Particles

Abstract

Particles of the invention relate to magnetic clusters of nanocrystals comprising iron oxide. The magnetic clusters comprise an aromatic carboxylic acid and / or the magnetic clusters are monodispersed. The magnetic clusters can be used in bioassays and other applications. The invention also relates to methods for making such magnetic clusters and methods of using the magnetic clusters, as well as kits comprising the magnetic clusters.

Description

[0001] The present invention relates to magnetic clusters of nanocrystals comprising iron oxide. The magnetic clusters comprise aromatic carboxylic acids and / or are monodisperse. The magnetic clusters can be used in bioassays and other applications. The present invention also relates to methods for preparing such particles and methods of using the particles, as well as other subjects. Background Art

[0002] Particles such as magnetic microparticles and magnetic nanoparticles are used in a wide variety of applications, such as bioassays and other applications. For example, nanoparticles and microparticles have been used in agglutination tests and assays, particle capture ELISA methods, lateral flow tests, solid phase assays, scintillation proximity assays, polymerase chain reaction (PCR) tests, superparamagnetic-based assays and magnetic separation systems and biosensors; as enhancers of Raman spectroscopy signals; in light scattering assays; in drug delivery applications; and as fluorescent markers or stains for detecting biomolecules.

[0003] In some applications, it is desirable to include a magnetic material in the particles. This allows for magnetic separation of polymer particles, as well as use of the particles in superparamagnetic based assays and magnetic separation systems and biosensors. It may also be desirable to use monodisperse particles, as monodisperse particles generally behave in a more consistent and reproducible manner in an assay than polydisperse particles.

[0004] Magnetic microparticles and nanoparticles are usually surface functionalized and / or coated prior to use. In addition, known particles may be polymer particles comprising magnetic material, such as porous polymer particles comprising magnetic material in the pores. Examples of such particles include magnetic Ugelstad-type particles, such as those described in WO 00 / 61647 or WO 2010 / 125170.

[0005] Known magnetic microparticles and magnetic nanoparticles used in applications such as bioassays are subject to many limitations. For example, for environmental reasons, it may be desirable to minimize the use of (non-biodegradable) plastics in microparticles and nanoparticles. In addition, it may also be desirable to provide particles with low CV. Therefore, new microparticles and nanoparticles are needed.

[0006] It is an object of the present invention to provide magnetic clusters comprising nanocrystals of iron oxide which can be used in applications such as bioassays. Summary of the invention

[0007] The present invention is based in part on the recognition that the formation of magnetic nanoclusters in the absence of significant polymers can reduce the level of microplastics, thereby providing a more environmentally compatible product. The present invention is also based on the recognition that monodisperse magnetic nanoclusters can be used in many different applications when appropriately functionalized.

[0008] According to a first aspect, a magnetic cluster is provided, wherein the magnetic cluster comprises nanocrystals of iron oxide and an aromatic carboxylic acid.

[0009] In one embodiment, the aromatic carboxylic acid is a compound of formula (I):

[0010]

[0011] L 1 Select from bonds, -C 1-6 Alkyl-, or -C 2-6 Alkenyl-. R 1 Selected from -H, -COOH, -(C 1-5 Alkyl)COOH, -(C 2-5 alkenyl)COOH or -C 1-4 Alkoxy. 2 Selected from -H, -halo, -C 1-4 Alkyl, -C 1-4 Alkoxy or -NO2. 3 Selected from -H, -halo, -C 1-12 Alkyl, -C 3-8 Cycloalkyl, -C 1-12 Alkoxy, -halogen (C 1-12 )alkyl, -NR 6 R 7 、-NO2、-C(O)OH、-C(O)OR 6 Or substituted or unsubstituted phenyl. 4 Selected from -H, -halo, -C 1-4 Alkyl, -C 1-4 Alkoxy or -NO2. 5 Select from -H or -C 1-4 Alkoxy. 6 Select from -H or -C 1-4 Alkyl. 7 Selected from -H, -C 1-4 Alkyl, -C(O)H, -C(O)C 1-4 In another embodiment, R1 and R2 together with the carbon atom to which they are attached form a substituted or unsubstituted phenyl group, and L 1 , R 3 , R 4 , R 5 , R 6 and R 7 As previously defined. In another embodiment, R2 and R3 together with the carbon atom to which they are attached form a substituted or unsubstituted phenyl group, and L 1 , R 1 , R 4 , R 5 , R6 and R 7 As previously defined;

[0012] In one embodiment, L 1 Select from bonds, -C 1-6 Alkyl-, or -C 2-6 Alkenyl-. R 1 Selected from -H, -COOH, -(C 1-5 Alkyl)COOH, -(C 2-5 alkenyl)COOH or -C 1-4 Alkoxy. 2 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy. 3 Selected from -H, -halo, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogen (C 1-6 )alkyl, -NR 6 R 7 Or substituted or unsubstituted phenyl. 4 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy. 5 Select from -H or -C 1-4 Alkoxy. 6 Select from -H or -C 1-4 Alkyl. 7 Selected from -H, -C 1-4 Alkyl, -C(O)H, -C(O)C 1-4 alkyl.

[0013] In one embodiment, L 1 Select from bonds, -C 1-6 Alkyl-, or -C 2-6 Alkenyl-. R 1 Selected from -H, -COOH, -(C 1-5 Alkyl)COOH, -(C 2-5 alkenyl)COOH or -C 1-4 Alkoxy. 2 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy. 3 Selected from -H, -C 1-6 Alkyl, -C 1-6 Alkoxy, -NR 6 R 7 Or substituted or unsubstituted phenyl. 4 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy. 5Select from -H or -C 1-4 Alkoxy. 6 Select from -H or -C 1-4 Alkyl. 7 Selected from -H, -C 1-4 Alkyl, -C(O)H, -C(O)C 1-4 alkyl.

[0014] A second aspect of the invention provides monodisperse magnetic clusters comprising nanocrystals of iron oxide, wherein the magnetic clusters have a mode diameter in the range of about 100 nm to about 2,500 nm and a coefficient of variation (CV) of no more than 5%. The mode diameter and the CV can be measured by a disc centrifuge. The magnetic clusters also contain an aromatic carboxylic acid, for example, at least one aromatic carboxylic acid of formula (I).

[0015] The third aspect provides a method for preparing magnetic nanoclusters, which comprises forming a solution comprising iron (III) ions, a protic solvent, an aromatic carboxylic acid and a precipitant; and reacting the solution to form a suspension comprising magnetic clusters.

[0016] A fourth aspect provides a magnetic cluster obtainable by the method of the third aspect.

[0017] The fifth aspect provides use of the magnetic cluster in an assay, wherein the magnetic cluster is of the first aspect, the second aspect or the third aspect.

[0018] A sixth aspect provides a kit comprising the magnetic cluster of the first aspect, the second aspect or the third aspect. The kit may comprise one or more buffers. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Embodiments of the present invention are further described below with reference to the accompanying drawings, in which:

[0020] Figure 1 is a schematic diagram showing a hypothesis of how magnetic clusters are formed from solution in the presence of citrate as a stabilizer.

[0021] Figure 2 : is an optical microscope image of magnetic clusters, showing magnetic clusters with five different aggregation levels. The scale bar in the lower right corner of each image represents 50 μm.

[0022] Figure 3 Provided are optical microscopy images of magnetic clusters obtained with various stabilizers with aggregation levels ranging from 0 to 2. The scale bar in the lower right corner of each image represents 50 μm.

[0023] Figure 4 The particle size distribution of magnetic clusters formed using citric acid or toluic acid as stabilizers at a molar ratio of 0.2, 0.4, 0.8 or 1.6 is provided.

[0024] Figure 5 Provided is the particle size distribution of magnetic clusters formed using 3,4,5-trimethoxybenzoic acid (eucalyptol), 4-acetamidobenzoic acid, biphenyl-4-carboxylic acid, trans-cinnamic acid, 4-(dimethylamino)benzoic acid, 3,5-dimethylbenzoic acid, 4-ethylbenzoic acid, 4-tert-butylbenzoic acid, benzoic acid, p-anisic acid, phthalic acid, homophthalic acid or 3-phenylpropionic acid.

[0025] Figure 6 Shown are the mode diameters (diamond markers) and CV% of the magnetic clusters as measured by disk centrifuge.

[0026] Figure 7 Scanning electron microscopy (SEM) images are provided comparing magnetic clusters prepared using biphenyl-4-carboxylic acid as a stabilizer (left) and magnetic clusters prepared using citrate as a stabilizer (right).

[0027] Figure 8 The particle size distribution of magnetic clusters formed using 4-(trifluoromethyl)benzoic acid, 4-iodobenzoic acid, or 4-chlorobenzoic acid is provided.

[0028] Fig. 9 Shown for Figure 7 Distribution of magnetic clusters as measured by disk centrifuge, mode diameter (diamond markers) and CV%.

[0029] Fig.10 is an image of a gel showing immunoprecipitation using protein G functionalized magnetic clusters.

[0030] Fig.11 A histogram showing data of cluster size measured by the scanning electron microscope (SEM) procedure provided herein is provided. The data is based on Figure 7 Particles depicted in , and compared to particles produced with biphenyl-4-carboxylic acid (dark grey) and monosodium citrate (light grey).

[0031] Fig.12 The hysteresis curves of magnetic clusters are provided. (A) Using 72.64Am 2 / kg provides the hysteresis curve of the magnetic cluster of Example 5. (B) Using a magnetic saturation of 72.27Am2 / kg provides the hysteresis curve of the magnetic cluster of Example 10. DETAILED DESCRIPTION

[0032] Throughout the description and claims of this specification, the words "include" and "contain" and variations of these words mean "including (but not limited to)", and are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood to contemplate the plural as well as the singular unless the context requires otherwise.

[0033] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except for at least some mutually exclusive combinations of such features and / or steps. The invention is not limited to the details of any foregoing embodiments. The invention extends to any novel feature or any novel combination of features disclosed in this specification (including any accompanying claims, abstract and drawings), or any novel step or any novel combination of steps of any method or process so disclosed.

[0034] The reader's attention is directed to all papers and documents which are filed concurrently with or before the present specification in connection with the present application and which are open to the public upon inspection of the present application, and the contents of all such papers and documents are incorporated herein by reference.

[0035] Unless otherwise indicated, the term "aromatic carboxylic acid" by itself or as part of another substituent refers to a molecule or moiety comprising an aromatic group (e.g., phenyl) covalently bonded to a carboxylic acid group. The carboxylic acid group can be directly attached to the aromatic group, such as in benzoic acid, or the carboxylic acid can be attached to the aromatic group via a linker.

[0036] "Magnetic" and "magnetic material" refer to the response to a magnetic field. When a magnetic material is paramagnetic, the magnetism is turned off when the magnetic field is removed. When a magnetic material is superparamagnetic, the magnetic material becomes saturated at relatively low magnetic fields, and the magnetic properties are turned off very quickly / instantaneously with the removal of the magnetic field. Paramagnetic and superparamagnetic materials can therefore be considered "magnetizable" because the magnetic properties depend on the application of an external magnetic field. When a magnetic material is ferromagnetic, all of its magnetic atoms within each magnetic domain add a positive contribution to the net magnetization. When a magnetic material is ferromagnetic, some of the magnetic atoms within each magnetic domain are offset, but the material as a whole exhibits a net magnetization. After the external magnetic field is removed, both ferromagnetic and ferrimagnetic materials remain magnetic. Above the Curie temperature of the material, ferromagnetic and ferromagnetic materials become paramagnetic materials. Magnetic properties may also be affected by the size of the magnetic particles in the magnetic material, with some materials being ferromagnetic and ferrimagnetic at larger particle sizes, but being superparamagnetic at a suitable small particle size (e.g., nm scale). For example, ferrimagnetic materials such as iron oxides form superparamagnetic crystals when the size of the crystals is sufficiently small (e.g., below about 15 nm scale for iron oxides).The magnetic clusters of the present disclosure and invention may be paramagnetic or superparamagnetic because they comprise nanocrystals of iron oxide.

[0037] "Magnetic material precursor" refers to a substance that can be transformed to provide a magnetic material. The magnetic material precursor may comprise solvated transition metal ions (e.g., multivalent cations of Fe, Ni, Co, or combinations thereof, optionally mixed with multivalent cations of Al, Mn, Cu, Zn, Ca, Ge, Te, Ti, or Sn and / or rare earths). The solvated transition metal ions and / or rare earth ions may be converted to a magnetic material by any process that causes precipitation of the ions (e.g., oxides). The precipitation may be caused, for example, by a change in pH, removal of a solvent, or a change in temperature. For example, a magnetic material precursor may be formed by a solution containing Fe at a pH of less than 6. 2+ and / or Fe 3+ ions are provided; and Fe 2+ and / or Fe 3+ The ions can be converted to magnetic material by precipitation, for example by raising the pH to greater than 8.

[0038] "Nucleic acid" refers to a molecule composed of ribonucleotides and / or deoxyribonucleotides and synthetic nucleotide residues that can participate in Watson-Crick type or similar base pair interactions, i.e., "hybridization" or "duplex" formation. Therefore, nucleic acid can be single-stranded or double-stranded DNA or RNA or any modification thereof, including conformationally restricted or oligomers with nucleobase analogs, such as "locked nucleic acids" (LNA) or "peptide nucleic acids" (PNA) or other derivatives containing non-nucleotide backbones. Nucleic acid can be a naturally occurring molecule, i.e., DNA or RNA, but also includes DNA / RNA hybrids, wherein the DNA is in an independent chain or in the same chain, wherein the 3' position of the pentose of one nucleotide is connected to the 5' position of the pentose of the next nucleotide by a phosphodiester bond. The nucleic acid used in various embodiments can include nucleotides or combinations thereof in chemical, enzymatic or metabolically modified forms, such as primers, probes, oligonucleotides or adapters.

[0039] "Coating" refers to a covering applied to the surface of a substrate. The coating can be a full coverage coating that completely covers the substrate, or it can be a partial coating that only covers a portion of the substrate surface. The coating can be applied to the substrate by any suitable type of combination, for example, by at least one of covalent bonding, metallic bonding, ionic bonding, hydrogen bonding, van der Waals interaction, hydrophobic interaction, etc. In the case where the substrate includes a magnetic material (such as a magnetic cluster), the coating (or at least a portion thereof) can be silicon dioxide, which can be formed on the surface of the magnetic material by the condensation of a silicate or an orthosilicate. The coating may include an organic coating that may include a spacer (such as oligoethylene glycol or polyethylene glycol) and / or a polymer. The coating may include a functional group, such as a hydroxyl, a carboxylic acid, a primary amine, a secondary amine, or an epoxy group.

[0040] As used herein, "cluster" refers to an agglomerate or particle composed of multiple smaller entities. For example, a magnetic nanocluster is a nanometer-sized particle composed or formed by multiple nanocrystals of iron oxide. Therefore, in many cases, the terms cluster and particle are used interchangeably herein. Particles or clusters with a spherical shape are also often referred to as beads.

[0041] "Functional group" or "reactive group" refers to a substituent capable of undergoing a characteristic chemical reaction. Exemplary functional groups / reactive groups of the present disclosure include hydroxyl groups, carboxylic acid groups, aldehyde groups, amine groups (e.g., primary or secondary aliphatic amine groups and aromatic amine groups), thiol groups, epoxy groups, amide groups, chloromethyl groups, or tosyl-activated groups. The functional group may be present on the surface of the substrate (such as a magnetic cluster), for example, the functional group may be present on the coating.

[0042] "Ligand" refers to a molecule that is capable of binding to another species. A ligand can be a capture ligand, such as a capture ligand useful in an assay. Exemplary ligands include antibodies, antibody fragments, peptides, carbohydrates, haptens, aptamers, and oligonucleotides. Standard techniques can be used to bind the ligand to a substrate (such as a magnetic cluster) containing a functional group. Examples of such techniques are described in Chapter 14 of GT Hermanson, Bioconjugate Techniques, Academic Press (3rd edition, 1996).

[0043] As used herein, the term "antibody" includes: (a) any of the various classes or subclasses of immunoglobulins (e.g., IgG, IgA, IgM, IgD or IgE, which are derived from any animal, for example, any of the animals conventionally used, such as sheep, rabbit, goat, mouse, camel or egg yolk), (b) monoclonal or polyclonal antibodies, (c) intact antibodies or antibody fragments, monoclonal or polyclonal, fragments being those containing the antibody binding region, such as fragments lacking the Fc portion (e.g., Fc fragments). b, Fab', f(ab')2, scFv, VHH antibodies, VHH antibody fragments, and other single domain antibodies), so-called "half-molecule" fragments obtained by reductive cleavage of the disulfide bonds linking the heavy chain components of an intact antibody (Fv can be defined as a fragment containing the light chain variable region and the heavy chain variable region, expressed as two chains), and (d) antibodies produced or modified by recombinant DNA or other synthetic techniques, including monoclonal antibodies, antibody fragments, "humanized antibodies", chimeric antibodies, or synthetic or altered antibody-like structures.

[0044] Unless otherwise stated, the "average" diameter referred to in this specification refers to the mode diameter of the magnetic clusters. The mode diameter can be measured by a disc centrifuge, such as a CPS disc centrifuge.

[0045] The term "monodisperse" means that for a plurality of particles (e.g., at least 100, more preferably at least 1000), the coefficient of variation (CV) or % polydispersity of the diameter of the particles is less than 20%, for example, less than 15%, typically less than 10%, optionally less than 8%, for example, less than 5%. CV can be measured by a disc centrifuge. Cv is preferably calculated on the main mode, for example based on the width of the mode peak at half height. Therefore, some particles below or above the mode size may be ignored in the calculation. The determination of this CV can be carried out on a CPS disc centrifuge. Exemplary determinations of the particle size and CV of the magnetic clusters of the present disclosure measured by a CPS disc centrifuge are provided below under the heading "Determination".

[0046] The term "alkyl" as used herein includes straight or branched chain alkyl moieties having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The term includes reference to, for example, methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl or tert-butyl), pentyl, hexyl, etc. In particular, the alkyl group may be a "C1-C6 alkyl", i.e. an alkyl group having 1, 2, 3, 4, 5 or 6 carbon atoms; or a "C1-C4 alkyl", i.e. an alkyl group having 1, 2, 3 or 4 carbon atoms. The term "lower alkyl" includes alkyl groups having 1, 2, 3 or 4 carbon atoms. Where chemically possible, the alkyl group may be optionally substituted with 1 to 5 substituents, each of which is independently selected at each occurrence from: oxo, =NR a , =NOR a , halogen, nitro, cyano, NR a R a NR a S(O)2R a NR a CONR a R a NR a CO2R a , OR a SR a 、S(O)R a 、S(O)2OR a 、S(O)2R a 、S(O)2NR a R a 、CO2R a 、C(O)R a ,CONR a R a , C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-haloalkyl; wherein R a is independently selected at each occurrence from: H and C1-C4 alkyl.

