NANO- OR MICROPARTICLE COMPRISING A POLYVINYL ALCOHOL MATRIX, AND, DISPERSED THEREIN, FERRITE, ITS PROCESS OF OBTAINING AND ITS USES

Abstract

The present invention relates to a nano- or microparticle comprising a matrix made of or comprising at least one polyvinyl alcohol (PVA), and ferrite dispersed therein, as well as a method for obtaining it. The present invention also relates to the use of these nano- or microparticles for the preparation and implementation of devices capable of being detected by giant magnetoresistance sensors (GMR sensors) as biological diagnostic tools.

Description

Title of the invention: NANO- OR MICROPARTICLE COMPRISING A POLYVINYL ALCOHOL MATRIX, AND, DISPERSED THEREIN, FERRITE, ITS PROCESS OF OBTAINING AND ITS USES

[0001] The present invention relates to a nano- or microparticle comprising a matrix made of or comprising at least one polyvinyl alcohol (PVA), and ferrite dispersed therein, as well as a method for obtaining it. The present invention also relates to the use of these nano- or microparticles for the preparation and implementation of devices capable of being detected by giant magnetoresistance sensors (GMR sensors) as biological diagnostic tools.

[0002] Clinical diagnostics is a field in which new analytical methods that are faster, more direct, more precise, selective, and, moreover, high-throughput and inexpensive, are in high demand. Due to their small size, ultrasensitive transduction, and potential integration into "lab-on-a-chip" systems, biosensing devices manufactured using nanotechnology are a potential candidate to meet all the above requirements. Over the past decade, much work has been done on biomagnetism and magnetic biosensors based on molecular processes, particularly on the application of magnetic nanoparticles in biomedicine, their synthesis, functionalization, and detection by magnetic sensors.

[0003] Magnetoresistive sensors appear to be among the best candidates for meeting these criteria. Since the late 1990s, ultra-sensitive giant magnetoresistive sensors (GMR sensors) have been excellent candidates for magnetic detection in a wide variety of fields, including healthcare. These are highly sensitive spin-electronic sensors. They offer the advantage of very high detectivity (detectiveness corresponds to the magnetic field for which the signal-to-noise ratio is 1; it is expressed in T / Hz and allows for easy comparison of the performance of different magnetic sensors), on the order of 50 to 200 pT / Hz, low cost, and the ability to be easily integrated into lab-on-a-chip devices.

[0004] In this field, biological targets are magnetically labeled by magnetic nano- or microparticles functionalized with specific antibodies directed against the biological target of interest. These magnetically marked objects are then detected dynamically (or statically) by GMR sensors. This technology is based on the detection of magnetic markers.

[0005] In recent years, GMR sensors have shown great potential as sensors for detecting biological targets (cells or bacteria). The resistance of a GMR sensor varies depending on the magnetic field applied to the sensor, so that a magnetically labeled biological object can induce a signal. Compared to traditional optical detection, widely used in biomedicine, GMR sensors allow working with complex, even opaque, matrices and enable the dynamic detection of magnetic objects one by one. Furthermore, a new biochip, based on GMR sensors arranged on either side of the microfluidic channel, makes it possible to determine the magnetic moment of the detected objects and thus identify a large number of false positives resulting from the presence of aggregates of magnetic nanoparticles (patent FR3082620).However, the hurdle to overcome in order to further increase the sensitivity of such a detection technique is to reduce the number of these aggregates of magnetic nanoparticles which still blend in with the labeled biological targets of interest.

[0006] Due to the advantages of GMR materials for magnetic field measurements, such as high sensitivity and fast response under a low magnetic field, increased attention has been paid to the development of these GMR materials for biosensors.

[0007] The use of magnetic particles for applications in biotechnology, the pharmaceutical industry, and medicine has become increasingly common. Magnetic particles emerged some twenty years ago and are currently experiencing significant growth, closely linked to the goal of miniaturizing analyses. Their size, ranging from nanometers to micrometers, and the possibility of manipulating them with a simple magnetic field, make them a tool well-suited to the manipulation and identification of biological molecules. Numerous companies supply a wide variety of microbeads onto the surface of which all kinds of molecules of interest can be specifically grafted.

[0008] In current applications, the magnetic particles used are mostly superparamagnetic, that is to say that they have no magnetic remanence, that the internal field is zero in the absence of an external magnetic field, which theoretically limits aggregation phenomena.

[0009] However, most commercial magnetic nanoparticles do not have high colloidal stability and form aggregates during analyses performed with them. And as mentioned above, the presence of magnetic nanoparticle aggregates leads to false positives that limit sensitivity and specificity. diagnostic tests. In particular, early diagnosis by GMR sensor is extremely sensitive, magnetically labeled biological objects can be detected one by one, but this sensitivity, as well as the specificity of the tests, is degraded by the formation of non-specific aggregates, i.e. not specifically linked to the biological target under study but only agglutinated for physico-chemical reasons.

