Superparamagnetic and highly porous polymer particles for diagnostic applications

Abstract

The present invention relates to magnetic particles, each particle comprising a polymer substrate and at least one magnetic core (M), wherein the polymer substrate comprises at least one cross-linked polymer and the magnetic particle has a particle size ranging from 1 to 60 micrometers. Furthermore, the present invention relates to a method for manufacturing such particles and to particles obtainable or obtainable by said method. Moreover, the present invention relates to the use of these magnetic particles for the qualitative and / or quantitative determination of at least one analyte in a fluid. Furthermore, the present invention relates to a method for determining at least one analyte in a fluid sample, comprising contact of the magnetic particles of the present invention or magnetic particles obtained by the present invention with a fluid sample that contains or is suspected of containing at least one analyte.

Description

Technology Field

[0001] The present invention relates to magnetic particles, each particle comprising a polymer substrate (P) and at least one magnetic core (M), wherein the polymer substrate comprises at least one cross-linked polymer, and the magnetic particles have a particle size ranging from 1 to 60 micrometers. Furthermore, the present invention relates to a method for manufacturing such particles and to particles obtainable or obtainable by said method. Moreover, the present invention relates to the use of these magnetic particles for the qualitative and / or quantitative determination of at least one analyte in a fluid. Furthermore, the present invention relates to a method for determining at least one analyte in a fluid sample, comprising contact of the magnetic particles of the present invention or magnetic particles obtained by the present invention with a fluid sample that contains or is suspected of containing at least one analyte. Background Technology

[0002] Magnetic particles are excellent tools for capturing analytes from human samples. For example, when coated with antibodies, these particles can specifically capture analytes that can be detected by optical techniques. Magnetic properties are very important because they enable easy, fast, and inexpensive automation in diagnostic systems and allow for the avoidance of additionally time-consuming centrifugation and filtration steps. Here, superparamagnetic materials receive more attention because they exhibit magnetization only when an external magnetic field is applied. In the absence of an external magnetic field, magnetization appears to be zero (no "memory effect"). For example, EP2003455A1 and EP2015074A1 describe the extraction of analytes from human samples using magnetic particles on an LC / MS system.

[0003] High specific surface areas on magnetic particles are required to concentrate analytes from human samples. To increase the surface area to over 1000 m² / g, magnetic particles must be coated with a porous substrate. This is typically achieved by embedding magnetic particles in a porous silica or titanium oxide substrate. One drawback is the high density of silica and titanium, which leads to a decrease in magnetization as the film thickness increases. Furthermore, while only mesoporous systems (pores > 2 nm) can be developed using silica or titanium oxide, they are only suitable for particularly small analytes, and materials with micropores (pores < 2 nm) are preferred. Additionally, proteins and phospholipids adsorb into large mesopores, causing problematic substrate effects in LC / MS systems.

[0004] One major requirement for the automation of particles within enrichment-workflow-MS technology is rapid magnetic separation (<5 s) for high throughput. Particle size and saturation magnetization are critical characteristics. Therefore, high saturation magnetization (>5 A m) 2 kg -1 Particles with high magnetization and large sizes (>2 μm) are required. Additionally, for the robustness of the system, particle carry-over must be avoided. Therefore, the particles must have high magnetization and particle sizes larger than 1 μm.

[0005] Particles having a polymer substrate with micropores are found, for example, in Yang et al. Polym. Chem., 2013, 4, 1425. According to Yang et al., iron oxide nanoparticles are first coated with oleic acid and then embedded in a polystyrene substrate via mini-emulsion polymerization. To achieve a high specific surface area, the nanoparticles are finally hypercrosslinked by a Friedel-Crafts reaction using FeCl3 as a catalyst and dimethoxymethane as a porosity-dependent crosslinking reagent. The resulting particles have an average size of 100 nm and a saturation magnetization of 4.1 A m² kg⁻¹. Although the particles are described as useful for extracting organic molecules from water and also as drug carriers for controlling ibuprofen drug delivery, they exhibit not only a relatively small particle size but also a low saturation magnetization that makes them unfavorable for automated processes.

[0006] Xu et al. describe the synthesis of citrate-stabilized iron oxide nanoparticles with grain sizes of approximately 300 nm (S. Xu et al. Polym. Chem., 2015, 6, 2892). These citrate-stabilized nanoparticles are coated with 3-(trimethoxysilyl)propyl methacrylate (MEMO) and covered with a polystyrene shell via soap-free emulsion polymerization. By this technique, a single nanoparticle is embedded within a polymer particle. In a second polymerization, another layer is formed on the particle. This polymerization is referred to as seed expansion polymerization and involves different monomer compositions. As a final step, porosity is formed by an over-crosslinking reaction with FeCl3 as a pore-forming catalyst. However, by this process, 14.1 A m 2 kg -1 Only sizes of about 400 nm are reached due to saturation magnetization.

[0007] Therefore, advantageous particles, particularly those useful for automation processes, are still in demand. means of solving the problem

[0008] Accordingly, the object of the present invention is to provide porous magnetic particles having a relatively large particle size as well as a relatively high saturation magnetization. This problem is solved by the present invention having the features of the independent patent claims. Advantageous developments of the present invention, which can be realized individually or in combination, are set forth in the dependent claims and / or in the following specification and detailed embodiments.

[0009] When used below, the terms "have," "comprise," or "include," or any grammatical variations thereof, are used in a non-exclusive manner. Thus, these terms may refer to both situations where, in addition to the features introduced by these terms, no additional features exist in the entity described in this context, and situations where one or more additional features exist. For example, the expressions "A has B," "A comprises B," and "A includes B" may refer to both situations where, in addition to B, no other element exists in A (i.e., A consists solely and exclusively of B), and situations where, in addition to B, one or more additional elements exist in entity A, such as element C, elements C and D, or even additional elements.

[0010] Also, it should be noted that terms such as “at least one,” “one or more,” or similar expressions indicating that a feature or element may typically exist one or more times will be used only once when introducing an individual feature or element. In the following, in most cases, when referring to an individual feature or element, the expressions “at least one” or “one or more” will not be repeated in light of the fact that an individual feature or element may exist one or more times.

[0011] Additionally, as used below, terms such as “preferably,” “more preferably,” “particularly,” “more specifically,” “specifically,” “more specifically,” or similar terms are used with optional features without limiting alternative possibilities. Accordingly, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As will be known to those skilled in the art, the present invention may be performed using alternative features. Similarly, features introduced by “in one embodiment of the present invention” or similar expressions are intended as optional features without any limitations regarding alternative embodiments of the present invention, any limitations regarding the scope of the present invention, and any limitations regarding the possibility of combining the features introduced in this manner with other optional or non-optional features of the present invention.

[0012] In a first aspect of the present invention, a magnetic particle comprising a polymer substrate (P) and at least one magnetic core (M) is disclosed, wherein the polymer substrate comprises at least one cross-linked polymer, and the magnetic particle has a particle size in the range of 1 to 60 micrometers when determined according to ISO 13320.

[0013] In a further aspect, the present invention relates to a method for producing magnetic particles comprising a polymer substrate (P) and at least one magnetic core (M), and magnetic particles obtained or obtainable by said method, wherein the polymer substrate (P) comprises at least one cross-linked polymer, and the magnetic particles have a particle size in the range of 1 to 60 micrometers as determined according to ISO 13320, and the present invention comprises the following:

[0014] (i) providing at least one magnetic core (M), preferably at least two magnetic cores (M),

[0015] (ii) a step of providing polymer precursor molecules,

[0016] (iii) polymerizing polymer precursor molecules according to (ii) in the presence of at least one magnetic core (M) to form a particle comprising at least one magnetic core (M), preferably at least two magnetic cores (M), embedded in a polymer substrate (P1), and

[0017] (iv) A step of overcrosslinking the polymer substrate (P1) of the polymer particles obtained in (iii) to provide magnetic particles.

[0018] In a third aspect, the present invention relates to the use of magnetic particles described above and below or magnetic particles obtainable by the method described above and below for the qualitative and / or quantitative determination of at least one analyte in a fluid.

[0019] In addition, the present invention relates to a method for determining at least one analyte in a fluid sample, comprising contact between the magnetic particles of the present invention or the magnetic particles obtained by the present invention and a fluid sample that contains or is suspected of containing at least one analyte.

[0020] magnetic particles

[0021] The magnetic particles according to the present invention have a particle size in the range of 1 to 60 micrometers when determined according to ISO 13320. More preferably, the particle size is in the range of 5 to 55 micrometers, more preferably in the range of 10 to 50 micrometers, more preferably in the range of 15 to 45 micrometers, more preferably in the range of 20 to 40 micrometers, and particularly in the range of 20 to 35 micrometers. According to a preferred embodiment, the magnetic particles according to the present invention have a particle size in the range of 5 to 40 micrometers when determined according to ISO 13320.

[0022] As described above, the magnetic particle according to the present invention comprises a polymer substrate (P) and at least one magnetic core (M). According to a preferred embodiment of the present invention, the magnetic particle comprises more than one magnetic core (M), that is, each particle comprises, preferably at least one, preferably at least two magnetic cores (M). The magnetic core (M) comprises one or more magnetic nanoparticles, such as, for example, 1 to 20 magnetic nanoparticles, preferably 1 to 10, more preferably 1 to 5, most preferably 1 to 3 magnetic nanoparticles. Alternatively, it may comprise more than 20 nanoparticles, preferably 1 to 1.5 million nanoparticles, more preferably 750 to 750,000 nanoparticles, more preferably 1,750 to 320,000 nanoparticles, particularly 90,000 to 320,000 nanoparticles.

[0023] Accordingly, the present invention also relates to magnetic particles as described above, as well as magnetic particles obtained or obtainable by the method described above, wherein the particles comprise at least two magnetic cores (M). According to a particularly preferred embodiment, magnetic particles, as well as magnetic particles obtained or obtainable by the method described above, consist of at least two magnetic cores (M) and a polymer substrate (P).

[0024] Preferably, the amount of magnetic cores (M) is selected so that the desired saturation magnetization of the final particle is achieved. Preferably, the magnetic particle according to the present invention, or the particle obtained or obtainable by the method described above, is at least 1 A m 2 It has a saturation magnetization of / kg. Preferably, when the saturation magnetization is determined according to ASTM A 894 / A 894M, it is at least 1 A m 2 / kg, more preferably at least 2 A m 2 / kg, more preferably at least 3 A m 2 / kg, more preferably at least 4 A m 2 / kg, more preferably at least 5 A m 2 / kg, more preferably at least 6 A m 2 / kg, more preferably at least 7 A m 2 / kg, more preferably at least 8 A m 2 / kg, more preferably at least 9 A m 2 / kg, especially, 10 A m 2 / kg to 20 A m 2 range of / kg, more preferably 10 A m 2 / kg to 30 A m 2 At least 10 A m, such as in the range of / kg 2 It is / kg.

[0025] In principle, the particles of the present invention may exhibit any geometric shape; however, preferably, the particles are substantially spherical. As used herein, the term “substantially spherical” preferably refers to particles having rounded shapes that are non-faceted or substantially free of sharp edges. In certain embodiments, substantially spherical particles typically have an average aspect ratio of 3:1 or less than 2:1, e.g., less than 1.5:1 or less than 1.2:1. In certain embodiments, substantially spherical particles may have an aspect ratio of about 1:1. Aspect ratio (A R ) is the maximum diameter (d max ) and the minimum diameter orthogonal thereto (d min It is defined as a function of (A R = d min / d max The diameters are determined through SEM or optical microscopy measurements.

[0026] The BET specific surface area of ​​magnetic particles obtained or obtainable by the method described above, as well as magnetic particles as described above, is preferably in the range of 50 to 2500 m² / g when determined according to ISO 9277. More preferably, the BET specific surface area of ​​magnetic particles is in the range of 100 to 1500 m² / g, particularly in the range of 300 to 1000 m² / g.

[0027] According to a preferred embodiment of the present invention, magnetic particles as described above, as well as magnetic particles obtained or obtainable by the method described above, are superparamagnetic. The term "superparamagnetism" is known to those skilled in the art and, in particular, refers to magnetic properties encountered for particles smaller than a single magnetic mono-domain. These particles are steadily oriented when an external magnetic field is applied until they reach a maximum value of global magnetization, called saturation magnetization. When the magnetic field is removed, they relax to no magnetic hysteresis (no residual magnetism) at room temperature. In the absence of an external magnetic field, superparamagnetic particles exhibit non-permanent magnetic moments due to thermal fluctuations in dipole orientation (Neel relaxation) and particle position (Brownian relaxation).

[0028] Magnetic core (M)

[0029] As described above, the magnetic particles according to the present invention comprise at least one magnetic core (M) and preferably at least two magnetic cores (M). Preferably, at least one magnetic core (M) comprises a compound selected from the group consisting of metals, metal carbides, metal nitrides, metal sulfides, metal phosphides, metal oxides, metal chelates, and mixtures of two of these. At least one magnetic core (M) may also comprise an alloy with a metal, such as gold, silver, platinum, or copper.

[0030] It should be understood that each magnetic core (M) may contain two or more of the groups mentioned above, namely metals, metal carbides, metal nitrides, metal sulfides, metal phosphides, metal oxides, metal chelates, and mixtures of two or more of these two. Also, mixtures of two or more different metals, two or more different metal oxides, two or more different metal carbides, two or more different metal nitrides, two or more different metal sulfides, two or more different metal phosphides, and two or more different metal chelates are conceivable.

[0031] Also, in the case where the magnetic particle according to the present invention comprises more than one magnetic core (M), it should be understood that each of the magnetic cores (M) present in a single particle may be identical or different from one another. Preferably, all magnetic cores (M) included in a single magnetic particle are identical.

[0032] More preferably, at least one magnetic core (M) comprises a metal oxide or a metal carbide.

