Monodisperse superparamagnetic particles and method for producing the same

Abstract

This invention provides a novel synthesis method for producing spherical monodisperse polymer beads that have adjustable diameter, good rigidity and good resistance to organic solvents, and contain sufficient hydrophilic functional groups to promote the uploading of a high percentage of superparamagnetic Fe3O4NP, enabling the production of new monodisperse superparamagnetic polymer beads in the 1-5 μm diameter range for IVD applications. [Solution] A monodisperse superparamagnetic bead having a core-shell structure. The bead includes: a core portion formed from a polystyrene polymer matrix material encapsulating a first batch of superparamagnetic Fe3O4 nanoparticles; a first shell portion directly located on the top of the core portion and formed from a crosslinked polymer matrix material composed of a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group; and a second shell portion directly located on the top of the first shell portion.

Description

[Technical Field]

[0001] (Field of Invention) The present invention relates to monodisperse superparamagnetic beads useful for in vitro diagnostic (or in-vitro diagnostic) assays and other applications, as well as to a method for producing said beads. [Background technology]

[0002] (background) Monodisperse superparamagnetic beads are widely used in in vitro diagnostics (IVD) and biotechnology. For example, in chemiluminescence assays, monodisperse superparamagnetic beads are applied to quantify the amount of antigen or antibody present in a sample via magnetically tagged antibodies or antigens. Monodisperse superparamagnetic beads are also commonly used in diagnostic kits for nucleic acid (DNA and RNA) extraction prior to polymerase chain reaction (PCR). Easily adaptable to automated processes, these beads are used in over 25,000 routine IVD instruments worldwide. Monodisperse superparamagnetic beads are also used for antibody purification and the isolation of a wide range of specific mammalian cells, bacteria, viruses, intracellular organelles, and individual proteins. Due to their superparamagnetic properties and their ability to be enriched with affinity groups (e.g., streptavidin) on their surface, monodisperse superparamagnetic beads are a powerful tool for antibody purification and separation. A typical separation procedure involves mixing a suspension of monodisperse superparamagnetic beads with a solution containing the target molecule (e.g., biotin-labeled antibody) or cells. After the incubation period, the target binds to an affinity ligand, and then a powerful magnet is used to capture the magnetic beads and their captured target molecules or cells. Unbound material or impurities are then removed by suction, and the bound material is washed for downstream application. Due to their ability to support the rapid development of IVD and biotechnology fields, monodisperse superparamagnetic beads have attracted considerable attention in recent years.

[0003] Monodisperse magnetic beads are a combination of magnetic particles and polymer materials(s). The beads benefit from the combination of properties inherently provided by these components. The magnetic properties (or magnetic properties) of the beads (arising from the magnetic particles) allow for rapid and easy separation of the beads upon application of an external magnetic field. Polymer materials(s) can be used to stabilize the magnetic particles, reduce the density of the beads, provide better dispersibility of the beads in various fluid buffers, confer functional groups to the particles, and enable desirable applications in immunoassays, nucleic acid sequences, cells, and microorganisms through the attachment of active sites via these functional groups. For a review of the applications and functionalization of these beads, see European Polymer Journal 2011, 47, 542-559.

[0004] Monodisperse superparamagnetic beads (typically 1–5 microns in diameter) are commonly used as a powerful tool in in vitro diagnostic (IVD) applications (see the review literature for a discussion of these applications). Good uniformity of size, shape, and surface area is crucial for reproducibility in biological systems and minimizes chemical aggregation during separation processes. Thus, the discovery of monodisperse polymer beads with superparamagnetic properties has significantly boosted the IVD industry globally over the past decade, and this industry is expected to continue growing rapidly in the next decade.

[0005] Magnetic beads typically have three different structural types (Figure 1, Types I-III). Type I structures have magnetic nanoparticles (NPs) distributed throughout the polymer matrix; Type II structures have a core-shell structure with a polymer core and a shell formed of magnetic NPs on the bead surface; and Type III structures have a polymer shell with a core containing magnetic NPs. Additionally, it is noteworthy that Type III structures generally tend to have poor superparamagnetic properties because the magnetic core is often too large, causing interference between the magnetic NPs and potentially reducing the beads' superparamagnetic properties. However, simply selecting one of these designs will not yield superparamagnetic properties. To obtain optimal superparamagnetic properties, the beads need to be supported with magnetic NPs in the 5-15 nm size range. Thus, the Type I-III composite beads shown in Figure 1 will be superparamagnetic when the magnetic NPs fall within this size range.

[0006] To achieve the aforementioned Type I design, there are two synthesis strategies, which will be discussed later.

[0007] The first strategy involves synthesizing superparamagnetic Fe3O4NPs, then dispersing them in a styrene emulsion polymerization system, and encapsulating the NPs in polystyrene beads [see, for example, U.S. Patent Application Publication No. 2009 / 0092837, Journal of Applied Polymer Science 2013, DOI: 10.1002 / APP. [38857(1726-1733), Journal of Polymer Science: Part A: Polymer Chemistry, 2007, 45, 5285-5295]]. However, this synthesis strategy has two challenges. The first challenge is that only superparamagnetic polystyrene beads with small diameters of typically 100-200 nm can be produced, which are too small for IVD applications. The second challenge is that the superparamagnetic Fe3O4NPs are not uniformly distributed throughout the polystyrene beads, and thus various beads contain different numbers of superparamagnetic Fe3O4NPs. As a result, the magnetic response of the magnetic spheres exhibits a broad distribution, which is undesirable for IVD applications. Furthermore, due to the properties of styrene emulsion polymerization, overcoming these two challenges has been impossible until now. Similar encapsulation strategies have also been applied to prepare type III magnetic beads.

[0008] A second strategy for synthesizing Type I monodisperse superparamagnetic beads is to first synthesize monodisperse polymer beads as carriers for superparamagnetic NPs, and then upload the superparamagnetic Fe3O4 NPs. In this synthesis strategy, the first step has at least three requirements: (1) The polymer beads are highly monodisperse with a reproducible diameter, e.g., 1.0 micron; (2) The polymer beads are crosslinked, and thus have good rigidity and resistance to organic solvents; and (3) Functionalizing polymer beads to promote on-site generation of superparamagnetic Fe3O4 nanoparticles. The objective is to synthesize highly monodisperse polymer beads that satisfy the following conditions.

[0009] To meet the above requirements, complex and expensive procedures are employed in state-of-the-art synthesis techniques. For example, U.S. Patent Application Publication No. 2017 / 0218095 (Ugelstad Process) discloses a three-step procedure for producing porous functionalized polystyrene beads used in the manufacture of superparamagnetic polymer beads. The first step is the synthesis of low molecular weight polystyrene seeds via emulsion polymerization. Sodium dodecyl sulfate (SDS) was used as a surfactant, ammonium persulfate (APS) as an initiator, and borax to enhance ionic strength. Styrene was extracted with 10 wt% sodium hydroxide to remove stabilizers (4-tert-butylcatechol) that could inhibit styrene polymerization. The resulting monodisperse polystyrene seed particles have a diameter in the range of 50 to 200 nm. The second step is active swelling and crosslinking. The polystyrene seeds were swelled at 25°C for 24 hours using an initiator emulsion containing dioctanoyl peroxide, SDS, and acetone. Next, the swollen seeds were mixed with a monomer emulsion containing toluene, DVB, styrene, PVP, SDS, and water, and the temperature was raised to 60°C to initiate crosslinking polymerization, which was carried out for 2 hours to obtain porous crosslinked polystyrene beads. The third step is to functionalize the porous crosslinked beads by introducing NO2 groups into these polystyrene spheres by nitrating the phenyl rings in the beads with a mixture of sulfuric acid and nitric acid (65%) (U.S. Patent No. 4,654,267). The nitrated beads are used in a subsequent magnetization step with ammonia (25%) and iron(II) sulfate heptahydrate. The authors have shown numerous methods for introducing various functional groups by using various monomers in the second step to increase the iron content in the third step. However, the magnetic content is less than 15% in all options. The only practical way to improve the iron content to more than 15% is to produce nitrided beads using a nitriding process. The problem with this synthesis strategy is that it is time-consuming and cumbersome to produce a highly controllable porous polystyrene support. Most importantly, to nitrate 5g of dry particles, a mixture of concentrated nitric acid and 125mL of concentrated sulfuric acid is required.Thus, the process requires the consumption of large quantities of concentrated acids that are both oxidizing and corrosive, which would cause serious environmental problems. In addition, scaling up this process would require strict reactors and process controls.

[0010] A Type II design can be obtained using the protocol of U.S. Patent No. 7,713,627, which discloses a method for producing non-magnetic nucleus particles having a diameter of 1.5 μm. First, primary large polystyrene beads were produced by seed emulsion polymerization of styrene with DVB at 75°C for 8 hours, using polystyrene beads with a diameter of 0.77 μm as seeds in an emulsion system containing di(3,5,5-trimethylhexanoyl) peroxide and SDS. The resulting primary beads were then separated by centrifugation, washed with water, dried, and pulverized to obtain non-magnetic nucleus particles with an average particle size of 1.5 microns. These were then used to produce magnetic beads by supporting ferrite-type fine magnetic material particles (average primary particle size: 0.01 μm) with a hydrophobic surface. The magnetic NPs then adhere to the surface of the spheres via hydrophobic interactions. The drawbacks of this process are that (1) the second step (grinding process) does not yield highly monodisperse beads, and the beads may not be spherical; (2) the smallest beads shown in this patent have a diameter of 1.5 μm; it appears difficult to produce smaller beads with a diameter of 1.0 μm or less; and (3) magnetic NPs simply adhere to the surface of the beads. Therefore, the outer surface area of ​​the beads is limited and determined by the core diameter of the polymer, making it difficult to achieve high magnetism or iron content.

[0011] It should be noted that crosslinked poly(styrene-DVB) beads can be produced by a one-pot two-step polymerization process. In the first step, polystyrene beads are formed in situ (or in the reaction mixture), and then DVB is added to form a core-shell structure. Such one-pot two-step polymerization can be carried out in an emulsion polymerization system or a dispersion polymerization system. In an emulsion styrene polymerization system using sodium styrenesulfonate as a surfactant and KPS as an initiator, the resulting polymer beads have a diameter of approximately 200 nm (Petroleum Science, 2008, 5, 375-378). On the other hand, in a dispersion styrene polymerization system using PVP as a stabilizer and AIBN as an initiator in ethanol, the resulting polymer beads have a diameter of approximately 2.3 μm (Journal of Applied Polymer Science, 2010, 115, 3092-3102). Regardless of the bead size, in both cases the resulting polystyrene-DVB beads are hydrophobic and lack hydrophilic functional groups that can be used to upload superparamagnetic Fe3O4 nanoparticles.

[0012] Therefore, there remains a need to develop a new synthetic strategy for producing spherical monodisperse polymer beads that have adjustable diameter, good rigidity and good resistance to organic solvents, and contain enough hydrophilic functional groups to facilitate the uploading of a high percentage of superparamagnetic Fe3O4NP, enabling the production of new monodisperse superparamagnetic polymer beads in the 1-5 μm diameter range for IVD applications. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] U.S. Patent Application Publication No. 2009 / 0092837 [Patent Document 2] U.S. Patent Application Publication No. 2017 / 0218095 [Patent Document 3] U.S. Patent No. 4,654,267

Patent Document 4

Non-Patent Document

[0014]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Means for Solving the Problems

[0015] (Summary of the Invention) Aspects and embodiments of the present invention are described in the following numbered paragraphs.

[0016] 1. A monodisperse superparamagnetic bead having a core - shell structure, wherein the bead is a core part formed from a polystyrene polymer matrix material, and the polystyrene polymer matrix material encapsulates (or encloses or encapsulates; encapsulate) the first batch of superparamagnetic Fe3O4 nanoparticles; A first shell portion located directly on the top of the core portion and formed from a crosslinked polymer matrix material formed from a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group, wherein the copolymer matrix material encapsulates a second batch of superparamagnetic Fe3O4 nanoparticles; and The second shell section is located directly on top of the first shell section. The second shell portion is formed as a first layer, a second layer, and a third layer, The first layer comprises a first layer polymer matrix material containing superparamagnetic Fe3O4 nanoparticles, as well as conjugated monomers and bulk monomers; The second layer extends beyond the first layer and is formed from a second layer polymer material containing bulk monomers; and The third layer is located on top of the second layer and is formed from a third layer polymer material comprising a bulk monomer and a second functional monomer having a functional group. The superparamagnetic Fe3O4 nanoparticles of the first layer directly bond to the functional groups present on the outer surface of the first shell portion; The first layer polymer matrix material surrounds the superparamagnetic Fe3O4 nanoparticles of the first layer; and Monodisperse superparamagnetic beads, wherein the second layer polymer matrix material extends from the first layer polymer matrix material and forms the outer surface of each monodisperse superparamagnetic bead.

[0017] 2. The beads according to paragraph 1, wherein the polymer matrix material of the second and third layers functions to prevent the elution of superparamagnetic magnetic nanoparticles when the beads are placed in a solvent.

[0018] 3. The beads according to the first or second paragraph, wherein the functional groups on the functional monomers of the third layer of the first shell portion and the second shell portion are independently selected from one or more amino, carboxyl, epoxy, and hydroxyl groups, and optionally, the functional groups on the functional monomers of the second layer of the first shell portion and the second shell portion are independently selected from a combination of a hydroxyl group and a carboxyl group, or a combination of an amino group and a carboxyl group.