[0010] The present invention aims to provide nano- or microparticles that avoid the aforementioned drawbacks.

[0011] Thus, one object of the invention is to provide magnetic nano- or microparticles having high colloidal stability, making it possible to overcome the problem of aggregation, in particular during early diagnostic tests involving them.

[0012] Another object of the invention is to provide nano- or microparticles having a magnetic moment equivalent to that of commercial beads, while making it possible to overcome the aggregation problem encountered with these commercial beads.

[0013] Another object of the invention is to provide monodisperse nano- or microparticles, by a simple and inexpensive process, which can be easily functionalized, in particular for the preparation and implementation of devices capable of being detected by giant magnetoresistance sensors (GMR sensors) as biological diagnostic tools.

[0014] Thus, according to a first aspect, the invention relates to a nano- or microparticle comprising a matrix made up of or comprising at least one polyvinyl alcohol (PVA), and, dispersed in the latter, ferrite.

[0015] By "nanoparticle", we mean in particular, unless otherwise stated, a particle having a size from 1 to 1000 nm, in particular from 50 to 500 nm, more particularly from 100 to 350 nm.

[0016] By "microparticle", we mean in particular, unless otherwise stated, a particle having a size from 1 to 100 pm, in particular from 1 to 10 pm, more particularly from 1 to 5 pm.

[0017] By “ferrite”, we mean in particular a compound of formula Fe3O4.

[0018] By "dispersion", it is understood in particular that the ferrite is distributed in the form of a plurality of particles in the PVA matrix.

[0019] According to a particular embodiment, the molecular mass of polyvinyl alcohol is from 10000 to 150000, in particular from 50000 to 90000.

[0020] A PVA that is too large may increase the viscosity of the particle formation medium, making it very heterogeneous. With a PVA of too low a molecular weight, it is possible that the Fe3O4 nuclei may coalesce, resulting in a mixture of particles of varying sizes, some of which may be larger than one micron.

[0021] According to a particular embodiment, the matrix is ​​devoid of polyvinylpyrrolidone (PVP).

[0022] By "devoid of PVP", it is meant that the matrix does not include PVP.

[0023] According to a particular embodiment, polyvinyl alcohol (PVA) is partially acetylated. In this case, it corresponds to a partially hydrolyzed polyvinyl acetate (or poly(vinyl acetate), or PVAC). More specifically, polyvinyl alcohol (PVA) is acetylated to less than 20% (that is, less than 20% of the -OH groups of PVA are acetylated), for example, less than 10% or about 10%, which corresponds to a poly(vinyl acetate) hydrolyzed to more than 80% (that is, more than 80% of the -OAc groups of PVAC are hydrolyzed), for example, more than 90% or about 90%.

[0024] According to another particular embodiment, the polyvinyl alcohol (PVA) is not acetylated. In this case, it corresponds to a totally hydrolyzed polyvinyl acetate (or poly(vinyl acetate), or PVAC).

[0025] According to a particular embodiment, the polyvinyl alcohol is crosslinked, for example, particularly on the surface of the nano- or microparticle, in particular using a crosslinking agent selected from the following compounds: boric acid, boronic acids, boric acid esters, boronic acid esters, borax (Na2B4O7.10H2O), and aldehydes, in particular from compounds of the following formulas: B(OR)3, RB(OH)2, B(OH)3, R-CHO, in which R is selected from aryls and heteroaryls, and compounds comprising at least two aldehyde functions, in particular compounds comprising two aldehyde functions, more particularly compounds of the formula HC(=O)-(CH2)nC(=O)-H, with n ranging from 0 to 6, for example n = 0, 1, 2 or 3, the crosslinking agent being, for example, 3-quinoline acid boronic. When PVA is crosslinked, it is likely to be insensitive to the presence of poor solvents, such as ethanol, particularly with regard to its volume.

[0026] According to a particular embodiment, the nano- or microparticle according to the present invention has a sphere or spheroid shape.

[0027] By "spheroid" is meant in particular a particle whose shape is close to the sphere, but not perfectly spherical, and in particular that all the dimensions of the particle are between 0.9 and 1 times the largest dimension of said particle.

[0028] According to a particular embodiment, the nano- or microparticle according to the present invention has a size of 50 to 1000 nm, in particular from 150 to 450 or 500 nm.

[0029] This size corresponds in particular to a nano- or microparticle whose PVA is free of solvent, in particular water, i.e. for example dried overnight at 100°C.

[0030] If necessary, the size of the nano- or microparticle can be modified: it can be decreased by adding a bad solvent such as ethanol, or increased (by hydration) with a good solvent, such as water.

[0031] By "size" we mean in particular the largest dimension of the particle.

[0032] According to a particular embodiment, at least one polyvinyl alcohol is present in the nano- or microparticle according to the present invention at a level of 30 to 50% by mass relative to the total mass of said nano- or microparticle.

[0033] According to a particular embodiment, the nano- or microparticle according to the present invention has a density of 1.7 to 2.5 g.cm3, the density being for example about 1.87 g.cm3.