[0033] In a preferred embodiment, at least one magnetic core (M) comprises a metal, metal carbide, metal nitride, metal sulfide, metal phosphide, metal oxide, or metal chelate comprising at least one transition metal. Preferred transition metals according to the present invention include, but are not limited to, chromium, manganese, iron, cobalt, nickel, zinc, cadmium, nickel, gadolinium, copper, and molybdenum. More preferably, the metal, metal carbide, metal nitride, metal sulfide, metal phosphide, metal oxide, or metal chelate comprises at least iron. More preferably, at least one magnetic core (M) comprises an iron oxide, in particular Fe3O4, α-Fe2O3, γ-Fe2O3, and MnFe x O y , CoFe xO y , NiFe x O y , CuFe x O y , ZnFe x O y , CdFe x O y , BaFe x O and SrFe x It comprises an iron oxide selected from the group consisting of O, wherein x and y differ depending on the method of synthesis, x is preferably an integer from 1 to 3, more preferably 2, and y is preferably 3 or 4. Most preferably, at least one magnetic core (M) comprises Fe3O4.

[0034] Accordingly, the present invention also relates to magnetic particles as described above, as well as magnetic particles obtained or obtainable by the method described above, wherein at least one magnetic core (M) comprises iron oxide. Most preferably, at least one magnetic core (M) comprises Fe3O4.

[0035] The magnetic core (M) preferably comprises nanoparticles and a coating C1, and more preferably is composed of these.

[0036] nanoparticles

[0037] Nanoparticles are preferably the parts that exhibit the magnetism, preferably superparamagnetism, of the particles. Nanoparticles are also often referred to herein as "magnetic nanoparticles".

[0038] Preferably, at least one nanoparticle comprises at least one magnetic, preferably superparamagnetic, nanoparticle and optionally one coating, e.g., coating C2, and preferably consists of these.

[0039] As used herein, the term “nanoparticle” refers to a particle having a diameter of less than 100 nanometers in at least one dimension, i.e., less than 100 nm. Preferably, the nanoparticle according to the present invention has a diameter in the range of 1 to 20 nm, preferably 4 to 15 nm, as determined by TEM measurements. Accordingly, according to a preferred embodiment, the present invention also relates to magnetic particles as described above, as well as magnetic particles obtained or obtainable by the method described above, wherein the magnetic particles comprise at least one nanoparticle and at least one magnetic core (M) comprising optionally one coating, e.g., coating C2.

[0040] Each nanoparticle(s) has a diameter in the range of 1 to 20 nm, preferably 4 to 15 nm, as determined by TEM measurements. Preferably, at least one magnetic nanoparticle is superparamagnetic.

[0041] The magnetic core (M) may contain only one nanoparticle or more than one nanoparticle. In one embodiment, it contains 1 to 20 nanoparticles. In another embodiment, it contains 1 to 1.5 million nanoparticles, more preferably 750 to 750,000 nanoparticles, more preferably 1,750 to 320,000 nanoparticles, particularly 90,000 to 320,000 nanoparticles. The nanoparticles may exist as individual (i.e., separate) particles as the magnetic core, or they may be aggregates composed of multiple nanoparticles. These aggregates may have different sizes depending on the number of nanoparticles included. Generally, so-called superparticles are formed, which is described further in more detail below. In the case of a magnetic core containing 100 or more nanoparticles, the nanoparticles typically form these superparticles.

[0042] Magnetic core (M) containing 1-20 nanoparticles

[0043] According to a first embodiment, the magnetic core (M) comprises 1 to 20 magnetic nanoparticles and optionally a coating C2, i.e., one magnetic nanoparticle forming the nanoparticles of the magnetic core (M), optionally a coating C2, and preferably consists of these. Generally, the magnetic core comprises 1 to 20 magnetic nanoparticles, preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to 3 nanoparticles.

[0044] Preferably, in this case, the nanoparticle comprises a compound selected from the group consisting of metals, metal carbides, metal nitrides, metal sulfides, metal phosphides, metal oxides, metal chelates, and mixtures of two of these, and more preferably, is composed of these. It should be understood that each nanoparticle may comprise, preferably be composed of, two or more of the above-mentioned groups, namely, metals, metal carbides, metal nitrides, metal sulfides, metal phosphides, metal oxides, metal chelates, and mixtures of two of these. Also, mixtures of two or more different metals, two or more different metal oxides, two or more different metal carbides, two or more different metal nitrides, two or more different metal sulfides, two or more different metal chelates, or two or more different metal phosphides are conceivable. More preferably, the nanoparticle comprises a metal oxide or a metal carbide, and more preferably is composed of these. In a preferred embodiment, the metal is a transition metal. Preferred transition metals according to the present invention include, but are not limited to, chromium, manganese, iron, cobalt, nickel, zinc, cadmium, nickel, gadolinium, copper, and molybdenum. Most preferably, the metal is iron.

[0045] Accordingly, according to a particularly preferred embodiment, the nanoparticles comprise a metal oxide, most preferably an iron oxide, particularly Fe3O4, and more preferably are composed of these.

[0046] According to this embodiment, when more than one magnetic core (M) is present in the magnetic particle, it is desirable that these magnetic cores (M) not aggregate with each other. Preferably, these particles are substantially uniformly distributed within the polymer substrate.

[0047] Magnetic core (M) containing superparticles

[0048] According to a second preferred embodiment, the magnetic core (M) comprises more than 20 nanoparticles, typically more than 100 nanoparticles, which are preferably aggregated together to form a superparticle. More preferably, in this case, the magnetic core (M) comprises a superparticle composed of aggregated, coated nanoparticles. Preferably, in this case, the magnetic core (M) comprises a superparticle comprising 100 to 150 million nanoparticles, more preferably 750 to 750,000 nanoparticles, more preferably 1,750 to 320,000 nanoparticles, particularly 90,000 to 320,000 nanoparticles. Preferably, each nanoparticle is coated with at least one coating C2. Preferably, in this case, the magnetic core (M) thus comprises a superparticle composed of coated nanoparticles aggregated together, preferably composed of these nanoparticles, and the nanoparticles are coated with at least one coating C2, and the coating is preferably deposited on the surface of the nanoparticles. The superparticle may also preferably be coated with coating C1.

[0049] Accordingly, according to this second preferred embodiment of the present invention, the magnetic particles according to the present invention comprise more than 20 magnetic nanoparticles, preferably 1 to 1.5 million nanoparticles, said nanoparticles form at least one superparticle. Each of the nanoparticles in the superparticle is typically coated with at least one coating C2, and the superparticle is typically coated with at least one coating C1.

[0050] Preferably, coating C2 is a coating that covers at least a portion, preferably the entire surface, of each nanoparticle. Preferably, in this case, each nanoparticle comprises a compound selected from the group consisting of metals, metal carbides, metal nitrides, metal sulfides, metal phosphides, metal oxides, metal chelates, and mixtures of two of these, and more preferably consists of these. It should be understood that each nanoparticle present in the superparticle may comprise two or more of the groups mentioned above, namely, metals, metal carbides, metal nitrides, metal sulfides, metal phosphides, metal oxides, metal chelates, and mixtures of two or more of these, and preferably may consist of these. Also, mixtures of two or more different metals, two or more different metal oxides, two or more different metal carbides, two or more different metal nitrides, two or more different metal sulfides, two or more different metal chelates, or two or more different metal phosphides are conceivable. More preferably, each nanoparticle in the superparticle comprises a metal oxide or a metal carbide, and more preferably consists of these. In a preferred embodiment, the metal is a transition metal. Preferred transition metals according to the present invention include, but are not limited to, chromium, manganese, iron, cobalt, nickel, zinc, cadmium, nickel, gadolinium, copper, and molybdenum. Most preferably, the metal is iron. According to a particularly preferred embodiment, each nanoparticle contained in the superparticle is a metal oxide nanoparticle, most preferably an iron oxide nanoparticle, in particular an Fe3O4 nanoparticle.

[0051] Accordingly, the present invention also relates to magnetic particles as described above, as well as magnetic particles obtained or obtainable by the method described above, wherein the magnetic core (M) comprises a superparticle composed of at least 20 aggregated magnetic nanoparticles, or preferably composed of these, and the nanoparticles are preferably coated with at least one coating C2.

[0052] Preferably, the magnetic core (M) comprising at least one optional coating C1 has a diameter in the range of 80 to 500 nm, more preferably 150 to 400 nm, and most preferably 200 to 300 nm, as determined according to DLS (ISO 22412).

[0053] Coating C2

[0054] As coating C2, any coating generally known to those skilled in the art can be conceivable. Preferably, however, coating C2 is selected from at least one member of the group consisting of dicarboxylic acids, tricarboxylic acids, polyacrylic acids, amino acids, surfactants, and fatty acids. Accordingly, it should be understood that the aforementioned group includes salts and derivatives of the mentioned compounds, such as esters and polymers. Accordingly, coating C2 is preferably selected from at least one member of the group consisting of dicarboxylic acids, dicarboxylic acid salts, dicarboxylic acid derivatives, tricarboxylic acids, tricarboxylic acid salts, tricarboxylic derivatives, polyacrylic acids, polyacrylic acid salts, polyacrylic acid derivatives, amino acids, amino acid salts, amino acid derivatives, surfactants, salts of surfactants, fatty acids, fatty acid salts, and fatty acid derivatives.

[0055] As used herein, the terms coated or coating are used to refer to processes of adsorption, van der Waals and / or non-polar group interactions (e.g., chemisorption or physisorption), or covalent bonding between a magnetic nanoparticle or superparticle core and a coating C2 or C1, or if present, two or more coatings.

[0056] Preferably, compounds capable of binding to the surface of the superparticle, such as fatty acids, fatty acid salts, or fatty acid derivatives, and thus preferably stabilizing the superparticle are selected. The fatty acid employed as the coating C2 is preferably a short chain of alkyl groups comprising 8 to 22 carbon atoms having a terminal carboxyl group (-COOH) and adsorption of high affinity to the surface of the magnetic particle (e.g., chemisorption or physisorption). The fatty acid has a number of functions including protecting the magnetic particle core from oxidation and / or hydrolysis in the presence of water, which can significantly reduce the magnetization of the nanoparticle (Hutten, et al. (2004) J. Biotech. 112:47-63); stabilizing the nanoparticle core; and others. The term "fatty acid" includes saturated or unsaturated, particularly unsaturated fatty acids. Exemplary saturated fatty acids include lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, propionic acid, butyric acid, valeric acid, caproic acid, enantic acid, caprylic acid, pelagonic acid, capric acid, undecylic acid, tridecylic acid, pentadecylic acid, margaric acid, nonadecylic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melizelic acid, henatriacontilic acid, laceroic acid, psylic acid, gedic acid, ceroplastic acid, hexatriacontilic acid, heptatriacontanic acid and octattriacontanic acid, and others.Exemplary unsaturated fatty acids are oleic acid, linoleic acid, linolenic acid, arachidonic acid, hexadecatrine acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentanoic acid, heneicosapentaenoic acid, docosapentaenoic acid, glupanodonic acid, docosahexaenoic acid, tetracosapentaenoic acid, tetracosahexaenoic acid, calendic acid, eicosadienoic acid, docosadienoic acid, adrenetic acid, docosapentaenoic acid, tetracosatetraenoic acid, tetracosapentaenoic acid, 5-dodeceneic acid, 7-tetradeceneic acid, palmitoleic acid, baksenic acid, paulinic acid, 15-docosenic acid, 17-tetracosenic acid, ellaidic acid, gondotic acid, midic acid, erucic acid, nervonic acid, and lumenic acid. It includes acid, calendric acid, jacaric acid, eleostearic acid, catal acid, punic acid, rumelenic acid, farina acid, boseopentaenoic acid, pinolenic acid, podocarpic acid, and others. Fatty acids may be synthesized from natural sources or isolated using established methods. Furthermore, fatty acids may be derivatives, such as fatty acid enol esters (i.e., fatty acids that react with acetone of the enol type), fatty esters (i.e., fatty acids in which active hydrogen is substituted with an alkyl group of a monohydric alcohol), fatty amines or fatty amides, or, in certain embodiments, fatty alcohols as described above. A particularly preferred fatty acid is oleic acid.

[0057] A surfactant is an organic compound that is amphiphilic when used in the context of the present invention, that is, containing both hydrophobic and hydrophilic groups. Preferably, surfactants capable of stabilizing superparticle surfactants having various chain lengths by binding to the surface of the superparticle are selected, and hydrophilic-lipophilic balance (HLB) values ​​and surface charges may be employed depending on the application. Preferably, the surfactant according to the present invention is a quaternary ammonium salt, alkylbenzenesulfonates, lignin sulfonates, polyoxyethoxylate, or sulfate ester. Non-limiting examples of surfactants include cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, nonylphenol polyethoxylates (i.e., NP-4, NP-40, and NP-7), sodium dodecylbenzenesulfonate, ammonium lauryl sulfate, sodium laureth sulfate, sodium myres sulfate, docusate, perfluorooctanesulfonate, perfluorobutanesulfonate, alkyl-aryl ether phosphates, alkyl ether phosphates, sodium stearate, 2-acrylamido-2-methylpropanesulfonic acid, ammonium perfluorononanoate, magnesium laureth sulfate, perfluorononanoic acid, perfluorooctanoic acid, phospholipids, potassium lauryl sulfate, sodium alkyl sulfate, sodium dodecyl sulfate, sodium laurate, and sodium lauroyl Sarcosinate, Sodium Nonanoyloxybenzenesulfonate, Sodium Pareth Sulfate, Behentrimonium Chloride, Benzalkonium Chloride, Benzethonium Chloride, Bronidox, Dimethyldioctadecylammonium Bromide, Dimethyldioctadecylammonium Chloride, Lauryl Methyl Gluceth-10 Hydroxypropyl Dimonium Chloride, Octenidine Dihydrochloride, Olaflu, N-Oleyl-1,3-Propanediamine, Stearconium Chloride, Tetramethylammonium Hydroxide, Tonzonium Bromide, Cetomacrogol 1000, Cetostearyl Alcohol, Cetyl Alcohol, Cocamide DEA, Cocamide MEA, Decyl Polyglucose, Disodium Cocoamphodiacetate, Glycerol Monostearate,Polyethylene glycol isocetyl ether, octylphenoxy-polyethoxyethanol, lauryl glucoside, maltosides, monolaurin, mycosubtylin, nonoxynols, octaethylene glycol monododecyl ether, N-octyl beta-D-thioglucopyranoside, octyl glucoside, oleyl alcohol, pentaethylene glycol monododecyl ether, polydocanol, poloxamer, polyethoxylated tallowamine, polyglycerol polyricinoleate, polysorbate, sorbitan, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, stearyl alcohol, surfactin, Triton X-100, Tween 80, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, dipalmitoylphosphatidylcholine, hydroxysultaine, lauryl dimethylamine oxide, They are lecithin, myristamine oxide, peptitergents, sodium lauroamphoacetate, and bis(2-ethylhexyl)sulfosuccine ester.

