a short time relative to the polymerization time. Second, the growth conditions need to be controlled to ensure that the growth of the polymer particles is the same in all particles. Third, because the process requires the transfer and swelling of monomer from a large reservoir droplet into the growing particle, the particles are not cross-linked, or have very low levels of cross-linking, as higher levels of cross-linking would prevent the particles from swelling.

[0005] Precipitation or dispersion polymerization can be used to produce larger particles of 0.5-10 microns. Precipitation polymerization is carried out in a solvent (e.g., alcohol) that dissolves the monomers but not the polymer. Monodispersity can be achieved when the polymer is precipitated from the solution and particle formation occurs in a short time. Relatively large amounts of steric stabilizers are required to stabilize the particles after precipitation. The amount and type of stabilizer can change the size of the particles, and porous particles can be obtained by using high levels of cross-linking agents. However, the conditions of precipitation polymerization are difficult to control, for example, it is difficult to control the porosity, which is a result of how the pore structure is formed when the polymer is precipitated from the solution. The difficulty of precipitation polymerization means that it is almost impossible in practice to produce porous particles with the same monomer composition and porosity for more than a very limited size variation.

[0006] One approach to solving some of the problems is to produce crosslinked porous or solid monodisperse polymer particles by a two-stage process, named the Ugelstad process after the late Professor John Ugelstad, which is described, for example, in EP-B-3905 and US-A-4530956. An improved Ugelstad process is described in WO00 / 61647. In the Ugelstad process, seed particles, suitably prepared by emulsion polymerization, are converted into monodisperse particles by seed suspension polymerization in two steps. In the first step, the seed particles are swollen by preparing a fine (e.g., submicron) aqueous emulsion of a relatively low molecular weight water-insoluble substance and then adding an organic solvent (e.g., acetone) miscible with water, so that the water-insoluble substance diffuses into the seed particles. It is convenient that the water-insoluble substance is a heat-activated polymerization initiator. In the second step, the solvent is then removed, the water-insoluble substance is locked in the seed particles, and the seed particles absorb a large amount of monomers and crosslinking agents. After initiating the polymerization, for example, by heating to activate the initiator, larger polymer particles are produced. Thus, the Ugelstad process comprises preparing seed particles by emulsion polymerization and swelling the seed particles by suspension polymerization.The average diameter of the smallest monodisperse particles described in the above-mentioned prior art is 1 μm.

[0007] In a simplified version of the Ugelstad process, enhanced swelling capacity can be achieved simply by using oligomeric seed particles, for example where the oligomer weight average molecular weight corresponds to up to 50 monomer units (in the case of polystyrene the molecular weight is about 5000). This is described in US 4530956. In another version of the Ugelstad process described in WO2010 / 125170, oligomeric seed particles can be used to prepare monodisperse particles with an average diameter in the submicron range.

[0008] Particles prepared according to the Ugelstad method as described above or variations thereof can be made magnetic, for example as described in WO00 / 61647 or WO2010 / 125170. However, particles prepared by the Ugelstad method and variations thereof are made of hydrophobic monomers such as styrene, usually using oil (discontinuous phase) in an aqueous (continuous phase) system. Therefore, the resulting polymer particles are hydrophobic. When used for biological applications, hydrophobic particles suffer from the problem of non-specific absorption. This means that hydrophobic polymer particles are usually surface-modified, for example with a coating, to increase the hydrophilicity of the surface before being used in biological applications.

[0009] US2014 / 0073715 describes a method for preparing monodisperse hydrophilic particles. The method uses a monomer with a hydrophobic protecting group, which is added to the monomer that is polymerized to form the particles, and the protecting group is subsequently removed. However, the addition and removal of the protecting group does increase the complexity of the method and may limit the number of different types of monomers that can be used in the method.

[0010] It is clear that the known magnetic monodisperse polymer particles and methods for preparing such particles are subject to a number of limitations. This also applies to coated monodisperse polymer particles. There is therefore a need for new monodisperse polymer particles and new methods for their production.

[0011] It is an object of the present invention to provide monodisperse magnetic polymer particles and methods for preparing monodisperse magnetic polymer particles having a low coefficient of variation (CV) and / or a low % polydispersity. It is also an object of the present invention to provide coated monodisperse polymer particles and methods for preparing coated monodisperse polymer particles having a low coefficient of variation (CV) and / or a low % polydispersity. Summary of the invention

[0012] The present invention is based in part on the recognition that seed particles comprising hydrophilic oligomers can be used in a new method of forming cross-linked monodisperse polymer particles. The present invention is also based on the recognition that the formed cross-linked monodisperse polymer particles can be made magnetic by depositing magnetic materials in the pores of the cross-linked monodisperse polymer particles. The present invention is also based on the recognition that the formed cross-linked monodisperse polymer particles can be coated with other substances.

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

[0014] In one embodiment, the hydrophilic vinyl monomer comprises at least one hydrophilic vinyl monomer selected from the following monomers:

[0015] (i) It contains -C(O)NH 2 、-C(O)NHCH 3 、-C(O)NHCH 2 CH 3 or -C(O)N(CH 3 ) 2 ;

[0016] (ii) Compounds of formula (Ib)

[0017]

[0018] (iii) Compounds of formula (Ic):

[0019]

[0020] in:

[0021] R 1 and R 1a Each independently selected from -H, -CH 3 、-CH 2 CH 3 or -CH 2 C(O)OH;

[0022] (iv) 4-acryloylmorpholine or 1-vinylimidazole; and

[0023] (v) It includes at least two vinyl groups that do not contain a primary amine, a secondary amine, a hydroxyl group or a carboxylic acid.

[0024] A second aspect of the present invention provides monodisperse coated hydrogel polymer particles comprising a polymer formed from: (a) a hydrophilic vinyl monomer having a log P oct / wat (log P) is less than about 0.6; and (b) a crosslinker comprising at least two vinyl groups; the particle further comprising a coating.

[0025] In one embodiment, the hydrophilic vinyl monomer comprises at least one hydrophilic vinyl monomer selected from the following monomers:

[0026] (i) It contains -C(O)NH 2 、-C(O)NHCH 3 、-C(O)NHCH 2 CH 3 or -C(O)N(CH 3 ) 2 ;

[0027] (ii) Compounds of formula (Ib)

[0028]

[0029] (iii) Compounds of formula (Ic):

[0030]

[0031] in:

[0032] R 1 and R 1a Each independently selected from -H, -CH 3 、-CH 2 CH 3 or -CH2 C(O)OH;

[0033] (iv) 4-acryloylmorpholine or 1-vinylimidazole; and

[0034] (v) It includes at least two vinyl groups that do not contain a primary amine, a secondary amine, a hydroxyl group or a carboxylic acid.

[0035] 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.

[0036] A fourth aspect of the present invention provides a method for forming monodisperse hydrogel polymer particles such as those of the third aspect. In one embodiment, the method comprises:

[0037] Forming a solution (a): an aqueous solution of at least 2% wt of a hydrophilic vinyl monomer, which comprises a monomer selected from 4-acryloylmorpholine and 1-vinylimidazole, and the solution further comprises a cross-linking agent containing at least two vinyl groups;

[0038] forming a solution (b) of a stabilizer in an organic solvent, wherein the organic solvent is immiscible with water and wherein at least one of the solution (a) and the solution (b) comprises a free radical initiator; mixing solutions (a) and (b) to form a water-in-oil emulsion (c) and adding monodisperse seed particles to the emulsion;

[0039] allowing monodisperse seed particles to form swollen particles in the emulsion; and

[0040] The swollen particles are polymerized to form monodisperse hydrogel polymer particles.

[0041] In one embodiment, the method comprises:

[0042] Forming a solution (a): an aqueous solution of at least 2% wt of a hydrophilic vinyl monomer, which comprises a monomer selected from 4-acryloylmorpholine and 1-vinylimidazole, and the aqueous solution further comprises a chain transfer agent;

[0043] forming a solution (b) of a stabilizer in an organic solvent, wherein the organic solvent is immiscible with water, and wherein at least one of solution (a) and solution (b) comprises a free radical initiator;

[0044] mixing solutions (a) and (b) to form a water-in-oil emulsion (c) and adding monodisperse seed particles to the emulsion;

[0045] allowing monodisperse seed particles to form swollen particles in the emulsion;

[0046] polymerizing the swollen particles to form monodisperse polymer particles;

[0047] forming a solution of the stabilizer in an organic solvent (d), wherein the organic solvent is immiscible with water;

[0048] Forming a solution (e): an aqueous solution of at least 2%wt of a hydrophilic vinyl monomer, which comprises a monomer selected from 4-acryloylmorpholine and 1-vinylimidazole, and the aqueous solution further comprises a cross-linking agent containing at least two vinyl groups, wherein at least one of the solution (d) and the solution (e) comprises a free radical initiator;

[0049] mixing solutions (d) and (e) to form a water-in-oil emulsion (f) and adding monodisperse polymer particles to the emulsion;

[0050] allowing monodisperse polymer particles to form swollen polymer particles in the emulsion; and

[0051] The swollen polymer particles are polymerized to form monodisperse hydrogel polymer particles.

[0052] A fifth aspect provides a method for forming monodisperse magnetic hydrogel polymer particles. The method comprises forming an aqueous suspension comprising monodisperse hydrogel polymer particles and a magnetic material or a magnetic material precursor. When the aqueous suspension comprises a magnetic material precursor, the magnetic material precursor is converted to a magnetic material. The magnetic material (regardless of whether the aqueous suspension initially contains a magnetic material precursor) is combined with the polymer particles. The monodisperse hydrogel polymer particles are composed of (a) log P oct / wat (log P) is less than about 0.6 hydrophilic vinyl monomer; and (b) comprises at least two vinyl crosslinking agent to form. In one embodiment, forming an aqueous suspension comprises forming an aqueous suspension having a pH less than 6, which comprises monodisperse hydrogel polymer particles and Fe 2+ and / or Fe 3+ ions; and converting the magnetic material precursor into the magnetic material comprises raising the pH to greater than 8; and allowing the magnetic material to precipitate.

[0053] A sixth aspect provides a method for forming coated monodisperse hydrogel polymer particles. The method comprises forming a suspension comprising monodisperse hydrogel polymer particles and at least one epoxide or at least one silicate or at least one silane; reacting the epoxy groups of the at least one epoxide with the surface functional groups of the particles; or reacting the silicate to form a silica coating. The monodisperse hydrogel polymer particles are composed of (a) log P oct / wat (log P) is less than 100 Å; and (b) a cross-linking agent comprising at least two vinyl groups is formed.

[0054] The seventh aspect comprises monodisperse magnetic hydrogel polymer particles or coated monodisperse hydrogel polymer particles obtainable by the method of the fifth or sixth aspect.Monodisperse Magnetic Hydrogel Polymer ParticlesMonodisperse or coated monodisperse hydrogel polymer particles may be obtainable by the method of the fifth or sixth aspect.

[0055] An eighth aspect comprises using the monodisperse magnetic hydrogel polymer particles of the first or seventh aspect in an assay.

[0056] A ninth aspect comprises using the coated monodisperse hydrogel polymer particles of the second or seventh aspect in an assay.

[0057] The tenth aspect comprises using the monodisperse hydrogel polymer particles of the third aspect, or the monodisperse hydrogel polymer particles formed by the method of the fourth aspect, in an assay.

[0058] One embodiment of the present invention provides particles obtained by the method described in this specification.

[0059] One embodiment of the present invention provides particles having the characteristics of particles obtained by the methods disclosed herein; although these particles can be obtained by the methods described herein, they are characterized only by their properties and not by their methods of manufacture, and therefore, the scope of protection of claims directed to particles specified by their characteristics is determined solely by their characteristics, excluding their actual methods of manufacture.

[0060] The products, methods and uses of the present invention are not limited to the subject matter just mentioned but are more fully described without limitation in the following description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Embodiments of the present invention are further described below with reference to the accompanying drawings, in which:

[0062] Figure 1 is a diagrammatic representation of the single-stage swollen particle formation process of the present disclosure, including an indication of example monomers.

[0063] Figure 2 is a diagrammatic representation of the single-stage particle formation process of the present disclosure.

[0064] Figure 3 is a schematic diagram of the two-stage swollen particle formation process of the present disclosure, including an indication of example monomers and chain transfer agents.

[0065] Figure 4 is a diagrammatic representation of the two-stage swollen particle formation method of the present disclosure.

[0066] Figure 5 The use of the particles of the invention in nucleotide amplification and / or detection is described.

[0067] Figure 6 Illustrated are the signals as a function of antigen concentration obtained in the assay of Example 67. Diamonds represent the results for the particles of Example 34, squares represent the results for the particles of Example 35 and triangles represent the results for the particles of Example 36.

[0068] Figure 7 Illustrated are the signals as a function of antigen concentration obtained in the assay of Example 68. Diamonds represent the results using the particles of Example 34, squares represent the results using the particles of Example 35 and triangles represent the results using the particles of Example 36.

[0069] Figure 8 Illustrated are the signals as a function of antigen concentration obtained in the assay of Example 69. Diamonds represent the results using the particles of Example 34, squares represent the results using the particles of Example 37 and triangles represent the results using the particles of Example 38.

[0070] Fig. 9 The particles of Examples 34, 35 and 36 are described for use with the commercial MyOne TM Nonspecific binding was obtained compared to epoxy particles. DETAILED DESCRIPTION

[0071] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of these words mean "including (but not limited to)", and are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood to contemplate the plural as well as the singular unless the context requires otherwise.

[0072] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All features disclosed in this specification (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 for at least some mutually exclusive combinations of such features and / or steps. The invention is not limited to the details of any embodiment disclosed herein. The invention extends to any novel feature or any novel combination of features disclosed in this specification (including any accompanying claims, abstract and drawings), or any novel step or any novel combination of steps of any method or process so disclosed.

[0073] The reader's attention is directed to all papers and documents which are filed concurrently with or before the present specification in connection with the present application and which are open to the public upon inspection of the present application, and the contents of all such papers and documents are incorporated herein by reference.

[0074] 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.

[0075] In one embodiment, the present invention provides novel magnetic polymer particles, and another embodiment provides methods by which the novel particles can be prepared. Other embodiments provide methods of using the polymer particles.

[0076] The polymer particle formation process comprises a water-in-oil emulsion, ie a discontinuous aqueous phase suspended in a continuous oil phase. The formed polymer particles may then be magnetized and / or coated.

[0077] Thus, the polymer particle formation method described herein involves two different particles, namely Seed The term "seed particles" and "polymer particles" are used herein as follows:

[0078] Unless the context requires otherwise, "seed particles" are particles obtainable by dispersion polymerization and used as intermediates in the formation of polymer particles.

[0079] "Polymer particles" refers to particles that can be prepared from seed particles by suspension polymerization in the methods described herein. After polymerization, magnetic material can be added to the particles. For example, in the case where the polymer particles contain pores, the magnetic material can be deposited in at least a portion of the pores. After polymerization (and optionally addition of magnetic material), the particles can be coated, for example, with an organic (e.g., polymer) or silica coating.

[0080] The polymer particles can be "formed" from a specific hydrophilic vinyl monomer and a crosslinking agent. The specific hydrophilic monomer can include one or more monomers. The specific crosslinking agent can include one or more crosslinking agents. "Formed from..." herein can mean that the polymer consists of a specific hydrophilic vinyl monomer and a crosslinking agent that react. "Formed from..." herein can mean that the polymer contains a specific hydrophilic vinyl monomer and a crosslinking agent, as well as a small amount of other vinyl monomers and / or crosslinking agents. For example, the polymer can contain at least 90% by weight of the specific monomer and the crosslinking agent, and no more than 10% by weight of other vinyl monomers and / or crosslinking agents; for example, the polymer can contain at least 95% by weight of the specific monomer and the crosslinking agent, and no more than 5% by weight of other vinyl monomers and / or crosslinking agents. For example, the polymer can contain at least 98% by weight of the specific monomer and the crosslinking agent, and no more than 2% by weight of other vinyl monomers and / or crosslinking agents; for example, the polymer can contain at least 99% by weight of the specific monomer and the crosslinking agent, and no more than 1% by weight of other vinyl monomers and / or crosslinking agents.

[0081] "Magnetic material" refers to a substance that responds to a magnetic field. When a magnetic material is paramagnetic, the magnetism is turned off when the magnetic field is removed. When a magnetic material is superparamagnetic, the magnetic material becomes saturated under a relatively low magnetic field, and the magnetic properties are turned off very quickly / instantaneously with the removal of the magnetic field. When a magnetic material is ferromagnetic, all of its magnetic atoms in each magnetic domain add a positive contribution to the net magnetization. When a magnetic material is ferromagnetic, some magnetic atoms in each magnetic domain are offset, but the material exhibits a net magnetization as a whole. After the external magnetic field is removed, both ferromagnetic and ferrimagnetic materials remain magnetic. Above the Curie temperature of the material, ferromagnetic and ferromagnetic materials become paramagnetic materials. Magnetic properties may 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 are superparamagnetic at a suitable small particle size (e.g., nm scale). For example, when the size of the crystal is sufficiently small (e.g., below about 15 nm scale for iron oxide), ferromagnetic materials such as iron oxides form superparamagnetic crystals.

[0082] "Magnetic material precursor" refers to a substance that can be transformed to provide a magnetic material. The magnetic material precursor may comprise solvated transition metal ions (e.g., multivalent cations of Fe, Ni, Co, or combinations thereof, optionally mixed with multivalent cations of Al, Mn, Cu, Zn, Ca, Ge, Te, Ti, or Sn and / or rare earths). The solvated transition metal ions and / or rare earth ions may be converted to a magnetic material by any process that causes precipitation of the ions (e.g., oxides). The precipitation may be caused, for example, by a change in pH, removal of a solvent, or a change in temperature. For example, a magnetic material precursor may be formed by a solution containing Fe at a pH of less than 6. 2+ and / or Fe 3+ions; and the Fe 2+ and / or Fe 3+ The ions are converted into magnetic material.

[0083] "Coating" refers to a covering applied to the surface of a substrate. The coating can be a full coverage coating that completely covers the substrate, or it can be a partial coating that only covers a portion of the substrate surface. The coating can be applied to the substrate by any suitable type of combination, such as by at least one of covalent bonding, metal bonding, ionic bonding, hydrogen bonding, van der Waals interaction, hydrophobic interaction, etc. Porous substrates can be coated so that at least a portion of the coating is present in the hole while maintaining a porous structure (for example, because the coating partially fills the hole). In the case where the substrate comprises a polymer, the coating (or at least a portion thereof) can be provided by grafting. In the case where the coating (or at least a portion thereof) is provided by grafting, the coating can be a partial coating. In the present disclosure, unless the context requires otherwise, the substrate is a polymer particle or a plurality of polymer particles. The polymer particles can be magnetic polymer particles or polymer particles that do not contain magnetic materials.

[0084] "Grafting" in the context of polymer chemistry includes reactions in which one or more blocks are attached to the backbone of a macromolecule as side chains, the side chains having different compositional or configurational characteristics than those in the backbone. Where the substrate is a polymer particle or 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 chain (or chains).

[0085] With respect to polymers, such as polymer particles, reference to a "hydrogel" refers to a polymer gel in which the swelling agent is water. A hydrogel polymer can absorb at least 20% of its weight in water. A 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, a hydrogel polymer can absorb at least 1000%, at least 1500%, or even at least 2000% of its weight in water.

[0086] Reference to "permeable" with respect to polymer particles (e.g., hydrogel polymer particles) means that the particles are porous and molecules or other agents of interest can easily diffuse through an aqueous solution in the pores of the particles. For example, the crosslinked hydrogel polymer particles of the present disclosure can be permeable to oligonucleotides and nucleic acid amplification and sequencing reagents, e.g., even if a polymerase is attached to the oligonucleotide, the oligonucleotide can be partially or completely located within the pores.

[0087] Unless otherwise indicated, the "average" diameter referred to in this specification refers to the mode diameter of the crosslinked polymer particles (e.g., crosslinked hydrogel polymer particles, magnetic hydrogel polymer particles, and / or coated hydrogel polymer particles) or refers to the z-average diameter of the seed particles. The mode diameter can be measured by a disc centrifuge, such as a CPS disc centrifuge. The z-average diameter can be the z-average average diameter measured by dynamic light scattering (also known as photon correlation spectroscopy). Throughout the range, embodiments are also disclosed in which the average diameter is the mode diameter, such as the mode diameter determined by optical microscopy.

[0088] The term "monodisperse" means that for a plurality of particles (e.g., at least 100, more preferably at least 1000), the coefficient of variation (CV) or % polydispersity of the diameter of the particles is less than 20%, such as less than 15%, typically less than 10%, optionally less than 8%, such as less than 5%.

[0089] The term "Mw" is the weight average molecular weight of a polymer. It is defined by the formula:

[0090]

[0091] Among them, M i is the molecular weight of a particular chain, N i is the number of chains of this molecular weight. Mw can be measured using gel permeation chromatography (GPC) relative to a set of standard polymers with a specific elution solvent system. For example, the Mw of an oligomer or polymer in a seed particle can be measured by GPC relative to a polystyrene standard using DMF with 0.01 M LiBr as an eluent.

[0092] References to "polydispersity" or "% polydispersity" in this specification refer to the value of the dynamic light scattering data derived from the "polydispersity index". The polydispersity index is a number calculated from a simple 2-parameter fit to related data, such as dynamic light scattering data, as defined in ISO standard documents 13321: 1996E and ISO22412: 2008. The polydispersity index is dimensionless and proportional, so that values ​​less than 0.05 are rarely seen except for highly monodisperse standards. A polydispersity index value of a particle sample greater than 0.7 indicates that the sample has a very broad size distribution, e.g., the particles are not monodisperse.

[0093] As used herein, the terms "alkyl" and "C x -C y"alkyl" (wherein x is at least 1 and less than 10 and y is a number greater than 10) includes reference to a straight or branched chain alkyl moiety having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The 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 "C 1 -C 6 "alkyl", i.e., an alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms; or "C 1 -C 4 The term "lower alkyl" includes alkyl groups having 1, 2, 3 or 4 carbon atoms. Where chemically possible, the alkyl group may be optionally substituted with 1 to 5 substituents, each of which is independently selected from: oxo, =NR a , =NOR a , halogenated, nitro, cyano, NR a R a , NR a S(O) 2 R a , NR a CONR a R a , NR a CO 2 R a , OR a SR a ,S(O)R a ,S(O) 2 OR a ,S(O) 2 R a ,S(O) 2 NR a R a , CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 alkyl.

[0094] As used herein, the terms "alcohol" and "Cx -C y "Alcohol" (wherein x is at least 1 and less than 10 and y is a number greater than 10) includes reference to a straight 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 group substituted with one or more hydroxyl groups (-OH). Thus, the term includes reference to a monohydroxy-substituted alcohol or a polyol (e.g., a diol or triol). In particular, the alcohol may be a "C 1 -C 6 alcohol”, i.e. an alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms and 1 or more hydroxyl groups; or “C 1 -C 4 "Alcohol", i.e. an alcohol having 1, 2, 3 or 4 carbon atoms and one or more hydroxyl groups.

[0095] symbol Indicates the point of attachment of a moiety to the rest of the compound.

[0096] As used herein, the term "log P" refers to the octanol-water partition coefficient (log P) of a compound, such as a hydrophilic vinyl monomer. oct / wat ). The log P of a compound can be determined by any of a variety of methods. In particular, for compounds used in the embodiments and other compounds disclosed herein, log P can be measured according to J. Sangster, "Octanol-water partition coefficients of simple organic compounds", J. Phys. Chem. Ref. Data, Vol. 18, No. 3, 1989, 1111-1227 (incorporated herein by reference in its entirety) page 1116, section 2.1.a. Log P can also be calculated from the structure of the compound using software, for example, Log P can be calculated using CambridgeSoft's Ultra 14.0 computing.

[0097] Since log P is the organic phase-aqueous phase partition coefficient, this value for a given compound is related to the fraction of monomers in the aqueous phase. In particular, the lower (including more negative) this value is, the higher the fraction of the compound (e.g., monomer or cross-linking agent) in water or aqueous phase. Compounds (e.g., monomers or cross-linking agents) used in the present invention should have a sufficiently low log P so that at equilibrium, the compound can be used in sufficient quantities in the aqueous phase for polymerization, for example according to the method for preparing particles disclosed herein. Therefore, Log P can be used to characterize the compounds used in the present invention.

[0098] Based on the molecular structure of the compound, it is also possible to estimate the tendency of the molecule to be in the organic phase or the aqueous phase. For example, a monomer containing more hydrophilic atoms such as nitrogen and oxygen and fewer carbon atoms may have more hydrophilic groups and have an increased tendency to partition into the aqueous phase.

[0099] Since the compounds of interest in the present invention (e.g., monomers and / or crosslinking agents) are typically polymerizable vinyl monomers, a formula that calculates the ratio between the number of hydrophobic and hydrophilic atoms in a compound can be used to provide an estimate of the relative tendency of a compound to partition into the aqueous phase. For a given class of compounds, the value calculated by such a formula can be (roughly) related to log P. Such a formula can be used instead of log P or in addition to log P to determine monomers that can be used in the present invention.

[0100] For example, suitable formulas of the hydrophilic vinyl monomers of the present invention (e.g., compounds of formula I, such as those wherein R 1 -H, -CH 3 )as follows:

[0101]

[0102] Where N C is the number of carbon atoms, N N is the number of nitrogen atoms, N O is the number of oxygen atoms in the monomer. 原子 <2.75, then acrylamide, vinylimidazole, acryloylmorpholine, vinylphosphonic acid and vinylsulfonic acid are suitable; if R 原子 ≤2 and the monomer has a water solubility (e.g. at 25°C) ≥50 g / L, then acrylates and other monomer classes are suitable. Acrylamide is generally more soluble in the aqueous phase than similar acrylates, and R 原子 The difference in passing standards reflects this. 原子 The formula and cut-off criteria may also apply to crosslinkers used in the present invention, such as compounds of formula (IIa) or (IIb). The R values ​​of exemplary monomers and crosslinkers are given in Table 1. 原子 value.

[0103] Polymer particles

[0104] The invention includes embodiments in which the particles are polymeric and monodisperse. The invention includes embodiments in which the particles are porous. According to the invention, the particles are magnetic and / or comprise a coating. Unless the context requires otherwise, references hereinafter to "particles" and "polymer particles" include references to monodisperse magnetic hydrogel polymer particles of the invention and / or monodisperse coated hydrogel polymer particles of the invention.

[0105] The particles may be in a population of at least 100, such as at least 1000. For example, for measurement purposes, the particles may be in a population of at least 100, such as at least 1000. For example, in certain end-use applications, the particles may conveniently be in a population of at least 100, such as at least 1000.

[0106] "Monodisperse" means that for a plurality of particles (e.g., at least 100, more preferably at least 1000), the coefficient of variation (CV) of the diameter of the particles is less than 20%, such as less than 15%, typically less than 10%, optionally less than 8%, such as less than 5%. A particular class of polymer particles has a CV of less than 10%. CV is defined as 100×(standard deviation) divided by the mean, where the "mean" is the average particle size and the standard deviation is the standard deviation of the particle size. The present invention also includes embodiments in which the "average" is the z-average or mode particle size. The CV is preferably calculated in the main mode. Therefore, some particles below or above the mode size can be discounted in a calculation of (detectable particles), which can be based, for example, on about 90% of the total number of particles. Such a determination of CV can be performed on a CPS disc centrifuge.

[0107] The polymer particles may be prepared by the polymer particle formation methods described later in this specification, for example by using monodisperse seed particles as described herein.

[0108] Returning now to the polymer particles, the specification discloses cross-linked hydrogel polymer particles. The cross-linked hydrogel polymer particles are characterized in that when placed in an aqueous solution that is a good solvent for the polymer, the particles swell rather than dissolve. For example, water is a good solvent for hydrogel particles containing acrylamide polymers. The hydrogel particles are hydrophilic and swell in water and can be made into a variety of different pores. The cross-linked hydrogel particles disclosed herein provide a combination of low non-specific binding, monodispersity and porosity, which provides advantages when the particles are used in bioassays. In addition, the non-specific binding and porosity properties can be changed by including magnetic materials in the particles and / or coating the particles.

[0109] The crosslinking level in the polymer particles prepared by the method can be expressed as the weight percentage (%wt) of the crosslinker monomer contained in the total monomers used in the suspension polymerization. The %wt of the crosslinker monomer can be equal to the %wt of the crosslinker in the matrix polymer (i.e., the %wt of the crosslinker in the dry weight of the crosslinked polymer particles). Therefore, when the monomers used in the suspension polymerization are, for example, monofunctional acrylamide and difunctional acrylamide, the percentage of difunctional acrylamide (crosslinker monomer) is calculated as a weight percentage based on the total weight of difunctional acrylamide plus monofunctional acrylamide. Typical crosslinking levels include>1%wt crosslinker, such as>2%wt crosslinker, for example,>5%wt crosslinker. For example, the crosslinking level can be>10%wt crosslinker, or>15%wt crosslinker, for example,>20%wt crosslinker or>25%wt crosslinker. The crosslinking level can also be, for example, 5-60%wt crosslinker, such as 10-50%wt crosslinker, such as 20-40%wt crosslinker or 20-30%wt crosslinker. The crosslinking level may also be 1-30% wt crosslinker, such as 1-20% wt crosslinker, such as 1-10% wt crosslinker; or the crosslinking level may also be 2-30% wt crosslinker, such as 2-20% wt crosslinker, such as 2-10% wt crosslinker; for example, levels suitable for porous hydrogel particles.

[0110] The crosslinking level may be >30% wt crosslinker or >40% wt crosslinker, for example in highly crosslinked particles. The crosslinking level may be 10-90% wt crosslinker, 20-80% wt crosslinker or 25-75% wt crosslinker, for example 25-60% wt crosslinker or 30-50% wt crosslinker. In highly crosslinked particles, the crosslinking level may be as high as 100% wt crosslinker, for example a hydrophilic vinyl monomer may be the crosslinker.

[0111] Where the particles are magnetic polymer particles (e.g. magnetic hydrogel polymer particles), the crosslinking level is preferably >10% wt crosslinker, such as >30% wt crosslinker or >40% wt crosslinker. These crosslinking levels may result in particles having better retention of magnetic material than particles with lower crosslinking levels. The crosslinking level may be 10-90% wt crosslinker, 20-80% wt crosslinker or 25-75% wt crosslinker, such as 25-60% wt crosslinker or 30-50% wt crosslinker.