[0019] 4. The beads according to any one of paragraphs 1 to 3, wherein the beads have a coefficient of variation based on their diameter of less than 15%, for example, less than 10%, for example, less than 5%.

[0020] 5. The beads according to paragraph 4, wherein the beads have a coefficient of variation based on their diameter of 2% or less.

[0021] 6. The beads according to any one of the first to fifth paragraphs, wherein the beads have an average diameter of 0.2 to 5.0 μm, for example, 0.5 to 4.0 μm.

[0022] 7. The beads according to any one of claims 1 to 6, wherein the polystyrene polymer matrix material is formed from one or more of the group consisting of styrene, styrene derivatives, and copolymers thereof, and optionally the styrene derivative is selected from one or more of the group consisting of 4-methylstyrene, 3-methylstyrene, and 4-tert-butylstyrene.

[0023] 8. Beads according to any one of items 1 to 7, wherein all of the superparamagnetic Fe3O4 nanoparticles in the beads account for 10 to 80% by weight, e.g., 20 to 70% by weight, e.g., 30 to 50% by weight, of the total weight of each bead.

[0024] 9.(a) The superparamagnetic Fe3O4 nanoparticles in the first batch account for 0.1–5% by weight of the total weight of each bead; and / or (b) The superparamagnetic Fe3O4 nanoparticles from the second batch account for 0.5–10% by weight of the total weight of each bead; (c) The beads according to item 8, wherein superparamagnetic Fe3O4 nanoparticles in the first layer of the second shell portion account for 9.4 to 79.4% by weight of each bead, for example, 19.4 to 69.4% by weight, for example, 29.4 to 49.4% by weight.

[0025] 10. The crosslinkable monomer is divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, butanediol dimethacrylate, tricyclodecanedimethanol diacrylate, pentaerythritol triacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol pentaacrylate, dipentaerythritol penta / hexaacrylate, tripropylene diacrylate, trimethylolpropane ethoxylate triacrylate, trimethylol Beads according to any one of claims 1 to 9, wherein the beads are selected from the group consisting of ropanepropoxylate triacrylate, di(trimethylolpropane)tetoacrylate, glycerolpropoxylate triacrylate, pentaerythritolpropoxylate triacrylate, poly(ethylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate, and optionally the crosslinkable monomer is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N'-methylenebis(acrylamide).

[0026] 11. The beads according to any one of claims 1 to 10, wherein the first functional monomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and 2-carboxyethyl acrylate oligomers.

[0027] 12. Beads according to any one of claims 1 to 11, wherein the styrene monomer is selected from one or more of the group consisting of styrene and styrene derivatives, and optionally the styrene derivative is selected from one or more of the group consisting of 4-methylstyrene, 3-methylstyrene, and 4-tert-butylstyrene.

[0028] 13. The first shell further comprises a first layer of the first polymer matrix composition and a second layer of the second polymer matrix composition. The first layer is formed from a copolymer of a styrene monomer and a crosslinkable monomer; and The beads according to any one of claims 1 to 12, wherein the second layer is formed from a copolymer of a styrene monomer, a crosslinkable monomer, and a functional monomer.

[0029] 14. The bead according to any one of items 1 to 13, wherein the superparamagnetic Fe3O4 nanoparticles have an average diameter of 5 to 15 nm.

[0030] 15. (a) The weight-to-weight ratio of styrene groups to crosslinking groups in the core and the first shell is 20:1 to 1:2, for example, 10:1 to 1:1, and / or (b) The beads according to any one of claims 1 to 14, wherein the weight-to-weight ratio of styrene groups to functional groups in the core portion and the first shell portion is 20:1 to 1:2, for example, 10:1 to 1:1.

[0031] 16. The beads according to any one of items 1 to 15, wherein the Fe3O4 nanoparticles further comprise Co3O4 and / or Mn3O4 nanoparticles.

[0032] 17. The beads according to any one of claims 1 to 16, wherein the conjugated monomer is selected from one or more of the group consisting of methacryloyl chloride, 3-ethoxyacryloyl chloride, acryloyl chloride, 4-pentenoyl chloride, methacrylic anhydride, 4-pentenoic anhydride, crotonic anhydride, valeric anhydride, 10-undecenoyl chloride, glycidol methacrylate, glycidol, and allyl glycidyl ether.

[0033] 18. The beads according to any one of claims 1 to 17, wherein the bulk monomer is selected from one or more of polyether monomers, polyester monomers, polyacrylamide monomers, and polyacid monomers, and optionally the bulk monomer is selected from one or more of the group consisting of methyl methacrylate, acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, 2-carboxyethyl acrylate oligomer, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, and maleic anhydride, and optionally the bulk monomer is selected from methacrylic acid and / or 2-hydroxyethyl methacrylate. Here,

[0034] 19. The beads according to any one of claims 1 to 18, wherein the second functional monomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and 2-carboxyethyl acrylate oligomers.

[0035] 20. The second shell portion further comprises a crosslinkable monomer in the first and / or second and / or third layer, optionally the crosslinkable monomer being divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, N,N'-methylenebis(acrylamide), bisphenol A epoxy acrylate, bisphenol diacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, propoxylated glycerol diacrylate, pentaerythritol propoxylate triacrylate, butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipene Beads according to any one of claims 1 to 19, wherein the crosslinkable monomer is selected from one or more of the group consisting of erythritol penta / hexaacrylate, tripropylene diacrylate, trimethylolpropane ethoxylate triacrylate, trimethylolpropane propoxylate triacrylate, di(trimethylolpropane)tetraacrylate, glycerol propoxylate triacrylate, poly(propylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate, for example, the crosslinkable monomer is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N'-methylenebis(acrylamide).

[0036] 21. A superparamagnetic bead according to any one of claims 1 to 20, wherein the combined weight of the second and third layers of the second shell accounts for 1 to 30% by weight, preferably 2 to 20% by weight, of the total weight of each bead.

[0037] 22. A method for preparing monodisperse superparamagnetic beads having a core-shell structure, (a) To provide monodisperse superparamagnetic precursor beads comprising a core portion formed from a polystyrene polymer matrix material, wherein the polystyrene polymer matrix material encapsulates a first batch of superparamagnetic Fe3O4 nanoparticles; A first shell portion located directly on the top of the core portion and formed from a crosslinked polymer matrix material formed from a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group, wherein the copolymer matrix material encapsulates a second batch of superparamagnetic Fe3O4 nanoparticles; and A second shell portion located directly on the top of the first shell portion, comprising superparamagnetic Fe3O4 nanoparticles and polymer precursor anchor points, both of which are bonded to functional groups on the surface of the first shell portion; and (b) Forming a functional coating layer on monodisperse superparamagnetic precursor beads by one-pot free radical polymerization, (i) Using a bulk monomer in the first step; and (ii) In the second step, monodisperse superparamagnetic beads are formed using a second functional monomer having a functional group, A method (or process) comprising bonding the functional coating layer to a first shell portion by polymer precursor anchor points.

[0038] 23. The method according to claim 22, wherein the bulk monomer is selected from one or more of the group consisting of polyether monomers, polyester monomers, polyacrylamide monomers, and polyacrylamide monomers.

[0039] 24. The method according to claim 23, wherein the bulk monomer is selected from one or more of the group consisting of methyl methacrylate, acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, 2-carboxyethyl acrylate oligomer, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, and maleic anhydride, and optionally, the bulk monomer is selected from methacrylic acid and / or 2-hydroxyethyl methacrylate.

[0040] 25. The method according to any one of claims 22 to 24, wherein the first step is to use a mixture comprising the bulk monomer and the initiator, and optionally the initiator is selected from one or more of the group consisting of tertial-amyl hydroperoxide, potassium persulfate, sodium persulfate, ammonium persulfate, 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide, 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine]hydrate, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2''-azobis[2-(2-imidazolin-2-yl)propane], and 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride.

[0041] 26. The mixture in the first step is further, (a) Crosslinking agent, optionally selected from divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, N,N'-methylenebis(acrylamide), bisphenol A epoxy acrylate, bisphenol diacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, propoxylated glycerol diacrylate, pentaerythritol propoxylate triacrylate, butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta / hexaacrylate A crosslinkable monomer is selected from one or more of the group consisting of di(b)acrylate, tripropylene diacrylate, trimethylolpropane ethoxylate triacrylate, trimethylolpropane propoxylate triacrylate, di(trimethylolpropane)tetraacrylate, glycerol propoxylate triacrylate, poly(ethylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate, for example, the crosslinkable monomer is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N'-methylenebis(acrylamide), and / or (b) Solvent, optionally selected from one or more of the group consisting of 1,4-dioxane, tetrahydrofuran, diglyme, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, and water. The method described in paragraph 25, including the method described in paragraph 25.

[0042] 27. The method according to any one of claims 22 to 26, wherein the second functional monomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and 2-carboxyethyl acrylate oligomers.

[0043] 28. (a) The polymerization of the first step and / or the second step is carried out at a temperature of 30 to 80°C, for example, 50 to 70°C; and / or (b) The method according to any one of claims 22 to 27, wherein the total polymerization time for the first and second steps is 10 to 30 hours, for example, 16 to 24 hours.

[0044] 29. The monodisperse superparamagnetic precursor beads are (i) To provide naked monodisperse superparamagnetic beads, the beads are A core portion formed from a polystyrene polymer matrix material, wherein the polystyrene polymer matrix material encapsulates a first batch of superparamagnetic Fe3O4 nanoparticles; A first shell portion located directly on the top of the core portion, formed from a crosslinked polymer matrix material composed of a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group, wherein the copolymer matrix material encapsulates a second batch of superparamagnetic Fe3O4 nanoparticles; and A second shell portion located directly on the top of the first shell portion, comprising superparamagnetic Fe3O4 nanoparticles bonded to functional groups on the surface of the first shell portion; and (ii) The method according to any one of paragraphs 22 to 28, wherein the naked monodisperse superparamagnetic beads are formed by a process comprising reacting the naked monodisperse superparamagnetic beads with an anchor material selected from one or more of the group consisting of methacryloyl chloride, 3-ethoxyacryloyl chloride, acryloyl chloride, 4-pentenoyl chloride, methacrylic anhydride, 4-pentenoic anhydride, crotonic anhydride, valeric anhydride, 10-undecenoyl chloride, glycidol methacrylate, glycidol, and allyl glycidyl ether to form polymer precursor anchor points on the first shell portion.

[0045] 30. The naked monodisperse superparamagnetic beads, (ai) To provide monodisperse beads, the monodisperse beads having a core formed from a polystyrene polymer matrix material; and The first shell portion is located directly on the top of the core portion and is formed from a crosslinked polymer matrix material made of a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group; and (aii) The naked monodisperse superparamagnetic beads are formed by placing the monodisperse beads in a solution containing Fe(III) salt and Fe(II) salt and adding a base. The method described in paragraph 29, formed by a process including the following.

[0046] 31. (a) The iron(III) salt is selected from FeCl3 and / or Fe2(SO4)3; and / or (b) The iron(II) salt is selected from one or more of the group consisting of FeCl2, FeSO4, and Fe(OAC)2; and / or (c) The base is selected from one or more of the group consisting of ammonium hydroxide, NaOH, KOH, and amines; and / or (d) The solution further comprises CoCl2 and / or MnCl2, The method described in paragraph 30.

[0047] 32. The monodisperse beads are formed by a "one-pot, three-stage" continuous process, and this process is (a) In the first step, a polystyrene core is produced by dispersion polymerization of styrene monomer with an initiator and a polymer stabilizer in a mixture of water and alcohol to form a nucleated polystyrene core; (b) In the second step, add a crosslinkable monomer to the mixture containing the nucleated polystyrene core; and (c) In the third step, a first functional monomer is added to the material obtained from the second step to provide monodisperse beads, and optionally, polystyrene beads are produced in Insights, and the subsequent addition of the crosslinking agent and functional monomer does not cause a second nucleation, leading to spherical monodisperse copolymer beads with functional groups (styrene / crosslinking agent / functional monomer) on the surface. The method described in paragraph 30 or 31, including the act described herein.

[0048] 33. (a) The initiator is selected from azo initiators, and optionally the azo initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN) and 2,2'-azobis(2-methylbutyronitrile) (AMBN); and / or (b) The polymer stabilizer is selected from one or more of the group consisting of poly(vinylpyrrolidone) (PVP), polyethyleneimine (PEI), polyacrylic acid (PAA), polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPC), and chitosan; and / or (c) The styrene monomer is selected from one or more of the group consisting of styrene and styrene derivatives, and optionally the styrene derivative is selected from one or more of the group consisting of 4-methylstyrene, 3-methylstyrene, and 4-tert-butylstyrene; and / or (d) The alcohol is selected from one or more of the group consisting of methanol, ethanol, isopropanol, or mixtures thereof; and / or (e) The method according to item 32, wherein the volume ratio of alcohol to water is 1:1 to 40:1, for example, 2:1 to 20:1.

[0049] 34. The crosslinkable monomer is divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, butanediol dimethacrylate, tricyclodecanedimethanol diacrylate, pentaerythritol triacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol pentaacrylate, dipentaerythritol penta / hexaacrylate, tripropylene diacrylate, trimethylolpropane ethoxylate triacrylate, trimethylolpropane The method according to claim 32 or 33, wherein one or more are selected from the group consisting of panpropoxylate triacrylate, di(trimethylolpropane)tetoacrylate, glycerolpropoxylate triacrylate, pentaerythritolpropoxylate triacrylate, poly(ethylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate, and optionally the crosslinkable monomer is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N'-methylenebis(acrylamide).