[0058] The term "amino acids," when used within the meaning of the present invention, refers to natural or non-natural amino acids or amino acid derivatives, as well as salts of amino acids. Preferably, amino acids capable of stabilizing the superparticle by binding to the surface of the superparticle are selected. Exemplary amino acids include cysteine, methionine, histidine, alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, selenocysteine, pyrrolicine, cysteine, dehydroalanine, enduracididin, lanthionine, norvaline, and derivatives thereof.

[0059] Within the meaning of the present invention, the term "dicarboxylic acid" refers to two carboxylic acid functional groups (i.e., R 1 It refers to a hydrocarbon containing -(C(O)OH)2) or a substituted hydrocarbon, where R 1It is (a) a linear hydrocarbon containing 0 to 18 carbon units or (b) an aromatic or non-aromatic ring, a cyclic hydrocarbon containing 3 to 8 carbon units. The term includes salts and derivatives of fatty acids, such as esters of fatty acids. Representative dicarboxylic acids are, for example, propanediic acid, butanediic acid, pentanedioic acid, hexanedioic acid, heptanediic acid, octanediic acid, nonandioic acid, decandioic acid, undecandioic acid, dodecandioic acid, hexadecandioic acid, maleic acid, fumaric acid, glutaconic acid, traumatic acid, muconic acid, glutinous acid, citraconic acid, mesaconic acid, malic acid, aspartic acid, glutamic acid, tartronic acid, tartaric acid, diaminopimel acid, sugar acid, mesoxalic acid, oxaloacetic acid, acetonedicarboxylic acid, arabinaric acid, phthalic acid, isophthalic acid, terephthalic acid, diphenic acid, and 2,6-naphthalenedicarboxylic acid.

[0060] Within the meaning of the present invention, the term "tricarboxylic acid" refers to three carboxylic acid functional groups (i.e., R 1 It refers to a hydrocarbon containing -(C(O)OH)3) or a substituted hydrocarbon, where R 1It is (a) a linear hydrocarbon containing 3 to 18 carbon units or (b) an aromatic or non-aromatic ring, a cyclic hydrocarbon containing 3 to 8 carbon units. The term includes salts and derivatives of fatty acids, such as esters of fatty acids. Representative tricarboxylic acids are, for example, citric acid (2-hydroxypropane-1,2,3-tricarboxylic acid), isocitric acid (l-hydroxypropane-1,2,3-tricarboxylic acid), aconitic acid (prop-l-en-1,2,3-tricarboxylic acid), propane-1,2,3-tricarboxylic acid, trimellitic acid (benzene-1,2,4-tricarboxylic acid), trimesic acid (benzene-l,3,5-tricarboxylic acid), oxalsuccinic acid (1-oxopropane-1,2,3-tricarboxylic acid) or hemimelitic acid (benzene-l,2,3-tricarboxylic acid). Preferably, the tricarboxylic acid is citric acid comprising citrates, i.e., salts and derivatives of citric acid.

[0061] Preferably, C2 is selected from the group consisting of citric acid, histidine, CTAB, CTAC, sodium oleate, polyacrylic acid, or mixtures of two or more of these (including individual salts or derivatives thereof). Accordingly, the present invention also relates to magnetic particles as described above, as well as magnetic particles obtained or obtainable by the method described above, wherein the magnetic core (M) preferably consists of a superparticle composed of aggregated magnetic nanoparticles having at least one coating C2, and at least one coating C2 is selected from the group consisting of citrate, histidine, CTAB, CTAC, sodium oleate, polyacrylic acid, or mixtures of two or more of these.

[0062] Preferably, the amount of coating C2 is in the range of 1 to 80 weight%, more preferably in the range of 5 to 70 weight%, more preferably in the range of 10 to 50 weight%, and most preferably in the range of 20 to 40%, based on the total weight of the sum of C2 and superparticles.

[0063] Coating C1

[0064] As described above, the magnetic core (M) preferably comprises magnetic nanoparticles and a coating C1, and more preferably consists of these. Accordingly, the present invention also relates to magnetic particles as described above, as well as magnetic particles obtained or obtainable by the method described above, wherein at least one magnetic core (M) further comprises a coating C1.

[0065] Coating C1 is preferably deposited on the surface of the magnetic core (M). It should be understood that additional separation layers may exist between coating C1 and the magnetic core (M), however, according to a preferred embodiment, C1 is coated directly on the magnetic core (M).

[0066] Preferably, coating C1 surrounds the entire surface of the magnetic core (M).

[0067] In principle, any suitable coating known to those skilled in the art may be employed. Preferably, coating C1 is selected from the group consisting of surfactants, silica, silicates, silanes, phosphates, phosphonates, phosphonic acids, and mixtures of two or more of these.

[0068] Accordingly, the present invention also relates to magnetic particles as described above, comprising at least one magnetic core (M), as well as magnetic particles obtained or obtainable by the method described above, wherein at least one magnetic core (M) comprises at least one coating C1, and the coating C1 is selected from the group consisting of surfactants, silica, silicates, silanes, phosphates, phosphonates, phosphonic acids and mixtures of two or more of these.

[0069] Preferably, coating C1 is silica, tetraethyl orthosilicate, 3-(trimethoxysilyl)propyl methacrylate, vinyltrimethoxysilane, vinyltriethoxysilane, allyltrimethoxysilane, allyltriethoxysilane, triethoxyvinylsilane, 3-(trimethoxysilyl)propyl acrylate, trimethoxy(7-octen-1-yl)silane, trimethoxymethylsilane, triethoxymethylsilane, ethyltrimethoxysilane, triethoxy(ethyl)silane, trimethoxyphenylsilane, trimethoxy(2-phenylethyl)silane, trimethoxy(propyl)silane, n-propyltriethoxysilane, isobutyl(trimethoxy)silane, isobutyltriethoxysilane, vinylphosphonic acid, dimethyl vinylphosphonate, diethyl vinylphosphonate, diethyl allylphosphonate, diethyl allyl phosphate, Diethyl (2-methylallyl)phosphonate, octylphosphonic acid, butylphosphonic acid, decylphosphonic acid, hexylphosphonic acid, hexadecylphosphonic acid, n-dodecylphosphonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, and arachidic acid, propionic acid, butyric acid, valeric acid, caproic acid, enantic acid, caprylic acid, pellagonal acid, capric acid, undecylic acid, tridecyl acid, pentadecylic acid, margaric acid, nonadecylic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melizelic acid, henatriacontylic acid, laceroic acid, psilic acid, gedic acid, ceroplastic acid, hexatriacontylic acid, Heptatriacontanic acid, octatriacontanic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, hexadecatrine acid, stearidonic acid, eicosatriene acid, eicosatetraene acid, eicosapentanic acid, heneicosapentaene acid, docosapentaene acid, glupanodonic acid, docosahexaenoic acid, tetracosapentaene acid, tetracosahexaene acid, calendic acid, eicosadiene acid, docosadiene acid, adrenetic acid, docosapentaene acid, tetracosatetraene acid, tetracosapentaene acid, 5-dodecene acid, 7-tetradecene acid, palmitoleic acid, baksenic acid, paulinic acid, 15-docosenic acid, 17-tetracosenic acid,It is selected from the group consisting of ellaid acid, gondolax acid, midi acid, erucic acid, nervon acid, lumenic acid, calendrone acid, zacar acid, eleostearic acid, catal acid, punic acid, rumelenic acid, farinaic acid, boseopentaenoic acid, pinolenic acid, podocarpic acid, and mixtures of two or more of these.

[0070] Preferably, each magnetic core (M) contains coating C1 in an amount of 1 to 40 weight%, preferably 2 to 15 weight%, more preferably 5 to 10 weight%, based on the total weight of at least one magnetic core (M).

[0071] According to one preferred embodiment of the present invention, coating C1 comprises vinyl or acrylic groups.

[0072] Polymer substrate (P)

[0073] As described above, each particle includes a polymer substrate (P) in addition to at least one magnetic core (M).

[0074] Preferably, the polymer substrate (P) is a porous polymer substrate, preferably, when determined according to ISO 15901, a porous polymer substrate comprising pores having a pore size of less than 100 nm, more preferably less than 100 nm, more preferably less than 90 nm, more preferably less than 80 nm, more preferably less than 70 nm, more preferably less than 60 nm, more preferably 50 nm or less, for example, in the range of 0.5 nm to 50 nm, preferably in the range of 1 to 20 nm.

[0075] Accordingly, the present invention also relates to magnetic particles as described above, as well as magnetic particles obtained or obtainable by the method described above, wherein the polymer substrate (P) is a porous polymer substrate comprising pores having a pore size of less than 100 nm, preferably 50 nm or less, when determined according to ISO 15901.

[0076] Preferably, at least 90% of all pores present in the polymer substrate have a pore size of less than 10 nm, and at least 50% of all pores present in the polymer substrate have a pore size of less than 5 nm when determined according to ISO 15901.

[0077] According to a particularly preferred embodiment, the polymer substrate does not contain large pores, i.e., pores having a pore size of less than 50 nm.

[0078] Preferably, the particles comprise a polymer substrate (P) in an amount ranging from 40 to 98 weight%, more preferably from 50 to 95 weight%, more preferably from 60 to 90 weight%, and most preferably from 70 to 85 weight%, based on the total weight of the particles.

[0079] The polymer substrate (P) preferably comprises a copolymer obtainable or obtainable by a method comprising the polymerization of at least two different monomer building blocks selected from the group consisting of styrene, functionalized styrenes, vinylbenzyl chloride, divinylbenzene, vinyl acetate, methyl methacrylate, and acrylic acid. Preferably, it comprises a copolymer obtainable or obtainable by a method comprising the polymerization of at least two different monomer building blocks selected from the group consisting of the following monomers:

[0080] and

[0081] Here, R 1 , R 2 , R 3 , R 4 and R 5 is, independently of each other, -N3, -NH2, -Br, -I, -F, -NR'R'', -NR'R''R''', -COOH, -CN, -OH, -OR', -COOR', -NO2, -SH2, -SO2, -R'(OH) x , -R'(COOH) x , -R'(COOR'') x , -R'(OR'') x , -R'(NH2) x , -R'(NHR'') x , -R'(NR''R''') x , -R'(Cl) x , -R'(I) x , -R'(Br) x , -R'(F) x , R'(CN) x , -R'(N3) x , -R'(NO2) x , -R'(SH2) x , -R'(SO2) x , selected from the group consisting of alkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, R', R'' and R''' are independently selected from the group consisting of alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, halides, hydrogen, sulfides, nitrates and amines, and x is an integer in the range of 1 to 3.

[0082] In principle, any polymer known to those skilled in the art may be employed. Preferably, the polymer substrate comprises a cross-linked polymer, which is more preferably obtained or obtainable by a method comprising the step of copolymerizing suitable monomer building blocks in the presence of at least one monomer building block, which is a crosslinking agent, thus, an agent through which crosslinking is achieved in the resulting polymer. Agents suitable for cross-linked polymers are known to those skilled in the art and include, but are not limited to, building blocks such as divinylbenzene, bis(vinylphenyl)ethane, bis(vinylbenzyloxy)hexane, bis(vinylbenzyloxy)dodecane and derivatives thereof.

[0083] According to a particularly preferred embodiment, divinylbenzene is employed as a crosslinking agent.

[0084] Preferably, the polymer substrate is obtained or can be obtained by a method comprising the step of copolymerizing monomer building blocks, and 5-90 vol% of all monomer building blocks are crosslinking agents.

[0085] Preferably, in the resulting polymer, at least 5% of crosslinking is obtained.

[0086] At least one magnetic core (M) is preferably embedded in a polymer substrate. In this context, the term "embedded" is used to mean that the magnetic core is preferably completely surrounded by the polymer substrate. Alternatively, it may be partially surrounded by the polymer substrate. In this case, however, the polymer substrate fixes the magnetic core.

[0087] As described above, according to a preferred embodiment, the particle comprises at least two magnetic cores (M). In this case, it should be understood that each magnetic core (M) present in the particle is embedded in a polymer substrate (P). Accordingly, the present invention also relates to magnetic particles as described above, as well as magnetic particles obtained or obtainable by the method described above, wherein at least two magnetic cores (M) are embedded in a polymer substrate.

[0088] Overcrosslinking

[0089] More preferably, the polymer substrate P comprises a cross-linked copolymer obtained or obtainable by a method comprising the polymerization of at least two different monomer building blocks as described above, thereby preferably obtaining a cross-linked polymer, and the cross-linked polymer is further hypercrosslinked. Thus, more preferably, the polymer substrate comprises, in particular, a hypercrosslinked polymer.

[0090] The term “hypercrosslinked” as used herein refers to a type of multiple crosslinks that form a rigid three-dimensional network. Preferably, hypercrosslinking is achieved by chemically reacting a crosslinked polymer to obtain a hypercrosslinked polymer. Accordingly, the polymer substrate (P) is a polymer substrate that is obtained or obtainable by an additional hypercrosslinked polymer with or without a hypercrosslinking agent by a chemical reaction. Agents suitable for hypercrosslinked polymers are known to those skilled in the art and include, but are not limited to, dichloroethane, dichloromethane, chloroform, carbon tetrachloride, dichlorobenzenes, trichlorobenzenes, dichloroalkanes, dibromoalkanes, diiodoalkanes, trichloroalkanes, tribromoalkanes, triiodoalkanes, dimethoxymethane, dimethoxyethane, and mixtures of two or more of these.

[0091] Preferably, over-crosslinking is achieved by treating the crosslinked polymer substrate with a Friedel-Crafts reaction, particularly the Friedel-Crafts reaction as described below.