[0112] As described above, when the crosslinked particles are placed in a good solvent for the polymer, the crosslinked particles swell. 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 generally swell less than particles made from a similar polymer, 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 known standards of varying crosslinking levels.

[0113] The particles suitably comprise an addition polymer prepared by polymerizing one or more ethylenically unsaturated monomers. The ethylenically unsaturated monomers may comprise an optionally substituted general vinyl group, -CR=CR'H, wherein R is H or an alkyl group (e.g. wherein R is -CH 3 or -CH 2 CH 3 ), and wherein R' is H, alkyl (e.g. -CH 3 or -CH 2 CH 3 ) or a functional group (e.g., a carboxylic acid, a phosphonic acid, a primary amine, or a secondary amine). The ethylenically unsaturated monomer may contain a general vinyl group, -CR=CH 2 , wherein R is H or alkyl (e.g., wherein R is -CH 3 or -CH 2 CH 3 The ethylenically unsaturated monomer may contain a vinyl group, -CH=CH 2 The monomer may be a hydrophilic vinyl monomer, such as a vinyl monomer having a log P value of less than about 1, such as a log P value of less than about 0.5. The hydrophilic vinyl monomer may include an optionally substituted topically vinyl group, -CR=CR'H, wherein R is H or an alkyl group (e.g., wherein R is -CH 3 or -CH 2 CH 3 ), and wherein R' is H, alkyl (e.g. -CH 3 or -CH 2 CH 3 ) or a functional group (e.g., a carboxylic acid, a phosphonic acid, a primary amine, or a secondary amine). The hydrophilic vinyl monomer may contain a general vinyl group, -CR=CH 2 , wherein R is H or alkyl (e.g., wherein R is -CH 3 or -CH 2 CH 3 ). The hydrophilic vinyl monomer may contain a vinyl group, -CH=CH 2. The monomer may 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. The monomer may be a vinyl monomer having a log P of less than about 0.5. For example, the monomer may be a vinyl monomer having a log P of less than about 0.3 or less than about 0.2, for example, the monomer may 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, a log P of less than about -0.2. The monomer may be a vinyl monomer having a log P of greater than about -3.8, for example, a log P of 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, for example, 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 that also contains a hydrophilic group, such as an acrylamide monomer or an acrylate monomer.

[0114] The monomer may have R 原子 When the monomer is acrylamide, vinylimidazole, acryloylmorpholine, vinylphosphonic acid and vinylsulfonic acid, the specific R 原子 It can be R 原子 <2.75. When the monomer is an acrylate of other monomer types, the specific R 原子 It can be R 原子 ≤2 and the monomer has a water solubility (eg at 25°C) ≥50 g / L.

[0115] The monomer used in the particles may be at least one compound of formula (I):

[0116]

[0117] in:

[0118] R 1 -H, -CH 3 、-CH 2 CH 3 or -CH 2 C(O)OH;

[0119] R 1a -H, -CH 3 、-CH 2 CH 3 、-CH 2 C(O)OH or -C(O)R 2 ;

[0120] R 1b -C(O)R 2 、–P(O)(OH) 2 、-S(O) 2OH or a heterocycle having 3 to 8 ring atoms, wherein the heteroatom is selected from N, O or S; or R 1b With R 1a or R 1 The combination is -C(O)-OC(O)-;

[0121] R 2 Select from -OR 3 , –N(R 4 )R 5 or -N-linked amino acids;

[0122] R 3 Selected from -H, -C 1 -C 6 Alkyl or -C 1 -C 6 alcohol; and

[0123] R 4 and R 5 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Alcohol; or R 4 and R 5 Together with the nitrogen to which they are attached they form a heterocyclic ring having 3 to 8 ring atoms, wherein the heteroatom is selected from N, O or S.

[0124] In one embodiment, R 1 -H, -CH 3 、-CH 2 CH 3 or -CH 2 C(O)OH;

[0125] R 1a -H or -C(O)R 2 ;

[0126] R 1b -C(O)R 2 、–P(O)(OH) 2 or -S(O) 2 OH; or R 1b With R 1a or R 1 The combination is -C(O)-OC(O)-;

[0127] R 2 Select from -OR 3 or –N(R 4 )R 5 ;

[0128] R 3 Selected from -H, -C 1 -C 6 Alkyl or -C 1 -C 6 alcohol; and

[0129] R 4 and R 5 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 alcohol.

[0130] When R 3 Yes-C 1 -C 6 Alkyl or -C 1 -C 6 In the case of alcohol, the alkyl group or alcohol may be substituted, where chemically possible, by 1 to 5 (e.g. 1, 2, 3 or 4) substituents, each of which is independently selected at each occurrence from: oxo, =NR a , =NOR a , halogenated, nitro, cyano, NR a R a , NR a S(O) 2 R a , NR a CONR a R a , NR a CO 2 R a , OR a SR a ,S(O)R a ,S(O) 2 OR a ,S(O) 2 R a ,S(O) 2 NR a R a ， CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 For example, when R 3 Yes-C 1 -C 6 Alkyl or -C 1 -C 6 In the case of alcohol, the alkyl group or alcohol may be substituted, where chemically possible, by 1 to 5 (e.g., 1, 2, 3 or 4) substituents, each of which is independently selected from OR a or CO 2 R a , optionally wherein R a It's H.

[0131] In R 4 and / or R 5 Yes-C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 In the case of alcohols, each may be independently substituted, where chemically possible, by 1 to 5 (e.g. 1, 2, 3 or 4) substituents, each of which is independently selected at each occurrence from: oxo, =NR a , =NOR a , halogenated, nitro, cyano, NR a R a , NR a S(O) 2 R a , NR a CONR a R a , NR a CO 2 R a , OR a SR a ,S(O)R a ,S(O) 2 OR a ,S(O) 2 R a ,S(O) 2 NR a R a , CO 2 R a C(O)R a ,CONR a R a , C 1 -C4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 For example, when R 4 and / or R 5 Yes-C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 alcohol, each may be independently substituted, where chemically possible, by 1 to 5 (e.g., 1, 2, 3 or 4) substituents, each of which is independently selected at each occurrence from OR a or CO 2 R a , optionally wherein R a It's H.

[0132] R 1 Can be -H, -CH 3 or -CH 2 CH 3 . R 1 Can be -H or -CH 3 For example, R 1 Can be -H. R 1 Can be -CH 2 C(O)OH.

[0133] R 1a Can be -H, -CH 3 or -CH 2 CH 3 . R 1a Can be -H. R 1a Can be -C(O)R 2 . R 1a Can be -CH 2 C(O)OH.

[0134] R 1b Can be -C(O)R 2 . R 1b It can be -P(O)(OH) 2 . R 1b Can be -S(O) 2 OH. When R 1b Yes-C(O)R 2When R 1a Can be -H. When R 1b Yes-C(O)R 2 When R 1a Can be -C(O)R 2 , and R 2 Can optionally be -OR 3 When R 1b Yes - P(O)(OH) 2 Time R 1a Can be -H, R 1 Can be -H, -CH 3 or -CH 2 CH 3 (e.g. –H). When R 1b Yes-S(O) 2 OH R 1a Can be -H, R 1 Can be -H, -CH 3 or -CH 2 CH 3 (e.g. –H).

[0135] R 1b It may be a heterocyclic ring having 3 to 8 ring atoms, wherein the heteroatom is selected from N, O or S. 1b It may be an imidazolyl group.

[0136] R 1b With R 1a or R 1的 The combination may be -C(O)-OC(O)-.

[0137] R 2 Can be -OR 3 . R 2 Can be -N(R 4 )R 5 . R 2 It can be -NH 2 、-NHCH 3 、-NHCH 2 CH 3 , or -NH(CH 3 ) 2 . R 2 It may be an N-linked amino acid. The N-linked amino acid may be selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, beta-alanine and methionine.

[0138] R 3 Can be -H. R 3 Can be -C 1 -C6 Alkyl. For example, R 3 -C may be substituted with 1 or 2 substituents 1 -C 6 Alkyl, each of which is independently selected at each occurrence from: oxo, halo, cyano, NR a R a , NR a CONR a R a , NR a CO 2 R a , OR a ;CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 Alkyl, for example, where R a Yes-H. R 3 Can be -C 1 -C 6 Alcohol. For example, R 3 -C may be substituted with 1 or 2 substituents 1 -C 6 alcohol, each of which is independently selected at each occurrence from: oxo, halo, cyano, NR a R a , NR a CONR a R a , NR a CO 2 R a , OR a ;CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 Alkyl, for example, where R a Yes -H.

[0139] R 4 Can be -H or -C 1 -C 6 Alkyl. 4 Can be -C 1 -C 6 Alkyl. For example, R 4 -C may be substituted by 1 or 2 substituents 1 -C 6 Alkyl, each of which is independently selected at each occurrence from: oxo, halo, cyano, NR a R a , NR a CONR a R a , NR a CO 2 R a , OR a ;CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 Alkyl, for example, where R a Yes -H.

[0140] R 5 Can be -H or -C 1 -C 6 Alkyl. 5 Can be -C 1 -C 6 Alkyl. For example, R 5 -C may be substituted by 1 or 2 substituents 1 -C 6 Alkyl, each of which is independently selected at each occurrence from: oxo, halo, cyano, NR aR a , NR a CONR a R a , NR a CO 2 R a , OR a ;CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 Alkyl, for example, where R a Yes -H.

[0141] R 4 and R 5 Together with the nitrogen to which they are attached they may form a heterocyclic ring having 3 to 8 ring atoms (e.g. 4, 5 or 6 ring atoms), wherein the heteroatoms are selected from N, O or S. 4 and R 5 Together with the nitrogen to which they are attached they form a morpholino heterocycle.

[0142] The monomer used in the particles may be at least one compound of formula (Ia):

[0143]

[0144] Where R 1 , R 2 and R 1a As defined elsewhere.

[0145] The monomer used in the particles may be at least one compound of formula (Ib):

[0146]

[0147] Where R 1 and R 1a is as defined elsewhere. In one embodiment, R 1 and R 1a Each independently selected from -H, -CH 3 、-CH 2 CH 3 or -CH2 C(O)OH.

[0148] The monomer used in the particles may be at least one compound of formula (Ic):

[0149]

[0150] Where R 1 and R 1a is as defined elsewhere. In one embodiment, R 1 and R 1a Each independently selected from -H, -CH 3 、-CH 2 CH 3 or -CH 2 C(O)OH.

[0151] The log P value of the compound of formula (I), (Ia), (Ib) or (Ic) may be less than about 1, for example, a log P of less than about 0.5. The log P of the compound of formula (I), (Ia), (Ib) or (1c) is 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. The log P of the compound of formula (I), (Ia), (Ib) or (Ic) is greater than about -3.8, for example, the log P is greater than about -3 or about -2. The log P of the compound of formula (I), (Ia), (Ib) or (1c) is 0.6 to -3. The log P of the compound of formula (I), (Ia), (Ib) or (1c) is 0.5 to -2, for example, the log P is 0 to -2, for example, the log P is -0.2 to -2.

[0152] The compound of formula (I) or (Ia) may have R 原子 <2.75, wherein the compound is acrylamide, vinylimidazole, acryloylmorpholine, vinylphosphonic acid or vinylsulfonic acid; or R 原子 ≤2 and water solubility ≥50 g / L, wherein the compound is an acrylate or other monomer. The compound of formula (Ib) or (Ic) may have an R<2.75 原 son.

[0153] The monomer may include at least one hydrophilic vinyl monomer comprising a monomer selected from -C(O)NH 2、-C(O)NHCH 3 、-C(O)NHCH 2 CH 3 or -C(O)N(CH 3 ) 2 The monomer may contain at least one primary amide group (-C(O)NH 2 )'s hydrophilic vinyl monomer.

[0154] In particular, acrylamide monomers and / or acrylate monomers may be mentioned. Suitable monomers include acrylamide (prop-2-enamide), N-(hydroxymethyl)acrylamide, N-hydroxyethylacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, 4-acryloylmorpholine, 3-acrylamidopropionic 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 having a log P value of less than about 1 (e.g., a log P value of less than about 0.5), acrylic acid monomers, methacrylamide monomers or methacrylic acid monomers; R 原子 Other acrylamide monomers or methacrylamide monomers with R < 2.75; 原子 Other acrylic acid monomers or methacrylic acid monomers with a molecular weight of ≤2 and a water solubility (for example, at 25°C) of ≥50 g / L.

[0155] The monomers may comprise a mixture of monomers. For example, the monomers may comprise at least one monomer as defined above and at least one compatible functional monomer. Exemplary functional monomers are hydrophilic vinyl monomers as defined herein, comprising carboxylic acid (—COOH), phosphonic acid (—P(O)(OH) 2 ), sulfonic acid (-S(O) 2 OH), primary amine or secondary amine. For example, the monomer may comprise at least one monomer of formula (I), (Ia), (Ib) or (Ic) which does not comprise a carboxylic acid (-COOH), a phosphonic acid (-P(O)(OH) 2 ), sulfonic acid (-S(O) 2 OH), a primary amine or a secondary amine; and a monomer of formula (I), (Ia) or (Ib) comprising at least one carboxylic acid (—COOH), a phosphonic acid (—P(O)(OH) 2 ), sulfonic acid (-S(O) 2 OH), a primary amine or a secondary amine (e.g., comprising at least one carboxylic acid (—COOH) or phosphonic acid (—P(O)(OH) 2 )).

[0156] In highly crosslinked particles, the monomer may be or comprise a crosslinker, such as a crosslinker as defined elsewhere herein. For example, the monomer may be or comprise at least one compound of formula (IIa) or (IIb).

[0157] The functional monomer can be a vinyl monomer having a log P value less than about 1, such as a log P less than about 0.5, which contains a carboxylic acid or a primary amine. The functional monomer can be a vinyl monomer having a log P less than about 0.6, such as a log P less than about 0.52. The functional monomer can be a vinyl monomer having a log P less than about 0.5. For example, the functional monomer can be a vinyl monomer having a log P less than about 0.3 or less than about 0.2, such as a functional monomer can be a vinyl monomer having a log P less than about 0.1. The functional monomer can be a vinyl monomer having a log P less than about 0, such as a log P less than about -0.2. The functional monomer can be a vinyl monomer having a log P greater than about -3.8, such as a log P 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, such as a log P of 0 to -2, such as a log P of -0.2 to -2. The functional monomer may be a compound of formula (I), (Ia) or (Ib) comprising a carboxylic acid, a phosphonic acid, a sulfonic acid or a primary amine. The functional monomer may comprise a carboxylic acid, a phosphonic acid or a sulfonic acid, for example, may comprise a carboxylic acid or a phosphonic acid. The functional monomer may be an acrylamide monomer comprising a carboxylic acid or a primary amine.

[0158] Suitable functional monomers include 3-acrylamidopropionic acid, 4-acrylamidobutyric acid, 5-acrylamidovaleric acid, N-(3-aminopropyl)methacrylamide, acrylic acid, fumaric acid, itaconic acid, vinylphosphonic acid, vinylsulfonic acid and 1-vinylimidazole. Other suitable functional monomers include anhydrides, such as maleic anhydride or itaconic anhydride, which can be hydrolyzed during the polymerization process to provide carboxylic acid groups.

[0159] When at least one functional monomer is present, the amount of the functional monomer may be from about 0.1 to about 100% mole, such as from about 0.2 to about 50% mole, such as from about 0.5 to about 40% mole or from about 1 to about 30% mole (e.g., from about 2 to about 20% mole). For example, the amount of the functional monomer may be at least about 2 to about 100% mole, such as from about 2 to about 50% mole, such as from about 4 to about 40% mole or from about 4 to about 30% mole (e.g., from about 2 to about 20% mole). The amount of the functional monomer may be from about 5 to about 100% mole, such as from about 5 to about 80% mole. % mole may refer to the amount of the functional monomer, in terms of mole % of the total hydrophilic vinyl monomer. The amount of the functional monomer may be from about 0.1 to about 60% wt, such as from about 0.2 to about 50% wt, such as from about 0.5 to about 40% wt. The amount of the functional monomer may be from about 1 to about 60% wt, such as from about 2 to about 50% wt, such as from about 2 or 3 to about 40% wt. The amount of the functional monomer can be from about 0.1 to about 10% wt, such as from about 0.2 to about 5% wt, such as from about 0.5 to about 2% wt. % wt can refer to the weight percentage of the functional monomer contained in the total monomers used in the polymerization process. The total monomers can, for example, include a hydrophilic vinyl monomer that is not at least one functional monomer, a crosslinking agent, and a functional monomer.

[0160] Crosslinking can be achieved by introducing a polyvinyl group comprising at least two (e.g., two) vinyl groups (—CH═CH 2 ) as a cross-linking agent for the comonomer. The cross-linking agent may be at least one compound of formula (IIa) or (IIb):

[0161]

[0162]

[0163] Where R 6 Selected from -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Hydroxyalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 Polyether of ether unit; R 7 and R 8 Each independently selected from -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 Ether; or R 7 and R 8 Connect together to form -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Hydroxyalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 Polyether of ether unit; R 9 Yes-N(R 11 )C(O)CH=CH 2 ; R 10 Selected from -H and -N(R 12 )C(O)CH=CH 2 ;and

[0164] R 11 and R 12 Each independently selected from -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 ether.

[0165] The crosslinking agent may be at least one compound of formula (IIa). The crosslinking agent may be at least one compound of formula (IIb).

[0166] Where chemically possible, R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 may be independently substituted by 1 to 5 substituents, each of which is independently selected from: oxo, =NR a , =NOR a , halogenated, nitro, cyano, NR a R a, NR a S(O) 2 R a , NR a CONR a R a , NR a CO 2 R a , OR a ;

[0167] SR a ,S(O)R a ,S(O) 2 OR a ,S(O) 2 R a ,S(O) 2 NR a R a , CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H, C 1 -C 4 Alkyl and C 1 -C 4 Alkenyl.

[0168] R 6 Can be selected from -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Hydroxyalkyl- and -C 1 -C 6 Ether-. R 6 Can be selected from -C 1 -C 6 Alkyl- and -C 1 -C 6 Hydroxyalkyl-. 6 Can be -C 1 -C 6 Alkyl-, such as -CH2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -or-(CH 2 ) 4 -, for example -(CH 2 ) 2 -. R 6 Can be -C 1 -C 6 Hydroxyalkyl-, such as -C(OH)H-, -(C(OH)H) 2 -、-(C(OH)H) 3 -, or -(C(OH)H) 4 -, for example -(C(OH)H) 2 -.

[0169] R 6 Can be -C 1 -C 6 Heteroalkyl-, optionally wherein the heteroatom is an amine (eg, a tertiary amine). For example, C(O)R a Replaced-C 1 -C 6 Heteroalkyl-, optionally wherein the heteroatom is an amine, such as R 6 Can be -CH 2 CH 2 N(C(O)CH=CH 2 )CH 2 CH 2 -.

[0170] When R 6 When it is a polyether, the polyether may be linear or branched. 6 It can contain 2 to 100 C 2 -C 3 Polyethers of ether units, for example containing 2 to 50 C 2 -C 3 Polyether of ether units. 6 It can contain 2 to 100 C 2 Polyethers of ether units, for example containing 2 to 50 C 2 For example, R 6 It can be -(CH 2 ) r (OCH 2 CH 2 ) n O(CH 2 ) s, wherein r and s are each independently 2 or 3 (e.g., 2); 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 polyethers (e.g., wherein R 6 The crosslinking agent (which is a polyether) has excellent solubility in the aqueous phase. This means that while such a crosslinking agent can be used to provide particles with a low level of crosslinking (e.g. 1-20%wt crosslinking agent, or 1-10%wt crosslinking agent), such a polyether comprising a crosslinking agent is particularly suitable for providing particles comprising a relatively high level of crosslinking, such as >20%wt crosslinking agent, >25%wt crosslinking agent, or >30%wt crosslinking agent. For example, the crosslinking level may be 10-90%wt crosslinking agent, 20-80%wt crosslinking agent, or 25-75%wt crosslinking agent, such as 25-60%wt crosslinking agent, or 30-50%wt crosslinking agent.

[0171] R 7 and / or R 8 and / or R 11 and / or R 12 It can be H. For example, R 7 and / or R 8 It can be H. For example, R 11 and / or R 12 It can be H.

[0172] R 7 and R 8 Can be connected together to form -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Hydroxyalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 Ether units. 7 and R 8 Connect together to form -C 1 -C 6 When alkyl-, R 7 and R 8 The defined groups can be combined with R 6 For example, R 6 Can be -C 1 -C 6 Alkyl- and R 7 and R 8 Can be -C 1 -C 6alkyl-.

[0173] R 6 Can be selected from -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 Polyether of ether unit; R 7 and R 8 can be independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Heteroalkyl, -C 3 -C 6 Cycloalkyl or -C 1 -C 6 Ether; or R 7 and R 8 Connect together to form -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 ether units; and R 11 and R 12 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Heteroalkyl, -C 3 -C 6 Cycloalkyl or -C 1 -C 6 ether.

[0174] The log P value of the compound of formula (IIa) or (IIb) may be less than about 1, for example, a log P of less than about 0.5. The log P of the compound of formula (IIa) or (IIb) is 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, a log P of less than about -0.2. The compound of formula (IIa) or (IIb) may have a log P greater than -3.8, for example, a log P greater than about -3 or about -2. The log P of the compound of formula (IIa) or (IIb) is 0.6 to -3. The compound of formula (IIa) or (IIb) has 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.

[0175] The compound of formula (IIa) or (IIb) may have R 原子 <2.75.

[0176] Exemplary crosslinking agents 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, 1,3-diethylene glycol glycerol 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 crosslinking agents N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW≤2000) and 4-arm PEG-acrylamide (MW≤2000), (particularly N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide) are particularly suitable for highly crosslinked particles and mechanically more stable particles, such as particles having a crosslinking level of at least 20% wt crosslinker (e.g., a crosslinking level of at least 30% wt crosslinker). One embodiment of the present invention includes the use of a combination of crosslinkers. As specific monomers, acrylamide (prop-2-enamide) may be mentioned, with N,N'-(1,2-dihydroxybisacrylamide) being a suitable crosslinker. As specific monomers, methylol acrylamide may be mentioned, of which N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is a suitable crosslinking agent. As specific monomers, ethylol acrylamide may be mentioned, of which N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is a suitable crosslinking agent.

[0177] Cross-linking agents 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.

[0178] The crosslinking agent may be a compound that does not contain a primary amine, a secondary amine, a hydroxyl group or a carboxylic acid. The crosslinking agent may be a compound of formula (IIa) or formula (IIb) that does not contain a primary amine, a secondary amine, a hydroxyl group or a carboxylic acid.

[0179] Many exemplary monomers and crosslinkers that can be used are listed in Table 1. Many other monomers are listed in Table 2. Software from CambridgeSoft was used to analyze the The Log P values ​​of the monomers listed in Tables 1 and 2 were determined by Ultra 14.0.原子 =N C / (N N +N O ) Calculate R 原子 Value, where N C 、N N and N O represents the number of carbon, nitrogen and oxygen atoms in the monomer or crosslinker, respectively. In some particles that are embodiments of the present invention, the monomers of Table 2 are not used, for example due to the log P value of the monomer. In addition, the monomers methyl acrylate and methacrylic acid of Table 2 can be used in conventional Ugelstad processes, so these monomers and other monomers with higher log P values ​​may be less preferred for use in the particles of the present invention that can be made using the polymer particle forming methods described herein.

[0180] Table 1: Exemplary Monomers and Crosslinkers

[0181]

[0182]

[0183]

[0184]

[0185] Table 2: Other monomers and crosslinkers

[0186]

[0187] The level of crosslinking in the crosslinked hydrogel polymer particles can be expressed as the weight percentage (wt%) of the crosslinker monomer included in the total monomers used in the polymerization. Typical crosslinking levels include >1% wt crosslinker, such as >2% wt crosslinker, such as >5% wt crosslinker. For example, the crosslinking level can be >10% wt crosslinker, or >15% wt crosslinker, such as >20% wt crosslinker (e.g. >30% wt crosslinker or >40 wt% crosslinker). The crosslinking level can also be 1-60% wt crosslinker, such as 5-30% wt crosslinker. For example, the crosslinking level can be 5-60% wt crosslinker, such as 10-50% wt crosslinker. The crosslinking level can also be, for example, 15-40% wt crosslinker, such as 20-40% wt crosslinker, such as 20-30% wt crosslinker. The crosslinking level may be 10-90% wt crosslinker, 20-80% wt crosslinker or 25-75% wt crosslinker, such as 25-60% wt crosslinker or 30-50% wt crosslinker; for example, in highly crosslinked particles. The crosslinking level may also be 1-30% wt crosslinker, such as 1-20% wt crosslinker, such as 1-10% wt crosslinker. The crosslinking level may also be 2-30% wt crosslinker, such as 2-20% wt crosslinker, such as 2-10% wt crosslinker. In highly crosslinked particles, the crosslinking level may be as high as 100% wt crosslinker, for example, a hydrophilic vinyl monomer may be the crosslinker.

[0188] In one embodiment, the particles may have an average diameter of at least 500 nm, for example at least 600 nm, optionally at least 800 nm, such as in the case of particles having a diameter of at least 1 μm.

[0189] In one embodiment, the particles may have an average diameter of no greater than 10 μm, such as no greater than 5 μm, optionally no greater than 3 μm, such as in the case of particles no greater than 2 μm in diameter.

[0190] In one embodiment, the present invention includes a class of polymer particles having an average diameter of 0.5 μm to 10 μm, such as 0.8 μm to 5 μm.

[0191] The size and size distribution of the polymer particles can be determined as described below under the heading "Analytical Methods".

[0192] The present disclosure includes porous polymer particles having a porosity of at least 5%, such as at least 10% or at least 30%. The present disclosure includes porous particles having a porosity of 20% to 95%, particularly 30% to 95%, such as 40% to 95%, such as 50% to 80%.

[0193] Porosity can be calculated after determining the density of the hydrogel polymer particles, since acrylamide and acrylate polymers have a density of about 1.3 g / mL, while water has a density of about 1 g / mL. Porous polymer particles can be permeable to solvated molecules, for example, porous polymer particles can be permeable to oligonucleotides and nucleic acid amplification reagents and sequencing reagents (e.g., primers, nucleotides, and polymerases). Although this method of determining porosity works well for intermediate hydrogel polymer particles, this method is ineffective for determining the porosity of magnetic polymer particles, since magnetic materials generally have a higher density than polymers. In addition, this method is only applicable to coated particles, where the coating has a density similar to that of acrylamide and acrylate polymers.

[0194] The porosity of magnetic or coated particles of known size (e.g., diameter) in a swollen (i.e., water-saturated hydrogel particles) state can be determined by comparing the mass of the swollen particles with the mass of the dried particles. The particles can be dried, for example, by placing the particles in a vacuum dryer for at least 24 hours. Since the pores of the swollen hydrogel particles primarily contain 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 particles (calculated by the diameter of the particles) to determine the porosity percentage of the particles. It is believed that this method is effective for magnetic polymer particles and coated polymer particles.

[0195] In one embodiment, the particles comprise functional groups. The functional groups may be selected from hydroxyl, carboxylic acid (-COOH), phosphonic acid (-P(O)(OH) 2 ), sulfonic acid (-S(O) 2 (OH)), primary amines and secondary amines. The functional group may be selected from carboxylic acids, phosphonic acids and sulfonic acids (-S(O) 2 (OH)), for example, the functional group may be selected from carboxylic acids and phosphonic acids. The functional group may comprise carboxylic acids and / or phosphonic acids and / or sulfonic acids (e.g., carboxylic acids and / or phosphonic acids). The functional group (e.g., carboxylic acids and / or phosphonic acids) may enhance the deposition level of the magnetic material in the polymer particles comprising the magnetic material. The functional group may enhance the binding of the coating to the polymer of the coated polymer particles, for example, the functional group may provide a chemical treatment for covalent bonding, ionic bonding, or hydrogen bonding.

[0196] In one embodiment, the functional groups are provided by a hydrophilic vinyl monomer (e.g., a compound of formula (Ia) or (Ib)) rather than a crosslinker (e.g., a compound of formula (IIa) or (IIb)). Functional groups can be enhanced to promote binding to a target analyte (e.g., an oligonucleotide) or a target receptor. Exemplary methods for enhancing the functional groups of particles of the invention are described herein in the section relating to "Uses of Particles".

[0197] 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, such as one or more magnetic materials, in the pores. The present invention is not limited to the properties of the magnetic material, for example, the magnetic material may comprise at least one of a paramagnetic, superparamagnetic, ferromagnetic, or 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.

[0198] 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 may be particularly advantageous because the lack of magnetic properties reduces the agglomeration of polymer particles and facilitates the dispersion of polymer particles in the absence of an applied magnetic field. When magnetic polymer particles are used for assays, this is beneficial because it allows the magnetic polymer particles to be evenly distributed during the assay, with a relatively strong magnetic field being applied only when the polymer particles need to be isolated from the bulk of the assay solution.

[0199] If the particles are porous, the superparamagnetic crystals of the polymer particles can be any material that can be deposited on the polymer particles or in their pores in the form of superparamagnetic crystals. The magnetic material may include or be a metal oxide or alloy. The magnetic material may include or be an iron oxide, such as a ferrite, such as magnetite or maghemite, or a combination thereof. A portion of the iron in an iron oxide, such as magnetite or maghemite, may be replaced (i.e., substituted) by Al, Mn, Ni, Cu, Co, Zn, Ca, Ge, Te, Ti or Sn or a combination thereof. In particular, a portion of the iron in magnetite or maghemite may be replaced by Mn. As described above, some particles contain a combination of magnetite and maghemite; in this case, the magnetite or maghemite or both may have a partial substitution of the iron content. Additionally or alternatively, the particles of the present disclosure may include iron-based metal nanoparticles and FeNi alloy nanoparticles, in either case increasing 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 up to 5 mol%, for example in the range 0.1 to 5 mol%, such as 0.5 to 4 mol%, for example 1 to 3 mol%.

[0200] In one embodiment, the polymer particles are ferrimagnetic, and in particular, the magnetic material may comprise or be ferrimagnetic iron oxide crystals. Thus, the present disclosure includes polymer particles as described herein, comprising a magnetic material selected from ferrimagnetic iron oxide crystals, superparamagnetic iron oxide crystals, and combinations thereof.

