Process for synthesizing hybrid core-shell microparticles comprising a polymer core and a silicon dioxide shell with controlled structure and surface

Abstract

Hybrid microparticle having a polymer core and a shell which surrounds the polymer core at least in sections and which has a silicon dioxide layer; characterized by an RF value, the RF value being defined as the ratio of an external surface area amenable to the adsorption of nitrogen to a surface area which is computable from an arithmetic mean diameter of the hybrid microparticle considered as an ideal sphere, where the shell has a structure selected from: closed and smooth, with the shell having an RF value of between 1 and 1.5; closed and hillocky, with the shell having an RF value of between 1.51 and 3; or open, with the shell having an RF value of greater than 3.01.

Claims

1. A hybrid microparticle comprising a polymer core and a shell which surrounds the polymer core and which comprises a silicon dioxide layer; characterized by an RF value, the RF value being defined as the ratio of an external surface area amenable to the adsorption of nitrogen to a surface area which is computable from an arithmetic mean diameter of the hybrid microparticle considered as an ideal sphere according to the formula $\mathrm{RF}=\frac{A_{\mathrm{hybrid}\mathrm{microparticle}\mathrm{external}}}{A_{\mathrm{hybrid}\mathrm{microparticle}\mathrm{ideal}}};$ wherein a polyvinylpyrrolidone having an average molecular weight is covalently linked to the polymer core; wherein the shell comprising the silicon dioxide layer is disposed directly on the polymer core; and wherein the shell has a structure selected from: closed and smooth, with the shell having an RF value of between 1 and 1.5 if the average molecular weight of the polyvinylpyrrolidone is 10 000 daltons; and closed and hillocky, with the shell having an RF value of between 1.51 and 3 if the average molecular weight of the polyvinylpyrrolidone is 40 000 daltons.

2. The hybrid microparticle according to claim 1, wherein the structure of the shell is closed and in a native state has no pores, has no micropores, characterized by a pore diameter of less than 2 nm; has no mesopores, characterized by a pore diameter of between 2 nm and 50 nm; and has no macropores, characterized by pore diameters of greater than 50 nm.

3. The hybrid microparticle according to claim 1, wherein the polymer core of the microparticle comprises a polymer component selected from polystyrene, a polystyrene derivative and/or a comonomer, where at least one polymer component has a polymerizable double bond, so that the polymer core of the microparticle can be formed by polymerization of at least one polymer component.

4. The hybrid microparticle according to claim 1, wherein the RF is in the range from 1 to 1.5 and the polyvinylpyrrolidone has an average molecular weight in the range from 7000 daltons to 11 000 daltons or wherein the RF is in the range from 1.51 to 3 and the polyvinylpyrrolidone has an average molecular weight from 25 000 daltons to 58 000 daltons.

5. The hybrid microparticle according to claim 1, the polyvinylpyrrolidone forming a layer between the polymer core and the silicon dioxide shell.

6. The hybrid microparticle according to claim 1, wherein the shell comprising the silicon dioxide layer is joined via hydrogen bonds to the PVP.

7. The hybrid microparticle according to claim 1, wherein the shell has an increased specific surface area by comparison with the polymer core, wherein an RF value of 1.33 for hybrid microparticles with a closed and smooth shell structure corresponds to a surface area increased by 33% relative to a surface area of the polymer cores, and an RF value of 1.72 for hybrid microparticles with a closed and hillocky shell structure corresponds to a surface area increased by 72% relative to a surface area of the polymer cores.

8. Method for the synthesis of spherical hybrid microparticles according to claim 1, the method comprises: preparation of polymer cores by means of a radical polymerization reaction; coating of the prepared polymer cores with a silicon dioxide layer, using a sol-gel process; and functionalization of an outer surface of the silicon dioxide layer with an organic chlorosilane or alkoxysilane derivative, where the preparation of the polymer cores comprises use of the homolytically cleavable initiator 4,4-azobis (4-cyanopentanoic acid).

9. Method according to claim 8, where the preparation of the polymer cores takes place in an organic solvent having a water fraction of 0 vol% up to 80 vol% and comprises the use of styrene as monomer with at least one styrene derivative and/or with at least one comonomer, and further comprises covalent bonding of a polyvinylpyrrolidone to the surface of the polymer cores, where an average molecular weight of the covalently bonded polyvinylpyrrolidone is selected so that: the silicon dioxide layer is closed and smooth, and an RF value of the closed and smooth silicon dioxide layer is adjustable in the range from 1 to 1.5; or the silicon dioxide layer is closed and hillocky and an RF value of the closed and hillocky silicon dioxide layer is adjustable in the range between 1.51 to 3; or the silicon dioxide layer is open, and an RF value of the open silicon dioxide layer is adjustable to a value of greater than 3.01.