[0034] According to a particular embodiment, the ferrite is in the form of crystallites.

[0035] By "crystallites", we mean in particular domains of matter having the same structure than a single crystal.

[0036] According to a particular embodiment, the ferrite is in the form of particles having an average size tA of 15 to 80 nm, in particular 15 to 60 nm, more particularly 25 to 30 nm.

[0037] By "size", we mean in particular the diameter of the particle when it is spherical, or the largest dimension when it is not.

[0038] The average size can for example be measured by image analysis carried out by scanning electron microscopy (SEM).

[0039] According to a particular embodiment, the ferrite is in the form of particles whose size distribution has a uniformity coefficient of 0.95 to 1.

[0040] According to a particular embodiment, the ferrite is in the form of particles of which more than 95% have a size of 28 nm + / - 10%.

[0041] According to a particular embodiment, the nano- or microparticle according to the present invention has a magnetic moment between 3.10 15 and 5.10 15Am2.

[0042] According to another aspect, the present invention also relates to a set of nano- or microparticles as defined above.

[0043] All embodiments described above relating to the nano- or microparticle can also be applied here, alone or in combination.

[0044] According to a particular embodiment, the size distribution of the set of nano- or microparticles of the present invention has a uniformity coefficient of 0.95 to 1.

[0045] According to a particular embodiment, the size distribution of the set of nano- or microparticles of the present invention is such that more than 95% of these nano- or microparticles have a size of 180 nm + / - 10%.

[0046] According to another aspect, the present invention also relates to a method for preparing a nano- or microparticle as defined above, or an assembly of nano- or microparticles as defined above, comprising:

[0047] (i) a step of contacting at least one polyvinyl alcohol (PVA) with a composition A is a ferrite precursor to obtain composition B;

[0048] (ii) a heating step of composition B to obtain said nano- or microparticles.

[0049] According to a particular embodiment, composition A comprises an iron (III) salt, optionally hydrated.

[0050] According to a more particular embodiment, composition A comprises a compound selected from FeCl3, Fe(NO3)3, Fe(C104)3, which are optionally hydrated, the compound being preferably selected from FeCl3, hydrated Fe(NO3)3 salts, and hydrated Fe(C104)3 salts.

[0051] In particular, composition A comprises said iron (III) salt, optionally hydrated, and is devoid of iron (II) salt, hydrated or not.

[0052] According to a particular embodiment, composition A comprises urea or hexamethylenetetramine, in particular urea.

[0053] According to a particular embodiment, composition A comprises a water-soluble iron chelator.

[0054] Said water-soluble iron chelating agent is in particular selected from citric acid, citrates, lactic acid, lactates, tartaric acid, and tartrates.

[0055] According to a particular embodiment, composition A comprises urea or hexamethylenetetramine, in particular urea, and a water-soluble iron chelator, in particular selected from citric acid, citrates, lactic acid, lactates, tartaric acid, and tartrates.

[0056] According to a particular embodiment, the contacting step (i) is carried out in the presence of water, the composition B obtained at the end of said step (i) being in particular an aqueous solution.

[0057] According to a particular embodiment, the heating step (ii) is carried out in an autoclave.

[0058] According to a particular embodiment, the heating step (ii) is carried out at a temperature between 180 and 230 °C, and / or for a duration of 6 to 24 hours.

[0059] According to a particular embodiment, after step (ii), and where appropriate before step (iii) as described below, at least one polyvinyl alcohol (PVA) is crosslinked, in particular using a crosslinking agent selected among the following compounds: boric acid, boronic acids, boric acid esters, boronic acid esters, borax (Na2B4O7.10H2O), and aldehydes, in particular among the compounds of the following formulas: B(OR)3, RB(OH)2, B(OH)3, R-CHO, in which R is selected from the aryls and heteroaryls, and compounds comprising at least two aldehyde functions, in particular compounds comprising two aldehyde functions, more particularly compounds of formula HC(=O)-(CH2)nC(=O)-H, with n ranging from 0 to 6, for example n = 0, 1, 2 or 3, the crosslinking agent being for example 3-quinoline boronic acid.

[0060] According to another aspect, the present invention also relates to the use of a nano- or microparticle as defined above, or of an assembly of nano- or microparticles as defined above, for the preparation of devices suitable for detection by a giant magnetoresistance sensor.

[0061] In all that follows, embodiments relating to a particle also apply to a set of such particles.

[0062] According to another aspect, the present invention also relates to a nano- or microparticle comprising a matrix made up of or comprising at least one polyvinyl alcohol (PVA), and, dispersed in the latter, ferrite, and further comprising an outer layer of silica.

[0063] According to a particular embodiment, said outer layer of silica has a thickness of 10 to 150 nm, in particular 15 to 30 nm, or 30 to 150 nm.

[0064] The presence of this outer layer of silica can allow the cores comprising PVA and ferrite to be kept apart, which is likely to give the particles, if necessary, an advantageous stability.

