Superparamagnetic microspheres and method for the production thereof

Abstract

Disclosed herein is a microsphere having a core-shell structure, the microsphere comprising a core portion formed from a polystyrene polymeric matrix material, a shell portion that encapsulates the core portion, the shell portion comprising a first layer, a first interlayer, a second interlayer, a third interlayer, and a second layer. Also disclosed herein is a method of preparing a microsphere having a core-shell structure.

Description

[0001] SUPERPARAMAGNETIC MICROSPHERES AND METHOD FOR THE PRODUCTION THEREOF

[0002] Field of Invention

[0003] The present invention generally relates to microspheres, and more particularly to superparamagnetic microspheres.

[0004] Background

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] Magnetic beads consist of a blend of magnetic particles and polymer, leveraging the advantageous traits of both constituents. Their magnetic nature facilitates swift and effortless separation via an external magnetic field. Meanwhile, the polymer component stabilizes the magnetic particles, decreases bead density, enhances bead dispersion in various fluid buffers, and provides functional groups crucial for diverse applications such as immunoassays, nucleic acid isolation, and cell / microorganism separation (O. Philippova et al., Eur. Polym. J. 2011 , 47, 542-559).

[0007] Superparamagnetic beads with rapid magnetic responsiveness and consistent size and surface area and minimal non-specific adsorption are indispensable for ensuring reproducibility during separation processes. The development of monodisperse polymer beads with superparamagnetic properties has markedly advanced the global in vitro diagnostics (IVD) industries over the past decade and is poised for continued rapid growth in the next decade.

[0008] Type (I): Magnetic nanoparticles dispersed in the polymer bead

[0009] In this design, magnetic nanoparticles are evenly dispersed throughout the polymer bead matrix. This configuration facilitates direct incorporation of magnetic particles. US 6,986,913 B2 and US 20170218095 A1 disclose reacting porous, surface-functionalized monodisperse cross-linked polymer particles. These particles have the potential to integrate magnetic material. However, the porous structure achievable through this method is constrained by the microsphere, as the polymeric network needs to be sufficiently strong to hold the original size and dispersity, thereby imposing limits on the maximum loading capacity of magnetic nanoparticles within the microspheres. As a result, the magnetic response may be limited.

[0010] Type (II): Magnetic shell - Polymer core

[0011] Type II magnetic beads exhibit a core-shell structure wherein a magnetic shell encases a nonmagnetic polymer core. This design relies on the concentrated magnetic material on the outer surface, while the polymer core provides structural support. To achieve the Type II design, US 7,713,627 discloses magnetic particles comprising: a non-magnetic material nucleus, a magnetic material layer covering the non-magnetic nucleus and an organic polymer layer atop the magnetic material layer. However, the magnetic particle's composition is contingent upon the surface area of the non-magnetic material nucleus. The attainable magnetic response using this method is restricted by the microsphere's surface area, as the thickness of the magnetic material layer is constrained by the outer organic layer. Consequently, the magnetic response may be constrained.

[0012] Type (III): Polymer shell - Magnetic core

[0013] In Type III magnetic beads, a non-magnetic polymer shell envelops a magnetic core, offering a layer to the core. This configuration ensures protection of magnetic nanoparticles from releasee, but also enables versatile surface functionalization and compatibility with biomolecules. US 20090092837 pertains to magnetic beads in the form of composite beads comprising a metal particle inner core coated successively with an inert synthetic polymer and a hydrophilic porous polymer, preferably dextran. However, this method may lead to non- uniform incorporation of the magnetic core, posing challenges in controlling the growth of the polymer shell and regulating the encapsulation of nanoparticles within each microsphere. Consequently, uneven distribution of nanoparticles and inconsistent magnetic properties among different beads may result.

[0014] US 7,713,627 outlines a method to produce 1.5-micron non-magnetic nuclear particles, used for making magnetic beads. Initially, large polystyrene beads are synthesized and ground to yield the particles. These particles are then loaded with fine magnetic material particles. However, drawbacks include potential lack of spherical shape and uniform size in the ground particles, limited smallest bead size (1.5 microns), and difficulty achieving high magnetic / Fe content due to surface area limitations.

[0015] The problem with many existing magnetic bead-based assay methods lies in their inability to achieve rapid and efficient magnetic separation while maintaining assay specificity and consistency. Some disadvantages include: 1. incomplete capture of target molecules. Existing methods may struggle to capture all target molecules efficiently during magnetic separation, leading to reduced assay sensitivity and accuracy. The coefficient of variation (CV) of the analysis tends to be high;

[0016] 2. background noise from nonspecific binding. Nonspecific binding of molecules to the magnetic beads can result in elevated background noise, reducing assay specificity and leading to false positive / false negative results.

[0017] 3. bead aggregation: aggregation of magnetic beads during separation processes may cause magnetic beads to precipitate unexpectedly, which can hinder the efficient capture of target molecules and compromise assay performance;

[0018] 4. batch-to- batch variation: inconsistent synthesis or functionalization of magnetic beads can lead to batch-to-batch variation, impacting assay reproducibility and reliability;

[0019] 5. limited tunability of magnetic properties: existing magnetic bead technologies may lack the ability to precisely control and adjust the magnetic properties of the beads, limiting their versatility and applicability in different assay conditions; and

[0020] 6. some IVD reagents may require or benefit from freeze-thaw stability, especially for shipping flexibility. Currently, the shipping and handling of such reagents are limited by the freeze sensitivity of magnetic beads. Due to weak interfacial bonding between nanoparticles and polymeric material, reducing stress concentrations during ice formation in voids are usually destructive to the structure and function of magnetic beads.

[0021] Therefore, to overcome at least one of the aforementioned problems, there exists a need for new superparamagnetic microspheres and methods of production thereof.

[0022] Summary of Invention

[0023] Aspects and embodiments of the invention are described in the following numbered clauses.

[0024] 1. A microsphere having a core-shell structure, the microsphere comprising: a core portion formed from a polystyrene polymeric matrix material, wherein superparamagnetic Fe3O4 nanoparticles are dispersed in the polystyrene polymeric matrix material; and a shell portion that encapsulates the core portion, the shell portion comprising a first layer, a first interlayer, a second interlayer, a third interlayer, and a second layer, wherein the first layer is disposed on the core portion, the first layer comprising a first crosslinked polymeric matrix material, wherein superparamagnetic FesC nanoparticles are dispersed in the first crosslinked polymeric matrix material, the first interlayer is disposed on the first layer, the first interlayer comprising a second crosslinked polymeric matrix material, wherein superparamagnetic FesC nanoparticles are dispersed in the second crosslinked polymeric matrix material, the second interlayer is disposed on the first interlayer, the second interlayer comprising an amphiphilic crosslinked polymeric matrix material, wherein superparamagnetic FeaCu nanoparticles are dispersed in the amphiphilic crosslinked polymeric matrix material, the third interlayer is disposed on the second interlayer, the third interlayer comprising a third crosslinked polymeric matrix material, and the second layer is disposed on the third interlayer, the second layer comprising a fourth crosslinked polymeric matrix material.

[0025] 2. The microsphere according to Clause 1 , wherein the first crosslinked polymeric matrix material is formed from a first styrene monomer, a first crosslinker, and a first functional monomer / oligomer that has at least one functional group.

[0026] 3. The microsphere according to Clause 2, wherein the first styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionally wherein the styrene derivative is selected from one or more of the group consisting of 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

[0027] 4. The microsphere according to Clause 2 or Clause 3, wherein the first crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, methylene acrylamide, bisphenol A dimethacrylate, N,N ' methylenebis(acrylamide), bisphenol A epoxy diacrylate, bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, propoxylated glycerol diacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, and tripropylene diacrylate.

[0028] 5. The microsphere according to any one of the preceding clauses, wherein the second crosslinked polymeric matrix material is formed from a second styrene monomer, and a second crosslinker.

[0029] 6. The microsphere according to Clause 5, wherein the second styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionally wherein the styrene derivative is selected from one or more of the group consisting of vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

[0030] 7. The microsphere according to Clause 5 or Clause 6, wherein the second crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, bisphenol A dimethacrylate, N,N' -methylenebis(acrylamide), bisphenol A epoxy diacrylate, bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, propoxylated glycerol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta-acrylate, hexa-acrylate, tripropylene diacrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, di(trimethylolpropane) tetraacrylate, trimethylolpropane triacrylate, poly(ethylene glycol) diacrylate, polypropylene glycol) diacrylate, and tri(propylene glycol) diacrylate.

[0031] 8. The microsphere according to any one of the preceding clauses, wherein the amphiphilic crosslinked polymeric matrix material is formed from a third crosslinker, and a second functional monomer / oligomer that has at least one functional group.

[0032] 9. The microsphere according to Clause 8, wherein the third crosslinker is selected from one or more of the group consisting of divinylbenzene, bisphenol A di methacrylate, bisphenol diacrylate, tricyclodecane dimethanol diacrylate, ditrimethylolpropane tetraacrylate, di(trimethylolpropane), tetraacrylate, ethylene glycol dimethacrylate, bisphenol A epoxy diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, pentaerythritol triacrylate, dipentaerythritol penta / hexa-acrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, trimethylolpropane triacrylate, diethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, trimethylolpropane ethoxylate diacrylate, isobornyl acrylate, 1 ,6-hexanediol diacrylate, cyclohexyl methacrylate, 1 ,4-butanediol dimethacrylate, cyclohexyl acrylate, 1 ,3-butylene glycol di methacrylate, and isodecyl acrylate.

[0033] 10. The microsphere according to any one of Clauses 2 to 4, 8 and 9, wherein the at least one functional group on the first functional monomer / oligomer and / or the second functional monomer / oligomer is independently selected from one or more of amino, carboxyl, epoxy, and hydroxyl, optionally wherein the at least one functional group on the first functional monomer / oligomer and / or the second functional monomer / oligomer is independently selected from a combination of hydroxyl and carboxyl groups or a combination of amino and carboxyl groups.

[0034] 11. The microsphere according to Clause 10, wherein the first functional monomer / oligomer is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the first functional monomer / oligomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, divinyl-benzene-trimethylamine, (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 oligomer.

[0035] 12. The microsphere according to Clause 10, wherein the second functional monomer / oligomer is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the second functional monomer / oligomer is selected from one or more of the group consisting of ethylenediamine, diethylenetriamine, triethylenetetramine, ethylene glycol diglycidyl ether, diglycidyl ether of bisphenol A, maleimide, N-(2-aminoethyl)methacrylamide, N,N- dimethylaminoethyl methacrylate, N-vinyl-2-pyrrolidone, 3-aminopropyltriethoxysilaneacrylic 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 oligomer.

[0036] 13. The microsphere according to any one of the preceding clauses, wherein the third crosslinked polymeric matrix material is formed from a third styrene monomer, and a fourth crosslinker.

[0037] 14. The microsphere according to Clause 13, where in the third styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionally wherein the styrene derivative is selected from one or more of the group consisting of vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene. 15. The microsphere according to Clause 13 or Clause 14, where in the fourth crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethacrylate, bisphenol A dimethacrylate , bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta / hexa-acrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, di(trimethylolpropane) tetraacrylate, and trimethylolpropane triacrylate.

[0038] 16. The microsphere according to any one of the preceding clauses, wherein the fourth crosslinked polymeric matrix material is formed from a fifth crosslinker, a third functional monomer that has at least one functional group, and a fourth functional monomer that has at least one functional group.

[0039] 17. The microsphere according to Clause 16, wherein the fifth crosslinker is selected from one or more of the group consisting of butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, di pentaerythritol pentaacrylate, dipentaerythritol penta / hexa-acrylate, tripropylene diacrylate, di(trimethylolpropane) tetraacrylate, polyethylene glycol) diacrylate, polypropylene glycol) diacrylate, bisphenol A dimethacrylate, and tri(propylene glycol) diacrylate, optionally wherein the fifth crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, bisphenol A dimethacrylate, and N,N' -methylenebis(acrylamide).

[0040] 18. The microsphere according to Clause 16 or Clause 17, wherein the third functional monomer that has at least one functional group is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the third functional monomer that has at least one functional group is selected from one or more of the group consisting of glycidyl methacrylate, allyl glycidyl ether, 2-hydroxyethyl methacrylate, poly(ethylene glycol) diacrylate, polypropylene glycol) diacrylate, and tripropylene glycol) diacrylate, acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, ethylenediamine, diethylenetriamine, triethylenetetramine, ethylene glycol diglycidyl ether, diglycidyl ether of bisphenol A, maleimide, N-(2-aminoethyl)methacrylamide, N,N- dimethylaminoethyl methacrylate, N-vinyl-2-pyrrolidone, and 3- aminopropyltriethoxysilaneacrylic acid. 19. The microsphere according to any one of Clauses 16 to 18, wherein the fourth functional monomer is selected from one or more of the group consisting of methyl methacrylate ethyl methacrylate, butyl methacrylate, methyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, butyl acrylate, methyl acrylate, and 2-ethylhexyl acrylate.

[0041] 20. The microsphere according to Clause 1, wherein the first and third crosslinked polymeric matrix materials function to prevent the superparamagnetic Fe3C>4 nanoparticles from leaching from the microsphere when the microsphere is placed into a solvent.

[0042] 21. The microsphere according to any one of the preceding clauses, wherein the polystyrene polymeric matrix material is formed from one or more of the group consisting of styrene, a styrene derivative, and copolymers thereof, optionally wherein the styrene derivative is selected from one or more of the group consisting of vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4-methylstyrene, 3-methylstyrene, and 4- tertbutylstyrene.

[0043] 22. The microsphere according to any one of the preceding clauses, wherein all of the superparamagnetic Fe3C>4 nanoparticles in a microsphere account for from 10 to 80 wt%, such as from 20 to 70 wt%, such as from 10 to 55 wt%, such as from 15 to 45% of the entire weight of each microsphere.

[0044] 23. The microsphere according to Clause 22, wherein:

[0045] (a) the superparamagnetic Fe3O4 nanoparticles in the core portion and in the first layer represent 20 to 40 wt% of the entire weight of each microsphere; and / or

[0046] (b) the superparamagnetic Fe3C>4 nanoparticles in the second interlayer represent 10 to 20 wt% of the entire weight of each microsphere.

[0047] 24. The microsphere according to any one of the preceding clauses, wherein the superparamagnetic Fe3O4 nanoparticles have an average diameter of from 5 nm to 25 nm.

[0048] 25. The microsphere according to any one of the preceding clauses, wherein:

[0049] (a) the weight to weight ratio of styrene groups to crosslinking groups in the first layer is from 20:1 to 1 :2, such as from 10:1 to 1:1 ;

[0050] (b) the weight to weight ratio of styrene groups to functional groups in the first layer is from 20:1 to 1 :2, such as from 10:1 to 1 :1 ; (c) the weight to weight of styrene groups to crosslinking groups in the first interlayer is from 20:1 to 1:2, such as from 10:1 to 1 :1 ;

[0051] (d) the weight to weight ratio of functional groups to crosslinking groups in the second interlayer is from 2:3 to 1 :20, such as from 1 :3 to 1:10; and / or

[0052] (e) the weight to weight ratio of functional groups to crosslinking groups in the second layer is from 2:3 to 1 :20, such as from 1 :3 to 1 :10.

[0053] 26. The microsphere according to any one of the preceding clauses, wherein the superparamagnetic FeaC nanoparticles dispersed in the polystyrene polymeric matrix material and in the first crosslinked polymeric matrix material have a total thickness of from 10 nm to 100 nm, such as from 45 nm to 85 nm, such as 71 nm.

[0054] 27. The microsphere according to any one of the preceding clauses, wherein the first interlayer and the second interlayer have a total thickness of from 10 nm to 50 nm, such as from 20 nm to 35 nm, such as 26 nm.

[0055] 28. The microsphere according to any one of the preceding clauses, wherein the second layer further comprises an organic polymer layer, wherein the organic polymer layer comprises a group shown by the following formula:

[0056] -CH2-CHR1-CH2-R2, wherein R1represents an alkoxy group and R2represents a carboxyl, tosyl or amine group, and wherein the carboxyl, tosyl and amine group is linear or branched.

[0057] 29. A method of preparing a microsphere having a core-shell structure, the method comprising:

[0058] (ai) providing a precursor microsphere comprising a core portion formed from a polystyrene polymeric matrix material,

[0059] (aii) disposing a first layer on the core portion by providing a first crosslinked polymeric matrix material through dispersion polymerisation and dispersing superparamagnetic Fe3O4 nanoparticles in the core portion and the first crosslinked polymeric matrix material;

[0060] (aiii) disposing a first interlayer on the first layer through precipitation coating, wherein the first interlayer comprises a second crosslinked polymeric matrix material;

[0061] (aiv) disposing a second interlayer on the first interlayer by providing an amphiphilic crosslinked polymeric matrix material through polymerisation and dispersing superparamagnetic Fe3C>4 nanoparticles in the amphiphilic crosslinked polymeric matrix material and the second crosslinked polymeric matrix material of the first interlayer; (av) disposing a third interlayer on the second interlayer through precipitation coating, wherein the third interlayer comprises a third crosslinked polymeric matrix material; and

[0062] (avi) disposing a second layer on the third interlayer through polymerisation, wherein the second layer comprises a fourth crosslinked polymeric matrix material.

[0063] 30. The method according to Clause 29, further comprising providing a porogen in step (ai).

