NANO- OR MICROPARTICLE COMPRISING A POLYVINYL ALCOHOL MATRIX AND DISPERSED THEREIN, FERRITE, METHOD FOR PRODUCING THE SAME AND USES THEREOF

Abstract

A nano- or microparticle comprising a matrix consisting of or comprising at least one polyvinyl alcohol (PVA) and dispersed therein, ferrite, and a method for producing the same. Further, the use of these nano- or micro-particles for the preparation and the implementation of devices that can be detected by giant magnetoresistance sensors (GMR sensors) as biological diagnostic tools.

Claims

1. A nano- or microparticle comprising a matrix consisting of or comprising at least one polyvinyl alcohol (PVA) and dispersed therein, ferrite.

2. The nano- or microparticle according to claim 1, wherein the polyvinyl alcohol is cross-linked, in particular with the aid of a crosslinking agent chosen from the following compounds: boric acid, boronic acids, boric acid esters, boronic acid esters, borax (Na.sub.2B.sub.4O.sub.7.Math.10H.sub.2O), and aldehydes, in particular from the compounds of the following formulae: B(OR).sub.3, RB(OH).sub.2, B(OH).sub.3, R-CHO, in which R is chosen from aryls and heteroaryls, and the compounds comprising at least two aldehyde functions, in particular the compounds comprising two aldehyde functions, more particularly the compounds of formula HC(O)(CH.sub.2).sub.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. The nano- or microparticle according to claim 1, wherein: is shaped like a sphere or spheroid; and/or has a size from 50 to 1000 nm, in particular from 150 to 450 or 500 nm.

4. The nano- or microparticle according to claim 1, wherein the at least one polyvinyl alcohol is present in an amount of 30 to 50% by mass relative to the total mass of said nano- or microparticle.

5. An assembly of nano- or microparticles according to claim 1, the size distribution of which has a uniformity coefficient from 0.95 to 1 and/or in which more than 95% of these nano- or microparticles have a size of 180 nm +/10%.

6. A method for preparing a nano- or microparticle according to claim 1, 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 the composition B to obtain said nano- or microparticles.

7. A method of preparation of devices capable of being detected by giant magnetoresistance sensors, said method comprising a step of using nano- or microparticle according to claim 1, or an assembly of nano- or microparticles according to claim 5.

8. The nano- or microparticle according to claim 1, further comprising an outer layer of silica, which is functionalised by compounds carrying a macromolecule, in particular a monoclonal antibody, said outer layer of silica being functionalised in particular by means of an amide function formed between a carboxylic acid carried by said compounds and an amine carried by said macromolecule.

9. The method of preparing a nano- or microparticle according to claim 8 comprising: (iii) a step of depositing a layer of silica on the nano- or microparticle according to claim 1 by sol-gel synthesis, in particular using a silica precursor chosen from tetraethylorthosilicate and tetramethylorthosilicate, optionally in the presence of aminopropyltriethoxysilane; (iv)a step of contacting the nano- or microparticle obtained at the end of the preceding step with an aluminium salt, chosen in particular from AlCl.sub.3, Al(NO.sub.3).sub.3, Al(ClO.sub.4).sub.3, which are optionally hydrated, the aluminium salt preferably being chosen from AlCl.sub.3, the hydrated Al(NO.sub.3).sub.3 salts and the hydrated Al(ClO.sub.4).sub.3 salts, to obtain a nano- or microparticle comprising an outer layer of silica, which carries on its surface a layer of Al(OH).sub.3; (v) a step of contacting the nano- or microparticle obtained at the end of the preceding step with a compound: consisting of or comprising a phosphonic acid in which the phosphor carries a linear or branched C.sub.1-C.sub.6 alkyl substituted by a carboxylic acid group (COOH); consisting of or comprising an arene carrying two carboxylic acid anhydrides (C(O)OC(O)); for example the following formula: ##STR00006## (vi)a step of forming an amide function between the carboxylic acid groups carried by the nano- or microparticle obtained at the end of the preceding 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 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

10. A method of diagnosis, in particular for the detection of bacteria, in particular in clinical samples without preculture, for example whole blood or other biological fluids; or of cancer cells, said method comprising a step of using nano- or microparticle according to claim.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0115] FIG. 1 is a scanning electron microscopy (SEM) image of the dry particles obtained in Example 1, as obtained by depositing an aqueous solution of said particles (mother solution) on a silicon plate, followed by SEM analysis.

[0116] FIG. 2 is a scanning electron microscopy (SEM) image of the particles obtained in example 1, at a smaller scale than that shown in FIG. 1.