[0050] 35. The method according to any one of claims 32 to 34, wherein the functional groups on the first functional monomer are independently selected from one or more amino, carboxyl, epoxy, and hydroxyl groups, and optionally, the functional groups on the functional monomer of the second layer of the first shell portion and the second shell portion are independently selected from a combination of a hydroxyl group and a carboxyl group, or a combination of an amino group and a carboxyl group.

[0051] 36. The method according to claim 35, wherein the first functional monomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and 2-carboxyethyl acrylate oligomers.

[0052] 37. The method according to any one of claims 32 to 36, wherein the weight ratio of the styrene monomer to the crosslinking agent is 20:1 to 1:2, for example, 10:1 to 1:1.

[0053] 38. The method according to any one of paragraphs 32 to 37, wherein the weight ratio of the styrene monomer to the first functional monomer is 20:1 to 1:2, preferably 10:1 to 1:1.

[0054] 39. The method according to any one of paragraphs 32 to 37, wherein the as-synthesized monodisperse beads are washed with one or more solvents before use in a subsequent process step, wherein the solvent is optionally selected from one or more of the group consisting of water, methanol, ethanol, isopropanol, and THF.

[0055] 40. The resulting monodisperse superparamagnetic beads are: (a) coefficient of variation based on their diameters of less than 15%, e.g., less than 10%, e.g., less than 5%, e.g., 2% or less; and / or (b) 0.2 to 5.0 microns, for example, an average diameter of 0.5 to 4.0 microns, A method according to any one of paragraphs 22 to 39, having the following characteristics.

[0056] 41. Superparamagnetic beads according to any one of paragraphs 1 to 21, which are used in an IVD assay. [Brief explanation of the drawing]

[0057] [Figure 1] Figure 1 schematically shows three types of magnetic polymer beads: Type (I): Magnetic NPs dispersed in polymer beads; Type (II): Magnetic shell and polymer core; Type (III): Polymer shell and magnetic core. [Figure 2] Figure 2 schematically illustrates the manufacturing process of spherical monodisperse superparamagnetic beads. [Figure 3] Figure 3 shows the SEM image of A-1. [Figure 4] Figure 4 shows the TEM image of C-1. [Figure 5] Figure 5 shows the hysteresis curve for C-1. [Figure 6] Figure 6 shows the TEM image of D-1. [Figure 7] Figure 7 shows the SEM image of A-2. [Figure 8] Figure 8 shows the TEM image of C-2. [Figure 9] Figure 9 shows the TEM image of D-2. [Figure 10] Figure 10 shows an SEM image of CE-2. [Figure 11] Figure 11 shows the TEM image of DE-2. [Figure 12] Figure 12 schematically shows the details of a well-formed monodisperse superparamagnetic bead. [Modes for carrying out the invention]

[0058] (explanation) We have surprisingly found that the above challenges can be solved, in whole or in part, by introducing a new type of monodisperse superparamagnetic beads. These monodisperse superparamagnetic beads have superparamagnetic NPs distributed throughout the core and shell(s) of the bead. Surprisingly, we have found that these beads can be loaded with Fe3O4 nanoparticles in a wide range of weight percentages (e.g., 30% to over 50% of the total weight of the beads) without suffering from the above problems. These beads are also expected to improve the efficiency of downstream separation processes and enhance the final application of IVD processes.

[0059] Thus disclosed herein are monodisperse superparamagnetic beads having a core-shell structure, the beads being A core portion formed from a polystyrene polymer matrix material, wherein the polystyrene polymer matrix material encapsulates a first batch of superparamagnetic Fe3O4 nanoparticles; A first shell portion located directly on the top of the core portion, formed from a crosslinked polymer matrix material composed of a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group, wherein the copolymer matrix material encapsulates a second batch of superparamagnetic Fe3O4 nanoparticles; and The second shell section is located directly on top of the first shell section. The second shell portion is formed as a first layer, a second layer, and a third layer, The first layer comprises a first layer polymer matrix material containing superparamagnetic Fe3O4 nanoparticles, as well as conjugated monomers and bulk monomers; A second layer extends beyond the first layer and is formed from a second layer polymer material containing bulk monomers; and The third layer is located on top of the second layer and is formed from a third layer polymer material containing a bulk monomer and a second functional monomer having a functional group. The superparamagnetic Fe3O4 nanoparticles in the first layer directly bond to the functional groups present on the outer surface of the first shell; The first layer polymer matrix material surrounds the superparamagnetic Fe3O4 nanoparticles of the first layer; and A second layer polymer matrix material extends from the first layer polymer matrix material and forms the outer surface of each monodisperse superparamagnetic bead.

[0060] The term “comprising” as used herein may be interpreted as requiring the mentioned features but not limiting the presence of other features. Alternatively, “comprising” may refer to a situation in which only the listed components / features are intended to exist (for example, “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is clearly conceivable that both the broader and narrower interpretations are applicable to all aspects and embodiments of the present invention. In other words, the term “comprising” and its synonyms may be replaced by the phrases “consists of” or “consists essentially of” or their synonyms, and vice versa.

[0061] As used herein, the term “particle” is intended to be synonymous with the terms “bead” and “microsphere.” As used herein, the term “polymer particle” is intended to be synonymous with the terms “polymer bead” and “polymer microsphere.” As used herein, the term “superparamagnetic polymer particle” is intended to be synonymous with the terms “superparamagnetic polymer bead” and “superparamagnetic polymer microsphere.”

[0062] As used herein, the term "monodisperse" refers to particles having a low coefficient of variation (CV) for a specific parameter (e.g., particle size), such as less than 20%, less than 15%, less than 10%, or less than 5%. More specifically, particles may have a CV of 2% or less, for example, 1% or less. Furthermore, the term "monodisperse" encompasses the term "highly monodisperse," which, as used herein, may refer to a CV of less than 5%, such as 2% or less, or less, or 1% or less.

[0063] As used herein, the term "coefficient of variation" refers to its statistical meaning. That is:

[0064]

number

[0065] It refers to.

[0066] The terms "standard deviation" and "mean" have their usual statistical meanings.

[0067] In this specification, "average" diameter refers to the average diameter obtained from a scanning electron microscope (SEM).

[0068] Magnetic NPs as used herein may include at least one of paramagnetic NPs, superparamagnetic NPs, ferromagnetic NPs, or ferrimagnetic NPs. Their definitions are listed below.

[0069] As used herein, "magnetism" refers to the property of a material that responds to a magnetic field. As used herein, "paramagnetism" refers to the fact that the magnetic properties of a material are turned off after the external magnetic field is removed. As used herein, "superparamagnetism" refers to the fact that the magnetism of a material disappears instantaneously when the external magnetic field is removed.

[0070] As used herein, "ferromagnetic" means that all magnetic atoms within each domain contribute positively to the pure magnetization of the entire material, and that the magnetic properties are retained even after the external magnetic field is removed. The material becomes paramagnetic above its Curie temperature.

[0071] As used herein, "ferrimagnetism" means that some magnetic atoms within each domain are opposed to each other, but the material as a whole exhibits net magnetization. After the removal of the external magnetic field, the material retains its magnetic properties. Above its Curie temperature, it becomes a paramagnetic material.

[0072] Figure 2D schematically shows a fully-formed monodisperse superparamagnetic bead according to the present invention. Figure 12 shows the fully-formed monodisperse superparamagnetic bead in more detail. Figure 12 shows a cross-section of a monodisperse superparamagnetic bead 100 according to the present invention. The bead has a core 110, a first shell 120, and a second shell 130. The core 110 is formed of a polystyrene polymer matrix material 111, and the core polymer matrix material contains pores 112 that encapsulate a portion of superparamagnetic Fe3O4 nanoparticles 115.

[0073] The first shell portion 120 may be in the form of a single layer (not shown) or two layers (121, 122). In the single layer form, the first shell portion has a crosslinkable polymer matrix material formed from a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group. In the two layer form, the first layer may be a crosslinkable polymer matrix material 123 formed from a copolymer of a styrene monomer and a crosslinkable monomer, and the second layer 124 may be formed from a copolymer of a styrene monomer, a crosslinkable monomer, and a functional monomer. Again, the polymer matrix material (multiple layers) of the first shell portion may encapsulate Fe3O4 nanoparticles 125.

[0074] The second shell portion 130 is formed from three layers (131, 132, and 133). The first layer contains Fe3O4 nanoparticles 134 bonded to functional groups provided on the surface of the first shell portion 120. In addition, the first layer includes a first layer of polymer matrix material 135 containing conjugated monomers and bulk monomers. Thus, the first layer of polymer matrix material extends from the surface of the first shell portion to near the top of the Fe3O4 nanoparticles 134. The second layer 132 extends beyond the first layer (and therefore beyond the top of the Fe3O4 nanoparticles 134), and this layer is formed from a second layer polymer material containing bulk monomers. The third layer 133 is formed on the top of the second layer, and this third layer is formed from a third layer polymer material containing bulk monomers and a second functional monomer having functional groups.

[0075] As used herein, "encapsulate" can mean either the complete encapsulation of one material within another, or the partial encapsulation of one material within another. It can also mean the state in which one material is trapped within the pores of another material.

[0076] It is noteworthy that the second and third layers of the polymer matrix material in the second shell portion have the function of preventing the superparamagnetic (Fe3O4) nanoparticles from leaching out when the beads are placed in a solvent. This is possible because, as described later, the polymer portion of the second shell is formed after the Fe3O4 nanoparticles are supported on the surface of the first shell (and core) of the newly formed beads. This allows the formed polymer shell to act as a barrier, protecting the Fe3O4 nanoparticles from leakage.

[0077] Any suitable functional group may be present on the functional monomer used for the first shell portion and the third layer of the second shell portion. As is understood, the desired functions of the functional groups of the first shell portion and the functional groups of the third layer of the second shell portion are different. The functional groups of the first shell portion (surface) are intended to bond to Fe3O4 nanoparticles so that Fe3O4 nanoparticles bond to the surface of the first shell portion. This bond may be by any suitable chemical interaction, such as coordination, covalent bonding, electrostatic force, or hydrogen bonding. The functional groups of the third layer of the second shell portion are intended to enable the easy functionalization of well-formed beads for any suitable intended application (e.g., covalent bonding of a ligand or antibody to the surface of well-formed beads). Nevertheless, the functional groups on the functional monomers of the first shell portion and the third layer of the second shell portion may be independently selected from one or more amino, carboxyl, epoxy, and hydroxyl groups. For example, the functional groups on the functional monomers of the first shell portion and the second layer of the second shell portion can be independently selected from a combination of a hydroxyl group and a carboxyl group, or a combination of an amino group and a carboxyl group.

[0078] The beads of the present invention are monodisperse as defined herein. More specifically, the beads disclosed herein may have a coefficient of variation based on their diameter of less than 15%, for example, less than 10%, for example, less than 5%. Even more specifically, the beads disclosed herein may be highly monodisperse as defined herein. For example, the beads may have a coefficient of variation based on their diameter of 2% or less.

[0079] The beads disclosed herein may have any suitable average diameter that is useful in the intended application (e.g., IVD). Examples of suitable average diameters are 0.2 to 5.0 μm, for example, 0.5 to 4.0 μm.

[0080] As described above, the core is formed from a polystyrene matrix material. This can be formed from a homopolymer of any suitable styrene monomer or a copolymer of two or more (e.g., 2, 3, 4, 5, or 6) styrene monomers. As should be understood, the term “styrene monomer” as used herein should be interpreted to generally cover the class of styrene monomers, i.e., styrene itself and its monomer derivatives. Examples of suitable styrene monomers that can be used to form a polystyrene polymer matrix material include, but are not limited to, styrene, styrene derivatives, and their copolymers. In certain embodiments that may be referenced herein, the styrene derivative may be selected from one or more of the group consisting of 4-methylstyrene, 3-methylstyrene, and 4-tert-butylstyrene. The polystyrene matrix material of the core may be an uncrosslinked material.

[0081] As described above, it has been surprisingly found that the beads disclosed herein can have a wide range of loading values ​​for superparamagnetic Fe3O4 nanoparticles without being plagued by the problems encountered in conventional designs. For example, all of the superparamagnetic Fe3O4 nanoparticles in a bead may account for 10-80% by weight, e.g., 20-70% by weight, e.g., 30-50% by weight, of the total weight of each bead. In certain embodiments that may be referenced herein, all of the superparamagnetic Fe3O4 nanoparticles in a bead may account for 30-50% by weight of the total weight of each bead. As can be understood, the amount of superparamagnetic Fe3O4 nanoparticles distributed in each part of the bead varies. For example, one or more of the following loading (or support) distributions may be applied: (a) The superparamagnetic Fe3O4 nanoparticles in the first batch may account for 0.1 to 5% by weight of the total weight of each bead. (b) The superparamagnetic Fe3O4 nanoparticles of the second batch may account for 0.5–10% by weight of the total weight of each bead; and (c) The superparamagnetic Fe3O4 nanoparticles in the first layer of the second shell portion may account for 9.4 to 79.4% by weight of the total weight of each bead, for example, 19.4 to 69.4% by weight, for example, 29.4 to 49.4% by weight.