[0064] 31 . The method according to Clause 30, wherein the porogen is selected from one or more of 1,4-dioxane, tetrahydrofuran, ethylene glycol, diglyme, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, toluene, ethylbenzene, xylene, benzene, and polyethylene glycol (400- 8000).

[0065] 32. The method according to Clause 30 or Clause 31 , wherein the weight to weight ratio of styrene groups to porogen in the core portion is from 50:1 to 1:2, such as from 20:1 to 1 :1.

[0066] 33. The method according to any one of Clauses 29 to 32, wherein step (ai) comprises providing:

[0067] (bi) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of methanol, ethanol, and isopropanol;

[0068] (bii) one or more polymeric stabilizers, optionally wherein the one or more polymeric stabilizers are selected from one or more of the group consisting of triton N-57, Triton X-100, poly(vinylpyrrolidone) (PVP), polyethylenimine (PEI), polyacrylic acid (PAA), polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPC), and chitosan, and optionally wherein the weight to weight ratio of styrene groups to one or more polymeric stabilizers in the core portion is from 50:1 to 1 :2, such as from 50:1 to 10:1 ; and / or

[0069] (biii) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2- methylbutyronitrile) (AM BN), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate and ammonia persulfate.

[0070] 34. The method according to any one of Clauses 29 to 33, wherein step (aii) comprises using a first mixture comprising a first styrene monomer, a first crosslinker, and a first functional monomer / oligomer that has at least one functional group, to provide the first crosslinked polymeric matrix material.

[0071] 35. The method according to Clause 34, wherein the first styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionally wherein the styrene derivative is selected from one or more of the group consisting of 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

[0072] 36. The method according to Clause 34 or Clause 35, wherein the first crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, methylene acrylamide, bisphenol A dimethacrylate, N,N ' methylenebis(acrylamide), bisphenol A epoxy diacrylate, bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, propoxylated glycerol diacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, and tripropylene diacrylate.

[0073] 37. The method according to any one of Clauses 34 to 36, wherein the first mixture in step (aii) further comprises a solvent, optionally wherein the solvent is selected from one or more of the group consisting of 1 ,4-dioxane, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, water, methanol, ethanol and isopropanol.

[0074] 38. The method according to any one of Clauses 29 to 37, wherein the dispersion polymerisation in step (aii) is conducted at a temperature of from about 40 °C to about 80 °C, such as from about 50 °C to about 70 °C, such as about 70 °C.

[0075] 39. The method according to any one of Clauses 29 to 38, wherein the dispersion polymerisation in step (aii) has a polymerisation time of from about 10 hours to about 72 hours, such as from about 16 hours to about 48 hours, such as about 16 hours.

[0076] 40. The method according to any one of Clauses 29 to 39, wherein step (aiii) comprises using a second mixture comprising a second styrene monomer, and a second crosslinker, to provide the second crosslinked polymeric matrix material.

[0077] 41 . The method according to Clause 40, wherein the second styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionally wherein the styrene derivative is selected from one or more of the group consisting of vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

[0078] 42. The method according to Clause 40 or Clause 41 , wherein the second crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, bisphenol A dimethacrylate, N,N' -methylenebis(acrylamide), bisphenol A epoxy diacrylate, bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, propoxylated glycerol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta-acrylate, hexa-acrylate, tripropylene diacrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, di(trimethylolpropane) tetraacrylate, trimethylolpropane triacrylate, polyethylene glycol) diacrylate, polypropylene glycol) diacrylate, and tri(propylene glycol) diacrylate.

[0079] 43. The method according to any one of Clauses 40 to 42, wherein the second mixture further comprises:

[0080] (ci) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of 1 ,4-dioxane, ethyl acetate, butyl acetate, acetone, hexane, octane, methyl ethyl ketone, and water;

[0081] (cii) a surfactant, optionally wherein the surfactant is selected from one or more of the group consisting of cetyltrimethylammonium bromide (CTAB), polyvinyl alcohol (PVA), Span 80, Span 60, and Span 20, Tween 20, Tween 80, Tween 60, sodium octyl sulfate, sodium dodecyl sulfate, sodium decyl sulfate, sodium tetradecyl sulfate, lauryl polyglucoside, decyl polyglucoside, and undecyl polyglucoside; and / or

[0082] (ciii) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2- methylbutyronitrile) (AM BN), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate and ammonia persulfate.

[0083] 44. The method according to any one of Clauses 29 to 43, wherein the precipitation coating in step (aiii) is conducted a temperature of from about 40 °C to about 85 °C, such as from about 60 °C to about 75 °C. 45. The method according to any one of Clauses 29 to 44, wherein the precipitation coating in step (aiii) has a polymerisation time of from about 1 hour to 24 hours, such as from about 3 hours to 20 hours.

[0084] 46. The method according to any one of Clauses 29 to 39, wherein step (aiii) comprises using a second mixture comprising an epoxide, to provide the second crosslinked polymeric matrix material.

[0085] 47. The method according to Clause 46, wherein the epoxide comprises phenol rings, optionally wherein the epoxide is selected from phenolic derivatives such as bis(epoxyethyl)benzene, 1 ,2-epoxy-4-(epoxyethyl)benzene, 1 ,3-bis(2,3-epoxypropoxy)- benzene, bisphenol A, AP, B, BP, C, E, F, G, M, P, PH, TMC, Z and its copolymer and propoxylated derivatives (e.g. bisphenol A propoxylate diglycidyl ether, bisphenol A diglycidyl ether-bisphenol A copolymer, poly(bisphenol A-co-epichlorohydrin), and glycidyl capped), triglycidyl p-amino-phenol, and diglycidyl aniline tetraglycidyl meta-xylene diamine.

[0086] 48. The method according to Clause 46 or Clause 47, wherein the second mixture further comprises:

[0087] (di) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of N-methyl-2-pyrrolidone, 1 ,4-dioxane, acetonitrile, acetone, bis(2- methoxyethyl) ether, methyl ethyl ketone, and ethanol; and / or

[0088] (dii) a catalyst, optionally wherein the catalyst is selected from one or more of the group consisting of pyridine, isoquinoline, quinoline, N,N-dimethyl-cyclohexylamine, tributylamine, N-ethyl morpholine, dimethylaniline, triethylamine (TEA), benzyl dimethylamine (BDMA), and 2,4,6-tris(dimethylaminomethyl)phenol (DMP-30).

[0089] 49. The method according to any one of Clauses 46 to 48, wherein the precipitation coating in step (aiii) is conducted a temperature of from about 40 °C to about 110 °C, such as from about 60 °C to about 95 °C.

[0090] 50. The method according to any one of Clauses 46 to 49, wherein the precipitation coating in step (aiii) has a polymerisation time of from about 1 hour to 24 hours, such as from about 3 hours to 17 hours.

[0091] 51. The method according to any one of Clauses 28 to 50, wherein step (aiv) comprises using a third mixture comprising a third crosslinker, and a second functional monomer / oligomer that has at least one functional group, to provide the amphiphilic crosslinked polymeric matrix material.

[0092] 52. The method according to Clause 51 , wherein the third crosslinker is selected from one or more of the group consisting of divinylbenzene, bisphenol A dimethacrylate, bisphenol diacrylate, tricyclodecane dimethanol diacrylate, ditrimethylolpropane tetraacrylate, di(trimethylolpropane), tetraacrylate, ethylene glycol dimethacrylate, bisphenol A epoxy diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, pentaerythritol triacrylate, dipentaerythritol penta / hexa-acrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, trimethylolpropane triacrylate, diethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, trimethylolpropane ethoxylate diacrylate, isobornyl acrylate, 1 ,6-hexanediol diacrylate, cyclohexyl methacrylate, 1 ,4-butanediol dimethacrylate, cyclohexyl acrylate, 1 ,3-butylene glycol di methacrylate, and isodecyl acrylate.

[0093] 53. The method according to any one of Clauses 34 to 37, 51 and 52, wherein the at least one functional group on the first functional monomer / oligomer and / or the second functional monomer / oligomer is independently selected from one or more of amino, carboxyl, epoxy, and hydroxyl, optionally wherein the at least one functional group on the first functional monomer / oligomer and / or the second functional monomer / oligomer is independently selected from a combination of hydroxyl and carboxyl groups or a combination of amino and carboxyl groups.

[0094] 54. The method according to Clause 53, wherein the first functional monomer / oligomer is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the first functional monomer / oligomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2- carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, divinyl-benzene- trimethylamine, (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 oligomer.

[0095] 55. The method according to Clause 53, wherein the second functional monomer / oligomer is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the second functional monomer / oligomer is selected from one or more of the group consisting of ethylenediamine, diethylenetriamine, triethylenetetramine, ethylene glycol diglycidyl ether, diglycidyl ether of bisphenol A, maleimide, N-(2-aminoethyl)methacrylamide, N,N-dimethylaminoethyl methacrylate, N-vinyl-2-pyrrolidone, 3-aminopropyltriethoxysilaneacrylic 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 oligomer.

[0096] 56. The method according to any one of Clauses 29 to 55, wherein the polymerisation in step (aiv) is conducted at a temperature of from about 40 °C to about 90 °C, such as from about 55 °C to about 80 °C, such as about 75 °C.

[0097] 57. The method according to any one of Clauses 29 to 56, wherein the polymerisation in step (aiv) has a polymerisation time of from about 1 hour to about 24 hours, such as from about 3 hours to about 17 hours.

[0098] 58. The method according to any one of Clauses 29 to 50, wherein step (aiv) comprises using a third mixture comprising:

[0099] (ei) an anhydride monomer, optionally wherein the anhydride monomer is selected from one or more of the group consisting of maleic anhydride, phthalic anhydride, succinic anhydride, trimellitic anhydride, octenyl succinic anhydride, polyisobutylene succinic anhydride, octadecenyl succinic anhydride, and dodecenyl succinic anhydride; and / or

[0100] (eii) a copolymer, optionally wherein the copolymer is selected from one or more of the group consisting of poly(ethylene-a / t-maleic anhydride), poly(isobutylene-a / f-maleic anhydride), poly(methyl vinyl ether-a / t-maleic anhydride), and poly[(isobutylene-a / f-maleic acid, ammonium salt)-co-(isobutylene-a / t-maleic anhydride)].

[0101] 59. The method according to Clause 58, wherein the third mixture further comprises:

[0102] (fi) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of N-methyl-2-pyrrolidone, 1 ,4-dioxane, acetonitrile, acetone, bis(2- methoxyethyl) ether, methyl ethyl ketone, and ethanol; and / or

[0103] (fii) a catalyst, optionally wherein the catalyst is selected from one or more of the group consisting of pyridine, isoquinoline, quinoline, N,N-dimethyl-cyclohexylamine, tributylamine, N-ethyl morpholine, dimethylaniline, triethylamine (TEA) , benzyl dimethylamine (BDMA), and 2,4,6-tris(dimethylaminomethyl)phenol (DMP-30). 60. The method according to Clause 58 or Clause 59, wherein the coating in step (aiv) is conducted a temperature of from about 25 °C to about 95 °C, such as from about 25 °C to about 75 °C.

[0104] 61 . The method according to any one of Clauses 58 to 60, wherein the coating in step (aiv) has a reaction time of from about 1 hour to 24 hours, such as from about 3 hours to 17 hours.

[0105] 62. The method according to any one of Clauses 29 to 61 , wherein dispersing superparamagnetic FeaC nanoparticles in steps (aii) and (aiv) comprise providing a solution that comprises a Fe(lll) salt, a Fe(ll) salt and a base.

[0106] 63. The method according to Clause 62, wherein:

[0107] (gi) the Fe(lll) salt is selected from one or more of the group consisting of FeCh and Fe2(SC>4)3, optionally wherein the Fe(lll) salt is FeCh;

[0108] (gii) the Fe(ll) salt is selected from one or more of the group consisting of FeCl2, FeSC and Fe(OAC)2, optionally wherein the Fe(ll) salt is FeCI2;and / or

[0109] (giii) the base is selected from one or more of the group consisting of ammonia hydroxide, NaOH, KOH, and amine, optionally wherein the base is aqueous ammonia.

[0110] 64. The method according to any of Clauses 29 to 63, wherein step (av) comprises using a fourth mixture comprising a third styrene monomer, and a fourth crosslinker, to provide the third crosslinked polymeric matrix material.

[0111] 65. The method according to Clause 64, wherein the third styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionally wherein the styrene derivative is selected from one or more of the group consisting of vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

[0112] 66. The method according to Clause 64 or Clause 65, wherein the fourth crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethacrylate, bisphenol A dimethacrylate , bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta / hexa-acrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, di(trimethylolpropane) tetraacrylate, and trimethylolpropane triacrylate.

[0113] 67. The method according to any one of Clauses 64 to 66, wherein the fourth mixture further comprises:

[0114] (hi) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2- methylbutyronitrile) (AMBN), 4,4'-azobis (4-cyanovaleric acid), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate, and ammonia persulfate; and / or

[0115] (hii) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of 1 ,4-dioxane, ethyl acetate, butyl acetate, acetone, hexane, octane, methyl ethyl ketone, and water.

[0116] 68. The method according to any one of Clauses 29 to 67, wherein the polymerisation in step (av) is conducted at a temperature of from about 40 °C to about 105 °C, such as from about 60 °C to about 95 °C, such as about 75 °C.

[0117] 69. The method according to any one of Clauses 29 to 68, wherein the polymerisation in step (av) has a polymerisation time of from about 1 hour to about 24 hours, such as from about 3 hours to about 17 hours.

[0118] 70. The method according to any of Clauses 29 to 69, wherein step (avi) comprises using a fifth mixture comprising a fifth crosslinker, a third functional monomer that has at least one functional group, and a fourth functional monomer that has at least one functional group, to provide the fourth crosslinked polymeric matrix material.

[0119] 71 . The method according to Clause 70, wherein the fifth crosslinker is selected from one or more of the group consisting of butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, di pentaerythritol pentaacrylate, dipentaerythritol penta / hexa-acrylate, tripropylene diacrylate, di(trimethylolpropane) tetraacrylate, polyethylene glycol) diacrylate, polypropylene glycol) diacrylate, bisphenol A dimethacrylate, and tri(propylene glycol) diacrylate, optionally wherein the fifth crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, bisphenol A dimethacrylate, and N,N -methylenebis(acrylamide). 72. The method according to Clause 70 or Clause 71 , wherein the third functional monomer that has at least one functional group is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the third functional monomer that has at least one functional group is selected from one or more of the group consisting of glycidyl methacrylate, allyl glycidyl ether, 2-hydroxyethyl methacrylate, poly(ethylene glycol) diacrylate, polypropylene glycol) diacrylate, and tri(propylene glycol) diacrylate, acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, ethylenediamine, diethylenetriamine, triethylenetetramine, ethylene glycol diglycidyl ether, diglycidyl ether of bisphenol A, maleimide, N-(2-aminoethyl)methacrylamide, N,N- dimethylaminoethyl methacrylate, N-vinyl-2-pyrrolidone, and 3- aminopropyltriethoxysilaneacrylic acid.

[0120] 73. The method according to any one of Clauses 70 to 72, wherein the fourth functional monomer is selected from one or more of the group consisting of methyl methacrylate ethyl methacrylate, butyl methacrylate, methyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, butyl acrylate, methyl acrylate, and 2-ethylhexyl acrylate.

[0121] 74. The method according to any one of Clauses 70 to 73, wherein the fifth mixture further comprises:

[0122] (ii) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2- methylbutyronitrile) (AMBN), 4,4'-azobis (4-cyanovaleric acid), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate, and ammonia persulfate; and / or

[0123] (iii) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of ethylene glycol, 1 ,4-dioxane, diglyme, octane, toluene, dimethylacetamide, dimethylformamide, xylene, n-methyl-2-pyrrolidone, n-butanol and water.

[0124] 75. The method according to any one of Clauses 29 to 69, wherein step (avi) comprises using a fifth mixture comprising a first epoxide, to provide the second crosslinked polymeric matrix material.

[0125] 76. The method according to Clause 75, wherein the first epoxide comprises phenolic derivatives, optionally wherein the first epoxide is selected from one or more of the group consisting of bisphenol A, AP, B, BP, C, E, F, G, M, P, PH, TMC, Z and its copolymer derivatives (e g. bisphenol A propoxylate diglycidyl ether, and bisphenol A diglycidyl etherbisphenol A copolymer).

[0126] 77. The method according to Clause 75 or Clause 76, wherein the fifth mixture further comprises:

[0127] (ji) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of N-methyl-2-pyrrolidone, 1 ,4-dioxane, acetonitrile, acetone, bis(2- methoxyethyl) ether, methyl ethyl ketone, and ethanol;

[0128] (jii) a catalyst, optionally wherein the catalyst is selected from one or more of the group consisting of pyridine, isoquinoline, quinoline, N,N-dimethyl-cyclohexylamine, tributylamine, N-ethyl morpholine, dimethylaniline, triethylamine (TEA), benzyl dimethylamine (BDMA), and 2,4,6-tris(dimethylaminomethyl)phenol (DMP-30); and / or

[0129] (jiii) a second epoxide selected from one or more of the group consisting of 1,2- epoxydodecane, 1 ,4-butanediol diglycidyl ether, 1 ,2-butylene oxide, tert-butyl glycidyl ether, butyl glycidyl ether, 1 ,2-epoxydecane,1 ,2-epoxydodecane,1,2-epoxyheptane,1 ,2- epoxyhexane, polyethylene glycol diglycidyl ether (n = 4 to 22), and glycerol triglycidyl ether.