10. Method according to claim 9, where the selected average molecular weight of the polyvinylpyrrolidone is 10 000 daltons and/or is between 7000 to 11 000 daltons, where the RF value achieved by the closed and smooth silicon dioxide layer is in the range from 1 to 1.5; or is 11 000 daltons to 58 000 daltons or is between 25 000 daltons and 58 000 daltons, where the RF value achieved by the closed and hillocky silicon dioxide layer is in the range between 1.51 to 3; or is more than 58 000 daltons, more particularly between 60 000 to 360 000 daltons, where the RF value achieved by the open silicon dioxide layer is above 3.01.

11. Method according to claim 8, where the preparation comprises washing of the polymer cores with an organic or with an aqueous solvent or with a solvent mixture and/or the coating and functionalization takes place in an alcohol/water mixture with an alkoxysilane and/or an organic chlorosilane derivative or an alkoxysilane derivative.

12. Method according to claim 8, where the coating takes place in an alcohol with a water fraction of 0 vol% up to 80 vol%, using a starting substance selected from an alkoxysilane and/or an organic chlorosilane or alkoxysilane derivative in the presence of a basic, an organic or an inorganic catalyst, where the catalyst is selected from ammonia, sodium hydroxide and/or an organic amine.

13. Method according to claim 8, where the functionalization comprises furnishing with amino groups and takes place without an additional catalyst.

14. Method according to claim 8, where the functionalization comprises furnishing with functional groups other than an amino group, and/or takes place using an organic chlorosilane or alkoxysilane derivative with a basic or an acidic catalyst.

15. Method according to claim 9, where the average molecular weight of the polyvinylpyrrolidone is selected from a monodisperse mixture, or a mixture substantially homogeneous in respect of the molecular weight, of PVP molecules of identical molecular weight, or from a heterogeneous mixture comprising PVP molecules having molecular weights which are different from one another.

16. Method according to claim 13, where the microparticles have an amine-modified surface, and the organic chlorosilane or alkoxysilane derivative, at least in sections, forms a closed monolayer or a crosslinked multilayer on the surface.

17. Method according to claim 13, where the amino groups or the other functional groups are bonded covalently on the outer surface of the silicon dioxide layer in each case via an Si-O-Si bond.

18. Method according to claim 8, further comprising: burning-out of the polymer core at a temperature above 200 C., to leave the silicon dioxide layer as a hollow sphere.

19. Use of 4,4-azobis(4-cyanopentanoic acid) as initiator for a radical polymerization of styrene, styrene derivatives and/or polystyrene derivatives.

20. Chromatographic support comprising a classified fraction of hybrid microparticles according to claim 1, where the hybrid microparticles are classified according to: a mean diameter or a range of mean diameters; a specific surface area or a range of specific surface areas; an outer structure or a roughness of their outer surface to which an RF value is assigned; the nature of a functional group present on the outer surface, or the nature of one or more ligands bound thereto.

21. Carrier particles for a particle-based assay, comprising at least one hybrid microparticle according to claim 1, where the at least one hybrid microparticle is characterized by at least one of the parameters set out below: a mean diameter or a range of mean diameters; a specific surface area or a range of specific surface areas; a morphological structure of the outer surface of the shell to which an RF value can be assigned, the RF value being in a range from 1 to 1.5 and the shell being closed and smooth; being in a range of 1.51-3 and the shell being closed and hillocky; or being above 3.01 and the shell being open, where the RF value is defined as the ratio of an external surface area amenable to the adsorption of nitrogen to a surface area which is computable from an arithmetic mean diameter of the hybrid microparticle considered as an ideal sphere; a density of the hybrid microparticle that is adjustable between a density value of the polymer core which is 1.05 0.1 g/ml and the density value of the shell comprising a silicon dioxide layer, which is between 1.8 g/ml to 2.2 g/ml; a nature of a functional group present on the shell, or the nature of one or more ligands bound thereto or polymer layer; an analytically analyzable signal which can be captured as a result of a specific interaction between an artificial and/or a biological receptor and/or an analyte-sensitive polymer layer, which are bound on the carrier particle, with the analyte to be detected by the assay, where the analytically analyzable signal comprises a fluorescence property.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The appended drawings illustrate embodiments and serve together with the description to elucidate the principles of the invention.