[0117] FIG. 3 shows the extraction of the magnetic moment from commercial particles (micromod 50 nm beads, ademtech 100 nm beads, ademtech 200 nm beads, micromod 500 nm beads and dyna-Myone 1000 nm beads) and particles according to the invention (example 1 and example 3).

EXAMPLES

Example 1: Synthesis of Fe.SUB.3.O.SUB.4.-PVA Beads

[0118] 1 g of PVA is added to 100 ml of DI water. The reaction medium is heated to 80 C. for approximately 1 hour, until the PVA is completely dissolved. The solution is clear and colourless. The temperature was allowed to fall to 50 C. and then 4.065 g of FeCl.sub.3, 6H.sub.2O was added. The dissolution is total. 4.05 g of FeCl.sub.3, 6H.sub.2O is then added. The solution is then translucent red.

[0119] At the same time, 8.82 g of sodium citrate 2H.sub.2O and 2.7 g of urea are added to 150 ml of DI water in another container.

[0120] The two solutions were mixed at room temperature for 10 minutes, the mixture was orange in colour and the medium cloudy. The resulting suspension is then poured into a Teflon-lined autoclave. 50 ml DI water is added. The autoclave is then heated to 200 C. for 24 hours. After 24 hours, the autoclave is left to cool. The reaction medium is a black suspension. The precipitate is recovered by centrifugation and washed 3 times with DI water, each wash being followed by a centrifugation cycle.

[0121] Approximately 2.2 g of dry matter is recovered (after one night at 100 C. in a ventilated oven). The powder is then recovered in 100 ml of anhydrous EtOH and centrifuged again. The precipitate is dried again overnight at 100 C. 1.9 g of powder is recovered. The atomic absorption of the powder gives a mass fraction of 34% Fe. This corresponds to a Fe.sub.3O.sub.4 mass fraction of 47.36%.

[0122] The resulting particles have a density of 1.87 kg.Math.m.sup.3 compared with 5.17 kg.Math.m.sup.3 for Fe.sub.3O.sub.4.

[0123] The SEM imaging (FIG. 1) allows to reveal the monodispersity in size of composite particles of the invention obtained in this way. This image also shows the absence of aggregation of the particles of the invention within their mother solution (a medium for biological use), and therefore their stability, which is particularly advantageous, in said medium. Indeed, if there had been aggregation in solution, this would also have been observed after evaporation of the solution when the SEM image was taken.

[0124] The SEM imaging also allows to highlight the monodispersity in size of the ferrite particles in a composite particle of the invention (FIG. 2).

Example 2: Magnetic Properties of the Particles of the Invention

[0125] The magnetic particles were characterised by measuring magnetisation as a function of the magnetic field at room temperature. It was thus possible to determine the magnetic moment of a magnetic particle of the invention and to compare it with that of commercial magnetic particles of equivalent size (ademtech beads of 200 nm in diameter and micromod beads of 500 nm in diameter). The magnetic moment obtained for the magnetic particles of the invention (according to example 1 or 3) have a moment equivalent to that of the commercial beads.

Example 3: Functionalisation of Fe.SUB.3.O.SUB.4.-PVA Particles According to the Invention

[0126] With mechanical stirring, 1 g of particles such those obtained in example 1 are dispersed in 200 ml of EtOH abs. The PVA in the composite is compacted in this anhydrous medium. Then, with stirring, 0.5 g of a mixture of tetraethylorthosilicate and aminopropyltriethoxysilane in a TEOS/APTES molar ratio=20/1 is added (the APTES is likely to act as a catalyst to initiate the polymerisation of the silica layer). After 10 minutes of homogenisation, 1 g of H.sub.2O is stirred. The medium was then left to stir mechanically 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 falls to 20-30 mV, illustrating that a layer of silica has been built up on the surface of the particles.

[0127] Optionally, the PVA of the Fe.sub.3O.sub.4-PVA core can be cross-linked by reacting a cross-linker of the PVA: for example B(OR).sub.3, RB(OH).sub.2, B(OH).sub.3 or Na.sub.2B.sub.4O.sub.7.Math.10H.sub.2O before growing the silica layer. The advantage of the compounds such as RB(OH).sub.2 is that, if desired, another organic function can be grafted onto the PVA of Fe.sub.3O.sub.4-PVA: in particular a fluorescent dye. 3-quinoline boronic acid was used successfully: 1 g of Fe.sub.3O.sub.4-PVA particles were dispersed in 200 ml of DI H.sub.2O with mechanical stirring. Then, with stirring, 0.2 g of 3-quinoline boronic acid was added to 50 ml of THF. The medium was then left to stir mechanically for 12 hours at room temperature. The product is recovered by centrifugation, washed three times with DI water, each wash being followed by a centrifugation. The product shows a fluorescence at 540 nm when excited at 400 nm.