[0201] The total amount of magnetic material present is generally greater than 20%, preferably greater than 25%, desirably greater than or equal to 30%, such as up to 85%wt or at least 50%wt, such as 30 to 80wt%. The percentages are weight percentages calculated based on the weight of the magnetic material (e.g., metal oxide) based on the total dry weight of the particles. Therefore, in the case where the magnetic material is composed of superparamagnetic material, the total amount of superparamagnetic material present is generally greater than 20%, preferably greater than 25%, desirably greater than or equal to 30%, such as up to 85%wt or at least 50%wt, such as 30 to 80wt%, and the percentages are each weight percentages calculated based on the weight of the magnetic material (e.g., metal oxide) based on the total dry weight of the particles.

[0202] 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 disposed on the outer surface of the particle, and for porous particles, the coating may also be disposed on the solvent-accessible surface 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 particles. The coating may cover a portion of the surface of the polymer particles. The use of the coating may provide many advantages. For example, where appropriate, the coating may be used to change the surface chemistry of the polymer particles for different applications. In the case where the polymer particles include a magnetic material, the coating may be used to isolate the magnetic material from a large portion of the solution in which the particles are suspended, for example to prevent the magnetic material from leaching from the particles and / or to prevent the magnetic material from reacting with components present in the solution.

[0203] In one embodiment, the coating is a polymer coating. In the case where 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 a mixture of epoxide monomers such as at least two or at least three epoxide monomers. The at least one epoxide monomer (or mixture of epoxide monomers) may be selected from 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, glycerol diglycidyl ether, glycidol, and glycidyl methacrylate, ethylhexyl glycidyl ether, methyl glycidyl ether, glycerol propoxylated triglycidyl ether, poly(propylene glycol) diglycidyl ether, 1,3-butanediol diglycidyl ether, tert-butyl glycidyl ether, 1,4-cyclohexanedimethanol diglycidyl ether, diethylene glycol diglycidyl ether, dodecyl glycidyl ether, O-(2,3-epoxypropoxy)butane 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-butylene oxide, 1,2-pentane oxide, 1,2-epoxy-5-hexene, 1,2-epoxyhexane, 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 a combination thereof.

[0204] In one embodiment, the coating comprises silicon dioxide. The silicon dioxide coating may be formed by a silicate (e.g., Na 2 SiO 3 ) 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., tetraethyl orthosilicate) 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.).

[0205] In one embodiment, the particles that can be magnetic and / or coated can be combined with affinity ligands, and the properties of affinity ligands will be selected based on its affinity for a specific analyte that is present or absent in the sample to be determined. Affinity ligands can include any molecule that can be connected to the particle, and the particle can also specifically identify a specific analyte. Affinity ligands include monoclonal antibodies, polyclonal antibodies, antibody fragments, nucleic acids, oligonucleotides, proteins, oligopeptides, polysaccharides, sugars, peptides, peptides encoding nucleic acid molecules, antigens, drugs and other ligands. The example of suitable affinity ligands can be obtained in disclosed literature and is well-known. The use of other binding partners, second affinity ligands and linking groups routinely used in this area will not be discussed further in this article, but it should be understood that these materials can be used together with particles of the present invention if necessary.

[0206] The particles of the present invention can be stable (i.e., resistant to polymer degradation) in aqueous solution over a typical temperature range of aqueous solutions. The monodisperse crosslinked hydrogel polymer particles can be stable at temperatures up to about 100° C. For example, the monodisperse crosslinked hydrogel polymer particles can be stable over a temperature range of about 0° C. to about 100° C.

[0207] Seed particles

[0208] Polymer particles can be prepared by a polymer particle formation process, starting from a specific seed particle. Applicants have determined that seed particles commonly used in other processes, such as polystyrene seed particles, are incompatible with the methods and disclosures of the present invention. Therefore, the present disclosure provides seed particles suitable for use in the present invention and the polymer particle formation methods of the present disclosure.

[0209] The seed particles may (and preferably are) monodisperse. The seed particles comprise a plurality of non-crosslinked oligomers of poly-N,N-dimethylacrylamide with a z-average diameter of 100 nm to 1,500 nm. Each seed particle may comprise more than 1×10 5 (For example, more than 1×10 6 ) of non-crosslinked oligomers of poly-N,N-dimethylacrylamide, for example, each seed particle may contain more than 5×10 6 A non-crosslinked oligomer of poly N,N-dimethylacrylamide.

[0210] The z-average diameter of the seed particles may be from 150 nm to 1,300 nm. For example, the seed particles may have a z-average diameter of from 300 nm to 1,100 nm.

[0211] The weight average molecular weight (Mw) of the oligomers is about 2,000 Da to about 100,000 Da when measured by GPC relative to polystyrene standards. Additionally or alternatively to the specific Mw range, each oligomer polymer may contain about 30 to 2,000 (e.g., about 60 to about 1,000 or about 80 to about 500) monomer units.

[0212] The seed particle oligomer may have a Mw of less than 100,000 Da, optionally less than 50,000 Da, further optionally less than 40,000 Da, for example less than 30,000 Da. The Mw may be greater than 4,000 Da, optionally greater than 5,000 Da, further optionally greater than 6,000 Da, such as greater than 8,000 Da, for example greater than 10,000 Da. For example, the Mw may be 5,000 Da to 70,000 Da, such as 6,000 Da to 60,000 Da, such as 7,000 Da to 50,000 Da or 8,000 Da to 40,000 Da.

[0213] Without wishing to be bound by any theory, it is believed that the Mw of the oligomer of the seed particles is an important feature of the seed particles. For example, it may be desirable to have a seed particle oligomer Mw within a specific range to provide seed particles that form monodisperse polymer particles in the particle formation process described herein. If the seed particle polymer has a higher molecular weight, for example because no chain transfer agent (e.g., monodisperse microspheres of M. Babic and D. Horak, Macromolecular Reaction Engineering, 2007, 1, 86-94) is used during seed particle formation, the particles are not suitable for use in the method of the present invention, for example because the particles obtained by this method may not be monodisperse, for example, the CV may be greater than 20%.

[0214] The seed particles may have characteristics of seed particles formed according to the methods for forming seed particles disclosed herein. For example, the seed particles may have characteristics of seed particles formed by free radical initiated polymerization of N,N-dimethylacrylamide in an organic solvent in the presence of a stabilizer and a chain transfer agent.

[0215] Preparation of seed pellets

[0216] Polymer particles can be prepared by the methods described herein, starting from specific seed particles. Thus, the present disclosure provides methods for preparing monodisperse seed particles.

[0217] The monodisperse seed particles may be prepared by a method comprising dissolving N,N-dimethylacrylamide, a stabilizer, a free 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 the monodisperse seed particles.

[0218] In this process, once the reaction mixture is formed in a suitable container, the reaction mixture is typically mixed, such as with a stirrer, during the heating step.

[0219] Preferably, the polymerization reaction will be carried out in a reaction mixture containing a minimum amount of oxygen. Therefore, before the reaction mixture is heated, the oxygen in the reaction mixture can be purged. Purging can include bubbling with a chemically inert gas. The inert gas can be nitrogen or an inert 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.

[0220] The method can be considered as reverse precipitation polymerization, wherein the hydrophilic seed particles are precipitated from an organic solvent. Stabilizers can prevent the aggregation of seed particles and help to form monodisperse seed particles. The selection of suitable stabilizers depends on the ability of the stabilizer to dissolve in an organic solvent. Stabilizers can be block copolymers of styrene and polyolefins, such as triblock copolymers based on styrene and ethylene / butylene, such as linear triblock copolymers composed of styrene and ethylene / butylene. Stabilizers can be triblock copolymers, which are composed of a midblock of poly(ethylene-to-butylene) and an outer block of polystyrene. Exemplary stabilizers include Kraton A1535H, Kraton G1650M, Kraton G1652M or Kraton G1657M, or a combination thereof.

[0221] The free radical initiator can be a peroxide initiator or an azo initiator, such as a peroxide initiator or an azo initiator that decomposes at an elevated temperature. Exemplary free radical initiators include 2,2'-azobis(2-methylpropionitrile), 2,2'-azobis(2-methylbutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), dibenzoyl peroxide, and the like. The free radical initiator can be 2,2'-azobis(2-methylpropionitrile). The free radical initiator can be 2,2'-azobis(2-methylbutyronitrile). The temperature at which the reaction mixture can be heated depends on the temperature at which the free 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, such as heating to a temperature of at least 50°C, such as heating to a temperature of at least 60°C, or, for example, heating to a temperature of at least 70°C. For example, when the free radical initiator is 2,2'-azobis(2-methylpropionitrile), the reaction mixture can be heated to a temperature of at least 50°C or a temperature of at least 60°C.

[0222] The addition of chain transfer agent terminates the chain by free radical reaction with the growing polymer chain and transfers a single electron to the free radical species derived from the chain transfer agent, thereby reducing the molecular weight of the polymer of the seed particles. Then, the free radical species derived from the chain transfer agent can react with the monomer to form a free radical from the monomer, and then the monomer can react with another monomer to start forming a new polymer chain. The chain transfer agent can be a mercaptan or a haloalkane. For example, the chain transfer agent can be selected from mercaptan (for example 1-octyl mercaptan, hexanethiol, 6-mercapto-1-hexanol, benzyl mercaptan), alkyl mercaptan (for example 1-octyl mercaptan, hexanethiol), carbon tetrachloride and bromotrichloromethane. For example, the chain transfer agent can be 1-octyl mercaptan. The total addition of the chain transfer agent can be 1mol per 10mol monomer to 1mol per 300mol monomer, for example 1mol per 20mol monomer to 1mol per 100mol monomer, for example the scope of about 1mol chain transfer agent per 30mol monomer. The timing of adding the chain transfer agent is important for obtaining 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 free radical initiator). This finding is unexpected, as for seed particles used in conventional Ugelstad processes (e.g. polystyrene seed particles), if a chain transfer agent is added, it should be added after the start of particle formation, as taught in WO2010 / 125170, which is incorporated herein by reference.

[0223] The organic solvent may include a mixture of an alkane component and an aromatic component. The organic solvent may include a single component, such as an alkane component or an aromatic component. The alkane component may be or include hexane, heptane or octane. The aromatic component may be or include C 1 -C 10 Alkyl substituted phenyl or C 1 -C 8 Dialkyl-substituted phenyl, such as C 1 -C 4 Alkyl substituted phenyl, such as toluene. When the organic solvent comprises a single component and the component is an aromatic component, the aromatic component may be C 4 -C 12 Alkyl substituted phenyl, or C 2 -C 8 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 about 0.5:1 to about 20:1, for example, a weight ratio of alkane component:aromatic component of about 1:1 to about 15:1, for example, about 1:1 to about 10:1.

[0224] The reaction mixture may contain: N,N-dimethylacrylamide in an amount of about 2% wt to about 5% wt; a stabilizer in an amount of about 1% wt to about 5% wt; a free radical initiator in an amount of about 0.01% wt to about 4% wt (e.g., about 0.05% wt to about 0.25% wt); and a chain transfer agent in an amount of about 0.05% wt to about 0.25% wt.

[0225] The monodisperse seed particles may then be subjected to a particle formation process, for example as follows:

[0226] Preparation of particles

[0227] The present disclosure provides methods for forming monodisperse cross-linked hydrogel polymer particles. The Ugelstad method cannot be used to directly form such particles, for example because the Ugelstad method requires that the particles be formed in the oil phase of an oil-in-water emulsion, whereas the hydrogel polymer particles and the corresponding hydrophilic monomers will preferentially dissolve in water (as determined by log P and / or R 原子 Applicants have solved this problem by providing the method of the present disclosure, which provides a particle formation method using an aqueous (discontinuous phase) in an oil (continuous phase) system. It is believed that this represents the first such method of forming monodisperse crosslinked polymer particles.

[0228] The present disclosure provides a method for forming monodisperse cross-linked hydrogel polymer particles. The method comprises: forming a solution (a): an aqueous solution of at least 2%wt of a hydrophilic vinyl monomer, the aqueous solution also comprising a cross-linking agent containing at least two vinyl groups; forming a solution (b) of a stabilizer in an organic solvent, wherein the organic solvent is immiscible with water, and wherein at least one of the solutions (a) and (b) comprises a free radical initiator; mixing solutions (a) and (b) to form an oil-in-water emulsion (c) and 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 cross-linked hydrogel polymer particles. As described below, the method can be considered a single-stage method because it includes a single swelling step and a single polymerization step.

[0229] Solution (a) can be formed by first forming a solution of at least 2% by weight of a hydrophilic vinyl monomer in aqueous solution and then adding a crosslinking agent; the crosslinking agent can be added before the hydrophilic vinyl monomer; or the hydrophilic vinyl monomer and the crosslinking agent can be added to the solution at about the same time. Similarly, the stabilizer and free radical initiator can be added to solution (b) at about the same time or sequentially.

[0230] In the context of the present disclosure, an organic solvent is considered to be immiscible with water when an amount of at least 5% wt of water is mixed with the organic solvent, and when the organic solvent and water separate into two separate phases.

[0231] The method involves swelling seed particles with an aqueous discontinuous phase in an oil continuous phase. A schematic diagram of the method is shown in Figure 1 (illustrated with specific monomers) and Figure 2 (more generally described), which can be considered a single-stage process since it comprises a single swelling step and a single polymerization step. Figure 1 and 2 The method illustrated in is conveniently divided into two steps, swelling of monodisperse seed particles 100 to form swollen particles 110 and polymerization of 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 comprising an exemplary monomer acrylamide 101 and an exemplary crosslinker 1,2-dihydroxybisacrylamide 102 with an oil phase comprising a steric stabilizer, the emulsion being formed upon agitation (e.g., by stirring) of the aqueous and oil phases. If (very) highly crosslinked particles are desired, the monomers may also be replaced by a crosslinker, such 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 typically also contains an initiator, which may be added to the oil phase. An initiator is a compound which, upon activation, will initiate polymerization of the monomers and the crosslinker. The monodisperse seed particles 100 contain non-crosslinked oligomers of poly-N,N-dimethylacrylamide 131 and are considered to be "activated seed particles". The seed particles 100 can be prepared as described elsewhere in this application. In the first step, the monodisperse seed particles 100 are added to an oil-in-water emulsion and the emulsion is stirred for a period of time (e.g., at least 30 minutes or at least 1 hour, typically 4-48 hours). During this period, the monomer 101 and the crosslinking agent 102 diffuse into the activated seed particles 100 to form swollen seed particles 110. The swollen seed particles 110 contain at least a mixture of monomers 101, crosslinking agents 102 and polymers from the activated seed particles 100. The swollen seed particles 110 may also include other components, such as one or more pore-forming agents, which can enter the particles if contained in the oil-in-water emulsion. For example, in the method shown, water is also present in the swollen seed particles 110, and the water can be considered to be a pore-forming agent. The second step comprises polymerizing the monomers 101 and the cross-linking agent 102 to form cross-linked hydrogel polymer particles 120. In the second step, polymerization is initiated by activating the initiator, for example by heating the emulsion.

[0232] Providing activated seed particles is a key feature of the successful Ugelstad process. Activation of the seed particles is usually provided by adding an organic compound with very low solubility as an emulsion to produce entropy activated seed particles. However, the applicants unexpectedly determined that in processes such as those in which the seed particles comprise oligomers of poly-N,N-dimethylacrylamide, no additional activation step is required.

[0233] Swelling is key to the particle formation process. In this method, it is required that the monomer and (when present) porogen have limited solubility in the continuous phase. If the monomer and porogen have too high solubility in the continuous phase, the solubility will not help drive the monomer (and optional porogen) into the seed and form swollen particles. On the other hand, if the solubility of the monomer (and optional porogen) in the continuous phase is insufficient, the diffusion through the continuous phase can be ignored, so it is impossible to transfer the monomer mass to the seed, thereby preventing the formation of swollen particles. Solubility can be conveniently measured with log P. The time for complete swelling, that is, the time until all monomers (and optional porogens) are co-located in the seed particles, will depend to a large extent on factors such as temperature, solubility and viscosity, and therefore will vary from system to system. Therefore, the typical time scale for the step of allowing monodisperse seed particles to form swollen particles will vary between 30 minutes and 48 hours. For example, the step of allowing monodisperse seed particles to swell can be carried out for at least 30 minutes, such as at least 1 hour. The step of allowing the monodisperse seed particles to swell 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, for example it is believed that swelling times of several days, such as 3 days or 2 days, may be used.

[0234] The step of allowing the monodisperse seed particles to form swollen particles may include mixing the emulsion, for example, mixing the emulsion for substantially all swelling time. The step of allowing the monodisperse seed particles to form swollen particles may be performed at a temperature of about 10°C to about 60°C, for example, at a temperature of about 10°C to about 40°C, for example, at a temperature between about 15°C to about 30°C. For example, mixing may be performed at a temperature of about 10°C to about 60°C, for example, at a temperature of about 10°C to about 40°C, for example, at a temperature of about 15°C to about 30°C.

[0235] Solution (a) comprises at least 2%wt of a hydrophilic vinyl monomer in an 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 C 1 -C 4 Alcohol, such as ethanol or methanol, such as methanol. The water-miscible organic solvent may be C 2 -C 4Nitriles, such as acetonitrile.

[0236] The hydrophilic vinyl monomer may comprise an optionally substituted vinyl group, -CR=CR'H, wherein R is H or an alkyl group (e.g., wherein R is -CH 3 or -CH 2 CH 3 ), and wherein R' is H, alkyl (e.g. -CH 3 or -CH 2 CH 3 ) or a functional group (e.g., a carboxylic acid, a phosphonic acid, a primary amine, or a secondary amine). The hydrophilic vinyl monomer may contain a general vinyl group, -CR=CH 2 , wherein R is an alkyl group (e.g., wherein R is -CH 3 or -CH 2 CH 3 ). The hydrophilic vinyl monomer may contain a vinyl group, -CH=CH 2 . The hydrophilic vinyl monomer can 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 can be a vinyl monomer having a log P of less than about 0.6 (for example, less than about 0.52). The monomer can 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, for example, 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, for example, a log P of less than about -0.2. The monomer can be a vinyl monomer having a log P of greater than about -3.8, for example, a log P of 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, for example, a log P of 0 to -2, for example, a log P of -0.2 to -2. In particular, the monomer can be a vinyl monomer that also contains a hydrophilic group, such as an acrylamide monomer or an acrylate monomer.

[0237] The monomer may have R 原子 When the monomer is acrylamide, vinylimidazole, acryloylmorpholine, vinylphosphonic acid and vinylsulfonic acid, the specific R 原子 It can be R 原子 <2.75. When the monomer is an acrylate of other monomer types, the specific R 原子 It can be R 原子 ≤2 and the monomer has a water solubility (eg at 25°C) ≥50 g / L.

[0238] The monomer used in the process may be at least one compound of formula (I) wherein R 1 -H, -CH 3 、-CH2 CH 3 or -CH 2 C(O)OH;R 1a -H, -CH 3 、-CH 2 CH 3 、-CH 2 C(O)OH or -C(O)R 2 ; R 1b -C(O)R 2 、–P(O)(OH) 2 、-S(O) 2 OH or a heterocycle having 3 to 8 ring atoms, wherein the heteroatom is selected from N, O or S; or R 1b With R 1a or R 1 The combination is -C(O)-OC(O)-; R 2 Select from -OR 3 , –N(R 4 )R 5 or -N-linked amino acid; R 3 Selected from -H, -C 1 -C 6 Alkyl or -C 1 -C 6 Alcohol; and R 4 and R 5 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Alcohol; or R 4 and R 5 Together with the nitrogen to which they are attached they form a heterocyclic ring having 3 to 8 ring atoms, wherein the heteroatom is selected from N, O or S.

[0239] The monomer used in the process may be at least one compound of formula (I) wherein R 1 -H, -CH 3 、-CH 2 CH 3 or -CH 2 C(O)OH;R 1a -H or -C(O)R 2 ; R 1b -C(O)R 2 、–P(O)(OH) 2 or -S(O) 2 OH; or R 1b With R 1a or R 1The combination is -C(O)-OC(O)-; R 2 Yes-OR 3 or –N(R 4 )R 5 ; R 3 Yes -H, -C 1 -C 6 Alkyl or -C 1 -C 6 Alcohol; and R 4 and R 5 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 alcohol.

[0240] When R 3 Yes-C 1 -C 6 Alkyl or -C 1 -C 6 In the case of alcohol, the alkyl group or alcohol may be substituted, where chemically possible, by 1 to 5 substituents (e.g., 1, 2, 3 or 4), each of which is independently selected at each occurrence from: oxo, =NR a , =NOR a , halogenated, nitro, cyano, NR a R a , NR a S(O) 2 R a , NR a CONR a R a , NR a CO 2 R a , OR a SR a ,S(O)R a ,S(O) 2 OR a ,S(O) 2 R a ,S(O) 2 NR a R a , CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 For example, when R 3 Yes-C 1 -C 6 Alkyl or -C 1 -C 6 In the case of alcohol, the alkyl group or alcohol may be substituted, where chemically possible, by 1 to 5 (e.g., 1, 2, 3 or 4) substituents, each of which is independently selected from OR a or CO 2 R a , optionally wherein R a It's H.

[0241] In R 4 and / or R 5 Yes-C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 In the case of alcohols, each may be independently substituted, where chemically possible, by 1 to 5 (e.g. 1, 2, 3 or 4) substituents, each of which is independently selected at each occurrence from: oxo, =NR a , =NOR a , halogenated, nitro, cyano, NR a R a , NR a S(O) 2 R a , NR a CONR a R a , NR a CO 2 R a , OR a SR a ,S(O)R a ,S(O) 2 OR a ,S(O) 2 R a ,S(O) 2 NR a R a , CO 2 R a C(O)R a ,CONR a Ra , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 For example, when R 4 and / or R 5 Yes-C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 alcohol, each may be independently substituted, where chemically possible, by 1 to 5 (e.g., 1, 2, 3 or 4) substituents, each of which is independently selected at each occurrence from OR a or CO 2 R a , optionally wherein R a It's H.

[0242] R 1 Can be -H, -CH 3 or -CH 2 CH 3 . R 1 Can be -H or -CH 3 For example, R 1 Can be -H. R 1 Can be -CH 2 C(O)OH.

[0243] R 1a Can be -H, -CH 3 or -CH 2 CH 3 . R 1a Can be -H. R 1a Can be -C(O)R 2 . R 1a Can be -CH 2 C(O)OH.

[0244] R 1b Can be -C(O)R 2 . R 1b It can be -P(O)(OH) 2 . R 1b Can be -S(O) 2 OH. When R1b Yes-C(O)R 2 When R 1a Can be -H. When R 1b Yes-C(O)R 2 When R 1a Can be -C(O)R 2 , and R 2 Can optionally be -OR 3 When R 1b Yes - P(O)(OH) 2 When R 1a Can be -H, R 1 Can be -H, -CH 3 or -CH 2 CH 3 (e.g. –H). When R 1b Yes-S(O) 2 OH, R 1a Can be -H, R 1 Can be -H, -CH 3 or -CH 2 CH 3 (e.g. –H).

[0245] R 1b It may be a heterocyclic ring having 3 to 8 ring atoms, wherein the heteroatom is selected from N, O or S. 1b It may be an imidazolyl group.

[0246] R 1b With R 1a or R 1的 The combination may be -C(O)-OC(O)-.

[0247] R 2 Can be -OR 3 . R 2 Can be -N(R 4 )R 5 . R 2 It can be -NH 2 、-NHCH 3 、-NHCH 2 CH 3 , or -NH(CH 3 ) 2 . R 2 It may be an N-linked amino acid. The N-linked amino acid may be selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, beta-alanine and methionine.

[0248] R 3 Can be -H. R3 Can be -C 1 -C 6 Alkyl. For example, R 3 -C may be substituted with 1 or 2 substituents 1 -C 6 Alkyl, each of which is independently selected at each occurrence from: oxo, halo, cyano, NR a R a , NR a CONR a R a , NR a CO 2 R a , OR a ;CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 Alkyl, for example, where R a Yes-H. R 3 Can be -C 1 -C 6 Alcohol. For example, R 3 -C may be substituted with 1 or 2 substituents 1 -C 6 alcohol, each of which is independently selected at each occurrence from: oxo, halo, cyano, NR a R a , NR a CONR a R a , NR a CO 2 R a , OR a ;CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 Alkyl, for example, where R a Yes -H.

[0249] R 4 Can be -H or -C 1 -C 6 Alkyl. 4 Can be -C 1 -C 6 Alkyl. For example, R 4 -C may be substituted with 1 or 2 substituents 1 -C 6 Alkyl, each of which is independently selected at each occurrence from: oxo, halo, cyano, NR a R a , NR a CONR a R a , NR a CO 2 R a , OR a ;CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 Alkyl, for example, where R a Yes -H.

[0250] R 5 Can be -H or -C 1 -C 6 Alkyl. 5 Can be -C 1 -C 6 Alkyl. For example, R 5 -C may be substituted with 1 or 2 substituents 1 -C 6Alkyl, each of which is independently selected at each occurrence from: oxo, halo, cyano, NR a R a , NR a CONR a R a , NR a CO 2 R a , OR a ;CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H and C 1 -C 4 Alkyl, for example, where R a Yes -H.

[0251] R 4 and R 5 Together with the nitrogen to which they are attached they may form a heterocyclic ring having 3 to 8 ring atoms (e.g. 4, 5 or 6 ring atoms), wherein the heteroatoms are selected from N, O or S. 4 and R 5 Together with the nitrogen to which they are attached they form a morpholino heterocycle.

[0252] The monomer used in the process may be at least one compound of formula (I), (Ia), (Ib) or (1c).

[0253] The log P value of the compound of formula (I), (Ia), (Ib) or (Ic) may be 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 (e.g., 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. The log P of the compound of formula (I), (Ia), (Ib) or (Ic) is greater than about -3.8, for example, the log P is greater than about -3 or about -2. The log P of the compound of formula (I), (Ia), (Ib) or (1c) is 0.6 to -3. The log P of the compound of formula (I), (Ia), (Ib) or (1c) is 0.5 to -2, for example, the log P is 0 to -2, for example, the log P is -0.2 to -2.

[0254] The compound of formula (I) or (Ia) may have R 原子 <2.75, wherein the compound is acrylamide, vinylimidazole, acryloylmorpholine, vinylphosphonic acid or vinylsulfonic acid; or R 原子 ≤2 and water solubility ≥50 g / L, wherein the compound is an acrylate or other monomer. The compound of formula (Ib) or (Ic) may have an R<2.75 原 son.

[0255] The monomer may include at least one hydrophilic vinyl monomer comprising a monomer selected from -C(O)NH 2 、-C(O)NHCH 3 、-C(O)NHCH 2 CH 3 or -C(O)N(CH 3 ) 2 The monomer may contain at least one primary amide group (-C(O)NH 2 )'s hydrophilic vinyl monomer.

[0256] Acrylamide monomers and / or acrylic acid monomers may be mentioned in particular. Suitable monomers include acrylamide (prop-2-enamide), N-(hydroxymethyl)acrylamide, N-hydroxyethylacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, 4-acryloylmorpholine, 3-acrylamidopropionic 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 having a log P value of less than about 1 (e.g., a log P value of less than about 0.5), acrylic acid monomers, methacrylamide monomers or methacrylic acid monomers; R 原子 Other acrylamide monomers or methacrylamide monomers with R < 2.75; 原子 Other acrylic acid monomers or methacrylic acid monomers with a molecular weight of ≤2 and a water solubility (for example, at 25°C) of ≥50 g / L.

[0257] Solution (a) may contain no more than 60%wt of hydrophilic vinyl monomers. For example, solution (a) may contain no more than 55%wt or 50%wt of hydrophilic vinyl monomers. For example, solution (a) may contain no more than 45%wt or 40%wt of hydrophilic vinyl monomers; for example, solution (a) may contain no more than 30%wt of hydrophilic vinyl monomers. Solution (a) may contain at least 2%wt of hydrophilic vinyl monomers. Solution (a) may contain at least 5%wt of hydrophilic vinyl monomers. Solution (a) may contain at least 8%wt of hydrophilic vinyl monomers. Solution (a) may contain at least 2%wt% (e.g., 5%wt) of hydrophilic vinyl monomers and no more than 60%wt of hydrophilic vinyl monomers, for example, solution (a) may contain at least 8%wt of hydrophilic vinyl monomers and no more than 60%wt of hydrophilic vinyl monomers. Solution (a) may contain at least 2% wt% (e.g. 5% wt) of hydrophilic vinyl monomer and no more than 50% wt of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% wt of hydrophilic vinyl monomer and no more than 50% wt of hydrophilic vinyl monomer. Solution (a) may contain at least 2% wt% (e.g. 5% wt) of hydrophilic vinyl monomer and no more than 45% wt of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% wt of hydrophilic vinyl monomer and no more than 45% wt of hydrophilic vinyl monomer. Solution (a) may contain at least 2% wt% (e.g. 5% wt) of hydrophilic vinyl monomer and no more than 30% wt of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% wt of vinyl monomer and no more than 30% wt of hydrophilic vinyl monomer. Solution (a) may contain at least 2% wt % (eg 5% wt ) of the hydrophilic vinyl monomer and no more than 15% of the hydrophilic vinyl monomer, for example, solution (a) may contain about 10% wt of the hydrophilic vinyl monomer.

[0258] The hydrophilic vinyl monomer may comprise a mixture of monomers. For example, the monomer may comprise at least one monomer as defined above and at least one compatible functional monomer. Exemplary functional monomers are hydrophilic vinyl monomers as defined herein that comprise carboxylic acids (—COOH), phosphonic acids (—P(O)(OH) 2 ), sulfonic acid (-S(O) 2 OH), primary amine or secondary amine. For example, the monomer may comprise at least one monomer of formula (I), (Ia) or (Ib) which does not comprise a carboxylic acid (-COOH), a phosphonic acid (-P(O)(OH) 2 ), sulfonic acid (-S(O) 2 OH), a primary amine or a secondary amine; and a monomer of formula (I), (Ia), (Ib) or (Ic) comprising at least one carboxylic acid (—COOH), a phosphonic acid (—P(O)(OH) 2 )、sulfonic acid (-S(O))2 OH), primary or secondary amines (e.g., containing at least one carboxylic acid (—COOH) or phosphonic acid (—P(O)(OH) 2 )).