[0130] 78. The method according to any one of Clauses 75 to 77, wherein the precipitation coating in step (avi) is conducted a temperature of from about 40 °C to about 110 °C, such as from about 60 °C to about 95 °C.

[0131] 79. The method according to any one of Clauses 75 to 78, wherein the precipitation coating in step (avi) has a reaction time of from about 1 hour to 24 hours, such as from about 3 hours to 20 hours.

[0132] 80. The method according to any one of Clauses 75 to 79, wherein step (avi) further comprises using a carboxylic acid to hydrolyse the epoxide groups.

[0133] 81 . The method according to any one of Clauses 29 to 80, wherein:

[0134] (ki) the weight to weight ratio of styrene groups to crosslinking groups in the first layer is from 20:1 to 1 :2, such as from 10:1 to 1:1 ; and / or

[0135] (kii) the weight to weight ratio of styrene groups to functional groups in the first layer is from 20:1 to 1 :2, such as from 10:1 to 1 :1 ;

[0136] (kiii) the weight to weight of styrene groups to crosslinking groups in the first interlayer is from 20:1 to 1:2, such as from 10:1 to 1 :1 ; (kiv) the weight to weight ratio of functional groups to crosslinking groups in the second interlayer is from 2:3 to 1 :20, such as from 1 :3 to 1:10; and / or

[0137] (kv) the weight to weight ratio of functional groups to crosslinking groups in the second layer is from 2:3 to 1 :20, such as from 1 :3 to 1 :10.

[0138] 82. The method according to any one of Clauses 29 to 81 , wherein step (avi) further comprises disposing an organic polymer layer, wherein the organic polymer layer comprises a group shown by the following formula:

[0139] -CH2-CHR1-CH2-R2, wherein R1represents an alkoxy group and R2represents a carboxyl, tosyl or amine group, and wherein the carboxyl, tosyl and amine group is linear or branched.

[0140] 83. Use of the microsphere of any one of Clauses 1 to 28 in a chemiluminescence immunoassay.

[0141] Drawings

[0142] Fig. 1 depicts a comparison of the different structures of magnetic polymer beads.

[0143] Figs. 2A to 2E depict a cartoon form of the preparation of a superparamagnetic microsphere according to embodiments of the present invention.

[0144] Description

[0145] It has been surprisingly found that the problems above can be solved, in whole or in part, by the introduction of a new type of superparamagnetic microspheres. These superparamagnetic microspheres have tunable magnetic properties and consistent magnetic output in magnetic particles, and reduce background noise from nonspecifically bound molecules.

[0146] Thus, in a first aspect of the invention, there is provided a microsphere having a core-shell structure, the microsphere comprising: a core portion formed from a polystyrene polymeric matrix material, wherein superparamagnetic FesC nanoparticles are dispersed in the polystyrene polymeric matrix material; and a shell portion that encapsulates the core portion, the shell portion comprising a first layer, a first interlayer, a second interlayer, a third interlayer, and a second layer, wherein the first layer is disposed on the core portion, the first layer comprising a first crosslinked polymeric matrix material, wherein superparamagnetic FesC nanoparticles are dispersed in the first crosslinked polymeric matrix material, the first interlayer is disposed on the first layer, the first interlayer comprising a second crosslinked polymeric matrix material, wherein superparamagnetic FesC nanoparticles are dispersed in the second crosslinked polymeric matrix material, the second interlayer is disposed on the first interlayer, the second interlayer comprising an amphiphilic crosslinked polymeric matrix material, wherein superparamagnetic Fe3O4 nanoparticles are dispersed in the amphiphilic crosslinked polymeric matrix material, the third interlayer is disposed on the second interlayer, the third interlayer comprising a third crosslinked polymeric matrix material, and the second layer is disposed on the third interlayer, the second layer comprising a fourth crosslinked polymeric matrix material.

[0147] The word “comprising” refers herein may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of’ or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0148] The term “microspheres” when used herein is intended to be synonymous with the terms “beads” and “particles”. The term “polymer particles” when used herein is intended to be synonymous with the terms “polymer beads” and “polymer microspheres”. The term “superparamagnetic polymer microspheres” when used herein is intended to be synonymous with the terms “superparamagnetic polymer beads” and “superparamagnetic polymer particles”.

[0149] The term “monodisperse” when used herein refers to particles having a low coefficient of variation (CV) of a specific parameter (e.g. particle diameter), for example a CV of less than 20%, such as less than 15%, such as less than 10%, such as less than 5%. More particularly, the particles may have a CV of less than or equal to 2%, such as less than or equal to 1%. The term “monodisperse” also encompasses the term “highly monodisperse”, which, when used herein, may refer to a CV of less than 5%, such as less than or equal to 2%, such as less than or equal to 1 %. When used herein, the term “coefficient of variation” refers to its statistical meaning. That is: standard deviation

[0150] CV (%) = - x 100 mean

[0151] The terms “standard deviation” and “mean” take their ordinary statistical meanings.

[0152] The magnetic NPs refers herein may comprise at least one kind of paramagnetic NPs, superparamagnetic NPs, ferromagnetic NPs or ferrimagnetic NPs. The definitions are listed below.

[0153] “Magnetic” when used herein means a property of responding to a magnetic field in a material.

[0154] “Paramagnetic” when used herein means that the magnetic property displayed by a material is switched off after the external magnetic field is removed. “Superparamagnetic” when used herein means that the magnetic property of a material is switched-off instantly upon removal of the external magnetic field.

[0155] “Ferromagnetic” when used herein means that all the magnetic atoms within each domain contribute a positive value to the overall net magnetization of the material, which retains magnetic properties after an external magnetic field is removed. Said material becomes paramagnetic material above its Curie temperature.

[0156] “Ferrimagnetic” when used herein means that some of magnetic atoms within each domain are opposed, but the material overall exhibits net magnetization. Said material retains its magnetic property after an external magnetic field is removed. Said material becomes a paramagnetic material above its Curie temperature.

[0157] “Hydrophilic” when used herein means a substance that has an affinity or attraction to water. Hydrophilic substances tend to interact well with water molecules, allowing them to be easily dissolved or dispersed in water.

[0158] “Hydrophobic” when used herein means a substance repel water and have little to no affinity for it. They tend to be non-polar or have low polarity, making them insoluble in water. Instead of dissolving in water, hydrophobic substances often aggregate together or repel water molecules. “Amphiphilic” when used herein means molecules or compounds that contain both hydrophilic (water-attracting) and hydrophobic (water-repelling) parts within their structure.

[0159] When used herein “encapsulate” may refer to the full encapsulation of a material within another material or it may also refer to the partial encapsulation of a material within another material. The term may also refer to the situation where a material is trapped within a pore of another material.

[0160] Figs. 2A-2E illustrate in cartoon form the preparation of a fully-formed microsphere according to embodiments of the present invention. As shown in Fig. 2E, there is a fully-formed microsphere 100 which has a core portion 110, and a shell portion that encapsulates the core portion and the shell portion has a first layer 120, a first interlayer 130, a second interlayer 140, a third interlayer 150, and a second layer 160.

[0161] As shown in Fig. 2A, there is a core portion 110 formed from a polystyrene polymeric matrix material 111 and superparamagnetic Fe3C>4 nanoparticles 112 are dispersed in the polystyrene polymeric matrix material 111. The first layer 120 is disposed on the core portion 110, the first layer 120 includes a first crosslinked polymeric matrix material 121 , and superparamagnetic Fe3O4 nanoparticles 122 are dispersed in the first crosslinked polymeric matrix material.

[0162] In some embodiments of the present invention that may be mentioned herein, the polystyrene polymeric matrix material may be formed from one or more of the group consisting of styrene, a styrene derivative such as vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4-methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene, and copolymers thereof.

[0163] In some embodiments of the present invention that may be mentioned herein, the first crosslinked polymeric matrix material may be formed from a first styrene monomer, a first crosslinker, and a first functional monomer / oligomer that has at least one functional group.

[0164] Any suitable crosslinker may be used as the first crosslinker. Examples of suitable crosslinkers include, but are not limited to divinylbenzene, ethylene glycol dimethylacrylate, methylene acrylamide, bisphenol A dimethacrylate, N,N' -methylenebis(acrylamide), bisphenol A epoxy diacrylate, bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, propoxylated glycerol diacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, and tripropylene diacrylate.

[0165] Any suitable styrene monomer may be used as the first styrene monomer. Examples of suitable styrene monomers include, but are not limited to styrene, and styrene derivatives such as 4-methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

[0166] Without wishing to be bound by theory, the first crosslinked polymeric matrix material may exhibit porosity, thus effectively encapsulating superparamagnetic Fe3C>4 nanoparticles within the first crosslinked polymeric matrix material and across its inner and outer surface layers.

[0167] As shown in Fig. 2B, the first interlayer 130 is disposed on the first layer 120. The first interlayer 130 is formed from a second crosslinked polymeric matrix material 131, and includes superparamagnetic Fe3O4 nanoparticles 132 that are dispersed in the amphiphilic crosslinked polymeric matrix material 131.

[0168] In some embodiments of the present invention that may be mentioned herein, the second crosslinked polymeric matrix material may be formed from a second styrene monomer and a second crosslinker, and by inter-crosslinking the second styrene monomer and the second crosslinker with the first crosslinked polymeric matrix material 121 of the first layer 120.

[0169] As will be appreciated, the first interlayer functions to encapsulate the superparamagnetic Fe3O4 nanoparticles, covering them on an outer surface of the first layer.

[0170] Without wishing to be bound by theory, the first interlayer may be formed through two mechanisms. First, the second styrene monomer may diffuse and swell the core portion and the first layer, creating an inter-crosslinking layer. Second, the majority of the styrene and crosslinker may undergo nucleation in solution, followed by coalescence and precipitation into the gaps and partially on the surface of the first layer. During nucleation, small polymeric nanoparticles may form. Due to their high surface energy, these nanoparticles may be unstable in the solution, causing them to precipitate and fill the gaps between the existing nanoparticles. This process may form a robust polymeric network that encapsulates clusters of nanoparticles and holds them within the polymer shell.

[0171] Any suitable styrene monomer may be used as the second styrene monomer. Examples of suitable styrene monomers include, but are not limited to styrene, styrene derivatives such as vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

[0172] Any suitable crosslinker may be used as the second crosslinker. Examples of suitable crosslinkers include, but are not limited to divinylbenzene, ethylene glycol dimethylacrylate, bisphenol A dimethacrylate, N,NZ-methylenebis(acrylamide), bisphenol A epoxy diacrylate, bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, propoxylated glycerol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta-acrylate, hexa-acrylate, tripropylene diacrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, di(trimethylolpropane) tetraacrylate, trimethylolpropane triacrylate, poly(ethylene glycol) diacrylate, polypropylene glycol) diacrylate, and tri(propylene glycol) diacrylate.

[0173] In some embodiments of the present invention that may be mentioned herein, the weight ratio of the second styrene monomer to the second crosslinker may be from 20:1 to 1:2, preferably from 10:1 to 1 :1.

[0174] As shown in Fig. 2C, the second interlayer 140 is disposed on the first interlayer 130. The second interlayer 140 includes an amphiphilic crosslinked polymeric matrix material 141, and superparamagnetic FeaC nanoparticles 142 are dispersed in the amphiphilic crosslinked polymeric matrix material 141.

[0175] Without wishing to be bound by theory, the amphiphilic crosslinked polymeric matrix material 141 may fill up gaps among the superparamagnetic FesC nanoparticles 142, hence forming an intervened network.

[0176] In some embodiments of the present invention that may be mentioned herein, the amphiphilic crosslinked polymeric matrix material may be formed from a third crosslinker, and a second functional monomer / oligomer that has at least one functional group. For example, the third crosslinker may be a hydrophobic crosslinker and the second functional monomer / oligomer that has at least one functional group may be a hydrophilic monomer / oligomer. It will be appreciated that the amphiphilic crosslinked polymeric matrix material facilitates the second interlayer’s structural integrity and functionality as the amphiphilic crosslinked polymeric matrix material not only capture superparamagnetic FesC nanoparticles, it also help to adhere the captured Fe3O4 nanoparticles on the surface of the first interlayer. Any suitable crosslinker may be used as the third crosslinker. Examples of suitable crosslinkers include, but are not limited to divinylbenzene, bisphenol A dimethacrylate, bisphenol diacrylate, tricyclodecane dimethanol diacrylate, ditrimethylolpropane tetraacrylate, di(trimethylolpropane), tetraacrylate, ethylene glycol dimethacrylate, bisphenol A epoxy diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, pentaerythritol triacrylate, dipentaerythritol penta / hexa-acrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, trimethylolpropane triacrylate, diethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, trimethylolpropane ethoxylate diacrylate, isobornyl acrylate, 1 ,6-hexanediol diacrylate, cyclohexyl methacrylate, 1 ,4-butanediol dimethacrylate, cyclohexyl acrylate, 1 ,3-butylene glycol di methacrylate, and isodecyl acrylate.

[0177] Without wishing to be bound by theory, the at least one functional group on the first functional monomer / oligomer and the second functional monomer / oligomer may capture superparamagnetic Fe3C>4 particles.

[0178] Without wishing to be bound by theory, amino, carboxyl, epoxy, and hydroxyl groups may form coordination bonds with Fe3O4 nanoparticles due to their ability to donate electron pairs to the iron ions (Fe2+ / Fe3+) on the nanoparticle surface. Amino groups may coordinate with Fe2+or Fe3+ions through the nitrogen atom, forming stable bonds that anchor them to the nanoparticle surface. Carboxyl groups, upon deprotonation, form negatively charged carboxylate anions (-COO-) that bind to Fe3+or Fe2+ions via their oxygen atoms, resulting in either bidentate or monodentate coordination. Although epoxy groups form weaker bonds compared to amino and carboxyl groups, the oxygen in the epoxy ring may still coordinate with Fe2+or Fe3+ions. Under acidic or basic conditions, epoxides may also open up to generate hydroxyl groups, which further enhance binding. Similarly, hydroxyl groups may form coordination bonds with Fe3O4 nanoparticles via their oxygen atoms.

[0179] In some embodiments of the present invention that may be mentioned herein, the at least one functional group on the first and second functional monomers / oligomers may be independently selected from one or more of amino, carboxyl, epoxy, and hydroxyl. For example, the first and second functional monomers / oligomers may be independently selected from a combination of hydroxyl and carboxyl groups or a combination of amino and carboxyl groups.

[0180] In some embodiments of the present invention that may be mentioned herein, the first functional monomer / oligomer may be selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer. For example, the first functional monomer / oligomer may be selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, divinyl-benzene-trimethylamine, (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 oligomer.

[0181] In some embodiments of the present invention that may be mentioned herein, the second functional monomer / oligomer may be selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer. For example, the second functional monomer / oligomer may be selected from one or more of the group consisting of ethylenediamine, diethylenetriamine, triethylenetetramine, ethylene glycol diglycidyl ether, diglycidyl ether of bisphenol A, maleimide, N-(2-aminoethyl)methacrylamide, N,N-dimethylaminoethyl methacrylate, N-vinyl-2-pyrrolidone, 3- aminopropyltriethoxysilaneacrylic 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 oligomer.

[0182] As shown in Fig. 2D, the third interlayer 150 is disposed on the second interlayer 140. The third interlayer 150 includes a third crosslinked polymeric matrix material 151. The third crosslinked polymeric matrix material 151 may penetrate through the second interlayer 140, and the third crosslinked polymeric matrix material 151 may intervene with the network of the second interlayer 140, thus partially or fully encapsulating around the surface of the exposed magnetic nanoparticles in the second interlayer 140. As will be appreciated, this prevents the nanoparticles from being exposed to a buffer solution, which may cause the nanoparticles to leach out or aggregate.

[0183] In some embodiments of the present invention that may be mentioned herein, the weight ratio of the second functional monomer to the third crosslinker may be from 2:3 to 1 :20, preferably from 1 :3 to 1 :10.

[0184] Without wishing to be bound by theory, a majority of the third crosslinker and the second functional monomer / oligomer may deposit or precipitate on the first interlayer. The majority of the third crosslinker and the second functional monomer / oligomer may form oligomer and polymer. Due to limited solubility, the amphiphilic crosslinked polymeric matrix material may precipitate on the surface of the first interlayer. Nevertheless, the amphiphilic crosslinked polymeric matrix material may be relatively soluble in the reaction mixture, which may prevent them from precipitating on the surface of the first interlayer. Another portion of the third crosslinker and the second functional monomer / oligomer (i.e. a minority of the third crosslinker and the second functional monomer / oligomer) may penetrate the first interlayer, then the amphiphilic crosslinked polymeric matrix material may be formed by polymerising the third crosslinker and the second functional monomer / oligomer with the second crosslinked polymeric material of the first interlayer. Thus, a network may be formed and the network may interpenetrate with the second crosslinked polymeric matrix material in the first interlayer.

[0185] Without wishing to be bound by theory, the third crosslinked polymeric matrix material may fill gaps among the superparamagnetic FesC nanoparticles in the second interlayer, thus preventing the superparamagnetic Fe3O4 nanoparticles in the second interlayer from dropping off or even merging into a larger size, and thereby avoiding the generation of undesired permanent magnetic properties.

[0186] In some embodiments of the present invention that may be mentioned herein, the third crosslinked polymeric matrix material may be formed from a third styrene monomer, and a fourth crosslinker.