[0065] According to another aspect, the present invention also relates to a method for preparing a nano- or microparticle as defined above, comprising:

[0066] (i) a step of contacting at least one polyvinyl alcohol (PVA) with a composition A is a ferrite precursor to obtain composition B;

[0067] (ii) a heating step of composition B to obtain said nano- or microparticles;

[0068] (iii) a step of depositing a layer of silica on the nano- or microparticle as obtained at the end of the previous step by sol-gel synthesis, in particular using a silica precursor selected from tetraethylorthosilicate and tetramethylorthosilicate, optionally in the presence of aminopropyltriethoxysilane.

[0069] According to another aspect, the present invention also relates to a nano- or microparticle comprising a matrix made of or comprising at least one polyvinyl alcohol (PVA), and, dispersed therein, ferrite, and including further an outer layer of silica, which has on its surface a layer of Al(OH)3.

[0070] According to a particular embodiment, said Al(OH)3 layer has a thickness of 1 to 10 nm, in particular 3 to 5 nm.

[0071] According to another aspect, the present invention also relates to a nano- or microparticle comprising a matrix made of or comprising at least one polyvinyl alcohol (PVA), and, dispersed therein, ferrite, and further comprising an outer layer of silica, which is functionalized by compounds bearing carboxylic acid (-COOH) or carboxylic acid anhydride (-C(=O)-OC(=O)-) groups, in particular compounds: - consisting of or comprising a phosphonic acid in which the phosphorus bears a linear or branched Ci-C6 alkyl, substituted by a carboxylic acid group (-COOH); - consisting of or comprising an arene bearing two carboxylic acid anhydrides (-C(=O)-OC(=O)-), possibly hydrolyzed.

[0072] Compounds comprising an arene are likely to be particularly rigid and thus to move the carboxylic acid group away from the surface of the particle, which may allow, if necessary, a particularly efficient coupling.

[0073] According to a particular embodiment, the present invention relates to a nano- or microparticle comprising a matrix made of or comprising at least one polyvinyl alcohol (PVA), and, dispersed therein, ferrite, and further comprising an outer layer of silica, which bears on its surface a layer of Al(OH)3 functionalized in part by compounds bearing carboxylic acid (-COOH) or carboxylic acid anhydride (-C(=O)-OC(=O)-) groups, in particular compounds: - consisting of or comprising a phosphonic acid in which the phosphorus bears a linear or branched Ci-C6 alkyl, substituted by a carboxylic acid group (-COOH); - consisting of or comprising an arene bearing two carboxylic acid anhydrides (-C(=O)-OC(=O)-), possibly hydrolyzed.

[0074] According to a particular embodiment, the present invention relates to a nano- or microparticle comprising an outer layer of silica, which has on its surface a layer comprising aluminium, in the form of Al(OH)3 or forming a complex of one of the following formulas:

[0075] [Chem.l] % / s Ai AI, ;p{CH2rC00H O—.....Z °'6' * , O

[0076] with n ranging from 1 to 6, in particular 1 or 2,

[0077] and mixtures thereof.

[0078] According to another aspect, the present invention also relates to a method for preparing a nano- or microparticle as defined above, comprising:

[0079] (i) a step of contacting at least one polyvinyl alcohol (PVA) with a composition A is a ferrite precursor to obtain composition B;

[0080] (ii) a heating step of composition B to obtain said nano- or microparticles;

[0081] (iii) a step of depositing a layer of silica on the nano- or microparticle as obtained at the end of the previous step by sol-gel synthesis, in particular using a silica precursor selected from tetraethylorthosilicate and tetramethylorthosilicate, optionally in the presence of aminopropyltriethoxysilane;

[0082] (iv) a step of contacting a nano- or microparticle such as obtained at the end of the previous step with an aluminium salt, in particular selected from A1C13, A1(NO3)3, A1(C1O4)3, which are optionally hydrated, the aluminium salt being preferably selected from A1C13, hydrated A1(NO3)3 salts, and hydrated A1(C1O4)3 salts, to obtain a nano- or microparticle comprising an outer layer of silica, which has on its surface a layer of Al(OH)3;

[0083] (v) a step of contacting the nano- or microparticle obtained at the end of the previous step with a compound: • consisting of or comprising a phosphonic acid in which the phosphorus bears a linear or branched Ci-C6 alkyl, substituted by a carboxylic acid group (-COOH); • consisting of or comprising an arene bearing two carboxylic acid anhydrides (-C(=O)-OC(=O)-); • for example, the following formula:

[0084] [Chem.2]

[0085] According to another aspect, the present invention also relates to a nano- or microparticle comprising a matrix made up of or comprising at least one polyvinyl alcohol (PVA), and, dispersed in the latter, ferrite, and further comprising an outer layer of silica, which is functionalized by compounds bearing a macromolecule, in particular a monoclonal antibody,

[0086] said nano- or microparticle comprising in particular an outer layer of silica, which is functionalized by compounds bearing a macromolecule, in particular a monoclonal antibody, via an amide function formed between a carboxylic acid borne by said compounds and an amine borne by said macromolecule.