[0259] The functional monomer can be a vinyl monomer having a log P value less than about 1 (e.g., log P less than about 0.5), which contains a carboxylic acid or a primary amine. The functional monomer can be a vinyl monomer having a log P less than about 0.6. The functional monomer can be a vinyl monomer having a log P less than about 0.52. The functional monomer can be a vinyl monomer having a log P less than about 0.5. For example, the functional monomer can be a vinyl monomer having a log P 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 less than about 0.1. The functional monomer can be a vinyl monomer having a log P less than about 0, for example, a log P less than about -0.2. The functional monomer can be a vinyl monomer having a log P greater than about -3.8, for example, a log P 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. Functional monomers can be compounds of formula (I), (Ia) or (Ib) comprising carboxylic acid or primary amine. Functional monomers can be acrylamide monomers comprising carboxylic acid, phosphonic acid, sulfonic acid or primary amine. Functional monomers can comprise carboxylic acid, phosphonic acid or sulfonic acid, for example, can comprise carboxylic acid or phosphonic acid. Suitable functional monomers include 3-acrylamidopropionic acid, 4-acrylamidobutyric acid, 5-acrylamidovaleric acid, N-(3-aminopropyl) methacrylamide, acrylic acid, fumaric acid, itaconic acid, vinylphosphonic acid, vinylsulfonic acid and 1-vinylimidazole. Other suitable functional monomers include anhydrides, such as maleic anhydride or itaconic anhydride, which can be hydrolyzed after the polymerization process to provide a carboxylic acid group.

[0260] When there is at least one functional monomer, the amount of the functional monomer may be from about 0.1 to about 100% mole, such as from about 0.2 to about 50% mole, such as from about 0.5 to about 40% mole or from about 1 to about 30% mole (e.g., from about 2 to about 20% mole). For example, the amount of the functional monomer may be at least about 2 to about 100% mole, such as from about 2 to about 50% mole, such as from about 4 to about 40% mole or from about 4 to about 30% mole (e.g., from about 2 to about 20% mole). The amount of the functional monomer may be from about 5 to about 100% mole, such as from about 5 to about 80% mole. % mole may refer to the amount of the functional monomer, in terms of mole % of the total hydrophilic vinyl monomer. The amount of the functional monomer may be from about 0.1 to about 60% mole, such as from about 0.2 to about 50% mole, such as from about 0.5 to about 40% mole. The amount of the functional monomer may be from about 1 to about 60% wt, such as from about 2 to about 50% wt, such as from about 2 or 3 to about 40% wt. The amount of the functional monomer may be from about 0.1 to about 10% wt, such as from about 0.2 to about 5% wt, such as from about 0.5 to about 2% wt. % mole may refer to the mole % of the functional monomer included in the total hydrophilic vinyl monomers in solution (a) (i.e., a solution comprising at least 2 wt % of the hydrophilic vinyl monomers in water).

[0261] In highly crosslinked particles, the monomer may be or comprise a crosslinking agent. For example, the monomer may be or comprise at least one compound of formula (IIa) or (IIb).

[0262] The crosslinking agent used in the method may include at least two (e.g., 2) vinyl groups (—CH═CH 2 The cross-linking agent used in the method may be at least one compound of formula (IIa) or formula (IIb), wherein R 6 Selected from -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Hydroxyalkyl-, -C 1 -C 6 Ether-, or containing 2 to 100 C 2 -C 3 ether units; and R 7 and R 8 Each independently selected from -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 Ether; or R 7 and R 8 Connect together to form -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Hydroxyalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 Polyether of ether unit; R 9 Yes-N(R 11 )C(O)CH=CH 2 ; R 10 Selected from -H and -N(R 12 )C(O)CH=CH 2 ; and R 11 and R 12 Each independently selected from -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 ether.

[0263] The crosslinking agent may be at least one compound of formula (IIa). The crosslinking agent may be at least one compound of formula (IIb).

[0264] Where chemically possible, R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 may be independently substituted by 1 to 5 substituents, each of which is independently selected from: oxo, =NR a , =NOR a , halogenated, nitro, cyano, NR a R a , NR a S(O) 2 R a , NR aCONR a R a , NR a CO 2 R a , OR a SR a ,S(O)R a ,S(O) 2 OR a ,S(O) 2 R a ,S(O) 2 NR a R a , CO 2 R a C(O)R a ,CONR a R a , C 1 -C 4 -alkyl, C 2 -C 4 -Alkenyl, C 2 -C 4 -Alkynyl, C 1 -C 4 haloalkyl; wherein R a is independently selected at each occurrence from: H, C 1 -C 4 Alkyl and C 1 -C 4 Alkenyl.

[0265] R 6 Can be selected from -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Hydroxyalkyl- and -C 1 -C 6 Ether-. R 6 Can be selected from -C 1 -C 6 Alkyl- and -C 1 -C 6 Hydroxyalkyl-. 6 Can be -C 1 -C 6 Alkyl-, such as -CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -or-(CH2 ) 4 -, for example -(CH 2 ) 2 -. R 6 Can be -C 1 -C 6 Hydroxyalkyl-, such as -C(OH)H-, -(C(OH)H) 2 -、-(C(OH)H) 3 -, or -(C(OH)H) 4 -, for example -(C(OH)H) 2 -.

[0266] R 6 Can be -C 1 -C 6 Heteroalkyl-, optionally wherein the heteroatom is an amine (eg, a tertiary amine). For example, C(O)R a Replaced-C 1 -C 6 Heteroalkyl-, optionally wherein the heteroatom is an amine, such as R 6 Can be -CH 2 CH 2 N(C(O)CH=CH 2 )CH 2 CH 2 -.

[0267] When R 6 When it is a polyether, the polyether may be linear or branched. 6 It can contain 2 to 100 C 2 -C 3 Polyethers of ether units, for example containing 2 to 50 C 2 -C 3 Polyether of ether units. 6 It can contain 2 to 100 C 2 Polyethers of ether units, for example containing 2 to 50 C 2 For example, R 6 It can be -(CH 2 ) r (OCH 2 CH 2 ) n O(CH 2 ) s , wherein r and s are each independently 2 or 3 (e.g., 2); 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 the polyether (e.g., wherein R 6The crosslinking agent (which is a polyether) has excellent solubility in the aqueous phase. This means that while such a crosslinking agent can be used to provide particles with a low level of crosslinking (e.g. 1-20%wt crosslinking agent, or 1-10%wt crosslinking agent), such a polyether comprising a crosslinking agent is particularly suitable for providing particles comprising a relatively high level of crosslinking, such as >20%wt crosslinking agent, >25%wt crosslinking agent, or >30%wt crosslinking agent. For example, the crosslinking level may be 10-90%wt crosslinking agent, 20-80%wt crosslinking agent, or 25-75%wt crosslinking agent, such as 25-60%wt crosslinking agent, or 30-50%wt crosslinking agent.

[0268] R 7 and / or R 8 and / or R 11 and / or R 12 It can be H. For example, R 7 and / or R 8 It can be H. For example, R 11 and / or R 12 It can be H.

[0269] R 7 and R 8 Can be connected together to form -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Hydroxyalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 Ether units. 7 and R 8 Connect together to form -C 1 -C 6 When alkyl-, R 7 and R 8 The defined groups can be combined with R 6 For example, R 6 Can be -C 1 -C 6 Alkyl- and R 7 and R 8 Can be -C 1 -C 6 alkyl-.

[0270] R 6 Can be selected from -C 1 -C 6 Alkyl-, -C1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 Polyether of ether unit; R 7 and R 8 can be independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Heteroalkyl, -C 3 -C 6 Cycloalkyl or -C 1 -C 6 Ether; or R 7 and R 8 Connect together to form -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 ether units; and R 11 and R 12 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Heteroalkyl, -C 3 -C 6 Cycloalkyl or -C 1 -C 6 ether.

[0271] The log P value of the compound of formula (IIa) or (IIb) may be less than about 1, for example, a log P of less than about 0.5. The log P of the compound of formula (IIa) or (IIb) is 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, a log P of less than about -0.2. The compound of formula (IIa) or (IIb) may have a log P greater than -3.8, for example, a log P greater than about -3 or about -2. The log P of the compound of formula (IIa) or (IIb) is 0.6 to -3. The compound of formula (IIa) or (IIb) has 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.

[0272] The compound of formula (IIa) or (IIb) may have R 原子 <2.75.

[0273] Exemplary crosslinking agents for use in embodiments of the present method include N,N'-(1,2-dihydroxyethylene)bisacrylamide, N,N'-methylenebis(acrylamide), N,N'-ethylenebis(acrylamide), piperazine diacrylamide, glycerol 1,3-diglycol 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 crosslinking agents N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW≤2000) and 4-arm PEG-acrylamide (MW≤2000), (e.g., N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide) are particularly suitable for highly crosslinked particles and mechanically more stable particles, i.e., particles having a crosslinking level of at least 20% wt crosslinker (e.g., a crosslinking level of at least 30% wt crosslinker). Embodiments may also include the use of a combination of crosslinking agents.

[0274] Cross-linking agents 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.

[0275] The crosslinking agent may be a compound that does not contain a primary amine, a secondary amine, a hydroxyl group or a carboxylic acid. The crosslinking agent may be a compound of formula (IIa) or formula (IIb) that does not contain a primary amine, a secondary amine, a hydroxyl group or a carboxylic acid.

[0276] The level of crosslinking in the crosslinked hydrogel polymer particles formed by the method can be expressed as a weight percentage (wt%) of the crosslinker monomer included in the total monomers used in the polymerization. Typical crosslinking levels include >5%wt crosslinker, such as >10%wt crosslinker, or >15%wt crosslinker, such as >20%wt crosslinker. The crosslinking level can also be, for example, 5-60%wt crosslinker, such as 10-50%wt crosslinker. The crosslinking level can also be, for example, 15-40%wt crosslinker, such as 20-40%wt crosslinker, such as 20-30%wt crosslinker. Thus, solution (a) can include a %wt amount of crosslinker corresponding to 5-60% of the amount of hydrophilic vinyl monomer, such as 10-50% of the amount of hydrophilic vinyl monomer. Thus, solution (a) can include an amount of crosslinker in %wt that corresponds to, for example, 15-40% of the amount of hydrophilic vinyl monomer, such as 20-40% of the amount of hydrophilic vinyl monomer, such as 20-30% of the amount of hydrophilic vinyl monomer.

[0277] The crosslinking level may be >30%wt crosslinker or >40%wt crosslinker (e.g. >50%wt crosslinker), e.g. in highly crosslinked particles. The crosslinking level may 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. In highly crosslinked particles, the crosslinking level may be as high as 100%wt crosslinker, e.g. the hydrophilic vinyl monomer may be the crosslinker, e.g. the hydrophilic vinyl monomer and the crosslinker may be the same compound. Thus, solution (a) may comprise a crosslinking amount (%wt) corresponding to 20-80% of the amount of hydrophilic vinyl monomer, e.g. 25-60% of the amount of hydrophilic vinyl monomer.

[0278] As a particular hydrophilic vinyl monomer, mention may be made of acrylamide (prop-2-enamide), for which 1,2-dihydroxybisacrylamide is a suitable crosslinking agent. As a particular hydrophilic vinyl monomer, mention may be made of methylol acrylamide, for which N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is a suitable crosslinking agent. As a particular hydrophilic vinyl monomer, mention may be made of hydroxyethyl acrylamide, for which N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is a suitable crosslinking agent. Other specific hydrophilic vinyl monomers and crosslinking agents are listed in Table 1.

[0279] Stabilizer can be or comprise nonionic surfactant, for example nonionic polymer surfactant.Nonionic surfactant can comprise at least one oligomeric surfactant.For example, nonionic surfactant can comprise oligomeric surfactant.Nonionic polymer or oligomeric surfactant can comprise at least one polyoxyethylene or at least one polyoxypropoxy.Nonionic polymer surfactant can comprise at least one polyethyleneoxy.Nonionic oligomeric surfactant can comprise at least one polyethyleneoxy or at least one polypropyleneoxy.Stabilizer can be selected from or comprise hypermer 2296, AbilWE09, Abil EM90 and sorbitan monooleate (Span 80).

[0280] Polymerization may include activating a free radical initiator. The free radical initiator is typically activated by heating to form free radicals that will initiate the polymerization reaction, however other activation methods, such as radiation, may be used. Activating the free radical initiator may include heating the emulsion containing the swollen particles. Heating typically includes heating the emulsion to a temperature above the temperature of the step that allows the monodisperse seed particles to form swollen particles. Heating may include heating to a temperature of at least 40°C, such as heating to a temperature of at least 50°C, such as a temperature of at least 60°C or a temperature of at least 70°C.

[0281] The free radical initiator can be or include a peroxide initiator or an azo initiator. For example, the free radical initiator can be a peroxide initiator. For example, the free radical initiator can be an azo initiator. An exemplary free radical initiator is 2,2'-azobis-2,4-dimethylvaleronitrile. An exemplary free radical initiator is 2,2'-azobis(2-methylbutyronitrile). The free radical initiator can be present in solution (a). The free radical initiator can be present in solution (b). The free radical initiator can be present in the emulsion in an amount of about 0.1 wt% to about 1.5 wt%. For example, the free radical initiator can be present in the emulsion in an amount of about 0.6 wt% to about 1.2 wt%; for example, the free radical initiator in the emulsion can be present in the emulsion in an amount of about 0.8%.

[0282] The organic solvent in the method for forming monodisperse cross-linked hydrogel polymer particles may contain (or consist of) at least one of aliphatic hydrocarbons, aliphatic carbonates, aliphatic esters, aliphatic ethers, aromatic hydrocarbons or siloxanes, or a combination thereof. For example, the organic solvent may contain (or consist of) at least two of aliphatic hydrocarbons, aliphatic carbonates, aliphatic esters, aliphatic ethers, aromatic hydrocarbons and siloxanes; or, the organic solvent may contain (or consist of) at least three of aliphatic hydrocarbons, aliphatic carbonates, aliphatic esters, aliphatic ethers, aromatic hydrocarbons and siloxanes. The organic solvent may contain (or consist of) a mixture of heptane and toluene. The organic solvent may contain (or consist of) a mixture of aliphatic hydrocarbons. The organic solvent may include (or consist of) bis(2-ethylhexyl) carbonate. The organic solvent may include (or consist of) bis(2-ethylhexyl) carbonate, aliphatic hydrocarbons and aromatic hydrocarbons. The organic solvent may contain (or consist of) bis(2-ethylhexyl) adipate.

[0283] 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.

[0284] In an embodiment, the present invention provides a method for forming monodisperse cross-linked hydrogel polymer particles. The method comprises: forming a solution (a): an aqueous solution of at least 2%wt of a hydrophilic vinyl monomer, the aqueous solution also comprising a chain transfer agent; forming a solution (b) of a stabilizer in an organic solvent, wherein the organic solvent is immiscible with water, and wherein at least one of the solution (a) and the solution (b) comprises a free radical initiator; mixing solutions (a) and (b) to form an oil-in-water 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; forming a stabilizer in an organic solvent; The invention relates to a method for preparing a hydrogel polymer particle by mixing a solution (d) in an organic solvent, wherein the organic solvent is immiscible with water; forming a solution (e): an aqueous solution of at least 2% wt of a hydrophilic vinyl monomer, the aqueous solution also comprising a crosslinking agent containing at least two vinyl groups, wherein at least one of the solution (d) and the solution (e) comprises a free radical initiator; mixing solutions (d) and (e) to form an oil-in-water 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, the method can be considered a two-stage 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 with the single-stage method, the two-stage method can provide large monodisperse crosslinked hydrogel crosslinked polymer particles.

[0285] Similar to the one-stage process, the two-stage process involves swelling the seed particles with an aqueous discontinuous phase in an oil continuous phase. A schematic diagram of the process is shown in Figure 3 (illustrated with specific monomers) and Figure 4 (more generally described) which can be considered a two-stage process since it comprises two swelling steps and two polymerization steps. Figure 3 and 4The illustrated method is conveniently divided into four steps: swelling monodisperse seed particles 100 with monomer 103 and chain transfer agent 105 to form swollen (monodisperse) seed particles 110; polymerizing monomer in the swollen seed particles to form (non-crosslinked) hydrophilic polymer particles 130; swelling hydrophilic polymer particles with monomer 103 and crosslinker 102 to form swollen hydrophilic polymer particles 140; and polymerizing monomer in 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 monomer, such as hydroxymethyl acrylamide 103 and a chain transfer agent, such as 1-thioglycerol 105, with an oil phase containing a steric stabilizer, wherein the emulsion is formed when the water and oil phases are agitated (e.g., by stirring). The water-in-oil emulsion also typically contains an initiator, which can be added to the oil phase and / or the water phase. An initiator is a compound that, upon activation, will initiate 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.

[0286] 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 4-48 hours). During this period, monomers 103 and chain transfer agents 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 monomers 103, chain transfer agents 105, and polymers from the activated seed particles 100. The swollen seed particles 110 may also include other components, such as one or more porogens, which can enter the particles if contained in the water-in-oil emulsion. For example, in the method shown, water is also present in the swollen seed particles 110, and the water can be considered a porogen. The second step includes polymerizing the monomers 103 to form (non-crosslinked) hydrophilic polymer particles 130. In the second step, polymerization is initiated by activating an initiator, such as by heating the emulsion. The presence of chain transfer agent 105 results in relatively short polymers of polyhydroxymethyl acrylamide 132 (e.g., oligomers) in the hydrogel polymer particles. These relatively short polymers, combined with the lack of crosslinking, mean that the hydrophilic polymer particles 130 can serve as larger "activated seed particles" suitable for further rounds of swelling and polymerization. The hydrophilic polymer particles will contain non-crosslinked, relatively short polymers of polyhydroxymethyl acrylamide 132 polymer, and (usually a smaller proportion) non-crosslinked oligomers of poly N, N-dimethyl acrylamide 131.

[0287] Prior to the third step, a water-in-oil emulsion is formed. The water-in-oil emulsion is formed by mixing an aqueous solution containing a monomer, such as hydroxymethyl acrylamide 103 and a crosslinker, such as 1,2-dihydroxybisacrylamide 102, with an oil phase containing a steric stabilizer. If (very) highly crosslinked particles are desired, the monomer can also be replaced by 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 typically also contains an initiator, which can be added to the oil phase or the water phase. An initiator is a compound that, when activated, will initiate polymerization of the monomer and the crosslinker. In the third step (second round of 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 for 4-48 hours). During this period, the monomer 103 and the 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 monomers 103, crosslinkers 102, and 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 may enter the particles if contained in a water-in-oil emulsion. For example, in the method shown, water is also present in the swollen seed particles 110, and this water can be considered a porogen. The fourth step includes polymerization of the monomers 103 and the crosslinkers 102 to form crosslinked hydrogel polymer particles 104. In the fourth step, polymerization is initiated by activating an initiator, such as by heating the emulsion.

[0288] Monomer, chain transfer agent, free radical initiator, stabilizer, crosslinking agent, organic solvent and optional porogen can each be as defined elsewhere herein. Monomer can be or include hydroxymethyl acrylamide or hydroxyethyl acrylamide. Chain transfer agent can be or include 1-thioglycerol. Free radical initiator can be or include azobisdimethylvaleronitrile, such as 2,2'-azobis(2-methylbutyronitrile) (AMBN). Stabilizer can be or include Hydropermer2296, Abil WE09 and Abil EM90. Crosslinking agent can be 1,2-dihydroxybisacrylamide or N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide.

[0289] Solution (a) may contain no more than 60%wt of hydrophilic vinyl monomers. For example, solution (a) may contain no more than 55%wt or 50%wt of hydrophilic vinyl monomers. For example, solution (a) may contain no more than 45%wt or 40%wt of hydrophilic vinyl monomers; for example, solution (a) may contain no more than 30%wt of hydrophilic vinyl monomers. Solution (a) may contain at least 2%wt of hydrophilic vinyl monomers. Solution (a) may contain at least 5%wt of hydrophilic vinyl monomers. Solution (a) may contain at least 8%wt of hydrophilic vinyl monomers. Solution (a) may contain at least 2%wt% (e.g., 5%wt) of hydrophilic vinyl monomers and no more than 60%wt of hydrophilic vinyl monomers, for example, solution (a) may contain at least 8%wt of hydrophilic vinyl monomers and no more than 60%wt of hydrophilic vinyl monomers. Solution (a) may contain at least 2% wt% (e.g. 5% wt) of hydrophilic vinyl monomer and no more than 50% wt of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% wt of hydrophilic vinyl monomer and no more than 50% wt of hydrophilic vinyl monomer. Solution (a) may contain at least 2% wt% (e.g. 5% wt) of hydrophilic vinyl monomer and no more than 45% wt of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% wt of hydrophilic vinyl monomer and no more than 45% wt of hydrophilic vinyl monomer. Solution (a) may contain at least 2% wt% (e.g. 5% wt) of hydrophilic vinyl monomer and no more than 30% wt of hydrophilic vinyl monomer, for example, solution (a) may contain at least 8% wt of vinyl monomer and no more than 30% wt of hydrophilic vinyl monomer. Solution (a) may contain at least 2% wt % (eg 5% wt ) of the hydrophilic vinyl monomer and no more than 15% of the hydrophilic vinyl monomer, for example, solution (a) may contain about 10% wt of the hydrophilic vinyl monomer.

[0290] Solution (e) may contain no more than 60%wt of hydrophilic vinyl monomers. For example, solution (e) may contain no more than 55%wt or 50%wt of hydrophilic vinyl monomers. For example, solution (e) may contain no more than 45%wt or 40%wt of hydrophilic vinyl monomers; for example, solution (e) may contain no more than 30%wt of hydrophilic vinyl monomers. Solution (e) may contain at least 2%wt of hydrophilic vinyl monomers. Solution (e) may contain at least 5%wt of hydrophilic vinyl monomers. Solution (e) may contain at least 8%wt of hydrophilic vinyl monomers. Solution (e) may contain at least 2%wt% (e.g., 5%wt) of hydrophilic vinyl monomers and no more than 60%wt of hydrophilic vinyl monomers, for example, solution (e) may contain at least 8%wt of hydrophilic vinyl monomers and no more than 60%wt of hydrophilic vinyl monomers. The solution (e) may contain at least 2% wt% (e.g. 5% wt) of a hydrophilic vinyl monomer and no more than 50% wt of a hydrophilic vinyl monomer, for example, the solution (e) may contain at least 8% wt of a hydrophilic vinyl monomer and no more than 50% wt of a hydrophilic vinyl monomer. The solution (e) may contain at least 2% wt% (e.g. 5% wt) of a hydrophilic vinyl monomer and no more than 45% wt of a hydrophilic vinyl monomer, for example, the solution (e) may contain at least 8% wt of a hydrophilic vinyl monomer and no more than 45% wt of a hydrophilic vinyl monomer. The solution (e) may contain at least 2% wt% (e.g. 5% wt) of a hydrophilic vinyl monomer and no more than 30% wt of a hydrophilic vinyl monomer, for example, the solution (e) may contain at least 8% wt of a vinyl monomer and no more than 30% wt of a hydrophilic vinyl monomer. Solution (e) may contain at least 2% wt % (eg 5% wt ) of the hydrophilic vinyl monomer and no more than 15% of the hydrophilic vinyl monomer, for example, solution (e) may contain about 10% wt of the hydrophilic vinyl monomer.

[0291] In highly crosslinked particles, the hydrophilic vinyl monomer of solution (e) may be or comprise a crosslinking agent. For example, the monomer may be or comprise at least one compound of formula (IIa) or (IIb).

[0292] The level of crosslinking in the crosslinked hydrogel polymer particles formed by the method can be expressed as the weight percentage (%wt) of the crosslinker monomer contained in the total monomers used in the polymerization (i.e., in the second polymerization step). The %wt of the crosslinker monomer can be equal to the %wt of the crosslinker in the matrix polymer (i.e., the %wt of the crosslinker in the dry weight of the crosslinked polymer particles). Typical crosslinking levels include >5%wt crosslinker, such as >10%wt crosslinker, or >15%wt crosslinker, such as >20%wt crosslinker (e.g., >30%wt crosslinker). The crosslinking level can also be, for example, 5-60%wt crosslinker, such as 10-50%wt crosslinker. The crosslinking level can also be, for example, 15-40%wt crosslinker, such as 20-40%wt crosslinker, such as 20-30%wt crosslinker. The degree of crosslinking can be 20-80%wt crosslinker or 25-75%wt crosslinker, such as 25-60%wt crosslinker or 30-50%wt crosslinker; for example, in high-density particles. Thus, solution (e) may comprise a %wt amount of crosslinker corresponding to 5-60% of the amount of hydrophilic vinyl monomer, such as 10-50% of the amount of hydrophilic vinyl monomer. Thus, solution (e) may comprise an amount of crosslinker in %wt corresponding to, for example, 15-40% of the amount of hydrophilic vinyl monomer, such as 20-40% of the amount of hydrophilic vinyl monomer, such as 20-30% of the amount of hydrophilic vinyl monomer.

[0293] The crosslinking level may be >30% wt crosslinker or >40% wt crosslinker (e.g. >50% wt crosslinker), e.g. in highly crosslinked particles. The crosslinking level may 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. In highly crosslinked particles, the crosslinking level may be as high as 100% wt crosslinker, e.g. the hydrophilic vinyl monomer in solution (e) may be the crosslinker, e.g. the hydrophilic vinyl monomer and the crosslinker may be the same compound. Solution (e) may comprise a % wt amount of crosslinker corresponding to 20-80% of the amount of hydrophilic vinyl monomer, e.g. 25-60% of the amount of hydrophilic vinyl monomer.

[0294] The free radical initiator may be present in each or at least one emulsion in an amount of about 0.1 wt % to about 1.5 wt %. For example, the free radical initiator may be present in each or at least one emulsion in an amount of about 0.6 wt % to about 1.2 wt %; for example, the free radical initiator may be present in each or at least one emulsion in an amount of about 0.8 wt % in each or at least one emulsion.

[0295] The addition of chain transfer agent terminates the chain by the free radical reaction with the growing polymer chain and transfers a single electron to the free radical species derived from the chain transfer agent, thereby reducing the molecular weight of the polymer of the monodisperse polymer particles. Then, the free radical species derived from the chain transfer agent can react with the monomer to form a free radical from the monomer, and then the monomer can react with another monomer to start forming a new polymer chain. The chain transfer agent can be a mercaptan or an alkyl halide. For example, the chain transfer agent can be selected from mercaptan (for example 1-thioglycerol, 1-octyl mercaptan, hexane mercaptan, 6-mercapto-1-hexanol and benzyl mercaptan), alkyl mercaptan (for example 1-octyl mercaptan, hexane mercaptan) and mercaptan (for example 1-thioglycerol). The chain transfer agent can be or include 1-thioglycerol. The total addition of the chain transfer agent can be 1mol per 10mol monomer to 1mol per 300mol monomer, for example 1mol per 20mol monomer to 1mol per 100mol monomer, for example the scope of about 1mol chain transfer agent per 30mol monomer.

[0296] The monodisperse cross-linked hydrogel polymer particles formed according to the method of the present invention may be porous. For example, the monodisperse cross-linked hydrogel polymer particles formed may have a porosity of at least 5%, such as at least 10%. The present disclosure includes porous monodisperse cross-linked hydrogel polymer particles having a porosity of 20% to 95%, particularly 30% to 90%, such as 40% to 90%, such as 50% to 80%. The porous monodisperse cross-linked hydrogel polymer particles formed may be permeable to solvated molecules, for example, the porous monodisperse cross-linked hydrogel polymer particles may be permeable to oligonucleotides and nucleic acid amplification reagents and sequencing reagents (e.g., primers, nucleotides, and polymerases).

[0297] The monodisperse cross-linked hydrogel polymer particles formed according to the method of the present invention may have a mode diameter of 0.5 μm to 10 μm, for example 0.5 μm to 5 μm. The monodisperse cross-linked hydrogel polymer particles formed according to the method of the present invention may have a mode diameter of at least 500 nm, for example at least 600 nm, optionally at least 800 nm, such as in the case of particles having a diameter of at least 1 micron. The monodisperse cross-linked hydrogel polymer particles formed according to the method of the present invention may have a mode diameter of no more than 10 μm, for example no more than 5 μm, optionally no more than 3 μm, such as in the case of particles having a diameter of no more than 2 μm. The size and size distribution of the monodisperse cross-linked hydrogel polymer particles formed according to the method of the present invention may be determined as described below under the heading "Analytical Methods".

[0298] Monodisperse crosslinked hydrogel polymer particles formed according to the method 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%, such as less than 8%, for example less than 5%.

[0299] The monodisperse cross-linked hydrogel polymer particles formed according to the method of the present invention may include 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 hydrophilic vinyl monomers (e.g., compounds of formula (I)) rather than cross-linking agents (e.g., compounds of formula (IIa) or formula (IIb)). The functional groups may be enhanced to promote binding to target analytes (e.g., oligonucleotides) or target receptors. Exemplary methods for enhancing the functional groups of particles are described herein in the section relating to "Uses of Particles".

[0300] The monodisperse cross-linked hydrogel polymer particles formed according to the inventive method may include oligonucleotides attached to the particles. The oligonucleotides may be connected to the monodisperse cross-linked hydrogel polymer particles by a joint. For example, each (or a portion) of the monodisperse hydrogel polymer particles may include a plurality of oligonucleotides attached to the particles. A plurality of oligonucleotides may be the same for each individual cross-linked hydrogel polymer particle. For example, the first cross-linked hydrogel polymer particle may include a plurality of oligonucleotides with a first sequence attached to the first particle, and the second cross-linked hydrogel polymer particle may include a plurality of oligonucleotides with a second sequence attached to the second cross-linked hydrogel polymer particle. When the monodisperse cross-linked hydrogel polymer particles are porous, the oligonucleotides may be connected to the outer surface of the particle or attached to the inside of the hole. The hole may have a sufficient size to make the particle permeable to the oligonucleotide, so that the oligonucleotide may be partially or completely located in the hole, even if the polymerase is connected to the oligonucleotide.

[0301] The monodisperse crosslinked hydrogel polymer particles formed according to the methods of the present invention can be stable in aqueous solution (i.e., resistant to polymer degradation) over a typical temperature range of aqueous solutions. The monodisperse crosslinked hydrogel polymer particles formed can be stable at temperatures up to about 100° C. For example, the monodisperse crosslinked hydrogel polymer particles formed can be stable over a temperature range of about 0° C. to about 100° C.

[0302] One embodiment of the present invention provides a particle obtained by the preparation method described herein or a particle having the characteristics of a particle obtained by the preparation method described herein.