[0082] As previously stated herein, crosslinkable monomers are used to form a crosslinked polymer matrix material in the first shell portion. The inclusion of crosslinks within the beads is thought to provide the beads with rigidity and / or mechanical strength, as well as resistance to swelling that may be caused by solvents. Any suitable crosslinkable monomer may be used to form the crosslinks in the first shell portion of the beads. Suitable crosslinkable monomers include divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dimethylolpropane tetraacrylate, dipentaerythritol pentaacrylate, and dipentaerythritol The crosslinkable monomer may be selected from one or more of the group consisting of trimethylolpropane / hexaacrylate, tripropylene diacrylate, trimethylolpropane ethoxylate triacrylate, trimethylolpropane propoxylate triacrylate, di(trimethylolpropane)tetraacrylate, glycerol propoxylate triacrylate, pentaerythritol propoxylate triacrylate, poly(ethylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate. In specific embodiments of the present invention that may be referenced herein, the crosslinkable monomer may be selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N-methylenebis(acrylamide).

[0083] Similar to the functional groups described above, the first functional monomer in the crosslinked polymer matrix material can be selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and 2-carboxyethyl acrylate oligomers. As can be understood, specific combinations of the functional groups described above can also be obtained by selection from these monomers.

[0084] Furthermore, the crosslinked polymer matrix material of the first shell portion is formed by the use of a styrene monomer. As described above, the styrene monomer can be selected from one or more of the group consisting of styrene and styrene derivatives. As described above, the styrene derivative can be selected from one or more of the group consisting of 4-methylstyrene, 3-methylstyrene, and 4-tert-butylstyrene.

[0085] As described above, the first shell portion may be formed from two layers of material instead of a single layer. That is, there may be a first layer of the first polymer matrix composition and a second layer of the second polymer matrix composition. In the two-layer embodiment, The first layer may be formed from a copolymer of styrene monomer and a crosslinkable monomer; and The second layer may be formed from a copolymer of styrene monomer, crosslinkable monomer, and functional monomer.

[0086] The styrene monomer, crosslinkable monomer, and functional monomer are as described above. It will be understood that the actual boundary between the two layers does not need to be perfectly clear. This is because the manufacturing method involves adding copolymers of styrene monomer and crosslinkable monomer to form a first layer, and then adding functional monomer in a later step to form a second layer. Also, therefore, a portion of the first layer may contain functional monomer. To avoid any doubt, the second layer is intended to form the outer surface of the first shell and is therefore desirable to present functional groups that can bond to superparamagnetic Fe3O4 nanoparticles and / or conjugated monomers (the latter enabling the formation of the polymer portion of the second shell). Further details of the manufacturing of the first shell will be provided below and in the experimental section.

[0087] As described above, the beads described herein include superparamagnetic Fe3O4 nanoparticles. These superparamagnetic Fe3O4 nanoparticles may have any suitable diameter that allows them to fall under the conventional definition of “nanoparticles”. However, in certain embodiments of the invention that may be referenced herein, the superparamagnetic Fe3O4 nanoparticles may have an average diameter of 5 to 15 nm.

[0088] In embodiments of the present invention as referred herein, the weight-to-weight ratio of styrene groups to crosslinking groups in the core and the first shell may be 20:1 to 1:2, for example, 10:1 to 1:1. In further or alternative embodiments as referred herein, the weight-to-weight ratio of styrene groups to functional groups in the core and the first shell may be 20:1 to 1:2, for example, 10:1 to 1:1. References to the core and the first shell are intended to mean that the combined weight of these parts should be taken into consideration when determining the overall load (or support or addition) of the components mentioned. For example, the core does not contain crosslinking groups or functional groups, but the weight of its styrene groups must be taken into consideration when determining the relative weight ratio of these components to the total weight of styrene groups in the core and the first shell.

[0089] Furthermore, in certain embodiments of the invention that may be referenced herein, the Fe3O4 nanoparticles include a portion of the Co3O4 and / or Mn3O4 nanoparticles. A suitable amount of either Co3O4 and / or Mn3O4 nanoparticles may be present in addition to the Fe3O4 nanoparticles. To avoid any doubt, where a weight percentage of Fe3O4 nanoparticles compared to the weight of the beads is provided herein, the weight of the Fe3O4 nanoparticles includes the weight of any present Co3O4 and / or Mn3O4 nanoparticles.

[0090] The second shell portion is formed from three separate material layers, as described above. The first layer is partially formed by Fe3O4 nanoparticles directly bonded to functional groups present on the outer surface of the first shell portion. This first layer also includes a first layer polymer matrix material containing a conjugated monomer and a bulk monomer. The conjugated monomer is a compound used as an anchor point on the surface of the first shell portion. Therefore, the conjugated monomer itself contains functional groups that can react with functional groups on the surface of the first shell portion, as well as functional groups that can be used in polymerization reactions. Any suitable conjugated monomer can be used herein. Examples of suitable conjugated monomers include, but are not limited to, methacryloyl chloride, 3-ethoxyacryloyl chloride, acryloyl chloride, 4-pentenoyl chloride, methacrylic anhydride, 4-pentenoic anhydride, crotonic anhydride, valeric anhydride, 10-undecenoyl chloride, glycidol methacrylate, glycidol, allyl glycidyl ether, and combinations thereof.

[0091] The bulk monomers forming the first and part of the second layer of the second shell portion may be selected from one or more polyether monomers, polyester monomers, polyacrylamide monomers, and polyacid monomers. Suitable bulk monomers include, but are not limited to, methyl methacrylate, acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, 2-carboxyethyl acrylate oligomer, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, and combinations thereof. In certain embodiments that may be referenced herein, the bulk monomer may be selected from methacrylic acid and / or 2-hydroxyethyl methacrylate.

[0092] The third layer of the second shell portion as described herein may be formed from a second functional monomer. The functional groups on this second functional monomer may be those described herein. Suitable examples of functional monomers include, but are not limited to, acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and one or more 2-carboxyethyl acrylate oligomers.

[0093] Furthermore, the polymer material used in the second shell portion may be crosslinked. For example, the first layer may be crosslinked, the second layer may be crosslinked, the third layer may be crosslinked, or any combination thereof. In embodiments that can be referenced herein, the first and second layers may be crosslinked, and the third layer may have no crosslinking or only slight crosslinking relative to the other layers.

[0094] The crosslinkable monomers used to form crosslinks in the second shell portion are divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, N,N'-methylenebis(acrylamide), bisphenol A epoxy acrylate, bisphenol diacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, propoxylated glycerol diacrylate, pentaerythritol propoxylate triacrylate, butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropanetetraacrylate, dipentaerythritol The crosslinkable monomer can be selected from one or more of the group consisting of polypenta / hexaacrylate, tripropylene diacrylate, trimethylolpropane ethoxylate triacrylate, trimethylolpropane propoxylate triacrylate, di(trimethylolpropane)tetraacrylate, glycerol propoxylate triacrylate, poly(ethylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate. For example, the crosslinkable monomer can be selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N'-methylenebis(acrylamide).

[0095] In embodiments disclosed herein, the combined weight of the second and third layers of the second shell may account for 1 to 30% by weight, preferably 2 to 20% by weight, of the total weight of each bead.

[0096] The advantages related to the beads disclosed herein include, but are not limited to, the following:

[0097] Of the three monomers, (a) styrene provides a nucleation template, leading to spherical monodisperse beads; (b) a crosslinking agent such as DVB provides crosslinking, making the beads rigid and resistant to organic solvents; and (c) a functional monomer facilitates the uploading of superparamagnetic nanoparticles (NPs). The copolymer beads obtained from the three monomers (styrene, crosslinking agent, and functional monomer) are spherical and highly monodisperse, and the bead diameter can be modulated by adjusting polymerization factors such as polymerization initiator concentration, PVP concentration, and monomer loading (or addition amount). In beads, various monomers supporting functional groups can be used on both the first and second shell surfaces. This allows for fine-tuning of the surface properties of the beads, making them easier to use in specific applications. The density of functional groups on the bead surface can be finely adjusted by adjusting the loading amount (or addition amount) of functional monomers (i.e., monomers that support functional groups). • High loading of superparamagnetic Fe3O4NPs onto beads (30-50 wt%) can be achieved by on-site generation of NPs. The size of Fe3O4NPs formed on the surface is well controlled by the well-controlled grafting of functional groups onto the PS core.

[0098] As should be understood, the structures and many advantages related to the beads described herein arise from the method of manufacturing the beads.

[0099] In the present invention, monodisperse superparamagnetic beads are obtained through four steps. Step 1: Synthesis of highly monodisperse copolymer beads of three monomers (styrene, crosslinking agent, and functional monomer) via "one-pot three-stage" polymerization. Step 2: Wash the as-synthesized copolymer beads with an organic solvent and perform surface modification (or surface modification). Step 3: On-site generation and upload of superparamagnetic Fe3O4NPs on copolymer beads. Step 4: Fabrication of functional coatings on superparamagnetic beads.

[0100] The inventors believe that the procedure outlined above is a novel process for producing monodisperse superparamagnetic beads, and is fundamentally different from conventional synthesis methods used to produce such beads. This process is outlined in Figure 2, where Figure 2A corresponds to the product obtained after step 1, Figure 2B corresponds to the product obtained after step 2, Figure 2C corresponds to the product obtained after step 3, and Figure 2D corresponds to the final product obtained after step 4. In Figure 2, 30 represents a pore, 40 represents Fe3O4NP, 60 represents the polymer portion of the first shell, and 80 represents the second shell.

[0101] More details on each step are provided below.

[0102] Step 1: Synthesis of highly monodisperse copolymer beads via a "one-pot three-step" procedure using three monomers (styrene, a crosslinking agent, and a functional monomer). This procedure provides the core and first shell polymer material (which does not contain Fe3O4 nanoparticles). The steps of this process are summarized below.

[0103] (a) The first step is dispersion polymerization of styrene in a solvent (e.g., ethanol containing 1-20 v% water). Nucleation is completed at this stage, consuming most of the styrene (e.g., 60-80 wt%), and thus the number of beads is fixed at this stage. The solvent may be a mixture of alcohols containing 1-20 v% water. The total polymerization time in the first step may be 2-12 hours, for example, 4-10 hours. (b) The second step is crosslinking by adding a crosslinking agent, and the crosslinked copolymerization of the crosslinking agent with the remaining styrene monomer forms a core-shell structure (see Figure 2A). The weight percentage of the crosslinking agent relative to the styrene can be 1 to 40% by weight, for example, 5 to 30%. In this step, the polymerization time can be 1 to 8 hours, for example, 2 to 6 hours. (c) In the third step, functional monomers are added and copolymerized with the remaining crosslinking agent and styrene to introduce hydrophilic functional groups to the bead surface. In addition, more than one functional monomer (e.g., two or three functional monomers) can be added at this stage to introduce polyfunctional groups in one step. The weight percentage of functional monomers relative to styrene can be 1 to 30% by weight, for example, 5 to 20% by weight. In this stage, the polymerization time can be 8 to 24 hours, for example, 10 to 20 hours.

[0104] This "one-pot, three-step" polymerization process can be carried out at any suitable temperature, for example, 40-80°C or 50-70°C.

[0105] Step 2: Thoroughly wash the as-synthesized copolymer beads from Step 1 with an organic solvent. The purpose of washing is to remove unreacted styrene, crosslinking agents and functional monomers, as well as stabilizers such as PVP (if used) from the beads. In addition, washing with a polar solvent can remove some linear polystyrene from the polymer matrix, loosening the beads and creating some pores to allow loading (or support) of superparamagnetic Fe3O4NP into the core and first shell of the beads. The washed beads can be redispersed in a solvent (e.g., water) for storage (see Figure 2B).

[0106] In this washing step, the beads can be washed with one or more of the following solvents: water, methanol, ethanol, isopropanol, THF, and mixtures thereof. The beads can be immersed in the solvent for several hours to remove the linear polystyrene and loosen the beads, or to create some pores inside the beads. During the washing or immersion process, the functional groups on the bead surface can be further modified to facilitate the uploading of superparamagnetic Fe3O4NPs. If epoxy groups are introduced onto the bead surface, the epoxy groups may undergo ring-opening hydrolysis, ring-opening aminolysis, or ring-opening polymerization during the immersion process to further modify (or modify) the bead surface. This will depend on the properties of the solvent used for this washing step or any reagents added to the solvent.

[0107] Step 3: Wash the beads, Fe 3+ Ions and Fe 2+ Ions in an appropriate molar ratio (for example, Fe 3+ :Fe 2+ The ions are dispersed in a solution containing them in a 2:1 molar ratio (for example, an aqueous solution such as a mixture of water and a polar solvent (e.g., THF)). The solution is then heated for several hours (e.g., up to 70°C) while stirring, allowing the two ions to interact with the functional groups on the bead surface. Some Fe 3+ Ions and Fe 2+ The ions will penetrate the polymer matrix of the beads, while some will be adsorbed onto the surface of the beads (through interaction with the functional groups on the surface of the beads as they are formed, i.e., the first shell surface). Then, water ammonia is added, and Fe 3+ Ions and Fe 2+ The ions are converted (or transformed) into Fe3O4NP using Insights. Before adding aqueous ammonia, a ligand that stabilizes Fe3O4NP, such as polyacrylic acid, tartaric acid, or citric acid, may be added to the solution to assist in the formation of superparamagnetic Fe3O4NP. The resulting superparamagnetic polymer beads (see C in Figure 2) are separated by centrifugation and washed with water and methanol to remove the loosened Fe3O4NP.

[0108] The added ligands that help stabilize the formation of Fe3O4NP can be polyacrylic acid, tartaric acid, citric acid, or a mixture thereof.