[0187] Any suitable styrene monomer may be used as the third styrene monomer. Examples of suitable styrene monomers include, but are not limited to styrene and styrene derivatives such as vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

[0188] Any suitable crosslinker may be used as the fourth crosslinker. Examples of suitable crosslinkers include, but are not limited to divinylbenzene, ethylene glycol dimethacrylate, bisphenol A di methacrylate , bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta / hexa-acrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, di(trimethylolpropane) tetraacrylate, and trimethylolpropane triacrylate.

[0189] As shown in Fig. 2E, the second layer 160 is disposed on the third interlayer 150. The second layer 160 includes a fourth crosslinked polymeric matrix material 161. The fourth crosslinked polymeric matrix material 161 may be linked with the third crosslinked polymeric matrix material 151 of the third interlayer 150. Further, the fourth crosslinked polymeric matrix material 161 may comprise functional groups conjugated on a surface of the second layer 160.

[0190] In some embodiments of the present invention that may be mentioned herein, the fourth crosslinked polymeric matrix material may be formed from a fifth crosslinker, a third functional monomer that has at least one functional group, and a fourth functional monomer that has at least one functional group.

[0191] Any suitable crosslinker may be used as the fifth crosslinker. Examples of suitable crosslinkers include, but are not limited to butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, di pentaerythritol pentaacrylate, dipentaerythritol penta / hexa-acrylate, tripropylene diacrylate, di(trimethylolpropane) tetraacrylate, polyethylene glycol) diacrylate, polypropylene glycol) diacrylate, bisphenol A dimethacrylate, and tri(propylene glycol) diacrylate, optionally wherein the fifth crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, bisphenol A dimethacrylate, and N,N' -methylenebis(acrylamide).

[0192] In some embodiments of the present invention that may be mentioned herein, the third functional monomer that has at least one functional group may be selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer. For example, the third functional monomer that has at least one functional group may be selected from one or more of the group consisting of glycidyl methacrylate, allyl glycidyl ether, 2-hydroxyethyl methacrylate, poly(ethylene glycol) diacrylate, polypropylene glycol) diacrylate, and tripropylene glycol) diacrylate, acrylic acid, methacrylic acid, 2- carboxyethyl acrylate, ethylenediamine, diethylenetriamine, triethylenetetramine, ethylene glycol diglycidyl ether, diglycidyl ether of bisphenol A, maleimide, N-(2- aminoethyl)methacrylamide, N,N-dimethylaminoethyl methacrylate, N-vinyl-2-pyrrolidone, and 3-aminopropyltriethoxysilaneacrylic acid.

[0193] In some embodiments of the present invention that may be mentioned herein, the third functional monomer that has at least one functional group may form a hydrophobic domain. In further embodiments of the present invention that may be mentioned herein, the third functional monomer may be selected from one or more of the group consisting of methyl methacrylate ethyl methacrylate, butyl methacrylate, methyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, butyl acrylate, methyl acrylate, and 2-ethylhexyl acrylate.

[0194] In some embodiments of the present invention that may be mentioned herein, the weight ratio of the third functional monomer to the fifth crosslinker may be from 2:3 to 1 :20, preferably from 1:3 to 1 :10.

[0195] Without wishing to be bound by theory, the first and third crosslinked polymeric matrix materials may function to prevent the superparamagnetic FeaCLt nanoparticles from leaching from the microsphere when the microsphere is placed into a solvent.

[0196] In some embodiments of the present invention that may be mentioned herein, the fourth crosslinked polymeric matrix material of the second layer may be crosslinked to the third crosslinked polymeric matrix material of the third interlayer through the fifth crosslinker. Thus, there may be a crosslinked network at an inner surface of the fourth crosslinked polymeric matrix material. In addition, the fourth crosslinked polymeric matrix material may have further functional groups conjugated on an outer surface of the fourth crosslinked polymeric matrix material.

[0197] Without wishing to be bound by theory, when a hydrophobic monomer and a hydrophilic monomer copolymerize, they may not form a uniform structure but instead, they may create distinct domains. Some domains may be enriched with the hydrophilic monomer containing functional groups, while others may be dominated by hydrophobic C-C chains. This results in the formation of two sublayers: the inner sublayer, primarily composed of hydrophobic domains, which serves to adhere to the polymer substrate; and the outer sublayer, enriched with hydrophilic domains. The hydrophobic domains may predominantly occupy the inner layer, while the hydrophilic domains may be oriented toward the outer layer.

[0198] In some embodiments of the present invention that may be mentioned herein, all of the superparamagnetic Fe3O4 nanoparticles in a microsphere may account for from 10 to 80 wt%, such as from 20 to 70 wt%, such as from 10 to 55 wt%, such as from 15 to 45% of the entire weight of each microsphere.

[0199] In some embodiments of the present invention that may be mentioned herein, the superparamagnetic Fe3C>4 nanoparticles in the core portion and in the first layer may represent 20 to 40 wt% of the entire weight of each microsphere, and / or the superparamagnetic Fe3C>4 nanoparticles in the second interlayer may represent 10 to 20 wt% of the entire weight of each microsphere.

[0200] In some embodiments of the present invention that may be mentioned herein, the superparamagnetic FesC nanoparticles may have an average diameter of from 5 nm to 25 nm.

[0201] In some embodiments of the present invention that may be mentioned herein, the weight to weight ratio of styrene groups to crosslinking groups in the first layer may be from 20:1 to 1 :2, such as from 10:1 to 1:1.

[0202] In some embodiments of the present invention that may be mentioned herein, the weight to weight ratio of styrene groups to functional groups in the first layer may be from 20:1 to 1 :2, such as from 10:1 to 1:1.

[0203] In some embodiments of the present invention that may be mentioned herein, the superparamagnetic Fe3C>4 nanoparticles dispersed in the polystyrene polymeric matrix material and in the first crosslinked polymeric matrix material may have a total thickness of from 10 nm to 100 nm, such as from 45 nm to 85 nm, such as 71 nm.

[0204] In some embodiments of the present invention that may be mentioned herein, the first interlayer and the second interlayer may have a total thickness of from 10 nm to 5 nm, such as from 20 nm to 35 nm, such as 26 nm.

[0205] Further details of the manufacture of the shell portion will be provided below and in the experimental section.

[0206] In some embodiments of the present invention that may be mentioned herein, the second layer further comprises an organic polymer layer disposed on the second layer, wherein the organic polymer layer comprises a group shown by the following formula:

[0207] -CH2-CHR1-CH2-R2, wherein R1represents an alkoxy group and R2represents a carboxyl, tosyl or amine group, and wherein the carboxyl, tosyl and amine group is linear or branched.

[0208] Without wishing to be bound by theory, a C-C backbone can be formed within the second layer - R1and R2are the results of further modifications of the second layer. In a second aspect of the invention, there is provided a method of preparing a microsphere having a core-shell structure, the method comprising:

[0209] (ai) providing a precursor microsphere comprising a core portion formed from a polystyrene polymeric matrix material,

[0210] (aii) disposing a first layer on the core portion by providing a first crosslinked polymeric matrix material through dispersion polymerisation and dispersing superparamagnetic FesCU nanoparticles in the core portion and the first crosslinked polymeric matrix material;

[0211] (aiii) disposing a first interlayer on the first layer through precipitation coating, wherein the first interlayer comprises a second crosslinked polymeric matrix material;

[0212] (aiv) disposing a second interlayer on the first interlayer by providing an amphiphilic crosslinked polymeric matrix material and dispersing superparamagnetic FesC nanoparticles in the amphiphilic crosslinked polymeric matrix material and the second crosslinked polymeric matrix material of the first interlayer;

[0213] (av) disposing a third interlayer on the second interlayer through precipitation coating, wherein the third interlayer comprises a third crosslinked polymeric matrix material; and

[0214] (avi) disposing a second layer on the third interlayer, wherein the second layer comprises a fourth crosslinked polymeric matrix material.

[0215] The core portion, the first layer, the first interlayer, the second interlayer, the third interlayer, and the second layer are as described hereinbefore.

[0216] In some embodiments of the present invention that may be mentioned herein, the core portion formed from a polystyrene polymeric matrix material of step (ai) may be provided by polymerising styrene monomers in a solvent comprising a mixture of alcohol containing 1-20 v% water. The alcohol may be selected from one or more of methanol, ethanol, and isopropanol. The polymerization time of the styrene monomers may be between 2 to 12 hours. For example, the polymerization time of the styrene monomers may be between 4 to 10 hours. Specific details for the preparation of the core portion are provided in the Examples section below.

[0217] In some embodiments of the present invention that may be mentioned herein, step (ai) may further comprise providing:

[0218] (bi) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of methanol, ethanol, and isopropanol; (bi i) one or more polymeric stabilizers, optionally wherein the one or more polymeric stabilizers are selected from one or more of the group consisting of triton N-57, Triton X-100, poly(vinylpyrrolidone) (PVP), polyethylenimine (PEI), polyacrylic acid (PAA), polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPC), and chitosan, and optionally wherein the weight to weight ratio of styrene groups to one or more polymeric stabilizers in the core portion is from 50:1 to 1 :2, such as from 50:1 to 10:1 ; and / or

[0219] (biii) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2- methylbutyronitrile) (AM BN), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate and ammonia persulfate.

[0220] In further embodiments of the present invention that may be mentioned herein, the solvent provided in step (ai) may be a mixture of alcohol containing 1-20 v% water. Any suitable alcohol may be used. Examples of suitable alcohols include, but are not limited to methanol, ethanol, isopropanol, and their mixture thereof.

[0221] In further embodiments of the present invention that may be mentioned herein, the polymerisation initiator provided in step (ai) may be any suitable polymerisation initiator. For example, the polymerisation initiator may be an azo initiator such as 2,2 -azobis(2- methylpropionitrile) (AIBN) and 2,2'-azobis(2-methylbutyronitrile) (AMBN), a peroxide initiator such as tertiary-amyl hydroperoxide, and a persulfate salt such as potassium persulfate, sodium persulfate and ammonia persulfate.

[0222] In further embodiments of the present invention that may be mentioned herein, the solvent provided in step (ai) may further contain one or more polymeric stabilizers. In such embodiments, the weight to weight ratio of styrene groups to one or more polymeric stabilizers in the core portion may be from 50:1 to 1 :2, such as from 50:1 to 10:1. Any suitable polymeric stabilizer may be used. Examples of suitable polymeric stabilizers include, but are not limited to triton N-57, Triton X-100, poly(vinylpyrrolidone) (PVP), polyethylenimine (PEI), polyacrylic acid (PAA), polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPC), chitosan, and their mixture thereof.

[0223] In some embodiments of the present invention that may be mentioned herein, dispersing superparamagnetic FeaC nanoparticles in steps (aii) and (aiv) may comprise providing a solution that comprises a Fe(lll) salt, a Fe(ll) salt and a base. In some embodiments of the present invention that may be mentioned herein, one or more of the following may apply:

[0224] (gi) the Fe(lll) salt is selected from one or more of the group consisting of FeC and Fe2(SC>4)3, optionally wherein the Fe(lll) salt is FeCh;

[0225] (gii) the Fe(ll) salt is selected from one or more of the group consisting of FeCl2, FeSCh and Fe(OAC)2, optionally wherein the Fe(ll) salt is FeCh;and / or

[0226] (giii) the base is selected from one or more of the group consisting of ammonia hydroxide, NaOH, KOH, and amine, optionally wherein the base is aqueous ammonia.

[0227] In further embodiments of the present invention that may be mentioned herein, the Fe(lll) salt may be FeCh, the Fe(ll) salt may be FeCl2, and the base may be aqueous ammonia.

[0228] The dispersing superparamagnetic Fe3O4nanoparticles in the core portion and the first crosslinked polymeric matrix material may be achieved by immersing the microsphere which has a first crosslinked polymeric matrix material disposed on the core portion into a solution containing a Fe(lll) salt, a Fe(ll) salt, and a base.

[0229] As mentioned above, dispersion polymerization is applied to form the first crosslinked polymeric matrix material, which involves the polymerization of styrene monomers along with a crosslinking monomer and a functional monomer containing specific functional groups.

[0230] In some embodiments of the present invention that may be mentioned herein, the dispersion polymerisation in step (aii) may be conducted at a temperature of from about 40 °C to about 80 °C, such as from about 50 °C to about 70 °C, such as about 70 °C.

[0231] In some embodiments of the present invention that may be mentioned herein, the dispersion polymerisation in step (aii) may have a polymerisation time of from about 10 hours to about 72 hours, such as from about 16 hours to about 48 hours, such as about 16 hours.

[0232] In some embodiments of the present invention that may be mentioned herein, the dispersion polymerisation in step (aii) may include providing a solvent for polymerisation. Any suitable solvent may be used. Examples of suitable solvents include, but are not limited to 1 ,4-dioxane, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, water, methanol, ethanol and isopropanol. In certain embodiments of the present invention that may be mentioned herein, the solvent may be a mixture of alcohol containing 1-20 v% water. Any suitable alcohol may be used. Examples of suitable alcohols include, but are not limited to methanol, ethanol, isopropanol, and their mixture thereof. In further embodiments of the present invention that may be mentioned herein, a porogen may be introduced into the solvent in step (ai). Subsequently, the porogen may be eliminated from the monodispersed porous polymeric matrix through purification in another solvent.

[0233] In some embodiments of the present invention that may be mentioned herein, the weight ratio of the first styrene monomer to the porogen may be from 50:1 to 1 :2, preferably from 20:1 to 1:1. In further embodiments, the porogen may be selected from one or more of 1,4-dioxane, tetrahydrofuran, ethylene glycol, diglyme, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, toluene, ethylbenzene, xylene, benzene, and polyethylene glycol (400-8000).

[0234] In some embodiments of the present invention that may be mentioned herein, a solvent may be used to purify the first crosslinked polymeric matrix. Examples of suitable solvents for purifying the first crosslinked polymeric matrix include, but are not limited to n-methyl-2- pyrrolidone, acetonitrile, tetrahydrofuran, tetrahydrofuran and diglyme.

[0235] Any suitable precipitation coating method may be applied in step (aiii). For example, the precipitation coating in step (aiii) may involve two steps:

[0236] (i) providing a second mixture (a coating solution) comprising a second styrene monomer and a second crosslinker, to provide the second crosslinked polymeric matrix material. The second mixture may penetrate through the first layer, a formed network intervenes with the network of the first layer through polymerization; and

[0237] (ii) depositing an oligomer and polymer material onto a surface of the microsphere formed in step (aii) through this reaction.

[0238] Without wishing to be bound by theory, a majority of the second styrene monomer and the second crosslinker may deposit or precipitate on the first layer. The majority of the second styrene monomer and the second crosslinker forms oligomer and polymer. Due to limited solubility, the second crosslinked polymeric matrix material may precipitate on the surface of the first layer. Another portion of the second styrene monomer and the second crosslinker (i.e. a minority of the second styrene monomer and the second crosslinker) may penetrate the first layer, then the second crosslinked polymeric matrix material may be formed by polymerising the second styrene monomer and the second crosslinker with the first crosslinked polymeric material of the first layer. Thus, a network may be formed and the network may interpenetrate with the first crosslinked polymeric material of the first layer. In some embodiments of the present invention that may be mentioned herein, the precipitation coating in step (aiii) may be conducted a temperature of from about 40 °C to about 85 °C, such as from about 60 °C to about 75 °C.

[0239] In some embodiments of the present invention that may be mentioned herein, the precipitation coating in step (aiii) may have a polymerisation time of from about 1 hour to 24 hours, such as from about 3 hours to 17 hours.

[0240] In some embodiments of the present invention that may be mentioned herein, the second mixture used in step (aiii) may further include:

[0241] (ci) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of 1 ,4-dioxane, ethyl acetate, butyl acetate, acetone, hexane, octane, methyl ethyl ketone, and water;

[0242] (cii) a surfactant, optionally wherein the surfactant is selected from one or more of the group consisting of cetyltrimethylammonium bromide (CTAB), polyvinyl alcohol (PVA), Span 80, Span 60, and Span 20, Tween 20, Tween 80, Tween 60, sodium octyl sulfate, sodium dodecyl sulfate, sodium decyl sulfate, sodium tetradecyl sulfate, lauryl polyglucoside, decyl polyglucoside, and undecyl polyglucoside; and / or

[0243] (ciii) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2- methylbutyronitrile) (AM BN), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate and ammonia persulfate.

[0244] In another embodiment of the present invention that may be mentioned herein, the precipitation coating in step (aiii) may involve providing a second mixture comprising an epoxide, to provide the second crosslinked polymeric matrix material. In such embodiment that may be mentioned herein, the precipitation coating may be conducted a temperature of from about 40 °C to about 110 °C, such as from about 60 °C to about 95 °C. In such embodiment that may be mentioned herein, the precipitation coating in step (aiii) may have a polymerisation time of from about 1 hour to 24 hours, such as from about 3 hours to 17 hours.

[0245] Any suitable epoxide may be used. For example, the epoxide may consist of phenol rings. Examples of epoxides include, but are not limited to phenolic derivatives such as bis(epoxyethyl)benzene, 1 ,2-epoxy-4-(epoxyethyl)benzene, 1 ,3-bis(2,3-epoxypropoxy)- benzene, bisphenol A, AP, B, BP, C, E, F, G, M, P, PH, TMC, Z and its copolymer and propoxylated derivatives (e g. bisphenol A propoxylate diglycidyl ether, bisphenol A diglycidyl ether-bisphenol A copolymer, poly(bisphenol A-co-epichlorohydrin), and glycidyl capped), triglycidyl p-amino-phenol, and diglycidyl aniline tetraglycidyl meta-xylene diamine.