[0303] The polymer particle formation method described herein can be processed to be highly reproducible and scalable. Therefore, the present invention can achieve consistency between batches and within batches, which is a prerequisite for industrial application. The present invention can also produce pilot-scale batches, such as at least 300g and kilogram-scale industrial batches.

[0304] The polymer particle formation methods described herein can be performed consistently without the problems that can arise from practical emulsion polymerization, such as agglomeration of particles and product variability.

[0305] Preparation of magnetic particles

[0306] In one embodiment, a method of 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. If present, the magnetic material precursor is converted into a magnetic material. The magnetic material is allowed to bind to the polymer particles. The magnetic material precursor can be provided, for example, by forming an aqueous suspension having a pH of less than 6, the aqueous suspension comprising monodisperse hydrogel polymer particles and Fe 2+ and / or Fe 3+ ions; converting it to a magnetic material by raising the pH above 8; and allowing the magnetic material to precipitate. Monodisperse hydrogel polymer particles can be prepared (eg, prepared) according to the methods of forming monodisperse hydrogel polymer particles disclosed herein.

[0307] Aqueous suspensions with a pH of less than 6 may contain Fe 2+ and Fe 3+ ions. Aqueous suspensions with a pH value less than 6 may contain FeCl 2 and FeCl 3 Aqueous suspensions with a pH of less than 6 may contain Fe 2+ and Fe 3+ ions, and may also contain polyvalent cations of a metal selected from Al, Mn, Ni, Cu, Co, Zn, Ca, Ge, Te, Ti or Sn or a combination thereof. When the aqueous suspension contains polyvalent cations of a metal selected from Al, Mn, Ni, Cu, Co, Zn, Ca, Ge, Te, Ti or Sn or a combination thereof, the amount of the polyvalent cations may include Fe 2+ and Fe 3+ The amount of ions is up to 5 mol%.

[0308] Raising the pH to greater than 8 may comprise adding ammonia (eg aqueous ammonia) to the aqueous suspension.

[0309] The magnetic material can be precipitated as a nanoparticulate magnetic material and / or a superparamagnetic material. The magnetic material can be precipitated as a paramagnetic material. The magnetic material can be precipitated as a superparamagnetic material. The magnetic material can be precipitated as a ferromagnetic material, a ferrimagnetic material, or both. In the case where the monodisperse hydrogel polymer particles are porous, the magnetic material can be precipitated in the pores, for example, the magnetic material can be precipitated in the pores as a superparamagnetic material.

[0310] The total amount of magnetic material present in the monodisperse magnetic hydrogel polymer particles formed by the method is generally greater than 20%, preferably greater than 25%, desirably greater than or equal to 30%, such as up to 85% wt or at least 50% wt, such as 30 to 80 wt%. The percentages are weight percentages calculated based on the weight of the magnetic material (e.g., metal oxide) based on the total dry weight of the particles. In the case where the magnetic material is composed of a superparamagnetic material, the total amount of superparamagnetic material present may be greater than 20%, preferably greater than 25%, desirably greater than or equal to 30%, such as up to 85% wt or at least 50% wt, such as 30 to 80 wt%, and the percentages are each weight percentages calculated based on the weight of the magnetic material (e.g., metal oxide) based on the total dry weight of the particles.

[0311] Preparation of coated particles

[0312] In one embodiment, a method of forming coated monodisperse hydrogel polymer particles is provided, comprising: forming a suspension comprising monodisperse hydrogel polymer particles and at least one epoxide or silicate or orthosilicate; reacting the epoxy groups of the at least one epoxide with the surface functional groups of the particles; or reacting a silicate or silane to form a silica coating. The monodisperse hydrogel polymer particles can be prepared according to the method of forming monodisperse hydrogel polymer particles disclosed herein. The monodisperse hydrogel polymer particles can be prepared according to the method of forming monodisperse magnetic hydrogel polymer particles disclosed herein.

[0313] In embodiments, the method comprises reacting the epoxy groups of the at least one epoxide with the surface functional groups of the particles. The suspension may comprise an organic solvent (e.g., an organic nitrile, such as acetonitrile; or an alcohol, such as isopropanol). The reaction may comprise a grafting reaction. The at least one epoxide monomer may comprise a mixture of epoxide monomers, such as at least two or at least three epoxide monomers. The at least one epoxide monomer (or mixture of epoxide monomers) may be selected from 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, glycerol diglycidyl ether, glycidol, and glycidyl methacrylate, ethylhexyl glycidyl ether, methyl glycidyl ether, glycerol propoxylated triglycidyl ether, poly(propylene glycol) diglycidyl ether, 1,3-butanediol diglycidyl ether, tert-butyl glycidyl ether, 1,4-cyclohexanedimethanol diglycidyl ether, diethylene glycol diglycidyl ether, dodecyl glycidyl ether, O-(2,3-epoxypropoxy)butane 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-butylene oxide, 1,2-pentane oxide, 1,2-epoxy-5-hexene, 1,2-epoxyhexane, 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 a combination thereof. The mass ratio of monodisperse hydrogel polymer particles: at least one epoxide monomer may be 1:10 to 10:1.

[0314] The surface functional groups may comprise amines. The amines may be derived from the functional monomers used in the method of forming the monodisperse hydrogel polymer particles, or the amines may be introduced using standard chemistry. For example, the (non-amine) functional groups of the polymer particles may be modified with reagents capable of converting the functional groups into reactive moieties that can undergo nucleophilic or electrophilic substitution, by selecting a suitable reagent comprising a nucleophile or an electrophile to introduce the amines.

[0315] In one embodiment, the method comprises reacting a silicate or a silane to form a silica coating. The hydrogel polymer particles may contain iron oxide crystals (e.g., the hydrogel polymer particles may contain a magnetic material containing iron oxide crystals) and / or hydroxyl groups. Without wishing to be bound by any theory, it is believed that the presence of iron oxide crystals and / or hydroxyl groups in the hydrogel polymer particles enhances the formation of the silica coating.

[0316] In one embodiment, the method comprises reacting a silicate to form a silica coating. The silicate may be a metasilicate, such as Na 2 SiO 3 The suspension may include an aqueous solvent (e.g., the solvent may be water). Reacting the silicate to form the silica coating may 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:silicate may be 1:10 to 10:1.

[0317] In one embodiment, the method comprises reacting a silane to form a silica coating. The silane may be an organosilane, such as tetraethyl orthosilicate. The suspension may comprise an organic solvent, such as the solvent may be or comprise dipropylene glycol dimethyl ether. Reacting the orthosilicate to form the silica coating may comprise 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 may comprise raising the temperature of the suspension to a temperature of 80°C to 155°C, such as a temperature of 100°C to 150°C, such as a temperature of 100°C to 150°C, such as a temperature of 110°C to 150°C (e.g., a temperature of about 130°C). The mass ratio of monodisperse hydrogel polymer particles: orthosilicate may be in the range of 1:10 to 10:1.

[0318] Uses of granules

[0319] 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 comprising magnetic materials are particularly useful in assays (e.g., where the polymer particles are conjugated to a target analyte such as an affinity ligand) because the magnetic material allows the particles (and any substances bound thereto) to be separated from the bulk solution by application of a magnetic field.

[0320] Polymer particles can be activated to promote conjugation with target analytes (e.g., polynucleotides or other affinity ligands). Affinity ligands include monoclonal antibodies, polyclonal antibodies, antibody fragments, nucleic acids, oligonucleotides, proteins, oligopeptides, polysaccharides, sugars, peptides, peptides encoding nucleic acid molecules, immobilized metal affinity chromatography (IMAC) agents, antigens, drugs and other ligands. When affinity ligands have clinical significance, polymer particles conjugated with affinity ligands can be used for in vitro diagnosis. In addition to the method described below, suitable methods for activation and bioconjugation are also described in GT Hermanson, Bioconjugate Techniques, 2013 (3rd edition), Academic Press, the contents of which are incorporated herein by reference in their entirety.

[0321] For example, the functional groups on the polymer particles can be enhanced to allow binding to the target analyte or analyte receptor. In a specific example, the functional groups of the polymer particles can be modified with reagents that can convert the functional groups into reactive parts that can undergo nucleophilic or electrophilic substitution. For example, the hydroxyl groups on the polymer particles can be activated by replacing at least a portion of the hydroxyl groups with sulfonic acid groups or chlorine. Exemplary sulfonic acid groups can be derived from trifluoroethane sulfonyl (tresyl), mesyl, tosyl or tosyl chloride, or any combination thereof. Sulfonates can play a role in making nucleophilic agents replace sulfonates. Sulfonates can further react with released chlorine to provide chlorinated groups, which can be used in the process of conjugating particles. In another example, wherein the polymer particles include amine groups or carboxylic acid groups (e.g., from functional monomers), amine groups or carboxylic acid groups can be activated.

[0322] For example, the target analyte or analyte receptor can be bound to the polymer particles by nucleophilic substitution of the sulfonate group or other activated group. In a specific example, the target analyte receptor terminated with a nucleophile (e.g., an amine or thiol) can undergo nucleophilic substitution to replace the sulfonate group on the surface of the polymer particle. As a result of activation, conjugated particles can be formed.

[0323] An example of such a target analyte receptor terminated with a nucleophilic agent is streptavidin, which is ideally suitable for many different assays. For example, a biotinylated single-stranded oligonucleotide probe bound to a particle conjugated with streptavidin can be used to separate sequence-specific DNA. By mixing an appropriate amount of polymer particles with an excess of biotinylated probe, the biotinylated probe is bound to the conjugated particle. Then, under conditions suitable for the length and sequence of the probe and DNA, the particles / probes are incubated with a DNA sample in a hybridization buffer (e.g., SSPE or SSC). In the case of using magnetic polymer particles, the magnetism of the polymer particles is utilized to wash away excess and unwanted DNA. The DNA captured can be detected / quantified by PCR or any other suitable technique. For example, a biotinylated double-stranded DNA fragment bound to a polymer particle conjugated with streptavidin can be used to separate DNA sequence-specific binding proteins. By mixing an appropriate amount of beads with an excess of biotinylated DNA fragments, the biotinylated DNA is bound to the conjugated polymer particles. Then, under conditions suitable for the protein under study, the particles / DNA are incubated in a hybridization buffer with a protein sample. In the case of magnetic polymer particles, the magnetic properties of the polymer particles are exploited to wash away excess and unwanted proteins. The captured proteins can be eluted from the probe (by high salt, low salt, high temperature, low pH, etc.) for downstream applications and detection.

[0324] In another example, the sulfonated particles can be further reacted with a monofunctional or polyfunctional mono- or poly-nucleophilic reagent that can form an attachment to the particle while retaining the nucleophilic activity of an oligonucleotide containing an electrophilic group (e.g., maleimide). In addition, the residual nucleophilic activity can be converted to electrophilic activity by attaching to an agent containing a poly-electrophilic group that is subsequently attached to an oligonucleotide containing a nucleophilic group.

[0325] In another example, monomers containing functional groups (functional monomers) can be included in the monomer mixture during polymerization. Functional monomers can include, for example, acrylamides containing carboxylic acid, phosphonic acid, sulfonic acid, ester, halogen or other amine reactive groups. The ester group can be hydrolyzed before reacting with an amine oligonucleotide or other amine-containing affinity ligand.

[0326] Exemplary amine containing affinity ligands are compounds of the formula

[0327]

[0328] wherein each R is independently hydrogen or a protecting group, and X represents C 2 -C 20 Alkylene linker (e.g. C 5 or C 6This affinity ligand provides a carboxymethylated aspartic acid (Cm-Asp) group that can be attached to the particle (MPP), deprotected (if R is a protecting group), and complexed with metal ions to form an IMAC reagent

[0329]

[0330] This ligand is thus capable of binding to His, Cys, Met, Gln, Asn, Lys or Tyr residues present in the histidine tag in a recombinant protein or in the active site of a metalloprotein in a native protein or peptide, as well as being capable of binding to phosphorylated proteins or polypeptides. Preparation, conjugation and use of the affinity ligand in assays (e.g., with magnetic polymer particles) are described in US2010 / 0222508A1, the contents of which are incorporated herein by reference in their entirety.

[0331] Other activation chemistries include combining multiple steps to convert specific functional groups to adapt to specific desired connections. For example, sulfonate-modified hydroxyls can be converted into nucleophilic groups by several methods. In one example, the reaction of sulfonates with azide anions produces azide-substituted hydrophilic polymers. Azides can be used directly for binding to acetylene-substituted biomolecules by "CLICK" chemistry, which can be performed with or without copper catalysis. Optionally, azides can be converted into amines by, for example, catalytic reduction with hydrogen or reduction with organic phosphine. The resulting amines can then be converted into electrophilic groups with various reagents, such as diisocyanates, bis-NHS esters, cyanuric chloride or combinations thereof. In one example, diisocyanates are used to generate urea bonds between polymers and joints, which generate residual isocyanate groups that can react with amino-substituted biomolecules to generate urea bonds between joints and biomolecules. In another example, bis-NHS esters are used to generate amide bonds and residual NHS ester groups between polymers and joints, which can react with amino-substituted biomolecules to generate amide bonds between joints and biomolecules. In another example, cyanuric chloride is used to generate an amino-triazine linkage between the polymer and the linker and two residual chloro-triazine groups, one of which is capable of reacting with an amino-substituted biomolecule to generate an amino-triazine linkage between the linker and the biomolecule. Other nucleophilic groups can be incorporated into the particles through sulfonate activation. For example, reaction of the sulfonated particles with thiobenzoate anions and subsequent hydrolysis of the thiobenzoate esters incorporates thiols into the particles, which can then react with maleimide-substituted biomolecules to generate thio-succinimide bonds to the biomolecule. Thiols can also react with bromo-acetyl groups.

[0332] The covalent attachment of biomolecules to resistant substrates or polymer substrates can be produced using an electrophilic portion on the substrate to a nucleophilic portion on the biomolecule or to a nucleophilic bond on the substrate to which the electrophilic bond is attached. Due to the hydrophilicity of the most common target biomolecules, the solvents selected for these couplings are water or water containing some water-soluble organic solvents so that the biomolecules are dispersed on the substrate. In particular, polynucleotides are usually coupled to substrates in aqueous systems due to their polyanionic properties. Because water competes with nucleophiles by hydrolyzing electrophiles into inactive parts for conjugation, aqueous systems usually result in low yields of coupled products, where the yield is based on the electrophilic portion of the pair. When a high yield of the electrophilic portion of the reaction pair is required, a high concentration of nucleophiles is required to drive the reaction and mitigate hydrolysis, resulting in inefficient use of nucleophiles. In the case of polynucleic acids, the metal counterions of the phosphate can be replaced with lipophilic counterions to help dissolve the biomolecules in polar, non-reactive, non-aqueous solvents. These solvents may 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 a combination thereof; carbonates such as dimethyl carbonate, propylene carbonate or a combination thereof; ethers such as tetrahydrofuran; sulfoxides and sulfones such as dimethyl sulfoxide, dimethyl sulfone or a combination thereof; hindered alcohols such as tert-butyl alcohol; or a combination thereof. Lipophilic cations may 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, tetraalkylarsine or tetraaryl onium such as tetraphenylarsine, and trialkylsulfonium cations such as trimethylsulfonium, or a combination thereof. The conversion of polynucleic acids into organic solvent soluble materials by exchanging metal cations with lipophilic cations can be performed by a variety of standard cation exchange techniques.

[0333] The polymer particles can be activated to facilitate conjugation with target analytes (e.g., polynucleotides or other affinity ligands). For example, the functional groups on the cross-linked hydrogel polymer particles can be enhanced to allow binding to target analytes or analyte receptors. In a specific example, the functional groups of the polymer can be modified with reagents that can convert hydrophilic polymer functional groups into reactive moieties that can undergo nucleophilic or electrophilic substitution. In particular, where the polymer particles have carboxyl functional groups, these can be activated to facilitate conjugation to, for example, biomolecules, such as nucleic acids.

[0334] In embodiments where particles are formed using a comonomer comprising a hydroxyl group, the hydroxyl groups on the hydrophilic particles can be activated by replacing at least a portion of the hydroxyl groups with a sulfonate group or chlorine. Exemplary sulfonic acid groups can be derived from trifluoroethanesulfonyl (tresyl), mesyl, tosyl or tosyl chloride, or any combination thereof. Sulfonates can serve to replace sulfonates with nucleophiles. Sulfonates can further react with released chlorine to provide chlorinated groups that can be used in the process of conjugating particles. In another example, the amine groups on the hydrophilic polymer can be activated.

[0335] For example, a target analyte or analyte receptor can be bound to a hydrophilic polymer by nucleophilic substitution of a sulfonate group. In a specific example, a target analyte receptor terminated with a nucleophile (e.g., an amine or thiol) can undergo nucleophilic substitution to replace the sulfonate group on the surface of the hydrophilic polymer. As a result of activation, conjugated particles can be formed.

[0336] In another example, where the particle comprises an amine (e.g., where the particle is formed from a monomer comprising an amine or comprises a coating comprising an amine), the nucleophilic amino group can be modified with a difunctional di-electrophilic moiety, such as a di-isocyanate or a bis-NHS ester, resulting in a hydrophilic particle that is reactive toward nucleophiles.

[0337] When conjugated to a polynucleotide, the polymer particle may include polynucleotides at a density, referred to as nucleotide density, of at least 7×10 4 / μm 3 For example, the nucleotide density can be at least 10 5 / μm 3 , such as at least 10 6 / μm 3 , at least 5×10 6 / μm 3 , at least 8×10 6 / μm 3 , at least 1×10 7 / μm 3 , or even at least 3×10 7 / μm 3 In a further example, the nucleotide density may be no greater than 10 15 / μm 3 .

[0338] Such polymer particles can be used in various separation techniques and analytical techniques. In particular, polymer particles can be used to bind polynucleotides. Such bound polynucleotides can be used to separate polynucleotides from a solution or can be used in analytical techniques, such as sequencing. Figure 5In the specific example shown in , such polymer particles can be used as carriers of polynucleotides during sequencing techniques. For example, the particles can immobilize polynucleotides for sequencing using fluorescent sequencing techniques.

[0339] In general, polymer particles can be treated to include 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 ends or any internal portions of biomolecules can be bound or attached to polymer particles. Polymer particles can be bound or attached to biomolecules using attachment chemistry. Attachment chemistry includes covalent or non-covalent bonds, including ionic bonds, hydrogen bonds, affinity bonds, dipole-dipole bonds, van der Waals bonds, and hydrophobic bonds. The linking chemistry includes affinity between binding partners, such as between: an avidin moiety and a biotin moiety; an antigenic epitope and an antibody or immunoreactive fragment thereof; an antibody and a hapten; a digoxigenin moiety and an anti-digoxigenin antibody; a fluorescein moiety and an anti-fluorescein antibody; an operator and an inhibitor; 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.

[0340] like Figure 5 As shown in FIG. 1 , a plurality of polymer particles 204 (e.g., monodisperse cross-linked hydrogel polymer particles of the present disclosure) can be placed in a solution with a plurality of polynucleotides 202. The plurality of particles 204 can be activated or otherwise prepared to bind to the polynucleotides 202. For example, the particles 204 can include oligonucleotides that are complementary to a portion of the plurality of polynucleotides 202.

[0341] In a specific embodiment, polymer particles and polynucleotides are subjected to polymerase chain reaction (PCR) amplification, such as emulsion PCR. For example, dispersed phase droplets 206 or 208 are formed as part of an emulsion and may include particles or polynucleotides. In an example, polynucleotides 202 and hydrophilic particles 204 are provided at low concentrations and ratios relative to each other so that a single polynucleotide 202 may be present in the same dispersed phase droplet together with a single polymer particle 204. Other droplets, such as droplet 208, may include a single polymer particle without including a polynucleotide. Each droplet 206 or 208 may include an enzyme, nucleotide, salt or other component sufficient to promote polynucleotide replication. Alternatively, an amplification technique may be used, such as recombinase polymerase amplification (RPA) with or without an emulsion.

[0342] In one aspect, the present invention provides use of the hydrogel polymer particles of the present invention in nucleic acid amplification. The nucleic acid amplification may be emulsion PCR.

[0343] In an embodiment, the method for nucleic acid amplification comprises: performing a primer extension reaction on a polynucleotide, the polynucleotide hybridizing with an oligonucleotide attached to a polymer particle. In an embodiment, the polymer particle is a monodisperse cross-linked hydrogel polymer particle of the present disclosure. In an embodiment, the method for nucleic acid amplification comprises: (a) providing a polymer particle connected to a single-stranded oligonucleotide (e.g., a primer oligonucleotide); (b) providing a single-stranded template polynucleotide; (c) hybridizing the single-stranded oligonucleotide with the single-stranded template polynucleotide; (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 to the single-stranded oligonucleotide, thereby producing an extended single-stranded oligonucleotide. In an embodiment, the method further comprises: (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 with a second single-stranded template polynucleotide; and (g) contacting the second single-stranded template polynucleotide with a second polymerase and a second at least one nucleotide under conditions suitable for a second polymerase to catalyze the polymerization of the second at least one nucleotide to the single-stranded oligonucleotide, thereby producing a subsequent extended single-stranded oligonucleotide. In an embodiment, steps (e), (f) and (g) may be repeated at least once. In an embodiment, the polymerase and the second polymerase comprise a thermostable polymerase. In an embodiment, conditions suitable for nucleotide polymerization include performing a nucleotide polymerization step (e.g., step (d) or (g)) at an elevated temperature. In an embodiment, conditions suitable for nucleotide polymerization include performing a nucleotide polymerization step (e.g., step (d) or (g)) at an alternating temperature (e.g., an elevated temperature and a relatively low temperature). In an embodiment, the alternating temperature range is 60-95°C. In embodiments, the temperature cycle may be from about 10 seconds to about 5 minutes, or about 10 minutes, or about 15 minutes, or longer. In embodiments, the method for nucleic acid amplification may produce one or more polymer particles each connected to a plurality of template polynucleotides, the template polynucleotides comprising a sequence complementary to a single-stranded template polynucleotide or a second single-stranded template polynucleotide. In embodiments, each polymer particle may be connected to a plurality of single-stranded oligonucleotides (e.g., capture oligonucleotides). In embodiments, steps (b), (c), (d), (e), (f) or (g) may be performed with a plurality of single-stranded polynucleotides. In embodiments, at least a portion of the single-stranded oligonucleotides comprises a nucleotide sequence complementary (or partially complementary) to at least a portion of the single-stranded polynucleotides. In embodiments, the method for nucleic acid amplification (as described above) may be performed in an aqueous solution of an oil phase (e.g., dispersed phase droplets).

[0344] After PCR, a particle is formed, such as particle 210, which may include a plurality of copies 214 of a polymer particle 212 and a polynucleotide. For the purpose of clarity, most of the polynucleotides 214 are shown on the outer surface of the particle 210. However, the polynucleotides may extend in the particle 210 (or be located in the particle 210), as also shown. For example, hydrogels and hydrophilic particles may have a low concentration of polymer relative to water, and therefore may be relatively porous. They may include polynucleotide segments in the inside of the particle 210 and in the entire particle 210, and the polynucleotides may be present in holes and other openings. In particular, the particle 210 may allow the diffusion of enzymes, nucleotides, primers, and reaction products for monitoring reactions. A large amount of polynucleotides of each particle may produce better signals.

[0345] In embodiments, the polymer particles from the demulsification process can be collected and washed in preparation for sequencing. The collection can be carried out by contacting the biotin portion (e.g., connected to the amplified polynucleotide template attached to the polymer particles) with the avidin portion and separating the polymer particles lacking the biotinylated template. The collected polymer particles carrying the double-stranded template polynucleotide can be denatured to produce a single-stranded template polynucleotide for sequencing. The denaturation step can include treatment with an alkali (e.g., NaOH), formamide, or pyrrolidone.

[0346] Another sequencing system is Applied Biosystems' SOLiD TM Sequencing system (sequencing by oligonucleotide connection and detection), which uses step-by-step cycle connection for high-throughput DNA sequencing. In this bead-based system, beads (i.e., polymer particles) loaded with DNA templates use 4-color fluorescently labeled octamer probes to perform continuous connection and cleavage reactions. These probes are continuously delivered and used to interrogate dinucleotide positions on the DNA chain. It is expected to support higher bead density, which promotes an increase in the number of bead events per instrument run and improved probe chemistry, thereby providing increased sequencing fidelity.

[0347] Sequencing by oligonucleotide ligation and detection involves attaching a nucleic acid target to cross-linked polymer particles (beads) and then 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).

[0348] The method of attaching beads to a support utilizes a flat glass microscope slide that is irreversibly coated with streptavidin. Beads carrying nucleic acids are contacted with biotinylated nucleotides (e.g., obtained by the action of biotinylated dNTPs and terminal deoxytransferase on a DNA target after attachment of the beads). Incubation of the biotinylated beads with the streptavidin-coated slide results in the beads being fixed to the slide through the interaction of biotin and streptavidin. Although kinetically this is a very efficient attachment scheme, movement of beads on the slide is sometimes observed under the conditions required for DNA sequencing. When beads are present at high density on the slide (e.g., up to 100,000 beads / mm 2 ) and interrogated multiple times (e.g., up to 25 times), any significant bead movement may prevent robust identification of specific beads in subsequent scans of densely packed beads.

[0349] US 2009 / 0099027 (corresponding to WO 2009 / 026546, both of which are incorporated herein by reference) thus describes a covalent system for immobilizing beads that reduces bead movement during sequencing and other forms of genetic analysis. The method comprises: reacting a nucleophilic group on a substrate surface with a molecule comprising a plurality of electrophilic groups, thereby providing one or more free electrophilic groups on the substrate surface; and reacting a nucleophilic group on a particle material surface with one or more free electrophilic groups on the substrate surface to covalently attach the particle material to the substrate.

[0350] US 2009 / 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)trialkoxysilanes.

[0351] DNA target nucleic acids that have been covalently attached to cross-linked 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 the residual electrophilic groups on the support surface to form multiple stable covalent bonds between the beads and the glass surface.

[0352] It has been found that stable covalent bonds can be formed between surfaces containing electrophilic groups and particles containing nucleophilic groups. In addition, beads containing amino-dNTPs and nucleophilic amino groups on the DNA target for the action of terminal deoxytransferase can be immobilized on the modified surface under aqueous alkaline conditions. For example, surfaces containing amino groups that have been activated with 1,4-benzene diisothiocyanate can be used to immobilize beads with nucleophilic groups. Furthermore, the covalent attachment appears to be very stable and no bead movement has been observed.

[0353] Surface-immobilized beads can be used in methods for analyzing nucleic acid sequences based on repeated cycles of duplex extension along single-stranded templates by ligation. This type of sequencing method is disclosed in U.S. Pat. Nos. 5,750,341; 5,969,119; and 6,306,597B1 and International Publication No. WO2006 / 084132A2. Each of these publications is incorporated herein by reference in its entirety. In addition, the techniques described in the above publications can be used to analyze (e.g., sequence) nucleic acid templates attached to particles bound to carriers described herein. Immobilized beads can be used in sequencing methods that do not necessarily employ a ligation step, such as sequencing of nucleotides using a label with a removable blocking group that prevents polynucleotide chain extension (e.g., U.S. Pat. Nos. 6,664,079; 6,232,465; and 7,057,026, each of which is incorporated herein by reference in its entirety). Immobilized beads can be used in a variety of techniques in which the signal on the beads is repeatedly detected by multiple cycles.

[0354] The beads used for SOLiD sequencing can be monodisperse magnetic and / or coated hydrogel particles of the present disclosure. Therefore, one embodiment includes using monodisperse particles in the methods and products disclosed in the publications mentioned in the previous paragraph, and the applicant of the present application believes that all these uses, methods and products belong to the present invention and retain the right to claim them. Submicron particles can be attached to glass surfaces (such as glass plates or microscope slides) with a larger density in SOLiD sequencing. Further included in the embodiment is a method for performing SOLiD sequencing, which uses monodisperse particles of the present disclosure, such as wherein the monodisperse submicron of the present disclosure is coupled to a nucleic acid target and fixed on a surface, such as a glass surface. The fixing method is not important and can be covalent or non-covalent, and an example of non-covalent coupling is by streptavidin / avidin-biotin binding. For example, covalent coupling can be as described in US2009 / 0099027 and WO2009026546, but any other suitable technology for covalent coupling can be used. Thus, in one embodiment, a method of forming a product (article of manufacture) is included, comprising coupling the monodisperse submicron particles of the present disclosure to a nucleic acid and optionally further comprising immobilizing the resulting nucleic acid-loaded particles on a surface, such as a glass surface. The nucleic acid can be used as a target in sequencing, such as using SOLiD sequencing.

[0355] For example, a method is provided, comprising:

[0356] (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, wherein the target polynucleotide is attached to a polymer particle, which is a member of a polymer particle population as disclosed herein, and wherein the particle is covalently attached to a surface of a solid support;

[0357] (b) ligating an extended oligonucleotide probe to the extendable probe end, thereby forming an extended duplex containing the extended oligonucleotide probe, wherein the extended oligonucleotide probe comprises a cleavage site and a detectable label;

[0358] (c) identifying one or more nucleotides of the target polynucleotide by detecting the label attached to the newly attached extended oligonucleotide probe;

[0359] (d) cleaving the just-ligated extended oligonucleotide probe at the cleavage site to generate an extendable probe end, wherein the cleavage removes a portion of the just-ligated extended oligonucleotide probe, the portion including the label from the probe-target duplex; and

[0360] (e) Repeating steps (b), (c) and (d) until the sequence of the nucleotides in the target polynucleotide is determined.

[0361] Also provided is a method for sequencing a nucleic acid, comprising:

[0362] (a) hybridizing a primer to a target polynucleotide to form a primer-target duplex, wherein the target polynucleotide is attached at the 5' end to a polymer particle that is a member of the polymer particle population disclosed herein, and wherein the polymer particle is covalently attached to a support surface;

[0363] (b) contacting the primer-target duplex with a polymerase and one or more different nucleotide analogs to incorporate the nucleotide analog into the 3' end of the primer, thereby forming an extended primer chain, wherein the incorporated nucleotide analog terminates the polymerase reaction and wherein 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 tag attached to the base or its analog via a cleavable linker; (iii) a deoxyribose sugar; and (iv) a cleavable chemical group that blocks an -OH group at the 3' position of the deoxyribose sugar;

[0364] (c) washing the support surface to remove any unincorporated nucleotide analogs;

[0365] (d) detecting a unique label attached to the just-incorporated nucleotide analog, thereby identifying the just-incorporated nucleotide analog;

[0366] (e) optionally, permanently blocking any unreacted -OH groups on the extended primer strand;

[0367] (f) cleaving the cleavable linker between the just incorporated nucleotide analog and the unique tag;

[0368] (g) cleaving the chemical group blocking the -OH group at the 3'-position of the deoxyribose of the nucleotide analogue just incorporated to unblock the -OH group;

[0369] (h) washing the support surface to remove the cleaved compound;

[0370] (i) Repeat steps (b)-(h).