[0109] Step 4: The coating layer is fabricated on superparamagnetic polymer beads by grafting a polymer network around Fe3O4NP. To achieve polymer grafting, one or more compounds capable of reacting with functional groups on the first shell surface are used. This material is bifunctional, having a first functional group capable of reacting with functional groups on the first shell surface and a second functional group capable of participating in the polymerization reaction. Once this compound is immobilized on the bead surface, it is subjected to free radical polymerization conditions using a bulk monomer and, optionally, a crosslinking agent. Subsequently, one or more monomers having desired functional groups or desired final functionalities for downstream processes are added to the polymerization mixture to provide the final part of the second shell.

[0110] In step 4 of the above process, the superparamagnetic Fe3O4NP is bonded together by a functional polymer network grafted from the surface in step 2. To achieve polymer grafting, the monomer is modified on the surface by reaction with functional groups remaining on the bead surface in step 2, such as excess hydroxyl or amine groups. An anhydrous solvent is used in this step, which can be selected from one or more of 1,4-dioxane, tetrahydrofuran, diglyme, ethyl acetate, butyl acetate, acetone, and methyl ethyl ketone. Specific solvents that may be used in this step later in this specification may be tetrahydrofuran and diglyme. The reaction time may be 12 to 36 hours with inert gas purging. The resulting beads are washed with an organic solvent to remove excess monomer.

[0111] If necessary, an initiator may be used in the above process. The initiator may be selected from azo initiators such as AIBN and AMBN. Alternatively, the initiator may be selected from peroxide initiators and persulfates, including tert-amyl hydroperoxide, potassium persulfate, sodium persulfate, and ammonium persulfate.

[0112] The weight percentage of the crosslinking agent relative to the bulk monomer may be 5 to 70% by weight, preferably 10 to 50% by weight. In step 4, the polymerization time may be 10 to 30 hours, for example, 16 to 24 hours. In step 4, free radical polymerization can be carried out at a temperature of 40 to 80°C, for example, 50 to 70°C.

[0113] Thus, a method for preparing monodisperse superparamagnetic beads having a core-shell structure is also disclosed, and this method is (a) To provide monodisperse superparamagnetic precursor beads comprising a core portion formed from a polystyrene polymer matrix material, wherein the polystyrene polymer matrix material encapsulates a first batch of superparamagnetic Fe3O4 nanoparticles; A first shell portion located directly on the top of the core portion, formed from a crosslinked polymer matrix material composed of a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group, wherein the copolymer matrix material encapsulates a second batch of superparamagnetic Fe3O4 nanoparticles; and A second shell portion located directly on the top of a first shell portion, comprising superparamagnetic Fe3O4 nanoparticles and polymer precursor anchor points, both of which are bonded to functional groups on the surface of the first shell portion; and (b) Forming a functional coating layer on monodisperse superparamagnetic precursor beads by one-pot free radical polymerization, (i) Using a bulk monomer in the first step; and (ii) In the second step, monodisperse superparamagnetic beads are formed using a second functional monomer having a functional group, The functional coating layer is bonded to the first shell portion by polymer precursor anchor points.

[0114] The bulk monomer can be selected from one or more of the group consisting of polyether monomers, polyester monomers, polyacrylamide monomers, and polyacid monomers. For example, the bulk monomer can be selected from one or more of the group consisting of methyl methacrylate, acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, 2-carboxyethyl acrylate oligomer, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, and maleic anhydride, and optionally, the bulk monomer can be selected from methacrylic acid and / or 2-hydroxyethyl methacrylate.

[0115] In the method described above, the first step may use a mixture comprising a bulk monomer and an initiator. In this case, the initiator may be selected from one or more of the group consisting of tert-amyl hydroperoxide, potassium persulfate, sodium persulfate, ammonium persulfate, 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide, 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine]hydrate, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2''-azobis[2-(2-imidazolin-2-yl)propane], and 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride. The mixture may also include the following: (a) Crosslinking agents. For example, crosslinking agents include divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, N,N'-methylenebis(acrylamide), bisphenol A epoxy acrylate, bisphenol diacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, propoxylated glycerol diacrylate, pentaerythritol propoxylate triacrylate, butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta / hexaacrylate, The crosslinkable monomer can be selected from one or more of the group consisting of tripropylene diacrylate, trimethylolpropane ethoxylate triacrylate, trimethylolpropane propoxylate triacrylate, di(trimethylolpropane)tetraacrylate, glycerol propoxylate triacrylate, poly(ethylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate, for example, the crosslinkable monomer can be selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N'-methylenebis(acrylamide), and / or (b) Solvent. For example, the solvent may be selected from one or more of the group consisting of 1,4-dioxane, tetrahydrofuran, diglyme, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, and water.

[0116] The second functional monomer described above can be selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and 2-carboxyethyl acrylate oligomers.

[0117] In the above process: (a) The polymerization of the first and / or second step may be carried out at a temperature of 30 to 80°C, for example, 50 to 70°C; and / or (b) The total polymerization time for the first and second steps may be 10 to 30 hours, for example, 16 to 24 hours.

[0118] To carry out the above process, it is necessary to provide monodisperse superparamagnetic precursor beads. These beads are: (i) To provide naked monodisperse superparamagnetic beads, the beads are A core portion formed from a polystyrene polymer matrix material, wherein the polystyrene polymer matrix material encapsulates a first batch of superparamagnetic Fe3O4 nanoparticles; A first shell portion located directly on the top of the core portion, formed from a crosslinked polymer matrix material composed of a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group, wherein the copolymer matrix material encapsulates a second batch of superparamagnetic Fe3O4 nanoparticles; and A second shell portion located directly on the top of a first shell portion, comprising a second shell portion containing superparamagnetic Fe3O4 nanoparticles bonded to functional groups on the surface of the first shell portion; and (ii) Naked monodisperse superparamagnetic beads can be formed by a process that includes reacting an anchor material selected from one or more of the group consisting of methacryloyl chloride, 3-ethoxyacryloyl chloride, acryloyl chloride, 4-pentenoyl chloride, methacrylic anhydride, 4-pentenoic anhydride, crotonic anhydride, valeric anhydride, 10-undecenoyl chloride, glycidol methacrylate, glycidol, and allyl glycidyl ether to form polymer precursor anchor points on a first shell portion, thereby forming monodisperse superparamagnetic precursor beads.

[0119] Naked monodisperse superparamagnetic beads are (ai) To provide monodisperse beads, a core portion formed from a polystyrene polymer matrix material; and It comprises a first shell portion located directly on the top of the core portion and formed from a crosslinked polymer matrix material composed of a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group; and (aii) Forming naked monodisperse superparamagnetic beads by placing monodisperse beads in a solution containing Fe(III) salt and Fe(II) salt and adding a base. It can be formed by a process that includes

[0120] In the process described above, (a) Iron(III) salts may be selected from FeCl3 and / or Fe2(SO4)3; and / or (b) The iron(II) salt may be selected from one or more of the group consisting of FeCl2, FeSO4, and Fe(OAC)2; and / or (c) The base may be selected from one or more of the group consisting of ammonium hydroxide, NaOH, KOH, and amines; and / or (d) The solution may further contain CoCl2 and / or MnCl2.

[0121] The monodisperse beads described above can be formed by a "one-pot, three-step" continuous process, and this process is (a) In the first step, a polystyrene core is produced by dispersion polymerization of styrene monomer with an initiator and a polymer stabilizer in a mixture of water and alcohol to form a nucleated polystyrene core; (b) In the second step, a crosslinkable monomer is added to the mixture containing the nucleated polystyrene core; and (c) In the third step, a first functional monomer is added to the material obtained from the second step to provide monodisperse beads, and optionally, polystyrene beads are produced in insights, and the subsequent addition of a crosslinking agent and a functional monomer leads to spherical monodisperse copolymer beads bearing functional groups on the surface (styrene / crosslinking agent / functional monomer) without causing a second nucleation.

[0122] In the process described above, (a) The initiator is selected from azo initiators, and optionally the azo initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN) and 2,2'-azobis(2-methylbutyronitrile) (AMBN); and / or (b) The polymer stabilizer may be selected from one or more of the following: poly(vinylpyrrolidone) (PVP), polyethyleneimine (PEI), polyacrylic acid (PAA), polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPC), and chitosan; and / or (c) The styrene monomer may be selected from one or more of the group consisting of styrene and styrene derivatives, and optionally the styrene derivative may be selected from one or more of the group consisting of 4-methylstyrene, 3-methylstyrene, and 4-tert-butylstyrene; and / or (d) The alcohol may be selected from one or more of the group consisting of methanol, ethanol, isopropanol, or mixtures thereof; and / or (e) The volume ratio of alcohol to water can be 1:1 to 40:1, for example, 2:1 to 20:1.

[0123] The crosslinkable monomers used above are divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, butanediol dimethacrylate, tricyclodecanedimethanol diacrylate, pentaerythritol triacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol pentaacrylate, dipentaerythritol penta / hexaacrylate, tripropylene diacrylate, and trimethylolpropane ethoxylate triacrylate. The crosslinkable monomer can be selected from one or more of the group consisting of trimethylolpropanepropoxylate triacrylate, di(trimethylolpropane)tetoacrylate, glycerolpropoxylate triacrylate, pentaerythritolpropoxylate triacrylate, poly(ethylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate, and optionally the crosslinkable monomer can be selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N'-methylenebis(acrylamide).

[0124] The functional group of the monomer having the first functional group can be independently selected from one or more amino, carboxyl, epoxy, and hydroxyl groups. For example, the functional groups on the functional monomer of the second layer of the first and second shell portions can be independently selected from a combination of a hydroxyl group and a carboxyl group, or a combination of an amino group and a carboxyl group. More specifically, the first functional monomer can be selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and 2-carboxyethyl acrylate oligomers.

[0125] In the process described above, the weight ratio of styrene monomer to crosslinking agent can be 20:1 to 1:2, for example, 10:1 to 1:1. For example, the weight ratio of styrene monomer to the first functional monomer is 20:1 to 1:2, preferably 10:1 to 1:1.

[0126] Before using monodisperse beads, they are washed with one or more solvents before being used in subsequent process steps. The solvents used for washing can be selected from one or more of the group consisting of water, methanol, ethanol, isopropanol, and THF.

[0127] In the process described above, the monodisperse superparamagnetic beads obtained are (a) coefficient of variation based on their diameters of less than 15%, e.g., less than 10%, e.g., less than 5%, e.g., 2% or less; and / or (b) 0.2 to 5.0 microns, for example, an average diameter of 0.5 to 4.0 microns It may have.

[0128] The present invention relates to monodisperse superparamagnetic beads useful for in vitro diagnostic (IVD) assays and other applications. These monodisperse superparamagnetic beads have a polystyrene core coated with a styrene-crosslinking agent-functional monomer crosslinking layer, further coated with a superparamagnetic Fe3O4 nanoparticle layer, and with minimal dispersion within the polymer matrix. Finally, a functional coating is fabricated on the bead surface to bind and coat the superparamagnetic Fe3O4 nanoparticles and provide functional groups for IVD assays.

[0129] Thus, as can be understood, the superparamagnetic beads described herein may be used in IVD assays.

[0130] The advantages of the present invention are described below.

[0131] • Manufacture highly monodisperse superparamagnetic beads. Highly monodisperse copolymer beads containing three monomers [styrene, a crosslinking agent (e.g., DVB), and a functional monomer] are produced by a "one-pot, three-step" polymerization procedure. This procedure is simple and practical compared to state-of-the-art methods that require multi-step synthesis processes involving complex operations and consuming very long polymerization times. In the "one-pot three-step" polymerization procedure, each monomer plays a role: (a) styrene provides a nucleation template, leading to spherical monodisperse beads; (b) a crosslinking agent such as DVB provides crosslinking, making the beads more rigid and resistant to organic solvents; and (c) a functional monomer facilitates the uploading of superparamagnetic nanoparticles (NPs). The copolymer beads obtained from the three monomers (styrene, crosslinking agent, and functional monomer) are spherical and highly monodispersible, and the bead diameter can be modulated by adjusting polymerization factors such as polymerization initiator concentration, PVP concentration, and monomer loading (or addition amount). Various functional monomers can be copolymerized into the beads, allowing for the introduction of functional groups onto the bead surface. Such functional monomers include 2-carboxyethyl acrylate, acrylic acid, allylamine, 1-vinylimidazole, and 4-vinylpyridine. In addition, more than one functional monomer can be added together in the third step, allowing for the introduction of multiple functional groups in a single process. The density of functional groups on the bead surface can be finely adjusted by changing the loading amount (or addition amount) of functional monomers. After polystyrene nucleation is complete, the subsequent addition of a crosslinking agent (e.g., DVB) and functional monomers does not cause secondary nucleation, which is one reason why highly monodisperse beads can be produced. High loading (or high support) of superparamagnetic Fe3O4NPs (e.g., 30-50 wt%) onto beads can be achieved by on-site generation of NPs. The size of Fe3O4NPs formed on the surface is well controlled by the well-controlled grafting of functional groups onto the PS core.

[0132] Further aspects and embodiments of the present invention will be described by reference to the following non-limiting examples. [Examples]

[0133] (Examples) This invention relates to monodisperse superparamagnetic beads having a core-shell structure. The beads can be manufactured in a four-step process schematicly shown in Figure 2, which is fundamentally different from conventional synthesis methods for manufacturing monodisperse superparamagnetic beads.