[0092] Surface sensoryization

[0093] It should be understood that magnetic particles comprising at least one magnetic core M and a polymer substrate (P) may further comprise surface modifications. The surface of the particle, and thus the surface of the polymer substrate (P), is preferably -OH, -COOH, diethylaminoethanol, R-SO2-OH, -NH2, R-SO2-OH, -RNH, -R2N, -R3N + -CH3, -C2H5, -C4H9, -C8H 17 , -C 18 H 37 It is functionalized with at least one group selected from the group consisting of -C6H5, -C6H9NO6, phenyl-hexyl, biphenyl, hydroxyapatite, boronic acid, biotin, azide, epoxide, alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, amino acids, -COOR, -COR, -OR, antibodies and fragments thereof, aptamers, nucleic acids, and receptor proteins or binding domains thereof. Preferably, these groups are covalently attached to suitable functional groups of the polymer substrate. Methods for carrying out these modifications are known to those skilled in the art.

[0094] Method for manufacturing magnetic particles

[0095] As described above, the present invention also relates to a method for manufacturing magnetic particles comprising a polymer substrate (P) and at least one magnetic core (M), preferably at least two magnetic cores (M), wherein the polymer substrate (P) comprises at least one cross-linked polymer, and the magnetic particles have a particle size in the range of 1 to 60 micrometers, and the present method comprises the following:

[0096] (i) providing at least one magnetic core (M), preferably at least two magnetic cores (M),

[0097] (ii) a step of providing polymer precursor molecules,

[0098] (iii) polymerizing polymer precursor molecules according to (ii) in the presence of at least one magnetic core (M) to form a particle comprising at least one magnetic core (M), preferably at least two magnetic cores (M), embedded in a polymer substrate (P1), wherein the polymer substrate (P1) preferably comprises a cross-linked polymer, more preferably composed thereof, and forming said particle; and

[0099] (iv) Preferably, a step of providing magnetic particles by overcrosslinking the polymer substrate (P1) of the particles obtained in (iii).

[0100] Preferably, in (iii), at least the magnetic core (M) is embedded in the substrate.

[0101] Step (i)

[0102] As described above, at least one magnetic core (M) preferably comprises a compound selected from the group consisting of metals, metal carbides, metal nitrides, metal sulfides, metal phosphides, metal oxides, metal chelates, and mixtures of two of these. At least one magnetic core (M) may also comprise an alloy with a metal, such as gold, silver, platinum, or copper. More preferably, at least one magnetic core (M) comprises a metal oxide or a metal carbide, and more preferably, at least one magnetic core (M) comprises an iron oxide, in particular Fe3O4, α-Fe2O3, γ-Fe2O3, and MnFe x O y , CoFe x O y , NiFe x Oy , CuFe x O y , ZnFe x O y , CdFe x O y , BaFe x O and SrFe x It comprises an iron oxide selected from the group consisting of O, wherein x and y differ depending on the method of synthesis, x is preferably an integer from 1 to 3, more preferably 2, and y is preferably 3 or 4, and most preferably, at least one magnetic core (M) comprises Fe3O4.

[0103] Accordingly, the present invention also relates to a method as described above, and to magnetic particles obtained or obtainable by said method, wherein at least one magnetic core (M) comprises a metal oxide or a metal carbide, more preferably, at least one magnetic core (M) comprises an iron oxide, in particular Fe3O4, α-Fe2O3, γ-Fe2O3, MnFe x O y , CoFe x O y , NiFe x O y , CuFe x O y , ZnFe x O y , CdFe x O y , BaFe x O and SrFe x It comprises an iron oxide selected from the group consisting of O, wherein x and y differ depending on the method of synthesis, x is preferably an integer from 1 to 3, more preferably 2, and y is preferably 3 or 4, and most preferably, at least one magnetic core (M) comprises Fe3O4.

[0104] As described above, the magnetic core (M) preferably comprises magnetic nanoparticles and a coating C1, and more preferably is composed of these.

[0105] Preferably, step (i) includes the following:

[0106] (i.1) a step of providing at least one magnetic nanoparticle, and

[0107] (i.2) a step of providing a magnetic core (M) by coating at least one magnetic nanoparticle with coating C1, wherein coating C1 is preferably selected from the group consisting of surfactants, silica, silicates, silanes, phosphates, phosphonates, phosphonic acids and mixtures of two or more of these.

[0108] The magnetic nanoparticles in (i.1) comprise at least one magnetic, preferably superparamagnetic, nanoparticle and optionally a coating C2, and are preferably composed of these.

[0109] Accordingly, step (i.1) includes providing at least one nanoparticle.

[0110] Methods for providing magnetic nanoparticles are known to those skilled in the art and are described in Lu, Salabas, Schuth, Angew. Chem Int. Ed. 2007, 46, 1222-1244, the individual contents of which are incorporated herein by reference.

[0111] In particular, in the case of Fe3O4 nanoparticles, Fe(II) and Fe(III), the oxide is precipitated from an aqueous, preferably alkaline aqueous, medium to provide individual nanoparticles.

[0112] According to the first preferred embodiment described above, each magnetic core (M) comprises at least one magnetic nanoparticle having a coating C1 optionally, and step (i.1) comprises providing at least one magnetic nanoparticle having a coating C1 optionally, which thus forms the magnetic core (M). Preferably, in this case, the magnetic nanoparticle does not have a coating C2. According to this first preferred embodiment of the present invention, the magnetic particles according to the present invention preferably comprise 1 to 20 magnetic nanoparticles.

[0113] According to this embodiment, when more than one magnetic core (M) is present in the magnetic particle, it is desirable that these magnetic cores (M) not aggregate with each other. Preferably, these particles are substantially uniformly distributed within the polymer substrate.

[0114] Accordingly, the present invention also relates to the method as described above, as well as to the magnetic particles as described above, and step (i) comprises the following:

[0115] (i.1) a step of providing at least one magnetic nanoparticle having optionally a coating C2, and

[0116] (i.2) a step of providing a magnetic core (M) by coating at least one magnetic nanoparticle with coating C1, wherein coating C1 is preferably selected from the group consisting of surfactants, silica, silicates, silanes, phosphates, phosphonates, phosphonic acids and mixtures of two or more of these.

[0117] According to the second preferred embodiment described above, the magnetic core (M), i.e., preferably each magnetic core (M), comprises more than 20 nanoparticles, preferably 100 to 150 million nanoparticles, more preferably 750 to 750,000 nanoparticles, more preferably 1,750 to 320,000 nanoparticles, in particular 90,000 to 320,000 nanoparticles. More preferably, the nanoparticles aggregate and form at least one superparticle composed of coated nanoparticles. Preferably, the superparticle is coated with at least one coating C1. Preferably, in this case, the superparticle is coated with at least one coating C1, and the coating is preferably deposited on the surface of the superparticle. Thus, the superparticle is preferably coated with coating C1. It should be understood that each nanoparticle within the superparticle is preferably coated, for example, with coating C2. Step (i.1) therefore comprises providing more than 20 nanoparticles, and the method further comprises the step of forming at least one superparticle by aggregating the nanoparticles and optionally coating each superparticle with at least one coating C1.

[0118] Accordingly, the present invention also relates to the method as described above, as well as to the magnetic particles as described above, and step (i) comprises the following:

[0119] (i.1) (i.1.1) providing more than 20 nanoparticles preferably having a coating C2, and (i.1.2) providing at least one superparticle by aggregating the nanoparticles to form a superparticle, and

[0120] (i.2) A step of providing a magnetic core (M) by coating at least one superparticle with coating C1, wherein coating C1 is preferably selected from the group consisting of surfactants, silica, silicates, silanes, phosphates, phosphonates, phosphonic acids and mixtures of two or more of these.

[0121] In this case, each magnetic core (M) comprises a superparticle composed of at least 21 coated nanoparticles, preferably 100 to 150 million nanoparticles, more preferably 750 to 750,000 nanoparticles, more preferably 1,750 to 320,000 nanoparticles, particularly 90,000 to 320,000 coated nanoparticles, which are preferably aggregated together.

[0122] (i.1.1) and (i.1.2) should be understood as potentially being performed in a single step, that is, through a reaction in which the initial particles are directly provided.

[0123] Methods for providing superparticles are known to those skilled in the art, for example, as described in Liu et al., Angew. Chem. Int. Ed. 2009, 48, 5875-5879, the individual contents thereof are incorporated herein by reference.

[0124] In particular, in the case of superparticles including Fe3O4 nanoparticles, the synthesis may involve partial reduction of FeCl3 to obtain Fe3O4 superparticles, preferably at elevated temperatures, e.g., in the range of 150 °C to 250 °C, and preferably at increased pressure, e.g., 2 to 10 bar.

[0125] As described above, coating C2 is therefore selected from at least one member of the group consisting of dicarboxylic acids, dicarboxylic acid salts, dicarboxylic acid derivatives, polyacrylic acid, polyacrylic acid salts, polyacrylic acid derivatives, tricarboxylic acids, tricarboxylic acid salts, tricarboxylic derivatives, amino acids, amino acid salts, amino acid derivatives, surfactants, salts of surfactants, fatty acids, fatty acid salts, and fatty acid derivatives.

[0126] Methods for coating nanoparticles or superparticles are known to those skilled in the art. The coating is preferably performed in situ during the synthesis of the superparticles.

[0127] Step (ii)

[0128] In step (ii), polymer precursor molecules are provided, that is, monomer building blocks that provide individual polymers after polymerization are provided.

[0129] Preferably, the polymer precursor molecules in (ii) are selected from the group consisting of styrene, functionalized styrenes, vinylbenzyl chloride, divinylbenzene, vinyl acetate, methyl methacrylate, and acrylic acid, and more preferably are selected from the group consisting of the following monomers:

[0130] and

[0131] Here, R 1 , R 2 , R 3 , R 4 and R 5 are independently of each other, -N3, -NH2, -Br, -I, -F, -NR'R'', -NR'R''R''', -COOH, -CN, -OH, -OR', -COOR', -NO2, -SH2, -SO2, -R'(OH) x , -R'(COOH) x , -R'(COOR'') x, -R'(OR'') x , -R'(NH2) x , -R'(NHR'') x , -R'(NR''R''') x , -R'(Cl) x , -R'(I) x , -R'(Br) x , -R'(F) x , R'(CN) x , -R'(N3) x , -R'(NO2) x , -R'(SH2) x , -R'(SO2) x , is selected from the group consisting of alkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, and R', R'' and R''' are independently selected from the group consisting of alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, halides, hydrogen, sulfides, nitrates and amines.

[0132] Step (iii)

[0133] In step (iii), the polymer precursor molecules according to (ii) are polymerized in the presence of at least one magnetic core (M) to form a particle comprising at least one magnetic core (M), preferably at least two magnetic cores (M), embedded in a polymer substrate (P1), wherein the polymer substrate (P1) preferably comprises a cross-linked polymer as described above and below, more preferably composed of these. This cross-linked polymer substrate P1 is then preferably further super-crosslinked in step (iv) to provide a polymer substrate P.

[0134] The polymerization in (iii) is preferably suspension polymerization. The term “suspension polymerization” refers to a system in which polymer precursor molecules that are relatively insoluble in water are suspended as droplets in an aqueous phase. Typically, a suspending agent is employed to maintain the suspension, and the final polymer is obtained as a dispersed solid phase. Monomer building blocks may be dispersed directly in the suspension polymerization system, but hydrocarbon solvents or diluents, including mixtures, are generally employed with monomers such as n-heptane, isooctane, cyclohexane, benzene, toluene, etc.

[0135] In a suspension polymerization system, the monomer mixture to be polymerized generally comprises monomers, or, if desired, a polymer-in-monomer solution, at least one magnetic core (M), a solvent, and, if employed, an initiator.

[0136] Accordingly, step (iii) preferably includes the following:

[0137] (iii.1) a step of providing a composition (A) comprising polymer precursor molecules according to (ii), at least one magnetic core (M) according to (i), at least one organic solvent, at least one initiator, and aqueous and optionally additives, wherein the organic solvent is not miscible with water, and

[0138] (iii.2) a step of stirring the composition (A) to provide an emulsion (B), wherein the emulsion is preferably an organic solvent-in-water emulsion.

[0139] Preferably, the polymerization in (iii) is carried out in the presence of an initiator selected from the group consisting of azobis(isobutyronitrile) (AIBN), 2,2'-azodi(2-methylbutyronitrile) (V-azobis 67), 1,1'-azobis(cyanocyclohexane) (V-azobis 88), benzoyl peroxide (BPO), 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), and 4,4′-azobis(4-cyanopentanoic acid) (ACVA).

[0140] The monomers and at least one magnetic core (M) are preferably suspended in an aqueous solution optionally containing at least one suspending agent. The amount of water used can vary widely depending on the type of reactor used, stirring means, etc., but the final suspension mixture preferably contains about 5 to 60 weight percent of monomer building blocks based on the total weight of the entire mixture containing water.

[0141] Since the present method involves a liquid-in-liquid dispersion and provides the final product in the form of separate solid particles, various suspending agents may be employed as additives in suspension polymerization systems. Suspending agents include insoluble carbonates, silicates, talc, gelatin, pectin, starch, cellulose derivatives, insoluble phosphates, PVA, salts, NaCl, KCl, PVP, and others. Preferably, the polymerization in (iii) is carried out in the absence of any surfactants.

[0142] The time employed for polymerization must be sufficient for the desired degree or range of transformation and can vary over a wide range, from just a few minutes to several hours, for example, 48 hours, depending on various reaction parameters, such as the temperature employed. Preferably, step (iii) is carried out for a time ranging from 1 hour to 30 hours, preferably from 1 hour to 8 hours.

[0143] The temperatures employed are at least sufficient to induce the decomposition of a free radical initiator, which provides the initiation of the reaction, when thermal polymerization is carried out or used, preferably at temperatures below which gel formation of the polymer may occur. The temperatures preferably employed are in the range of about 0°C to 100°C, preferably 40 to 90°C.

[0144] Stirring is preferably performed using an overhead stirrer.

[0145] Preferably, the polymer substrate (P1) comprises a cross-linked polymer, which is more preferably obtained or obtainable by copolymerizing the polymer with a cross-linking agent. Suitable agents for cross-linked polymers are known to those skilled in the art and include, but are not limited to, divinylbenzene, bis(vinylphenyl)ethane, bis(vinylbenzyloxy)hexane, bis(vinylbenzyloxy)dodecane and derivatives thereof.

[0146] According to a particularly preferred embodiment, divinylbenzene is employed as a crosslinking agent. Preferably, the polymer substrate (P1) is obtained or can be obtained by crosslinking the polymer with 5-90 vol% of the crosslinking agent based on the total amount of the polymer.