(2) FIG. 1 shows, from top to bottom, SEM micrographs of particles with different surface structures, synthesized by the method proposed.

(3) FIG. 2 shows results of the thermogravimetric analysis with synthetic air. The particles with smooth and hillocky shells exhibit increased thermal stability; the particles with a closed and smooth shell exhibit the greatest stability.

(4) FIG. 3 shows nitrogen desorption isotherms and adsorption isotherms on particles from working examples P1, P2 and P3;

(5) FIG. 4 shows micrographs of the surface of the silicate shell of typical particles, by means of scanning electron microscopy (SE detector);

(6) FIG. 5 shows, from top to bottom, SEM micrographs of thermally treated microparticles; particles with smooth appearance as per working example P1-H, whose RF values in the native state, i.e. before thermal treatment, were around 1.3. The middle picture shows particles from working example P2-H. The native particles (P2) are prepared using PVP with an average molecular weight of 40 000 daltons, and before the thermal treatment had a hillocky surface with an average RF value of 1.7. The bottom picture depicts particles as per working example P3-H, which in the native state, before the thermal treatment, a silicate shell, described here as open and riven, and an RF value of about 4.8. The particles P3 were produced with PVP having an average molecular weight of 360 000 daltons, and then subjected to thermal treatment. The thermal treatment, identical for all three particle classes, encompassed a gradual combustion of the polymer core in synthetic air at a heating rate of 5 K/min up to 800 C. with a subsequent constant combustion time of 1 hour at 800 C.

(7) FIG. 6 shows a scheme of the synthesis pathways proposed in accordance with the invention for the core-shell particles described here.

(8) FIG. 7 shows scattered light signals (FSC) of the hybrid particles in comparison to the scattered light signals (FSC) of the corresponding polymer cores, in each case in a flow cytometer. The abbreviation MCI here stands for the mean channel intensity.

DETAILED DESCRIPTION

(9) In particular, FIG. 1 shows in each case typical particles when imaged by means of scanning electron microscopy using an in-lens secondary-electron detector. The top picture shows particles with a smooth appearance (working example P1) whose silicate shell has an RF value of between 1 and 1.5, produced using PVP having an average molecular weight of 10 000 daltons. The middle picture shows microparticles whose silica shell has a hillocky structure (working example P2) and whose roughness factors lies substantially in the range between 1.51 and 3. Such particles were produced using PVP with an average molecular weight of 40 000 daltons. The bottom picture shows particles with open silicate shells (working example P3), in which mesopores and macropores are visible by means of the imaging techniques. These particles were preparedas elucidated in more detail belowusing PVP with an average molecular weight of 360 000 daltons. The silicate shell of the particles, formed by coagulation of different-sized silicate nanoparticles, features a structure which changes significantly from top to bottom. The three micrographs show exclusively native particles, i.e. particles not subjected to an additional heat treatment, described in detail below.

(10) The thermogravimetric analysis in accordance with FIG. 2 comprises thermal exposure of the particles in synthetic air at a heating rate of 5 K/min. It is assumed that in the case both of P2 and of P3, PVP is intercalated between the silicate nanoparticles. If PVP is removed thermally, then corresponding pores can be formed. From the TGA plot it can be seen that the cores are protected by the shell from thermal decomposition and release, in a manner such that, in the case of P1 in particular, a part of the core undergoes measurable escape only at a temperature of 500 C. and above.

(11) FIG. 3 shows adsorption and desorption isotherms by the method of Brunauer-Emmet-Teller (BET) of nitrogen at 77 kelvins, recorded using an ASAP 2010 (Micromeritics Instrument Corp., Norcross, Ga. (USA)). The samples, with an initial mass of at least 400 mg, were dried at 60 C. for 5 seconds in a high vacuum prior to the measurement. The external surface areas of the hybrid microparticles are calculated using the t method, for which the required conversion of (p/p0) into the adsorbed layer thickness in Angstroms took place with the aid of the Harkins and Jura approximation

(12) $t\left(\right)=\sqrt{\frac{13.99}{0.034-\log \left(\frac{p}{p_0}\right)}}$
(cf. Harkins W D, Jura G, (1944). The adsorption data are then plotted against the layer thickness. The external surface areas per gram of the particles P1, P2 and P3 were then calculated on the basis of the slope of the linear line of best fit through the points from the resulting adsorption plot between 3.5 and 5 , and of the equation A.sub.external=Slope15 468. To determine the surface area per particle, the number of particles in one gram is calculated from the mass of the polymer cores contained therein (determined from values obtained by thermogravimetry), which have an average density of 1.05 g/ml, on the assumption of an ideal sphere. The resulting external surface area per gram is then divided by the number of hybrid microparticles per gram. The figure obtained is designated in abbreviated form as A.sub.hybrid microparticle, external.