[0134] For details on the four steps, please refer to Figure 2, which is explained in more detail below.

[0135] Step 1: Synthesis of highly monodisperse terpolymer beads via a "one-pot, three-step" polymerization procedure from three monomers (styrene, crosslinking agent, and functional monomer). (a) The first step is dispersion polymerization of styrene in ethanol containing 0-20 v% water. Nucleation is completed in this step to obtain a polystyrene core 20. Most of the styrene monomer (e.g., 60-80 wt%) is consumed, and thus the number of beads is fixed at this stage. The polymerization time is 2-12 hours, preferably 4-10 hours. (b) The second step is crosslinking by adding a crosslinking agent. Copolymerization of the crosslinking agent with the remaining styrene forms a core-shell structure (A). The polymerization time is 1 to 8 hours, preferably 2 to 6 hours. (c) In the third step, functional monomers are added to copolymerize the remaining small amount of crosslinking agent with styrene. The resulting terpolymer shell 40 contains hydrophilic functional groups on the bead surface. In addition, more than one functional monomer (e.g., two or three functional monomers) can be added at this stage to introduce multiple functional groups in a single run. The polymerization time is 8 to 24 hours, preferably 10 to 20 hours.

[0136] We have remarkably discovered that this "one-pot, three-step" continuous process can produce spherical, highly monodisperse terpolymer beads from three types of monomers (styrene, DVB, and functional monomers). This "one-pot, three-step" continuous process has three outstanding features. (a) Styrene provides a nucleation template, leading to spherical monodisperse beads. (b) A crosslinking agent (e.g., DVB) provides crosslinking and determines the rigidity and resistance of the beads to organic solvents. (c) Functional monomers can be further modified by introducing hydrophilic functional groups, such as -COOH, -NH2, epoxy groups, and acid anhydride groups, to facilitate the uploading of superparamagnetic Fe3O4NPs.

[0137] Furthermore, surprisingly, we found that the addition of crosslinking agents and functional monomers did not induce secondary nucleation. In addition, the addition of functional monomers at this stage achieved a high functional group density on the surface, enabling precipitation (or deposition) of superparamagnetic Fe3O4NP at insights.

[0138] Step 2: Washing and surface modification of as-synthesized terpolymer beads with an organic solvent. Wash the as-synthesized terpolymer beads (A) from Step 1 thoroughly with an organic solvent. The purpose of the washing is to remove unreacted styrene, crosslinking agent, functional monomers, and stabilizers such as PVP in the solution. In addition, washing with a polar solvent (example below) can also remove some of the linear polystyrene present in the polymer matrix, thereby loosening the beads and creating some pores (shown as 30) that are favorable for on-site loading of superparamagnetic Fe3O4 NPs. Redisperse the washed beads and store them in water to obtain (B).

[0139] During the washing or immersion process, the functional groups on the bead surface can be further modified to facilitate the upload of superparamagnetic Fe3O4 NPs. When epoxy groups are introduced onto the bead surface, the epoxy groups can undergo ring-opening hydrolysis, ring-opening aminolysis, and ring-opening polymerization to further modify the bead surface.

[0140] Step 3: On-site generation and upload of superparamagnetic Fe3O4 NPs onto terpolymer beads. Disperse the washed beads (B) in an aqueous solution (a mixture of water and a polar solvent such as THF) containing Fe 3+ and Fe 2+ in a molar ratio of 2:1, and heat the dispersion while stirring for several hours (e.g., up to 70 °C) to allow the two ions to interact with the functional groups of the beads. Without being bound by theory, during this process, some Fe 3+ ions and Fe 2+ ions are thought to penetrate the polymer matrix of the beads and some are adsorbed onto the bead surface. Then, add aqueous ammonia to Fe 3+ ions and Fe 2+The ions are converted into Fe3O4 nanoparticles (Fe3O4NP;60). After adding aqueous ammonia, a ligand that stabilizes Fe3O4NP, such as polyacrylic acid, tartaric acid, or citric acid, can be added to the bead solution to assist in the formation of superparamagnetic Fe3O4NP. The resulting superparamagnetic polymer beads (C) are separated by centrifugation and washed with water and methanol to remove loose Fe3O4NP 60.

[0141] Step 4: Fabrication of functional coatings on superparamagnetic beads. A coating layer 80 is fabricated on superparamagnetic polymer beads (C). The coating layer is achieved by reacting an anchor material with (C), followed by free radical polymerization of a bulk monomer, a second functional monomer, and an optional crosslinking agent. Furthermore, functional groups such as hydroxyl groups and carboxyl groups can be introduced onto the surface at the desired density.

[0142] In a specific example, the superparamagnetic Fe3O4NP 40 is encapsulated within a functional polymer network formed in step 2. To achieve polymer grafting, an anchoring material is introduced via a reaction with functional groups remaining on the bead surface in step 2, such as hydroxyl groups or amine groups. The anchoring material can be selected from those listed in the detailed description. The anhydrous solvent is selected from 1,4-dioxane, tetrahydrofuran, diglyme, ethyl acetate, butyl acetate, acetone, and methyl ethyl ketone. Tetrahydrofuran and diglyme are preferred. The reaction time is 12 to 36 hours with inert gas purging. The resulting beads are washed with an organic solvent to remove excess monomer.

[0143] Furthermore, the coating layer is grafted by reacting anchor groups (e.g., unsaturated groups) on the bead surface with a bulk monomer in the first step, a second functional monomer in the second step, and optionally a crosslinking agent. Specific materials are listed in the detailed description. In this step, the polymerization time is 10 to 30 hours, preferably 16 to 24 hours. Free radical polymerization can be carried out at a temperature of 40 to 80°C, preferably 50 to 70°C.

[0144] material: The materials were purchased from the following suppliers. Styrene: Stabilized with TBC (4-tert-butylcatechol), Tokyo Chemical Industry Co., Ltd. (TCI), >99.0% (GC). Azobisisobutyronitrile (AIBN): Sigma-Aldrich (12% by weight in acetone). Sodium persulfate (Na2S2O8, SPS): Alfa Aesar, crystalline, 98%. Polyvinylpyrrolidone (PVP, K30, MW=40,000): TCI, total nitrogen 12.0%~12.8% (calculated for anhydrous substance); water up to 7.0%, K value 26.0~34.0. Divinylbenzene (DVB, m- and p-mixture): TCI, 50.0% (GC) (containing ethyl vinylbenzene and diethylbenzene) (stabilized with 4-tert-butylcatechol). Acrylic acid: TCI, >99.0% (GC) (stabilized with monomethyl ether hydroquinone). Glycidyl methacrylate: Sigma-Aldrich, ≥97.0% (GC). 2-Hydroxyethyl methacrylate: Sigma-Aldrich, ≥99.0% 2-carboxyethyl acrylate oligomer: Sigma-Aldrich, MEHQ at 2000 ppm as an inhibitor. Ethanol: 99%. Citric acid: Sigma-Aldrich, 98%. Trimethylolpropane triacrylate: Sigma-Aldrich Methyl methacrylate: TCI 2-(2-aminoethoxy)ethanol: Sigma-Aldrich, 98%. Deionized water: Available from ELGA Ultrapure Water Treatment Systems (PURELAB Option). Scanning electron microscopy (SEM) imaging was performed using a JEOL JSM6700F. Transmission electron microscopy (TEM) imaging was performed using a JEOL 2100F. Mechanical Stirrer: Wiggens, WB2000-M Overhead Stirrer

[0145] Thermogravimetric analysis (TGA) was performed using a Shimadzu DTG-60. A sample containing approximately 15 mg (dry mass) of beads was dried in an oven at 70°C. An empty crucible containing a reference (or reference) crucible and the sample crucible was placed in parallel on a holder. The weight difference between the reference crucible and the sample crucible was eliminated. The bead sample was then filled into the sample crucible. The temperature profile was set in increments of 20°C / min from room temperature to 700°C, with a holding time of 10 minutes at 100°C. The entire process was carried out under N2 purging. The magnetic content was calculated using the following formula.

[0146]

number

[0147] (In the formula, W1 is the weight at 100°C after holding for 10 minutes, and W2 is the weight at 700°C.)

[0148] Superparamagnetic properties were measured using Quantum Design's 6000PPMS (Physical Property Measurement System). A sample containing approximately 15 mg (dry mass) of beads was dried in a vacuum oven at room temperature. The magnetization curve [MH] was obtained using a physical property measurement system (Quantum Design, PPMS 6000) equipped with a vibrating sample magnetometer (P525) and a vibrating sample magnetometer (Micro Sense, EV9). MH was measured at 300 K with an appropriate electric field strength applied.

[0149] The coefficient of variation (CV) of polystyrene beads was calculated from SEM images (10,000x magnification). 100 beads were measured using IMAGE J, and the average diameter and CV were calculated.

[0150] General Considerations Mechanical stirring (200 rpm) was used for all polymerization processes. Before use, DI water and ethanol were bubbling with nitrogen for 20 minutes to remove oxygen. All chemicals (including AIBN, SPS, PVP, acrylic acid, allylamine, DVB, styrene, glycidol, glycidyl methacrylate, bisphenol A diglycidyl ether, and 2-hydroxyethyl methacrylate) were used as received without purification. All polymerization and reactions were carried out under nitrogen protection using the standard Schlenk wire technique.

[0151] Example 1: Synthesis of monodisperse beads supporting -COOH and -OH groups The following describes a "one-pot, three-step" polymerization process for synthesizing monodisperse beads supporting -COOH and -OH groups.

[0152] In a 250 mL three-necked round-bottom glass reactor equipped with a mechanical stirrer, AIBN solution (2 g, 12 wt% in acetone) was added, and nitrogen was introduced through one of the necks to remove the acetone solvent. After 10 minutes of nitrogen flow, dried AIBN powder was observed at the bottom of the reactor. Next, PVP (0.2 g), ethanol (80 mL), and DI water (20 mL) were added. This mixture was stirred at room temperature for 5 minutes to obtain a clear solution, which was then heated to 60°C in an oil bath, and styrene (7.5 mL) was added. After 4 hours, a white colloidal solution was formed, indicating polymerization of styrene. Next, a solution of DVB (3 mL of DVB in 7 mL of ethanol) was gradually added over 30 minutes using a constant-pressure dropping funnel. After the addition of the DVB solution was complete, the reaction (crosslinking polymerization) was continued for 3 hours. Next, monomer solutions of 2-hydroxyethyl methacrylate (0.75 g in 5 mL of ethanol) and 2-carboxyethyl acrylate oligomer (0.75 g in 5 mL of ethanol, neutralized with aqueous ammonia, 25% w / w) were added using a syringe. The reaction (polymerization) was continued for 6 hours to obtain a milky colloidal solution. SEM images showed that the as-synthesized beads were spherical and monodisperse with a diameter of approximately 1.0 micron (Figure 3). The obtained beads were named "A-1".

[0153] Example 2: Cleaning and surface modification of beads A-1 The beads of A-1 (50 mL dispersion, mass concentration approximately 8 wt%) were separated by centrifugation and redispersed in ethanol by sonication. The beads were washed with a mixture of ethanol (50 mL x 2), THF, and water (1:1 volume ratio; 50 mL x 2). The resulting beads were centrifuged and redispersed in THF (50 mL) to obtain B-1. SEM images showed that the beads were spherical and monodisperse with a diameter of approximately 1.0 micron.

[0154] Example 3: Magnetization of Bead B-1 In a 250 mL three-necked round-bottom glass reactor equipped with a mechanical stirrer, 8.0 g of FeCl3 and 100 mL of water were added and stirred to form a brown solution. Then, 3.1 g of FeCl2 and 20 mL of THF were added. This mixture was stirred for 30 minutes to form a solution, to which an aqueous solution of beads B-1 (50 mL, mass concentration approximately 8 wt%) was added and stirred at 70°C for 3 hours. After the mixture cooled to room temperature, 30 mL of aqueous ammonia (25 wt%) was added under vigorous stirring to immediately obtain a black slurry. 0.5 g of citric acid solution (in 10 mL of water) was added to the reactor, and the black slurry was heated at 70°C for 1 hour to complete the reaction. These superparamagnetic beads were purified by several centrifugal shifts to remove unbound superparamagnetic NPs, and finally redispersed in DI water (100 mL, approximately 4 wt%). The resulting dark brown superparamagnetic beads were named "C-1". The TGA results showed that the beads contained 43 wt% NPs. The resulting magnetic beads were monodisperse, with some Fe3O4NPs uniformly scattered within the core and a very thick layer of fluffy Fe3O4NPs on the surface. The thickness of the Fe3O4NP layer was approximately 70–100 nm around the bead (Figure 4). Superparamagnetic properties were measured using a Quantum Design 6000 PPMS (Physical Property Measurement System). Saturation magnetization (MS) values ​​were normalized using the mass of the beads measured by atomic absorption spectrometry. Figure 5 shows a typical hysteresis curve of sample C-1 at room temperature. The hysteresis loop exhibits superparamagnetic behavior, as evidenced by the zero coercivity and remanence on the magnetization loop.

[0155] Example 4: Fabrication of functional coatings Bonding step: Superparamagnetic beads C-1 (30 mL, 4 wt% in anhydrous THF) and methacryloyl chloride (100 mg, dissolved in 10 mL of anhydrous THF) were added to a 250 mL three-necked round-bottom glass reactor equipped with a mechanical stirrer. The mixture was heated to 60°C under nitrogen purge and maintained for 18 hours, then cooled to room temperature. The beads were washed with THF to remove excess methacryloyl chloride, and then washed with water. The beads were stored in water under nitrogen protection.