[0371] The polymer particles of the present invention can be used in any nucleic acid sequencing method involving polymer particles. One embodiment includes particles of the present disclosure coupled to nucleic acids and nucleic acid sequencing methods, which include coupling nucleic acids to a population of particles of the present disclosure. The nucleic acids can be DNA or RNA.

[0372] The present disclosure includes products (e.g., articles) comprising a plurality of monodisperse particles of the present disclosure coupled to a substrate, such as a glass surface, such as by streptavidin-biotin linkage, avidin-biotin linkage, or by covalent linkage, such as described in US2009 / 0099027 and WO2009 / 026546. The particles can be coupled to the substrate via nucleic acids. The present disclosure includes the use of monodisperse particles of the present disclosure to prepare such products. One embodiment includes attaching monodisperse particles of the present disclosure to a substrate using the linkage chemistry described in US2009 / 0099027 and WO2009 / 026546, and the applicant reserves the right to claim the use of methods and products thereof using such chemistry. Therefore, the disclosure of US2009 / 0099027 and WO2009026546 is incorporated by reference into this specification.

[0373] Thus, embodiments include methods wherein the functionalized monodisperse polymer particles of the present disclosure are subjected to one or more further reactions to obtain a desired product. Other embodiments include using these products in applications.

[0374] In view of the consistency of quality and characteristics that the particles of the present disclosure can have, they can be used in methods including performing processes related to conjugated substances, such as selected from labels, biomolecules and biostructures, such as biomolecules prepared by condensing two or more such monomers (such as polypeptides, proteins, polysaccharides, oligonucleotides and nucleic acids) together, such as amino acids, sugars, nucleotides and nucleosides and polymers. As labels, dyes such as fluorescent dyes, quenchers, enzymes and semiconductor nanocrystals can be mentioned. Embodiments of the present invention include such uses as well as:

[0375] i) a conjugate comprising a population of particles of the present disclosure, at least a portion of which is coupled to a conjugating substance, such as that just described

[0376] ii) a method comprising coupling at least a portion of the particle population of the present disclosure to a substance, such as that just described

[0377] iii) A method comprising coupling at least a portion of the disclosed particle population to a substrate.

[0378] The present invention includes the following aspects / embodiments / features in any order and / or in any combination:

[0379] 1. Monodisperse magnetic hydrogel polymer particles comprising a magnetic material and a polymer formed from:

[0380] (a) Hydrophilic vinyl monomers, whose log P oct / wat (log P) less than about 0.6; and

[0381] (b) a cross-linking agent comprising at least two vinyl groups, wherein the hydrophilic vinyl monomer comprises at least one hydrophilic vinyl monomer selected from the group consisting of:

[0382] (i) It contains -C(O)NH 2 、-C(O)NHCH 3 、-C(O)NHCH 2 CH 3 or -C(O)N(CH 3 ) 2 ;

[0383] (ii) Compounds of formula (Ib)

[0384]

[0385] (iii) Compounds of formula (Ic):

[0386]

[0387] in:

[0388] R 1 and R 1a Each independently selected from -H, -CH 3 、-CH 2 CH 3 or -CH 2 C(O)OH;

[0389] (iv) 4-acryloylmorpholine or 1-vinylimidazole; and

[0390] (v) It includes at least two vinyl groups, wherein the monomer does not contain a primary amine, a secondary amine, a hydroxyl group or a carboxylic acid.

[0391] 2. The polymer particle of any preceding or following embodiment / feature / aspect, wherein monomer (i) comprises -C(O)NH 2 .

[0392] 3. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 1a Yes -H.

[0393] 4. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the at least one hydrophilic monomer is selected from monomers (i), (ii) or (iii).

[0394] 5. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the hydrophilic vinyl monomer has a log P of less than about 0.3, optionally wherein the log P is less than about 0.

[0395] 6. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the hydrophilic vinyl monomer has a log P of 0.5 to -2, optionally wherein the log P is 0 to -2.

[0396] 7. The polymer particles of any preceding or following embodiment / feature / aspect, wherein log P is calculated using CambridgeSoft's Ultra 14.0 is calculated from the structure of the monomer.

[0397] 8. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the hydrophilic vinyl monomer is or comprises an acrylamide monomer and / or an acrylate monomer.

[0398] 9. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the hydrophilic vinyl monomer comprises a vinyl monomer having R according to the formula 原子 Monomer:

[0399]

[0400] Where N C is the number of carbon atoms in the hydrophilic vinyl monomer, N N is the number of nitrogen atoms and N O is the number of oxygen atoms; wherein when the hydrophilic vinyl monomer is acrylamide, vinyl imidazole, acryloyl morpholine, vinyl phosphonic acid or vinyl sulfonic acid, R 原子 <2.75; and when the hydrophilic vinyl monomer is an acrylate or other monomer, R 原子 ≤2 and the water solubility of the hydrophilic vinyl monomer at 25°C is ≥50g / L.

[0401] 10. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the hydrophilic vinyl monomer comprises at least one compound of formula (I):

[0402]

[0403] in:

[0404] R 1 -H, -CH 3 、-CH 2 CH 3 or -CH 2 C(O)OH;

[0405] R 1a -H or -C(O)R 2 ;

[0406] R 1b -C(O)R 2 、–P(O)(OH) 2 、-S(O) 2 OH or a heterocycle having 3 to 8 ring atoms, wherein the heteroatom is selected from N, O or S; or R 1b With R 1a or R 1 The combination is -C(O)-OC(O)-;

[0407] R 2 For-OR 3 , –N(R 4 )R 5 or -N-linked amino acids;

[0408] R 3 -H, -C 1 -C 6 Alkyl or -C 1 -C 6 alcohol; and

[0409] R 4 and R5 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 Alcohol; or

[0410] R 4 and R 5 Together with the nitrogen to which they are attached they form a heterocyclic ring having 3 to 8 ring atoms, wherein the heteroatom is selected from N, O or S.

[0411] 11. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 1 -H, -CH 3 、-CH 2 CH 3 or -CH 2 C(O)OH;

[0412] R 1a -H or -C(O)R 2 ;

[0413] R 1b -C(O)R 2 、–P(O)(OH) 2 or -S(O) 2 OH; or R 1b With R 1a or R 1 The combination is -C(O)-OC(O)-;

[0414] R 2 Yes-OR 3 or –N(R 4 )R 5 ;

[0415] R 3 Yes -H, -C 1 -C 6 Alkyl or -C 1 -C 6 alcohol; and

[0416] R 4 and R 5 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Haloalkyl, -C 1 -C 6 alcohol.

[0417] 12. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 1a Is -H and R 1b Yes-C(O)R 2 , optionally wherein R 2 Yes-OR 3 .

[0418] 13. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 1b Yes - P(O)(OH) 2 .

[0419] 14.-C 1 -C 6 alcohol.

[0420] 15. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 3 Yes -H.

[0421] 16. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 4 and / or R 5 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Alcohol; or R 4 and R 5 Together with the nitrogen to which they are attached they form a heterocyclic ring having 3 to 8 ring atoms, wherein the heteroatom is selected from N, O or S.

[0422] 17. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 4 Is -H or -C 1 -C 6 Alkyl, optionally wherein R 4 Yes -H.

[0423] 18. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 5 Is -H or -C 1 -C 6 Alkyl, optionally wherein R 5 Yes -H.

[0424] 19. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 1a Yes -C(O)NH 2 、-C(O)NHCH 3 、-C(O)NHCH 2 CH 3 or -C(O)N(CH3 ) 2 ; and / or R 1b Yes -C(O)NH 2 、-C(O)NHCH 3 、-C(O)NHCH 2 CH 3 or -C(O)N(CH 3 ) 2 、-P(O)(OH) 2 、-S(O) 2 OH.

[0425] 20. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 3 , R 4 or R 5 At least one of them is -C 1 -C 6 Polyols.

[0426] 21. The polymer particles of any preceding or following embodiment / feature / aspect, wherein the hydrophilic vinyl monomer is selected from or comprises acrylamide, hydroxymethyl acrylamide, [tris(hydroxymethyl)methyl] acrylamide, 4-acryloylmorpholine, 3-acrylamidopropionic acid, methacrylamide, 2-hydroxyethyl methacrylamide, (3-aminopropyl) methacrylamide, or 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, acrylic acid, itaconic acid, fumaric acid, vinylphosphonic acid, vinylsulfonic acid, itaconic anhydride, 1-vinylimidazole and maleic anhydride.

[0427] 22. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the hydrophilic vinyl monomer is selected from or comprises acrylamide, 4-acryloylmorpholine, methacrylamide and vinylphosphonic acid.

[0428] 23. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the hydrophilic vinyl monomer comprises a primary amide group (-C(O)NH 2 ).

[0429] 24. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the crosslinking agent comprises at least one compound of formula (IIa) or (IIb):

[0430]

[0431]

[0432] Where R 6 Selected from -C 1 -C 6 Alkyl-, -C1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Hydroxyalkyl-, -C 1 -C 6 Ether-, or containing 2 to 100 C 2 -C 3 Polyethers of ether units;

[0433] R 7 and R 8 Each independently selected from -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 Ether; or R 7 and R 8 Connect together to form -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Hydroxyalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 Polyethers of ether units;

[0434] R 9 Yes-N(R 11 )C(O)CH=CH 2 ;

[0435] R 10 Selected from -H and -N(R 12 )C(O)CH=CH 2 ;and

[0436] R 11 and R 12 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Heteroalkyl, -C 3 -C 6 Cycloalkyl, -C1 -C 6 Hydroxyalkyl or -C 1 -C 6 ether.

[0437] 25. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 6 Selected from -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 Polyethers of ether units;

[0438] R 7 and R 8 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Heteroalkyl, -C 3 -C 6 Cycloalkyl or -C 1 -C 6 Ether; or R 7 and R 8 Connect together to form -C 1 -C 6 Alkyl-, -C 1 -C 6 Heteroalkyl-, -C 1 -C 6 Cycloalkyl-, -C 1 -C 6 Ether-, containing 2 to 100 C 2 -C 3 polyethers of ether units; and

[0439] R 11 and R 12 Each independently selected from -H, -C 1 -C 6 Alkyl, -C 1 -C 6 Heteroalkyl, -C 3 -C 6 Cycloalkyl or -C 1 -C 6 ether.

[0440] 26. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 6for (CH 2 ) r (OCH 2 CH 2 ) n O(CH 2 ) s , wherein r and s are each independently 2 or 3, and n is an integer from 1 to 100; optionally wherein r and s are each 2 and n is an integer from 1 to 50.

[0441] 27. The polymer particle of any preceding or following embodiment / feature / aspect, wherein R 7 and / or R 8 It's H.

[0442] 28. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the crosslinking agent has a log P value of less than about 1, optionally a log P value of less than about 0.5, further optionally a log P value of less than about 0.

[0443] 29. The polymer particle of any preceding or following embodiment / feature / aspect, wherein log P is calculated using CambridgeSoft's Ultra 14.0 is calculated from the structure of the cross-linker.

[0444] 30. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the crosslinker is or comprises N,N'-(1,2-dihydroxyethylene)bisacrylamide, N,N'-methylenebis(acrylamide), N,N'-ethylenebis(acrylamide), 1,3-diethylene glycol 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 wherein the cross-linking agent is or comprises 1,2-dihydroxybis-acrylamide; or wherein the cross-linking agent is or comprises N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide (MW≤2000) and 4-arm PEG-acrylamide (MW≤2000), optionally wherein the cross-linking agent is or comprises N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide.

[0445] 31. The polymer particle of any preceding or subsequent embodiment / feature / aspect, wherein the crosslinker is or comprises N,N'-methylenebis(acrylamide), N,N'-ethylenebis(acrylamide), piperazinediacrylamide, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycoldiacrylamide (MW≤2000), 4-arm PEG-acrylamide (MW≤2000), N,N-bis(2-acrylamidoethyl)acrylamide.

[0446] 32. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the crosslinker does not comprise a primary amine, a secondary amine, a hydroxyl group, or a carboxylic acid.

[0447] 33. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the crosslinking level is 1-60%wt crosslinker, optionally 1-30%wt crosslinker; or wherein the crosslinking level is 10-90%wt crosslinker, optionally 25-60%wt crosslinker.

[0448] 34. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the hydrophilic vinyl monomer is a cross-linker as defined in any preceding or following embodiment / feature / aspect.

[0449] 35. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the polymer comprises pores, and the pores comprise at least a portion of the magnetic material, optionally wherein the magnetic material is substantially located in the pores.

[0450] 36. The polymer particle of any preceding or following embodiment / feature / aspect, wherein said pores provide said polymer with a porosity level of 40% to 95%.

[0451] 37. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the magnetic material is a nanoparticle magnetic material; and / or wherein the magnetic material is a superparamagnetic material.

[0452] 38. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the magnetic material is a metal oxide or alloy; and / or wherein the magnetic material comprises ferromagnetic and / or superparamagnetic magnet oxide crystals.

[0453] 39. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the polymer particle further comprises a coating.

[0454] 40. The polymer particle of any preceding or following embodiment / feature / aspect, wherein said coating is a polymer coating formed from at least one epoxide monomer.

[0455] 41. The polymer particle of any preceding or following embodiment / feature / aspect, wherein said coating comprises silica.

[0456] 42. Monodisperse coated hydrogel polymer particles comprising a polymer formed from

[0457] (a) Hydrophilic vinyl monomers, whose log P oct / wat (log P) less than about 0.6; and

[0458] (b) a cross-linking agent comprising at least two vinyl groups; and

[0459] The coating, wherein the hydrophilic vinyl monomer comprises at least one hydrophilic vinyl monomer selected from the group consisting of:

[0460] (i) It contains -C(O)NH 2 、-C(O)NHCH 3 、-C(O)NHCH 2 CH 3 or -C(O)N(CH 3 ) 2 ;

[0461] (ii) Compounds of formula (Ib):

[0462]

[0463] (iii) Compounds of formula (Ic):

[0464]

[0465] in:

[0466] R 1 and R 1a Each independently selected from -H, -CH 3 、-CH 2 CH 3 or -CH 2 C(O)OH;

[0467] (iv) 4-acryloylmorpholine or 1-vinylimidazole; and

[0468] (v) It includes at least two vinyl groups, wherein the monomer does not contain a primary amine, a secondary amine, a hydroxyl group or a carboxylic acid.

[0469] 43. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the coating is or comprises an organic coating or a silica coating.

[0470] 44. The polymer particle of any preceding or following embodiment / feature / aspect, wherein said coating comprises an organic coating formed from at least one epoxide monomer.

[0471] 45. The polymer particle of any preceding or following embodiment / feature / aspect, wherein said coating comprises silica.

[0472] 46. ​​The polymer particle of any preceding or following embodiment / feature / aspect, wherein the hydrophilic vinyl monomer is as further defined in any preceding or following embodiment / feature / aspect.

[0473] 47. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the crosslinker is as further defined in any preceding or following embodiment / feature / aspect.

[0474] 48. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the polymer comprises pores.

[0475] 49. Monodisperse hydrogel polymer particles comprising a polymer formed from: (a) a hydrophilic vinyl monomer comprising a monomer selected from the group consisting of 4-acryloylmorpholine and 1-vinylimidazole; and (b) a crosslinker comprising at least two vinyl groups.

[0476] 50. The monodisperse hydrogel polymer particle of any preceding or following embodiment / feature / aspect, wherein the crosslinker is as further defined in any preceding or following embodiment / feature / aspect.

[0477] 51. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the polymer comprises pores, optionally wherein the pores provide the polymer with a porosity level of 40% to 95%.

[0478] 52. The polymer particles of any preceding or following embodiment / feature / aspect, wherein the polymer particles have an average diameter of 0.5 μm to 10 μm, optionally 0.5 μm to 5 μm.

[0479] 53. The polymer particles of any preceding or following embodiment / feature / aspect, wherein the polymer particles have 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.

[0480] 54. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the coefficient of variation (CV) is less than 20%, optionally wherein the CV is less than 15%, further optionally wherein the CV is less than 10%.

[0481] 55. The polymer particle of any preceding or following embodiment / feature / aspect, wherein the polymer particle comprises functional groups, optionally wherein the functional groups are selected from carboxylic acids (—COOH), primary amines or secondary amines.

[0482] 56. A method of forming monodisperse magnetic hydrogel polymer particles, comprising:

[0483] forming an aqueous suspension comprising monodisperse hydrogel polymer particles and a magnetic material or a precursor of a magnetic material;

[0484] When the aqueous suspension includes a magnetic material precursor, converting the magnetic material precursor into a magnetic material; and

[0485] allowing the magnetic material to bind to the polymer particles, wherein the monodisperse hydrogel polymer particles are formed from:

[0486] (a)log P oct / wat A hydrophilic vinyl monomer having a (log P) of less than about 0.6; and

[0487] (b) A cross-linking agent comprising at least two vinyl groups.

[0488] 57. The method of any preceding or following embodiment / feature / aspect, wherein:

[0489] Forming an aqueous suspension comprises forming an aqueous suspension having a pH less than 6, comprising monodisperse hydrogel polymer particles and Fe 2+ and / or Fe 3+ ions; and

[0490] Converting the magnetic material precursor to the magnetic material comprises raising the pH to greater than 8; and allowing the magnetic material to precipitate.

[0491] 58. The method of any preceding or subsequent embodiment / feature / aspect, wherein the aqueous suspension having a pH less than 6 comprises Fe 2+ and Fe 3+ ions, optionally wherein the suspension comprises FeCl 2 and FeCl 3 .

[0492] 59. The method of any preceding or following embodiment / feature / aspect, further comprising forming a polymer coating and / or a silica coating on the magnetic hydrogel polymer particles.

[0493] 60. The method of any preceding or subsequent embodiment / feature / aspect, wherein 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.

[0494] 61. The method of any preceding or subsequent embodiment / feature / aspect, wherein forming the silica coating comprises forming a suspension comprising monodisperse magnetic hydrogel polymer particles and a silicate or orthosilicate, and reacting the silicate or silane to form the silica coating.

[0495] 62. The method of any preceding or following embodiment / feature / aspect, wherein reacting the silicate to form the silica coating comprises lowering the pH of the suspension to less than 11.

[0496] 63. The method of any preceding or following embodiment / feature / aspect, wherein reacting the orthosilicate to form the silica coating comprises raising the temperature of the suspension to at least 80°C.

[0497] 64. A method of forming coated monodisperse hydrogel polymer particles comprising:

[0498] forming a suspension comprising monodisperse hydrogel polymer particles and at least one epoxide or silicate or orthosilicate; and

[0499] reacting epoxy groups of the at least one epoxide with surface functional groups of the particles; or

[0500] reacting a silicate or orthosilicate to form a silicon dioxide coating;

[0501] Wherein the monodisperse hydrogel polymer particles are formed from:

[0502] (a)log P oct / wat A hydrophilic vinyl monomer having a (log P) of less than about 0.6; and

[0503] (b) A cross-linking agent comprising at least two vinyl groups.

[0504] 65. The method of any preceding or following embodiment / feature / aspect, wherein said surface functional group is an amine.

[0505] 66. The method of any preceding or following embodiment / feature / aspect, wherein reacting the silicate to form the silica coating comprises lowering the pH of the suspension to less than 11.

[0506] 67. The method of any preceding or following embodiment / feature / aspect, wherein reacting the orthosilicate to form the silica coating comprises raising the temperature of the suspension to greater than 90°C.

[0507] 68. The method of any preceding or following embodiment / feature / aspect, wherein said monodisperse hydrogel polymer particles are formed according to a method comprising:

[0508] Forming a solution (a): an aqueous solution of at least 2% by weight of a hydrophilic vinyl monomer, the solution further comprising a cross-linking agent containing at least two vinyl groups;

[0509] forming a solution (b) of a stabilizer in an organic solvent, wherein the organic solvent is immiscible with water and wherein at least one of the solution (a) and the solution (b) comprises a free radical initiator; mixing solutions (a) and (b) to form a water-in-oil emulsion (c) and adding monodisperse seed particles to the emulsion;

[0510] allowing the monodisperse seed particles to form swollen particles in the emulsion; and polymerizing the swollen particles to form monodisperse hydrogel polymer particles.

[0511] 69. The method of any preceding or following embodiment / feature / aspect, wherein the hydrophilic vinyl monomer is as further defined in any of any preceding or following embodiment / feature / aspect.

[0512] 70. The method of any preceding or following embodiment / feature / aspect, wherein the cross-linking agent is as further defined in any preceding or following embodiment / feature / aspect.

[0513] 71. The method of any preceding or following embodiment / feature / aspect, wherein the stabilizer is a nonionic surfactant, optionally wherein the nonionic surfactant is a nonionic polymeric surfactant;

[0514] Further optionally wherein the nonionic polymeric surfactant comprises at least one polyoxyethylene group or at least one polyoxypropoxy group; and / or

[0515] The nonionic polymeric surfactant is an oligomeric surfactant.

[0516] 72. The method of any preceding or following embodiment / feature / aspect, wherein said stabilizer is selected from the group consisting of hypermer 2296, Abil EM90, and sorbitan monooleate.

[0517] 73. The method of any preceding or following embodiment / feature / aspect, wherein the free radical initiator is a peroxide initiator or an azo initiator, optionally wherein the free radical initiator is 2,2'-azobis-2,4-dimethylvaleronitrile.

[0518] 74. The method of any preceding or subsequent embodiment / feature / aspect, wherein 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 siloxane, or a combination thereof; and / or

[0519] The organic solvent comprises a mixture of heptane and toluene; a mixture of aliphatic hydrocarbons; bis(2-ethylhexyl) carbonate, optionally mixed with aliphatic and aromatic hydrocarbons; or (2-ethylhexyl) adipate.

[0520] 75. The method of any preceding or following embodiment / feature / aspect, wherein solution (a) comprises no more than 60% wt of hydrophilic vinyl monomer.

[0521] 76. The method of any preceding or subsequent embodiment / feature / aspect, wherein solution (a) comprises at least 2% wt hydrophilic vinyl monomer and no more than 60% wt hydrophilic vinyl monomer, optionally wherein solution (a) comprises at least 5% wt hydrophilic vinyl monomer and no more than 45% wt hydrophilic vinyl monomer.

[0522] 77. The method of any preceding or following embodiment / feature / aspect, wherein solution (a) comprises about 10% wt of the hydrophilic vinyl monomer.

[0523] 78. The method of any preceding or subsequent embodiment / feature / aspect, wherein the content of the initiator in the emulsion is about 0.1%wt to about 1.5%wt, optionally wherein the content of the initiator in the emulsion is about 0.6%wt to about 1.2%wt, further optionally wherein the content of the initiator in the emulsion is about 0.8%wt.

[0524] 79. The method of any preceding or following embodiment / feature / aspect, wherein said polymerizing comprises activating said free radical initiator.

[0525] 80. The method of any preceding or following embodiment / feature / aspect, wherein said monodisperse hydrogel polymer particles are formed according to a method comprising:

[0526] Forming a solution (a): an aqueous solution of at least 2% by weight of a hydrophilic vinyl monomer, the aqueous solution further comprising a chain transfer agent;

[0527] forming a solution (b) of a stabilizer in an organic solvent, wherein the organic solvent is immiscible with water, and wherein at least one of the solution (a) and the solution (b) comprises a free 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; forming a solution (d) of a stabilizer in an organic solvent, wherein the organic solvent is immiscible with water;

[0528] forming a solution (e): an aqueous solution of at least 2%wt of a hydrophilic vinyl monomer, the aqueous solution further comprising a crosslinking agent containing at least two vinyl groups, wherein at least one of the solution (d) and the solution (e) comprises a free radical initiator;

[0529] mixing solutions (d) and (e) to form a water-in-oil emulsion (f) and adding monodisperse polymer particles to the emulsion;

[0530] allowing the monodisperse polymer particles to form swollen polymer particles in the emulsion; and polymerizing the swollen polymer particles to form monodisperse hydrogel polymer particles.

[0531] 81. The method of any preceding or following embodiment / feature / aspect, wherein the monodisperse seed particles have a z-average diameter of 100 nm to 1500 nm, wherein each seed particle comprises a plurality of non-crosslinked oligomers of poly-N,N-dimethylacrylamide.

[0532] 82. The method of any preceding or following embodiment / feature / aspect, wherein the monodisperse seed particles are formed by free radical initiated polymerization of N,N-dimethylacrylamide in an organic solvent in the presence of a stabilizer and a chain transfer agent.

[0533] 83. The method of any preceding or subsequent embodiment / feature / aspect, wherein the monodisperse seed particles are formed by: dissolving N,N-dimethylacrylamide, a stabilizer, a free 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.

[0534] 84. The method of any preceding or subsequent embodiment / feature / aspect, wherein the monodisperse magnetic hydrogel polymer particles formed or the coated monodisperse hydrogel polymer particles formed have a mode diameter of 0.5 μm to 10 μm or 1 μM to 10 μm, optionally a mode diameter of 0.5 μm to 5 μm or 1 μm to 5 μm.

[0535] 85. The method of any preceding or subsequent embodiment / feature / aspect, wherein the coefficient of variation (CV) of the monodisperse magnetic hydrogel polymer particles formed or the coated monodisperse hydrogel polymer particles formed is less than 20%, optionally wherein the CV is less than 15%, further optionally wherein the CV is less than 10%.

[0536] 86. The method of any preceding or subsequent embodiment / feature / aspect, wherein the monodisperse magnetic hydrogel polymer particles formed or the coated monodisperse hydrogel polymer particles formed comprise functional groups; optionally wherein the functional groups are selected from hydroxyl, carboxylic acid (—COOH), primary amine or secondary amine; further optionally wherein the functional groups are selected from carboxylic acid (—COOH), primary amine or secondary amine.

[0537] 87. Monodisperse magnetic hydrogel polymer particles or coated monodisperse hydrogel polymer particles obtainable by the method of any preceding or subsequent embodiment / feature / aspect.

[0538] 88. Use of the monodisperse magnetic hydrogel polymer particles of any preceding or following embodiment / feature / aspect in an assay.

[0539] 89. Use of the monodisperse coated hydrogel polymer particles of any preceding or following embodiment / feature / aspect in an assay.

[0540] 90. Use of the monodisperse hydrogel polymer particles of any preceding or following embodiment / feature / aspect in an assay.

[0541] 91. Use of monodisperse magnetic hydrogel polymer particles of any preceding or following embodiment / feature / aspect in nucleic acid amplification.

[0542] 92. Use of monodisperse coated hydrogel polymer particles of any preceding or following embodiment / feature / aspect in nucleic acid amplification.

[0543] 93. Use of the monodisperse hydrogel polymer particles of any preceding or following embodiment / feature / aspect in nucleic acid amplification.

[0544] 94. The use of any preceding or subsequent embodiment / feature / aspect, wherein the nucleic acid amplification is an emulsion

[0545] PCR.

[0546] Analytical methods

[0547] Molecular weight measurement

[0548] The weight average molecular weight (Mw) of the oligomer in the seed particles can be determined by the measurement 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 using DMF with 0.01M LiBr as an eluent relative to a polystyrene standard. Since these Mw values ​​are calculated relative to a standard of a polymer (polystyrene) different from the standard of the seed particles, the calculated Mw represents a relative value, not an absolute value. Therefore, the measurement result is repeatable, but the actual Mw will not be provided.

[0549] An overview of the GPC method used in the examples provided herein is as follows. The following experimental conditions were used:

[0550] Eluent: DMF with 0.01M LiBr

[0551] Pre-column: PSS GRAM, 10μm, protected ID 8.0mm x 50mm

[0552] Column: PSS GRAM, 10μm, linear M ID 8.0mm x 300mm PSS GRAM, 10μm, linear M ID 8.0mm x 300mm

[0553] Temperature: 70℃

[0554] Pump: PSS SECurity 1260HPLC pump

[0555] Flow rate: 1.0mL / min injection

[0556] System: PSS SECurity 1260 Autosampler

[0557] Injection volume: 50 μL sample

[0558] Concentration: 3.0g / L

[0559] Detector: PSS SECurity 1260 Refractive Index Detector (RID)

[0560] Chromatography data system: PSS WinGPC UniChrom version 8.2

[0561] Polystyrene standards with different molecular weights were measured under the above experimental conditions to obtain a calibration curve. The samples were then run. The Mw of the samples was then calculated based on the PS calibration curve.

[0562] Size and size distribution

[0563] The size distribution of a sample can be measured using disc centrifugation, using the protocol provided by the instrument manufacturer, such as the CPS Disc Centrifugation on a disc centrifuge model DC20000. TM Accurate results require calibration using standards of similar density to the sample being analysed and are therefore only used where suitable polymer standards are available, such as a set of compact polystyrene particle standards for the particles of the present invention, which comprise predominantly polystyrene. In cases where the sample being measured has an unknown density, such as for porous particles, measurements obtained by CPS disc centrifugation will be reproducible but will not provide the actual diameter.

[0564] CPSDisc Centrifugation used in the examples provided herein TM A summary of the method is as follows. One skilled in the art will be able to readily adapt this method, where appropriate, to other samples, for example by selecting appropriate gradients, disk speeds, and standard particles based on the size and porosity of the particles to be analyzed.

[0565] Disc centrifuge analysis was performed on a CPS DC20000 from CPS Instruments with a disc speed of 10000 rpm and a gradient of 8-24 wt% sucrose in 1.5 g / L SDS (aq). The gradient was formed using an Auto Gradient pump from a CPS instrument and the volume of the injected gradient was 16-17 mL. The sample was diluted to about 0.01 wt% in MilliQ-H2O before injection.

[0566] The method used for 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 non-sphericity 1, calibration standard diameter 1.098, calibration standard density 1.6, standard half width 0.2 μm, liquid density 1.06 g / mL, liquid refractive index 1.355.

[0567] The reported sizes are the absorbance peak diameters and the CVs are determined by setting bounds around the main peak.

[0568] Photon correlation spectroscopy (PCS) can be used to obtain the hydrodynamic diameter of the particles in the form of z-means. 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 manual (incorporated herein by reference in its entirety).