[0246] In such embodiment of the present invention that may be mentioned herein, the second mixture comprising an epoxide may further comprise:

[0247] (di) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of N-methyl-2-pyrrolidone, 1 ,4-dioxane, acetonitrile, acetone, bis(2- methoxyethyl) ether, methyl ethyl ketone, and ethanol; and / or

[0248] (dii) a catalyst, optionally wherein the catalyst is selected from one or more of the group consisting of pyridine, isoquinoline, quinoline, N,N-dimethyl-cyclohexylamine, tributylamine, N-ethyl morpholine, dimethylaniline, triethylamine (TEA), benzyl dimethylamine (BDMA), and 2,4,6-tris(dimethylaminomethyl)phenol (DMP-30).

[0249] Any suitable precipitation coating method may be applied in step (aiv). For example, the precipitation coating in step (aiv) may involve providing a third mixture (a coating solution) comprising a third crosslinker (e g. a hydrophobic crosslinker), and a second functional monomer / oligomer that has at least one functional group (e.g. a hydrophilic monomer / oligomer), to provide the amphiphilic crosslinked polymeric matrix material. The third mixture may penetrate through the first interlayer, and a formed network intervenes with the network of the first interlayer through polymerization.

[0250] As mentioned above, the third mixture may penetrate through the first interlayer. As such, superparamagnetic FesC nanoparticles may be incorporated into the first interlayer.

[0251] In some embodiments of the present invention that may be mentioned herein, the polymerisation in step (aiv) may be conducted at a temperature of from about 40 °C to about 90 °C, such as from about 55 °C to about 80 °C, such as about 75 °C.

[0252] In some embodiments of the present invention that may be mentioned herein, the polymerisation in step (aiv) may have a polymerisation time of from about 1 hours to about 24 hours, such as from about 3 hours to about 17 hours.

[0253] In another embodiment of the present invention that may be mentioned herein, the precipitation coating in step (aiv) may involve providing a third mixture comprising:

[0254] (ei) an anhydride monomer, optionally wherein the anhydride monomer is selected from one or more of the group consisting of maleic anhydride, phthalic anhydride, succinic anhydride, trimellitic anhydride, octenyl succinic anhydride, polyisobutylene succinic anhydride, octadecenyl succinic anhydride, and dodecenyl succinic anhydride; and / or

[0255] (eii) a copolymer, optionally wherein the copolymer is selected from one or more of the group consisting of poly(ethylene-a / f-maleic anhydride), poly(isobutylene-a / t-maleic anhydride), poly(methyl vinyl ether-a / t-maleic anhydride), and poly[(isobutylene-a / f-maleic acid, ammonium salt)-co-(isobutylene-a / f-maleic anhydride)].

[0256] In such embodiment of the present invention that may be mentioned herein, the copolymer may have a molecular weight of from 2,000 to 300,000.

[0257] In such embodiment of the present invention that may be mentioned herein, the third mixture may further comprise:

[0258] (fi) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of N-methyl-2-pyrrolidone, 1 ,4-dioxane, acetonitrile, acetone, bis(2- methoxyethyl) ether, methyl ethyl ketone, and ethanol; and / or

[0259] (fii) a catalyst, optionally wherein the catalyst is selected from one or more of the group consisting of pyridine, isoquinoline, quinoline, N,N-dimethyl-cyclohexylamine, tributylamine, N-ethyl morpholine, dimethylaniline, triethylamine (TEA) , benzyl dimethylamine (BDMA), and 2,4,6-tris(dimethylaminomethyl)phenol (DMP-30).

[0260] In such embodiment of the present invention that may be mentioned herein, the coating in step (aiv) may be conducted a temperature of from about 25 °C to about 95 °C, such as from about 25 °C to about 75 °C. In such embodiment that may be mentioned herein, the coating in step (aiv) may have a reaction time of from about 1 hour to 24 hours, such as from about 3 hours to 17 hours.

[0261] Any suitable precipitation coating method may be applied in step (av). For example, the precipitation coating in step (av) may be achieved by providing a fourth mixture (a coating solution) comprising a third styrene monomer, and a fourth crosslinker, to provide the third crosslinked polymeric matrix material. A formed network intervenes with the network of the second interlayer through polymerization, thus partially or fully encapsulating around the surface of the exposed magnetic nanoparticles in the second interlayer. As will be appreciated, this prevents the nanoparticles from being exposed to a buffer solution, which may cause the nanoparticles to leach out or aggregate.

[0262] In some embodiments of the present invention that may be mentioned herein, the fourth mixture used in step (av) may further comprise: (hi) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2- methylbutyronitrile) (AMBN), 4,4'-azobis (4-cyanovaleric acid), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate, and ammonia persulfate; and / or

[0263] (hii) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of 1 ,4-dioxane, ethyl acetate, butyl acetate, acetone, hexane, octane, methyl ethyl ketone, and water.

[0264] In some embodiments of the present invention that may be mentioned herein, the polymerisation in step (av) may be conducted at a temperature of from about 40 °C to about 105 °C, such as from about 60 °C to about 95 °C, such as about 75 °C.

[0265] In some embodiments of the present invention that may be mentioned herein, the polymerisation in step (av) may have a polymerisation time of from about 1 hours to about 24 hours, such as from about 3 hours to about 17 hours.

[0266] Without wishing to be bound by theory, the third crosslinked polymeric matrix material fills the gaps among superparamagnetic Fe3C>4 nanoparticles, preventing them from dropping off or even merging into a larger size, and thereby avoiding the generation of undesired permanent magnetic properties.

[0267] In some embodiments of the present invention that may be mentioned herein, disposing a third interlayer on the second interlayer through polymerisation in step (avi) may comprise using a fifth mixture comprising a fifth crosslinker, a third functional monomer that has at least one functional group, and a fourth functional monomer that has at least one functional group, to provide the fourth crosslinked polymeric matrix material.

[0268] In some embodiments of the present invention that may be mentioned herein, the fifth mixture used in step (avi) may further comprise:

[0269] (ii) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2- methylbutyronitrile) (AMBN), 4,4'-azobis (4-cyanovaleric acid), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate, and ammonia persulfate; and / or (iii) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of ethylene glycol, 1 ,4-dioxane, diglyme, octane, toluene, dimethylacetamide, dimethylformamide, xylene, n-methyl-2-pyrrolidone, n-butanol and water.

[0270] In another embodiment of the present invention that may be mentioned herein, step (avi) may comprise using a fifth mixture comprising a first epoxide, to provide the second crosslinked polymeric matrix material. For the avoidance of doubt, the first epoxide may be as described hereinbefore. As such, a full description of the first epoxide is not included here again for the sake of brevity.

[0271] In such embodiment of the present invention that may be mentioned herein, the fifth mixture may further comprise:

[0272] (ji) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of N-methyl-2-pyrrolidone, 1 ,4-dioxane, acetonitrile, acetone, bis(2- methoxyethyl) ether, methyl ethyl ketone, and ethanol;

[0273] (jii) a catalyst, optionally wherein the catalyst is selected from one or more of the group consisting of pyridine, isoquinoline, quinoline, N,N-dimethyl-cyclohexylamine, tributylamine, N-ethyl morpholine, dimethylaniline, triethylamine (TEA), benzyl dimethylamine (BDMA), and 2,4,6-tris(dimethylaminomethyl)phenol (DMP-30); and / or

[0274] (jiii) a second epoxide selected from one or more of the group consisting of 1,2- epoxydodecane, 1 ,4-butanediol diglycidyl ether, 1 ,2-butylene oxide, tert-butyl glycidyl ether, butyl glycidyl ether, 1 ,2-epoxydecane,1 ,2-epoxydodecane,1,2-epoxyheptane,1 ,2- epoxyhexane, polyethylene glycol diglycidyl ether (n = 4 to 22), and glycerol triglycidyl ether.

[0275] In such embodiment of the present invention that may be mentioned herein, the precipitation coating in step (avi) may be conducted a temperature of from about 40 °C to about 110 °C, such as from about 60 °C to about 95 °C.

[0276] In such embodiment of the present invention that may be mentioned herein, the precipitation coating in step (avi) may have a reaction time of from about 1 hour to 24 hours, such as from about 3 hours to 20 hours.

[0277] In such embodiment of the present invention that may be mentioned herein, step (avi) may further comprise using a carboxylic acid to hydrolyse the epoxide groups. Any suitable carboxylic acid may be used. For example, the carboxylic acid may be an aliphatic carboxylic or an aromatic carboxylic acid. Examples of carboxylic acids include, but are not limited to formic acid, acetic acid, propionic acid and butyric acid. In some embodiments of the present invention that may be mentioned herein:

[0278] (ki) the weight to weight ratio of styrene groups to crosslinking groups in the first layer may be from 20:1 to 1 :2, such as from 10:1 to 1:1 ;

[0279] (kii) the weight to weight ratio of styrene groups to functional groups in the first layer may be from 20:1 to 1 :2, such as from 10:1 to 1 :1;

[0280] (kiii) the weight to weight of styrene groups to crosslinking groups in the first interlayer may be from 20:1 to 1 :2, such as from 10:1 to 1 :1 ;

[0281] (kiv) the weight to weight ratio of functional groups to crosslinking groups in the second interlayer may be from 2:3 to 1 :20, such as from 1 :3 to 1 :10; and / or

[0282] (kv) the weight to weight ratio of functional groups to crosslinking groups in the second layer may be from 2:3 to 1 :20, such as from 1 :3 to 1 :10.

[0283] In a third aspect of the invention, there is provided a use of the microsphere as described hereinbefore in a chemiluminescence immunoassay. The microsphere as described hereinbefore may be used in any suitable chemiluminescence immunoassay.

[0284] It will be appreciated that the present invention overcomes the challenge of achieving consistent magnetic output and tunable magnetic properties in microspheres. This is accomplished via meticulous design and synthesis techniques aimed at preventing nanoparticle aggregation and preserving superparamagnetic behavior. Achieving this balance entails managing the hydrophobicity / hydrophilicity of adjacent layers and optimizing the charge distribution across different layers.

[0285] As also will be appreciated, the present invention provides the following advantages.

[0286] • Enhanced efficiency: by optimizing magnetic separation processes, the present invention accelerates assay procedures, leading to increased throughput and efficiency in diagnostic testing and biotechnology applications.

[0287] • Improved specificity: through tailored formulations and surface modifications, the present invention reduces background noise from nonspecifically bound molecules, enhancing assay specificity and accuracy.

[0288] • Consistency: the synthesis of monodisperse polymer beads as carriers for superparamagnetic nanoparticles ensures consistent magnetic output and tunable magnetic properties, promoting reliable and reproducible assay results. • Versatility: the present invention's adaptability to various high-throughput applications in in vitro diagnostics and biotechnology enables its widespread use across different laboratory settings and assay platforms.

[0289] • Addressing challenges: by addressing critical challenges such as bead aggregation, incomplete capture of target molecules, and batch-to-batch variation, the invention facilitates smoother and more reliable assay workflows, ultimately improving overall laboratory productivity and quality of results.

[0290] With increasing demand for high-throughput diagnostic testing in healthcare and biotechnology sectors, there is a growing need for technologies that can enhance assay efficiency, accuracy, and throughput. The present disclosure may directly address these demands by improving magnetic bead-based assay processes.

[0291] The present disclosure may provide a competitive edge to companies operating in the in vitro diagnostics (IVD) and biotechnology industries by offering enhanced assay performance, reliability, and consistency. This may lead to market differentiation and increased market share.

[0292] By streamlining assay workflows and reducing the likelihood of assay failures or inconsistencies, the present disclosure may contribute to cost savings for laboratories and diagnostic companies. This may be particularly important in high-throughput settings where efficiency and productivity are paramount.

[0293] The present disclosure's ability to improve assay accuracy and reproducibility may facilitate compliance with regulatory requirements in the healthcare industry.

[0294] Overall, the present disclosure’s commercial importance lies in its ability to address critical needs in the market, enhance assay performance, and drive innovation in the fields of diagnostics and biotechnology.

[0295] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0296] Examples

[0297] Materials

[0298] Styrene: Tokyo Chemical Industry, stabilized with TBC (4-tert-butylcatechol), >99.0%(GC). Azobisisobutyronitrile (AIBN): Sigma-Aldrich (12 wt% in acetone). Sodium persulfate (Na2S20s, SPS): Alfa Aesar, crystalline, 98%.

[0299] Polyvinylpyrrolidone (PVP, K30, MW=40,000): TCI, total nitrogen 12.0% to 12.8% (calcd.on anhydrous substance); water max. 7.0 %, K value 26.0 to 34.0.

[0300] Divinylbenzene (DVB, m- and p- mixture): Tokyo Chemical Industry, 50.0%(GC) (contains Ethylvinylbenzene, Diethylbenzene) (stabilized with 4-tert-butylcatechol).

[0301] 1 -ethyl 3-dimethylaminopropylcarbodiimide hydrochloride (EDC), Tokyo Chemical Industry Acrylic Acid: tokyo chemical industry, >99.0%(GC) (stabilized with monomethyl ether hydroquinone).

[0302] Glycidyl methacrylate: Sigma-Aldrich, >97.0% (GC).

[0303] 2-hydroxyethyl methacrylate: Sigma-Aldrich, >99.0%.

[0304] Methacrylic Acid (stabilized with MEHQ), tokyo chemical industry, >99.0%(GC).

[0305] 2-Carboxyethyl acrylate oligomers: Sigma-Aldrich, 2000 ppm MEHQ as inhibitor.

[0306] 2-hydroxyethyl methacrylate, Sigma-Aldrich.

[0307] Ethanol: Aik Moh, 99%.

[0308] Sodium dodecyl sulfate: Sigma-Aldrich, 98%.

[0309] Trimethylolpropane triacrylate: Sigma-Aldrich.

[0310] Methyl methacrylate: Tokyo Chemical Industry.

[0311] 2-(2-Aminoethoxy)ethanol: Sigma-Aldrich, 98%.

[0312] MES Hydrate>=99.5% (titration): Sigma-Aldrich.

[0313] N-Methyl Pyrrolidone (NMP), 99%, Aik Moh Paints & Chemicals Pte Ltd.

[0314] Neopentyl glycol diacrylate, Sigma-Aldrich

[0315] Trimethylolpropane triacrylate, Tee Hai Chem Pte. Ltd

[0316] Bisphenol A epoxy diacrylate, Tee Hai Chem Pte. Ltd

[0317] Benzyl methacrylate, Tee Hai Chem Pte. Ltd

[0318] Polyethylene glycol, Tee Hai Chem Pte. Ltd.

[0319] Acetonitrile, (>=99.9%), VWR Singapore Pte Ltd

[0320] FeCIs (IRON(III)CHOLORIDE, REGENT Grade, 97%), Sigma-Aldrich

[0321] FeCI2(IRON (ll)CHLORIDE TETRHYDRATE) 99.99%, Sigma-Aldrich

[0322] Ammonium Hydroxide Solution, 28-30%, Sigma-Aldrich

[0323] 4,4'-Azobis (4-cyanovaleric acid), 98%, Sigma-Aldrich

[0324] Bisphenol A diglycidyl ether, >85%, Tee Hai Chem Pte. Ltd.

[0325] Succinic anhydride, >95%, Tee Hai Chem Pte. Ltd.

[0326] Triethylamine, >99.5% Tee Hai Chem Pte. Ltd. p-Toluenesulfonyl Chloride, >99% Tee Hai Chem Pte. Ltd.

[0327] Pyridine, >99%, Sigma-Aldrich

[0328] Sodium hydroxide, Sigma-Aldrich Acetic acid, glacial, >99%, Sigma-Aldrich

[0329] Borate buffer, 20X, Thermofisher rStreptavidin, Thermofisher

[0330] Phosphate buffered saline (PBS), Sigma-Aldrich

[0331] Tween 20, Sigma-Aldrich

[0332] C-2 commerical beads Dyna T1 , Thermofisher.

[0333] Biotinylated anti-thyroid stimulating hormone (TSH) antibody is a common reagent and can be obtained from commercial sources. IL-6 Chemiluminescent Immunoassay Kit is a common diagnostic kit and can be obtained from commercial sources. CA19-9 Chemiluminescent Immunoassay Kit is a common diagnostic kit and can be obtained from commercial sources. Deionized (DI) water was obtained from ELGA Ultrapure Water Treatment Systems (PURELAB Option).

[0334] Analytical techniques

[0335] Thermal Gravimetric Analysis (TGA) was performed using Shimadzu, DTG-60.

[0336] Mechanical stirrer: Wiggens, WB2000-M overhead stirrer.

[0337] Size measurement was performed using Multisizer from Beckman Coulter.

[0338] TEM (Transmission Electron Microscopy) - JEOL 2010

[0339] Example 1

[0340] In a 250 mL three-necked round-bottom glass reactor equipped with a mechanical stirrer, PVP (0.1 g), ethanol (75 mL), polyethylene glycol (Mw = 400, 5 mL) were added. The mixture was stirred at room temperature for 5 minutes until a clear solution was obtained. This solution was then heated to 60 °C using an oil bath, styrene (7.5 mL) was added to the mixture, followed by the addition of AIBN (0.25 mL).