[0087] According to another aspect, the present invention also relates to a method for preparing a nano- or microparticle as defined above, comprising:

[0088] (i) a step of contacting at least one polyvinyl alcohol (PVA) with a composition A is a ferrite precursor to obtain composition B;

[0089] (ii) a heating step of composition B to obtain said nano- or microparticles;

[0090] (iii) a step of depositing a layer of silica on the nano- or microparticle as obtained at the end of the previous step by sol-gel synthesis, in particular using a silica precursor selected from tetraethylorthosilicate and tetramethylorthosilicate, optionally in the presence of aminopropyltriethoxysilane;

[0091] (iv) a step of contacting a nano- or microparticle such as obtained at the end of the previous step with an aluminium salt, in particular selected from A1C13, A1(NO3)3, A1(C1O4)3, which are optionally hydrated, the aluminium salt being preferably selected from A1C13, hydrated A1(NO3)3 salts, and hydrated A1(C1O4)3 salts, to obtain a nano- or microparticle comprising an outer layer of silica, which has on its surface a layer of Al(OH)3;

[0092] (v) a step of contacting the nano- or microparticle obtained at the end of the previous step with a compound: • consisting of or comprising a phosphonic acid in which the phosphorus bears a linear or branched Ci-C6 alkyl, substituted by a carboxylic acid group (-COOH); • consisting of or comprising an arene bearing two carboxylic acid anhydrides (-C(=O)-OC(=O)-); • for example, the following formula:

[0093] [Chem.3]

[0094] (vi) a step of forming an amide function between the carboxylic acid groups carried by the nano- or microparticle as obtained at the end of the previous step and an amine carried by said macromolecule, in particular by the formation of intermediate activated esters from said carboxylic acids, for example by using l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0095] According to a particular embodiment, step (vi) is carried out at a pH between 4 and 5, in particular around 4.5.

[0096] According to a particular embodiment, the preparation processes as described above are partially or totally carried out in the absence of magnetic fields. More specifically, the steps described above, in particular step (ii) as described above, are carried out in the absence of magnetic fields.

[0097] By "absence of magnetic fields" is meant in particular the fact of not approaching a magnet, in particular not using magnetic stirring or magnetized devices such as a magnetized autoclave.

[0098] According to another aspect, the present invention also relates to a nano- or microparticle as defined above, or an assembly of nano- or microparticles as defined above, for its use in diagnostics, in particular for the detection of bacteria, in particular in clinical samples without preculture, for example whole blood or other biological fluids; or cancer cells.

[0099] The bacteria are in particular pathogenic bacteria well known to those skilled in the art, in particular of environmental and / or public health interest, for example Yersinia enterocolitica (likely to be responsible in particular for gastroenteritis, food poisoning) and Legionella pneumophila (likely to be responsible in particular for pneumonia by inhalation of microdroplets).

[0100] DEFINITIONS

[0101] As used in this description, the term "approximately" refers to a range of values ​​within ±10% of a specific value. For example, the expression "approximately 20" includes values ​​within 20 ±10%, that is, values ​​from 18 to 22.

[0102] For the purposes of this description, percentages refer to percentages by mass relative to the total mass of the formulation, unless otherwise stated.

[0103] As understood here, value ranges in the form of "xy" or "from x to y" or "between x and y" include, in particular, the bounds x and y as well as the integers between these bounds. For example, "1-5" or "from 1 to 5" refers, in particular, to the integers 1, 2, 3, 4, and 5. Preferred embodiments include each integer taken individually in the value range, as well as any subcombination of these integers. For example, preferred values ​​for "1-5" may include the integers 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, etc.

[0104] As used herein, the term "alkyl" refers to a linear or branched alkyl group having the number of carbon atoms indicated before the term, in particular 2 to 6 carbon atoms, such as ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, neopentyl, 1-ethylpropyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, hexyl, etc. Thus, an expression such as "C1-C4 alkyl" refers to an alkyl radical containing 1 to 4 carbon atoms. The same applies to the term "alkane".

[0105] Cycloalkyls are in particular alkyls (as defined above) comprising a ring. An example is cyclohexyl.

[0106] As used herein, the term "arene" refers to a mono- or bicyclic, substituted or unsubstituted, aromatic hydrocarbon cyclic system having 6 to 32 carbon atoms in the ring. Examples include benzene, naphthalene, anthracene, perylene, chryene, corannulene, phenanthrene, triphenylene, benzo[a]pyrene, coronene, tetracene, pentacene, pyrene, and ovalene. Preferred arenes include benzene, naphthalene, and perylene, whether unsubstituted or substituted. The definition of "arene" includes condensed cyclic systems, including, for example, cyclic systems in which an aromatic ring is condensed to a cycloalkyl ring. Examples of such condensed cyclic systems include, for example, indane, indene, and tetrahydronaphthalene.