(13) The surface area which can be calculated starting from a mean diameter of the hybrid microparticles and on assumption of an ideal spherical form for all the hybrid microparticles (referred to hereinafter as A.sub.hybrid microparticle, ideal) is determined as follows: a particle sample comprising more than 100 hybrid microparticles is dried at room temperature on a carbon sample-support film for transmission electron microscopy. Thereafter the sample support with the dried particle sample is imaged by means of scanning electron microscopy at 15 000 magnification and 20 kV in transmission mode. The sample does not need to be sputtered for this purpose, and so the size of the microparticles is not distorted. Micrographs obtained in a sense in transmitted-light mode for a cohort of >100 particles are represented as monochrome micrographs (bit depth: 1 bit) with the aid of suitable image analysis software, ImageJ, for example, and the area of the particles, which appear black against a white ground, is ascertained. From the area ascertained from the particles imaged, a particle diameter is computed, on the assumption that all of the particles are ideal spheres, and an arithmetic mean value of the diameter is determined from all the particles measured. This arithmetic mean particle diameter is used to calculate the ideal surface area of the hybrid microparticles in accordance with A.sub.hybrid microparticle, ideal=.Math.d.sup.2.

(14) Ultimately here, as elucidated above, an RF value of 1.33 was obtained for particles from working example P1, an RF value of 1.72 for particles as per working example P2, and an RF value of 4.84 for particles as per working example P3.

(15) FIG. 4 shows scanning electron micrographs of the surface of the silicate shell of typical particles (SE detector).

(16) FIG. 5 shows, from top to bottom, scanning electron micrographs of thermally treated microparticles. The topmost image shows particles with a smooth appearance as per working example P1-H, whose RF values in the native state, i.e. before thermal treatment, were in the region of around 1.3. The middle picture shows particles from working example P2-H, in the form in which they are present after a thermal treatment of native particles produced in accordance with working example P2 using PVP with an average molecular weight of 40 000 daltons. Before the thermal treatment, they had a hillocky surface with an average RF value of 1.7. The bottom picture depicts particles as per working example P3-H, which in the native state before the thermal treatment have a silicate shell, referred to here as open and riven, and an RF value of about 4.84. The particles P3 were prepared using PVP K360 (PVP with an average molecular weight of 360 000 daltons) and were then thermally treated. The thermal treatment, identical for all three particle classes, encompassed a gradual combustion of the polymer core in synthetic air at a heating rate of 5 K/min up to 800 C. with a subsequent combustion taking place constantly at 800 C. over a time of 1 hour.

(17) FIG. 7 shows scattered light signals of forward-scattered light (FSC) of the microparticles (comprising polymer core and silicate shell) in comparison to the respective polymer cores, in a flow cytometer. Comparing the samples, it becomes apparent that the scattered light intensity increases at constant distribution, and is therefore at least above that of the pure polymer core.

(18) Hybrid particles are known from publications U.S. Pat. No. 6,103,379 and Bamnolker, Nitzan et al. 1997. Described exclusively, however, is the use of long-chain PVP (360 000 daltons), leading to the formation of hybrid microparticles having an open, riven structure to the silicate shell. From the TGA data, the average size of the silicate nanoparticles and of the polymer core, which is employed here as a reference value for determining the average surface area of the hybrid microparticles, it is possible to calculate RF values of 3.66, 5.92 and 8.15. The authors Hong, Han et al. (2008) describe the use of PVP with an average molecular weight of 40 000 daltons for the direct coating of polymer cores using the toxic and environmentally damaging AIBN. Core-shell particles produced with short-chain PVP (Mw less than 40 000 daltons) are unknown. None of the aforesaid publications provides any indication of an influence on the structure of the silicate shell through the PVP, which is used exclusively to stabilize the core particles. And there are certainly no indications of any influence by the molecular weight of the PVP used on the fine structure of the shell. Instead, PVP is used merely as a stabilizer. An effect of the stabilizer on the processes which occur during silicate coating was unknown.