[0341] A white colloidal solution was formed after 3 hours of styrene polymerization. Subsequently, a solution of DVB (1.5 mL DVB in 10 mL ethanol) was added slowly drop by drop over 30 minutes. After the DVB solution was fully added, the cross-linking polymerization was continued for another 3 hours. Thus, a core portion is formed from a polystyrene polymeric matrix material.

[0342] Then, a solution of 2-carboxyethyl acrylate oligomers (1.5 g in 5 mL ethanol) was added using a syringe. The copolymerization of 2-carboxyethyl acrylate oligomers with excess styrene and DVB was then carried out for another 16 hours, resulting in a crosslinked polymeric matrix material disposed on the core portion. The produced beads are named beads A-1. Beads A- 1 were washed with acetonitrile 3 times.

[0343] To a 250 ml_ three-necked round bottom glass reactor equipped with a mechanical stirrer, FeCI3(15.6 g) and 100 mL water were added and stirred to form a brown solution, followed by addition of FeCl2 (9.6 g). The mixture was stirred for 30 min to form a solution, to which a solution of beads A-1 in water (200 mL, mass concentration -3.75 wt%) was added and aqueous ammonia (25 wt%, 230 mL) was added under vigorous stirring, affording a black slurry immediately. The black slurry was heated to 70 °C for 1 hour to complete the reaction. The superparamagnetic beads were purified to remove unbound superparamagnetic nanoparticles, and finally re-dispersed into DI water (200 mL, -4 wt%). Without wishing to be bound by theory, the superparamagnetic nanoparticles were dispersed in the polystyrene polymeric matrix material and in the crosslinked polymeric matrix material layer disposed on the core portion.

[0344] As will be appreciated, the superparamagnetic Fe3C>4 nanoparticles were partially dispersed in beads A-1 and a layer of the superparamagnetic FesCu nanoparticles was formed around beads A-1 . The size of the Fe3O4 nanoparticles, as measured using TEM, ranged from 5 to 20 nm. The sizes of beads A-1 before and after the addition of superparamagnetic Fe3C>4 nanoparticles were 0.924 pm and 0.995 pm, respectively, as measured using a Multisizer. The net size increase was 71 nm.

[0345] Example 2

[0346] The modified superparamagnetic beads from the previous step (250 mL, -5 wt% in water) were diluted with 250 mL of DI water. This mixture was then transferred to a 2 L double-jacket glass reactor equipped with a mechanical stirrer. The reactor was purged with nitrogen to eliminate oxygen.

[0347] Following this, a monomer mixture containing styrene (1200 mg), trimethylolpropane triacrylate (600 mg) and sodium dodecyl sulfate (SDS) was added in. The dispersion was heated to 75 °C and stirred at this temperature for 30 minutes. SPS (36 mg, dissolved in 2 mL of water) was then added to the reactor. The polymerization was allowed to continue for an additional 6 hours at 75 °C. The coated superparamagnetic beads were separated using a magnetic separator and washed twice with 500 mL of ethanol and twice with 500 mL of DI water. These coated beads were designated as B-1. Example 3

[0348] The coated superparamagnetic beads B-1 from the previous step (250 mL, ~4 wt% in water) were diluted with 400 L of DI water. This mixture was then transferred to a 2 L double-jacket glass reactor equipped with a mechanical stirrer. The reactor was purged with nitrogen to eliminate oxygen. SPS (36 mg, dissolved in 2 mL of water) was then added to the reactor. The dispersion was heated to 55 °C and stirred at this temperature for 10 minutes. A 10 mL water solution containing neopentyl glycol diacrylate (1000 mg) was added drop-by-drop at 20- minute intervals over the course of 30 min. Once the addition of the neopentyl glycol diacrylate mixture was completed, the reaction mixture was reacted for 2 hours. Following this, a 2 mL of water solution containing 2-hydroxyethyl methacrylate (75 mg) and 2-carboxyethyl acrylate (175 mg) was added, and the polymerization was allowed to continue for an additional 6 hours at 75 °C. The coated superparamagnetic beads were separated using a magnetic separator and washed twice with 500 mL of ethanol and twice with 500 mL of DI water and then diluted with 400 mL of DI water and added into a 2 L double-jacket glass reactor equipped with a mechanical stirrer. FeCh (8.0 g) and water (100 mL) were added and stirred to form a brown solution, followed by addition of FeCl2 (4.9 g). The mixture was stirred 30 minutes to form a solution, to which a solution of beads in water (460 mL, mass concentration ~2 wt%) was added and aqueous ammonia (25 wt%, 400 mL) was added under vigorous stirring, affording a black slurry immediately. The black slurry was heated to 70 °C for 1 hour to complete the reaction. The superparamagnetic beads were purified to remove unbound superparamagnetic NPs, and finally re-dispersed into N-Methyl Pyrrolidone (NMP) (100 mL, ~4 wt%).

[0349] The modified superparamagnetic beads C-1 had a size of 1.021 pm, as measured using a Multisizer. Compared to the size of A-1, this represents a net increase of 26 nm.

[0350] Example 4

[0351] The modified superparamagnetic beads C-1 is dispersed in water from the previous step (10 mL, ~4 wt%). This mixture was then transferred to a 2 L double-jacket glass reactor equipped with a mechanical stirrer. The reactor was purged with nitrogen to eliminate oxygen. An initiator, 4,4'-Azobis (4-cyanovaleric acid) (0.1g), was then added to the reactor. The dispersion was heated to 75 °C and stirred at this temperature for 10 minutes.

[0352] Following this, a monomer mixture containing benzyl methacrylate (2000 mg) and trimethylolpropane triacrylate (1100 mg) was added in the beads solution drop-by-drop. Once the addition of the monomer mixture was complete, the polymerization was allowed to continue for an additional 6 hours at 75 °C. The coated superparamagnetic beads were separated using a magnetic separator and washed twice with 500 mL of ethanol and twice with 500 mL of DI water. These coated beads were designated as D-1.

[0353] Example 5

[0354] The modified superparamagnetic beads D-1 prepared in Example 4 were dispersed in ethanol (10 mL, ~4 wt%). This mixture was then transferred to a 2 L double-jacket glass reactor equipped with a mechanical stirrer. The reactor was purged with nitrogen to eliminate oxygen.

[0355] Following this, a mixture containing methyl methacrylate (100 mg), bisphenol A epoxy diacrylate (180 mg) and methacrylic acid (50 mg) was added in beads solution and mixing for 2 hours. AIBN (0.25 mL) was then added to the reactor. The dispersion was heated to 65 °C and reacted for 16 hours. The coated superparamagnetic beads were washed twice with 500 mL of ethanol and twice with 500 mL of DI water. These coated beads were designated as E- 1.

[0356] Example 6

[0357] The coated superparamagnetic beads B-1 from the previous step (250 mL, ~4 wt% in water) were diluted with 400 mL of DI water. This mixture was then transferred to a 2 L double-jacket glass reactor equipped with a mechanical stirrer. The reactor was purged with nitrogen to eliminate oxygen. SPS (36 mg, dissolved in 2 mL of water) was then added into the reactor. The dispersion was heated to 55 °C and stirred at this temperature for 10 minutes. Divinyl benzene (1100 mg) and sodium dodecyl sulfate (50ml, 0.5% wt%) were homogenised and added into the dispersion. Once the addition of the styrene mixture was completed, the reaction mixture was reacted for 6 hours at 75 °C. Following this, the coated superparamagnetic beads were separated using a magnetic separator and washed with acetonitrile. The washed beads were dispersed in a 200 ml mixture composed of Bisphenol A diglycidyl ether (1.0 wt%) and triethylamine (1.0 wt%) in acetonitrile added into a 2 L doublejacket glass reactor equipped with a mechanical stirrer. The reaction mixture was reacted for 6 hours at 65 °C. Following this, the coated superparamagnetic beads were separated using a magnetic separator and washed with NMP. The washed beads were dispersed in 200ml mixture composed of succinic anhydride (5.0 wt%) and triethylamine (0.3 wt%) in NMP added into a 2 L double-jacket glass reactor equipped with a mechanical stirrer. The reaction mixture was reacted for 20 hours at 65 °C. FeCla (8.0 g) and water (100 mL) were added and stirred to form a brown solution, followed by addition of FeCh (4.9 g). The mixture was stirred 30 minutes to form a solution, to which a solution of beads in water (460 mL, mass concentration ~2 wt%) was added and aqueous ammonia (25 wt%, 400 mL) was added under vigorous stirring, affording a black slurry immediately. The black slurry was heated to 70 °C for 1 hour to complete the reaction. The superparamagnetic beads were purified to remove unbound superparamagnetic NPs, and finally re-dispersed into DI water (100 mL, ~4 wt%).

[0358] The modified superparamagnetic beads C-3 had a size of 1.065 pm, as measured using a Multisizer. Compared to the size of A-1 , this represents a net increase of 71 nm.

[0359] Example 7

[0360] The modified superparamagnetic beads C-3 prepared in Example 6 are dispersed in 10 mL of water (~4 wt%) from the previous step and then processed according to the procedure described in Example 4 to prepare the modified superparamagnetic beads D-2.

[0361] The modified superparamagnetic beads D-2 were then dispersed in acetonitrile (10 mL, ~4 wt%). This mixture was then transferred to a 250 mL round bottom flask equipped with a mechanical stirrer.

[0362] The coated superparamagnetic beads were washed with acetonitrile. The washed beads were dispersed in 80 mL acetonitrile mixture composed of Bisphenol A diglycidyl ether (1.0 wt%) and triethylamine (1.0 wt%). The reaction mixture was reacted for 3 hours at 65 °C.

[0363] Following this, the coated superparamagnetic beads were separated using a magnetic separator and washed with NMP. The washed beads were dispersed in a 200 ml mixture composed of succinic anhydride (2 wt%) and triethylamine (0.3 wt%) in NMP added into a 250 mL round bottom flask equipped with a mechanical stirrer. The reaction mixture was reacted for 17 hours at 65 °C. The coated superparamagnetic beads were washed twice with 400 mL of ethanol and twice with 400 mL of DI water. These coated beads were designated as E-2.

[0364] Example 8 The modified superparamagnetic beads C-3 prepared in Example 6 were dispersed in 10 mL of water (~4 wt%) from the previous step and then processed according to the procedure described in Example 4 to prepare the modified superparamagnetic beads D-2.

[0365] The modified superparamagnetic beads D-2 were then dispersed in acetonitrile (10 mL, ~4 wt%). This mixture was then transferred to a 250 mL round bottom flask equipped with a mechanical stirrer.

[0366] The coated superparamagnetic beads were separated using a magnetic separator, followed by solvent exchange with acetonitrile. The washed beads were dispersed in a 80 ml acetonitrile mixture composed of poly(bisphenol A-co-epichloro hydrin), capped with glycidyl (0.1 wt%) and triethylamine (0.05 wt%). The reaction mixture was reacted for 3 hours at 65 °C. Following this, the coated superparamagnetic beads were separated using a magnetic separator and washed with acetonitrile thrice.

[0367] Following this, the coated superparamagnetic beads were hydrolysed with 0.4 M acetic acid and wash with DI water. The coated superparamagnetic beads were washed with acetonitrile. This mixture was then transferred to a 100 mL round bottom flask equipped with a mechanical stirrer. The washed beads were dispersed in 40 mL of acetonitrile mixture containing tosyl chloride (38 mg) and pyridine (0.016 mL). The reaction mixture was reacted for 3 hours at room temperature.

[0368] The coated superparamagnetic beads were washed twice with 400 mL of ethanol and twice with 400 mL of DI water. These coated beads were designated as E-3.

[0369] Example 9

[0370] The modified superparamagnetic beads E-1 prepared in Example 5 were dispersed in MES pH = 6.5 (10 mL, ~4 wt%). EDC solution 10 mg / mL was added into the mixture for activation of 15 minutes. After 15 minutes, the supernatant was removed and Streptavidin was added. The magnetic beads were mixed for 2 hours. This mixture was then washed with 1xPBS buffer and redispersed in 1xPBS buffer. These coated beads were designated as “F-1”. The unreacted Streptavidin (SA) was removed to obtain particles for bonding biotins with streptavidin immobilized on the surface of F-1. 1 mg of the biotin-bonding particles were dispersed in a PBS / 0.01% Tween 20 solution and 10 microgram of a biotinization anti-TSH antibody was added, followed by reaction at room temperature for one hour. The beads were then coated with anti-TSH antibody, designated as G-1. Example 10

[0371] The modified superparamagnetic beads E-1 and E-2 prepared in Examples 6 and 7 , respectively, were dispersed in MES pH = 6.5 (10 ml_, ~4 wt%). EDC solution 10 mg / mL was added into the mixture for activation of 15 minutes. After 15 minutes, the supernatant was removed and Streptavidin was added. The magnetic beads were mixed for 2 hours. This mixture was then washed with 1xPBS buffer and redispersed in 1xPBS buffer. These coated beads were designated as “F-1” and “F-2”. The unreacted Streptavidin (SA) was removed to obtain particles for bonding biotins with streptavidin immobilized on the surface of F-1 and F- 2. 1 mg of the biotin-bonding particles were dispersed in a PBS / 0.01 % Tween 20 solution and 10 microgram of a biotinization anti-TSH antibody was added into F-1 , followed by reaction at room temperature for one hour. The beads were then coated with anti-TSH antibody, designated as G-1. 1 mg of F-2 were dispersed in 5 ml_ of 1xPBS / 0.01 % Tween 20 solution and designated as G-2.

[0372] Comparative Example 1

[0373] The purpose of this comparative example is to compare how FejC content and surface hydrophilic to hydrophobic ratio will affect the final performance of the magnetic beads. Following examples 5 to 8 described in WO2021 / 225519 A1 , the magnetic particles were synthesized and designated as CA-1.

[0374] A one-pot, three-stage polymerization was used to create monodisperse beads from styrene, DVB, and GMA. AIBN solution (2.0 g, 12 wt% in acetone) was added to a nitrogen-flushed 250 mL reactor, and the acetone was evaporated. PVP (0.3 g), ethanol (80 mL), and DI water (16 mL) were added and stirred at room temperature for 5 minutes. The solution was heated to 60°C, and styrene (7.5 mL) was added. After 6 hours, a white colloidal solution formed. DVB (1.5 mL in 8.5 mL ethanol) was added over 30 minutes, and the reaction continued for 2 hours. GMA (2.0 g in 10 mL ethanol) was added, and the mixture reacted for 24 hours, forming a milk-like solution, designated as “CA-1.”

[0375] The CA-1 beads (~8 wt%) were centrifuged, re-dispersed in ethanol, and washed with ethanol and a THF / water mixture. They were then stirred in a THF solution with 2-(2- aminoethoxy)ethanol for 20 hours, washed with water, and re-dispersed, designated as “CB- 1.” FeCh (8.0 g) and water (100 mL) were mixed in a 250 mL reactor, followed by FeCh (3.1 g) and THF (20 mL). CB-1 beads (50 mL, ~8 wt%) were added and stirred at 65 °C for 3 hours. After cooling, aqueous ammonia was added, forming a black slurry. The beads were purified and re-dispersed in DI water, yielding superparamagnetic beads “CC-1.”

[0376] In a 250 mL three-necked reactor with a stirrer, superparamagnetic beads CC-1 (10 mL, 4 wt% in anhydrous THF), methacrylic anhydride (120 mg in 30 mL anhydrous THF), and triethylamine (20 pL in 10 mL anhydrous THF) were added. The mixture was heated to 65 °C for 3 hours under nitrogen, then cool to room temperature. The beads were washed with THF and water, then stored in water under nitrogen.

[0377] The modified beads (10 mL, ~4 wt% in water) were transferred to a 50 mL centrifuge tube with 30 mL DI water, and added to a 250 mL three-necked reactor with a stirrer. The mixture was flushed with nitrogen, SPS (30 mg in 2 mL water) was added to the mixture, and the mixture was heated to 60 °C for 10 minutes. A monomer mixture (methyl methacrylate 400 mg, bisphenol A epoxy diacrylate 150 mg) was added in 4 batches over 4 hours. Polymerization was continued for 4 hours at 60 °C. Then, acrylic acid monomers (100 mg in water, neutralized with ammonia) were added. The reaction mixture was reacted for 16 hours at 60 °C. After cooling, the beads were isolated with a magnetic separator, washed with ethanol and DI water, yielding "CD-1" beads.

[0378] Comparative Example 2

[0379] The modified superparamagnetic beads CD-1 were coated with streptavidin according to the same protocol in Example 6, yielding SA coated beads, designated as “CE-1”. 1 mg of the biotin-bonding particles were dispersed in a PBS / 0.01% Tween 20 solution and 10 microgram of a biotinization anti-TSH antibody was added, followed by reaction at room temperature for one hour. The beads were then coated with anti-TSH antibody, designated as “CF-1”.

[0380] Example 11

[0381] 1 mg of the commercial beads C-2, CF-1 and G-1 were dispersed in a 1xPBS / 0.01% Tween 20 solution respectively and tested using a commercial IL-6 Chemiluminescent Immunoassay Kit by following the manufacturer’s protocol but without the addition of the antigen. The signal obtained is attributed solely to detector noise and nonspecific adsorption between the magnetic beads and the antibody in the kit. As shown in Table 1 , Sample CF-1 exhibited unstable and significantly high nonspecific adsorption, causing background fluctuations over 15 samples. In contrast, sample G-1 showed much lower background noise compared to both the control and CF-1 , indicating exceptional performance.

[0382] Table 1. Non-specific adsorption of IL-6.