[0107] As used here, the term "heteroarene" refers in particular to an aromatic group containing 5 to 10 ring carbon atoms, in which one or more ring carbon atoms are replaced by at least one heteroatom such as -O-, -N-, or -S-. Examples of heteroarene groups include pyrrole, furan, thiene, pyrazole, imidazole, thiazole, and isothiazole. isoxazole, oxazole, oxathiole, oxadiazole, triazole, oxatriazole, furazane, tetrazole, pyridine, pyrazine, pyrimidine, pyridazine, triazine, indole, isoindole, indazole, benzofuran, isobenzofuran, purine, quinazoline, quinole, isoquinole, benzimidazole, benzothiazole, benzothiophene, thianaphthene, benzoxazole, benzisoxazole, cinnoline, phthalazine, naphthyridine, and quinoxaline. The definition of the term "heteroarene" includes fused cyclic systems, including, for example, cyclic systems in which an aromatic ring is fused to a heterocycloalkyl ring. Examples of such fused cyclic systems include, for example, phthalamide, phthalic anhydride, indoline, isoindoline, tetrahydroisoquinoline, chromane, isochromane, chromene and isochromene. FIGURES

[0108] [Fig.1] corresponds to a scanning electron microscopy (SEM) image of the particles obtained in Example 1, dry, as obtained by depositing an aqueous solution of said particles (mother solution) on a silicon plate, then SEM analysis.

[0109] [Fig.2] corresponds to a scanning electron microscopy (SEM) image of the particles obtained in example 1, at a smaller scale than that of [Fig.1].

[0110] [Fig.3] relates to the extraction of the magnetic moment of commercial particles (micromod 50nm beads, ademtech lOOnm beads, ademtech 200nm beads, ademtech 200nm beads, micromod 500nm beads and dyna-Myone lOOOnm) and of particles according to the invention (example 1 and example 3). [YES] EXAMPLES

[0112] Example 1: Synthesis of Fe3O4-PVA beads

[0113] 1 g of PVA is added to 100 ml of DI water. The reaction mixture is heated to 80°C for approximately 1 hour, until the PVA is completely dissolved. The solution is clear and colorless. The temperature is allowed to drop to 50°C, then 4.065 g of FeCl3, 6H2O is added. Dissolution is complete. Another 4.05 g of FeCl3, 6H2O is then added. The solution is now translucent red.

[0114] In parallel, in another container, 8.82g of sodium citrate 2H2O and 2.7g of urea are added to 150ml of DI water.

[0115] The two solutions are mixed at room temperature for 10 minutes. The mixture is orange and cloudy. The resulting suspension is then poured into a Teflon-lined autoclave. 50 mL of dielectric water is added. The autoclave is then heated to 200°C for 24 hours. After 24 hours, the autoclave is allowed to cool. The reaction mixture is a black suspension. The precipitate is collected by centrifugation and washed three times with dielectric water, each wash followed by a centrifugation cycle.

[0116] Approximately 2.2 g of dry matter is recovered (after overnight drying at 100°C in a ventilated oven). The powder is then resuspended in 100 mL of anhydrous EtOH and centrifuged again. The precipitate is again dried overnight at 100°C. 1.9 g of powder is recovered. The atomic absorption spectrometry of the powder gives a mass fraction of 34% Fe, which corresponds to a mass fraction of 47.36% Fe3O4.

[0117] The particles thus obtained have a density of 1.87 kg.m3 compared to 5.17 kg.m3 for Fe3O4.

[0118] SEM imaging ([Fig. 1]) highlights the monodispersity in size of the composite particles of the invention thus obtained. This image also shows the absence of aggregation of the particles of the invention within their mother solution (a biological medium), and therefore their particularly advantageous stability in said medium. Indeed, if aggregation had occurred in solution, it would also have been observed after evaporation of the solution during the acquisition of the SEM image.

[0119] SEM imaging also makes it possible to highlight the size monodispersity of ferrite particles in a composite particle of the invention ([Fig.2]).

[0120] Example 2: Magnetic properties of the particles of the invention

[0121] The magnetic particles were characterized using magnetization measurements as a function of the magnetic field at room temperature. This made it possible to determine the magnetic moment of a magnetic particle of the invention and to compare it to that of commercial magnetic particles of equivalent size (Ademtech beads with a diameter of 200 nm and Micromod beads with a diameter of 500 nm). The magnetic moment obtained for the magnetic particles of the invention (according to Example 1 or 3) is equivalent to that of the commercial beads.