[0147] Step (iv)

[0148] The polymer substrate (P1) is preferably supercrosslinked in an additional step (iv). If the additional step (iv) is not performed, the polymer substrate (P1) preferably corresponds to a polymer. However, according to a particularly preferred embodiment, the supercrosslinking in (iv) is preferably performed via a Friedel-Crafts reaction. For the purposes of this application, the term "Friedel-Crafts reaction" refers to a widely known type of reaction developed by Charles Friedel and James Crafts to attach substituents to an aromatic ring by electrophilic aromatic substitution, and includes two main types of Friedel-Crafts reactions: alkylation reactions and acylation reactions. Preferably, alkylation may be used in this invention. These reactions are usually carried out in the presence of a suitable catalyst, such as a Lewis acid.

[0149] Preferably, (iv) is carried out in the presence of a catalyst comprising a Lewis acid selected from the group consisting of FeCl3, ZnCl2, AlCl3, BF3, SbCl5, SnCl4, TiCl4, SiCl4, and mixtures of two or more of these, more preferably composed of these. More preferably, the catalyst comprises FeCl3 or ZnCl2 or a mixture thereof, more preferably composed of these.

[0150] Preferably, the over-crosslinking in (iv) is performed at a temperature in the range of -30°C to 120°C, and more preferably, the over-crosslinking in (iv) is performed at a temperature of 80°C or lower, for example, at a temperature in the range of -30°C to 80°C.

[0151] More preferably, (iv) is carried out at a temperature of less than 70°C, more preferably less than 60°C, more preferably less than 50°C, more preferably less than 40°C, more preferably less than 30°C, for example, preferably in the range of -30°C to 30°C, more preferably in the range of -20°C to 30°C, more preferably in the range of -20°C to 30°C, more preferably in the range of -10°C to 30°C, more preferably in the range of 0°C to 30°C, most preferably in the range of 10°C to 30°C. During the reaction, the temperature may be constant or varied in steps, or maintained essentially constant. In the event that the temperature changes, it should be understood that the temperature is always maintained at a temperature of 120°C or less, less than 100°C, less than 80°C, preferably less than 70°C, more preferably less than 60°C, more preferably less than 50°C, more preferably less than 40°C, more preferably less than 30°C, for example, preferably in the range of -30°C to 30°C, more preferably in the range of -20°C to 30°C, more preferably in the range of -20°C to 30°C, more preferably in the range of -10°C to 30°C, more preferably in the range of 0°C to 30°C, most preferably in the range of 10°C to 30°C.

[0152] Crosslinking may be performed in any suitable solvent known to those skilled in the art, preferably in a solvent comprising dichloroethane, toluene, acetonitrile, DMF, ethyl ether, THF, benzene, xylene, dioxane, alkanes, dichloromethane, chloroform, chlorobenzene, carbon tetrachloride, NMP, dichlorobenzenes, trichlorobenzenes, ethers, cycloalkanes, organic halides, or a mixture of two or more of these. More preferably, crosslinking in (iv) is performed in a solvent selected from the group consisting of dichloroethane, toluene, acetonitrile, DMF, ethyl ether, THF, benzene, xylene, dioxane, alkanes, dichloromethane, chloroform, chlorobenzene, and carbon tetrachloride.

[0153] Preferably, the reaction in (iv) is not carried out in dichloroethane or other organic halides, and more preferably, the reaction in (iv) is not carried out in a solvent containing dichloroethane or other organic halides.

[0154] In particular, the reaction in (iv) is carried out in a solvent comprising at least THF, acetonitrile, DMF, dioxane, or toluol. More preferably, (iv) is carried out in a solvent selected from the group consisting of THF, acetonitrile, DMF, dioxane, toluol, and mixtures of two or more of these.

[0155] Preferably, the reaction in (iv) is carried out for a reaction time of 4 h or less, for example, in the range of 10 minutes to 2 h, more preferably 30 minutes to 1.5 h, more preferably 45 minutes to 1 h.

[0156] Preferably, the reaction in (iv) is carried out under an inert atmosphere, and even more preferably, an inert gas is streamed through the mixture during the reaction in (iv). Preferably, the inert gas is nitrogen and / or argon.

[0157] Optional step (v)

[0158] In addition to the steps described above, the present method may include one or more additional steps. In particular, the present method may include a step of functionalizing the surface of the polymer particles obtained according to (iii) or (iv).

[0159] Accordingly, the present invention also relates to magnetic particles obtained or obtainable by the method described above, as well as the method described above, and the method further comprises the following:

[0160] (v) A step of functionalizing the surface of polymer particles according to (iii) or (iv).

[0161] Preferably, at this step, the polymer particles are -OH, -COOH, diethylaminoethanol, R-SO2-OH, -NH2, R-SO2-OH, -RNH, -R2N, -R3N + -CH3, -C2H5, -C4H9, -C8H 17 , -C 18 H 37 It is functionalized with a group selected from the group consisting of -C6H5, -C6H9NO6, phenyl-hexyl, bi-phenyl, hydroxyapatite, boronic acid, biotin, azide, epoxide, alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, amino acids, -COOR, -COR, -OR, antibodies and fragments thereof, aptamers, nucleic acids, polymers and their receptor proteins or binding domains.

[0162] Methods for functionalizing polymer particles are known to those skilled in the art, for example, Bioconjugate Techniques 2 nd It is described in Edition, GT Hermanson, and the disclosures thereof are incorporated herein by reference.

[0163] Preferably, this step involves at least the treatment of particles with a suitable base, such as KOH (OH functionalization). Alternatively, the functionalization may be COOH-functionalization, Cx-functionalization, or epoxy-functionalization. Further details regarding the functionalizations are also provided in the attached examples below.

[0164] Applications / Analysis Methods

[0165] The magnetic particles described above and the magnetic particles obtainable or obtained by the method described above are preferably used for the qualitative and / or quantitative determination of at least one analyte in a fluid.

[0166] As used herein, the term "qualitative" determination refers to determining the presence or absence of at least one analyte in a fluid. Furthermore, the term may also encompass the evaluation of the properties of the analyte, namely, the identification of the analyte or the identification of the class of chemical molecules to which the analyte belongs.

[0167] The presence or absence of at least one analyte can be determined by contacting a fluid sample with magnetic particles for a sufficient time and under conditions to enable the binding of at least one analyte to the magnetic particles, then removing the remaining fluid sample from the magnetic particles and determining whether at least one analyte is bound to the magnetic particles. To determine whether an analyte is bound to the magnetic particles, compounds bound to the particles may be eluted by suitable techniques, and the presence or absence of at least one analyte may then be determined from the eluted material. Alternatively, the binding of at least one analyte may be achieved directly, i.e., by binding to the magnetic particles.

[0168] Identification of at least one analyte or the chemical class to which it belongs may be performed after said analyte has been eluted from the magnetic particles by suitable analytical methods, such as mass spectrometry, UV-vis, NMR, IR, or biochemical methods, such as ELISA, RIA, and others. Alternatively, depending on the type of agent used for surface functionalization, only specific analytes may be bound. For example, in the case of antibodies, the chemical properties of the analyte are predetermined due to the specificity of the antibody.

[0169] The term “quantitative” as used herein refers to determining the absolute or relative amount of at least one analyte contained in a fluid sample.

[0170] The amount of at least one analyte can be determined for qualitative determination as described above. However, after the elution of the analyte from the magnetic particles, the amount must be determined from the eluent. Alternatively, the amount of the combined analyte may be determined directly.

[0171] In light of the above, the present invention also considers a method for determining at least one analyte in a fluid sample, comprising the following steps:

[0172] (a) a step of contacting magnetic particles according to the present invention or magnetic particles obtained or obtainable by the present invention with a fluid sample that comprises or is suspected of comprising at least one analyte; and

[0173] (b) A step of determining at least one analyte eluted from the magnetic particles.

[0174] Generally, the decision mentioned in this situation is a qualitative or quantitative decision.

[0175] Generally, step (a) of the method is performed for a sufficient time and under conditions to enable the binding of at least one analyte to the particle. Thus, preferably, in step (a), at least a portion, preferably all, of the analyte is bound to the particle. If the determination is a quantitative determination, preferably substantially all of the analyte present in the fluid sample is bound to the particle.

[0176] Preferably, step (a) further includes the following steps:

[0177] (a1) Preferably, a step of washing a magnetic particle to which at least a portion of at least one analyte is bound, under conditions in which at least one analyte is not eluted; and / or

[0178] (a2) A step of eluting at least one analyte from magnetic particles under conditions suitable for enabling the elution of at least one analyte.

[0179] More specifically, the qualitative or quantitative determination in (b) may include the determination of the presence or absence of the analyte bound to the magnetic particle or the determination of the amount of the analyte bound to the magnetic particle.

[0180] It should be understood that the cleaning step in (a1) may be performed as a single cleaning step. Alternatively, more than one cleaning step may be performed.

[0181] More specifically, the qualitative decision may include the following additional steps as part of steps (a) and / or (b):

[0182] - A step of determining whether at least one analyte is bound to a magnetic particle.

[0183] Using the magnetic particles of the present invention or the magnetic particles obtainable by the present invention advantageously reduces substrate carry-over of said fluids in applications such as those described above.

[0184] The analytes to be determined by the magnetic particles of the present invention or the magnetic particles obtainable by the present invention, or the analytes to be determined according to the aforementioned uses, are preferably solutions of chemical compounds or mixtures of chemical compounds present in biological fluid samples or environmental samples. Accordingly, the fluid samples cited according to the present invention are preferably selected from the group of fluids consisting of body fluids, liquids or dissolved environmental samples, and solutions of mixtures of chemical compounds.

[0185] In a preferred embodiment, the fluid sample, as used herein, refers to a biological sample obtained for the purpose of in vitro evaluation. In the methods of the present invention, the fluid sample or patient sample may preferably comprise any body fluid.

[0186] Desirable fluid samples are whole blood, serum, plasma, bronchoalveolar lavage fluid (BAL), epithelial endothelial fluid (ELF), urine, or sputum, and plasma or serum is most preferred.

[0187] The term fluid sample includes biological samples that have been manipulated in any way after acquisition, such as treatment with reagents, lysis, or concentration of specific components like proteins or polynucleotides. Generally, a fluid sample is a liquid sample.

[0188] The fluid sample may be, for example, whole blood, serum, antibodies recovered from a patient, or plasma. The fluid sample is preferably whole blood, serum, or plasma. In one embodiment, the sample is a clinical sample. In another embodiment, the sample is used in a diagnostic test.

[0189] Depending on the properties of the fluid sample, different classes of chemical compounds will be detected. Preferably, the analytes according to the present invention may be selected from the group of steroids, sugars, vitamins, drugs including pharmaceuticals and abused drugs, organic compounds, proteins, nucleic acids, and mixtures of two or more of these. These analytes occur pivotally in biological samples. However, they may also be present in environmental samples.

[0190] The aforementioned applications for determining analytes in fluid samples may preferably be applied to or related to diagnostic purposes, drug abuse testing, environmental control, food safety, quality control, purification, or manufacturing processes. In diagnostic applications, the qualitative or quantitative determination of an analyte may enable the assistance of diagnosis, for example, if the analyte is a biomarker for a disease or medical condition. Similarly, the qualitative or quantitative evaluation of an analyte as an indicator of environmental changes may help identify contamination or assess environmental changes. Food safety, as well as manufacturing or purification processes, may be controlled by the qualitative or quantitative determination of indicator analytes. These indicators may also be determined in relation to general aspects of quality control, for example, the assessment of storage stability of products and others.

[0191] Preferably, the analyte is determined by mass spectrometry, UV-vis, NMR, and IR.

[0192] To summarize the research results of the present invention, the following embodiments are particularly preferred:

[0193] 1. A magnetic particle comprising a polymer substrate (P) and at least one magnetic core (M), preferably at least two magnetic cores, wherein the polymer substrate comprises at least one cross-linked polymer and the magnetic particle has a particle size in the range of 1 to 60 micrometers when determined according to ISO 13320.

[0194] 2. In Embodiment 1, the polymer substrate comprises pores having a pore size of less than 100 nm, preferably 50 nm or less, when determined according to ISO 15901-3, a magnetic particle.

[0195] 3. In embodiment 2, at least 90% of all pores present in the polymer substrate (P) have a pore size of less than 10 nm as determined by ISO 15901-3 and at least 50% of all pores present in the polymer substrate have a pore size of less than 5 nm, and preferably, the polymer substrate (P) does not contain large pores having a pore size greater than 50 nm.

[0196] 4. In embodiment 2 or 3, the particles are magnetic particles having a BET specific surface area in the range of 50 to 2500 m² / g when determined according to ISO 9277.

[0197] 5. In any one of embodiments 1 to 4, the magnetic particle is at least 1 A m when determined according to ASTM A 894 / A 894M 2 / kg, preferably at least 10 A m 2 Magnetic particle having a saturation magnetization of / kg.

[0198] 6. In any one of embodiments 1 to 5, at least one magnetic core (M) comprises a compound selected from the group consisting of metal, metal carbide, metal nitride, metal sulfide, metal phosphide, metal oxide, metal chelate, and mixtures of two of these, a magnetic particle.

[0199] 7. In any one of embodiments 1 to 6, at least one magnetic core (M) is a metal oxide or metal carbide, more preferably an iron oxide, in particular Fe3O4, α-Fe2O3, γ-Fe2O3, MnFe x O y , CoFe x O y , NiFe x O y , CuFe x O y , ZnFe x O y , CdFe x O y , BaFe x O and SrFe x A magnetic particle comprising an iron oxide selected from the group consisting of O, most preferably Fe3O4, wherein x and y differ depending on the method of synthesis, x is preferably an integer from 1 to 3, more preferably 2, and y is preferably 3 or 4.

[0200] 8. In any one of embodiments 1 to 7, the magnetic particle is a superparamagnetic magnetic particle.

[0201] 9. In any one of embodiments 1 to 8, the magnetic core (M) comprises at least one magnetic nanoparticle, preferably at least one iron oxide nanoparticle, more preferably Fe3O4 nanoparticle.

[0202] 10. In embodiment 9, the at least one magnetic core (M) comprises magnetic nanoparticles and a coating C1, more preferably composed of these magnetic particles.