[0569] 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 an image of the beads at a suitable magnification (e.g., 100x or more), and analyzing the size of the beads using image analysis software.

[0570] An overview of the optical microscopy methods used in the examples provided herein is as follows. TM A 0.12 mm, 9 mm diameter Secure-Seal was sandwiched between the hybridization chamber gaskets. TM spacer to prepare microscopy samples. The formed cavity was filled with 8 μL of beads diluted in 150 mM NaCl, then briefly centrifuged and attached to 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. Out-of-focus images were collected as background and subtracted from the phase contrast images. The background-subtracted images were then thresholded using the Otsu method to identify individual beads for size analysis. The measured bead sizes were calibrated by fluorescence microscopy using green fluorescent polystyrene beads with standard sizes. A series of Alexa 488-labeled hydrogel beads were then used to calibrate the phase contrast size to the fluorescence size.

[0571] Optical microscopy is the preferred method for measuring particle size because it provides measurements that are independent of the density of the particles.Disk centrifugation 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 lead to artificially high CVs.

[0572] magnetic susceptibility

[0573] The magnetic susceptibility was determined using an MS3 meter, a magnetic susceptibility system from Bartington Instruments equipped with an MS2B dual frequency sensor. The samples for the measurement were prepared by weighing 100.0 ± 20.0 mg of particles diluted in 7.0000 ± 0.1000 g of alumina. Each batch was analyzed in duplicate using the following settings, with mass-specific calibration: measurement period 1 s, number of iterations 1 and number of cycles 100.

[0574] Vibrating Sample Magnetometer (VSM)

[0575] VSM was performed on a LakeShore 7400. 3-10 mg of sample was weighed out and placed in a locked ring gelatin capsule (Torpac). To prevent movement during the measurement, the capsule was fixed with glue and allowed to dry overnight. Hysteresis curves were collected by setting the magnetic field initially to 10000 Oe and then gradually changing to -10000 Oe and finally gradually changing to 10000 Oe.

[0576] Iron content

[0577] Quantitative determination of the iron content of the magnetic particles was performed by Alcontrol Laboratories Norway by dissolving 0.11 g of the particles in 5 mL of aqua regia followed by microwave heating. 2 The samples were diluted to 50 mL and finally diluted 250 times. The iron concentration was then determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis using a Varian 720-ES instrument.

[0578] Example

[0579] Synthesis of seed particles

[0580] Example 1

[0581] 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 were added to a 2L jacketed round bottom flask equipped with a mechanical anchor stirrer and condenser. Once all the materials were 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 heated for 16 hours while stirring at 400 rpm. The resulting dispersion was analyzed by dynamic light scattering to obtain a z-average diameter of 360 nm.

[0582] Example 2

[0583] 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 were added to a 2L jacketed round bottom flask equipped with a mechanical anchor stirrer and condenser. Once all the materials were 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 heated for 16 hours while stirring at 400 rpm. The resulting dispersion was analyzed by dynamic light scattering to obtain a z-average diameter of 970 nm.

[0584] Synthesis of polymer particles from low molecular weight seed particles

[0585] Example 3

[0586] Solution 1 (monomer phase): 7.4 g acrylamide (AAm), 2.5 g N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBAAm) and 14.9 g H 2 O. The mixture was shaken at 61 °C until everything was dissolved.

[0587] Solution 2 (stabilizer): Add 33 g Abil WE09, 36 g mineral oil and 145 g diethylhexyl carbonate (TegosoftDEC) to a 400 mL beaker and mix with a magnetic stir bar until all dissolved.

[0588] Solution 3 (emulsion): 66 g of solution 2 was added to solution 1 and mixed using an Ultraturrax for 1 min. Further emulsification was performed using an UP 200s from Hielscher Ultrasound Technology with sonication set to 0.9 cycles and 40% amplitude. In the next step, solution 3 and 9.5 g of PDMAAm seeds (Example 1) were charged into a 250 mL 3-neck round bottom flask equipped with a condenser, mechanical stirrer and gas inlet and stirred at 100 rpm for 1 hour at 60°C. Finally, the flask was purged with Ar(g) for 15 minutes and then 2.6 g of 2,2'-azobis(2-methylbutyronitrile) (AMBN) dissolved in 2.6 g of toluene was added and heated to 70°C while stirring at 50 rpm for 7 hours.

[0589] Redispersion: 30 g of the crude product was added to a 250 mL centrifuge flask and filled with 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 washed twice with NMP and washed with H 2 O for 3 times and finally dispersed in HO 2 In O.

[0590] Example 4

[0591] Solution 1 (monomer phase): 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 H 2 O. The mixture was shaken at 61 °C until everything was dissolved.

[0592] Solution 2 (stabilizer), solution 3 (emulsion) and redispersion: as described in Example 3.

[0593] Example 5

[0594] Solution 1 (monomer phase): 7.4 g hydroxyethyl acrylamide (HEAAm), 2.5 g N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBAAm) and 14.9 g H 2 O. The mixture was shaken at 61 °C until everything was dissolved.

[0595] Solution 2 (stabilizer), solution 3 (emulsion) and redispersion: as described in Example 3.

[0596] Example 6

[0597] Solution 1 (monomer phase): 7.5 g hydroxymethyl acrylamide (HMAAm), 5.0 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 12.4 g HMAAm were added to a 250 mL Duran flask. 2 O. Stir the mixture until everything is dissolved.

[0598] Solution 2 (stabilizer): Add 35 g Abil WE09, 38 g mineral oil and 152 g diethylhexyl carbonate (TegosoftDEC) to a 400 mL beaker and mix with a magnetic stir bar until all dissolved.

[0599] Solution 3 (emulsion): 71 g of solution 2 was added to solution 1 and then mixed using an Ultraturrax for 1 minute. Further emulsification was performed using an UP 200s from Hielscher Ultrasound Technology with a cycle 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 3-neck round bottom flask equipped with a condenser, mechanical stirrer and gas inlet and stirred at 100 rpm for 1 hour at 19°C. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C while stirring at 50 rpm for 7 hours.

[0600] Redispersion: 50 g of the crude product was added to a 250 mL centrifuge flask and filled with 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 washed twice with NMP and washed with H 2 O for 5 times and finally dispersed in HO 2 In O.

[0601] Example 7

[0602] Solution 1 (monomer phase): 7.5 g hydroxymethyl acrylamide (HMAAm), 2.5 g piperazine diacrylamide and 15.0 g HMAAm were added to a 250 mL Duran flask. 2 O. Stir the mixture until everything is dissolved.

[0603] Solution 2 (stabilizer), solution 3 (emulsion) and redispersion: as described in Example 6.

[0604] Example 8

[0605] Solution 1 (monomer phase): 7.5 g hydroxymethyl acrylamide (HMAAm), 5.0 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 12.4 g HMAAm were added to a 250 mL Duran flask. 2 O. Stir the mixture until everything is dissolved.

[0606] Solution 2 (stabilizer): Add 24 g Abil WE09, 26 g mineral oil and 104 g diethylhexyl carbonate (TegosoftDEC) to a 250 mL beaker and mix with a magnetic stir bar until all dissolved.

[0607] Solution 3 (emulsion): as described in Example 6.

[0608] Redispersion: 25 g of the crude product was added to a 250 mL centrifuge flask and filled with 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 washed twice with NMP and washed with H 2 O for 4 times and finally dispersed in HO 2 In O.

[0609] Example 9

[0610] Solution 1 (monomer phase): To a 500 mL Duran flask was added 36.8 g hydroxymethyl acrylamide (HMAAm), 2.6 g acrylic acid, 39.4 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 118.3 g H 2 O. The mixture was shaken until all dissolved.

[0611] Solution 2 (stabilizer): 7.6 g 2,2'-azobis(2-methylbutyronitrile) (AMBN), 95.6 g Abil WE09, 104.3 g mineral oil, and 417.2 g diethylhexyl carbonate (Tegosoft DEC) were added to a 1 L beaker and mixed with a magnetic stir bar until all dissolved.

[0612] Solution 3 (emulsion): 520 g of solution 2 was added to solution 1 and then mixed using an Ultraturrax for 1 minute. Further emulsification was performed using an UP 200s from Hielscher Ultrasound Technology with a cycle 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 added to a 1 L jacketed cylindrical reactor equipped with a condenser, mechanical stirrer and gas inlet and stirred at 100 rpm for 80 minutes at 22°C. Finally, the flask was purged with Ar(g) for 30 minutes and then heated to 70°C while stirring at 55 rpm for 20 hours.

[0613] Redispersion and analysis: The crude product was divided into 2 × 1 L centrifuge flasks and centrifuged at 3600 rpm for 15 min. The supernatant was removed and the particles were redispersed in isopropanol. The redispersed particles were then divided into a total of 4 × 1 L centrifuge bottles, washed 4 times with isopropanol, and washed with H 2 O for 8 times and finally dispersed in HO 2 The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter relative to the standard was determined to be 1.62 μm, with a CV of 16.1.

[0614] Example 10

[0615] Solution 1 (monomer phase): 3.7 g acrylic acid, 3.7 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 17.3 g H 2 O. Shake the mixture until all dissolved.

[0616] Solution 2 (stabilizer): 2.6 g 2,2'-azobis(2-methylbutyronitrile) (AMBN), 33 g Abil WE09, 36 g mineral oil, and 144 g diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stir bar until all dissolved.

[0617] Solution 3 (emulsion): 66 g of solution 2 was added to solution 1 and then mixed using an Ultraturrax for 1 minute. Further emulsification was performed using an UP 200s from Hielscher Ultrasound Technology with a cycle 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 3-neck round bottom flask equipped with a condenser, mechanical stirrer and gas inlet and stirred at 100 rpm for 1 hour at 20°C. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C while stirring at 62 rpm for 7 hours.

[0618] Redispersion and analysis: 77 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 min. The supernatant was removed and the particles were redispersed in isopropanol and washed three times in isopropanol and then washed. 2 HO, transfer the redispersed particles to a 1 L centrifuge flask and rinse with HO. 2 The batch was then transferred to 3 x 250 mL centrifuge flasks and finally redispersed in HO. 2 The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter was determined to be 0.37 μm with a CV of 5.4%.

[0619] Embodiment 11

[0620] Solution 1 (monomer phase): 1.9 g hydroxymethyl acrylamide (HMAAm), 1.9 g acrylic acid, 3.7 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 17.3 g HMAAm were added to a 250 mL Duran flask. 2 O. Shake the mixture until all dissolved.

[0621] Solution 2 (stabilizer), solution 3 (emulsion): as in Example 10.

[0622] Redispersion and analysis: As described in Example 10, but using 78 g of crude product. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter relative to the standard value was 0.43 μm, CV = 5.6%.

[0623] Example 12

[0624] Solution 1 (monomer phase): 2.8 g hydroxymethyl acrylamide (HMAAm), 0.9 g acrylic acid, 3.7 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 17.3 g HMAAm were added to a 250 mL Duran flask. 2 O. Shake the mixture until all dissolved.

[0625] Solution 2 (stabilizer), solution 3 (emulsion): as in Example 10.

[0626] Redispersion and analysis: As described in Example 10, but using 78 g of crude product. The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter correlated with the standard value of 0.43 μm, CV = 5.2%.

[0627] Embodiment 13

[0628] Solution 1 (monomer phase): 3.7 g hydroxymethyl acrylamide (HMAAm), 3.7 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 17.3 g HMAAm were added to a 250 mL Duran flask. 2 O. Shake the mixture until all dissolved.

[0629] Solution 2 (stabilizer): 2.6 g 2,2'-azobis(2-methylbutyronitrile) (AMBN), 33 g Abil WE09, 36 g mineral oil, and 144 g diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stir bar until all dissolved.

[0630] Solution 3 (emulsion): 66 g of solution 2 was added to solution 1 and then mixed using an Ultraturrax for 1 minute. Further emulsification was performed using an UP 200s from Hielscher Ultrasound Technology with a cycle 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 3-neck round bottom flask equipped with a condenser, mechanical stirrer and gas inlet and stirred at 100 rpm for 1 hour at 20°C. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C while stirring at 62 rpm for 7 hours.

[0631] Redispersion and analysis: 78 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 min. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 min. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H 2 O and divided into a total of 3 × 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed with HO. 2 O for 3 times and finally dispersed in HO 2 The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter relative to the standard was determined to be 0.43 μm, with a CV of 5.4%.

[0632] Embodiment 14

[0633] Solution 1 (monomer phase): 3.4 g hydroxyethyl acrylamide (HEAAm), 0.3 g acrylic acid (AA), 3.7 g N,N'-((ethane-1,2-diylbis)(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 17.3 g H 2 O. Shake the mixture until all dissolved.

[0634] Solution 2 (stabilizer), solution 3 (emulsion): as in Example 13.

[0635] Redispersion and analysis: 77 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 min. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 min. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H 2 O and divided into a total of 3 × 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed with HO. 2 O for 3 times and finally dispersed in HO 2 The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter relative to the standard was determined to be 0.41 μm, with a CV of 6.1%.

[0636] Embodiment 15

[0637] Solution 1 (monomer phase): 3.4 g N-[tris(hydroxymethyl)methyl]acrylamide (THMAAm), 0.3 g acrylic acid (AA), 3.7 g N,N'-((ethane-1,2-diylbis)(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 17.3 g H 2 O. Shake the mixture until all dissolved.

[0638] Solution 2 (stabilizer), solution 3 (emulsion): as in Example 13.

[0639] Redispersion and analysis: 78 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 min. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 min. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H 2 O and divided into a total of 3 × 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed with HO. 2 O for 3 times and then dispersed in HO. 2 The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter relative to the standard was determined to be 0.42 μm, with a CV of 5.7%.

[0640] Example 16

[0641] Solution 1 (monomer phase): 3.2 g hydroxymethyl acrylamide (HMAAm), 0.5 g fumaric acid, 3.7 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 17.3 g HMAAm were added to a 250 mL Duran flask. 2 O. Heat the mixture to 40 °C and mix with a magnetic stir bar until everything is dissolved.

[0642] Solution 2 (stabilizer): 2.6 g 2,2'-azobis(2-methylbutyronitrile) (AMBN), 33 g Abil WE09, 35 g mineral oil, and 140 g diethylhexyl carbonate (Tegosoft DEC) were added to a 250 mL beaker and mixed with a magnetic stir bar until all dissolved.

[0643] Solution 3 (emulsion): 66 g of solution 2 was added to solution 1 and then mixed using an Ultraturrax for 1 minute. Further emulsification was performed using an UP 200s from Hielscher Ultrasound Technology with a cycle 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 3-neck round bottom flask equipped with a condenser, mechanical stirrer and gas inlet and stirred at 100 rpm for 1 hour at 20°C. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C while stirring at 65 rpm for 7 hours.

[0644] Redispersion and analysis: 77 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 min. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 min. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H 2 O and divided into a total of 3 × 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed with HO. 2 O for 3 times and finally dispersed in HO 2 The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter relative to the standard was determined to be 0.44 μm, with a CV of 5.5%.

[0645] Embodiment 17

[0646] Solution 1 (monomer phase): 3.1 g hydroxymethyl acrylamide (HMAAm), 0.6 g itaconic acid, 3.7 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 17.3 g HMAAm were added to a 250 mL Duran flask. 2 O. Shake the mixture until all dissolved.

[0647] Solution 2 (stabilizer), solution 3 (emulsion): as in Example 16.

[0648] Redispersion and analysis: 77 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 min. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 min. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H 2 O and divided into a total of 3 × 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed with HO. 2 O for 3 times and finally dispersed in HO 2 The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter relative to the standard was determined to be 0.47 μm, with a CV of 5.4%.

[0649] Embodiment 18

[0650] Solution 1 (monomer phase): 3.2 g hydroxymethyl acrylamide (HMAAm), 0.5 g vinylphosphonic acid, 3.7 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 17.3 g HMAAm were added to a 250 mL Duran flask. 2 O. Shake the mixture until all dissolved.

[0651] Solution 2 (stabilizer), solution 3 (emulsion): as in Example 16.

[0652] Redispersion and analysis: 77 g of crude product was added to a 250 mL centrifuge flask and centrifuged at 5000 rpm for 20 min. The supernatant was removed and the particles were redispersed in isopropanol and centrifuged at 3000 rpm for 15 min. The redispersed particles were washed three times with isopropanol. The particles were redispersed in H 2 O and divided into a total of 3 × 250 mL centrifuge bottles and centrifuged at 5000 rpm for 20 minutes. The redispersed particles were washed with HO. 2 O for 3 times and then dispersed in HO. 2 The product was analyzed using an optical microscope and a disc centrifuge. The relative disc centrifuge diameter relative to the standard was determined to be 0.42 μm, with a CV of 5.3%.

[0653] Embodiment 19

[0654] Solution 1 (monomer phase): Add 0.6 g of hydroxymethyl acrylamide to a 250 mL Duran flask.

[0655] (HMAAm), 0.7 g acrylamidopropionic acid (AAPA), 1.2 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 22.2 g H 2 O. Shake the mixture until all dissolved.

[0656] Solution 2 (stabilizer): 2.6 g 2,2'-azobis(2-methylbutyronitrile) (AMBN), 32 g Abil WE09, 35 g mineral oil, and 140 g diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stirring bar until all dissolved.

[0657] Solution 3 (emulsion): 65 g of solution 2 was added to solution 1 and then mixed using an Ultraturrax for 1 minute. Further emulsification was performed using an UP 200s from Hielscher Ultrasound Technology with a cycle 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 3-neck round bottom flask equipped with a condenser, mechanical stirrer and gas inlet and stirred at 100 rpm for 1 hour at 20°C. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C while stirring at 57 rpm for 7 hours.

[0658] Redispersion and analysis: Add 71 g crude product, 500 mL H2 O and 300 mL 2-BuOH. Place the flask on a shaker and centrifuge. Remove the 2-BuOH and add 150 mL 2-BuOH. Resuspend the pellet in H 2 Repeat this process once more before adding 4 × 250 mL centrifuge bottles and centrifuging. 2 O and then washed three times, and finally dispersed in H 2 O. 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, giving a mode diameter of 0.85 μm.

[0659] Embodiment 20

[0660] Solution 1 (monomer phase): 1.8 g hydroxymethyl acrylamide (HMAAm), 0.7 g acrylamidopropionic acid (AAPA), 2.5 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 19.7 g HMAAm were added to a 250 mL Duran flask. 2 O. Shake the mixture until all dissolved.

[0661] Solution 2 (stabilizer), solution 3 (emulsion): as in Example 19.

[0662] Redispersion and analysis: Add 70 g crude product, 500 mL H 2 O and 300 mL 2-BuOH. Place the flask on a shaker and centrifuge. Remove the 2-BuOH and add 150 mL 2-BuOH. Repeat 2 more times, then divide the particle suspension into a total of 6 x 250 mL centrifuge bottles and centrifuge. Redisperse the particles in H 2 O and centrifuge. The redispersed particles were 2 O for 5 times and finally dispersed in HO 2 O. Bright field microscopy was performed by diluting the redispersed product to a concentration of 0.01 wt% in 150 mM NaCl solution and collecting image statistics from 25 images, yielding a mode diameter of 0.83 μm.

[0663] Embodiment 21

[0664] Solution 1 (monomer phase): 4.2 g hydroxymethyl acrylamide (HMAAm), 0.7 g acrylamidopropionic acid (AAPA), 4.9 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 14.8 g HMAAm were added to a 250 mL Duran flask.2 O. Shake the mixture until all dissolved.

[0665] Solution 2 (stabilizer): 2.6 g 2,2'-azobis(2-methylbutyronitrile) (AMBN), 33 g Abil WE09, 35 g mineral oil, and 140 g diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stirring bar until all dissolved.

[0666] Solution 3 (emulsion): 66 g of solution 2 was added to solution 1 and then mixed using an Ultraturrax for 1 minute. Further emulsification was performed using an UP 200s from Hielscher Ultrasound Technology with a cycle 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 3-neck round bottom flask equipped with a condenser, mechanical stirrer and gas inlet and stirred at 100 rpm for 1 hour at 20°C. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C while stirring at 58 rpm for 7 hours.

[0667] Redispersion and analysis: Add 71 g crude product, 500 mL H 2 O and 300 mL 2-BuOH. Place the flask on a shaker and centrifuge. Remove the 2-BuOH and add 150 mL 2-BuOH. Repeat 2 more times, then divide the particle suspension into a total of 6 x 250 mL centrifuge bottles and centrifuge. Redisperse the particles in H 2 O and centrifuge. The redispersed particles were 2 O for 5 times and finally dispersed in HO 2 O. 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, giving a mode diameter of 0.82 μm.

[0668] Embodiment 22

[0669] Solution 1 (monomer phase): 5.5 g hydroxymethyl acrylamide (HMAAm), 0.7 g acrylamidopropionic acid (AAPA), 6.2 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 12.3 g HMAAm were added to a 250 mL Duran flask. 2 O. Shake the mixture until all dissolved.

[0670] Solution 2 (stabilizer), solution 3 (emulsion): as in Example 21.

[0671] Redispersion and analysis: Add 68 g crude product, 500 mL H 2 O and 300 mL 2-BuOH. Place the flask on a shaker and centrifuge. Remove the 2-BuOH and add 150 mL 2-BuOH. Repeat 2 more times, then divide the particle suspension into a total of 6 x 250 mL centrifuge bottles and centrifuge. Redisperse the particles in H 2 O and centrifuge. The redispersed particles were 2 O for 5 times and finally dispersed in HO 2 O. 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, giving a mode diameter of 0.86 μm.

[0672] Embodiment 23

[0673] Solution 1 (monomer phase): 3.1 g hydroxymethyl acrylamide (HMAAm), 0.7 g acrylamidopropionic acid (AAPA), 3.7 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 17.3 g HMAAm were added to a 250 mL Duran flask. 2 O. Shake the mixture until all dissolved.

[0674] Solution 2 (stabilizer): 2.6 g 2,2'-azobis(2-methylbutyronitrile) (AMBN), 32 g Abil WE09, 35 g mineral oil, and 140 g diethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker and mixed with a magnetic stirring bar until all dissolved.

[0675] Solution 3 (emulsion): 66 g of solution 2 was added to solution 1 and then mixed using an Ultraturrax for 1 minute. Further emulsification was performed using an UP 200s from Hielscher Ultrasound Technology with a cycle 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 3-neck round bottom flask equipped with a condenser, mechanical stirrer and gas inlet and stirred at 100 rpm for 1 hour at 20°C. Finally, the flask was purged with Ar(g) for 15 minutes and then heated to 70°C while stirring at 58 rpm for 7 hours.

[0676] Redispersion and analysis: Add 70 g crude product, 500 mL H 2O and 300 mL 2-BuOH. Place the flask on a shaker and centrifuge. Remove the 2-BuOH and add 150 mL 2-BuOH. Repeat 2 more times, then divide the particle suspension into a total of 6 x 250 mL centrifuge bottles and centrifuge. Redisperse the particles in H 2 O and centrifuge. The redispersed particles were 2 O for 5 times and finally dispersed in HO 2 O. 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, giving a mode diameter of 0.84 μm.

[0677] Embodiment 24

[0678] Solution 1 (monomer phase): 3.1 g hydroxymethyl acrylamide (HMAAm), 0.7 g acrylamidopropionic acid (AAPA), 3.7 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 17.3 g HMAAm were added to a 250 mL Duran flask. 2 O. Shake the mixture until all dissolved.

[0679] Solution 2 (stabilizer), solution 3 (emulsion): as in Example 23.

[0680] Redispersion and analysis: Add 71 g crude product, 500 mL H 2 O and 300 mL 2-BuOH. Place the flask on a shaker and centrifuge. Remove the 2-BuOH and add 150 mL 2-BuOH. Repeat 2 more times, then divide the particle suspension into a total of 6 x 250 mL centrifuge bottles and centrifuge. Redisperse the particles in H 2 O and centrifuge. The redispersed particles were 2 O for 5 times and finally dispersed in HO 2 O. Bright field microscopy was performed by diluting the redispersed product to a concentration of 0.01 wt% in 150 mM NaCl solution and collecting image statistics from 25 images, giving a mode diameter of 0.85 μm.

[0681] Embodiment 25

[0682] Solution 1 (monomer phase): 28.6 g hydroxymethyl acrylamide (HMAAm), 0.95 g acrylamidopropionic acid (AAPA), 19.7 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 29.4 g HMAAm were added to a 500 mL Duran flask. 2O. Shake the mixture until all dissolved.

[0683] Solution 2 (stabilizer): 3.8 g 2,2'-azobis(2-methylbutyronitrile) (AMBN), 48 g Abil WE09, 53 g mineral oil, and 210 g diethylhexyl carbonate (Tegosoft DEC) were added to a 600 mL beaker and mixed with a magnetic stirring bar until all dissolved.

[0684] Solution 3 (emulsion): 262 g of solution 2 was added to solution 1 and then mixed using an Ultraturrax for 1 minute. Further emulsification was performed using an UP 200s from Hielscher Ultrasound Technology with a cycle 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 added to 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. while stirring at 64 rpm for 7 hours.

[0685] Redispersion and analysis: The crude product was divided into 2 × 250 mL centrifuge flasks and centrifuged at 5000 rpm for 15 min and 7000 rpm for 10 min. The supernatant was removed and the particles were redispersed in isopropanol. The redispersed particles were then divided into a total of 6 × 250 mL centrifuge bottles, washed 4 times with isopropanol and washed with H 2 O and then dispersed in HO for 6 times. 2 O. The particles were then filtered through three different filter fabrics: SefarNitex 03-64 / 32, SefarNitex 03-30 / 18 and SefarNitex03-1 / 1. The products were 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 collecting image statistics from 18 images, giving a mode diameter of 0.91 μm. The relative disc centrifuge diameter using CPS method 3 was determined to be 0.54 μm, CV = 6.2%.

[0686] Embodiment 26

[0687] Solution 1 (monomer phase): 27.0 g hydroxymethyl acrylamide (HMAAm), 2.6 g acrylamidopropionic acid (AAPA), 19.7 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 49.3 g HMAAm were added to a 500 mL Duran flask. 2O. Shake the mixture until all dissolved.

[0688] Solution 2 (stabilizer), Solution 3 (emulsion): as described in Example 25.

[0689] Redispersion and analysis: The crude product was divided into 2 × 250 mL centrifuge flasks and centrifuged at 5000 rpm for 25 min. The supernatant was removed and the particles were redispersed in isopropanol. The redispersed particles were then divided into a total of 6 × 250 mL centrifuge bottles, washed 4 times with isopropanol, and washed with H 2 O for 5 times and finally dispersed in HO 2 O. The particles were then filtered through two different filter fabrics: SefarNitex 03-30 / 18 and SefarNitex 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, giving a mode diameter of 0.90 μm. The relative disc centrifuge diameter using CPS method 3 was determined to be 0.53 μm, CV = 5.2%.

[0690] Embodiment 27

[0691] Solution 1 (monomer phase): 24.8 g hydroxymethyl acrylamide (HMAAm), 5.0 g acrylamidopropionic acid (AAPA), 19.9 g N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide (EGBEAAm) and 49.7 g HMAAm were added to a 500 mL Duran flask. 2 O. Shake the mixture until all dissolved.

[0692] Solution 2 (stabilizer): 3.8 g 2,2'-azobis(2-methylbutyronitrile) (AMBN), 52 g Abil WE09, 57 g mineral oil, and 228 g diethylhexyl carbonate (Tegosoft DEC) were added to a 1000 mL beaker and mixed with a magnetic stirring bar until all dissolved.

[0693] Solution 3 (emulsion): 284 g of solution 2 was added to solution 1 and then mixed for 1 minute using an Ultraturrax. Further emulsification was performed using an UP 200s from Hielscher Ultrasound Technology with a cycle 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 added to 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. while stirring at 62 rpm for 7 hours.

[0694] Redispersion and analysis: The crude product was divided into 2 × 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 6 × 250 mL centrifuge bottles, washed 4 times with isopropanol, and washed with H 2 O and then dispersed in HO for 6 times. 2 O. The particles were then filtered through two different filter fabrics: 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 collecting image statistics from 30 images, giving a mode diameter of 1.0 μm. The relative disc centrifuge diameter using CPS method 3 was determined to be 0.57 μm, CV = 6.3%.

[0695] Magnetization of polymer particles

[0696] Embodiment 28

[0697] Magnetization: Add 9.8 g of H 2 O particle suspension (Example 3, dry content 3.1%) and purged with Ar(g) for 20 min, then 0.18 g FeCl 2 x 4H 2 O and 0.20 g FeCl 3 x6H 2 The mixture was stirred at 250 rpm for 10 min and 1.24 g NH 3 (25%) and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0698] Workup and analysis: The batch was added to a 250 mL centrifuge flask and washed with H 2O and washed 12 times. 1 mL batch sample was added to a 2 mL Eppendorf tube and placed in the DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0699] Embodiment 29

[0700] Magnetization: Add 7.5 g of H 2 O particle suspension (Example 4, dry content 4.0%) and 2.4 mL H 2 O and purged with Ar(g) for 20 min, then 0.18 g FeCl 2 x 4H 2 O and 0.20 g FeCl 3 x 6H 2 The mixture was stirred at 250 rpm for 10 min and 1.24 g NH 3 (25%) and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0701] Workup and analysis: The batch was added to a 250 mL centrifuge flask and washed with H 2 O and washed 12 times. 1 mL batch sample was added to a 2 mL Eppendorf tube and placed in the DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0702] Embodiment 30

[0703] Magnetization: Add 6.0 g of H 2 O particle suspension (Example 5, dry content 5.0%) and 3.8 mL H 2 O and purged with Ar(g) for 20 min, then 0.18 g FeCl 2 x 4H 2 O and 0.20 g FeCl 3 x 6H 2 The mixture was stirred at 250 rpm for 10 min and 1.24 g NH 3 (25%) and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0704] Workup and analysis: The batch was added to a 250 mL centrifuge flask and washed with H 2 O and washed 12 times. 1 mL batch sample was added to a 2 mL Eppendorf tube and placed in the DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0705] Embodiment 31

[0706] Magnetization: Add 9.7 g of H 2 O particle suspension (Example 6, dry content 3.1%), 0.18 g FeCl 2 x 4H 2 O and 0.20 g FeCl 3 x 6H 2 The mixture was stirred at 250 rpm for 5 min and then purged with Ar(g) for 25 min. Finally, 1.24 mL of NH 3 (25%) and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0707] Workup and analysis: The batch was added to a 250 mL centrifuge flask and washed with H 2 O and washed 7 times. 1.5 mL batch sample was added to a 1.5 mL Eppendorf tube and placed in DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0708] Embodiment 32

[0709] Magnetization: Add 9.5 g of H 2 O particle suspension (Example 7, dry content 3.1%), 0.18 g FeCl 2 x 4H 2 O and 0.20 g FeCl 3 x 6H 2 The mixture was stirred at 250 rpm for 5 min and then purged with Ar(g) for 25 min. Finally, 1.24 mL of NH 3 (25%) and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0710] Workup and analysis: The batch was added to a 250 mL centrifuge flask and washed with H 2 O and washed 7 times. 1.5 mL batch sample was added to a 1.5 mL Eppendorf tube and placed in DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0711] Embodiment 33

[0712] Magnetization: Add 9.1 g of H 2 O particle suspension (Example 8, dry content 3.1%), 0.18 g FeCl 2x 4H 2 O and 0.20 g FeCl 3 x 6H 2 The mixture was stirred at 250 rpm for 5 minutes and then purged with Ar(g) for 25 minutes. Finally, 1.24 mL of NH 3 (25%) and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0713] Workup and analysis: The batch was added to a 250 mL centrifuge flask and washed with H 2 O and washed 7 times. 1.5 mL batch sample was added to a 1.5 mL Eppendorf tube and placed in DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0714] Embodiment 34

[0715] Magnetization: Add 72 g of H 2 O particle suspension (Example 25, dry content 5.6%), 2.4 g FeCl 2 x 4H 2 O and 2.7 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 15 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0716] Workup and analysis: The batch was divided into 2 × 250 mL centrifuge flasks and washed with H 2 O was washed 15 times. 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, the saturation magnetization of 10000Oe is 16.0Am 2 / kg.