[0122] Example 3: Functionalization according to the invention of Fe3O4-PVA particles

[0123] 1 g of particles such as those obtained in Example 1 are dispersed under mechanical stirring in 200 ml of EtOH abs. In this anhydrous medium, the PVA of the composite is compacted. Then, 0.5 g of a mixture of tetraethyl orthosilicate and aminopropyltriethoxysilane in a molar ratio TEOS / APTES = 20 / 1 is added under stirring (F APTES is likely to serve as a catalyst to initiate the polymerization of the silica layer). After 10 minutes of homogenization, 1 g of H2O is stirred. The medium is then left under mechanical stirring for 12 hours at room temperature. The product is recovered by centrifugation and washed three times with DI water. The zeta potential of the particles goes to -20 -30 mV, illustrating that a silica layer has been built up on the surface of the particles.

[0124] Optionally, the PVA of the Fe3O4-PVA core can be crosslinked by reacting a PVA crosslinking agent: for example, B(OR)3, RB(OH)2, B(OH)3, or Na2B4O7·10H2O before growing the silica layer. The advantage of compounds such as RB(OH)2 is It is possible, if desired, to graft another organic function onto the Fe3O4-PVA PVA, notably a fluorescent dye. 3-Quinoline boronic acid has thus been used successfully: 1 g of Fe3O4-PVA particles are dispersed under mechanical stirring in 200 ml of H2O DI. Then, 0.2 g of 3-quinoline boronic acid in 50 ml of THF is added under stirring. The mixture is then left under mechanical stirring for 12 hours at room temperature. The product is recovered by centrifugation, washed three times with DI water, each wash followed by centrifugation. The product exhibits fluorescence at 540 nm when excited at 400 nm.

[0125] Magnetic particles can carry surface carboxylic acid functions, which can then react, for example with EDC which will allow coupling, with a biological molecule.

[0126] To achieve this, the zeta potential of the silica layer can be changed by hydrolyzing an aluminum salt (for example, Al1C13, Al1(NO3)3). Without limiting ourselves to any particular theory, aminopropyltriethoxysilane, used as a catalyst, is likely to participate in the silica network by leaving dangling primary amine groups onto which aluminum salts can hydrolyze to form an Al(OH)3 layer.

[0127] One gram of SiO2-modified PVA-Fe3O4 particles is dispersed under mechanical stirring in 200 ml of DI water. Then, 0.3 g of a hydrated aluminum salt is added under stirring. The mixture is then left under mechanical stirring for 12 hours at room temperature. The product is recovered by centrifugation and washed three times with DI water. The zeta potential of the particles rises to +15 mV, proving that an Al(OH)3 layer has indeed been built up on the surface of the particles. Without limiting ourselves to any particular theory, the amorphous silica surface layer is likely to inhibit the formation of crystalline gibbsite (Al(OH)3). Without this layer, gibbsite crystals could begin to grow alongside the Fe3O4-PVA particles.

[0128] The surface layer is then functionalized by functional phosphonic acid grafts, so that the magnetic particles carry pendant carboxylic acid functions on the surface.

[0129] To do this, 1 g of SiO2-modified Fe3O4-PVA particles with an Al(OH)3 surface are dispersed under mechanical stirring in 200 ml of H2O DI. Then, 0.2 g of HOOCCH2PO(OH)2 and / or HOOC(CH2)2PO(OH)2 are added under stirring. The mixture is then left under mechanical stirring for 12 hours at room temperature. The product is recovered by centrifugation and washed three times with water DI. The zeta potential of the particles drops to -10 mV, proving that a portion of the Al(OH)3 layer on the surface of the particles has been successfully passivated.

[0130] Alternatively, the surface layer can be functionalized by aromatic carboxylic polyanhydrides. For example, the reaction of an aromatic carboxylic acid dianhydride such as perylene-3,4,9,10-tetracarboxylic dianhydride with surface Al-OH leads to the formation of the following complex:

[0131] [Chem.4]

[0132] This is a bidentate very firmly attached to the surface A1(OH)3.

[0133] If the pH of the colloidal suspension becomes acidic between 4 and 5, the second anhydride function opens, forming the following complex which carries two dangling carboxylic acid functions at the end of a rigid sp2 segment.

[0134] [Chem.5]

[0135] This graft is fluorescent (exc 420 nm, therefore at low energies, fluorescence in the red). Moreover, when the pendant carboxyl groups react, the fluorescence varies. It is therefore a probe that allows evaluation of the quality of the EDC coupling as described below.

[0136] To this end, 1 g of SiO2-modified Fe3O4-PVA particles with an Al(OH)3 surface are dispersed under mechanical stirring in 200 ml of H2O DI. Then, 0.1 g of perylene-3,4,9,10-tetracarboxylic dianhydride is added under stirring in 15 ml of THF. The mixture is then left under mechanical stirring for 12 hours at room temperature. The product is recovered by centrifugation and washed three times with water DI. The zeta potential of the particles drops to -5 mV, proving that a portion of the Al(OH)3 layer on the surface of the particles has been passivated. The particles are red.

[0137] To functionalize the magnetic beads, the surface carboxylic groups are first modified into activated esters by reaction with EDC. Then, in a second step, in the presence of the primary amines of the antibody side chains, an amide bond is formed, thus ensuring the covalent grafting of the antibodies to the surface of the magnetic beads.