(19) With regard to the utilization of the hybrid microparticles obtained, applications contemplated are those hitherto-typical applications for surface coatings or the generation of colloidal crystals. The hybrid microparticles have been used much more rarely in analytical applications based on individual particles. Examples in this case are lanthanoid-doped polymer beads, provided with a silicate shell for better functionalization (Abdelrahman 2011). The polymer cores are prepared here with a hydrophobic initiator, PVP (MW 54 000 daltons) and additional costabilizer, and feature silicate shells with a thickness of above around 150 nm. Other examples are quantum dot-functionalized beads (Cao, Huang et al. 2006) or Raman-coded beads (Jun, Kim et al. 2007), which are coated with a silicate shell and then used in analytical assays. Here, no PVP is used in the synthesis of the polymer core, and the coating takes place after the sulfonation of the polymer cores, with no control over the structure, density or light-scattering qualities of the hybrid microparticles.

(20) The aforesaid approaches do not offer any synthesis route with general validity for the controlled preparation of native hybrid microparticles having an adjustable size within the relevant range between 0.5 and 10 m and a narrow size distribution, structure, density and light-scattering properties for the application of the hybrid microparticles in flow-cytometric methods or related miniaturized variants. The initiator normally used for the radical polymerization of the polymer cores is AIBN ((2,2-azobisisobutyronitrile), which is environmentally burdensome and toxic. Moreover, it is not possible here to produce monodisperse particles with PVP (less than 25 000 daltons), which is needed for the preparation of closed and smooth surfaces and hence for the acquisition of the entire structural range with RF values of between 1 and greater than 3, as for example between 1 and 10.

(21) Proposed in accordance with the invention, via the provision of hybrid microparticles comprising a polymer core and a silicon dioxide shell (here also called silicate shell) at least partly surrounding the polymer core, is the combining of the advantages of the organic polymer of the core and of the silicon dioxide of the shell in an assembly of materials, in order:

(22) a) to utilize the advantages of the polymer core synthesis in a radical polymerization by means of ACVA for preparing particles with different sizes of between 0.5 and 10 m with a sufficiently narrow size distribution;

(23) (b) to control the effective density of the hybrid microparticles, which is authoritatively determined by the mass fraction of the polymer core and of the silicate shell and is between 1.05 and 1.8, but preferably between 1.09 and 1.53;

(24) (c) to maintain at least the scattered-light intensity of the particles in comparison to polystyrene, but also, moreover, to increase the intensity by the grown addition of a silicate shell in accordance with the mass fraction;

(25) (d) to be able to bring about a fluid adjustment to the structure of the silicate shell, in a range between closed and smooth to hillocky, corresponding to RF values of the microparticles of between 1 and 3, through to open and riven, with RF values of greater than 3.01, on the basis of the selection of the average molecular weight of PVP;

(26) (e) to authoritatively determine the external surface area accessible by a potential analyte, and the functional groups of the hybrid microparticles that are anchored to that external surface via a linker or are directly present thereon, through the structure and associated surface area of the silicate shell. The word authoritatively in this context also describes the option whereby, in addition to a silicon dioxide shell at least partly covering the core, with the surface area thereof accessible from the outside, there are also fractions of the surface of the polymer core that are freely accessible. The freely accessible fractions of the surface of the polymer core can be modified chemically in accordance with the methods known for the respective polymer, and, as and when required, may be furnished with at least one kind or two or more kinds of ligands or with a mixture thereof. Ligands are understood to include biological receptors (antibodies, antibody fragments or peptides, reactive proteins, DNA or DNA-based biopolymers, (poly)sugars, or mixed conjugates thereof), analyte-selective groups, analyte-sensitive groups or groups with analyte affinity, fluorescent dyes, molecular probes, atom clusters, quantum dots, organic or further inorganic nanoparticles such as, for example metal nanoparticles or metal-oxidic nanoparticles.

(27) (f) to raise the chemical and physical stability of the particles, as a result of the at least partial envelopment with the silicon dioxide shell, relative to that of the pure polymer core.

(28) (g) to furnish the accessible external silicatic surface and the functional groups anchored thereto via a linker, or present directly bound thereon, as and when required, with at least one kind or two or more kinds of ligands or with a mixture thereof. Ligands are understood to include biological receptors (antibodies, antibody fragments or peptides, reactive proteins, DNA or DNA-based biopolymers, (poly)sugars, or mixed conjugates thereof), analyte-selective groups, analyte-sensitive groups or groups with analyte affinity, fluorescent dyes, molecular probes, atom clusters, quantum dots, organic or further inorganic nanoparticles such as, for example, quantum dots, metal nanoparticles or metal-oxidic nanoparticles. Furthermore, the grown addition of a further polymer layer on the silicate shell is proposed, such as the growing-on of a molecularly imprinted polymer layer (MIP), for example.