[0156] Emulsion polymerization coating step: The modified superparamagnetic beads from the previous step (10 mL, 4 wt% in water) were added to a 50 mL centrifuge tube containing 30 mL of DI water. This mixture was then added to a 250 mL three-necked round-bottom glass reactor equipped with a mechanical stirrer. The reactor was flushed with nitrogen to remove oxygen, and then SPS (30 mg, dissolved in 2 mL of water) was added. The mixed dispersion was heated to 60°C and stirred for 10 minutes. Subsequently, a monomer mixture containing methyl methacrylate (400 mg) and trimethylolpropane triacrylate (200 mg) was added in four batches over 1 hour (thus at 20-minute intervals). After the addition of the monomer mixture was complete, the reaction (coating polymerization) was continued at 60°C for 6 hours. After cooling to room temperature, the coated superparamagnetic beads were separated using a magnetic separator and washed with ethanol (50 mL x 2) and DI water (50 mL x 2). The coated superparamagnetic bead was named D-1. The TEM image in Figure 6 clearly shows that the bead structure corresponds to the one proposed in Figure 2(D). The edges of the bead were sufficiently covered with a polymer layer that could prevent the elution of magnetic particles.

[0157] Example 5: Synthesis of monodisperse beads supporting epoxy groups The following describes a "one-pot, three-step" polymerization process for synthesizing monodisperse beads made from three types of monomers (styrene, DVB, and GMA), and the beads supporting epoxy groups.

[0158] In a 250 mL three-necked round-bottom glass reactor equipped with a mechanical stirrer, AIBN solution (2.0 g, 12 wt% in acetone) was added, and nitrogen was introduced through one of the necks to remove the acetone solvent. After 10 minutes of nitrogen flow, dried AIBN powder was observed at the bottom of the reactor. Next, PVP (0.3 g), ethanol (80 mL), and DI water (16 mL) were added. This mixture was stirred at room temperature for 5 minutes to obtain a clear solution, which was then heated to 60°C in an oil bath, after which styrene (7.5 mL) was added.

[0159] After 6 hours, a white colloidal solution was formed, indicating styrene polymerization. Next, a solution of DVB (1.5 mL dissolved in 8.5 mL of ethanol) was gradually added over 30 minutes using a constant-pressure dropping funnel. After the addition of the DVB solution was complete, the reaction (crosslinking polymerization) was continued for 2 hours. Then, a solution of glycidyl methacrylate (GMA) (2.0 g dissolved in 10 mL of ethanol) was added using a syringe. The reaction (polymerization) was continued for 24 hours to obtain a milky colloidal solution. SEM images showed that the synthesized beads were spherical and monodisperse with a diameter of approximately 1.0 micron (Figure 7). The obtained beads were named "A-2".

[0160] Example 6: Cleaning and surface modification of A-2 The A-2 bead dispersion (50 mL, mass concentration approximately 8 wt%) was separated by centrifugation and redispersed in ethanol by sonication. The beads were washed with ethanol (50 mL x 2) and a mixture of THF and water (1:1 volume ratio; 50 mL x 2), and the beads were redispersed in a THF solution containing 1 g of 2-(2-aminoethoxy)ethanol and stirred at room temperature for 20 hours. The beads were then washed five times with water and redispersed in water to obtain B-2. SEM images showed that the beads were spherical monodisperse with a diameter of approximately 1.0 micron.

[0161] Example 7: Magnetization of B-2 beads In a 250 mL three-necked round-bottom glass reactor equipped with a mechanical stirrer, FeCl3 (8.0 g) and 100 mL of water were added and stirred to form a brown solution. Then FeCl2 (3.1 g) and THF (20 mL) were added. This mixture was stirred for 30 minutes to form a solution, to which the solution of beads B-2 (50 mL, approximately 8 wt%) in water was added, and the mixture was stirred at 65 °C for 3 hours. After the mixture was cooled to room temperature, ammonia water (25 wt%, 25 mL) was added under vigorous stirring to immediately obtain a black slurry. Citric acid solution (0.5 g, in 10 mL of water) was added to the reactor, and the black slurry was heated to 70 °C for 1 hour to complete the reaction. These superparamagnetic beads were purified by several centrifugal shifts to remove unbound superparamagnetic NPs, and finally redispersed in DI water (100 mL, approximately 4 wt%). The resulting blackish-brown superparamagnetic beads were named "C-2". The TGA results showed that the beads contained 41 wt% NPs. The TEM image of C-2 is shown in Figure 8.

[0162] Example 8: Fabrication of functional coatings Bonding step: Superparamagnetic beads C-2 (10 mL, 4 wt% in anhydrous THF), methacrylic anhydride (120 mg, dissolved in 30 mL of anhydrous THF), and triethylamine (TEA) (20 μL, dissolved in 10 mL of anhydrous THF) were added to a 250 mL three-necked round-bottom glass reactor equipped with a mechanical stirrer. The mixture was heated to 65 °C under nitrogen purge and maintained for 3 hours, then cooled to room temperature. The beads were washed with THF to remove excess methacrylic anhydride, and then washed with water. The beads were stored in water under nitrogen protection.

[0163] Coating step by graft polymerization: The modified superparamagnetic beads from the previous step (10 mL, 4 wt% in water) were added to a 50 mL centrifuge tube containing 30 mL of DI water. This mixture was added to a 250 mL three-necked round-bottom glass reactor equipped with a mechanical stirrer. The reactor was flushed with nitrogen to remove oxygen, and then SPS (30 mg, dissolved in 2 mL of water) was added. The mixed dispersion was heated to 60°C and stirred for 10 minutes. Subsequently, a monomer mixture containing methyl methacrylate (400 mg) and bisphenol A type epoxy acrylate (150 mg) was added in four batches (with 1 hour intervals) over 4 hours. After the addition of the monomer mixture was complete, the reaction (coating polymerization) was continued at 60°C for 4 hours. Acrylic acid monomer (100 mg, diluted with water and further neutralized with aqueous ammonia, 25% (w / w)) was added, and the reaction was continued at 60°C for 16 hours. After cooling to room temperature, the coated superparamagnetic beads were separated using a magnetic separator and washed with ethanol (50 mL x 2) and DI water (50 mL x 2). The coated superparamagnetic beads were named D-2. The TEM image in Figure 9 shows a smoother outer layer compared to C-1, indicating that the nanoparticles are effectively encapsulated within the polymer network.

[0164] Comparative Example 1. Synthesis of monodisperse beads without functional groups The following describes a "one-pot, two-step" polymerization process for synthesizing monodisperse beads from two monomers (styrene-DVB) that do not contain functional groups.

[0165] In a 250 mL three-necked round-bottom glass reactor equipped with a mechanical stirrer, AIBN solution (2.2 g, 12 wt% in acetone) was added, and nitrogen was introduced through one of the necks to remove the acetone. After 10 minutes of nitrogen flow, dried AIBN powder was observed at the bottom of the reactor. Next, PVP (0.9 g), ethanol (90 mL), and DI water (10 mL) were added. The mixture was stirred at room temperature for 5 minutes to obtain a clear solution, which was then heated to 70°C in an oil bath, after which styrene (7.5 mL) was added.

[0166] After 6 hours, a white colloidal solution was formed, indicating styrene polymerization. Next, a solution of DVB (1.5 mL dissolved in 8.5 mL of ethanol) was gradually added over 30 minutes using a constant-pressure dropping funnel. After the addition of the DVB solution was complete, the reaction (crosslinking polymerization) was continued for 20 hours to obtain a milky colloidal solution. SEM images showed that the synthesized beads were spherical and monodisperse with a diameter of approximately 0.8 microns (Figure 10). The obtained beads were named "CE-1". Since no functional monomers were added, the CE-1 beads do not have functional groups on their surface, and the beads are hydrophobic.

[0167] Comparative Example 2: Cleaning and immersion of CE-1 The as-synthesized poly(styrene-DVB) beads of CE-1 (50 mL dispersion, mass concentration approximately 8 wt%) were separated by centrifugation and redispersed in ethanol by sonication. The beads were washed with ethanol (50 mL x 2), a mixture of THF and water (1:1 volume ratio; 50 mL x 2), and THF (50 mL x 2). The washed beads were named "CE-2". SEM images showed that the beads were spherical and monodisperse with a diameter of approximately 0.8 microns.

[0168] Comparative Example 3: Magnetization of CE-2 beads In a 250 mL three-necked round-bottom glass reactor equipped with a mechanical stirrer, FeCl3 (8.0 g) and 100 mL of water were added and stirred to form a brown solution. Then FeCl2 (3.1 g), citric acid (8.5 g), and THF (20 mL) were added. This mixture was stirred for 30 minutes to form a solution, to which a solution of beads CE-2 in THF (50 mL, mass concentration approximately 8 wt%) was added, and the mixture was stirred at 60°C for 3 hours. After the mixture was cooled to room temperature, aqueous ammonia (25 wt%, 30 mL) was added under vigorous stirring to immediately obtain a black slurry. The black slurry was heated at 60°C for 1 hour to complete the reaction. These superparamagnetic beads were purified by several centrifugal shifts to remove unbound superparamagnetic NPs, and finally redispersed in DI water (100 mL, approximately 4 wt%).

[0169] The resulting beads were still white and were named "CE-3". TGA results showed that the beads contained 1 wt% NPs. This comparative experiment showed that poly(styrene-DVB) beads could not bind a sufficient amount of superparamagnetic Fe3O4 NPs. This was further confirmed by TEM images (Figure 11), which showed that only a small amount of magnetic NPs were formed on the surface of CE-3. Magnetic nanoparticles aggregate only in the gaps between microspheres.

Claims

1. A monodisperse superparamagnetic bead having a core-shell structure, wherein the bead is A core portion formed from a polystyrene polymer matrix material, wherein the polystyrene polymer matrix material is a first batch of superparamagnetic Fe ３ O ４ Core section containing nanoparticles; A first shell portion is directly located on the top of the core portion and is formed from a crosslinked polymer matrix material formed from a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group, wherein the copolymer matrix material is a second batch of superparamagnetic Fe ３ O ４ A first shell portion for encapsulating nanoparticles; and The second shell section is located directly on top of the first shell section. The second shell portion is formed as a first layer, a second layer, and a third layer, The first layer is superparamagnetic Fe ３ O ４ The material comprises nanoparticles and a first layer polymer matrix material containing conjugated monomers and bulk monomers; The second layer extends beyond the first layer and is formed from a second layer polymer material containing a bulk monomer; and The third layer is located on top of the second layer and is formed from a third layer polymer material comprising a bulk monomer and a second functional monomer having a functional group. The superparamagnetic Fe of the first layer ３ O ４ The nanoparticles directly bond to the functional groups present on the outer surface of the first shell portion; The first layer polymer matrix material surrounds the superparamagnetic Fe ３ O ４ nanoparticles; and Monodisperse superparamagnetic beads, wherein the second layer polymer matrix material extends from the first layer polymer matrix material and forms the outer surface of each monodisperse superparamagnetic bead.

2. The beads according to claim 1, wherein the polymer matrix material of the second and third layers functions to prevent the dissolution of superparamagnetic magnetic nanoparticles when the beads are placed in a solvent.

3. The beads according to claim 1 or 2, wherein the functional groups on the functional monomers of the third layer of the first shell portion and the second shell portion are independently selected from one or more amino, carboxyl, epoxy, and hydroxyl groups, and optionally, the functional groups on the functional monomers of the second layer of the first shell portion and the second shell portion are independently selected from a combination of a hydroxyl group and a carboxyl group, or a combination of an amino group and a carboxyl group.

4. The beads according to any one of claims 1 to 3, wherein the beads have a coefficient of variation based on their diameter of less than 15%, for example, less than 10%, for example, less than 5%.

5. The beads according to claim 4, wherein the beads have a coefficient of variation based on their diameter of 2% or less.

6. The beads according to any one of claims 1 to 5, wherein the beads have an average diameter of 0.2 to 5.0 μm, for example, 0.5 to 4.0 μm.

7. The beads according to any one of claims 1 to 6, wherein the polystyrene polymer matrix material is formed from one or more of the group consisting of styrene, styrene derivatives, and copolymers thereof, and optionally the styrene derivative is selected from one or more of the group consisting of 4-methylstyrene, 3-methylstyrene, and 4-tert-butylstyrene.

8. Superparamagnetic Fe in beads ３ O ４ The beads according to any one of claims 1 to 7, wherein all of the nanoparticles account for 10 to 80% by weight, for example, 20 to 70% by weight, or for example, 30 to 50% by weight, of the total weight of each bead.

9. (a) Superparamagnetic Fe of the first batch ３ O ４ Nanoparticles make up 0.1 to 5% by weight of the total weight of each bead; and / or (b) Second batch of superparamagnetic Fe ３ O ４ Nanoparticles account for 0.5 to 10% by weight of the total weight of each bead; (c) Superparamagnetic Fe in the first layer of the second shell ３ O ４ The beads according to claim 8, wherein nanoparticles account for 9.4 to 79.4% by weight of the total weight of each bead, for example, 19.4 to 69.4% by weight, for example, 29.4 to 49.4% by weight.