[0203] 11. In embodiment 9 or 10, at least one magnetic nanoparticle is a magnetic particle having a diameter in the range of 1 to 20 nm, preferably 4 to 15 nm, when determined according to TEM measurements.

[0204] 12. In any one of embodiments 1 to 8, at least one magnetic core (M) comprises a superparticle, preferably composed thereof, and optionally comprises a coating C1, a magnetic particle.

[0205] 13. In embodiment 12, the superparticle is a magnetic particle composed of aggregated nanoparticles, preferably more than 20 aggregated nanoparticles, more preferably 1 million to 1.5 million nanoparticles.

[0206] 14. In embodiment 13, each nanoparticle is coated with at least one coating C2, which is preferably deposited on the surface of the nanoparticle, and the coating C2 is preferably selected from the group consisting of dicarboxylic acids, dicarboxylic acid salts, dicarboxylic acid derivatives, tricarboxylic acids, tricarboxylic acid salts, tricarboxylic derivatives, amino acids, amino acid salts, amino acid derivatives, surfactants, salts of surfactants, polyacrylic acids, polyacrylic acid salts, polyacrylic acid derivatives, fatty acids, fatty acid salts, and fatty acid derivatives, magnetic particles.

[0207] 15. In any one of embodiments 12 to 14, the superparticle comprising at least one coating C2 has a diameter in the range of 80 to 500 nm, preferably 150 to 400 nm, most preferably 200 to 300 nm, when determined according to DLS, a magnetic particle.

[0208] 16. In any one of embodiments 10 to 15, at least one coating C1 is a magnetic particle selected from the group consisting of surfactants, silica, silicates, silanes, phosphates, phosphonates, phosphonic acids, and mixtures of two or more of these.

[0209] 17. In any one of embodiments 10 to 16, coating C1 comprises silica, tetraethyl orthosilicate, 3-(trimethoxysilyl)propyl methacrylate, vinyltrimethoxysilane, vinyltriethoxysilane, allyltrimethoxysilane, allyltriethoxysilane, triethoxyvinylsilane, 3-(trimethoxysilyl)propyl acrylate, trimethoxy(7-octen-1-yl)silane, trimethoxymethylsilane, triethoxymethylsilane, ethyltrimethoxysilane, triethoxy(ethyl)silane, trimethoxyphenylsilane, trimethoxy(2-phenylethyl)silane, trimethoxy(propyl)silane, n-propyltriethoxysilane, isobutyl(trimethoxy)silane, isobutyltriethoxysilane, vinylphosphonic acid, dimethyl vinylphosphonate, diethyl vinylphosphonate, Diethyl allyl phosphonate, diethyl allyl phosphate, diethyl (2-methylallyl) phosphonate, octylphosphonic acid, butylphosphonic acid, decylphosphonic acid, hexylphosphonic acid, hexadecylphosphonic acid, n-dodecylphosphonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, and arachidic acid, propionic acid, butyric acid, valeric acid, caproic acid, enantic acid, caprylic acid, pellagonal acid, capric acid, undecylic acid, tridecyl acid, pentadecyl acid, margaric acid, nonadecylic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosyl acid, melizelic acid, henatriacontylic acid, laceroic acid, psilic acid, gedic acid, Seroplastic acid, hexatriacontyl acid, heptatriacontanic acid, octatriacontanic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, hexadecatrine acid, stearidonic acid, eicosatriene acid, eicosatetraenoic acid, eicosapentanic acid, heneicosapentaenoic acid, docosapentaenoic acid, glupanodonic acid, docosahexaenoic acid, tetracosapentaenoic acid, tetracosahexaenoic acid, calendic acid, eicosadiene acid, docosadiene acid, adrenetic acid, docosapentaenoic acid, tetracosatetraenoic acid, tetracosapentaenoic acid, 5-dodeceneic acid, 7-tetradeceneic acid, palmitoleic acid, baksenic acid, Paulinic acid,Magnetic particles selected from the group consisting of 15-tokosenic acid, 17-tetracosenic acid, ellaidic acid, gondoic acid, midic acid, erucic acid, nervonic acid, lumenic acid, calendic acid, jacaric acid, eleostearic acid, catalic acid, punic acid, rumelenic acid, farinaic acid, boseopentaenoic acid, pinolenic acid, podocarpic acid, and mixtures of two or more of these.

[0210] 18. In embodiment 16 or 17, the magnetic particle, wherein coating C1 is preferably a surfactant or silica coating.

[0211] 19. In any one of embodiments 10 to 18, the magnetic particle comprises at least one magnetic core (M) in an amount of 1 to 40 weight%, preferably 2 to 15 weight%, more preferably 5 to 10 weight%, based on the total weight of at least one magnetic core (M).

[0212] 20. In any one of embodiments 10 to 12, the coating C1 is a surfactant, and at least one magnetic core (M) preferably comprises the coating C1 in an amount of 1 to 15 weight%, preferably 5 to 10 weight%, based on the total weight of at least one magnetic core (M), preferably an oleic acid coating.

[0213] 21. In any one of embodiments 10 to 16, the coating C1 is a magnetic particle comprising vinyl or acrylic groups.

[0214] 22. In any one of embodiments 1 to 21, the particle is a magnetic particle comprising at least two magnetic cores (M).

[0215] 23. In any one of embodiments 1 to 22, the polymer substrate (P) comprises a crosslinked copolymer obtained or obtainable by a method comprising the step of copolymerizing suitable monomer building blocks in the presence of at least one monomer building block that is a crosslinking agent, wherein preferably 5-90 vol% of all monomer building blocks are crosslinking agents, more preferably divinylbenzenes, magnetic particles.

[0216] 24. In embodiment 23, a magnetic particle in which a polymer having a degree of crosslinking of at least 5% is obtained in copolymerization.

[0217] 25. In any one of embodiments 1 to 24, the polymer substrate comprises a copolymer obtained or obtainable by a method comprising copolymerizing at least two different monomer building blocks selected from the group consisting of styrene, functionalized styrenes, vinylbenzyl chloride, divinylbenzene, vinyl acetate, methyl methacrylate, and acrylic acid, wherein the monomer building blocks are preferably selected from the group consisting of the following monomers:

[0218] and

[0219] Here, R 1 , R 2 , R 3 , R 4 and R 5 are independently -N3, -NH2, -Br, -I, -F, -NR'R'', -NR'R''R''', -COOH, -CN, -OH, -OR', -COOR', -NO2, -SH2, -SO2, -R'(OH) x , -R'(COOH) x , -R'(COOR'') x , -R'(OR'') x , -R'(NH2) x , -R'(NHR'') x , -R'(NR''R''')x , -R'(Cl) x , -R'(I) x , -R'(Br) x , -R'(F) x , R'(CN) x , -R'(N3) x , -R'(NO2) x , -R'(SH2) x , -R'(SO2) x A magnetic particle selected from the group consisting of alkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, wherein R', R'' and R''' are independently selected from the group consisting of alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, halides, hydrogen, sulfides, nitrates, and amines.

[0220] 26. In embodiment 25, one or more monomer building blocks are cross-linked with each other, forming magnetic particles.

[0221] 27. A magnetic particle in any one of embodiments 1 to 26, wherein at least one magnetic core (M), preferably at least two magnetic cores (M), is embedded in a polymer substrate.

[0222] 28. In any one of embodiments 1 to 27, the particle is a magnetic particle comprising at least two magnetic cores (M) and a polymer substrate (P).

[0223] 29. In any one of embodiments 1 to 28, the surface of the particle is -OH, -COOH, diethylaminoethanol, R-SO2-OH, -NH2, R-SO2-OH, -RNH, -R2N, -R3N + -CH3, -C2H5, -C4H9, -C8H 17 , -C 18 H 37A magnetic particle functionalized with at least one group selected from the group consisting of -C6H5, C6H9NO6, phenyl-hexyl, bi-phenyl, hydroxyapatite, boronic acid, biotin, azide, epoxide, alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, amino acids, -COOR, -COR, -OR, antibodies and fragments thereof, aptamers, nucleic acids, and receptor proteins or binding domains thereof.

[0224] 30. In any one of embodiments 1 to 29, the particle is a magnetic particle that is substantially spherical.

[0225] 31. A method for manufacturing magnetic particles comprising a polymer substrate (P) and at least one magnetic core (M), preferably at least two magnetic cores (M), wherein the polymer substrate (P) comprises at least one cross-linked polymer, and the magnetic particles have a particle size in the range of 1 to 60 micrometers when determined according to ISO 13320, and the method comprises

[0226] (i) providing at least one magnetic core (M), preferably at least two magnetic cores (M),

[0227] (ii) a step of providing polymer precursor molecules,

[0228] (iii) polymerizing polymer precursor molecules according to (ii) in the presence of at least one magnetic core (M) to form a particle comprising at least one magnetic core (M), preferably at least two magnetic cores (M), embedded in a polymer substrate (P1), wherein the polymer substrate (P1) preferably comprises a cross-linked polymer, more preferably composed thereof, and forming said particle; and

[0229] (iv) Preferably, a method for manufacturing magnetic particles comprising the step of overcrosslinking the polymer substrate (P1) of the polymer particles obtained in (iii) to provide magnetic particles.

[0230] 32. A method for manufacturing magnetic particles in embodiment 31, wherein, in (iii), at least the magnetic core (M) is embedded in the substrate.

[0231] 33. A method for producing magnetic particles in which, in either embodiment 31 or 32, the over-crosslinking in (iv) is performed through a Friedel-Crafts reaction.

[0232] 34. A method for manufacturing magnetic particles in which, in embodiment 33, the over-crosslinking in (iv) is performed at a temperature in the range of -30 to 120°C.

[0233] 35. A method for producing magnetic particles, wherein in any one of embodiments 31 to 34, the over-crosslinking in (iv) is performed in the presence of a catalyst comprising a Lewis acid selected from the group consisting of FeCl3, ZnCl2, AlCl3, BF3, SbCl5, SnCl4, TiCl4, SiCl4 and mixtures of two or more of these, more preferably in the presence of a catalyst comprising FeCl3 or ZnCl2 or a mixture thereof, more preferably in the presence of a catalyst comprising these.

[0234] 36. In any one of embodiments 31 to 35, the over-crosslinking in (iv) is in a solvent selected from the group consisting of dichloroethane, toluene, acetonitrile, dioxane, DMF, ethyl ether, THF, benzene, xylene, alkanes, dichloromethane, chloroform, chlorobenzene, carbon tetrachloride, NMP, dichlorobenzenes, trichlorobenzenes, ethers, cycloalkanes, organic halides, and mixtures of two or more of these, more preferably in a solvent selected from the group consisting of dichloroethane, toluene, acetonitrile, DMF, ethyl ether, THF, benzene, xylene, alkanes, dichloromethane, chloroform, chlorobenzene, carbon tetrachloride, and dioxane, more preferably in a solvent consisting of THF, acetonitrile, DMF, dioxane, and toluene A method for manufacturing magnetic particles, performed in a solvent selected from the group.

[0235] 37. A method for producing magnetic particles, wherein in any one of embodiments 31 to 36, the over-crosslinking in (iv) is performed with a crosslinking agent selected from the group consisting of dichloroethane, dichloromethane, chloroform, carbon tetrachloride, dichlorobenzenes, trichlorobenzenes, dichloroalkanes, dibromoalkanes, diiodoalkanes, trichloroalkanes, tribromoalkanes, triiodoalkanes, dimethoxymethane, dimethoxyethane, and mixtures of two or more of these.

[0236] 38. In any one of embodiments 31 to 37, the method

[0237] (v) A method for manufacturing magnetic particles, further comprising the step of functionalizing the surface of the polymer particles according to (iii) or (iv).

[0238] 39. In Example 38, at step (v), the polymer particles are -OH, -COOH, diethylaminoethanol, R-SO2-OH, -NH2, R-SO2-OH, -RNH, -R2N, -R3N +-CH3, -C2H5, -C4H9, -C8H 17 , -C 18 H 37 A method for preparing magnetic particles functionalized with a group selected from the group consisting of -C6H5, C6H9NO6, phenyl-hexyl, bi-phenyl, hydroxyapatite, boronic acid, biotin, azide, epoxide, alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, amino acids, -COOR, -COR, -OR, antibodies and fragments thereof, aptamers, nucleic acids, and their receptor proteins or binding domains.

[0239] 40. A method for producing magnetic particles, wherein, in any one of embodiments 31 to 39, the polymerization in (iii) is suspension polymerization.

[0240] 41. A method for producing magnetic particles, wherein in any one of embodiments 31 to 40, the polymerization in (iii) is carried out in the presence of an initiator selected from the group consisting of azobis(isobutyronitrile) (AlBN), 2,2'-azodi(2-methylbutyronitrile) (V-Azo 67), 1,1'-azobis(cyanocyclohexane) (V-Azo 88), benzoyl peroxide (BPO), 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), and 4,4′-azobis(4-cyanopentanoic acid) (ACVA).

[0241] 42. In any one of embodiments 31 to 41, step (iii) is

[0242] (iii.1) a step of providing a composition (A) comprising polymer precursor molecules according to (ii), at least one magnetic core (M) according to (i), at least one organic solvent, at least one initiator, and an aqueous phase, wherein the organic solvent is immiscible with water, and optionally, the water comprises at least one additive; and

[0243] (iii.2) A method for producing magnetic particles, comprising the step of stirring composition (A) to provide an emulsion (B), wherein the emulsion is preferably an organic solvent emulsion in water.

[0244] 43. A method for producing magnetic particles according to Embodiment 42, wherein at least one additive is selected from the group consisting of insoluble carbonates, silicates, talc, gelatin, pectin, starch, cellulose derivatives, insoluble phosphates, PVA, salts, NaCl, KCl, and PVP.

[0245] 44. A method for producing magnetic particles according to embodiment 42 or 43, wherein stirring is performed with an overhead stirrer.

[0246] 45. A method for producing magnetic particles in any one of embodiments 29 to 43, wherein the polymerization in (iii) is carried out in the absence of any surfactants.

[0247] 46. ​​A method for manufacturing magnetic particles, wherein in any one of embodiments 31 to 45, step (iii) is performed for a time ranging from 1 hour to 30 hours, preferably from 1 hour to 8 hours.