(29) The method proposed provides spherical hybrid microparticles whose surface, on the basis of its physical nature as a silicate surface, is readily amenable to a respectively desired chemical modification with organic chlorosilane or alkoxysilane derivatives, exhibiting a sufficiently high difference in refractive index relative to water, an optimized propensity toward sedimentation in aqueous solutions, and a narrow size distribution.

(30) Advantageously, the proposed concept of the hybrid microparticles makes possible a more readily manageable synthesis, in accordance with the proposed method comprising the use of the initiator ACVA, since the initiator is less environmentally burdensome and toxic by comparison with AIBN. Moreover, in the case of transport, additional costs for the transmission of hazardous goods, which are incurred in the case of AIBN, are not incurred. The synthesis presented here allows precise control over the fine structure of the silicate shell. In particular, it is possible to establish a surface of the outer particle surface area from closed and smooth (RF values of between 1 and 1.5) through closed and hillocky (RF values of between 1.51 and 3) up to open with RF values of greater than 3.01. Likewise, with the production method proposed here, it is also possible to produce open structures with lower RF values of less than 3, provided an incomplete monolayer of silicate nanoparticles is applied to polymer cores which have been produced using PVP with an average molecular weight of 10 000 Da or PVP with an average molecular weight of 40 000 Da. Otherwise, the RF values may also be greater than 3, but not more than 4, if a complete monolayer is present. This is possible with a relatively small amount of TEOS (ratio of TEOS to amount of PS used: less than 3:1). These structures, however, are not very suitable for analytical application, since silicate nanoparticles which have not fused to form closed shells have a tendency to part from the core. Lastly, such monolayers can also not be referred to as smooth, and would, in analogy to P3, suffer from reduced accessibility by large molecules.

(31) In accordance with practical working examples, the core particles are prepared in a dispersion polymerization in EtOH (96%). For this purpose, first 1.7 g of the PVP (equal amounts in the case of PVP with different molecular weights) are dissolved in 100 ml of EtOH in a 250 ml three-neck flask, equipped with an oval magnetic stirrer, at 75 with stirring at 250 rpm. At the same time, in a 50 ml glass beaker, 5 ml of styrene (alternatively other amounts of styrene, between 5 and 50 ml, corresponding to a volume fraction of 4-42% in comparison to the amount of EtOH used, for the preparation of larger particles) and 95.4 mg of ACVA (2.1% by weight to the amount of styrene used) are dissolved in 20 ml of EtOH. The two solutions are then flushed simultaneously with argon for 30 minutes. Subsequently the ACVA-styrene solution is added to the preheated PVP solution. The polymerization is carried out over 24 hours at 75 C. with a stirring speed of 250 rpm. The reaction mixture is then left to cool for 30 minutes, after which the particles obtained are separated from the reaction medium by centrifuging. Here, the reaction mixture is divided between 50 ml centrifuge vessels and centrifuged at 4000 RCF (relative centrifugation force) for 10 minutes. The supernatant is removed and the particles are redispersed in 30 ml of methanol in each centrifuge vessel. The methanol is subsequently replaced 2 in each case in order to separate the particles from impurities. The polymer cores are subsequently dried in a vacuum oven for 12 hours.

(32) For the preparation of closed and smooth hybrid particles, PVP with an average molecular weight of 10 000 daltons is used in the polymer core synthesis. For the preparation of closed and hillocky hybrid particles, PVP with an average molecular weight of 40 000 daltons is used in the polymer core synthesis. For the preparation of open and riven hybrid particles, PVP with an average molecular weight of 360 000 daltons is used in the polymer core synthesis.