[0047] As used herein, the term "alkoxy" includes -O-alkyl, wherein the alkyl is linear or branched, and contains 1, 2, 3, 4, 5 or 6 carbon atoms. In one class of embodiments, the alkoxy has 1, 2, 3 or 4 carbon atoms, such as 1, 2 or 3 carbon atoms. The term includes, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, etc. The term "lower alkoxy" includes alkoxy having 1, 2, 3 or 4 carbon atoms.

[0048] As used herein, the term "aromatic carboxylic acid" refers to a carboxylic acid containing at least one carboxylic acid moiety (e.g., -COOH, -(C 1-5 alkyl)COOH or -(C 2-5The aromatic carboxylic acid may also contain additional substituents such as -halo, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogen (C 1-6 ) alkyl, -halogen (C 3-6 )cycloalkyl, -NR 6 R 7 , -NO2, -C(O)OH. The protonation level of the aromatic carboxylic acid depends on the pH. Therefore, unless the context requires otherwise, reference to an aromatic carboxylic acid includes reference to the acid, its salt form, or a mixture of the acid and one or more salt forms thereof. Exemplary aromatic carboxylic acids of the present disclosure are provided by compounds of formula (I).

[0049] As used herein, the term "halo" or "halogen" includes fluorine, chlorine, bromine or iodine atoms. As used herein, the term "halide" includes fluorine, chloride, bromide or iodine. In addition, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C1-C6) alkyl" is intended to include (but not limited to) trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, etc.

[0050] The term "phenyl" as used herein includes a benzene ring. Where possible, the phenyl group may be optionally substituted with 1 to 5 substituents, each of which is independently selected from the group consisting of: -halo, -C1-C6 alkyl, -C2-C6 alkenyl, -C1-C6 haloalkyl, and -C2-C6 haloalkenyl.

[0051] As used herein, the term "substituted" refers to one or more of the moieties, especially up to 5, more especially 1, 2 or 3 hydrogen atoms, which are independently substituted by the corresponding number of substituents. Unless otherwise indicated, exemplary substituents include -OH, -CN, -NH2, =O, -halo, -C1-C6 alkyl, -C2-C6 alkenyl, -C1-C6 haloalkyl, -C1-C6 haloalkoxy and -C2-C6 haloalkenyl, -C1-C6 alkylcarboxylic acid (e.g., -CH3COOH or -COOH). When the substituent is -C1-C6 alkyl or -C1-C6 haloalkyl, the C1-C6 chain is optionally interrupted by an ether bond (-O-) or an ester bond (-C(O)O-). Exemplary substituents for substituted alkyl groups may include -OH, -CN, -NH2, =O, -halo, -CO2H, -C1-C6 haloalkyl, -C1-C6 haloalkoxy and -C2-C6 haloalkenyl, -C1-C6 alkylcarboxylic acid (e.g., -CH3COOH or -COOH). For example, exemplary substituents for alkyl groups may include -OH, -CN, -NH2, =O, -halo.

[0052] Of course, it is understood that substituents are only located where they are chemically possible, and one skilled in the art will have no difficulty determining (experimentally or theoretically) whether a particular substitution is possible. For example, an amino group or a hydroxyl group with a free hydrogen may be unstable if bound to a carbon atom by an unsaturated (e.g., olefinic) bond. In addition, of course, it is understood that the substituents described herein may themselves be substituted with any substituent, as long as the above-mentioned restrictions on suitable substituents recognized by those skilled in the art are met.

[0053] When steric considerations dictate the position of substituents on a group, the isomer with the lowest conformational energy may be preferred.

[0054] When a compound, moiety, process, or product is described as "optionally" having a feature, the disclosure includes such compounds, moieties, processes, or products having that feature, as well as such compounds, moieties, processes, or products not having that feature. Thus, when a moiety is described as "optionally substituted," the disclosure includes both unsubstituted and substituted moieties.

[0055] When two or more moieties are described as being "independently" or "each independently" selected from a series of atoms or groups, this means that the moieties may be the same or different. Thus, the identity of each moiety is independent of the identity of one or more other moieties.

[0056] Magnetic clusters

[0057] The present invention includes embodiments in which the magnetic clusters comprise nanocrystals of iron oxide and aromatic carboxylic acids. The present invention also includes embodiments in which the magnetic clusters are monodisperse and comprise nanocrystals of iron oxide. Unless the context requires otherwise, references to magnetic clusters hereinafter include magnetic clusters of the present invention comprising aromatic carboxylic acids and / or monodisperse magnetic clusters of the present invention.

[0058] The aromatic carboxylic acid may be a compound of formula (I):

[0059]

[0060] L 1 Select from bonds, -C 1-6 Alkyl-, or -C 2-6 Alkenyl-. R 1 Selected from -H, -COOH, -(C 1-5 Alkyl)COOH, -(C 2-5 alkenyl)COOH or -C 1-4 Alkoxy. 2 Selected from -H, -halo, -C 1-4 Alkyl, -C 1-4 Alkoxy or -NO2. 3Selected from -H, -halo, -C 1-12 Alkyl, -C 3-8 Cycloalkyl, -C 1-12 Alkoxy, -halogen (C 1-12 )alkyl, -NR 6 R 7 、-NO2、-C(O)OH、-C(O)OR 6 Or substituted or unsubstituted phenyl. 4 Selected from -H, -halo, -C 1-4 Alkyl, -C 1-4 Alkoxy or -NO2. 5 Select from -H or -C 1-4 Alkoxy. 6 Select from -H or -C 1-4 Alkyl. 7 Selected from -H, -C 1-4 Alkyl, -C(O)H, -C(O)C 1-4 Alternatively, R1 and R2 together with the carbon atom to which they are attached form a substituted or unsubstituted phenyl group, and L 1 , R 3 , R 4 , R 5 , R 6 and R 7 As defined herein. In another alternative, R2 and R3 together with the carbon atom to which they are attached form a substituted or unsubstituted phenyl group, and L 1 , R 1 , R 4 , R 5 , R 6 and R 7 As defined elsewhere.

[0061] In one embodiment, L 1 Select from bonds, -C 1-6 Alkyl-, or -C 2-6 Alkenyl-. R 1 Selected from -H, -COOH, -(C 1-5 Alkyl)COOH, -(C 2-5 alkenyl)COOH or -C 1-4 Alkoxy. 2 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy. 3 Selected from -H, -halo, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogen (C 1-6 )alkyl, -NR 6 R 7Or substituted or unsubstituted phenyl. 4 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy. 5 Select from -H or -C 1-4 Alkoxy. 6 Select from -H or -C 1-4 Alkyl. 7 Selected from -H, -C 1-4 Alkyl, -C(O)H, -C(O)C 1-4 alkyl.

[0062] L 1 Can be selected from key, -C 1-4 Alkyl-, or -C 2-4 Alkenyl-. L 1 Can be a key. 1 Can be selected from -C 1-4 Alkyl- or -C 2-4 Alkenyl-. L 1 Can be -C 1-6 Alkyl-, optionally -C 1-4 Alkyl-.L 1 Can be -C 2-6 Alkenyl-, optionally -C 2-4 Alkenyl-.

[0063] R 1 Can be selected from -H, -COOH, -(C 1-5 alkyl)COOH or -(C 2-5 R1 can be selected from -H or -COOH. 1 Can be -H, R 1 It can be -COOH. R1 can be -H-(C 1-5 alkyl)COOH. R1 can be -(C 2-5 (Alkenyl)COOH. 1 Can be -C 1-4 Alkoxy.

[0064] R 2 Can be selected from -H, -C 1-4 Alkyl or -C 1-4 Alkoxy. 2 Can be selected from -H or -C 1-4 Alkoxy. 2 R may be selected from -H or -OCH3. 2 Can be -H. R 2 Can be -C 1-4 Alkyl groups, such as R 2 It can be -CH3. 2 Can be -C 1-4Alkoxy, for example, R 2 It can be -OCH3.

[0065] R1 and R2 together with the carbon atoms to which they are attached may form a substituted or unsubstituted phenyl group.

[0066] R 3 Can be selected from -H, -halogen, -C 1-6 Alkyl, -C 3-6 Cycloalkyl, -C 1-6 Alkoxy, -halogen (C 1-6 )alkyl, -NR 6 R 7 , -NO2, -C(O)OH, or substituted or unsubstituted phenyl. 3 Can be selected from -H, -halogen, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogen (C 1-6 )alkyl, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3 Can be selected from -H, -C 1-4 Alkyl, halogen, -C 1-4 Alkoxy, -halogen (C 1-4 )alkyl, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3 Can be selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy, -halogen (C 1-4 )alkyl, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3 Can be selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3 Can be selected from -C 1-4 Alkoxy, -halogen (C 1-4 )alkyl, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3 Can be selected from -C 1-4 Alkoxy, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3 Can be selected from -C 1-4 Alkoxy, -halogen (C 1-4 )alkyl, -NR 6 R7 or unsubstituted phenyl. 3 Can be selected from -C 1-4 Alkoxy, -NR 6 R 7 or unsubstituted phenyl. 3 Can be selected from -H, -C 1-4 Alkyl, -C1, -I, -OCH3, -CF3, -N(CH3)2, -NHC(O)CH3 or phenyl. 3 Can be selected from -H, -C 1-4 Alkyl, -OCH3, -N(CH3)2, -NHC(O)CH3 or phenyl. 3 R may be selected from -OCH3, -CF3, -N(CH3)2, -NHC(O)CH3 or phenyl. 3 It can be selected from -OCH3, -N(CH3)2, -NHC(O)CH3 or phenyl.

[0067] R 3 Can be selected from -H, -C 1-10 Alkoxy, -NR 6 R 7 、-C(O)OR 6 Or substituted or unsubstituted phenyl. 3 Can be selected from -H, -C 1-4 Alkyl, -OCH3, -OCH2(CH2)8CH3, -N(CH3)2, -NHC(O)CH3, -C(O)OCH3 or phenyl.

[0068] R 3 Can be -H. R 3 Can be -C 1-6 Alkyl groups, such as R 3 Can be -C 1-4 Alkyl. 3 Can be -C 1-6 Alkoxy, for example, R 3 Can be -C 1-4 Alkoxy. 3 It can be -OCH3. 3 Can be -NR 6 R 7 . R 3 It may be a substituted or unsubstituted phenyl group, for example, R 3 It may be an unsubstituted phenyl group. 3 It can be -halo, for example, R 3 It can be -Cl. 3 It can be -halogen (C 1-6 ) alkyl, for example, R 3 It can be -halogen (C 1-4) alkyl. 3 It can be -CF3. 3 Can be C 1-12 Alkyl groups, such as R 3 It can be -OCH2(CH2)8CH3 or -OCH3. 3 Can be -C(O)OR 6 , for example, R 3 It can be -C(O)OCH3.

[0069] R2 and R3 together with the carbon atoms to which they are attached may form a substituted or unsubstituted phenyl group.

[0070] R 4 Can be selected from -H, -C 1-4 Alkyl or -C 1-4 Alkoxy. 4 Can be selected from -H or -C 1-4 Alkoxy. 4 R may be selected from -H or -OCH3. 4 Can be -H. R 4 Can be -C 1-4 Alkyl groups, such as R 4 It can be -CH3. 4 Can be -C 1-4 Alkoxy, for example, R 4 It can be -OCH3.

[0071] In an embodiment, R 2 , R 3 and R 4 No more than one of the may be -halo.

[0072] R 5 R may be selected from -H or -OCH3. 5 Can be -H. R 5 Can be -C 1-4 Alkoxy, for example, R 5 It can be -OCH3.

[0073] R 6 It can be -H or -CH3. 6 Can be -H. R 6 Can be -C 1-4 Alkyl groups, such as R 6 It can be -CH3.

[0074] R 7 It can be -CH3 or -C(O)CH3. 7 Can be -H. R 7 Can be -C 1-4 Alkyl groups, such as R 7It can be -CH3. 7 It can be -C(O)H. 7 Can be -C(O)C 1-4 Alkyl groups, such as R 7 It can be -C(O)CH3.

[0075] The aromatic carboxylic acid may be selected from benzoic acid, toluic acid (e.g., p-toluic acid), 4-ethylbenzoic acid, 3,5-dimethylbenzoic acid, 4-tert-butylbenzoic acid, p-anisic acid, 3,4,5-trimethoxybenzoic acid (eucalyptol), trans-cinnamic acid, phthalic acid, homophthalic acid, 4-(trifluoromethyl)benzoic acid, 4-iodobenzoic acid, 4-chlorobenzoic acid, 3-phenylpropionic acid, 4-(dimethylamino)benzoic acid, 4-acetamidobenzoic acid, diphenyl-4-carboxylic acid, methyl hydrogen terephthalate, 3-dimethylamino benzoic acid, 4-methyl-3,5-dinitrobenzoic acid, 3-bromobenzoic acid, 4-aminobenzoic acid, 4-dimethylaminobenzoic acid, 4-(deepoxy)benzoic acid, 3-bromobenzoic acid, 1-naphthoic acid, 4-dimethylaminobenzoic acid, 4-nitrobenzoic acid, terephthalic acid (and / or disodium terephthalate), 4-bromobenzoic acid, 3,5-dimethyl-p-anisic acid, 4-epoxybenzoic acid, 4-n-pentylbenzoic acid, 4-n-heptylbenzoic acid, 4-n-hexylbenzoic acid, 4-n-butylbenzoic acid and 2-naphthoic acid.

[0076] The aromatic carboxylic acid may be selected from 4-dimethylaminobenzoic acid, 4-nitrobenzoic acid, terephthalic acid (and / or disodium terephthalate), 4-bromobenzoic acid, 3,5-dimethyl-p-anisic acid, 4-epoxybenzoic acid, 4-n-pentylbenzoic acid, 4-n-heptylbenzoic acid, 4-n-hexylbenzoic acid, 4-n-butylbenzoic acid and 2-naphthoic acid.

[0077] The aromatic carboxylic acid can be selected from benzoic acid, toluic acid (e.g., p-toluic acid), 4-ethylbenzoic acid, 3,5-dimethylbenzoic acid, 4-tert-butylbenzoic acid, p-anisic acid, 3,4,5-trimethoxybenzoic acid (eucalyptol), trans-cinnamic acid, phthalic acid, homophthalic acid, 4-(trifluoromethyl)benzoic acid, 4-iodobenzoic acid, 4-chlorobenzoic acid, 3-phenylpropionic acid, 4-(dimethylamino)benzoic acid, 4-acetamidobenzoic acid and bisphenyl-4-carboxylic acid.

[0078] The presence and optionally the identity of the aromatic carboxylic acid in the magnetic clusters of the invention can be confirmed using a suitable mass spectrometry method, such as analyzing the magnetic clusters using electrospray ionization mass spectrometry.

[0079] The particles may be in a population of at least 100, such as at least 1000. For example, for measurement purposes, the particles may be in a population of at least 100, such as at least 1000. For example, in certain end-use applications, the particles may conveniently be in a population of at least 100, such as at least 1000.

[0080] The magnetic clusters can have a mode diameter of at least about 100 nm. For example, the magnetic clusters can have a mode diameter of at least about 150 nm, at least about 200 nm, or at least about 250 nm.

[0081] The magnetic clusters can have a mode diameter of no more than about 2,500 nm.For example, the magnetic clusters can have a mode diameter of no more than about 2,000 nm, no more than about 1,500 nm, or no more than about 1,000 nm.

[0082] The magnetic clusters may have a mode diameter in the range of about 100 nm to about 2,500 nm. The magnetic clusters have a mode diameter in the range of about 150 nm to about 2,000 nm. The magnetic clusters have a mode diameter in the range of about 150 nm or about 200 nm to about 1,500 nm. The magnetic clusters have a mode diameter in the range of about 200 nm to about 1,000 nm.

[0083] When measured by a disc centrifuge, the magnetic clusters may have a coefficient of variation (CV) of no more than about 20%. When measured by a disc centrifuge, the magnetic clusters may have a coefficient of variation (CV) of no more than about 15%. When measured by a disc centrifuge, the magnetic clusters may have a coefficient of variation (CV) of no more than about 10%. When measured by a disc centrifuge, the magnetic clusters may have a coefficient of variation (CV) of no more than about 8%. When measured by a disc centrifuge, the magnetic clusters may have a coefficient of variation (CV) of no more than about 5%.

[0084] The magnetic clusters may have a coefficient of variation (CV) of no more than 5% when measured by a disc centrifuge.The magnetic clusters may have a mode diameter in the range of about 100 nm to about 2,500 nm and a coefficient of variation (CV) of no more than about 5% when measured by a disc centrifuge.

[0085] The nanocrystals of iron oxide can comprise a mode average diameter of about 5nm to about 20nm. The nanocrystals can comprise a mode average diameter of about 10nm to about 15nm. The nanocrystals can comprise a mode average diameter of about 12nm. Providing nanocrystals in magnetic clusters within this size range can provide advantages. For example, the nanocrystals within this size range can be superparamagnetic, resulting in magnetic clusters with negligible magnetic properties when there is no external magnetic field. The lack of this remanence minimizes the aggregation of magnetic particles when there is no external magnetic field. In addition, relatively high levels of iron oxide in the magnetic clusters provide effective magnetic separation / collection of magnetic clusters during determination.

[0086] The magnetic clusters may comprise a coating. The coating may comprise at least one layer of silicon dioxide. The coating may comprise at least one layer of an organic coating. The organic coating may comprise a spacer (e.g., oligoethylene glycol or polyethylene glycol) and / or a polymer. Hydrophilic spacers, such as oligoethylene glycol or polyethylene glycol, may be particularly suitable for use in magnetic clusters in bioassays because the hydrophilic spacer arms are able to interact with aqueous environments and tend to limit non-specific protein adsorption. The coating may comprise at least one layer of silicon dioxide and at least one layer of an organic coating, optionally wherein the at least one layer of the organic coating is coated with at least one layer of silicon dioxide.

[0087] The coating may include functional groups, such as functional groups on the spacer. The functional groups may be selected from hydroxyl groups, carboxylic acid groups, aldehyde groups, amine groups (e.g., primary or secondary aliphatic amine groups and aromatic amine groups), thiol groups, epoxy groups, amide groups, chloromethyl groups and tosyl activated groups. The functional groups may be selected from hydroxyl groups, carboxylic acid (-COOH), primary amines, secondary amines and epoxy groups. For example, the functional groups may be selected from carboxylic acids, primary amines, secondary amines and epoxy groups; for example, the functional groups may be selected from carboxylic acids, primary amines and secondary amines.

[0088] Ligands can be attached to functional groups using methods known to the skilled person. Examples of such methods are described in Chapter 14 of GT Hermanson, Bioconjugate Techniques, Academic Press (3rd edition, 1996). Thus, the magnetic clusters may include ligands, for example ligands attached to the magnetic clusters via reactive groups that react.