[0128] The magnetic particles can carry surface carboxylic acid functions, which can then react, for example with EDC to allow the coupling, with a biological molecule.

[0129] This can be achieved by changing the zeta potential of the silica layer by hydrolysing an aluminium salt (e.g. AlCl.sub.3, Al(NO.sub.3).sub.3). Without wishing to limit ourselves to any one theory, the aminopropyltriethoxysilane used as a catalyst is likely to participate in the silica network by leaving pending primary amine functions on which the aluminium salts can hydrolyse to form an Al(OH).sub.3 layer.

[0130] 1 g of SiO.sub.2-modified PVA-Fe.sub.3O.sub.4 particles are dispersed in 200 ml of DI H.sub.2O with mechanical stirring. Then 0.3 g of a hydrated aluminium salt is added with stirring. The medium was then left to stir mechanically 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 rose to +15 mV, proving that a layer of Al(OH).sub.3 had been built up on the surface of the particles. Without wishing to limit ourselves to any one theory, the amorphous silica layer on the surface is likely to allow to inhibit the formation of crystallised gibbsite (Al(OH).sub.3). Without this layer, the gibbsite crystals could start to grow alongside the Fe.sub.3O.sub.4-PVA particles.

[0131] The surface layer is then functionalised with functional phosphonic acid grafts, so that the magnetic particles carry pending carboxylic acid functions on their surface.

[0132] To do this, 1 g of SiO.sub.2-modified Fe.sub.3O.sub.4-PVA particles with an Al(OH).sub.3 surface is dispersed in 200 ml of H.sub.2O DI with mechanical stirring. Then 0.2 g of HOOCCH.sub.2PO(OH).sub.2 and/or HOOC(CH.sub.2).sub.2PO(OH).sub.2 is added with stifling. The medium was then left to stir mechanically 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 10 mV, proving that a part of the Al(OH).sub.3 layer on the surface of the particles has been passivated.

[0133] Alternatively, the surface layer can be functionalised with aromatic carboxylic polyanhydrides. For example, the reaction of an aromatic carboxylic acid dianhydride such as perylene-3,4,9,10-tetracarboxylic dianhydride with the surface AlOH leads to the formation of the following complex:

##STR00004##

[0134] It is a bidentate very firmly attached to the Al(OH).sub.3 surface.

[0135] If the pH of the colloidal suspension becomes acidic, between 4 and 5, the second anhydride function opens to form the following complex, which has two pending carboxylic acid functions from the end of a rigid sp.sup.2 segment.

##STR00005##

[0136] This graft is fluorescent (exc 420 nm, i.e. at low energies, fluoresces in the red). In addition, when the pending carboxyl groups react, the fluorescence varies. It is therefore a probe that allows to evaluate the quality of the EDC coupling as described below.

[0137] To do this, 1 g of SiO.sub.2-modified Fe.sub.3O.sub.4-PVA particles with an Al(OH).sub.3 surface is dispersed in 200 ml of DI H.sub.2O with mechanical stirring. Then, with stifling, 0.1 g of perylene-3,4,9,10-tetracarboxylic dianhydride was added to 15 ml of THF. The medium was then left to stir mechanically 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 rose to 5 mV, proving that a part of the Al(OH).sub.3 layer on the surface of the particles had been passivated. The particles are red.

[0138] To functionalize the magnetic beads, the surface carboxyl functions are first modified into activated esters by reaction with EDC. Then, in a second step, in the presence of the primary amines of the lateral chains of the antibodies, an amide bond is formed, ensuring the covalent grafting of the antibodies to the surface of the magnetic beads.

[0139] To this end, 80 L of a solution of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) prepared extemporaneously 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 minutes at 37 C. with stirring at 150 rpm to obtain the intermediate activated esters from the carboxylic acids. Next, 10 to 50 g of purified antibody are added per mg of beads for conjugation reaction for 2 h at 37 C. with stifling at 150 rpm. Finally, 2 mL of 0.5 mg/mL BSA in 0.1 M MES buffer pH 4.75 is added per mL of functionalised beads. The mixture was incubated for a final 30 minutes at 37 C. with stirring at 150 rpm to ensure the saturation of the non-specific or unreacted sites. The magnetic beads functionalized in this way were washed twice by centrifugation in PBS pH7.4+0.1% BSA and recovered in the latter buffer at a final concentration of 5 mg/mL for a storage at 4 C. until use.