[0248] 47. A method for manufacturing magnetic particles, wherein in any one of embodiments 31 to 46, step (iii) is performed at a temperature in the range of 0°C to 100°C, preferably 40°C to 90°C.

[0249] 48. In any one of embodiments 31 to 47, step (i) is

[0250] (i.1) a step of providing at least one magnetic nanoparticle, and

[0251] (i.2) a step of providing at least one magnetic core (M) by coating at least one magnetic nanoparticle with coating C1, and

[0252] A method for manufacturing magnetic particles, wherein coating C1 is preferably selected from the group consisting of surfactants, silica, silicates, silanes, phosphates, phosphonates, phosphonic acids, and mixtures of two or more of these.

[0253] 49. In embodiment 48, (i) is

[0254] (i.1.1) A step of providing more than 20 nanoparticles having at least one coating C2, preferably at least 21 nanoparticles,

[0255] (i.1.2) A step of aggregating more than 20 nanoparticles to form a superparticle, and

[0256] (i.1.3) Optionally, a method for manufacturing magnetic particles comprising the step of coating a superparticle according to (i.2) with at least one coating C1 to provide at least one magnetic core (M).

[0257] 50. A method for manufacturing magnetic particles in embodiment 49, wherein at least one coating C2 is selected from the group consisting of dicarboxylic acids, dicarboxylic acid salts, dicarboxylic acid derivatives, polyacrylic acid, polyacrylic acid salts, polyacrylic acid derivatives, tricarboxylic acids, tricarboxylic acid salts, tricarboxylic derivatives, amino acids, amino acid salts, amino acid derivatives, surfactants, salts of surfactants, fatty acids, fatty acid salts, and fatty acid derivatives.

[0258] 51. A method for producing magnetic particles, wherein the particles are preferably substantially spherical particles, the magnetic particles being obtained or obtainable by any one of the methods of embodiments 31 to 50.

[0259] 52. Use of magnetic particles according to any one of embodiments 1 to 30 or magnetic particles according to embodiment 51 for qualitative and / or quantitative determination of at least one analyte in a fluid.

[0260] 53. In embodiment 52, the analyte is selected from the group of steroids, sugars, vitamins, drugs, organic compounds, proteins, nucleic acids, sugars, and mixtures of two or more of these.

[0261] 54. In embodiment 52 or 53, the analyte is used to be concentrated by magnetic particles according to embodiments 1 to 30 or magnetic particles according to embodiment 51.

[0262] 55. In any one of embodiments 52 to 54, the analyte is used, determined by mass spectrometry, UV-vis, NMR, IR.

[0263] 56. In any one of embodiments 52 to 55, the fluid is selected from the group of fluids comprising body fluids, liquids or solutions of mixtures of dissolved environmental samples and chemical compounds.

[0264] 57. In any one of embodiments 52 to 56, the magnetic particle of any one of embodiments 1 to 30 or the magnetic particle according to embodiment 51 is used to reduce substrate carry-over of the fluids.

[0265] 58. In embodiment 57, the qualitative and / or quantitative determination of at least one analyte in a fluid is for use related to diagnostic purposes, drug abuse testing, environmental control, food safety, quality control, purification or manufacturing processes.

[0266] 59. A method for determining at least one analyte in a fluid sample,

[0267] a) a step of contacting the magnetic particles of the present invention or the magnetic particles obtained by the present invention with a fluid sample that comprises or is suspected of comprising at least one analyte; and

[0268] b) A method for determining at least one analyte in a fluid sample, comprising the step of determining at least one analyte bound to the magnetic particle.

[0269] 60. A method for determining at least one analyte in a fluid sample, wherein, in embodiment 59, step a) of the method is performed for a sufficient time and under conditions to enable binding of at least one analyte to a magnetic particle.

[0270] 61. In embodiment 59 or 60, step a) is

[0271] a1) preferably, a step of washing magnetic particles to which at least a portion of at least one analyte is bound, under conditions in which at least one analyte is not eluted; and / or

[0272] a2) A method for determining at least one analyte in a fluid sample, further comprising the step of eluting at least one analyte from a magnetic particle under conditions suitable for enabling the elution of at least one analyte.

[0273] 62. A method for determining at least one analyte in a fluid sample, wherein in any one of embodiments 59 to 61, the determination comprises determining the presence or absence of an analyte bound to magnetic particles or determining the amount of analyte bound to magnetic particles.

[0274] Additional optional features and embodiments of the invention will be disclosed in more detail in the subsequent description of the preferred embodiments, preferably together with dependent claims. Herein, each optional feature may be realized in any feasible combination as well as in a separate manner, as will be known to a person skilled in the art. The scope of the invention is not limited by the preferred embodiments. Embodiments are schematically illustrated in the drawings. Herein, the same reference numerals in these drawings refer to the same or functionally comparable elements.

[0275] All references cited throughout this specification are incorporated herein by reference in connection with their entire contents as well as specifically mentioned in the disclosures. Brief explanation of the drawing

[0276] FIG. 1 is a schematic diagram of a magnetic particle comprising 1-20 nanoparticles (10a), a single magnetic core (M) (10a + 10b) with coating C1 (10b), and a polymer substrate (P) (11). FIG. 2 is a schematic diagram of a more preferred magnetic particle comprising more than one, namely three magnetic cores (M) (10a + 10b) as well as a polymer substrate (P) (11), each having 1-20 nanoparticles (10a) and a coating C1 (10b). FIG. 3 is a schematic diagram of a magnetic particle having one magnetic core (M) and a polymer substrate (P) (11), which is composed of superparticles formed upon aggregation of a plurality of nanoparticles (10a) coated with coating C2 (10c) and additionally has coating C1 (10b). FIG. 4 is a schematic diagram of a more preferred magnetic particle having more than one magnetic core (M) and polymer substrate (P) (11), which is composed of superparticles formed upon aggregation of a plurality of nanoparticles (10a) coated with coating C2 (10c) and additionally has coating C1 (10b). Figure 5 shows the calculated saturation magnetization for particles of different sizes synthesized by Xu et al. Figure 6 shows a concentration workflow using beads as described herein for sample analysis. Specific details for implementing the invention

[0277] Examples

[0278] The following examples are merely illustrative of the invention. In any case, they should not be construed as limiting the scope of the invention.

[0279] Example 1: Preparation of Beads

[0280] Silica-coated magnetic nanoparticles (1)

[0281] In a standard procedure, 43 g FeCl2·4 H2O (0.22 mol) and 70 g FeCl3 (0.43 mol) were added to 2 L of water under stirring and heated to 70 °C. 125 mL of NH4OH (28% of H2O) was added, and after 10 minutes, the black precipitate was separated by magnet. The supernatant was discarded, and the magnetic nanoparticles were washed 5 times with water. The nanoparticles were resuspended in 2 L of ethanol under sonication and transferred to a 4 L reactor. The total volume was filled with ethanol to 3.2 L, and a mixture of tetraethoxysilane (TEOS; 50 mL; 0.22 mol) and ethanol (350 mL) was added dropwise under stirring. After stirring for 24 hours at 25 °C, the nanoparticles were separated by magnet, and the supernatant was discarded. The TEOS-coated nanoparticles were washed three times with ethanol and resuspended in 2 L of ethanol under sonication. The nanoparticles were transferred to a 4 L reactor, and the total volume was filled to 3.1 L with ethanol. The mixture was heated to 50 °C, and a mixture of [3-(methacryloyloxy)propyl]trimethoxysilane (MEMO; 77 mL; 0.32 mol), isobutyl(trimethoxy)silane (ISO; 33 mL; 0.17 mol), and ethanol (390 mL) was added dropwise while stirring. After stirring for 24 hours at 50 °C, the nanoparticles were separated by magnet, and the supernatant was discarded. The MEMO / ISO-coated nanoparticles were washed twice with ethanol and stored in ethanol Silica-coated magnetic nanoparticles (1) (50 g) was obtained.

[0282] Surfactant-coated magnetic nanoparticles (2)

[0283] In a standard procedure, 126 g FeCl2·4 H2O (0.63 mol) and 248 g FeCl3 (1.53 mol) were added to 3 L of water under stirring and heated to 55 °C. 460 mL of NH4OH (28% of H2O) was added, and after 15 minutes, a black precipitate was separated by magnet. The supernatant was discarded, and the magnetic nanoparticles were washed three times with water. The magnetic nanoparticles were resuspended in 2000 mL, and the pH was adjusted to 7–9 with NaOH (10 M). After sonication for 30 minutes, the suspension was transferred to a 4 L reactor, and 1 L of water was added. 120 mL of oleic acid was added under stirring, and the suspension was stirred at 25 °C for 45 minutes. The magnetic nanoparticles were separated by magnet, and the supernatant was discarded. Surfactant-coated nanoparticles were washed three times with water and ethanol and stored in ethanol, Surfactant-coated magnetic nanoparticles (2) (203 g) was obtained.

[0284] Silica-coated magnetic superparticles (3)

[0285] In a standard procedure, 44 g of FeCl3·6H2O (0.16 mol) was dissolved in 800 mL of ethylene glycol and transferred to a high-pressure reactor. 9.7 g of sodium citrate (0.037 mol) and 51.9 g of sodium acetate (0.63 mol) were added, and the high-pressure reactor was sealed. The mixture was stirred and processed at 160 °C for 2 h and at 200 °C for 18 h. The formed nanoparticles were separated by a magnet, and the supernatant was discarded. The magnetic nanoparticles were washed three times with ethanol and five times with water to obtain magnetic superparticles (13.3 g). 10 g of the superparticles were resuspended in 1500 mL of ethanol under sonication in a 2 L reactor, and a mixture of tetraethoxysilane (TEOS; 15 mL; 68 mmol) and ethanol (50 mL) was added dropwise while stirring. After stirring for 20 hours at 25 °C, the supernatants were separated by magnet, and the supernatant was discarded. The TEOS-coated supernatants were washed twice with ethanol and resuspended in 1300 mL of ethanol under sonication. The mixture was heated to 65 °C, and a mixture of [3-(methacryloyloxy)propyl]trimethoxysilane (MEMO; 14 mL; 59 mmol), isobutyl(trimethoxy)silane (ISO; 6 mL; 31 mmol), and ethanol (180 mL) was added dropwise under stirring. After stirring for 16 hours at 50 °C, the supernatants were separated by magnet, and the supernatant was discarded. The MEMO / ISO-coated supernatants were washed three times with ethanol and stored in ethanol Silica-coated magnetic superparticles (3) (10 g) was obtained.

[0286] Self-polymer particles (4)

[0287] In a standard procedure, 650 mL of water was added to a 2 L glass reactor equipped with a mechanical stirrer. 13.5 g of PVA was added and stirred until a complete solution was formed.

[0288] 10 g of (1), (2), or (3) was separated by magnet, and the supernatant was discarded. The magnetic nanoparticles were washed once with toluene and resuspended in 168 mL of toluene. 23.6 mL of divinylbenzene (0.17 mol) and 23.6 mL of vinylbenzyl chloride (0.17 mol) were added, and the mixture was sonicated for 1 h. 3.84 g of 2,2′-azobis(2-methylbutyronitrile) (20 mmol) was added, and the mixture was transferred to a PVA solution under rapid stirring. The mixture was heated to 80 °C, and the reaction was continued for 4 h. The formed magnetic polymer particles were separated by magnet, and the supernatant was discarded. The particles were washed three times with water and methanol and resuspended in isopropanol / water (10 / 90 v / v), Self-polymer particles (4) (52.3 g) was obtained.

[0289] porous magnetic polymer particles (5)

[0290] In a standard procedure, 5 g of (4) was separated by magnet and the supernatant was discarded. The magnetic nanoparticles were washed three times with a hypercrosslinking solvent (dichloroethane, toluene, DMF, MeCN, dioxane, or THF) and then resuspended in 60 mL of a selected solvent. The suspension was stirred for 30 minutes and then heated to 80 °C. When the temperature was reached, a catalyst (FeCl3 or ZnCl2; 12 mmol) was added and nitrogen was bubbled through the suspension. After 1 h, the particles were separated by magnet and the supernatant was discarded. The particles were washed five times with ethanol porous magnetic polymer particles (5) (4.8 g) was obtained.

[0291] OH-functionalized porous magnetic polymer particles (6)

[0292] In a standard procedure, 4.8 g of (5) as synthesized was suspended in 60 mL of KOH solution (6 M in H2O). The suspension was stirred at 60 °C for 16 h. The particles were separated by magnet and the supernatant was discarded. The particles were washed several times with water until the pH reached 7. OH-functionalized porous magnetic polymer particles (6) (4.8 g) was obtained.

[0293] COOH-functionalized porous magnetic polymer particles (7)

[0294] In a standard procedure, 1 g of (6) as synthesized was suspended in 20 mL of NaClO solution (10-15% available chlorine in H2O). The suspension was stirred at 60 °C for 1.5 h. The particles were separated by a magnet and the supernatant was discarded. The particles were washed three times with water. COOH-functionalized porous magnetic polymer particles (7) (1 g) was obtained.

[0295] C x - Functionalized porous magnetic polymer particles (8)

[0296] In a standard procedure, 1 g of (7) was used as synthesized and washed twice with MES-buffer (2-morpholine-4-ylethanesulfonic acid; 0.1 M; pH 5.0). The particles were resuspended in 40 mL of MES-buffer (0.1 M; pH 5.0), and 6.6 mL of EDC-solution (3-(ethyliminomethyleneamino)-N,N-dimethylpropane-1-amine; 10% w / w in H2O) and 39.6 mL of sulfo-NHS-solution (N-hydroxysulfosuccinimide; 0.03 M in H2O) were added. The suspension was stirred at 11 °C for 35 minutes. The particles were separated by magnet, and the supernatant was discarded. The particles were washed twice with MES-buffer, and 46 mL of C x H 2x+1-NH2- solution (x = 4, 6, 8, or 10; 1 / 6 v / v in ethanol) and 49.5 mL of potassium phosphate-buffer (0.1 M; pH 7.5) were added. The suspension was stirred at 25 °C for 2.5 h. The particles were separated by magnet, and the supernatant was discarded. The particles were washed three times with ethanol and twice with potassium phosphate-buffer (0.02 M; pH 7.0), C x - Functionalized porous magnetic polymer particles (8) (1 g) was obtained.