[0089] The magnetic clusters may comprise at least 80% by dry weight, such as at least 85% by dry weight, of iron oxide.

[0090] Magnetic clusters can be characterized by other features. An example of such a feature is specific surface area. Specific surface area represents the total external and internal specific surface area of ​​a sample obtained by measuring the amount of physically adsorbed gas (usually nitrogen or argon), which is based on Brunauer, Emmett and Teller (BET) method (Brunauer, Stephen; Emmett, PH; Teller, Edward (1938). "Adsorption of Gases in Multimolecular Layers". Journal of the American Chemical Society. 60 (2): 309-319). Therefore, the specific surface area is a function of the size, porosity and surface roughness of the magnetic clusters. As the technician will appreciate, the coated magnetic clusters can reduce porosity and / or surface roughness, reducing the surface area, compared to the corresponding uncoated magnetic clusters. An exemplary magnetic cluster may have a surface area of ​​about 5 m 2 / g to about 100m 2 For example, the specific surface area can be about 10 m 2 / g to about 75m 2 / g.

[0091] Magnetic clusters may be obtainable or obtained by the methods disclosed herein.

[0092] Preparation of magnetic clusters

[0093] The present disclosure provides a method for forming magnetic clusters. The method involves providing a solution of iron (III) ions in a protic solvent, and precipitating iron from the solution to form magnetic nanoclusters. Although methods for precipitating iron (III) ions from solution are known in the art, the resulting magnetic particles may be highly polydisperse and / or ferromagnetic or ferrimagnetic, both of which are undesirable for particles used to measure. The inventors have determined that precipitation reactions in the presence of certain aromatic carboxylic acids provide (highly) monodisperse and paramagnetic or superparamagnetic magnetic clusters. Such magnetic clusters are useful in measuring.

[0094] C. Cheng et al., J. Mater. Chem., 2009, 19, 8782-8788 have prepared magnetic nanoclusters by precipitating iron from solution in the presence of sodium citrate as a stabilizer. Figure 1(Adapted from Scheme 1 of C. Cheng et al.) provides a hypothetical schematic diagram of how magnetic nanoclusters are formed, and additionally indicates that the resulting clusters may be relatively uniform in size. For example, it was determined that by changing the concentration of sodium citrate in the reaction from 0.001M to 0.1M, the diameter of the magnetic nanoclusters could be tuned from about 300nm to 40nm.

[0095] Without wishing to be bound by any theory, Figure 1 As indicated, it is believed that the process of forming nanoclusters begins with the formation of iron oxide cores in an ethylene glycol solution. These cores become iron oxide nanocrystals, with citrate stabilizers adsorbed to the surface. Adjacent nanocrystals interact with each other. The aggregation of nanocrystals to form magnetic nanoclusters is believed to be governed by a balance between surface tension and electrostatic repulsion. Stabilizers adsorbed on the surface of nanocrystals provide electrostatic repulsion between adjacent nanocrystals. The surface tension of nanocrystals has the opposite effect, because nanocrystals want to aggregate to reduce the surface area to volume ratio (and therefore surface energy) in ethylene glycol. Therefore, as Cheng et al. observed, increasing the concentration of sodium citrate acts to increase electrostatic repulsion as more citrate is adsorbed on the surface of nanocrystals, which has the effect of reducing the size of magnetic nanoclusters.

[0096] We have determined that aromatic carboxylic acids, particularly those of formula (I), provide good nanocrystal stabilizers when magnetic clusters are prepared by precipitation of iron from solution. Surprisingly, increasing the concentration of aromatic carboxylic acid stabilizers, unlike the case of citrate stabilizers, generally increases the size of the magnetic nanoclusters formed. Without wishing to be bound by any theory, we believe that aromatic carboxylic acid stabilizers do not impart significant electrostatic charge to the particles. On the contrary, the main effect of adsorbed aromatic carboxylic acid stabilizers is to increase surface tension, and therefore we observe that increasing the amount of stabilizer generally results in larger magnetic clusters. Therefore, regulating the amount of stabilizer allows tuning the size of the magnetic clusters, from submicron to several micrometers. In addition, the presence of aromatic rings in the stabilizer can promote preferential orientation between stabilizer molecules (e.g., due to π-π interactions), which helps the stabilizer to uniformly cover the nanocrystals. A more uniform coverage of the stabilizer also provides a more uniform distribution of electrostatic repulsion and surface tension between the nanocrystals and the clusters. This may result in a given batch of magnetic clusters having a high level of homogeneity, ie having a low CV (eg a CV of less than 20% or 15%, such as a CV of less than 10%).

[0097] The embodiment provides a method for preparing magnetic nanoclusters. The method includes forming a solution comprising iron (III) ions, a protic solvent, an aromatic carboxylic acid, and a precipitant; and reacting the solution to form a suspension comprising magnetic clusters.

[0098] The iron (III) ions may be provided in the form of a soluble iron (III) salt. The soluble iron (III) salt may be an iron (III) halide. The soluble iron (III) salt may be iron (III) chloride.

[0099] The protic solvent can be or comprise an alcohol. The alcohol can be a diol or a polyol (such as a triol). The alcohol can be a diol, such as propylene glycol or ethylene glycol. For example, the alcohol can be ethylene glycol. Compared with propylene glycol, it may be preferred to use ethylene glycol because ethylene glycol is believed to provide a solvent system that provides slower particle orientation and aggregation, which can promote a more uniform size distribution when used in combination with an aromatic carboxylic acid stabilizer in the method of the present invention. The alcohol can be a polyol, such as diethylene glycol (DEG) or tetraethylene glycol (TEG). In an exemplary method, a diol (e.g., ethylene glycol) may be more preferred than a polyol.

[0100] The presence of a significant amount of water (e.g., >1% water) in the protic solvent may have a negative impact on the method of the present invention, for example, by affecting the uniformity of surface tension and / or stabilizer coverage. Without wishing to be bound by any theory, it is believed that this may be due to water preferentially binding to the surface of the nanocrystals, which is believed to inhibit the coordination ability of the stabilizer (see, e.g., S. W Cao et al., New J. Chem., 2008, 32, 1526-1530; A.; A. Kostopoulou et al., Nanoscale, 2014, 6, 3764-3776). Therefore, the protic solvent may contain less than 1% water, for example, the protic solvent may be (substantially) anhydrous.

[0101] The aromatic carboxylic acid may be a compound of formula (I):

[0102]

[0103] L 1 Select from bonds, -C 1-6 Alkyl-, or -C 2-6 Alkenyl-. R 1 Selected from -H, -COOH, -(C 1-5 Alkyl)COOH, -(C 2-5 alkenyl)COOH or -C 1-4 Alkoxy. 2 Selected from -H, -halo, -C 1-4 Alkyl, -C 1-4 Alkoxy or -NO2. 3 Selected from -H, -halo, -C 1-12 Alkyl, -C 3-8 Cycloalkyl, -C 1-12 Alkoxy, -halogen (C 1-12 )alkyl, -NR6 R 7 、-NO2、-C(O)OH、-C(O)OR 6 Or substituted or unsubstituted phenyl. 4 Selected from -H, -halo, -C 1-4 Alkyl, -C 1-4 Alkoxy or -NO2. 5 Select from -H or -C 1-4 Alkoxy. 6 Select from -H or -C 1-4 Alkyl. 7 Selected from -H, -C 1-4 Alkyl, -C(O)H, -C(O)C 1-4 Alternatively, R1 and R2 together with the carbon atom to which they are attached form a substituted or unsubstituted phenyl group, and L 1 , R 3 , R 4 , R 5 , R 6 and R 7 As defined herein. In another alternative, R2 and R3 together with the carbon atom to which they are attached form a substituted or unsubstituted phenyl group, and L 1 , R 1 , R 4 , R 5 , R 6 and R 7 As defined elsewhere.

[0104] In one embodiment, L 1 Select from bonds, -C 1-6 Alkyl-, or -C 2-6 Alkenyl-. R 1 Selected from -H, -COOH, -(C 1-5 Alkyl)COOH, -(C 2-5 alkenyl)COOH or -C 1-4 Alkoxy. 2 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy. 3 Selected from -H, -halo, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogen (C 1-6 )alkyl, -NR 6 R 7 Or substituted or unsubstituted phenyl. 4 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy. 5 Select from -H or -C 1-4Alkoxy. 6 Select from -H or -C 1-4 Alkyl. 7 Selected from -H, -C 1-4 Alkyl, -C(O)H, -C(O)C 1-4 alkyl.

[0105] L 1 Can be selected from key, -C 1-4 Alkyl-, or -C 2-4 Alkenyl-. L 1 Can be a key. 1 Can be selected from -C 1-4 Alkyl- or -C 2-4 Alkenyl-. L 1 Can be -C 1-6 Alkyl-, optionally -C 1-4 Alkyl-.L 1 Can be -C 2-6 Alkenyl-, optionally -C 2-4 Alkenyl-.

[0106] R 1 Can be selected from -H, -COOH, -(C 1-5 alkyl)COOH or -(C 2-5 R1 can be selected from -H or -COOH. 1 Can be -H, R 1 It can be -COOH. 1 It can be -H-(C 1-5 Alkyl)COOH. R 1 Can be -(C 2-5 (Alkenyl)COOH. 1 Can be -C 1-4 Alkoxy.

[0107] R 2 Can be selected from -H, -C 1-4 Alkyl or -C 1-4 Alkoxy. 2 Can be selected from -H or -C 1-4 Alkoxy. 2 R may be selected from -H or -OCH3. 2 Can be -H. R 2 Can be -C 1-4 Alkyl groups, such as R 2 It can be -CH3. 2 Can be -C 1-4 Alkoxy, for example, R 2 It can be -OCH3.

[0108] R1 and R2 together with the carbon atoms to which they are attached may form a substituted or unsubstituted phenyl group.

[0109] R 3 Can be selected from -H, -halogen, -Ci 1-6 Alkyl, -C 3-6 Cycloalkyl, -C 1-6 Alkoxy, -halogen (C 1-6 )alkyl, -NR 6 R 7 , -NO2, -C(O)OH or substituted or unsubstituted phenyl. 3 Can be selected from -H, -halogen, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogen (C 1-6 )alkyl, -NR 6 R 7 or a substituted or unsubstituted phenyl group.

[0110] R 3 Can be selected from -H, -C 1-4 Alkyl, halogen, -C 1-4 Alkoxy, -halogen (C 1-4 )alkyl, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3 Can be selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy, -halogen (C 1-4 )alkyl, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3 Can be selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3 Can be selected from -C 1-4 Alkoxy, -halogen (C 1-4 )alkyl, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3 Can be selected from -C 1-4 Alkoxy, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3 Can be selected from -C 1-4 Alkoxy, -halogen (C 1-4 )alkyl, -NR 6 R 7 Or substituted or unsubstituted phenyl. 3Can be selected from -C 1-4 Alkoxy, -NR 6 R 7 or unsubstituted phenyl. 3 Can be selected from -H, -C 1-4 Alkyl, -C1, -I, -OCH3, -CF3, -N(CH3)2, -NHC(O)CH3 or phenyl. 3 Can be selected from -H, -C 1-4 Alkyl, -OCH3, -N(CH3)2, -NHC(O)CH3 or phenyl. 3 R may be selected from -OCH3, -CF3, -N(CH3)2, -NHC(O)CH3 or phenyl. 3 It can be selected from -OCH3, -N(CH3)2, -NHC(O)CH3 or phenyl.

[0111] R 3 Can be selected from -H, -C 1-10 Alkoxy, -NR 6 R 7 、-C(O)OR 6 Or substituted or unsubstituted phenyl. 3 Can be selected from -H, -C 1-4 Alkyl, -OCH3, -OCH2(CH2)8CH3, -N(CH3)2, -NHC(O)CH3, -C(O)OCH3 or phenyl.

[0112] R 3 Can be -H. R 3 Can be -C 1-6 Alkyl groups, such as R 3 Can be -C 1-4 Alkyl. 3 Can be -C 1-6 Alkoxy, for example, R 3 Can be -C 1-4 Alkoxy. 3 It can be -OCH3. 3 Can be -NR 6 R 7 . R 3 It may be a substituted or unsubstituted phenyl group, for example, R 3 It may be an unsubstituted phenyl group. 3 It can be -halo, for example, R 3 It can be -Cl. 3 It can be -halogen (C 1-6 ) alkyl, for example, R 3 It can be -halogen (C 1-4 ) alkyl. 3 It can be -CF3.3 Can be C 1-12 Alkyl groups, such as R 3 It can be -OCH2(CH2)8CH3 or -OCH3. 3 Can be -C(O)OR 6 , for example, R 3 It can be -C(O)OCH3.

[0113] R2 and R3 together with the carbon atoms to which they are attached may form a substituted or unsubstituted phenyl group.

[0114] R 4 Can be selected from -H, -C 1-4 Alkyl or -C 1-4 Alkoxy. 4 Can be selected from -H or -C 1-4 Alkoxy. 4 R may be selected from -H or -OCH3. 4 Can be -H. R 4 Can be -C 1-4 Alkyl groups, such as R 4 It can be -CH3. 4 Can be -C 1-4 Alkoxy, for example, R 4 It can be -OCH3.

[0115] R 5 R may be selected from -H or -OCH3. 5 Can be -H. R 5 Can be -C 1-4 Alkoxy, for example, R 5 It can be -OCH3.

[0116] R 6 It can be -H or -CH3. 6 Can be -H. R 6 Can be -C 1-4 Alkyl groups, such as R 6 It can be -CH3.

[0117] R 7 It can be -CH3 or -C(O)CH3. 7 Can be -H. R 7 Can be -C 1-4 Alkyl groups, such as R 7 It can be -CH3. 7 It can be -C(O)H. 7 Can be -C(O)C 1-4 Alkyl groups, such as R 7 It can be -C(O)CH3.

[0118] In an embodiment, R 2 , R 3 and R 4 No more than one of the may be -halo.

[0119] The aromatic carboxylic acid may be selected from benzoic acid, toluic acid (e.g., p-toluic acid), 4-ethylbenzoic acid, 3,5-dimethylbenzoic acid, 4-tert-butylbenzoic acid, p-anisic acid, 3,4,5-trimethoxybenzoic acid (eucalyptol), trans-cinnamic acid, phthalic acid, homophthalic acid, 4-(trifluoromethyl)benzoic acid, 4-iodobenzoic acid, 4-chlorobenzoic acid, 3-phenylpropionic acid, 4-(dimethylamino)benzoic acid, 4-acetamidobenzoic acid, diphenyl-4-carboxylic acid, methyl hydrogen terephthalate, 3-dimethylamino benzoic acid, 4-methyl-3,5-dinitrobenzoic acid, 3-bromobenzoic acid, 4-aminobenzoic acid, 4-dimethylaminobenzoic acid, 4-(deepoxy)benzoic acid, 3-bromobenzoic acid, 1-naphthoic acid, 4-dimethylaminobenzoic acid, 4-nitrobenzoic acid, terephthalic acid (and / or disodium terephthalate), 4-bromobenzoic acid, 3,5-dimethyl-p-anisic acid, 4-epoxybenzoic acid, 4-n-pentylbenzoic acid, 4-n-heptylbenzoic acid, 4-n-hexylbenzoic acid, 4-n-butylbenzoic acid and 2-naphthoic acid.

[0120] The aromatic carboxylic acid may be selected from 4-dimethylaminobenzoic acid, 4-nitrobenzoic acid, terephthalic acid (and / or disodium terephthalate), 4-bromobenzoic acid, 3,5-dimethyl-p-anisic acid, 4-epoxybenzoic acid, 4-n-pentylbenzoic acid, 4-n-heptylbenzoic acid, 4-n-hexylbenzoic acid, 4-n-butylbenzoic acid and 2-naphthoic acid.

[0121] The aromatic carboxylic acid can be selected from benzoic acid, toluic acid (e.g., p-toluic acid), 4-ethylbenzoic acid, 3,5-dimethylbenzoic acid, 4-tert-butylbenzoic acid, p-anisic acid, 3,4,5-trimethoxybenzoic acid (eucalyptol), trans-cinnamic acid, phthalic acid, homophthalic acid, 4-(trifluoromethyl)benzoic acid, 4-iodobenzoic acid, 4-chlorobenzoic acid, 3-phenylpropionic acid, 4-(dimethylamino)benzoic acid, 4-acetamidobenzoic acid, and bisphenyl-4-carboxylic acid.

[0122] The magnetic clusters formed contain both iron oxide and aromatic carboxylic acid.The presence and optionally the identity of the aromatic carboxylic acid in the magnetic clusters can be confirmed using a suitable mass spectrometry method, such as analyzing the magnetic clusters using electrospray ionization mass spectrometry.

[0123] The iron oxide of the magnetic clusters may include divalent iron ions and trivalent iron ions. For example, the iron oxide magnetic clusters may include magnetite (having about Fe 2+ (Fe 3+ )2(O 2-)4). Thus, allowing the solution to react to form a suspension comprising magnetic clusters and / or precipitating iron from the solution to form magnetic nanoclusters may include reducing some of the iron (III) ions to iron (II) ions. The reducing agent providing the reduction may be or include a protic solvent (such as an alcohol, e.g., ethylene glycol). The precipitant may act as a reduction aid; and, in the case where the precipitant includes an ionic species, the pre-precipitant may be an electrostatic stabilizer that reduces or prevents particle agglomeration.

[0124] The precipitant may be urea or an acetate, for example the precipitant may be an acetate. For example, the precipitant may be an acetate, such as an alkali metal acetate. The precipitant may be sodium acetate.

[0125] The molar ratio of iron (III) ions to aromatic carboxylic acid may be from about 1:0.1 to about 1:5. For example, the molar ratio of iron (III) ions to aromatic carboxylic acid may be from about 1:0.2 to about 1:2; for example, the molar ratio may be from about 1:0.2 to about 1:1.5.

[0126] The molar ratio of iron (III) ions to the precipitant may be about 1:1 to about 1:10. For example, the molar ratio of iron (III) ions to the aromatic carboxylic acid may be about 1:2 to about 1:5; for example, the molar ratio may be about 1:3.5.

[0127] The molar ratio of iron (III) ions to aromatic carboxylic acid and precipitation agent may be from about 1:0.1:1 to about 1:5:10. For example, the molar ratio of iron (III) ions to aromatic carboxylic acid and precipitation agent may be from about 1:0.2:2 to about 1:2:5; for example, the molar ratio may be from about 1:0.2:3.5 to about 1:1.5:3.5.

[0128] The molar ratio of iron (III) ions to protic solvents may be from about 1:30 to about 1:250. For example, the molar ratio of iron (III) ions to protic solvents may be from about 1:30 to about 1:200; for example, the molar ratio of iron (III) ions to protic solvents may be from about 1:30 to about 1:150. The molar ratio of iron (III) ions to protic solvents may be from about 1:50 to about 1:100.