(33) In accordance with practical working examples, the core particles are coated with a silicate shell in a sol-gel process under Stber-like conditions (ethanol-water mixture and ammonia as catalyst). For this purpose, 50 mg of polymer cores are dispersed in 5 ml of EtOH and 0.1 ml of water in a glass beaker with a capacity of 15-20 ml. Then 150 l (in a ratio of 3:1 to the amount of polymer core used) of TEOS and 150 l of ammonia (concentrated, 32%) are added. The coating reaction is carried out with stirring using a magnetic stirrer at 500 rpm. After 18 hours, the hybrid core particles obtained are transferred to a 5 ml vessel with a snap-fastening lid (Eppendorf) and separated from the reaction medium by centrifuging at 4000 RCF for 5 minutes. The particles are washed twice with 3 ml of water and once with 3 ml of EtOH (96%) by centrifuging (4000 RCF, 5 minutes) and redispersing in the wash medium. Following centrifugation and separation from EtOH, finally, the particles are dried in a vacuum oven at room temperature for 4 hours.

(34) In accordance with the practical working examples P1, P2 and P3, described above in paragraph [0042], different particles P1, P2 or P3 are obtained, according to which polymer cores were used in the coating reaction.

(35) TABLE-US-00001 TABLE 1 Working example P1 P2 P3 Calculated surface area 2.74 m.sup.2 3.25 m.sup.2 3.18 m.sup.2 (hybrid microparticle) Measured surface area 3.65 m.sup.2 5.60 m.sup.2 15.40 m.sup.2 hybrid particle (t-plot) RF value ~1.33 ~1.72 ~4.84 RF value range (class) 1-1.5 1.51-3 greater than 3.01

(36) A categorization of the microparticles in greater detail may be undertaken in accordance with the micropores, mesopores and macropores that are observed and are verified by means of nitrogen adsorption, for example (cf. Tab. 2):

(37) TABLE-US-00002 TABLE 2 Presence of Working Presence of RF meso- and/or Topography example micropores value macropores smooth P1 No 1.33 No C value 160 hillocky P2 No 1.72 No C value 158 open P3 Yes 4.84 Yes C value 369

(38) The C value is determined by means of nitrogen adsorption and provides information on the presence of micropores, since only at values between C=50 and C=150 is it possible to assume a nitrogen adsorption which can be described reliably by a B.E.T. adsorption isotherm, and the absence of micropores (Rouquerol et al., 1999).

(39) The values determined for working examples P1 and P2 are just above this. Minimally, therefore, there could be pores present, since the C value here is in each case a little over 150 (see Table 2). Up to a value of 200, therefore, closed particles without micropores are defined. P3 exhibits increased microporosity and also, independently thereof, meso-/macropores which are detectable by means of imaging methods (cf. FIG. 4-f), the particle being consequently categorized as open.

(40) Micropores are also accessible by nitrogen. The critical factor here is that the surface area is characterized by means of nitrogen adsorption, but in that case by the t method for determining the fraction of the external surface area, or by assessing the surface area by means of imaging analyses. A distinction is to be made here between closed shells, where only the bulging of the silicate nanoparticles is assessed, or open shells, where the external surface area is provided by the summing of the surface areas of the individual silicate nanoparticles. As elucidated below, comparable results are achieved here. An objective assessment and categorization is also possible in this way for the particles stated in other patents and publications.

(41) On the basis of the desorption isotherms (cf. FIG. 3), however, no mesopores as such are measurable, since otherwise a type IV isotherm would have been expected. Nevertheless, pore sizes of below 50 nm are visible by means of imaging methods (cf. FIG. 4-f). An open assembly of silicate nanoparticles can therefore be assumed, surrounding the silicate shell.

(42) As shown in FIG. 3, the hybrid particles have an increased measurable specific surface area. The surface areas here were determined on the basis of the T method, which differentiates external surfaces from surfaces resulting from micropores. For this purpose, using the Harkins-Jura approximation, the relative pressures are converted into layer thickness of the adsorbed nitrogen molecules. The data obtained by means of nitrogen adsorption are then plotted against the calculated layer thicknesses. On the basis of the slope, obtained by linear regression of the data points between 3.5 and 5 , corresponding to the range between the formation of a monolayer and the onset of capillary condensation, the external surface area is calculated. For determining the surface area per particle, the number of particles in 1 gram is calculated from the mass of polymer core contained therein (determined via the TGA values) on assumption of an ideal sphere. The external surface area per gram that is obtained is then divided by the number of hybrid microparticles per gram, to give the external surface area per particle.

(43) The hybrid microparticles according to P1, identified here as closed and smooth, have a surface area increased by 33% relative to the pure polymer cores (according to an RF value of 1.33); the closed hillocky hybrid microparticles according to P2 have a surface area increased by about 72% (according to an RF value of 1.72). In the case of ideal occupancy of the polymer cores with silicate nanoparticles which have fused to form a closed, hillocky silicate shell, and which have undergone bulging up to half of their diameter, a maximum RF value of 2 is anticipated. RF values for a closed, hillocky structure of the silicate shell that are above 2 and below 3 are obtained if silicate nanoparticles with more than half the diameter protrude in isolation from the closed shell.