[0717] Embodiment 35

[0718] Magnetization: Add 72 g of H 2 O particle suspension (Example 25, dry content 5.6%), 4.8 g FeCl 2 x 4H 2 O and 5.4 g FeCl 3 x 6H 2The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 30 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0719] Workup and analysis: The batch was divided into 2 × 250 mL centrifuge flasks and washed with H 2 O was washed 30 times. 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, the saturation magnetization of 10000Oe is 21.5Am 2 / kg.

[0720] Embodiment 36

[0721] Magnetization: Add 72 g of H 2 O particle suspension (Example 25, dry content 5.6%), 7.3 g FeCl 2 x 4H 2 O and 8.2 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 45 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0722] Workup and analysis: The batch was divided into 2 × 250 mL centrifuge flasks and washed with H 2 O was washed 45 times. 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, the saturation magnetization of 10000Oe is 30.5Am 2 / kg.

[0723] Embodiment 37

[0724] Magnetization: Add 71 g of H 2 O particle suspension (Example 26, dry content 5.6%), 7.3 g FeCl 2 x 4H 2 O and 8.2 g FeCl 3 x 6H 2The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 45 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0725] Processing and analysis: The batch was divided into 2 x 250 mL centrifuge bottles and washed a total of 65 times (with H 2 O for 5 times, then washed once with 1 M acetic acid and washed with H 2 O was washed 59 times). 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, the saturation magnetization of 10000Oe is 20.6Am 2 / kg.

[0726] Embodiment 38

[0727] Magnetization: Add 39 g of H 2 O particle suspension (Example 27, dry content 5.1%), 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0728] Workup and analysis: The batch was divided into 2 x 250 mL centrifuge bottles and washed 65 times (with H 2 O for 5 times, then washed once with 1 M acetic acid and washed with H 2 O was washed 59 times). The magnetic susceptibility is 72×10 -5 m 3 / kg, the saturation magnetization of 10000Oe is 24.7Am 2 / kg.

[0729] Embodiment 39

[0730] Magnetization: Add 35 g of H 2 O particle suspension (Example 19, dry content 1.7%), 33 g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0731] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 The cells were washed 9 times with 0.01 M citrate buffer (pH 5.3), twice with HO, and washed 2 times with 1% HCl. 2 O was washed 28 times more. The Fe content in the dried pellets was determined to be 478 mg / g and the magnetic susceptibility was 72×10 -5 m 3 / kg, the saturation magnetization of 10000Oe is 37.8Am 2 / kg.

[0732] Embodiment 40

[0733] Magnetization: Add 42 g of H 2 O particle suspension (Example 20, dry content 1.8%), 1.5 g H 2 O, 2.3 g FeCl 2 x 4H 2 O and 2.6 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 14 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0734] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 The cells were washed 9 times with 0.01 M citrate buffer (pH 5.3), twice with HO, and washed 2 times with 1% HCl. 2 O was washed 27 times more. The Fe content in the dried pellets was determined to be 363 mg / g and the magnetic susceptibility was 67×10 -5 m 3 / kg, the saturation magnetization of 10000Oe is 31.0Am 2 / kg.

[0735] Embodiment 41

[0736] Magnetization: Add 58 g of H 2O particle suspension (Example 23, dry content 4.1%), 9.4 g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0737] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 The cells were washed 9 times with 0.01 M citrate buffer (pH 5.3), twice with HO, and washed 2 times with 1% HCl. 2 O was washed 27 times more. The Fe content in the dried pellets was determined to be 233 mg / g and the magnetic susceptibility was 53×10 -5 m 3 / kg, the saturation magnetization of 10000Oe is 20.2Am 2 / kg.

[0738] Embodiment 42

[0739] Magnetization: Add 68 g of H 2 O particle suspension (Example 21, dry content 3.5%), 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0740] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 The cells were washed 9 times with 0.01 M citrate buffer (pH 5.3), twice with HO, and washed 2 times with 1% HCl. 2 O was washed 38 times more. The Fe content in the dried pellets was determined to be 177 mg / g and the magnetic susceptibility was 42×10 -5 m 3 / kg, the saturation magnetization of 10000Oe is 15.0Am 2 / kg.

[0741] Embodiment 43

[0742] Magnetization: Add 58 g of H 2 O particle suspension (Example 22, dry content 5.1%), 9.0 g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0743] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 The cells were washed 9 times with 0.01 M citrate buffer (pH 5.3), twice with HO, and washed 2 times with 1% HCl. 2 O was washed 38 times more. The Fe content in the dried pellets was determined to be 205 mg / g and the magnetic susceptibility was 52×10 -5 m 3 / kg, the saturation magnetization of 10000Oe is 16.6Am 2 / kg.

[0744] Embodiment 44

[0745] Magnetization: Add 51 g of H 2 O particle suspension (Example 24, dry content 3.5%), 17g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0746] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 The cells were washed 9 times with 0.01 M citrate buffer (pH 5.3), twice with HO, and washed 2 times with 1% HCl. 2 O was washed 27 times more. The Fe content in the dried pellets was determined to be 288 mg / g and the magnetic susceptibility was 68 × 10 -5 m3 / kg, the saturation magnetization of 10000Oe is 23.8Am 2 / kg.

[0747] Embodiment 45

[0748] Magnetization: 252 g of H 2 O particle suspension (Example 9, 5.6% dry content), 18.4 g FeCl 2 x 4H 2 O and 20.5 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 112 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0749] Workup and analysis: The batch was divided into 2 × 1 L centrifuge flasks and washed with H 2 O and washed 33 times.

[0750] Embodiment 46

[0751] Magnetization: Add 44 g of H 2 O particle suspension (Example 10, 4.5% dry content), 31 g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0752] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 O and washed 21 times. 1.5 mL batch sample was added to a 1.5 mL Eppendorf tube and placed in the DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0753] Embodiment 47

[0754] Magnetization: Add 37 g of H 2O particle suspension (Example 11, 5.6% dry content), 39g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0755] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 O and wash 30 times.

[0756] Embodiment 48

[0757] Magnetization: Add 34 g of H 2 O particle suspension (Example 12, 5.6% dry content), 41g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0758] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 O and washed 30 times. 1.5 mL batch sample was added to a 1.5 mL Eppendorf tube and placed in the DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0759] Embodiment 49

[0760] Magnetization: Add 42 g of H 2 O particle suspension (Example 13, 4.7% dry content), 33g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x6H 2The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0761] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 O and washed 34 times. 1.5 mL batch sample was added to a 1.5 mL Eppendorf tube and placed in the DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0762] Embodiment 50

[0763] Magnetization: Add 44 g of H 2 O particle suspension (Example 14, 4.5% dry content), 31 g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0764] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 O and washed 40 times. 1.5 mL batch sample was added to a 1.5 mL Eppendorf tube and placed in the DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0765] Embodiment 51

[0766] Magnetization: Add 44 g of H 2 O particle suspension (Example 15, 4.5% dry content), 31g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x 6H 2The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0767] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 O and washed 40 times. 1.5 mL batch sample was added to a 1.5 mL Eppendorf tube and placed in the DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0768] Embodiment 52

[0769] Magnetization: Add 35 g of H 2 O particle suspension (Example 16, 5.65% dry content), 40 g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0770] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 O and washed 27 times. 1.5 mL batch sample was added to a 1.5 mL Eppendorf tube and placed in the DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0771] Embodiment 53

[0772] Magnetization: Add 45 g of H 2 O particle suspension (Example 17, 4.47% dry content), 30 g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x 6H 2The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0773] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 O and washed 27 times. 1.5 mL batch sample was added to a 1.5 mL Eppendorf tube and placed in the DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0774] Embodiment 54

[0775] Magnetization: Add 49 g of H 2 O particle suspension (Example 18, 4.12% dry content), 26g H 2 O, 3.7 g FeCl 2 x 4H 2 O and 4.1 g FeCl 3 x 6H 2 The mixture was stirred at 150 rpm for 10 minutes and then purged with Ar(g) for 30 minutes. Finally, a stirring speed of 250 rpm and 22 g NH 3 (25%), and the reaction was allowed to proceed under Ar(g) for 1 hour.

[0776] Workup and analysis: The batch was transferred to a 250 mL centrifuge flask and washed with H 2 O and washed 27 times. 1.5 mL batch sample was added to a 1.5 mL Eppendorf tube and placed in the DynaMag TM -2 magnet, and the sample completely migrated to the magnet within 1 minute.

[0777] Epoxy coated polymer particles

[0778] Example 55 - Tosyl Activation of Polyacrylamide Particles

[0779] To a solution of 13w% of the particles of Example 9 in acetone (46.7 mL) was added 0.7 g of pyridine and 1.4 g of p-toluenesulfonyl chloride under stirring. The reaction was then further stirred at room temperature overnight. Upon completion, the supernatant was removed and post-processed by washing with 4×250 mL of acetone.

[0780] Example 56 - Amination of Tosylated Polyacrylamide Particles

[0781] 5 g of the tosylated particles of Example 55 were added to acetone to remove the solvent and then resuspended in a total of 75 g of 2,2'-(ethylenedioxy)bis(ethylamine) (EDEA). The mixture was stirred at 72°C for 4 hours. The reaction product was washed with 3×300 mL H 2 O, 3×300 mL 10 mM acetic acid (aq), 3×300 mL H 2 O and finally washed with 4 x 300 mL N-methylpyrrolidone.

[0782] Example 57 - Epoxy Coating of Aminated Polyacrylamide Particles

[0783] 235 g of solvent were removed from 4.1 g of aminated particles of Example 56 in 265.5 g of NMP, and 8.5 g of 1,4-butanediol diglycidyl ether, 3.3 g of glycidol, and 6.1 g of glycidyl methacrylate were added to the residue under 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, 2×200 mL of methanol, 2×200 mL of 30:70 methanol:isopropanol, and 4×200 mL of isopropanol.

[0784] Example 58 - Acrylic acid grafted onto polyacrylamide particles

[0785] 52 mg of 2,2'-azobis(2-methylpropionitrile) (AIBN) was dissolved in 20 g of isopropanol by stirring and added to a solution of 4.3 g of particles of Example 57 in 47 g of isopropanol. 1.1 mL of acrylic acid was introduced and the mixture was heated to 72° C. under stirring and the reaction was continued for 20 hours after the desired temperature was reached. Subsequently, it was allowed to cool for 20 minutes and washed with 2×300 mL of isopropanol and 3×200 mL of methanol.

[0786] Example 59 - One-step epoxy coating of polyacrylamide particles

[0787] To a 10 wt% solution of Example 35 particles 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. Thereafter, 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 4×5 mL of acetone.

[0788] Silica coated polymer particles

[0789] Embodiment 60

[0790] 1.5 g Na2 SiO 3 and 10 mL H 2 O. The pH of the solution was adjusted to pH 12-13 with 1M HCl before adding 7.8 g of the particles of Example 35 (dry content 6.4 wt%). The mixture was stirred at 200 rpm for 10 minutes and then the flask was lowered into an oil bath preheated to 60°C. The pH was then adjusted to pH 6 by dropwise addition of 1M HCl. The mixture was then stirred for 15 minutes and then the reaction mixture was transferred to a 100 mL Duran flask and placed in H 2 O was subjected to five magnetic shifts. The FTIR spectrum performed on the dried sample was -1 The peak at 2 coating.

[0791] Embodiment 61

[0792] 0.5 g Na 2 SiO 3 and 10 mL H 2 O. The pH of the solution was adjusted to pH 12-13 with 1M HCl before adding 7.8 g of the particles of Example 35 (dry content 6.4 wt%). The mixture was stirred at 200 rpm for 5 minutes and then the flask was lowered into an oil bath preheated to 60°C. The pH was then adjusted to pH 2.5 by dropwise addition of 1M HCl. The mixture was then stirred for 5 minutes and then the reaction mixture was transferred to a 250 mL Duran flask and placed in H 2 O was subjected to 7 magnetic shifts. The FTIR spectrum performed on the dried sample was -1 The peak at 2 coating.

[0793] Embodiment 62

[0794] 0.5 g Na 2 SiO 3 and 10 mL H 2 O. The pH of the solution was adjusted to pH 12-13 with 1M HCl before adding 8.9 g of the particles of Example 26 (dry content 5.6 wt%). The mixture was stirred at 200 rpm for 5 minutes and then the flask was lowered into an oil bath preheated to 60°C. The pH was then adjusted to pH 2.5 by dropwise addition of 1M HCl. The mixture was then stirred for 5 minutes and then the reaction mixture was transferred to a 250 mL centrifuge flask and subjected to HCl. 2O by centrifugation 5 times. By FTIR spectroscopy performed on the dried samples at 1094 cm -1 The peak at 2 coating.

[0795] Example 63 - Silane Route

[0796] 7.8 g of the particles of Example 35 (dry content 6.4 wt%) were added to a 20 mL screw cap vial and magnetically pipetted 3 times with methanol and 5 times 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 H 2 O. The reaction was then heated to 130 °C and stirred overnight. The reaction mixture was subsequently transferred to a 20 mL screw cap vial, magnetically pipetted 5 times in dipropylene glycol dimethyl ether, and heated in H 2 The particle suspension was finally stirred for 3 days. The FTIR spectrum performed on the dried sample was -1 The peak at 2 coating.

[0797] Antibody conjugation and functional assays

[0798] Embodiment 64

[0799] Anti-TnI mAb was coupled to the particles of Examples 34, 35 and 36 according to the following procedure. 5 mg of beads (500 μl of 1% stock solution) were added to 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 wash step was repeated 3 times and then resuspended in 205 μL of MES buffer. Subsequently, 45 μL of EDC (20 mg / mL, in H 2 0), the tube is vortexed, and then incubated on a roller at room temperature for 30 minutes. The tube is placed on a magnet, the supernatant is removed, and then resuspended in 200 μL of MES buffer. 52 μL of anti-TnI mAb SDIX (3.8 mg / mL) is added to the tube, the tube is vortexed, and then incubated on a roller at room temperature for 2 hours. The particles are then washed twice with 1 mL TBST buffer (50 mM Tris-HCl pH 7.4, containing 140 mM NaCl and 0.1% Tween-20), and then resuspended in TBST buffer on a roller at 37 ° C overnight. Finally, the particles are washed for the last time with 1 mL TBST buffer, and then resuspended in 985 μL TBST buffer to obtain a 5 mg / mL particle suspension.

[0800] Embodiment 65

[0801] The anti-hGH mAb GhG2 was coupled to the particles of Examples 34, 35 and 36 according to the following procedure. 5 mg of particles (500 μl of 1% stock solution) were added to 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 wash step was repeated 3 times and then resuspended in 205 μl of MES buffer. Subsequently, 45 μl of EDC (20 mg / mL, in H 2 O), vortex the tube, and then incubate on a roller at room temperature for 30 minutes. Place the tube on a magnet, remove the supernatant, and then resuspend in 190 μL of MES buffer. 60 μL of anti-hGH mAb GhG2 (3.5 mg / mL) is added to the tube, vortex the tube, and then incubate on a roller at room temperature for 2 hours. The particles are then washed twice with 1 mL TBST buffer (50 mM Tris-HCl pH 7.4, containing 140 mM NaCl and 0.1% Tween-20), and then resuspended in TBST buffer on a roller at 37°C overnight. Finally, wash the particles for the last time with 1 mL TBST buffer and then resuspend in 985 μL TBST buffer to obtain a 5 mg / mL particle suspension.

[0802] Embodiment 66

[0803] Anti-TnI mAb was coupled to the particles of Examples 34, 37, and 38 according to the following procedure. 5 mg of particles (500 μl of 1% stock solution) were added to 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 wash step was repeated 3 times and then resuspended in 205 μL of MES buffer. Subsequently, 45 μL of EDC (20 mg / mL, in H 2 0), the tube is vortexed, and then incubated on a roller at room temperature for 30 minutes. The tube is placed on a magnet, the supernatant is removed, and then resuspended in 205 μL of MES buffer. 42.5 μL of anti-TnI mAb SDIX (4.7 mg / mL) is added to the tube, the tube is vortexed, and then incubated on a roller at room temperature for 2 hours. The particles are then washed twice with 1 mL TBST buffer (50 mM Tris-HCl pH 7.4, containing 140 mM NaCl and 0.1% Tween-20), and then resuspended in TBST buffer on a roller at 37 ° C overnight. Finally, the particles are washed for the last time with 1 mL TBST buffer and then resuspended in 985 μL TBST buffer to obtain a 5 mg / mL bead suspension.

[0804] Embodiment 67

[0805] A 96 microplate (Greiner-bio-one) was used for the experiment, and each batch of particles was screened in triplicate at 4 different antigen concentrations (0, 5 ng / mL, 50 ng / mL, and 100 ng / mL recombinant human growth hormone in bovine serum). 50 μL of antigen solution and 50 μL of detection antibody (mAb GhB9-AP) were added to each well, the plate was sealed with tape and incubated on a shaker at 37°C for 15 minutes. Subsequently, 100 μL of suspended particles coupled to the anti-hGH mAb GhG2 prepared according to Example 65 were added, 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, the beads were washed with 3×200 μL of TBST buffer, and finally resuspended in 20 μL of TBST buffer. Finally, the substrate (DynaLightSubstrate w / RapidGlow) was added and the plate was incubated on a BioTek Synergy TM 4. Read the plate on a plate reader. The results obtained are as follows Figure 6 shown.

[0806] Embodiment 68

[0807] 96 microplates (Greiner-bio-one) were used for the experiment, and four different antigen concentrations (0, 0.5 ng / mL, 5 ng / mL, and 50 ng / mL human cardiac troponin ITC complex in bovine serum) of each batch of particles were screened in triplicate. 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 incubated on an oscillator 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 incubated on an oscillator at 37°C for 30 mi...

Claims

1. Monodisperse magnetic hydrogel polymer particles comprising a magnetic material and a polymer formed from: (a) Hydrophilic vinyl monomers, whose log P oct / wat (log P) less than 0.6; and (b) a cross-linking agent comprising at least two vinyl groups; wherein the particles are monodisperse, wherein a coefficient of variation (CV) of a plurality of at least 100 of the particles as measured by a disc centrifuge analysis is less than 20%; The log P is calculated using CambridgeSoft's Ultra 14.0 calculation; and wherein the hydrophilic vinyl monomer comprises an acrylamide monomer and / or an acrylate monomer or the hydrophilic vinyl monomer is selected from fumaric acid, itaconic acid, vinylphosphonic acid and vinylimidazole. 2 . The polymer particles according to claim 1 , wherein the hydrophilic vinyl monomer is an acrylamide monomer and / or an acrylate monomer.

3. The polymer particles of claim 1, wherein the hydrophilic vinyl monomer comprises an R 原子 Monomer: Where N C is the number of carbon atoms in the hydrophilic vinyl monomer, N N is the number of nitrogen atoms and N O is the number of oxygen atoms; When the hydrophilic vinyl monomer is acrylamide, vinyl imidazole or vinyl phosphonic acid, R 原子 <2.75; and when the hydrophilic vinyl monomer is an acrylate or other monomer, R 原子 ≤2 and the water solubility of the hydrophilic vinyl monomer at 25°C is ≥50g / L.

4. The polymer particle according to any one of claims 1 to 3, wherein the hydrophilic vinyl monomer is selected from acrylamide, hydroxymethyl acrylamide, [tris(hydroxymethyl)methyl] acrylamide, 4-acryloylmorpholine, methacrylamide, 2-hydroxyethyl methacrylamide, (3-aminopropyl) methacrylamide, or 2-hydroxyethyl acrylate, and 2-hydroxyethyl methacrylate.

5. The polymer particles according to claim 4, wherein the hydrophilic vinyl monomer is selected from acrylamide, 4-acryloylmorpholine and methacrylamide.

6. The polymer particle according to any one of claims 1 to 3, wherein the hydrophilic vinyl monomer comprises acrylamide, hydroxymethyl acrylamide, [tris(hydroxymethyl)methyl] acrylamide, 4-acryloylmorpholine, methacrylamide, 2-hydroxyethyl methacrylamide, (3-aminopropyl) methacrylamide, or 2-hydroxyethyl acrylate, and 2-hydroxyethyl methacrylate.

7. The polymer particles according to claim 6, wherein the hydrophilic vinyl monomer comprises acrylamide, 4-acryloylmorpholine and methacrylamide.

8. The polymer particle according to any one of claims 1 to 3, wherein the crosslinking agent comprises at least one compound of formula (IIa) or (IIb): Where R 6 Selected from -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl- excluding one-membered and two-membered rings, or polyethers containing 2 to 100 C2-C3 ether units; R 7 and R 8 Each is independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, or -C3-C6 cycloalkyl; or R 7 and R 8 linked together to form -C1-C6 alkyl-, -C1-C6 heteroalkyl-, excluding one-membered and two-membered rings -C1-C6 cycloalkyl-, or polyethers containing 2 to 100 C2-C3 ether units; R 9 Yes-N(R 11 )C(O)CH=CH2; R 10 Selected from -H and -N(R 12 )C(O)CH=CH2; and R 11 and R 12 Each is independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, or -C3-C6 cycloalkyl.

9. The polymer particles according to claim 8, wherein -C1-C6 heteroalkyl- is -C1-C6 hydroxyalkyl- or -C1-C6 ether-.

10. The polymer particles according to claim 8, wherein the -C1-C6 heteroalkyl group is a -C1-C6 hydroxyalkyl group or a -C1-C6 ether.

11. The polymer particles according to claim 8, wherein R 6 Selected from -C1-C6 alkyl-, -C1-C6 heteroalkyl-, -C1-C6 cycloalkyl- excluding one-membered and two-membered rings, or polyethers containing 2 to 100 C2-C3 ether units; R 7 and R 8 Each is independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, or -C3-C6 cycloalkyl; or R 7 and R 8 Polyethers comprising 2 to 100 C2-C3 ether units linked together to form -C1-C6 alkyl-, -C1-C6 heteroalkyl-, excluding one- and two-membered rings -C1-C6 cycloalkyl-; and R 11 and R 12 Each is independently selected from -H, -C1-C6 alkyl, -C1-C6 heteroalkyl, or -C3-C6 cycloalkyl; and / or Where R 6 For (CH2) r (OCH2CH2) n O(CH2) s , wherein r and s are each independently 2 or 3, and n is an integer from 1 to 100.

12. The polymer particles according to claim 11, wherein -C1-C6 heteroalkyl- is -C1-C6 ether-.

13. The polymer particles according to claim 11, wherein the -C1-C6 heteroalkyl group is a -C1-C6 ether.

14. The polymer particle of claim 11, wherein r and s are each 2 and n is an integer from 1 to 50.

15. The polymer particles according to any one of the preceding claims 1 to 3, wherein the log P value of the crosslinking agent is less than 1; wherein the log P value is calculated using CambridgeSoft Ultra 14.0 computing.

16. The polymer particle of claim 15, wherein the crosslinking agent has a log P value of less than 0.

5.

17. The polymer particle of claim 15, wherein the crosslinking agent has a log P value of less than 0.

18. The polymer particles according to any one of claims 1 to 3, wherein the crosslinking agent comprises N,N'-(1,2-dihydroxyethylene)bisacrylamide, N,N'-methylenebis(acrylamide), N,N'-ethylenebis(acrylamide), 1,3-diethylene glycol glycerol diacrylate, piperazine bisacrylamide, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)) bisacrylamide, polyethylene glycol bisacrylamide with a weight average molecular weight Mw ≤ 2000, 4-arm PEG-acrylamide with a weight average molecular weight Mw ≤ 2000 and / or N,N-bis(2-acrylamidoethyl)acrylamide; or wherein the crosslinking agent comprises 1,2-dihydroxybis-acrylamide; and / or wherein the crosslinking agent does not contain a primary amine, a secondary amine, a hydroxyl group or a carboxylic acid, and The molecular weight is determined according to the method disclosed in the specification.

19. The polymer particles according to claim 18, wherein the crosslinking agent is N,N'-(1,2-dihydroxyethylene)bisacrylamide, N,N'-methylenebis(acrylamide), N,N'-ethylenebis(acrylamide), 1,3-diglycol glycerol diacrylate, piperazine diacrylamide, N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide with a weight average molecular weight Mw ≤ 2000, 4-arm PEG-acrylamide with a weight average molecular weight Mw ≤ 2000 and / or N,N-bis(2-acrylamidoethyl)acrylamide.

20. The polymer particle of claim 18, wherein the crosslinking agent is 1,2-dihydroxybis-acrylamide.

21. The polymer particle of claim 18, wherein the crosslinking agent comprises N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide having a weight average molecular weight Mw ≤ 2000, and 4-arm PEG-acrylamide having a weight average molecular weight Mw ≤ 2000.

22. The polymer particle of claim 18, wherein the crosslinking agent is N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide, polyethylene glycol diacrylamide with a weight average molecular weight Mw ≤ 2000, and 4-arm PEG-acrylamide with a weight average molecular weight Mw ≤ 2000.

23. The polymer particle of claim 18, wherein the crosslinker comprises N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide.

24. The polymer particle of claim 18, wherein the crosslinker is N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide.

25. The polymer particle of any one of claims 1 to 3, wherein the polymer comprises pores, and the pores comprise at least a portion of the magnetic material.

26. The polymer particle of claim 25, wherein the magnetic material is substantially located in the pores.

27. The polymer particle of claim 25, wherein the pores provide the polymer with a porosity level of 40% to 95%, the porosity being determined as disclosed in the specification from the volume of water present in the pores compared to the total volume of the polymer particle when saturated with water.

28. The polymer particle of any one of claims 1 to 3, wherein the magnetic material is a nanoparticle magnetic material; and / or wherein the magnetic material is a superparamagnetic material; and / or wherein the magnetic material is a metal oxide or alloy.

29. The polymer particle of claim 28, wherein the magnetic material comprises ferromagnetic and / or superparamagnetic magnetic oxide crystals.

30. The polymer particle of any one of claims 1 to 3, wherein the polymer particle further comprises a coating.

31. The polymer particle of claim 30, wherein the coating is a polymer coating formed from at least one epoxide monomer.

32. The polymer particle of claim 30, wherein the coating comprises silicon dioxide.

33. A method of forming monodisperse magnetic hydrogel polymer particles, comprising: forming an aqueous suspension comprising monodisperse hydrogel polymer particles and a magnetic material or a precursor of a magnetic material; When the aqueous suspension includes a magnetic material precursor, converting the magnetic material precursor into a magnetic material; and allowing the magnetic material to bind to the polymer particles, Wherein the monodisperse hydrogel polymer particles are formed by: (a)log P oct / wat A hydrophilic vinyl monomer having a (log P) of less than 0.6; and (b) a cross-linking agent comprising at least two vinyl groups; wherein the particles are monodisperse, wherein a coefficient of variation (CV) of a plurality of at least 100 of the particles as measured by a disc centrifuge analysis is less than 20%; The log P is calculated using CambridgeSoft's Ultra 14.0 calculation; and wherein the hydrophilic vinyl monomer comprises an acrylamide monomer and / or an acrylate monomer or the hydrophilic vinyl monomer is selected from fumaric acid, itaconic acid, vinylphosphonic acid and vinylimidazole.

34. The method of claim 33, wherein the hydrophilic vinyl monomer is an acrylamide monomer and / or an acrylate monomer.

35. The method of claim 33, wherein: Forming an aqueous suspension comprises forming an aqueous suspension having a pH less than 6, comprising monodisperse hydrogel polymer particles and Fe 2+ and / or Fe 3+ ions; and Converting the magnetic material precursor to the magnetic material comprises raising the pH to greater than 8; and allowing the magnetic material to precipitate.

36. The method of claim 35, wherein the aqueous suspension having a pH of less than 6 comprises Fe 2+ and Fe 3+ ion.

37. The method of claim 35, wherein the suspension comprises FeCl2 and FeCl3.

38. The method of any one of claims 33 to 37, further comprising forming a polymer coating and / or a silica coating on the magnetic hydrogel polymer particles.

39. The method of claim 38, wherein 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.

40. The method of claim 39, wherein the surface functional group is an amine.

41. The method of claim 38, wherein forming the silica coating comprises forming a suspension comprising monodisperse magnetic hydrogel polymer particles and a silicate or an orthosilicate, and reacting the silicate or the orthosilicate to form the silica coating.

42. The method of claim 41, wherein reacting the silicate to form the silica coating comprises lowering the pH of the suspension to less than 11.

43. The method of claim 41, wherein reacting the orthosilicate to form the silica coating comprises raising the temperature of the suspension to at least 80°C.

44. Use of monodisperse magnetic hydrogel polymer particles according to any one of claims 1 to 32 in an assay or in nucleic acid amplification.

45. Use according to claim 44, wherein the nucleic acid amplification is emulsion PCR.

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