[0129] The step of allowing the solution to react may include allowing the solution to react at a high temperature (e.g., greater than about 50° C.). The high temperature may be a temperature of about 150° C. to about 250° C. For example, the high temperature may be a temperature of about 170° C. to about 250° C., such as a temperature of about 180° C. to about 230° C.

[0130] The step of allowing the solution to react may include allowing the solution to react at a given pressure, wherein the pressure represents the total (non-partial) pressure. The step of allowing the solution to react may include allowing the solution to react at a pressure of about 1 bar to about 15 bar. For example, the pressure may be about 1 bar to about 10 bar, for example, the pressure may be about 3 bar to about 10 bar.

[0131] The exact reaction time is not critical, although it is important to allow sufficient time to form the magnetic clusters, for example, allowing a reaction time of at least about 6 hours (e.g., at least about 8 hours) may be suitable. The step of allowing the solution to react may include allowing the solution to react for a reaction time of about 6 hours to about 36 hours. For example, the reaction time may be about 8 hours to about 24 hours.

[0132] The method may further comprise separating the magnetic clusters from the suspension. The separation may be accomplished by any suitable technique, such as decantation (optionally after centrifugation or magnetic precipitation of the magnetic clusters), filtration, etc.

[0133] The method may further comprise washing the magnetic clusters after separation. Washing may be performed with any suitable washing solution, such as alcohol (e.g., ethanol, methanol), water, aqueous citric acid solution (e.g., aqueous sodium citrate), acetone, (1S,5R)-6,8-dioxabicyclo[3.2.1]octan-4-one (Cyrene TM ), etc., or mixtures thereof. Washing may include one or more applications of the same or different washing solutions. For example, an exemplary step of washing the magnetic clusters may include washing with ethanol, then washing with water, then washing with aqueous citric acid, then washing with water.

[0134] The method may also include adding a silica coating to the separated magnetic clusters, including: forming a suspension comprising the separated magnetic clusters and a silicate or an orthosilicate; and reacting the silicate or orthosilicate to form the silica coating. The silica coating may be formed by reacting a silicate (e.g., Na2SiO3) in the presence of the separated magnetic clusters at a pH below about 11. The silica coating may be formed by reacting an orthosilicate (e.g., tetraethyl orthosilicate) in the presence of the separated magnetic clusters. Although the reaction may be carried out at room temperature, in one embodiment, the reaction is carried out at a higher temperature, such as a temperature of at least 40°C (e.g., a temperature of at least 60°C or at least 90°C).

[0135] The method may also include coating the silica coating with an organic coating. The overcoating may be performed in the presence of a water miscible solvent, such as Cyrene TM((1S,5R)-6,8-dioxabicyclo[3.2.1]octan-4-one). The organic coating may include a spacer and / or a polymer. The spacer may be or include oligoethylene glycol or polyethylene glycol. Hydrophilic spacers, such as oligoethylene glycol or polyethylene glycol, may be particularly suitable for magnetic clusters used in bioassays because the hydrophilic spacer arms are able to interact with aqueous environments and tend to limit nonspecific protein adsorption.

[0136] The coating may include functional groups. The functional groups may be selected from hydroxyl groups, carboxylic acid groups, aldehyde groups, amine groups (e.g., primary or secondary aliphatic amine groups and aromatic amine groups), thiol groups, epoxy groups, amide groups, chloromethyl groups and tosyl activated groups. The functional groups may be selected from hydroxyl groups, carboxylic acid (-COOH), primary amines, secondary amines and epoxy groups. For example, the functional groups may be selected from carboxylic acids, primary amines, secondary amines and epoxy groups; for example, the functional groups may be selected from carboxylic acids, primary amines and secondary amines.

[0137] The ligand can be attached to the functional group using methods known to the skilled person. Examples of such methods are described in Chapter 14 of GT Hermanson, Bioconjugate Techniques, Academic Press (3rd edition, 1996). The method may include attaching the ligand to the functional group of the magnetic cluster. For example, the method may include attaching the ligand to the functional group of the magnetic cluster coating.

[0138] The magnetic clusters can have a mode diameter of at least about 100 nm. For example, the magnetic clusters can have a mode diameter of at least about 150 nm, at least about 200 nm, or at least about 250 nm.

[0139] The magnetic clusters can have a mode diameter of no more than about 2,500 nm.For example, the magnetic clusters can have a mode diameter of no more than about 2,000 nm, no more than about 1,500 nm, or no more than about 1,000 nm.

[0140] The magnetic clusters may have a mode diameter in the range of about 100 nm to about 2,500 nm. The magnetic clusters have a mode diameter in the range of about 150 nm to about 2,000 nm. The magnetic clusters have a mode diameter in the range of about 150 nm or about 200 nm to about 1,500 nm. The magnetic clusters have a mode diameter in the range of about 200 nm to about 1,000 nm.

[0141] The magnetic clusters can be monodisperse.

[0142] When measured by a disc centrifuge, the magnetic clusters may have a coefficient of variation (CV) of no more than about 20%. When measured by a disc centrifuge, the magnetic clusters may have a coefficient of variation (CV) of no more than about 15%. When measured by a disc centrifuge, the magnetic clusters may have a coefficient of variation (CV) of no more than about 10%. When measured by a disc centrifuge, the magnetic clusters may have a coefficient of variation (CV) of no more than about 8%. When measured by a disc centrifuge, the magnetic clusters may have a coefficient of variation (CV) of no more than about 5%.

[0143] The magnetic clusters may have a coefficient of variation (CV) of no more than 5% when measured by a disc centrifuge.The magnetic clusters may have a mode diameter in the range of about 100 nm to about 2,500 nm and a coefficient of variation (CV) of no more than about 5% when measured by a disc centrifuge.

[0144] The nanocrystals of iron oxide can comprise a mode average diameter of about 5nm to about 20nm. The nanocrystals can comprise a mode average diameter of about 10nm to about 15nm. The nanocrystals can comprise a mode average diameter of about 12nm. Providing nanocrystals in magnetic clusters within this size range can provide advantages. For example, the nanocrystals within this size range can be superparamagnetic, resulting in magnetic clusters with negligible magnetic properties when there is no external magnetic field. The lack of this remanence minimizes the aggregation of magnetic particles when there is no external magnetic field. In addition, relatively high levels of iron oxide in the magnetic clusters provide effective magnetic separation / collection of magnetic clusters during determination.

[0145] The magnetic clusters may comprise at least 80% by dry weight, such as at least 85% by dry weight, of iron oxide.

[0146] Uses of Magnetic Clusters

[0147] Magnetic clusters can be used in many applications, such as polynucleotide sequencing, bioassays, information storage, color imaging, in vitro diagnostics, bioprocessing, diagnostic microbiology, biosensors, and drug delivery. Magnetic clusters are particularly useful in assays (e.g., where polymer particles are conjugated to target analytes such as affinity ligands) because the magnetic material allows the particles (and any species bound thereto) to be separated from the bulk solution by application of a magnetic field.

[0148] In particular, the coated magnetic clusters of the present invention (e.g., comprising silica and / or functional groups) can be used as reagents for separating and / or detecting biological materials, particularly nucleic acids, and can separate and purify nucleic acids in high yields. In addition, highly active silica magnetic nanoparticles can also be used for protein purification, antibody purification, enzyme immunoassay, peptide purification, endotoxin removal, etc.

[0149] Nucleic acid purification and operation are basic processes in all molecular biology laboratories. Nucleic acid can be separated from a variety of sources, including samples containing viruses, bacteria, plants or animals or human cells or tissues. Nucleic acid can also be from acellular sources, clinical samples, such as blood, blood plasma, serum, swabs, biopsies, tissues, various environmental sources or from in vitro reactions. Therefore, nucleic acid is generally used as a marker for detecting biological entities (e.g., viruses, bacteria), and therefore can be used for the diagnosis of various diseases. The method is also applicable to separating free circulating nucleic acids from blood plasma and / or serum, such as tumor DNA and RNA and fetal DNA.

[0150] In some cases, the sample can be used directly without pretreatment. However, nucleic acids must usually be separated and amplified from their environment before they can be effectively detected or further manipulated. Therefore, in many cases, the sample must first be digested by a suitable method and the biological material contained in the sample must be released. Methods for breaking samples and cells are well known in the art and can be chemical, enzymatic or physical in nature. Combinations of these methods are also possible.

[0151] In this case, there may be various factors that are found to be beneficial to different biomaterials. In many cases, the sample is contacted with a lysis reagent, which includes, for example, ionic detergents and non-ionic detergents, such as, for example, SDS, lid or sodium lauryl sarcosine in an appropriate buffer, a chaotropic salt, such as, for example, guanidine hydrochloride (GHCL), guanidine thiocyanate (GTC), guanidine isothiocyanate (GITC), sodium iodide, sodium perchlorate, etc. In some cases, mechanical tearing may also be performed, such as, for example, by French pressing, ultrasound, grinding with glass beads, nickel beads, aluminum or in liquid nitrogen. Other treatments, such as enzymatic lysis, for example, with lysozyme, protease, proteinase K or cellulase or by other commercially available enzymes for lysis; lysis of cells by phage or virus infection; freeze drying; osmotic shock; microwave treatment; heat treatment; for example, by heating or boiling or freezing, for example, in dry ice or liquid nitrogen, and thawing; alkaline lysis is possible.

[0152] In one example, the silica-coated magnetic clusters of the present invention can be used to separate nucleic acids by using a widely known method for separating nucleic acids on a silica surface using a chaotropic agent (R. Boom et al., J. Clin. Microbiol., Vol 28 (3), p495-503 (1990)). In this method, when the magnetic clusters coated with silica bind to nucleic acids through a chaotropic agent, the nucleic acids are separated by separating the silica magnetic particles using an external magnetic force.

[0153] In the first step of this exemplary method, the silica-coated magnetic clusters of the present invention are added to a sample containing nucleic acids to be separated to induce binding of the nucleic acids to the silica magnetic nanoparticles.

[0154] In this article, a binding buffer is used. As a binding buffer, a chaotropic agent can be used. Chaotropic agents include guanidine salts, urea, chlorides, iodides, perchlorates, (iso)thiocyanates, etc., and specific examples include but are not limited to sodium perchlorate, guanidine hydrochloride, guanidine isothiocyanate, potassium iodide, potassium thiocyanate, sodium chloride, sodium isothiocyanate, magnesium chloride, sodium iodide, etc. Chaotropic agents are preferably used at a concentration of 1mol / L-8mol / L.

[0155] Alternatively or in addition, the binding buffer may comprise branched or unbranched alcohols, such as, for example, ethanol or isopropanol. WO91 / 12079 describes a method for capturing nucleic acids on a solid surface by precipitation. Generally, alcohols and salts are used as precipitants. Depending on the size of the nucleic acid to be separated, during the binding step, the alcohol may be present in a concentration greater than 5% (v / v) and less than 60% (v / v), preferably less than 40% (v / v). In some cases, during the binding step, the alcohol may be present in a concentration of about 10% (v / v) to about 30% (v / v).

[0156] U.S. Patent Nos. 5,705,628 and 5,898,071 describe methods for separating nucleic acid fragments using a combination of a high molecular weight polyalkylene glycol (e.g., polyethylene glycol) at a concentration of 7%-13% with a salt in the range of 0.5M-5M to achieve binding to functional groups on a solid support that acts as a bioaffinity adsorbent for DNA. In other examples, tetraethylene glycol can be used to precipitate nucleic acids on a solid phase, as described in U.S. Patent No. 8,569,477. Any of these known methods can be used to bind nucleic acids to the silica-coated particles of the present invention.

[0157] The second step of the nucleic acid separation method is a step of separating the silica-coated magnetic clusters bound with nucleic acid. In this step, the silica-coated magnetic clusters bound with nucleic acid are collected on the container wall by an external magnetic force, and unbound species are separated, followed by washing.

[0158] The third step is a step of removing the external magnetic force and separating the nucleic acid from the silica magnetic nanoparticles bound with the nucleic acid. In this step, the nucleic acid bound to the silica magnetic particles is separated using an elution buffer (such as a tris(hydroxymethyl)aminomethane buffer).

[0159] Thus, the silica-coated particles of the present invention can be used for general capture of nucleic acids. Alternatively, specific capture of nucleic acids can be achieved by hybridization with oligonucleotides immobilized on the particles. The isolated nucleic acids can be used for further manipulation or detection by amplification techniques.

[0160] One embodiment provides the use of the magnetic clusters of the present invention in assays such as bioassays. The magnetic clusters are particularly suitable for various types of assays due to the following advantageous properties: 1) the small size of the nanoparticles ensures a large surface area available for target capture; 2) a large number of particles per sample volume ensures rapid and efficient capture of the target; 3) the magnetic properties described herein allow for simple automation of the assay / separation protocol, and 4) the high iron content ensures efficient magnetic separation in viscous samples.

[0161] The magnetic clusters used in the assay may comprise a coating. The coating may comprise at least one layer of silica. The coating may comprise at least one layer of organic coating. The organic coating may comprise a spacer (e.g., oligoethylene glycol or polyethylene glycol) and / or a polymer. Hydrophilic spacers, such as oligoethylene glycol or polyethylene glycol, may be particularly suitable for use in magnetic clusters in biological assays because the hydrophilic spacer arms are able to interact with the aqueous environment and tend to limit non-specific protein adsorption. The coating may comprise at least one layer of silica and at least one layer of organic coating, optionally wherein at least one layer of organic coating is coated with at least one layer of silica.

[0162] The coating may include functional groups, such as functional groups on the spacer. The functional groups may be selected from hydroxyl groups, carboxylic acid groups, aldehyde groups, amine groups (e.g., primary or secondary aliphatic amine groups and aromatic amine groups), thiol groups, epoxy groups, amide groups, chloromethyl groups and tosyl activated groups. The functional groups may be selected from hydroxyl groups, carboxylic acid (-COOH), primary amines, secondary amines and epoxy groups. For example, the functional groups may be selected from carboxylic acids, primary amines, secondary amines and epoxy groups; for example, the functional groups may be selected from carboxylic acids, primary amines and secondary amines.

[0163] The ligands can be attached to the functional groups using methods known to the skilled person. Examples of such methods are described in Chapter 14 of GT Hermanson, Bioconjugate Techniques, Academic Press (3rd edition, 2013). Thus, the magnetic clusters used in the assay may comprise ligands, for example ligands attached to the magnetic clusters via reactive groups that react.

[0164] In one embodiment, the magnetic clusters comprise (surface) capture ligands. The capture ligands may be selected from antibodies, antibody fragments, peptides, carbohydrates, haptens, aptamers and oligonucleotides.

[0165] The magnetic clusters may comprise surface functionalization for capturing a class of molecules. For example, the surface functionalization may comprise ionic groups or hydrophobic groups.

[0166] The assay may comprise a process selected from the group consisting of: immunoprecipitation, nucleic acid capture, immunoassay, and / or lateral flow assay.

[0167] For example, immunoprecipitation can be performed by binding antibodies directly or indirectly (usually with protein A or G, streptavidin, or a second antibody first bound to a particle) to capture a class of proteins or protein complexes. The captured proteins can be analyzed by mass spectrometry, gel electrophoresis, or protein array technology.

[0168] Magnetic nanoparticles can also be used in immunoassays using direct or indirect binding of antibodies or antigens and detecting analytes using sandwich immunoassays or competitive immunoassays known in the art. Detection systems can include chemiluminescent, electrochemiluminescent, colorimetric or electrochemical detection devices.

[0169] In addition, magnetic particles can be used as reporter molecules in immunoassays by binding detection antibodies to the particles. The particles can then be used in assays where capture antibodies are immobilized on a sensor surface that is able to detect changes in the magnetic field.

[0170] Due to the small size of the particles of the present invention, they can also be used in lateral flow assays, where the particles will serve as reporters by colorimetric detection, detection using magnetic-based sensors, and the like.

[0171] The assay may include a process selected from the group consisting of protein purification, antibody purification, enzyme immunoassay, peptide purification, and endotoxin removal.

[0172] One embodiment provides a method for isolating nucleic acid from a sample containing biological material, the method comprising:

[0173] (a) contacting a sample containing biological material with a lysis solution;

[0174] (b) contacting the sample from (a) with silica-coated magnetic clusters (as defined herein) under conditions suitable for adsorbing nucleic acids in the sample to the silica-coated magnetic clusters;

[0175] (c) washing the silica coated magnetic particles from (b); and

[0176] (d) Desorption (elution) of nucleic acids from silica-coated magnetic particles.

[0177] An exemplary lysis solution comprises:

[0178] (a) buffer (e.g., Tris-HCl);

[0179] (b) chelating agents (e.g., EDTA);

[0180] (c) chaotropic agents (e.g., guanidine salts such as guanidine isothiocyanate);

[0181] (d) a detergent (e.g., 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100) or sodium lauroyl sarcosinate (sodium lauryl sarcosinate)); and optionally

[0182] (e) a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)); and / or

[0183] (f) Nucleic acid carrier (eg, a polymer such as glycogen).

[0184] One embodiment provides an in vitro method for detecting nucleic acid from an infectious agent in a biological sample (e.g., an oral and / or nasal sample), the method comprising:

[0185] (a) isolating nucleic acids from a biological sample suspected of containing an infectious agent (e.g., a virus such as a coronavirus) using the methods disclosed herein with the magnetic clusters of the invention; and

[0186] (b) analyzing the nucleic acid from (a) to determine the presence of nucleic acid from an infectious agent.

[0187] Another embodiment provides a method for determining in vitro whether a subject is infected with an infectious agent (e.g., a virus, such as a coronavirus), the method comprising:

[0188] (a) isolating nucleic acid from a biological sample (e.g., oral and / or nasal sample) of a subject suspected of being infected with the magnetic clusters of the present invention using the methods disclosed herein; and

[0189] (b) analyzing the nucleic acid from (a) for the presence of nucleic acid from an infectious agent (e.g., a virus such as a coronavirus), wherein detection of nucleic acid from an infectious agent indicates that the subject has an infection.

[0190] The particles and methods can be used to separate various types of nucleic acids, such as DNA or RNA, whether obtained from biological samples or synthesized in vitro or de novo. The magnetic clusters of the present invention are particularly suitable for separating or purifying or enriching gDNA, cell-free nucleic acids (cfDNA, cfRNA), total RNA, mRNA, microRNA (miRNA), long non-coding RNA (IncRNA) or circular RNA (circRNA).

[0191] The nucleic acids obtained from the method may be further processed (e.g., by PCR) or analyzed, e.g., to detect infectious agents as described above. The step of analyzing the nucleic acids may use any nucleic acid analysis. For example, the nucleic acids may be analyzed by mass spectrometry or their sequence determined (e.g., by nucleic acid sequencing), although actual sequence determination may not be required - any method of analyzing sequence may be used.