[0138] To this end, 80 µL of a freshly prepared EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide) solution at 4 mg / mL in 0.1 M MES buffer, pH 4.7, is added to each milligram of magnetic beads. The mixture is incubated for 10 min at 37°C with stirring at 150 rpm to obtain the intermediate activated esters from the carboxylic acids. Then, 10 to 50 µg of purified antibody is added per mg of beads for conjugation reaction for 2 h at 37°C with stirring at 150 rpm. Finally, 2 mL of BSA at 0.5 mg / mL in 0.1 M MES buffer, pH 4.75, is added per mL of functionalized beads. The mixture is incubated a final time for 30 minutes at 37°C with stirring at 150 rpm to ensure saturation of non-specific or unreacted sites. The functionalized magnetic beads are then washed twice by centrifugation in PBS pH 7.4 + 0.1% BSA and resuspended in the same buffer to a final concentration of 5 mg / mL for storage at 4°C until use.

Claims

Demands

1. Nano- or microparticle comprising a matrix made of or comprising at least one polyvinyl alcohol (PVA), and, dispersed therein, ferrite, the ferrite being distributed in the form of a plurality of particles in the PVA matrix, said nano- or microparticle further comprising an outer layer of silica, which is functionalized by compounds bearing a macromolecule.

2. Nano- or microparticle according to claim 1, wherein polyvinyl alcohol is crosslinked, in particular using a crosslinking agent selected from the following compounds: boric acid, boronic acids, boric acid esters, boronic acid esters, borax (Na2B4O7.10H2O), and aldehydes, in particular from compounds of the following formulas: B(OR)3, RB(OH)2, B(OH)3, R-CHO, in which R is selected from aryls and heteroaryls, and compounds comprising at least two aldehyde functions, in particular compounds comprising two aldehyde functions, more particularly compounds of formula HC(=O)-(CH2)nC(=O)-H, with n ranging from 0 to 6, for example n = 0, 1, 2 or 3, the crosslinking agent being for example 3-quinoline boronic acid.

3. Nano- or microparticle according to any one of the preceding claims, which, excluding the outer layer of silica: - has the shape of a sphere or spheroid; and / or - has a size of 50 to 1000 nm, in particular 150 to 450 or 500 nm.

4. Nano- or microparticle according to any one of the preceding claims, wherein at least one polyvinyl alcohol is present therein at a mass of 30 to 50% relative to the total mass of said nano- or microparticle excluding outer silica layer.

5. Nano- or microparticle according to any one of the preceding claims, wherein the outer silica layer is functionalized: by compounds bearing a monoclonal antibody; and / or via an amide function formed between a carboxylic acid carried by said compounds and an amine carried by said macromolecule.

6. An assembly of nano- or microparticles according to any one of the preceding claims, the size distribution of which, excluding the outer silica layer, has a uniformity coefficient of 0.95 to 1 and / or of which more than 95% of these nano- or microparticles have a size of 180 nm + / - 10% excluding the outer silica layer.

7. A method for preparing a nano- or microparticle according to any one of claims 1 to 5, or an assembly of nano- or microparticles according to claim 6, comprising: (i) a step of contacting at least one polyvinyl alcohol (PVA) with a ferrite precursor composition A to obtain a composition B; (ii) a step of heating composition B to obtain said nano- or microparticle; (iii) a step of depositing a silica layer on the nano- or microparticle by sol-gel synthesis, in particular using a silica precursor selected from tetraethyl orthosilicate and tetramethyl orthosilicate, optionally in the presence of aminopropyltriethoxysilane;(iv) a step of contacting the nano- or microparticle obtained at the end of the previous step with an aluminium salt, in particular selected from A1C13, A1(NO3)3, A1(C1O4)3, which are optionally hydrated, the aluminium salt being preferably selected from A1C13, hydrated A1(NO3)3 salts, and hydrated A1(C1O4)3 salts, to obtain a nano- or microparticle comprising an outer layer of silica, which has on its surface a layer of Al(OH)3; (v) a step of contacting the nano- or microparticle obtained at the end of the previous step with a compound: • consisting of or comprising a phosphonic acid having a linear or branched Ci-C6 alkyl group, substituted by a carboxylic acid group (-COOH); • consisting of or comprising an arene bearing two carboxylic acid anhydrides (-C(=O)-OC(=O)-); • for example, of the following formula:; [Chem.6] HO, , >Ich4cooh HO" d -n M or

8. (vi) a step of forming an amide function between the carboxylic acid groups on the nano- or microparticle obtained at the end of the preceding step and an amine on said macromolecule, in particular by the formation of intermediate activated esters from said carboxylic acids, for example by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Nano- or microparticle according to any one of claims 1 to 5, or assembly of nano- or microparticles according to claim 6, for use in diagnostics, in particular for the detection of bacteria, especially in clinical samples without preculture, for example whole blood or other biological fluids; or cancer cells.

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