10. The crosslinkable monomers include divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, butanediol dimethacrylate, tricyclodecanedimethanol diacrylate, pentaerythritol triacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol pentaacrylate, dipentaerythritol penta / hexaacrylate, tripropylene diacrylate, trimethylolpropane ethoxylate triacrylate, and trimethylolpropane acrylate. Beads according to any one of claims 1 to 9, wherein the beads are selected from the group consisting of panpropoxylate triacrylate, di(trimethylolpropane)tetoacrylate, glycerolpropoxylate triacrylate, pentaerythritolpropoxylate triacrylate, poly(ethylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate, and optionally the crosslinkable monomer is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N'-methylenebis(acrylamide).

11. The beads according to any one of claims 1 to 10, wherein the first functional monomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and 2-carboxyethyl acrylate oligomers.

12. The beads according to any one of claims 1 to 11, wherein the styrene monomer is selected from one or more of the group consisting of styrene and styrene derivatives, and optionally the styrene derivative is selected from one or more of the group consisting of 4-methylstyrene, 3-methylstyrene, and 4-tert-butylstyrene.

13. The first shell further comprises a first layer of the first polymer matrix composition and a second layer of the second polymer matrix composition. The first layer is formed from a copolymer of a styrene monomer and a crosslinkable monomer; and The beads according to any one of claims 1 to 12, wherein the second layer is formed from a copolymer of a styrene monomer, a crosslinkable monomer, and a functional monomer.

14. The superparamagnetic Fe ３ O ４ The beads according to any one of claims 1 to 13, wherein the nanoparticles have an average diameter of 5 to 15 nm.

15. (a) The weight-to-weight ratio of styrene groups to crosslinking groups in the core and the first shell is 20:1 to 1:2, for example, 10:1 to 1:1, and / or (b) The beads according to any one of claims 1 to 14, wherein the weight-to-weight ratio of styrene groups to functional groups in the core portion and the first shell portion is 20:1 to 1:2, for example, 10:1 to 1:

1.

16. The Fe ３ O ４ Nanoparticles further Co ３ O ４ and / or Mn ３ O ４ Beads according to any one of claims 1 to 15, comprising nanoparticles.

17. The beads according to any one of claims 1 to 16, wherein the conjugated monomer is selected from one or more of the group consisting of methacryloyl chloride, 3-ethoxyacryloyl chloride, acryloyl chloride, 4-pentenoyl chloride, methacrylic anhydride, 4-pentenoic anhydride, crotonic anhydride, valeric anhydride, 10-undecenoyl chloride, glycidol methacrylate, glycidol, and allyl glycidyl ether.

18. The beads according to any one of claims 1 to 17, wherein the bulk monomer is selected from one or more of polyether monomers, polyester monomers, polyacrylamide monomers, and polyacid monomers, and optionally the bulk monomer is selected from one or more of the group consisting of methyl methacrylate, acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, 2-carboxyethyl acrylate oligomer, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, and maleic anhydride, and optionally the bulk monomer is selected from methacrylic acid and / or 2-hydroxyethyl methacrylate.

19. The beads according to any one of claims 1 to 18, wherein the second functional monomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and 2-carboxyethyl acrylate oligomers.

20. The second shell portion further comprises a crosslinkable monomer in the first and / or second and / or third layer, optionally the crosslinkable monomer being divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, N,N'-methylenebis(acrylamide), bisphenol A epoxy acrylate, bisphenol diacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, propoxylated glycerol diacrylate, pentaerythritol propoxylate triacrylate, butanediol dimethacrylate, tricyclodecanedimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipenta Beads according to any one of claims 1 to 19, wherein the crosslinkable monomer is selected from one or more of the group consisting of erythritol penta / hexaacrylate, tripropylene diacrylate, trimethylolpropane ethoxylate triacrylate, trimethylolpropane propoxylate triacrylate, di(trimethylolpropane)tetraacrylate, glycerol propoxylate triacrylate, poly(propylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate, for example, the crosslinkable monomer is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N'-methylenebis(acrylamide).

21. The superparamagnetic bead according to any one of claims 1 to 20, wherein the combined weight of the second and third layers of the second shell accounts for 1 to 30% by weight, preferably 2 to 20% by weight, of the total weight of each bead.

22. A method for preparing monodisperse superparamagnetic beads having a core-shell structure, (a) To provide monodisperse superparamagnetic precursor beads comprising a core portion formed from a polystyrene polymer matrix material, wherein the polystyrene polymer matrix material is a first batch of superparamagnetic Fe ３ O ４ Encapsulating nanoparticles; A first shell portion is directly located on the top of the core portion and is formed from a crosslinked polymer matrix material formed from a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group, wherein the copolymer matrix material is a second batch of superparamagnetic Fe ３ O ４ A first shell portion for encapsulating nanoparticles; and A second shell portion located directly on the top of the first shell portion, wherein superparamagnetic Fe ３ O ４ A second shell portion comprising nanoparticles and polymer precursor anchor points, both of which are bonded to functional groups on the surface of the first shell portion; and (b) Forming a functional coating layer on monodisperse superparamagnetic precursor beads by one-pot free radical polymerization. (i) Using a bulk monomer in the first step; and (ii) In the second step, monodisperse superparamagnetic beads are formed using a second functional monomer having a functional group, A method comprising bonding the functional coating layer to a first shell portion by polymer precursor anchor points.

23. The method according to claim 22, wherein the bulk monomer is selected from one or more of the group consisting of polyether monomers, polyester monomers, polyacrylamide monomers, and polyacid monomers.

24. The method according to claim 23, wherein the bulk monomer is selected from one or more of the group consisting of methyl methacrylate, acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, 2-carboxyethyl acrylate oligomer, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, and maleic anhydride, and optionally the bulk monomer is selected from methacrylic acid and / or 2-hydroxyethyl methacrylate.

25. The method according to any one of claims 22 to 24, wherein the first step uses a mixture comprising the bulk monomer and an initiator, and optionally the initiator is selected from one or more of the group consisting of tert-amyl hydroperoxide, potassium persulfate, sodium persulfate, ammonium persulfate, 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide, 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] hydrate, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2''-azobis[2-(2-imidazolin-2-yl)propane], and 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride.

26. The mixture in the first step is further, (a) Crosslinking agent, optionally selected from divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, N,N'-methylenebis(acrylamide), bisphenol A epoxy acrylate, bisphenol diacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, propoxylated glycerol diacrylate, pentaerythritol propoxylate triacrylate, butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta / hexaacrylate The crosslinkable monomer is selected from one or more of the group consisting of di(b)acrylate, tripropylene diacrylate, trimethylolpropane ethoxylate triacrylate, trimethylolpropane propoxylate triacrylate, di(trimethylolpropane)tetraacrylate, glycerol propoxylate triacrylate, poly(ethylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate, for example, the crosslinkable monomer is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N'-methylenebis(acrylamide), and / or (b) Solvent, optionally selected from one or more of the group consisting of 1,4-dioxane, tetrahydrofuran, diglyme, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, and water. The method according to claim 25, including the method described in claim 25.

27. The method according to any one of claims 22 to 26, wherein the second functional monomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and 2-carboxyethyl acrylate oligomers.

28. (a) The polymerization of the first step and / or the second step is carried out at a temperature of 30 to 80°C, for example, 50 to 70°C; and / or (b) The method according to any one of claims 22 to 27, wherein the total polymerization time for the first and second steps is 10 to 30 hours, for example, 16 to 24 hours.

29. The monodisperse superparamagnetic precursor beads are (i) To provide naked monodisperse superparamagnetic beads, the beads are A core portion formed from a polystyrene polymer matrix material, wherein the polystyrene polymer matrix material is a first batch of superparamagnetic Fe ３ O ４ The core portion that encapsulates nanoparticles; A first shell portion is directly located on the top of the core portion and is formed from a crosslinked polymer matrix material formed from a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group, wherein the copolymer matrix material is a second batch of superparamagnetic Fe ３ O ４ A first shell portion for encapsulating nanoparticles; and A second shell portion located directly on the top of the first shell portion, wherein superparamagnetic Fe is bonded to the functional groups on the surface of the first shell portion. ３ O ４ It comprises a second shell portion containing nanoparticles; and (ii) The method according to any one of claims 22 to 28, wherein the naked monodisperse superparamagnetic beads are formed by a process comprising reacting the naked monodisperse superparamagnetic beads with an anchor material selected from one or more of the group consisting of methacryloyl chloride, 3-ethoxyacryloyl chloride, acryloyl chloride, 4-pentenoyl chloride, methacrylic anhydride, 4-pentenoic anhydride, crotonic anhydride, valeric anhydride, 10-undecenoyl chloride, glycidol methacrylate, glycidol, and allyl glycidyl ether to form polymer precursor anchor points on the first shell portion.

30. The naked monodisperse superparamagnetic beads are (ai) To provide monodisperse beads, wherein the monodisperse beads have a core portion formed from a polystyrene polymer matrix material; and The first shell portion is located directly on the top of the core portion and is formed from a crosslinked polymer matrix material made of a styrene monomer, a crosslinkable monomer, and a first functional monomer having a functional group; and (aii) The naked monodisperse superparamagnetic beads are formed by placing the monodisperse beads in a solution containing Fe(III) salt and Fe(II) salt and adding a base. The method according to claim 29, formed by a process including the following.

31. (a) The iron(III) salt is FeCl ３ and / or Fe ２ (SO ４ ) ３ Selected from; and / or (b) The iron(II) salt is FeCl ２ FeSO ４ Fe(OAC) ２ Selected from one or more of the following groups; and / or (c) The base is selected from one or more of the group consisting of ammonium hydroxide, NaOH, KOH, and amines; and / or (d) The solution contains CoCl ２ and / or MnCl ２ Further including, The method according to claim 30.

32. The monodisperse beads are formed by a "one-pot, three-stage" continuous process, and this process is (a) In the first step, a polystyrene core is formed by dispersing polymerization of a styrene monomer in a mixture of water and alcohol with an initiator and a polymer stabilizer; (b) In the second step, add a crosslinkable monomer to the mixture containing the nucleated polystyrene core; and (c) In the third step, a first functional monomer is added to the material obtained from the second step to provide monodisperse beads, and optionally, polystyrene beads are produced in Insights, and the subsequent addition of the crosslinking agent and functional monomer does not cause a second nucleation, leading to spherical monodisperse copolymer beads with functional groups on the surface (styrene / crosslinking agent / functional monomer). The method according to claim 30 or claim 31, including the action of:

33. (a) The initiator is selected from azo initiators, and optionally the azo initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN) and 2,2'-azobis(2-methylbutyronitrile) (AMBN); and / or (b) The polymer stabilizer is selected from one or more of the group consisting of poly(vinylpyrrolidone) (PVP), polyethyleneimine (PEI), polyacrylic acid (PAA), polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPC), and chitosan; and / or (c) The styrene monomer is selected from one or more of the group consisting of styrene and styrene derivatives, and optionally the styrene derivative is selected from one or more of the group consisting of 4-methylstyrene, 3-methylstyrene, and 4-tert-butylstyrene; and / or (d) The alcohol is selected from one or more of the group consisting of methanol, ethanol, isopropanol, or mixtures thereof; and / or (e) The method according to claim 32, wherein the volume ratio of alcohol to water is 1:1 to 40:1, for example, 2:1 to 20:

1.

34. The crosslinkable monomers include divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, butanediol dimethacrylate, tricyclodecanedimethanol diacrylate, pentaerythritol triacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol pentaacrylate, dipentaerythritol penta / hexaacrylate, tripropylene diacrylate, trimethylolpropane ethoxylate triacrylate, and trimethylolpropane The method according to claim 32 or claim 33, wherein one or more are selected from the group consisting of propoxylate triacrylate, di(trimethylolpropane)tetoacrylate, glycerol propoxylate triacrylate, pentaerythritol propoxylate triacrylate, poly(ethylene glycol) diacrylate, poly(propylene glycol) diacrylate, and tri(propylene glycol) diacrylate, and optionally the crosslinkable monomer is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethyl acrylate, bisphenol A dimethacrylate, and N,N'-methylenebis(acrylamide).

35. The method according to any one of claims 32 to 34, wherein the functional group on the first functional monomer is independently selected from one or more of amino, carboxyl, epoxy, and hydroxyl, and optionally, the functional groups on the functional monomer of the second layer of the first shell portion and the second shell portion are independently selected from a combination of a hydroxyl group and a carboxyl group, or a combination of an amino group and a carboxyl group.

36. The method according to claim 35, wherein the first functional monomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1,2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, and 2-carboxyethyl acrylate oligomers.

37. The method according to any one of claims 32 to 36, wherein the weight ratio of the styrene monomer to the crosslinking agent is 20:1 to 1:2, for example, 10:1 to 1:

1.

38. The method according to any one of claims 32 to 37, wherein the weight ratio of the styrene monomer to the first functional monomer is 20:1 to 1:2, preferably 10:1 to 1:

1.

39. The method according to any one of claims 32 to 37, wherein the as-synthesized monodisperse beads are washed with one or more solvents before use in a subsequent process step, wherein the solvent is optionally selected from one or more of the group consisting of water, methanol, ethanol, isopropanol, and THF.

40. The resulting monodisperse superparamagnetic beads are: (a) coefficient of variation based on their diameters of less than 15%, e.g., less than 10%, e.g., less than 5%, e.g., 2% or less; and / or (b) 0.2 to 5.0 microns, for example, an average diameter of 0.5 to 4.0 microns, The method according to any one of claims 22 to 39, comprising:

41. The superparamagnetic beads according to any one of claims 1 to 21, which are applied in an IVD assay.

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