[0192] The particles described herein can also be used to separate, purify, concentrate or remove a variety of different targets from a given sample, including cells, organelles, exosomes, vesicles, apoptotic bodies, liposomes, viruses, bacteria, parasites, plant cells, etc. Depending on the target, various types of samples can be used. For example, the sample can be derived from various sources, such as blood (whole blood, plasma, serum), bone marrow, tissue, feces, urine, tears, saliva, cell culture, perilymph or cerebrospinal fluid, milk water, sewage, etc. In addition, biological samples can be obtained from different species, such as microorganisms, viruses, plants, mammals or humans.

[0193] In some cases, the target to be separated, purified, concentrated or consumed from the sample can be one or more cells. In some cases, the cell can be a cancer cell, CTC, stem cell, neuron, immune cell, endothelial cell, epithelial cell or organ-specific cell (β cell, hepatocyte, glial cell). In some examples, immune cells such as T cells, B cells or natural killer cells.

[0194] In some embodiments, as described in WO2022 / 081519A1, the particles of the present invention can be used in a magnetic particle processing system for use with biological cells. For example, such a system can be used to attach magnetic clusters to biological cells in a sample and / or to separate magnetic beads attached to cells from a sample.

[0195] In some embodiments, the magnetic clusters may be coated with an antibody having binding affinity for a protein selected from the group consisting of CD3, CD4, CD5, CD6, CD8, CD25, CD27, CD28, CD137, and CD278, or a protein comprising CD3 or CD28.

[0196] In some embodiments, the cells to be separated or removed from the sample can be T cells, wherein the cells are attached to the magnetic particles by antigen-antibody interactions. The cells can then be collected or removed from the sample (e.g., by magnetic forces), and then separated from the magnetic particles by disrupting the antigen-antibody interactions (e.g., by cleaving the antibodies and separating the antibodies from the particles).

[0197] In some embodiments, the antibody can be attached to the magnetic particle via a ligand. The bound cells can then be separated from the magnetic particle by disrupting the interaction of the ligand with the antibody or the magnetic particle.

[0198] Cells isolated by using the particles of the present invention can be used for a variety of downstream analyses or applications, such as single cell analysis (scRNAseq) or CAR-T cell-based applications.

[0199] In other embodiments, the magnetic clusters described herein can be used for mRNA in vitro transcription and / or purification methods. For example, a first type of magnetic cluster (e.g., functionalized with streptavidin) can be used to fix a template (e.g., a biotinylated DNA template) for in vitro transcription. Then, a second type of magnetic cluster (e.g., coated with carboxylic acid) can be used to capture or purify in vitro transcribed mRNA. Such uses are described in, for example, WO 2022 / 101304 A1.

[0200] In some examples, a method for producing purified mRNA molecules may include: (a) fixing a first magnetic particle in place by a magnetic field, wherein an in vitro transcription (IVT) template is attached to the first magnetic particle; (b) contacting the first magnetic particle of step (a) with a reagent mixture suitable for template IVT under conditions where IVT occurs, thereby producing mRNA molecules, and (c) separating the mRNA molecules from the first magnetic particle, thereby producing purified mRNA molecules. The method may also include: (d) contacting the purified RNA molecules of step (c) with second magnetic particles under conditions that allow the purified mRNA molecules to remain bound to the second magnetic beads during washing, (e) washing the second magnetic beads while the second magnetic beads are fixed in place by a magnetic field, and (f) releasing the purified RNA molecules from the binding to the second magnetic beads, thereby producing highly purified RNA molecules. In some cases, the IVT template can be produced by polymerase chain reaction (PCR), optionally wherein one or more biotinylated primers are used in the PCR, and resulting in the formation of a biotinylated IVT template. The biotinylated IVT template can be attached to the magnetic particle by the interaction between the biotin of the biotinylated IVT template and the group on the magnetic particle that has affinity for biotin. In addition, in many cases, the IVT template may comprise an open reading frame encoding a protein and a promoter operably linked to the open reading frame. In some embodiments, free carboxylic acid groups may be present on the surface of the second magnetic particle. The purified mRNA molecules may encode proteins or pathogens, such as, for example, viral proteins. The RNA molecules produced and / or purified using the particles of the present invention can be used in various types of applications, such as vaccine compositions.

[0201] In additional embodiments, the magnetic clusters described herein can be used to separate organelles, vesicles, virions, virus-like particles or exosomes from a sample based on the attachment of antibody or ligand functionalized particles to surface markers. Such targets can be derived from living organisms, or can be grown in / derived from cell culture.

[0202] For example, surface markers that can be used for separation, purification or classification of exosomes or vesicles include, but are not limited to, tumor markers and MHC class II markers. Class II markers that have been associated with exosomes include HLA DP, DQ and DR haplotypes. Other surface markers associated with exosomes include CD9, CD81, CD63 and CD82 (Thery et al., Nat. Rev. Immunol. 2 (2002) 569-579; Valadi et al., Nat. Cell. Biol.). 9 (2007) 654-659. Using magnetic particles that form affinity connections with CD3 receptors, CD4 receptors and / or CD8 receptors, vesicles (e.g., exosomes) generated by T cells can be separated from other biological materials. Antibody-coated magnetic clusters can be added to samples containing exosomes and incubated at 2°C-8°C or room temperature for 5 minutes to overnight. Particles bound to exosomes can then be collected with a magnet. The isolated particle-bound exosomes can then be resuspended in a suitable buffer, such as phosphate buffered saline, and used for downstream analysis (qRT-PCR, sequencing, Westerns, flow cytometry, etc.). Similar protocols can be used for any other surface marker on exosomes or other vesicles for which antibodies or other specific ligands are available. Indirect binding methods, such as those using biotin-avidin, can also be used. Once an isolated exosome sample has been prepared, the contents of the exosomes can be extracted for study and characterization. Biological materials that can be extracted from exosomes include proteins, peptides, RNA and DNA, lipids. For example, mirVana TM The PARIS kit (AM 1556, Life Technologies) can be used to recover native protein and RNA species, including small RNAs such as miRNA, snRNA, and snoRNA, from exosomes.

[0203] In additional embodiments, the magnetic clusters described herein can be used to separate peptides, proteins, or other macromolecules.

[0204] Proteins purified by methods using magnetic clusters can also be bound to affinity reagents that bind to naturally occurring regions of the protein or exogenously added tags. In addition to examples such as protein A binding antibodies, such methods include methods in which antibodies specific to the protein being purified are attached to particles. For example, when the protein is an antibody (e.g., IgG, IgA, IgD, IgM, IgE, etc.) or a mixture of antibodies (e.g., IgG antibodies present in serum, etc.), the ligand can be protein A, protein G, protein L, or one or more functional variants of one or more of these proteins.

[0205] Therefore, protein purification methods can also be based on the binding of exogenously added affinity tags. This means that affinity tags are not usually present in naturally occurring proteins. Exemplary tags and binding partners that may be present in the composition and used for the methods described herein include maltose binding protein (MBP) / amylose, glutathione-S-transferase (GST) / glutathione tags, polyhistidine (His) / metal ions (e.g., copper and cobalt), streptavidin / biotin (e.g., N-ethyl-biotin) and antigen-antibody reaction (epitope) tags (e.g., c-Myc tags / anti-c-Myc antibodies, FLAG / anti-FLAG antibodies and hemagglutinin (HA) tags / anti-HA antibodies). The methods in the protein purification workflow and other workflows may vary greatly, but in some cases, the magnetic particles described herein can be kept in place by a magnetic field and then contacted with a protein binding partner, or can be contacted with a protein binding partner and then kept in place by a magnetic field. In both cases, the carrier can be kept in place and washed. It may be desirable to release the protein from the particle, for example, after the protein has been separated from other materials (e.g., cell debris). The process by which the protein is released from the particle will be determined by the nature of the protein and / or the binding between the protein and the carrier. The release of the protein from the magnetic particle can be mediated by the use of a protease cleavage within the protein or between the protein and an exogenously added tag. Exemplary proteases that can be used include rhinovirus 3C protease, TVMV protease, plum poxvirus protease, turnip mosaic virus protease, tobacco etch virus (TEV) protease, thrombin, factor Xa, and enteropeptidase.

[0206] One protein purification method that allows purification and release of proteins from supports uses biotin and biotin derivatives (e.g., biotin, desthiobiotin, N-ethyl-biotin) and biotin binding proteins. Suitable biotin derivatives are described, for example, in U.S. Pat. No. 9,567,346.

[0207] In some cases, antibodies or other proteins with ligand binding activity are associated with magnetic particles via low affinity biotin derivatives (e.g., desthiobiotin, N-ethylbiotin, etc.) / biotin binding protein (e.g., avidin, streptavidin, neutravidin, etc.) association. Release of the low affinity biotin derivative from the biotin binding protein is mediated by competition with high affinity biotin (e.g., d-biotin).

[0208] Cell capture and release methods that can be used for protein purification as well as cells, viruses, virus-like particles, and other biomolecules are those that use CAPTURESELECT TM N-ethyl biotin (NEB) anti-CD4 conjugate (Thermo Fisher Scientific, catalog number 7113762100) and CAPTURESELECT TM N-ethyl biotin (NEB) anti-CD8 conjugate (Thermo Fisher Scientific, catalog number 7113772100). In this case, the biotinylated anti-CD4 antibody or anti-CD8 antibody is bound to the magnetic particles through biotin / streptavidin binding. In summary, the method of using the magnetic clusters of the present invention can be described as follows: First, the anti-CD4 antibody or anti-CD8 antibody biotinylated by NEB is contacted with the magnetic particles coated with streptavidin. Then, under conditions that allow T cells to bind by NEB / streptavidin, the particles are contacted with CD4+ or CD8+T cells. After a short period of incubation, the particles are washed. In many cases, the particles are fixed by a magnetic field during washing. After washing, the particles are contacted with a release reagent containing d-biotin. The supernatant containing CD4+ or CD8+T cells is then removed. In many cases, the particles are fixed by a magnetic field during washing. The result is a purified population of CD4+T or CD8+T cells with little or no particles present.

[0209] Viruses and virus-like particles can be separated by binding to carriers (e.g., magnetic beads). The manner in which viruses and virus-like particles bind to carriers can vary depending on the structure of the individual viruses and virus-like particles. For example, adeno-associated virus (AAV) is non-enveloped, and antibodies with binding affinity to AAV capsid proteins have been developed. In addition, AAV capsid variations (serotypes) are known and result in different AAV serotypes having different cell and tissue specificities.

[0210] Envelope viruses and virus-like particles can also be purified using the particles provided herein. Envelope viruses and virus-like particles, as well as cells and exosomes can be purified, for example, by using affinity reagents. As part of these methods, enveloped viruses and virus-like particles can be purified based on the binding of ligands to proteins present in the envelope of the virus and virus-like particles that people seek to purify. An example of such a method is in "Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell" by Mekkaoui et al., Mol. Ther. Methods Clin. Dev., 11: 155-165 (2018).

[0211] In other embodiments, magnetic clusters as described herein can be used to selectively remove small molecules, analyte cations, anions, ions or environmental toxins (such as selenium) from drainage or other samples. For such uses, magnetic clusters can be functionalized with certain coatings as described above. For example, dextran, sugar, PEG, PEG-OH, other modified PEG moieties, polyvinyl alcohol, gold, azide, carboxyl, activated carbon, zeolite, amine, polyacrylic acid, charged polymers or other can be used as surface functionalization.

[0212] Reagent test kit

[0213] The magnetic clusters of the invention may be provided as part of a kit. For example, where the magnetic clusters of the invention are intended for an assay, it may be convenient to provide a kit comprising the magnetic clusters.

[0214] Thus, one embodiment of the present invention provides a kit comprising the magnetic cluster of the present invention.

[0215] The kit may include one or more buffers. The buffer may include a lysis buffer, and / or a binding buffer, and / or a washing buffer, and / or an elution buffer. As will be appreciated by the skilled person, the specific composition of a given buffer will depend on the assay and the target analyte. For example, in the case where the assay includes nucleic acid separation, the buffer may include a lysis buffer (e.g., an exemplary lysis solution described herein), and / or a nucleic acid binding buffer, and / or a washing buffer, and / or an elution buffer.

[0216] The kit may include a magnet for isolating the magnetic clusters.

[0217] The kit may also include instructions. "Instructions" are printed materials describing how to use the kit, for example, methods for preparing reagents, recommended preparation conditions, etc. Instructions for use include instructions affixed to the kit label, the kit packaging, etc., and operating manuals in the form of brochures or leaflets. In addition, instructions include information disclosed or provided through electronic media such as the Internet.

[0218] Also provided are kits comprising components for use in methods of isolating nucleic acids from biological samples, such as lysis buffer and silica-coated magnetic particles.

[0219] Additional embodiments

[0220] This disclosure includes implementations of the following numbered clauses:

[0221] 1. A magnetic cluster, wherein the magnetic cluster comprises nanocrystals of iron oxide and an aromatic carboxylic acid.

[0222] 2. The magnetic cluster according to clause 1, wherein the aromatic carboxylic acid is a compound of formula (I):

[0223]

[0224] in:

[0225] L 1 Select from bonds, -C 1-6 Alkyl-, or -C 2-6 alkenyl-;

[0226] R 1 Selected from -H, -COOH, -(C 1-5 Alkyl)COOH, -(C 2-5 alkenyl)COOH or -C 1-4 Alkoxy;

[0227] R 2 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy;

[0228] R 3 Selected from -H, -halo, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogen (C 1-6 )alkyl, -NR 6 R 7 or substituted or unsubstituted phenyl;

[0229] R 4 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy;

[0230] R 5 Select from -H or -C 1-4 Alkoxy;

[0231] R 6 Select from -H or -C 1-4 Alkyl; and

[0232] R 7 Selected from -H, -C 1-4 Alkyl, -C(O)H, -C(O)C 1-4 alkyl.

[0233] 3. The magnetic cluster according to clause 2, wherein L 1 Select from the key, -C 1-4 Alkyl-, or -C 2-4 Alkenyl-.

[0234] 4. The magnetic cluster according to clause 2 or clause 3, wherein L 1 is the key.

[0235] 5. The magnetic cluster according to clause 2, wherein L 1 Selected from -C 1-4 Alkyl or -C 2-4 alkenyl; optionally wherein L 1 Yes-C 2-4 Alkenyl-.

[0236] 6. A magnetic cluster according to any one of clauses 2 to 5, wherein R 1 Selected from -H, -COOH, -(C 1-5 alkyl)COOH or -(C 2-5 alkenyl)COOH.

[0237] 7. A magnetic cluster according to any one of clauses 2 to 6, wherein R 1 Selected from -H or -COOH.

[0238] 8. A magnetic cluster according to any one of clauses 2 to 7, wherein R 2 Select from -H or -C 1-4 Alkoxy.

[0239] 9. A magnetic cluster according to any one of clauses 2 to 8, wherein R 2 Selected from -H or -OCH3.

[0240] 10. The magnetic cluster according to any one of clauses 2 to 9, wherein R 3 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy, -NR 6 R 7or substituted or unsubstituted phenyl;

[0241] Optionally, R 3 Selected from -C 1-4 Alkoxy, -NR 6 R 7 or a substituted or unsubstituted phenyl group.

[0242] 11. A magnetic cluster according to any one of clauses 2 to 10, wherein R 6 is -H or -CH3, and / or wherein R 7 It is -CH3 or -C(O)CH3.

[0243] 12. A magnetic cluster according to any one of clauses 2 to 11, wherein R 3 Selected from -H, -C 1-4 alkyl 、 -OCH3, -N(CH3)2, -NHC(O)CH3 or phenyl.

[0244] Optionally, R 3 Selected from -OCH3, -N(CH3)2, -NHC(O)CH3 or phenyl.

[0245] 13. A magnetic cluster according to any one of clauses 2 to 12, wherein R 4 Select from -H or -C 1-4 Alkoxy.

[0246] 14. A magnetic cluster according to any one of clauses 2 to 13, wherein R 4 Selected from -H or -OCH3.

[0247] 15. A magnetic cluster according to any one of clauses 2 to 14, wherein R 5 Yes -H.

[0248] 16. A magnetic cluster according to any one of clauses 2 to 16, wherein R 1 and R 5 Both are -H.

[0249] 17. A magnetic cluster according to any one of clauses 2 to 16, wherein R 3 Yes -OH, R 2 Yes-C 1-4 Alkoxy, and R 3 Yes-C 1-4 Alkoxy.

[0250] 18. A magnetic cluster according to any of the preceding clauses, wherein the aromatic carboxylic acid is selected from benzoic acid, toluic acid (e.g., p-toluic acid), 4-ethylbenzoic acid, 3,5-dimethylbenzoic acid, 4-tert-butylbenzoic acid, p-anisic acid, 3,4,5-trimethoxybenzoic acid (eucalyptol), trans-cinnamic acid, phthalic acid, homophthalic acid, 3-phenylpropionic acid, 4-(dimethylamino)benzoic acid, 4-acetamidobenzoic acid, and bisphenyl-4-carboxylic acid.

[0251] 19. A magnetic cluster according to any preceding clause, wherein the magnetic cluster is monodisperse.

[0252] 20. The magnetic cluster according to any preceding clause, wherein the magnetic cluster has a mode diameter in the range of about 100 nm to about 2,500 nm;

[0253] Optionally wherein the magnetic clusters have a mode diameter in the range of about 200 nm to about 1,000 nm.

[0254] 21. Monodisperse magnetic clusters comprising nanocrystals of iron oxide, wherein the magnetic clusters have a mode diameter in the range of about 100 nm to about 2,500 nm and a coefficient of variation (CV) of no more than 5% when measured by a disc centrifuge.

[0255] 22. The monodisperse magnetic cluster according to clause 21, wherein the magnetic cluster further comprises an aromatic carboxylic acid, optionally wherein the aromatic carboxylic acid is according to any one of clauses 2 to 18.

[0256] 23. A magnetic cluster according to any one of clauses 1 to 19, or a monodisperse magnetic cluster according to clause 21 or clause 22, wherein the nanocrystals of iron oxide comprise a mode average diameter of about 5 nm to about 20 nm, optionally wherein the nanocrystals have a mode average diameter of about 10 nm to about 15 nm, further optionally wherein the nanocrystals have a mode average diameter of about 12 nm.

[0257] 24. The magnetic cluster according to any one of clauses 1 to 19 or 23, or the monodisperse magnetic cluster according to any one of clauses 21 to 23, further comprising a coating.

[0258] 25. The magnetic cluster or monodisperse magnetic cluster according to clause 24, wherein the coating comprises at least one layer of silicon dioxide.

[0259] 26. A magnetic cluster or monodisperse magnetic cluster according to clause 23 or clause 24, wherein the coating comprises at least one organic coating, optionally wherein the at least one organic coating is overcoated with at least one layer of silicon dioxide.