[0383] Example 12

[0384] Precision testing in chemiluminescence assays is crucial for ensuring the reliability and reproducibility of the assay results. To provide an insight of how magnetic content could affect reproducibility, Intra-Assay Precision (Repeatability) was evaluated by using three different magnetic beads.

[0385] 1 mg of the commercial beads C-2, CF-1 and G-1 were dispersed in a PBS / 0.01 % Tween 20 solution respectively and tested using a commercial TSH Chemiluminescent Immunoassay Kit by following the manufacturer’s protocol but without two antigen concentration levels, Cal_L and Cal_H. As shown in Table 3, Sample CF-1 exhibited a CV over 10 repeats. This variability is due to the differing iron content of the beads, as shown in Table 2. The magnetic separation time for the auto-chemiluminescence platform is a fixed parameter, leading to inconsistent recovery of the magnetic beads during repeated washing. Consequently, the signal fluctuates randomly due to the loss of magnetic beads in each washing step and it is challenging to achieve CV lower than 5%.

[0386] Table 2. Percentage of FesCV / o in beads analysed by TGA.

[0387] Table 3. Repeatability of 3 magnetic beads.

[0388] Example 13

[0389] 20 mg of modified superparamagnetic beads E-3, prepared as described in Example 8, were dispersed in 25mM borate buffer (pH 9.0). Streptavidin was added at a ratio of 0.02 mg per mg of E-3. The mixture was incubated at room temperature with gentle mixing for 24 hours. After incubation, the supernatant was removed, and the beads were resuspended in 1x PBS containing 0.1% BSA to block nonspecific binding. The coated beads were designated as F- 3. 1 mg of F-3 were dispersed in 5 mL of 1xPBS / 0.01 % Tween 20 solution and designated as G-3.

[0390] Example 14

[0391] 1 mg each of the commercial beads G-2 and G-3 prepared in Examples 10 and 13, respectively, was dispersed in 1xPBS containing 0.01% Tween 20 and tested using a commercial CA19-9 Chemiluminescent Immunoassay Kit, following the manufacturer’s protocol with the exception of using only two calibration levels. 'Cal_0' consisted of 1xPBS with 0.01% Tween 20, and 'Cal_2' was obtained from the commercial kit. The control was performed using the original CA19-9 testing kit without modification. The control showed a signal ratio of 597, reflecting the baseline performance of the unmodified commercial kit. Beads G-3 achieved the highest signal ratio of 817, suggesting enhanced sensitivity and / or lower background, and outperforming both the control and G-2. Different surface functionalization strategies can significantly impact the performance of various assays.

[0392] Table 4. Chemiluminescent Immunoassay Results.

[0393] Example 15

[0394] Freeze-thaw properties of beads

[0395] CF-1 and G-2 prepared in Comparative Example 2 and Example 10, respectively, were dispersed in a suspension buffer consisting of 1 x PBS with 0.1% BSA at a bead concentration of 10 mg / mL. All samples were subjected to freezing at -20 °C, followed by thawing to room temperature and vortexing at 2000 rpm for approximately 15 seconds. This freeze-thaw process was repeated for a total of three cycles. After three cycles, visible precipitates were observed in the CF-1 sample, whereas G-2 remained well-dispersed in the suspension buffer.

[0396] Table 5 compares the signal intensities of commercial beads without freeze-thaw cycles to beads G-2 after undergoing three freeze-thaw cycles, across three TSH antigen concentration levels (Cal_S1 , Cal_S2, and Cal_S3). Cal_S1 represents zero antigen concentration, G-2 shows acceptable noise level compared to the control. At Cal_S2, G-2 maintained similar reading as compared with the control signal. These results suggest that G-2 maintains good functional stability after three freeze-thaw cycles, indicating its potential applications requiring repeated freeze-thaw handling.

[0397] Table 5. Freeze-thaw properties of G-2.

Claims

Claims1. A microsphere having a core-shell structure, the microsphere comprising: a core portion formed from a polystyrene polymeric matrix material, wherein superparamagnetic FesC nanoparticles are dispersed in the polystyrene polymeric matrix material; and a shell portion that encapsulates the core portion, the shell portion comprising a first layer, a first interlayer, a second interlayer, a third interlayer, and a second layer, wherein the first layer is disposed on the core portion, the first layer comprising a first crosslinked polymeric matrix material, wherein superparamagnetic FesCu nanoparticles are dispersed in the first crosslinked polymeric matrix material, the first interlayer is disposed on the first layer, the first interlayer comprising a second crosslinked polymeric matrix material, wherein superparamagnetic Fe3O4nanoparticles are dispersed in the second crosslinked polymeric matrix material, the second interlayer is disposed on the first interlayer, the second interlayer comprising an amphiphilic crosslinked polymeric matrix material, wherein superparamagnetic Fe3C>4 nanoparticles are dispersed in the amphiphilic crosslinked polymeric matrix material, the third interlayer is disposed on the second interlayer, the third interlayer comprising a third crosslinked polymeric matrix material, and the second layer is disposed on the third interlayer, the second layer comprising a fourth crosslinked polymeric matrix material.

2. The microsphere according to Claim 1 , wherein the first crosslinked polymeric matrix material is formed from a first styrene monomer, a first crosslinker, and a first functional monomer / oligomer that has at least one functional group.

3. The microsphere according to Claim 2, wherein the first styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionally wherein the styrene derivative is selected from one or more of the group consisting of 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

4. The microsphere according to Claim 2 or Claim 3, wherein the first crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, methylene acrylamide, bisphenol A dimethacrylate, N,N ’ methylenebis(acrylamide), bisphenol A epoxy diacrylate, bisphenol diacrylate, tripropyleneglycol diacrylate, neopentyl glycol diacrylate, propoxylated glycerol diacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, and tripropylene diacrylate.

5. The microsphere according to any one of the preceding claims, wherein the second crosslinked polymeric matrix material is formed from a second styrene monomer, and a second crosslinker.

6. The microsphere according to Claim 5, wherein the second styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionally wherein the styrene derivative is selected from one or more of the group consisting of vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

7. The microsphere according to Claim 5 or Claim 6, wherein the second crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, bisphenol A dimethacrylate, N,N' -methylenebis(acrylamide), bisphenol A epoxy diacrylate, bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, propoxylated glycerol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta-acrylate, hexa-acrylate, tripropylene diacrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, di(trimethylolpropane) tetraacrylate, trimethylolpropane triacrylate, poly(ethylene glycol) diacrylate, polypropylene glycol) diacrylate, and tri(propylene glycol) diacrylate.

8. The microsphere according to any one of the preceding claims, wherein the amphiphilic crosslinked polymeric matrix material is formed from a third crosslinker, and a second functional monomer / oligomer that has at least one functional group.

9. The microsphere according to Claim 8, wherein the third crosslinker is selected from one or more of the group consisting of divinylbenzene, bisphenol A dimethacrylate, bisphenol diacrylate, tricyclodecane dimethanol diacrylate, ditrimethylolpropane tetraacrylate, di(trimethylolpropane), tetraacrylate, ethylene glycol dimethacrylate, bisphenol A epoxy diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, pentaerythritol triacrylate, dipentaerythritolpenta / hexa-acrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, trimethylolpropane triacrylate, diethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, trimethylolpropane ethoxylate diacrylate, isobornyl acrylate, 1 ,6-hexanediol diacrylate, cyclohexyl methacrylate, 1 ,4-butanediol dimethacrylate, cyclohexyl acrylate, 1 ,3-butylene glycol di methacrylate, and isodecyl acrylate.

10. The microsphere according to any one of Claims 2 to 4, 8 and 9, wherein the at least one functional group on the first functional monomer / oligomer and / or the second functional monomer / oligomer is independently selected from one or more of amino, carboxyl, epoxy, and hydroxyl, optionally wherein the at least one functional group on the first functional monomer / oligomer and / or the second functional monomer / oligomer is independently selected from a combination of hydroxyl and carboxyl groups or a combination of amino and carboxyl groups.

11. The microsphere according to Claim 10, wherein the first functional monomer / oligomer is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the first functional monomer / oligomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2- carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, divinyl-benzene- trimethylamine, (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 oligomer.

12. The microsphere according to Claim 10, wherein the second functional monomer / oligomer is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the second functional monomer / oligomer is selected from one or more of the group consisting of ethylenediamine, diethylenetriamine, triethylenetetramine, ethylene glycol diglycidyl ether, diglycidyl ether of bisphenol A, maleimide, N-(2-aminoethyl)methacrylamide, N,N- dimethylaminoethyl methacrylate, N-vinyl-2-pyrrolidone, 3-aminopropyltriethoxysilaneacrylic 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 oligomer.

13. The microsphere according to any one of the preceding claims, wherein the third crosslinked polymeric matrix material is formed from a third styrene monomer, and a fourth crosslinker.

14. The microsphere according to Claim 13, where in the third styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionally wherein the styrene derivative is selected from one or more of the group consisting of vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

15. The microsphere according to Claim 13 or Claim 14, where in the fourth crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethacrylate, bisphenol A dimethacrylate , bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta / hexa-acrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, di(trimethylolpropane) tetraacrylate, and trimethylolpropane triacrylate.

16. The microsphere according to any one of the preceding claims, wherein the fourth crosslinked polymeric matrix material is formed from a fifth crosslinker, a third functional monomer that has at least one functional group, and a fourth functional monomer that has at least one functional group.

17. The microsphere according to Claim 16, wherein the fifth crosslinker is selected from one or more of the group consisting of butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, di pentaerythritol pentaacrylate, dipentaerythritol penta / hexa-acrylate, tripropylene diacrylate, di(trimethylolpropane) tetraacrylate, polyethylene glycol) diacrylate, polypropylene glycol) diacrylate, bisphenol A dimethacrylate, and tri(propylene glycol) diacrylate, optionally wherein the fifth crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, bisphenol A dimethacrylate, and N,N' -methylenebis(acrylamide).

18. The microsphere according to Claim 16 or Claim 17, wherein the third functional monomer that has at least one functional group is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the third functional monomer that has at least one functional group is selected from one or more of the group consisting of glycidyl methacrylate, allyl glycidyl ether, 2-hydroxyethyl methacrylate, poly(ethylene glycol) diacrylate, polypropylene glycol) diacrylate, and tri(propylene glycol) diacrylate, acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, ethylenediamine, diethylenetriamine, triethylenetetramine, ethylene glycol diglycidyl ether, diglycidyl ether of bisphenol A, maleimide, N-(2-aminoethyl)methacrylamide, N,N- dimethylaminoethyl methacrylate, N-vinyl-2-pyrrolidone, and 3- aminopropyltriethoxysilaneacrylic acid.

19. The microsphere according to any one of Claims 16 to 18, wherein the fourth functional monomer is selected from one or more of the group consisting of methyl methacrylate ethyl methacrylate, butyl methacrylate, methyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, butyl acrylate, methyl acrylate, and 2-ethylhexyl acrylate.

20. The microsphere according to Claim 1 , wherein the first and third crosslinked polymeric matrix materials function to prevent the superparamagnetic Fe3O4 nanoparticles from leaching from the microsphere when the microsphere is placed into a solvent.

21. The microsphere according to any one of the preceding claims, wherein the polystyrene polymeric matrix material is formed from one or more of the group consisting of styrene, a styrene derivative, and copolymers thereof, optionally wherein the styrene derivative is selected from one or more of the group consisting of vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4-methylstyrene, 3-methylstyrene, and 4- tertbutylstyrene.

22. The microsphere according to any one of the preceding claims, wherein all of the superparamagnetic Fe3O4 nanoparticles in a microsphere account for from 10 to 80 wt%, such as from 20 to 70 wt%, such as from 10 to 55 wt%, such as from 15 to 45% of the entire weight of each microsphere.

23. The microsphere according to Claim 22, wherein:(a) the superparamagnetic Fe3O4 nanoparticles in the core portion and in the first layer represent 20 to 40 wt% of the entire weight of each microsphere; and / or(b) the superparamagnetic FesC nanoparticles in the second interlayer represent 10 to 20 wt% of the entire weight of each microsphere.

24. The microsphere according to any one of the preceding claims, wherein the superparamagnetic FeaC nanoparticles have an average diameter of from 5 nm to 25 nm.

25. The microsphere according to any one of the preceding claims, wherein:(a) the weight to weight ratio of styrene groups to crosslinking groups in the first layer is from 20:1 to 1 :2, such as from 10:1 to 1:1 ;(b) the weight to weight ratio of styrene groups to functional groups in the first layer is from 20:1 to 1 :2, such as from 10:1 to 1 :1 ;(c) the weight to weight of styrene groups to crosslinking groups in the first interlayer is from 20:1 to 1:2, such as from 10:1 to 1 :1 ;(d) the weight to weight ratio of functional groups to crosslinking groups in the second interlayer is from 2:3 to 1 :20, such as from 1 :3 to 1:10; and / or(e) the weight to weight ratio of functional groups to crosslinking groups in the second layer is from 2:3 to 1 :20, such as from 1 :3 to 1 :10.

26. The microsphere according to any one of the preceding claims, wherein the superparamagnetic Fe3C>4 nanoparticles dispersed in the polystyrene polymeric matrix material and in the first crosslinked polymeric matrix material have a total thickness of from 10 nm to 100 nm, such as from 45 nm to 85 nm, such as 71 nm.

27. The microsphere according to any one of the preceding claims, wherein the first interlayer and the second interlayer have a total thickness of from 10 nm to 50 nm, such as from 20 nm to 35 nm, such as 26 nm.

28. The microsphere according to any one of the preceding claims, wherein the second layer further comprises an organic polymer layer, wherein the organic polymer layer comprises a group shown by the following formula:-CH2-CHR1-CH2-R2, wherein R1represents an alkoxy group and R2represents a carboxyl, tosyl or amine group, and wherein the carboxyl, tosyl and amine group is linear or branched.

29. A method of preparing a microsphere having a core-shell structure, the method comprising:(ai) providing a precursor microsphere comprising a core portion formed from a polystyrene polymeric matrix material,(aii) disposing a first layer on the core portion by providing a first crosslinked polymeric matrix material through dispersion polymerisation and dispersing superparamagnetic Fe3O4 nanoparticles in the core portion and the first crosslinked polymeric matrix material;(aiii) disposing a first interlayer on the first layer through precipitation coating, wherein the first interlayer comprises a second crosslinked polymeric matrix material;(aiv) disposing a second interlayer on the first interlayer by providing an amphiphilic crosslinked polymeric matrix material through polymerisation and dispersing superparamagnetic Fe3C>4 nanoparticles in the amphiphilic crosslinked polymeric matrix material and the second crosslinked polymeric matrix material of the first interlayer;(av) disposing a third interlayer on the second interlayer through precipitation coating, wherein the third interlayer comprises a third crosslinked polymeric matrix material; and(avi) disposing a second layer on the third interlayer through polymerisation, wherein the second layer comprises a fourth crosslinked polymeric matrix material.

30. The method according to Claim 29, further comprising providing a porogen in step (ai).31 . The method according to Claim 30, wherein the porogen is selected from one or more of 1,4-dioxane, tetrahydrofuran, ethylene glycol, diglyme, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, toluene, ethylbenzene, xylene, benzene, and polyethylene glycol (400- 8000).

32. The method according to Claim 30 or Claim 31 , wherein the weight to weight ratio of styrene groups to porogen in the core portion is from 50:1 to 1 :2, such as from 20:1 to 1 :1.

33. The method according to any one of Claims 29 to 32, wherein step (ai) comprises providing:(bi) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of methanol, ethanol, and isopropanol;(bii) one or more polymeric stabilizers, optionally wherein the one or more polymeric stabilizers are selected from one or more of the group consisting of triton N-57, Triton X-100, poly(vinylpyrrolidone) (PVP), polyethylenimine (PEI), polyacrylic acid (PAA), polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPC), and chitosan, and optionally wherein the weightto weight ratio of styrene groups to one or more polymeric stabilizers in the core portion is from 50:1 to 1 :2, such as from 50:1 to 10:1 ; and / or(biii) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2- methylbutyronitrile) (AM BN), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate and ammonia persulfate.

34. The method according to any one of Claims 29 to 33, wherein step (aii) comprises using a first mixture comprising a first styrene monomer, a first crosslinker, and a first functional monomer / oligomer that has at least one functional group, to provide the first crosslinked polymeric matrix material.

35. The method according to Claim 34, wherein the first styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionally wherein the styrene derivative is selected from one or more of the group consisting of 4-methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

36. The method according to Claim 34 or Claim 35, wherein the first crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, methylene acrylamide, bisphenol A dimethacrylate, N,N ' -methylenebis(acrylamide), bisphenol A epoxy diacrylate, bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, propoxylated glycerol diacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, and tripropylene diacrylate.

37. The method according to any one of Claims 34 to 36, wherein the first mixture in step (aii) further comprises a solvent, optionally wherein the solvent is selected from one or more of the group consisting of 1 ,4-dioxane, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, water, methanol, ethanol and isopropanol.

38. The method according to any one of Claims 29 to 37, wherein the dispersion polymerisation in step (aii) is conducted at a temperature of from about 40 °C to about 80 °C, such as from about 50 °C to about 70 °C, such as about 70 °C.

39. The method according to any one of Claims 29 to 38, wherein the dispersion polymerisation in step (aii) has a polymerisation time of from about 10 hours to about 72 hours, such as from about 16 hours to about 48 hours, such as about 16 hours.