[0297] Epoxy-functionalized porous magnetic polymer particles (9)

[0298] In a standard procedure, 1 g of (6) was used as synthesized and suspended in 10 mL of DMF. 20 mL of epichlorohydrin and 10 mL of NaOH solution (3.5 M in H2O) were added. The suspension was stirred at 40 °C for 22 h. The particles were separated by a magnet and the supernatant was discarded. The particles were washed several times with water, Epoxy-functionalized porous magnetic polymer particles (9) (1 g) was obtained.

[0299] The following particles were manufactured:

[0300] Table 1: Manufactured particles

[0301]

[0302]

[0303] Characterization methods

[0304] For particle size analysis, a Mastersizer 2000 (Malvern Instruments) with Hydro 2000 was used. Samples were suspended in water or ethanol before measurement.

[0305] Autosorb iQ (Quantachrome Instruments) was used for surface area and pore size analysis. For activation, the samples were degassed under vacuum at 95 °C for 12 h.

[0306] For magnetization measurements, a 7404-S magnetometer (Lake Shores Cryotronics) was used.

[0307] One major requirement for the automation of particles within enrichment-workflow-MS technology is rapid magnetic separation (<5 s) for high throughput. Particle size and saturation magnetization are critical characteristics. Therefore, high saturation magnetization (>1 A m) 2 kg -1 Particles with high magnetization and large sizes (>2 μm) are required. Additionally, for the robustness of the system, particle carry-over must be avoided. Therefore, the particles must have high magnetization and particle sizes larger than 1 μm.

[0308] This is a major problem with the synthesis routes of magnetic polymer particles just described. The method of Yang et al. is based on low saturation magnetization (4,1 A m 2 kg -1 This describes the synthesis of small particles (< 100 nm) with ). These particles do not satisfy the requirements for automation due to their small particle size and low magnetization characteristics. Since it is difficult to produce particles larger than 1 μm by emulsion polymerization, this synthesis method presents low potential for the production of particles with required characteristics. Xu et al. [demonstrated] high saturation magnetization (14,1 A m 2 kg -1This represents the synthesis of small particles (approx. 400 nm) having ). According to this method, due to the fact that only one Fe3O4-NP core is embedded per polymer particle (mono-core), larger particles can be fabricated simply by increasing the polymer shell, but this will result in a significant reduction in saturation magnetization. Figure 5 shows the calculated saturation magnetization for particles of different sizes synthesized by Xu et al. For example, for the synthesis of particles with a size of 2 μm, the saturation magnetization is 1 A m 2 kg -1 Less than, and 0.01 A m for 10 μm 2 kg -1 It will be less. Such significant loss of magnetization would result in extremely long separation times, making it unacceptable for high-throughput automation. Therefore, these particles do not satisfy the requirements for automation due to their small particle size and the synthesis of mono-core submicron particles, which result in low magnetization characteristics.

[0309] After polymerization, there is a hypercrosslinking reaction necessary to create porosity within the particles and, consequently, high surface areas. One disadvantage is the formation of HCl during this chemical reaction. HCl attacks Fe3O4-NPs and thus reduces the magnetization of the particles. For this reaction step, the coating thickness and type of the coating on the Fe3O4-NPs are essential. Yang et al. use a coating with oleic acid, while Xu et al. protect Fe3O4-NPs with citrate.

[0310] Example 2: Analyte capturing using magnetic beads

[0311] Analytical capture

[0312] For bead evaluation, the beads underwent a concentration workflow as exemplified in Fig. 6. Spiked serum pools were used as test samples, and the spiked analytes are listed in the following Table 2.

[0313] Table 2: List of analytes tested with CPS particles

[0314]

[0315] Preparation of samples

[0316] Samples were prepared by spiked 13 analytes of interest into an analyte-free human serum pool. The internal standard solution was a 50:50 v / v methanol / water mixture containing isotope-labeled analogs of the target analytes.

[0317] Bead extraction

[0318] For each sample, 100 μL of serum was mixed with 50 μL of bead suspension and water to a concentration of 50 mg / mL and equilibrated at room temperature for 5 minutes under appropriate rolling conditions to allow the analytes to access the entire surface of the access particles. In the cases of analyzing 25OH-Vitamin D3, an additional step was included in which 100 μL of pre-treatment solution was added to the mixture and incubated for 15 minutes. Afterward, the supernatant was discarded, and the magnetic beads were washed twice with 200 μL of water. Elution was performed with 100 μL of acetonitrile / 2% formic acid in water at 70:30 v / v. In the next step, 80 μL of the eluent was recovered from the vial and transferred to an HPLC vial, and 5 μL of internal standard solution was added prior to LC-MS / MS analysis.

[0319] recovery rate

[0320] Quantification was performed by external calibration. To this end, the calibration curve was recorded in a pure solution. The recovery rate was calculated by comparing the calculated concentration in the eluent fraction with the spiked amount.

[0321] An additional aspect to be characterized was the eluent purity for residual proteins as classified according to the following:

[0322] Residual protein: Excellent < 1.0% (< 855 μg / mL)

[0323] Intermediate 1.0 - 2.0% (855 - 1710 μg / mL)

[0324] Defective > 2.0% (> 1710 μg / mL)

[0325] For almost all samples, residual protein was less than 1%, showing excellent results for the removal of substrate effects.

[0326] Table 3 below shows the capture characteristics of the different bead types presented above. As can be seen, the capture efficiency for different analyte classes varies with different monomer compositions, providing the possibility for customized bead design.

[0327] Table 3: Capture characteristics of different beads

[0328]

[0329]

Claims

Claim 1 A magnetic particle comprising a polymer substrate (P) and at least two magnetic cores (M), wherein the polymer substrate comprises a hypercrosslinked polymer, the magnetic particle has a particle size in the range of 10 to 40 micrometers, the surface of the particle is functionalized with -OH, and the polymer substrate (P) comprises a copolymer obtained by a method comprising: (a) copolymerizing suitable monomer building blocks in the presence of one monomer building block that is a crosslinking agent, wherein 5-90 vol% of all monomer building blocks are divinylbenzenes, and in the copolymerization a polymer having at least 5% crosslinking is obtained, wherein divinylbenzene is the crosslinking agent, and the suitable monomer building blocks are vinylbenzyl chloride and divinylbenzene, and these form a crosslinked polymer substrate formed from monomer building blocks composed of vinylbenzyl chloride and divinylbenzene, and the at least two magnetic cores (M) are at least two iron A step comprising oxide nanoparticles and a coating C1, wherein the coating C1 is a surfactant or a silica coating; (b) a step of over-crosslinking a crosslinked polymer substrate, wherein the over-crosslinking in (b) is performed via a Friedel-Crafts reaction, the reaction in (b) is not performed in a solvent containing dichloroethane or other organic halides, the reaction in (b) is performed in a solvent containing at least THF, acetonitrile, DMF, dioxane, or toluene, the reaction in (b) is performed for a reaction time of 4 h or less, and the over-crosslinking in (b) is performed at a temperature in the range of -30 to 120°C; wherein the magnetic particles are at least 5 A m 2 Magnetic particle having a saturation magnetization of / kg. Claim 2 In claim 1, the polymer substrate comprises pores having a pore size of less than 100 nm or 50 nm or less, a magnetic particle. Claim 3 A magnetic particle according to claim 2, wherein at least 90% of all pores present in the polymer substrate (P) have a pore size smaller than 10 nm, and at least 50% of all pores present in the polymer substrate have a pore size smaller than 5 nm. Claim 4 In claim 2, the polymer substrate (P) is a magnetic particle that does not contain large pores having a pore size larger than 50 nm. Claim 5 In claim 1, the magnetic particle is at least 10 A m 2 Magnetic particle having a saturation magnetization of / kg. Claim 6 In claim 1, the magnetic particle is a superparamagnetic magnetic particle. Claim 7 In claim 1, the at least two magnetic cores (M) are magnetic particles comprising Fe3O4 nanoparticles. Claim 8 In claim 1, the at least two magnetic cores (M) comprise a superparticle and a coating C1, or are composed of magnetic particles. Claim 9 In claim 8, the superparticle is a magnetic particle composed of aggregated nanoparticles, or more than 20 aggregated nanoparticles, or 1 million to 1.5 million nanoparticles. Claim 10 In claim 9, at least one coating C2 is deposited on the surface of nanoparticles, said coating C2 is selected from the group consisting of dicarboxylic acids, dicarboxylic acid salts, dicarboxylic acid derivatives, polyacrylic acid, polyacrylic acid salts, polyacrylic acid derivatives, tricarboxylic acids, tricarboxylic acid salts, tricarboxylic derivatives, amino acids, amino acid salts, amino acid derivatives, surfactants, salts of surfactants, fatty acids, fatty acid salts, and fatty acid derivatives. Claim 11 In claim 1, the reaction in (b) is carried out in a solvent selected from the group consisting of THF, acetonitrile, DMF, dioxane, toluene, and mixtures of two or more of these, magnetic particles. Claim 12 In claim 1, the reaction in (b) is carried out in a solvent selected from the group consisting of THF, acetonitrile, dioxane, toluene, and mixtures of two or more of these, magnetic particles. Claim 13 In claim 1, the magnetic particle, wherein the over-crosslinking in (b) is performed in the presence of a catalyst selected from the group consisting of Lewis acids. Claim 14 In claim 1, the magnetic particle, wherein the over-crosslinking in (b) is performed in the presence of a catalyst selected from the group consisting of FeCl3, ZnCl2, AlCl3, BF3, SbCl5, SnCl4, TiCl4, SiCl4, and mixtures of two or more of these. Claim 15 In claim 1, the magnetic particle, wherein the over-crosslinking in (b) is performed at a temperature in the range of -30 to 80°C. Claim 16 A method for manufacturing magnetic particles comprising a polymer substrate (P) and at least one magnetic core (M) or at least two magnetic cores (M), wherein the polymer substrate (P) comprises at least one cross-linked polymer, and the magnetic particles have a particle size in the range of 5 to 40 micrometers, and the method comprises: (i) providing at least one magnetic core (M) or at least two magnetic cores (M); (ii) providing polymer precursor molecules; (iii) polymerizing the polymer precursor molecules according to (ii) in the presence of the at least one magnetic core (M) to form a particle comprising the at least one magnetic core (M) or at least two magnetic cores (M) embedded in a polymer substrate (P1); and (iv) over-crosslinking the polymer substrate (P1) of the polymer particles obtained in (iii) through a Friedel-Crafts reaction to provide the magnetic particles, wherein the at least one magnetic core (M) comprises at least one iron oxide A method for producing magnetic particles comprising nanoparticles and coating C1, wherein coating C1 is a surfactant or silica coating, and the reaction in (iv) is not carried out in a solvent comprising dichloroethane or other organic halides, and the method further comprises the step of functionalizing the surface of the polymer particles obtained according to (v) (iii) or (iv), wherein in (iii), the polymer substrate (P1) comprises a crosslinked polymer obtained by crosslinking the polymer with a divinylbenzene crosslinking agent in an amount of 5-90 vol% based on the total amount of polymer, wherein at least 5% degree of crosslinking is obtained in the prepared polymer, and divinylbenzene is the crosslinking agent, and the over-crosslinking in (iv) is carried out at a temperature in the range of -30 to 120°C, and the reaction in (iv) is carried out in a solvent comprising at least THF, acetonitrile, DMF, dioxane, or toluene, and in (v), the polymer particles are functionalized with -OH. method. Claim 17 A method for manufacturing magnetic particles according to claim 16, wherein the polymer substrate (P1) comprises a cross-linked polymer or is composed thereof. Claim 18 A magnetic particle comprising a polymer substrate (P) and at least two magnetic cores (M), wherein the polymer substrate (P) comprises at least one cross-linked polymer, and the magnetic particle has a particle size in the range of 10 to 40 micrometers, and is obtained or obtainable by the following method: (i) providing at least two magnetic cores (M); (ii) providing polymer precursor molecules; (iii) polymerizing the polymer precursor molecules according to (ii) in the presence of the at least two magnetic cores (M) to form a particle comprising at least two magnetic cores (M) embedded in a polymer substrate (P1); and (iv) super-crosslinking the polymer substrate (P1) of the polymer particle obtained in (iii) through a Friedel-Crafts reaction to provide the magnetic particle; wherein the at least two magnetic cores (M) comprise at least two iron oxide nanoparticles and a coating C1; and wherein the coating C1 is a surfactant or silica coating; (iv) the reaction is not carried out in a solvent containing dichloroethane or other organic halides; the method further comprises the step of (v) functionalizing the surface of the polymer particles obtained according to (iii) or (iv); (iii) the polymer substrate (P1) comprises a crosslinked polymer obtained by crosslinking the polymer with 5-90 vol% of a divinylbenzene crosslinking agent based on the total amount of polymer, wherein at least 5% degree of crosslinking is obtained in the prepared polymer; and divinylbenzene is the crosslinking agent; and (iii) the monomer building blocks are vinylbenzyl chloride and divinylbenzene, and these form a crosslinked polymer substrate formed from monomer building blocks composed of vinylbenzyl chloride and divinylbenzene;(iv) Over-crosslinking is performed in a solvent containing at least THF, acetonitrile, DMF, dioxane, or toluene, and (iv) over-crosslinking is performed for a reaction time of 4 h or less; (iv) over-crosslinking is performed at a temperature in the range of -30 to 120°C; and (v) polymer particles are functionalized with -OH.; Claim 19 In any one of claims 1 to 15 or 18, the magnetic particle is a magnetic particle used for the qualitative and / or quantitative determination of at least one analyte in a fluid. Claim 20 In claim 19, the analyte is a magnetic particle selected from the group of steroids, vitamins, drugs, sugars, organic compounds, proteins, nucleic acids, and mixtures of two or more of these. Claim 21 A method for determining at least one analyte in a fluid sample, comprising: a) contacting a magnetic particle of any one of claims 1 to 15 or 18 with a fluid sample that contains or is suspected of containing said at least one analyte; and b) determining said at least one analyte bound to said magnetic particle. Claim 22 A method for determining at least one analyte in a fluid sample, wherein the analyte is selected from the group of steroids, vitamins, drugs, sugars, organic compounds, proteins, nucleic acids, and mixtures of two or more of these. Claim 23 delete Claim 24 delete Claim 25 delete

Images