[0260] 27. The magnetic cluster or monodisperse magnetic cluster according to any one of clauses 23 to 26, wherein the coating comprises functional groups;

[0261] Optionally wherein the functional group is selected from a hydroxyl group, a carboxylic acid group, an aldehyde group, an amine group, a thiol group, an epoxy group, an amide group, a chloromethyl group and a tosyl-activated group;

[0262] Further optionally wherein the functional group is selected from hydroxyl, carboxylic acid (—COOH), primary amine, secondary amine or epoxy; further optionally wherein the functional group is selected from carboxylic acid (—COOH), primary amine, secondary amine or epoxy.

[0263] 28. A method for preparing a magnetic cluster, the method comprising:

[0264] forming a solution comprising iron (III) ions, a protic solvent, an aromatic carboxylic acid, and a precipitant; and

[0265] The solution is reacted to form a suspension comprising magnetic clusters.

[0266] 29. A method according to clause 28, wherein the iron (III) ions are provided as a soluble iron (III) salt, optionally wherein the soluble iron (III) salt is an iron (III) halide, such as an iron (III) chloride salt.

[0267] 30. The process according to clause 28 or clause 29, wherein the protic solvent is an alcohol, optionally a diol or a polyol, further optionally wherein the alcohol is ethylene glycol.

[0268] 31. The process according to any one of clauses 28 to 30, wherein the aromatic carboxylic acid is according to any one of clauses 2 to 18.

[0269] 32. A method according to any one of clauses 28 to 31 wherein the precipitating agent is an acetate salt, optionally sodium acetate.

[0270] 33. A method according to any one of clauses 28 to 32. wherein reacting the solution comprises

[0271] One or more of the following conditions:

[0272] (a) elevated temperature, optionally 150°C to 250°C; and / or

[0273] (b) a pressure of about 1 bar to about 15 bar; and / or

[0274] (c) a reaction time of about 6 hours to about 36 hours.

[0275] 34. The method according to any one of clauses 28 to 33, further comprising separating the magnetic clusters from the suspension.

[0276] 35. The method according to clause 34, further comprising washing the magnetic clusters.

[0277] 36. The method according to clause 34 or clause 35, further comprising adding a silica coating to the separated magnetic clusters, comprising:

[0278] forming a suspension comprising the separated magnetic clusters and a silicate or orthosilicate; and

[0279] The silicate or the orthosilicate is reacted to form a silicon dioxide coating.

[0280] 37. The method of clause 36, wherein reacting the silicate to form a silica coating comprises lowering the pH of the suspension to less than 11.

[0281] 38. The method of clause 36, wherein reacting the orthosilicate to form a silica coating comprises raising the temperature of the suspension to above 40°C, optionally to above 60°C or 90°C.

[0282] 39. The method according to any one of clauses 36 to 38, further comprising overcoating the silica coating with an organic coating, optionally wherein the organic coating comprises a spacer and / or a polymer;

[0283] 40. The method according to clause 39, wherein the coating is carried out in the presence of a water-miscible solvent, optionally wherein the water-miscible solvent is Cyrene TM .

[0284] 41. A method according to any one of clauses 28 to 40, wherein the coating comprises functional groups;

[0285] Optionally wherein the functional group is selected from a hydroxyl group, a carboxylic acid group, an aldehyde group, an amine group, a thiol group, an epoxy group, an amide group, a chloromethyl group and a tosyl-activated group;

[0286] Further optionally wherein the functional group is selected from hydroxyl, carboxylic acid (—COOH), primary amine, secondary amine or epoxy; further optionally wherein the functional group is selected from carboxylic acid (—COOH), primary amine, secondary amine or epoxy.

[0287] 42. A method according to any one of clauses 28 to 41, wherein the magnetic clusters comprise nanocrystals comprising iron oxide having an average diameter of about 5 nm to about 40 nm, optionally wherein the nanocrystals have a diameter of about 5 nm to about 20 nm, further optionally wherein the nanocrystals have a diameter of about 10 nm to about 15 nm, still further optionally wherein the nanocrystals have a diameter of about 12 nm.

[0288] 43. A method according to any one of clauses 28 to 42, wherein the magnetic clusters are monodisperse.

[0289] 44. A method according to any one of clauses 28 to 43, wherein the magnetic clusters have a diameter in the range of about 100 nm to about 2,500 nm;

[0290] Optionally wherein the magnetic clusters have a diameter in the range of about 200 nm to about 1,000 nm.

[0291] 45. Magnetic clusters obtainable by a method according to any one of clauses 28 to 44.

[0292] 46. ​​Use of a magnetic cluster according to any one of clauses 1 to 27 or according to clause 45 in an assay.

[0293] 47. Use of a magnetic cluster according to any one of clauses 1 to 27 or according to clause 45 for separating, purifying, fractionating, depleting, concentrating and / or analyzing cells, viruses, vesicles, organelles, exosomes, small molecules, analyte cations, anions, ions, toxins, pathogens, nucleic acids, proteins or peptides.

[0294] 48. Use according to clause 46 or 47, wherein the magnetic cluster comprises a (surface) capture ligand, optionally wherein the capture ligand is selected from the group consisting of: an antibody, an antibody fragment, a peptide, a carbohydrate, a hapten, an aptamer and an oligonucleotide.

[0295] 49. Use according to clause 46 or 47, wherein the magnetic cluster comprises a surface functionalization for capturing a class of molecules, optionally wherein the surface functionalization comprises ionic groups or hydrophobic groups.

[0296] 50. Use according to any one of clauses 46 or 48 to 49, wherein the assay comprises immunoprecipitation, nucleic acid capture, immunoassay and / or lateral flow assay.

[0297] 51. A kit, comprising:

[0298] A magnetic cluster according to any one of clauses 1 to 27, or according to clause 45.

[0299] 52. The kit according to clause 51, further comprising one or more buffers.

[0300] 53. A kit according to clause 52, wherein the one or more buffers comprise a lysis and / or binding buffer and optionally a wash buffer and / or an elution buffer.

[0301] 54. A kit according to clause 53, wherein the lysis and / or binding buffer may contain one or more of a chaotropic agent, a precipitating agent such as an alcohol or ethylene glycol.

[0302] The following numbered clauses provide additional implementation options:

[0303] 1. A magnetic cluster, wherein the magnetic cluster comprises nanocrystals of iron oxide and an aromatic carboxylic acid.

[0304] 2. The magnetic cluster according to clause 1, wherein the aromatic carboxylic acid is a compound of formula (I):

[0305]

[0306] in:

[0307] L 1 Select from bonds, -C 1-6 Alkyl-, or -C 2-6 alkenyl-;

[0308] R 1 Selected from -H, -COOH, -(C 1-5 Alkyl)COOH, -(C 2-5 alkenyl)COOH or -C 1-4 Alkoxy;

[0309] R 2 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy;

[0310] R 3 Selected from -H, -halo, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogen (C 1-6 )alkyl, -NR 6 R 7 or substituted or unsubstituted phenyl;

[0311] R 4 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy;

[0312] R 5Select from -H or -C 1-4 Alkoxy;

[0313] R 6 Select from -H or -C 1-4 Alkyl; and

[0314] R 7 Selected from -H, -C 1-4 Alkyl, -C(O)H, -C(O)C 1-4 alkyl.

[0315] 3. The magnetic cluster according to clause 2, wherein L 1 Select from the key, -C 1-4 Alkyl-, or -C 2-4 alkenyl-;

[0316] Optionally, L 1 Select from bond or -C 2-4 Alkenyl-.

[0317] 4. The magnetic cluster according to clause 2 or clause 3, wherein R 1 Selected from -H, -COOH, -(C 1-5 alkyl)COOH or -(C 2-5 alkenyl)COOH;

[0318] Optionally, R 1 Selected from -H or -COOH.

[0319] 5. The magnetic cluster according to any one of clauses 2 to 4, wherein R 2 Select from -H or -C 1-4 Alkoxy;

[0320] Optionally, R 2 Selected from -H or -OCH3.

[0321] 6. A magnetic cluster according to any one of clauses 2 to 5, wherein R 3 Selected from -H, -halo, -C 1-4 Alkyl, -C 1-4 Alkoxy, -halogen (C 1-4 )alkyl, -NR 6 R 7 or substituted or unsubstituted phenyl;

[0322] Optionally, R 3 Selected from -C 1-4 Alkoxy, -halogen (C 1-4 )alkyl, -NR 6 R 7 or a substituted or unsubstituted phenyl group.

[0323] 7. A magnetic cluster according to any one of clauses 2 to 6, wherein R 6 is -H or -CH3, and / or wherein R 7 It is -CH3 or -C(O)CH3.

[0324] 8. A magnetic cluster according to any one of clauses 2 to 7, wherein R 4 Select from -H or -C 1-4 Alkoxy;

[0325] Optionally, R 4 Selected from -H or -OCH3.

[0326] 9. A magnetic cluster according to any one of clauses 2 to 8, wherein R 5 is -H; and / or

[0327] Where R 1 and R 5 are all -H; and / or

[0328] Where R 3 Yes -OH, R 2 Yes-C 1-4 Alkoxy, and R 3 Yes-C 1-4 Alkoxy.

[0329] 10. A magnetic cluster according to any preceding clause, wherein the aromatic carboxylic acid is selected from benzoic acid, toluic acid, 4-ethylbenzoic acid, 3,5-dimethylbenzoic acid, 4-tert-butylbenzoic acid, p-anisic acid, 3,4,5-trimethoxybenzoic acid (eucalyptol), trans-cinnamic acid, phthalic acid, homophthalic acid, 4-(trifluoromethyl)benzoic acid, 4-iodobenzoic acid, 4-chlorobenzoic acid, 3-phenylpropionic acid, 4-(dimethylamino)benzoic acid, 4-acetamidobenzoic acid, and bisphenyl-4-carboxylic acid.

[0330] 11. A magnetic cluster according to any preceding clause, wherein the magnetic cluster is monodisperse.

[0331] 12. A magnetic cluster according to any preceding clause, wherein the magnetic cluster has a mode diameter in the range of about 100 nm to about 2,500 nm;

[0332] Optionally wherein the magnetic clusters have a mode diameter in the range of about 200 nm to about 1,000 nm.

[0333] 13. Monodisperse magnetic clusters comprising nanocrystals of iron oxide, wherein the magnetic clusters have a mode diameter in the range of about 100 nm to about 2,500 nm and a coefficient of variation (CV) of no more than 5% when measured by a disc centrifuge.

[0334] 14. The monodisperse magnetic cluster according to clause 13, wherein the magnetic cluster further comprises an aromatic carboxylic acid, optionally wherein the aromatic carboxylic acid is according to any one of clauses 2 to 12.

[0335] 15. A magnetic cluster according to any one of clauses 1 to 12, or a monodisperse magnetic cluster according to clause 13 or clause 14, wherein the nanocrystals of iron oxide comprise a mode average diameter of about 5 nm to about 20 nm, optionally wherein the nanocrystals have a mode average diameter of about 10 nm to about 15 nm, further optionally wherein the nanocrystals have a mode average diameter of about 12 nm.

[0336] 16. The magnetic cluster according to any one of clauses 1 to 12 or 15, or the monodisperse magnetic cluster according to any one of clauses 13 to 16, further comprising a coating.

[0337] 17. The magnetic cluster or monodisperse magnetic cluster according to clause 16, wherein the coating comprises at least one layer of silicon dioxide; and / or

[0338] The coating comprises at least one organic coating, and optionally the at least one organic coating is coated with at least one layer of silicon dioxide.

[0339] 18. The magnetic cluster or monodisperse magnetic cluster according to clause 16 or clause 17, wherein the coating comprises functional groups;

[0340] Optionally wherein the functional group is selected from a hydroxyl group, a carboxylic acid group, an aldehyde group, an amine group, a thiol group, an epoxy group, an amide group, a chloromethyl group and a tosyl-activated group;

[0341] Further optionally wherein the functional group is selected from hydroxyl, carboxylic acid (—COOH), primary amine, secondary amine or epoxy; further optionally wherein the functional group is selected from carboxylic acid (—COOH), primary amine, secondary amine or epoxy.

[0342] 19. A method for preparing a magnetic cluster, the method comprising:

[0343] forming a solution comprising iron (III) ions, a protic solvent, an aromatic carboxylic acid, and a precipitant; and

[0344] The solution is reacted to form a suspension comprising magnetic clusters.

[0345] 20. The process according to clause 19, wherein the protic solvent is an alcohol, optionally a diol or a polyol, further optionally wherein the alcohol is ethylene glycol.

[0346] 21. The process according to clause 19 or clause 20, wherein the aromatic carboxylic acid is according to any one of clauses 2 to 10.

[0347] 22. The method according to any one of clauses 19 to 21, wherein the precipitating agent is an acetate salt, optionally sodium acetate.

[0348] 23. The method according to any one of clauses 19 to 22, further comprising separating the magnetic clusters from the suspension;

[0349] Optionally, the method further comprises washing the magnetic clusters.

[0350] 24. The method according to clause 23, further comprising adding a silica coating to the separated magnetic clusters, comprising:

[0351] forming a suspension comprising the separated magnetic clusters and a silicate or orthosilicate; and

[0352] The silicate or the orthosilicate is reacted to form a silicon dioxide coating.

[0353] 25. The method according to clause 24, further comprising overcoating the silica coating with an organic coating, optionally wherein the organic coating comprises a spacer and / or a polymer;

[0354] 26. A method according to any one of clauses 19 to 25, wherein the coating comprises functional groups;

[0355] Optionally wherein the functional group is selected from a hydroxyl group, a carboxylic acid group, an aldehyde group, an amine group, a thiol group, an epoxy group, an amide group, a chloromethyl group and a tosyl-activated group;

[0356] Further optionally wherein the functional group is selected from hydroxyl, carboxylic acid (—COOH), primary amine, secondary amine or epoxy; further optionally wherein the functional group is selected from carboxylic acid (—COOH), primary amine, secondary amine or epoxy.

[0357] 27. A method according to any one of clauses 19 to 26, wherein the magnetic clusters are monodisperse; and / or

[0358] wherein the magnetic clusters have a diameter in the range of about 100 nm to about 2,500 nm;

[0359] Optionally wherein the magnetic clusters have a diameter in the range of about 200 nm to about 1,000 nm.

[0360] 28. Use of a magnetic cluster according to any one of clauses 1 to 18 in an assay.

[0361] 29. The use according to 28, wherein the magnetic cluster comprises a (surface) capture ligand, optionally wherein the capture ligand is selected from the group consisting of: an antibody, an antibody fragment, a peptide, a carbohydrate, a hapten, an aptamer and an oligonucleotide; and / or

[0362] wherein the magnetic cluster comprises surface functionalization for capturing a class of molecules, optionally wherein the surface functionalization comprises ionic groups or hydrophobic groups; and / or

[0363] Wherein the assay comprises immunoprecipitation, nucleic acid capture, immunoassay and / or lateral flow assay.

[0364] 30. A kit, comprising:

[0365] A magnetic cluster according to any one of clauses 1 to 18.

[0366] 31. The kit according to clause 30, further comprising one or more buffers;

[0367] Optionally wherein the one or more buffers comprise a lysis and / or binding buffer, and optionally a wash buffer and / or an elution buffer.

[0368] Analytical methods

[0369] Particle size and CV measurement using CPS disc centrifuge

[0370] The size distribution of a sample can be measured using disc centrifugation, using the protocol provided by the instrument manufacturer, such as the CPSDisc Centrifugation on a disc centrifuge model DC20000. TM Accurate results require calibration using standards of similar density to the sample being analyzed, for example a set of compact polystyrene particle standards can be used for particles consisting primarily of polystyrene. If the density of the sample being measured is unknown, or a similar density standard is not available, the measurements obtained by CPS disk centrifugation are reproducible and therefore can be used to determine the CV, but will not provide the actual diameter.

[0371] The CPS disk centrifugation methods used in the examples provided herein are summarized below. Those skilled in the art will be readily able to adapt these methods appropriately, for example by selecting appropriate gradients, disk speeds, and standard particles based on the size of the particles to be analyzed.

[0372] Disc centrifuge analysis was performed on a CPSDC20000 from CPS instruments with a disc speed of 5000 rpm and a gradient of 3 wt% to 7 wt% sucrose in trisodium citrate dihydrate (Na3Cit·2H2O) solution (2 mM in water). The gradient was formed using an Auto Gradient pump from CPS instruments and the volume of the injected gradient was 16 mL to 17 mL. Prior to injection, the sample was diluted to about 0.01 wt% (or as low as 0.0025 wt% to reduce secondary peak artifacts) in nanogram-H2O. The method used for analysis had the following settings: maximum diameter 3.0 μm, minimum diameter 0.2 μm, particle density 3.5 g / mL, particle refractive index 2.344, particle absorbance 0.13, particle non-sphericity 1, calibration standard diameter 1.099 g / mL, calibration standard density 1.6, standard semi-absorbent 0.2 g / mL, liquid density 1.016 g / mL, and liquid refractive index 1.343.

[0373] We have observed that insufficient sample dilution may result in secondary peak artifacts that appear as apparently larger particle sizes due to agglomeration of two or more (e.g., 2, 3, 4, or more) particles during disc centrifuge analysis. Such artifacts may artificially increase the CV and / or particle size determined by the disc centrifuge analysis. Therefore, in cases where secondary peak artifacts are suspected, it is preferred to perform the disc centrifuge analysis at a relatively high sample dilution, for example, the sample may be diluted to as low as about 0.0025 wt%.

[0374] Suitable particles for use as disc centrifuge standards for the particles of the present invention are cross-linked polystyrene particles. For example, in some of the examples provided herein, the standard particles used are magnetizable porous 1 micron polystyrene particles cross-linked with 50% divinylbenzene and coated with a cross-linked hydrophilic polyether. The density of the standard particles in water is about 1.6 g / cm 3 .

[0375] The reported sizes are based on the mode absorption peak diameter. The CV is determined by taking the upper and lower diameters of the mode peak at half height (also called half height width), dividing by the mode peak diameter, and multiplying by 100 to obtain the CV (%).

[0376] Using the mode peak at its half-height to determine the CV can be advantageous because it reduces the risk of secondary or tertiary peak artifacts influencing the CV determination. Such artifacts can be caused by the aggregation of a few particles during the disc centrifuge analysis.

[0377] Measurement of aggregation and agglomeration degree, size and morphology of magnetic clusters using optical microscopy

[0378] Qualitative characterization of magnetic clusters can be performed by optical microscopy. Images obtained with optical microscopy can be used to study particle aggregation and agglomeration level (AL) and give an indication of size. However, the resolution of optical microscopy is not high enough to provide good images of particles with a size / diameter less than 1 μm.

[0379] Bulk magnetic clusters (or other particles) as FW suspensions (undiluted or further diluted in H2O) imaged at 1.6x, 10x, 20x, and 40x (100x) magnification. Aggregation and agglomeration levels of these batches are shown in Figure 2. Figure 2 The 5-level scale shown is visually inspected and characterized, and exemplary results for these batches are given in Figure 3 The scale bar at the bottom right of each individual figure represents 50 μm.