40. The method according to any one of Claims 29 to 39, wherein step (aiii) comprises using a second mixture comprising a second styrene monomer, and a second crosslinker, to provide the second crosslinked polymeric matrix material.

41. The method according to Claim 40, wherein the second styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionally wherein the styrene derivative is selected from one or more of the group consisting of vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

42. The method according to Claim 40 or Claim 41 , wherein the second crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, bisphenol A dimethacrylate, N,N' -methylenebis(acrylamide), bisphenol A epoxy diacrylate, bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, propoxylated glycerol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta-acrylate, hexa-acrylate, tripropylene diacrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, di(trimethylolpropane) tetraacrylate, trimethylolpropane triacrylate, poly(ethylene glycol) diacrylate, polypropylene glycol) diacrylate, and tri(propylene glycol) diacrylate.

43. The method according to any one of Claims 40 to 42, wherein the second mixture further comprises:(ci) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of 1 ,4-dioxane, ethyl acetate, butyl acetate, acetone, hexane, octane, methyl ethyl ketone, and water;(cii) a surfactant, optionally wherein the surfactant is selected from one or more of the group consisting of cetyltrimethylammonium bromide (CTAB), polyvinyl alcohol (PVA), Span 80, Span 60, and Span 20, Tween 20, Tween 80, Tween 60, sodium octyl sulfate,sodium dodecyl sulfate, sodium decyl sulfate, sodium tetradecyl sulfate, lauryl polyglucoside, decyl polyglucoside, and undecyl polyglucoside; and / or(ciii) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2- methylbutyronitrile) (AM BN), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate and ammonia persulfate.

44. The method according to any one of Claims 29 to 43, wherein the precipitation coating in step (aiii) is conducted a temperature of from about 40 °C to about 85 °C, such as from about 60 °C to about 75 °C.

45. The method according to any one of Claims 29 to 44, wherein the precipitation coating in step (aiii) has a polymerisation time of from about 1 hour to 24 hours, such as from about 3 hours to 20 hours.

46. The method according to any one of Claims 29 to 39, wherein step (aiii) comprises using a second mixture comprising an epoxide, to provide the second crosslinked polymeric matrix material.

47. The method according to Claim 46, wherein the epoxide comprises phenol rings, optionally wherein the epoxide is selected from phenolic derivatives such as bis(epoxyethyl)benzene, 1 ,2-epoxy-4-(epoxyethyl)benzene, 1 ,3-bis(2,3-epoxypropoxy)- benzene, bisphenol A, AP, B, BP, C, E, F, G, M, P, PH, TMC, Z and its copolymer and propoxylated derivatives (e g. bisphenol A propoxylate diglycidyl ether, bisphenol A diglycidyl ether-bisphenol A copolymer, poly(bisphenol A-co-epichlorohydrin), and glycidyl capped), triglycidyl p-amino-phenol, and diglycidyl aniline tetraglycidyl meta-xylene diamine.

48. The method according to Claim 46 or Claim 47, wherein the second mixture further comprises:(di) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of N-methyl-2-pyrrolidone, 1 ,4-dioxane, acetonitrile, acetone, bis(2- methoxyethyl) ether, methyl ethyl ketone, and ethanol; and / or(dii) a catalyst, optionally wherein the catalyst is selected from one or more of the group consisting of pyridine, isoquinoline, quinoline, N,N-dimethyl-cyclohexylamine,tributylamine, N-ethyl morpholine, dimethylaniline, triethylamine (TEA), benzyl dimethylamine (BDMA), and 2,4,6-tris(dimethylaminomethyl)phenol (DMP-30).

49. The method according to any one of Claims 46 to 48, wherein the precipitation coating in step (aiii) is conducted a temperature of from about 40 °C to about 110 °C, such as from about 60 °C to about 95 °C.

50. The method according to any one of Claims 46 to 49, wherein the precipitation coating in step (aiii) has a polymerisation time of from about 1 hour to 24 hours, such as from about 3 hours to 17 hours.

51. The method according to any one of Claims 28 to 50, wherein step (aiv) comprises using a third mixture comprising a third crosslinker, and a second functional monomer / oligomer that has at least one functional group, to provide the amphiphilic crosslinked polymeric matrix material.

52. The method according to Claim 51 , wherein the third crosslinker is selected from one or more of the group consisting of divinylbenzene, bisphenol A dimethacrylate, bisphenol diacrylate, tricyclodecane dimethanol diacrylate, ditrimethylolpropane tetraacrylate, di(trimethylolpropane), tetraacrylate, ethylene glycol dimethacrylate, bisphenol A epoxy diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, pentaerythritol triacrylate, dipentaerythritol penta / hexa-acrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, trimethylolpropane triacrylate, diethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, trimethylolpropane ethoxylate diacrylate, isobornyl acrylate, 1 ,6-hexanediol diacrylate, cyclohexyl methacrylate, 1 ,4-butanediol dimethacrylate, cyclohexyl acrylate, 1 ,3-butylene glycol di methacrylate, and isodecyl acrylate.

53. The method according to any one of Claims 34 to 37, 51 and 52, wherein the at least one functional group on the first functional monomer / oligomer and / or the second functional monomer / oligomer is independently selected from one or more of amino, carboxyl, epoxy, and hydroxyl, optionally wherein the at least one functional group on the first functional monomer / oligomer and / or the second functional monomer / oligomer is independently selected from a combination of hydroxyl and carboxyl groups or a combination of amino and carboxyl groups.

54. The method according to Claim 53, wherein the first functional monomer / oligomer is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the first functional monomer / oligomer is selected from one or more of the group consisting of acrylic acid, methacrylic acid, 2- carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, divinyl-benzene- trimethylamine, (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 oligomer.

55. The method according to Claim 53, wherein the second functional monomer / oligomer is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the second functional monomer / oligomer is selected from one or more of the group consisting of ethylenediamine, diethylenetriamine, triethylenetetramine, ethylene glycol diglycidyl ether, diglycidyl ether of bisphenol A, maleimide, N-(2-aminoethyl)methacrylamide, N,N-dimethylaminoethyl methacrylate, N-vinyl-2-pyrrolidone, 3-aminopropyltriethoxysilaneacrylic 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 oligomer.

56. The method according to any one of Claims 29 to 55, wherein the polymerisation in step (aiv) is conducted at a temperature of from about 40 °C to about 90 °C, such as from about 55 °C to about 80 °C, such as about 75 °C.

57. The method according to any one of Claims 29 to 56, wherein the polymerisation in step (aiv) has a polymerisation time of from about 1 hour to about 24 hours, such as from about 3 hours to about 17 hours.

58. The method according to any one of Claims 29 to 50, wherein step (aiv) comprises using a third mixture comprising:(ei) an anhydride monomer, optionally wherein the anhydride monomer is selected from one or more of the group consisting of maleic anhydride, phthalic anhydride, succinic anhydride, trimellitic anhydride, octenyl succinic anhydride, polyisobutylene succinic anhydride, octadecenyl succinic anhydride, and dodecenyl succinic anhydride; and / or(eii) a copolymer, optionally wherein the copolymer is selected from one or more of the group consisting of poly(ethylene-a / t-maleic anhydride), poly(isobutylene-a / f-maleicanhydride), poly(methyl vinyl ether-a / f-maleic anhydride), and poly[(isobutylene-a / f-maleic acid, ammonium salt)-co-(isobutylene-a / t-maleic anhydride)].

59. The method according to Claim 58, wherein the third mixture further comprises:(fi) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of N-methyl-2-pyrrolidone, 1 ,4-dioxane, acetonitrile, acetone, bis(2- methoxyethyl) ether, methyl ethyl ketone, and ethanol; and / or(fii) a catalyst, optionally wherein the catalyst is selected from one or more of the group consisting of pyridine, isoquinoline, quinoline, N,N-dimethyl-cyclohexylamine, tributylamine, N-ethyl morpholine, dimethylaniline, triethylamine (TEA) , benzyl dimethylamine (BDMA), and 2,4,6-tris(dimethylaminomethyl)phenol (DMP-30).

60. The method according to Claim 58 or Claim 59, wherein the coating in step (aiv) is conducted a temperature of from about 25 °C to about 95 °C, such as from about 25 °C to about 75 °C.61 . The method according to any one of Claims 58 to 60, wherein the coating in step (aiv) has a reaction time of from about 1 hour to 24 hours, such as from about 3 hours to 17 hours.

62. The method according to any one of Claims 29 to 61, wherein dispersing superparamagnetic Fe3O4 nanoparticles in steps (aii) and (aiv) comprise providing a solution that comprises a Fe(lll) salt, a Fe(ll) salt and a base.

63. The method according to Claim 62, wherein:(gi) the Fe(lll) salt is selected from one or more of the group consisting of FeCh and Fe2(SC )3, optionally wherein the Fe(lll) salt is FeCh;(gii) the Fe(ll) salt is selected from one or more of the group consisting of FeCl2, FeSC and Fe(OAC)2, optionally wherein the Fe(ll) salt is FeChiand / or(giii) the base is selected from one or more of the group consisting of ammonia hydroxide, NaOH, KOH, and amine, optionally wherein the base is aqueous ammonia.

64. The method according to any of Claims 29 to 63, wherein step (av) comprises using a fourth mixture comprising a third styrene monomer, and a fourth crosslinker, to provide the third crosslinked polymeric matrix material.

65. The method according to Claim 64, wherein the third styrene monomer is selected from one or more of the group consisting of styrene, and a styrene derivative, optionallywherein the styrene derivative is selected from one or more of the group consisting of vinyltoluene, chlorostyrene, bromostyrene, vinylbenzyl chloride, a-methylstyrene, vinyl naphthalene, acenaphthylene, benzyl methacrylate, phenylacetylene, phenyl vinyl sulfide, 4- methylstyrene, 3-methylstyrene, and 4-tertbutylstyrene.

66. The method according to Claim 64 or Claim 65, wherein the fourth crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethacrylate, bisphenol A dimethacrylate , bisphenol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol propoxylate triacrylate, butandiol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, dipentaerythritol penta / hexa-acrylate, trimethylol propane ethoxylate triacrylate, trimethylol propane propoxylate triacrylate, di(trimethylolpropane) tetraacrylate, and trimethylolpropane triacrylate.

67. The method according to any one of Claims 64 to 66, wherein the fourth mixture further comprises:(hi) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2- methylbutyronitrile) (AMBN), 4,4'-azobis (4-cyanovaleric acid), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate, and ammonia persulfate; and / or(hii) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of 1 ,4-dioxane, ethyl acetate, butyl acetate, acetone, hexane, octane, methyl ethyl ketone, and water.

68. The method according to any one of Claims 29 to 67, wherein the polymerisation in step (av) is conducted at a temperature of from about 40 °C to about 105 °C, such as from about 60 °C to about 95 °C, such as about 75 °C.

69. The method according to any one of Claims 29 to 68, wherein the polymerisation in step (av) has a polymerisation time of from about 1 hour to about 24 hours, such as from about 3 hours to about 17 hours.

70. The method according to any of Claims 29 to 69, wherein step (avi) comprises using a fifth mixture comprising a fifth crosslinker, a third functional monomer that has at least onefunctional group, and a fourth functional monomer that has at least one functional group, to provide the fourth crosslinked polymeric matrix material.

71. The method according to Claim 70, wherein the fifth crosslinker is selected from one or more of the group consisting of butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, tripropylene glycol diacrylate, neopentyl glycol diacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, di pentaerythritol pentaacrylate, dipentaerythritol penta / hexa-acrylate, tripropylene diacrylate, di(trimethylolpropane) tetraacrylate, polyethylene glycol) diacrylate, polypropylene glycol) diacrylate, bisphenol A dimethacrylate, and tri(propylene glycol) diacrylate, optionally wherein the fifth crosslinker is selected from one or more of the group consisting of divinylbenzene, ethylene glycol dimethylacrylate, bisphenol A dimethacrylate, and N,N' -methylenebis(acrylamide).

72. The method according to Claim 70 or Claim 71 , wherein the third functional monomer that has at least one functional group is selected from one or more of a polyether monomer, a polyester monomer, a polyacrylamide monomer, and a polyacid monomer, optionally wherein the third functional monomer that has at least one functional group is selected from one or more of the group consisting of glycidyl methacrylate, allyl glycidyl ether, 2-hydroxyethyl methacrylate, poly(ethylene glycol) diacrylate, polypropylene glycol) diacrylate, and tripropylene glycol) diacrylate, acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, ethylenediamine, diethylenetriamine, triethylenetetramine, ethylene glycol diglycidyl ether, diglycidyl ether of bisphenol A, maleimide, N-(2-aminoethyl)methacrylamide, N,N- dimethylaminoethyl methacrylate, N-vinyl-2-pyrrolidone, and 3- aminopropyltriethoxysilaneacrylic acid.

73. The method according to any one of Claims 70 to 72, wherein the fourth functional monomer is selected from one or more of the group consisting of methyl methacrylate ethyl methacrylate, butyl methacrylate, methyl methacrylate, 2-ethylhexyl methacrylate, methyl acrylate, ethyl acrylate, butyl acrylate, methyl acrylate, and 2-ethylhexyl acrylate.

74. The method according to any one of Claims 70 to 73, wherein the fifth mixture further comprises:(ii) a polymerisation initiator, optionally wherein the polymerisation initiator is selected from one or more of the group consisting of an azo initiator, a peroxide initiator, and a persulfate salt, and more optionally, the polymerisation initiator is selected from one or more of the group consisting of 2,2'-azobis(2-methylpropionitrile) (AIBN), 2,2'-azobis(2-methylbutyronitrile) (AMBN), 4,4'-azobis (4-cyanovaleric acid), tertiary-amyl hydroperoxide, potassium persulfate, sodium persulfate, and ammonia persulfate; and / or(iii) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of ethylene glycol, 1 ,4-dioxane, diglyme, octane, toluene, dimethylacetamide, dimethylformamide, xylene, n-methyl-2-pyrrolidone, n-butanol and water.

75. The method according to any one of Claims 29 to 69, wherein step (avi) comprises using a fifth mixture comprising a first epoxide, to provide the second crosslinked polymeric matrix material.

76. The method according to Claim 75, wherein the first epoxide comprises phenolic derivatives, optionally wherein the first epoxide is selected from one or more of the group consisting of bisphenol A, AP, B, BP, C, E, F, G, M, P, PH, TMC, Z and its copolymer derivatives (e.g. bisphenol A propoxylate diglycidyl ether, and bisphenol A diglycidyl etherbisphenol A copolymer).

77. The method according to Claim 75 or Claim 76, wherein the fifth mixture further comprises:(ji) a solvent, optionally wherein the solvent is selected from one or more of the group consisting of N-methyl-2-pyrrolidone, 1 ,4-dioxane, acetonitrile, acetone, bis(2- methoxyethyl) ether, methyl ethyl ketone, and ethanol;(jii) a catalyst, optionally wherein the catalyst is selected from one or more of the group consisting of pyridine, isoquinoline, quinoline, N,N-dimethyl-cyclohexylamine, tributylamine, N-ethyl morpholine, dimethylaniline, triethylamine (TEA), benzyl dimethylamine (BDMA), and 2,4,6-tris(dimethylaminomethyl)phenol (DMP-30); and / or(jiii) a second epoxide selected from one or more of the group consisting of 1,2- epoxydodecane, 1 ,4-butanediol diglycidyl ether, 1 ,2-butylene oxide, tert-butyl glycidyl ether, butyl glycidyl ether, 1 ,2-epoxydecane,1 ,2-epoxydodecane,1,2-epoxyheptane,1 ,2- epoxyhexane, polyethylene glycol diglycidyl ether (n = 4 to 22), and glycerol triglycidyl ether.

78. The method according to any one of Claims 75 to 77, wherein the precipitation coating in step (avi) is conducted a temperature of from about 40 °C to about 110 °C, such as from about 60 °C to about 95 °C.

79. The method according to any one of Claims 75 to 78, wherein the precipitation coating in step (avi) has a reaction time of from about 1 hour to 24 hours, such as from about 3 hours to 20 hours.

80. The method according to any one of Claims 75 to 79, wherein step (avi) further comprises using a carboxylic acid to hydrolyse the epoxide groups.81 . The method according to any one of Claims 29 to 80, wherein:(ki) the weight to weight ratio of styrene groups to crosslinking groups in the first layer is from 20:1 to 1 :2, such as from 10:1 to 1:1 ; and / or(kii) the weight to weight ratio of styrene groups to functional groups in the first layer is from 20:1 to 1 :2, such as from 10:1 to 1 :1 ;(kiii) the weight to weight of styrene groups to crosslinking groups in the first interlayer is from 20:1 to 1:2, such as from 10:1 to 1 :1 ;(kiv) the weight to weight ratio of functional groups to crosslinking groups in the second interlayer is from 2:3 to 1 :20, such as from 1 :3 to 1:10; and / or(kv) the weight to weight ratio of functional groups to crosslinking groups in the second layer is from 2:3 to 1 :20, such as from 1 :3 to 1 :10.

82. The method according to any one of Claims 29 to 81 , wherein step (avi) further comprises disposing an organic polymer layer, wherein the organic polymer layer comprises a group shown by the following formula:-CH2-CHR1-CH2-R2, wherein R1represents an alkoxy group and R2represents a carboxyl, tosyl or amine group, and wherein the carboxyl, tosyl and amine group is linear or branched.

83. Use of the microsphere of any one of Claims 1 to 28 in a chemiluminescence immunoassay.

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