[0380] Cluster size measurement by scanning electron microscopy (SEM)

[0381] Magnetic clusters, including those with a size / diameter below 1 micron, can be imaged by SEM to obtain high resolution images for precise determination of the diameter of the particles.

[0382] A suitable SEM analysis method is as follows. The magnetic clusters may be provided as an aqueous suspension, for example with a particle dry content of approximately 5%, or as vacuum dried particles. Prior to analysis, a drop of the aqueous suspension is deposited on a carbon tape and dried under a 100W warm lamp. Preferably, the particles (dried under vacuum) are trapped in an epoxy resin and then cured at 60°C for 1-2 days. SEM images may be taken using any suitable SEM instrument at an appropriate magnification. For analysis of the example particles, the SEM instrument was a Hitachi S-4800FEG at a V of 2kV. acc At a working distance of 2.8 mm, the magnification is between 5 000 and 100 000X.

[0383] The average size of particles in the image can be quantified in the following manner. The images were analyzed using the open platform image processing program "Fiji IsJust-Image J" (Schindelin, J. et al., (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676-682). The scale bar was used for calibration, and at least 200 particles were manually selected for each batch before calculating the number average and surface average size.

[0384] Measurement of magnetic properties

[0385] The magnetic properties of magnetic nanoclusters, such as remnant mass magnetization, saturation mass magnetization and initial magnetic mass susceptibility, can be measured according to the procedures explained in sections 5.2.6 and 5.2.7 of the standard Nanotechnology - Magnetic Nanomaterials Part 2, ISO / TS19807-2:2021(E).

[0386] Residual mass magnetization

[0387] The remnant mass magnetization of a magnetic cluster is proportional to the mechanical force acting on the cluster when it is in a gradient magnetic field with zero absolute magnetic field. Therefore, the remnant species magnetization affects the speed and efficiency of the extraction process in many bioassays, as well as the possible aggregation of beads. When solid magnetizable surfaces are present in the reaction environment, the remnant mass magnetization may also lead to unwanted aggregation of clusters on these surfaces in the absence of an external magnetic field.

[0388] For the measurement of the remnant mass magnetization, the magnetic clusters should be cleaned and dried in an oven. Their mass is determined by weighing. The magnetic moment of the dried cluster sample is measured using a suitable device, such as a superconducting quantum interference device (SQUID) or a vibrating sample magnetometer (VSM). During the measurement, the dried magnetic cluster sample is first exposed to a strong magnetic field in the range of the saturation magnetization field. Then, the magnetic field is monotonically changed to zero and the remnant magnetic moment of the sample is measured. The remnant mass magnetization is calculated as the ratio of the zero-field magnetic moment to the mass of the beads and is expressed in units of m 2 / kg report.

[0389] Saturation mass magnetization

[0390] The saturation mass magnetization of the magnetic clusters has an impact on the speed and efficiency of the extraction process during the assay and on possible agglomeration of the beads during the extraction process. This is an important feature of the magnetic behavior of the clusters and can be used to monitor the quality of the clusters.

[0391] To measure the saturation mass magnetization, the magnetic clusters should be cleaned and dried in an oven. Their mass is determined by weighing. Using a suitable device, such as a SQUID or VSM, the magnetic moment of the dried cluster sample is measured. During the measurement, the dried magnetic cluster sample is subjected to an enhanced magnetic field until the magnetic moment value no longer changes with the magnetic field enhancement. At this field strength, the magnetic moment of the sample is measured. The saturation mass magnetization is calculated as the ratio of the measured magnetic moment to the mass of the beads and is expressed in units of m 2 / kg report.

[0392] Initial magnetic mass susceptibility

[0393] Initial magnetic mass susceptibility is an important property of magnetic clusters used for assays such as biological assays that include at least one extraction step because it has a significant impact on extraction performance. In the absence of an external magnetic field, the net magnetization of the magnetic clusters is very small due to the random orientation of the magnetic moments of the nanoparticles within the cluster. However, when the external magnetic field is turned on, the magnetic moments of the nanoparticles within the cluster will obtain a preferential orientation, so the magnetic clusters will produce a net magnetization. The ratio between the change in magnetization intensity and the corresponding change in the magnetic field is called magnetic susceptibility. The magnetic susceptibility measured under a very small absolute magnetic field is called initial magnetic susceptibility.

[0394] Before the measurement, the magnetic clusters should be cleaned and dried in an oven. Their mass is determined by weighing. For the measurement of the initial magnetic susceptibility, the dry magnetic cluster sample should be demagnetized by a sufficiently small absolute magnetic field whose amplitude disappears over time. Then, the magnetic moment of the dry magnetic cluster sample is measured using a VSM or SQUID at a small absolute magnetic field amplitude, where the relationship between the magnetic moment of the sample and the magnetic field is still sufficiently linear. The initial magnetic mass susceptibility of the magnetic cluster sample is calculated by dividing the magnetic moment of the sample by the product of the applied magnetic field and the mass of the dry cluster. The measurement result is expressed in units of m 3 / kg.

[0395] Detection of aromatic carboxylic acids in magnetic clusters

[0396] The presence and / or identity of the aromatic carboxylic acids used as stabilizers in the magnetic clusters of the invention can be confirmed using suitable mass spectrometry methods, such as analyzing the magnetic clusters using electrospray ionization mass spectrometry. Exemplary methods are provided below.

[0397] The magnetic cluster sample is washed with 96% ethanol (3 times) and then with water (3 times) and decanted on a magnet. After removing the solvent, the magnetic cluster is dissolved in a hydrochloric acid solution. The acid is then diluted with water and the aqueous phase is extracted three times with chloroform. The organic phases are combined and evaporated in vacuo to obtain a sample. The sample is dissolved in a suitable solvent and the resulting solution is then introduced into an ESI-MS instrument. A skilled operator can then use, for example, high resolution analysis or tandem MS / MS analysis to confirm the presence and / or identity of the aromatic carboxylic acid.

[0398] In the case where the magnetic cluster samples are coated, the coating can be removed or disrupted using standard methods prior to analysis as described in the previous paragraph. For example, for silica-coated particles, the silica coating can be removed by reaction with an alkaline solution prior to analysis.

[0399] Measurement of specific surface area

[0400] Specific surface area is a characteristic of finely divided samples, such as magnetic clusters. The determination of specific surface area relies on measuring the amount of gas adsorbed at different partial pressures. There are two types of adsorption: physical adsorption and chemical adsorption. Physical adsorption (or physisorption) is weaker than chemical adsorption (chemisorption) and is only observed at low temperatures. This means that physical adsorption is reversible when the partial pressure is reduced. The adsorption and desorption of physically adsorbed gas molecules are measured during the specific surface area measurement.

[0401] The specific surface area can be measured according to the Brunauer-Emmett-Teller (BET) method, such as the method specified in the relevant standards (e.g., Determination of the specific surface area of ​​solids by gas adsorption - BET method, second edition, BS ISO 9277: 2010). An exemplary procedure as used herein is provided below.

[0402] Samples for measurement were prepared using Micromeritics VacPrep 061. Measurements were performed using a Micromeritics TriStar 300 surface area and porosity analyzer with argon as the analytical molecule.

[0403] First weigh the sample tube and filler rod. Then weigh and add 0.2500g0.5000g of the pre-weighed sample (make sure the sample reaches the bottom of the glass tube). Place the filler rod in the sample tube, and then weigh the sample tube with the filler rod and sample. The weighed sample is then connected to the vacuum preparation station and then treated at 160°C for 16 hours-18 hours. When the vacuum preparation is complete, the sample tube is reweighed before being connected to the analysis port and the Dewar flask filled with liquid nitrogen. The program is then run to select the adsorption and desorption points based on the sample being measured. For non-porous and mesoporous materials, it is important for BET measurements to measure the linear region between (p / p0)=0.05 and 0.35.

[0404] Example

[0405] Synthesis of magnetic clusters

[0406] Example 1

[0407] Ethylene glycol (150mL) and iron (III) chloride hexahydrate (8.11g, 30mmol) are loaded into a conical flask and stirred for 15 minutes. Then sodium citrate (1.28g, 6mmol) is added, the flask is sealed, flushed with argon and stirred for 15 minutes. The mixture is then ultrasonicated for 15 minutes, sodium acetate (8.61g, 105mmol) is added afterwards, the solution is stirred for 15 minutes (under argon), then ultrasonicated for 30 minutes, stirred for 15 minutes, and finally ultrasonicated for 30 minutes. Thereafter, the solution is loaded into a Teflon liner, covered with argon, sealed and placed in a steel-lined autoclave. The autoclave is heated to 210°C in 60 minutes, kept at 210°C for 10 hours, and then cooled to room temperature. The reactant is filtered with a wire mesh to remove any large particles and transferred to a bottle using ethanol (96%). The suspension was washed once with 96% ethanol (3 times), water (3 times), 1 M sodium citrate solution (including 30 minutes of shaking and 15 minutes of sonication) and finally with water (3 times) and decanted on a magnet.

[0408] Example 2

[0409] Ethylene glycol (150mL) and iron (III) chloride hexahydrate (8.11g, 30mmol) are loaded into a conical flask and stirred for 15 minutes. Then p-toluic acid (3.27g, 24mmol) is added, the flask is sealed, flushed with argon and stirred for 15 minutes. The mixture is then ultrasonicated for 15 minutes, sodium acetate (8.61g, 105mmol) is added afterwards, the solution is stirred for 15 minutes (under argon), then ultrasonicated for 30 minutes, stirred for 15 minutes, and finally ultrasonicated for 30 minutes. Thereafter, the solution is loaded into a Teflon liner, covered with argon, sealed and placed in a steel-lined autoclave. The autoclave is heated to 210°C in 60 minutes, kept at 210°C for 10 hours, and then cooled to room temperature. The reactant is filtered with a wire mesh to remove any large particles, and transferred to a bottle using ethanol (96%). The suspension was washed once with 96% ethanol (3 times), water (3 times), 1 M sodium citrate solution (including 30 minutes of shaking and 15 minutes of sonication) and finally with water (3 times) and decanted on a magnet.

[0410] According to the method for detecting aromatic carboxylic acids in magnetic clusters provided herein, the presence of toluic acid in the obtained magnetic clusters was confirmed using high-resolution analysis on an ESI-MS instrument.

[0411] The synthesis method was repeated, but the molar ratio of toluic acid to iron (III) chloride was adjusted to levels of 0.2, 0.4 and 1.6. The magnetic clusters obtained in Example 2 (toluic acid stabilizer) and Example 1 (citric acid stabilizer) were analyzed by a disc centrifuge. The results were as follows: Figure 4This indicates that increasing the concentration of toluic acid increases the size of the particles relative to iron (III) chloride. In addition, using toluic acid as a stabilizer provides magnetic nanoclusters with smaller CVs than when using citric acid as a stabilizer.

[0412] Table 1: Synthesis using toluic acid as stabilizer

[0413]

[0414] Example 3-15

[0415] In Examples 3-15, the procedure of Example 2 was followed, but different organic aromatic acids were used as stabilizers.

[0416] In a typical procedure, ethylene glycol (150 mL) and iron (III) chloride hexahydrate (8.11 g, 30 mmol) were loaded into a conical flask and stirred for 15 minutes. Stabilizer (24 mmol, mass listed in Table 2) was then added, the flask was sealed, flushed with argon and stirred for 15 minutes. The mixture was then ultrasonicated for 15 minutes, after which sodium acetate (8.61 g, 105 mmol) was added, the solution was stirred for 15 minutes (under argon), then ultrasonicated for 30 minutes, stirred for 15 minutes, and finally ultrasonicated for 30 minutes. Thereafter, the solution was loaded into a Teflon liner, covered with argon, sealed and placed in a steel-lined autoclave. The autoclave was heated to 210° C. in 60 minutes, kept at 210° C. for 10 hours, and then cooled to room temperature. The reactants were filtered with a wire mesh to remove any large particles and transferred to a bottle using ethanol (96%). The suspension was washed with 96% ethanol (3 times), water (3 times), 1 M sodium citrate solution (including 30 min shaking and 15 min sonication) once, and finally water (3 times) and decanted on a magnet.

[0417] Table 2: Stabilizers used in Examples 2-15

[0418]

[0419] The magnetic clusters obtained by disk centrifuge analysis showed Figure 5 The mode peak provided in . Figure 6 An indication of the mode diameter (diamond markers) and CV (bar markers) of the magnetic clusters determined from the disk centrifuge results is provided. Results for the citrate reference example are also included. Figure 5 and Figure 6 SEM images of magnetic clusters obtained with biphenyl-4-carboxylic acid (Example 5) and citrate reference examples were also obtained ( Figure 7 ).

[0420] Examples 16-18

[0421] In Examples 16-18, the procedure of Example 2 was followed, but the following organic aromatic acids were used as stabilizers: 4-(fluoromethyl)formic acid (Example 16), 4-iodobenzoic acid (Example 17), or 4-chlorobenzoic acid (Example 18).

[0422] The magnetic clusters obtained by disk centrifuge analysis showed Figure 8 The mode peak provided in . Fig. 9 Provides an indication of the mode diameter (circle markers) and CV (bar markers) of the magnetic clusters determined from the disk centrifuge results

[0423] Coating magnetic clusters

[0424] Example 19: Silica coating of magnetic clusters

[0425] Using magnetic separation, the magnetic clusters (2.0 g dry content) in water were washed with EtOH (96%, 3x100 mL). Before ultrasonic treatment for 15 minutes, clusters, EtOH (99.8%, 170 mL) and NH3 (2 mL, 25% in H2O) were added to a round-bottomed flask. Ethanol (99.8%, 30 mL) and TEOS (1.40 mL) were mixed in a separate flask and quickly added to the above suspension. The flask was stirred at room temperature for 18 hours, the final product was washed with ethanol (96%, 3x100 mL) and H2O (5x100 mL), and stored as a H2O suspension.

[0426] Example 20: Epoxy coating on silica coated clusters

[0427] Using magnetic separation, Cyrene TM (3 x 100 ml) Wash the silica coated magnetic clusters (2.0 g dry content) in water. TM (16.73 g) was sonicated for 15 minutes. (3-Glycidyloxypropyl)trimethoxysilane (21.27 g) was added, and the mixture was heated to 130°C and reacted for 18 hours. After cooling, H2O (2 x 100 mL) and Cyrene TM (5x100mL) to wash the epoxy functionalized particles. The particles were suspended in Cyrene TM (65mL).

[0428] Example 21: Coupling of Protein G to Silica-Epoxy Coated Clusters

[0429] Cyrene TMA suspension of silica-epoxy coated clusters in (10 mg dry weight) was transferred to a 2 mL tube and placed on a magnetic separation device. The supernatant was removed, the clusters were washed twice in water (1 mL), once in 0.1 M phosphate buffer pH 7.4 (C1) (1 mL), resuspended by vortexing and sonicating in a water bath for 5 minutes, and then magnetically separated after each wash. The particles were resuspended in C1 (195 μl). Protein A / G (Thermo Fisher Scientific #77677) (10 mg / mL, 55 μl) of C1 was added to the clusters, and the mixture was incubated on a roller for 5 minutes. 3 M ammonium sulfate (C2) (250 μl) was added, and the particles were incubated on a roller at 37°C overnight.

[0430] As above, the clusters were washed three times with 50 mM Tris, 140 mM NaCl, 0.1% Tween-20 (TBST) (1 mL) and incubated overnight on a roller drum at 37° C. The clusters were washed again in TBST (1 mL) and then resuspended to 20 mg / mL in TBST and stored at 2-8° C. until use.

[0431] Example 22: Immunoprecipitation (IP) using clusters

[0432] The protein G functionalized clusters of Example 21 can be used for IP by replacing the beads provided in the kit with the clusters of Example 21, following the protocol described in the Dynabeads Protein G Immunoprecipitation Kit (Thermo Fisher Scientific #10007D). The process can also be automated using suitable equipment, such as the KingFisher Flex instrument (Thermo Fisher Scientific #5400640) with the script provided for the above-mentioned kit. A brief description of an exemplary manual process is given below.

[0433] Sample preparation: Jurkat cells (10x10∧6) were lysed by adding RIPA buffer (1000 μl). The samples were warmed on ice for 15 minutes and then centrifuged at 14000 g, 4°C for 15 minutes. The supernatant was carefully transferred to a new tube.

[0434] Cluster preparation: Anti-CD81 antibody (Thermo Fisher Scientific #10630d) (0.5 mg / mL, 5 μl) was added to the particles of Example 17 (25 μl, 0.5 mg) in PBS containing 0.05% Tween-20. Antibody binding was achieved by incubation on a roller for 30 minutes, then the supernatant was removed, the particles were washed and resuspended in PBS containing 0.05% Tween-20 (200 μl).

[0435] Immunoprecipitation: The antibody-bound clusters (200 μl) were placed on a magnet and the supernatant was removed. The sample (cell lysate) (200 μl) was added and the particles were resuspended by pipetting and then incubated on a roller for 30 minutes at room temperature. The particles were then washed three times using magnetic separation and resuspended by gentle pipetting in PBS containing 0.05% Tween-20 (200 μl).

[0436] Elution: Remove the supernatant using magnetic separation and add elution buffer (LDS sample buffer, ThermoFisher Scientific #B0007) (25 μl). Resuspend the particles by pipetting and incubate at 70°C for 10 minutes. Separate the particles using a magnet and transfer the eluate to a new tube.

[0437] Analysis of eluates: The eluates were analyzed using precast polyacrylamide gels and Coomassie staining. Fig.10 An image of this gel at the end of the analysis is provided, and Table 3 provides information on the samples loaded into each column of the gel. The column "Bead Batch" refers to minor variations in coating and antibody conjugate preparation. In all cases, there is a clear band for the target CD81 antigen. In addition, antibodies that bind to Protein A / G by affinity will be eluted due to the denaturing conditions used.

[0438] Table 3: Samples loaded on the gel

[0439] sample Beads describe Gel 2 wash 1 MA236 Standard buffer + CTAC TBST+BSA 2 MA236 Standard buffer TBST+BSA 3 MA406 Standard buffer + CTAC TBST+BSA 4 MA406 Standard buffer TBST+BSA 5 MA407 Standard buffer + CTAC TBST+BSA 6 MA407 Standard buffer TBST+BSA 7 MA371 Standard buffer + CTAC TBST+BSA 8 MA371 Standard buffer TBST+BSA 9 MA372 Standard buffer + CTAC TBST+BSA 10 MA372 Standard buffer TBST+BSA

[0440] Further synthesis of magnetic clusters

[0441] Embodiment 23-42

[0442] In Examples 23-42, the procedure of Example 2 was followed, but different organic aromatic acids were used as stabilizers.

[0443] In a typical procedure, ethylene glycol (150 mL) and iron (III) chloride hexahydrate (8.11 g, 30 mmol) were loaded into a conical flask and stirred for 15 minutes. Stabilizer (24 mmol, mass listed in Table 2) was then added, the flask was sealed, flushed with argon and stirred for 15 minutes. The mixture was then ultrasonicated for 15 minutes, after which sodium acetate (8.61 g, 105 mmol) was added, the solution was stirred for 15 minutes (under argon), then ultrasonicated for 30 minutes, stirred for 15 minutes, and finally ultrasonicated for 30 minutes. Thereafter, the solution was loaded into a Teflon liner, covered with argon, sealed and placed in a steel-lined autoclave. The autoclave was heated to 210° C. in 60 minutes, kept at 210° C. for 10 hours, and then cooled to room temperature. The reactants were filtered with a wire mesh to remove any large particles and transferred to a bottle using ethanol (96%). The suspension was washed once with 96% ethanol (3 times), water (3 times), 1 M sodium citrate solution (including 30 minutes of shaking and 15 minutes of sonication) and finally with water (3 times) and decanted on a magnet.

[0444] According to the analytical method provided herein, the magnetic clusters obtained were analyzed by disk centrifuge, and the samples were diluted to as low as 0.0025 wt % before injection to reduce secondary peak artifacts. The mode diameters and CVs are shown in Table 4.

[0445] Table 4: Stabilizers used in Examples 23-42

[0446]

[0447]

[0448] Embodiment 43

[0449] In Example 43, the procedure of Example 2 was followed, but a lower concentration of an organic aromatic acid was used as a stabilizer. The mode diameter and CV are shown in Table 5.

[0450] Table 5: Stabilizers used in Example 43

[0451]

[0452] Further analysis of magnetic clusters

[0453] Embodiment 44

[0454] The effect of stabilizer concentration on cluster size was further investigated for different stabilizer concentrations of the stabilizer used in Example 5 (biphenyl-4-carboxylic acid), Example 2 (toluic acid) and Example 35 (4-tert-butylbenzoic acid). The clusters obtained were analyzed by disc centrifuge and the results are shown in Table 6. This shows that for each specific aromatic carboxylic acid, the concentration of the relevant aromatic carboxylic acid tends to increase the size of the particles relative to iron (III) chloride. However, the mode diameter also depends on the identity of the aromatic carboxylic acid. Therefore, the mode diameter of the obtained magnetic clusters can be adjusted by selecting a suitable aromatic carboxylic acid and adjusting the concentration of the aromatic carboxylic acid relative to the iron (III) ion.

[0455] Table 6: stable Effect of dosage concentration on particle size

[0456]

[0457] Embodiment 45

[0458] Using the procedures outlined in the Analytical Methods, analyze Figure 7 SEM images were taken to determine the size of the clusters obtained with biphenyl-4-carboxylic acid (Example 5) and the citrate reference example. Fig.11 The histogram for biphenyl-4-carboxylic acid is represented by dark grey columns, while the histogram for monosodium citrate is represented by light grey columns. This further confirms that the clusters obtained using biphenyl-4-carboxylic acid are significantly more monodisperse than those obtained using citrate.

[0459] Embodiment 46

[0460] The specific surface areas of the magnetic clusters of Examples 2, 10 and 5 were measured according to the exemplary analytical procedures provided herein. The results obtained are shown in Table 7.

[0461] Table 7: Specific surface area measurements

[0462]

[0463] Embodiment 47

[0464] Use 72.64Am 2 The hysteresis curve of the magnetic clusters produced in Example 5 was obtained using a magnetic saturation of 72.27Am / kg. 2 The hysteresis curve of the magnetic clusters produced in Example 10 was obtained by measuring the magnetic saturation of the magnetic clusters produced in Example 10. Fig.12Hysteresis curves are provided, where (A) shows the results for particles produced using biphenyl-4-carboxylic acid as a stabilizer (Example 5), and (B) shows the results for particles produced using 4-tert-butylbenzoic acid as a stabilizer (Example 10).

[0465] The magnetic susceptibility is concentrated on the portion of the hysteresis curve where the magnetizing force decreases from the magnetic saturation point. The value of the magnetic susceptibility can be derived by taking the slope around the point where the slope intersects the y-axis. Fig.12 The magnetic clusters produced using biphenyl-4-carboxylic acid (Example 5) have a value of 80.55×10-5 m 3 / kg magnetic susceptibility. Based on Fig.12 The magnetic clusters produced using 4-tert-butylbenzoic acid (Example 10) have a B value of 120.39×10-5 m 3 / kg magnetic susceptibility.

Claims

1. A magnetic cluster, wherein the magnetic cluster comprises nanocrystals of iron oxide and an aromatic carboxylic acid.

2. The magnetic cluster according to claim 1, wherein the aromatic carboxylic acid is a compound of formula (I): in: L 1 Select from bonds, -C 1-6 Alkyl-, or -C 2-6 alkenyl-; R 1 Selected from -H, -COOH, -(C 1-5 alkyl)COOH, -(C 2-5 alkenyl)COOH or -C 1-4 Alkoxy; R 2 Selected from -H, -halo, -C 1-4 Alkyl, -C 1-4 Alkoxy or -NO2; R 3 Selected from -H, -halo, -C 1-12 Alkyl, -C 3-8 Cycloalkyl, -C 1-12 Alkoxy, -halogen (C 1-12 )alkyl, -NR 6 R 7 、-NO2、-C(O)OH、-C(O)OR 6 or substituted or unsubstituted phenyl; R 4 Selected from -H, -halo, -C 1-4 Alkyl, -C 1-4 Alkoxy or -NO2; R 5 Select from -H or -C 1-4 Alkoxy; R 6 Select from -H or -C 1-4 Alkyl; and R 7 Selected from -H, -C 1-4 Alkyl, -C(O)H, -C(O)C 1-4 Alkyl; or R1 and R2 together with the carbon atoms to which they are attached form a substituted or unsubstituted phenyl group, and L 1 , R 3 , R 4 , R 5 , R 6 and R 7 as previously defined; or R2 and R3 together with the carbon atoms to which they are attached form a substituted or unsubstituted phenyl group, and L 1 , R 1 , R 4 , R 5 , R 6 and R 7 As defined previously.

3. The magnetic cluster according to claim 2, wherein L 1 Select from the key, -C 1-4 Alkyl-, or -C 2-4 Alkenyl-.

4. The magnetic cluster according to claim 2 or claim 3, wherein L 1 is the key.

5. The magnetic cluster according to claim 2, wherein L 1 Selected from -C 1-4 Alkyl or -C 2-4 alkenyl; optionally wherein L 1 Yes-C 2-4 Alkenyl-.

6. The magnetic cluster according to any one of claims 2 to 5, wherein: R1 and R2 together with the carbon atom to which they are attached form a substituted or unsubstituted phenyl group; or R2 and R3 together with the carbon atom to which they are attached form a substituted or unsubstituted phenyl group.

7. The magnetic cluster according to any one of claims 2 to 5, wherein R 1 Selected from -H, -COOH, -(C 1-5 alkyl)COOH or -(C 2-5 alkenyl)COOH.

8. The magnetic cluster according to any one of claims 2 to 5 or 7, wherein R 1 Selected from -H or -COOH.

9. The magnetic cluster according to any one of claims 2 to 5, 7 or 8, wherein R 2 Selected from -H, -C 1-4 Alkyl or -C 1-4 Alkoxy.

10. The magnetic cluster according to any one of claims 2 to 5 or 7 to 9, wherein R 2 Select from -H or -C 1-4 Alkoxy.

11. The magnetic cluster according to any one of claims 2 to 5 or 7 to 10, wherein R 2 Selected from -H or -OCH3.

12. The magnetic cluster according to any one of claims 2 to 5 or 7 to 11, wherein R 3 Selected from -H, -halo, -C 1-6 Alkyl, -C 1-6 Alkoxy, -halogen (C 1-6 )alkyl, -NR 6 R 7 or substituted or unsubstituted phenyl; 13. The magnetic cluster according to any one of claims 2 to 5 or 7 to 12, wherein R 3 Selected from -H, -C 1-4 Alkyl, -C 1-4 Alkoxy, -NR 6 R 7 or substituted or unsubstituted phenyl; Optionally, R 3 Selected from -C 1-4 Alkoxy, -NR 6 R 7 or a substituted or unsubstituted phenyl group.

14. The magnetic cluster according to any one of claims 2 to 13, wherein R 6 is -H or -CH3, and / or wherein R 7 It is -CH3 or -C(O)CH3.

15. The magnetic cluster according to any one of claims 2 to 5 or 7 to 14, wherein R 3 Selected from -H, -C 1-4 Alkyl, -OCH3, -N(CH3)2, -NHC(O)CH3 or phenyl; Optionally, R 3 Selected from -OCH3, -N(CH3)2, -NHC(O)CH3 or phenyl.

16. The magnetic cluster according to any one of claims 2 to 15, wherein R 4 Selected from -H, -C 1-4 Alkyl or -C 1-4 Alkoxy.

17. The magnetic cluster according to any one of claims 2 to 16, wherein R 4 Select from -H or -C 1-4 Alkoxy.

18. The magnetic cluster according to any one of claims 2 to 17, wherein R 4 Selected from -H or -OCH3.

19. The magnetic cluster according to any one of claims 2 to 18, wherein R 2 , R 3 and R 4 No more than one of the groups is -halo.

20. The magnetic cluster according to any one of claims 2 to 19, wherein R 5 Yes -H.

21. The magnetic cluster according to any one of claims 2 to 20, wherein R 1 and R 5 Both are -H.

22. The magnetic cluster according to any one of claims 2 to 21, wherein R 3 Yes -OH, R 2 Yes-C 1-4 Alkoxy, and R 3 Yes-C 1-4 Alkoxy.

23. A magnetic cluster according to any preceding claim, wherein the aromatic carboxylic acid is selected from benzoic acid, toluic acid (e.g. p-toluic acid), 4-ethylbenzoic acid, 3,5-dimethylbenzoic acid, 4-tert-butylbenzoic acid, p-anisic acid, 3,4,5-trimethoxybenzoic acid (eucalyptol), trans-cinnamic acid, phthalic acid, homophthalic acid, 4-(trifluoromethyl)benzoic acid, 4-iodobenzoic acid, 4-chlorobenzoic acid, 3-phenylpropionic acid, 4-(dimethylamino)benzoic acid, 4-acetamidobenzoic acid, bisphenyl-4-carboxylic acid, methyl hydrogen terephthalate, 3-dimethylaminobenzoic acid, 4-methyl-3,5-dinitrobenzoic acid, 3-bromobenzoic acid, 4-aminobenzoic acid, 4-dimethylaminobenzoic acid, 4-(deepoxy)benzoic acid, 3-bromobenzoic acid, 1-naphtholic acid, 4-dimethylaminobenzoic acid, 4-nitrobenzoic acid, terephthalic acid (and / or disodium terephthalate), 4-bromobenzoic acid, 3,5-dimethyl-p-anisic acid, 4-epoxybenzoic acid, 4-n-pentylbenzoic acid, 4-n-heptylbenzoic acid, 4-n-hexylbenzoic acid, 4-n-butylbenzoic acid and 2-naphthoic acid.

24. A magnetic cluster according to any preceding claim, wherein the aromatic carboxylic acid is selected from 4-dimethylaminobenzoic acid, 4-nitrobenzoic acid, terephthalic acid (and / or disodium terephthalate), 4-bromobenzoic acid, 3,5-dimethyl-p-anisic acid, 4-epoxybenzoic acid, 4-n-pentylbenzoic acid, 4-n-heptylbenzoic acid, 4-n-hexylbenzoic acid, 4-n-butylbenzoic acid, 2-naphtholic acid.

25. The magnetic cluster according to any preceding claim, wherein the magnetic cluster is monodisperse.

26. The magnetic cluster of any preceding claim, wherein the magnetic cluster has a mode diameter in the range of about 100 nm to about 2,500 nm; Optionally wherein the magnetic clusters have a mode diameter in the range of about 200 nm to about 1,000 nm.

27. Monodisperse magnetic clusters comprising nanocrystals of iron oxide, wherein the magnetic clusters have a mode diameter in the range of about 100 nm to about 2,500 nm and a coefficient of variation (CV) of no more than 5% when measured by a disc centrifuge.

28. The monodisperse magnetic clusters according to claim 27, wherein the magnetic clusters further comprise an aromatic carboxylic acid, optionally wherein the aromatic carboxylic acid is according to any one of claims 2 to 24.

29. The magnetic cluster of any one of claims 1 to 25, or the monodisperse magnetic cluster of claim 27 or claim 28, wherein the nanocrystals of iron oxide comprise a mode average diameter of about 5 nm to about 20 nm, optionally wherein the nanocrystals have a mode average diameter of about 10 nm to about 15 nm, further optionally wherein the nanocrystals have a mode average diameter of about 12 nm.

30. The magnetic cluster of any one of claims 1 to 25 or 29, or the monodisperse magnetic cluster of any one of claims 27 to 29, further comprising a coating.

31. The magnetic cluster or monodisperse magnetic cluster according to claim 30, wherein the coating comprises at least one layer of silicon dioxide.

32. The magnetic cluster or monodisperse magnetic cluster according to claim 30 or claim 31, wherein the coating comprises at least one organic coating, optionally wherein the at least one organic coating is overcoated with at least one layer of silicon dioxide.

33. The magnetic cluster or monodisperse magnetic cluster according to any one of claims 30 to 32, wherein the coating comprises functional groups; Optionally wherein the functional group is selected from a hydroxyl group, a carboxylic acid group, an aldehyde group, an amine group, a thiol group, an epoxy group, an amide group, a chloromethyl group and a tosyl-activated group; Further optionally wherein the functional group is selected from hydroxyl, carboxylic acid (—COOH), primary amine, secondary amine or epoxy; further optionally wherein the functional group is selected from carboxylic acid (—COOH), primary amine, secondary amine or epoxy.

34. A method for preparing a magnetic cluster, the method comprising: forming a solution comprising iron (III) ions, a protic solvent, an aromatic carboxylic acid, and a precipitant; as well as The solution is reacted to form a suspension comprising magnetic clusters.

35. A method according to claim 34, wherein the iron (III) ions are provided as a soluble iron (III) salt, optionally wherein the soluble iron (III) salt is an iron (III) halide, such as an iron (III) chloride salt.

36. A method according to claim 34 or claim 35, wherein the protic solvent is an alcohol, optionally a diol or polyol, further optionally wherein the alcohol is ethylene glycol.

37. The method according to any one of claims 34 to 36, wherein the aromatic carboxylic acid is according to any one of claims 2 to 24.

38. The method of any one of claims 34 to 37, wherein the precipitation agent comprises at least one of acetate, benzoate or urea, optionally mixed with an alkali metal halide salt.

39. A method according to any one of claims 34 to 38, wherein the precipitation agent comprises sodium acetate, sodium benzoate or urea, optionally mixed with sodium chloride.

40. The method of any one of claims 34 to 38, wherein the precipitating agent is an acetate salt, optionally sodium acetate.

41. The method of any one of claims 34 to 40. wherein reacting the solution comprises one or more of the following conditions: (a) elevated temperature, optionally 150°C to 250°C; and / or (b) a pressure of about 1 bar to about 15 bar; and / or (c) a reaction time of about 6 hours to about 36 hours.

42. The method of any one of claims 34 to 41 further comprising separating the magnetic clusters from the suspension.

43. The method of claim 42, further comprising washing the magnetic clusters.

44. The method of claim 42 or claim 43, further comprising adding a silica coating to the separated magnetic clusters, comprising: forming a suspension comprising the separated magnetic clusters and a silicate or orthosilicate; as well as The silicate or the orthosilicate is reacted to form a silicon dioxide coating.

45. The method of claim 44, wherein reacting the silicate to form a silica coating comprises lowering the pH of the suspension to less than 11.

46. ​​The method of claim 44, wherein reacting the orthosilicate to form a silica coating comprises raising the temperature of the suspension to above 40°C, optionally to above 60°C or 90°C.

47. The method of any one of claims 44 to 46, further comprising overcoating the silica coating with an organic coating, optionally wherein the organic coating comprises a spacer and / or a polymer; Further optionally wherein in Cyrene TM The coating is carried out in the presence of 48. The method of any one of claims 34 to 47, wherein the coating comprises functional groups; Optionally wherein the functional group is selected from a hydroxyl group, a carboxylic acid group, an aldehyde group, an amine group, a thiol group, an epoxy group, an amide group, a chloromethyl group and a tosyl-activated group; Further optionally wherein the functional group is selected from hydroxyl, carboxylic acid (—COOH), primary amine, secondary amine or epoxy; further optionally wherein the functional group is selected from carboxylic acid (—COOH), primary amine, secondary amine or epoxy.

49. The method of any one of claims 34 to 48, wherein the magnetic clusters have an average diameter of nanocrystals of iron oxide of about 5 nm to about 40 nm, optionally wherein the nanocrystals have a diameter of about 5 nm to about 20 nm, further optionally wherein the nanocrystals have a diameter of about 10 nm to about 15 nm, still further optionally wherein the nanocrystals have a diameter of about 12 nm.

50. A method according to any one of claims 34 to 49, wherein the magnetic clusters are monodisperse.

51. The method of any one of claims 34 to 50, wherein the magnetic clusters have a diameter in the range of about 100 nm to about 2,500 nm; Optionally wherein the magnetic clusters have a diameter in the range of about 200 nm to about 1,000 nm.

52. Magnetic clusters obtainable by a method according to any one of claims 34 to 51.

53. Use of a magnetic cluster according to any one of claims 1 to 33 or claim 52 in an assay.

54. Use of a magnetic cluster according to any one of claims 1 to 33 or according to claim 52 for separating, purifying, fractionating, depleting, concentrating and / or analyzing cells, viruses, vesicles, organelles, exosomes, small molecules, analyte cations, anions, ions, toxins, pathogens, nucleic acids, proteins or peptides.

55. Use according to claim 53 or claim 54, wherein the magnetic cluster comprises a (surface) capture ligand, optionally wherein the capture ligand is selected from the group consisting of: an antibody, an antibody fragment, a peptide, a carbohydrate, a hapten, an aptamer and an oligonucleotide.

56. Use according to claim 53 or claim 54, wherein the magnetic cluster comprises surface functionalisation for capturing a class of molecules, optionally wherein the surface functionalisation comprises ionic groups or hydrophobic groups.

57. Use according to any one of claims 53 or 55 to 56, wherein the assay comprises immunoprecipitation, nucleic acid capture, immunoassay and / or lateral flow assay.

58. A kit, comprising: A magnetic cluster according to any one of claims 1 to 33 or according to claim 52.

59. The kit of claim 58, further comprising one or more buffers.

60. The kit of claim 59, wherein the one or more buffers comprise a lysis and / or binding buffer, and optionally a wash buffer and / or an elution buffer.

61. The kit of claim 60, wherein the lysis and / or binding buffer may contain one or more of a chaotropic agent, a precipitating agent such as an alcohol or ethylene glycol.

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