(44) Open, riven structures according to P3 have a surface area increased by 384% relative to pure polymer cores (according to the RF value of 4.84). The surface area determined by means of nitrogen adsorption using the t method corresponds approximately here to the sum of the surface areas of the core and an accretion of around 3520 silicate nanoparticles with a size of around 35 nm. The number of silicate nanoparticles was calculated on the basis of the silicate fraction, measured by TGA, for a particle at a level of 28.7% with the average diameter of 893.5 nm. Determination of the RF value on the basis of the T method or by means of imaging methods therefore leads to comparable results, thus enabling an objective categorization of hybrid core-shell particles using nitrogen adsorption data or image analyses.

(45) The surface area adjustably increased in each case can be exploited for analytical use, by the possibility, for example, of covalent anchoring of small and of larger molecules, utilizable as scavengers and/or receptors, or of a further polymer layer with a high density/microparticle and/or with a defined density per unit area.

(46) Furthermore, however, the specifically established particle density can also be utilized in order to provide particles having properties adapted in each case to a specific analytical task. For instance, particles with a density of greater than 1.05 undergo more rapid sedimentation in water, and/or have a significantly more rapid sedimentation behavior on centrifuging, and this may mean considerable time savings especially for washing steps during production, functionalization and use.

(47) Lastly, a feature of the hybrid particles is that their light-scattering properties are authoritatively influenced by the polystyrene core, the light-scattering intensity therefore corresponding at least to that of the polystyrene core. This can be confirmed (FIG. 7) on the basis of the scattered-light intensity distribution as measured by means of flow cytometry. Measured in this case at each instance is an increase in scattered-light intensity, with retention of the scattering properties and of the scattered-light distribution. It can be assumed that the mass fraction of silicate in the hybrid microparticle correlates with the increase in the scattered-light intensity.

(48) In accordance with the invention, the polymer core is prepared in the presence of polyvinylpyrrolidone (PVP) by radical polymerization in alcoholic solvent with a possible water fraction of 0-80%, starting from styrene and/or from a mixture of styrene and other polymerizable comonomers. The radical polymerization initiator used at 75 C. is a homolytically cleavable initiator, preferably ACVA (ACVA=4,4-azobis(4-cyanovaleric acid), or 4,4-azobis(4-cyanopentanoic acid)). Advantageously, ACVA is less toxic to humans and the environment and, moreover, it equips the polymer core with carboxylic acid groups, which ensure additional stabilization during the polymerization, and for this reason polymer cores with PVP of relatively low molecular weight, of less than 25 000 Da, can also be produced in narrow size distributions. The coating of silicon dioxide takes place after washing of the core particles with organic or aqueous solvent in a sol-gel process in alcohol/water mixtures with alkoxysilanes in pure form, or in a mixture of these as starting substances and alkaline initiators such as ammonia, sodium hydroxide or organic amines.

(49) Surprisingly it has emerged that the molecular weight of the polyvinylpyrrolidone used as a stabilizer during the polymerization authoritatively influences the structure of the silicate shell. In particular, depending on the average molecular weight of the PVP, it is possible to configure the surface of the silicate shell, alternatively, from a closed and smooth or hillocky surface to an open surface, in which case short chains (average molecular weight 10 000 daltons) lead to a closed and smooth shell structure, medium chain lengths (average molecular weight 40 000 daltons) lead to a closed and hillocky shell structure, and long chains (average molecular weight 360 000 daltons) lead to an open and riven shell structure (cf. FIGS. 1-a to 1-c, and FIGS. 4-a, d or FIGS. 4-b,e and FIGS. 4-c,f).

(50) From a scientific and technical standpoint, the proposed hybrid microparticles with adjustable size in conjunction with narrow size distributions, precisely controllable densities, suitable light-scattering properties, and also structure and surface of the silicate shells represent an innovative and improved spherical platform particularly for particle-based analytical applications.

(51) By means of the size of the polymer cores it is possible to code the particles for a multiplex application. An illumination property can also be used for coding, by using fluorescent comonomers.

(52) Fluorescent molecules or fluorescent organic and/or inorganic nanoparticles may also be integrated into the core, for dye coding, statistically and not covalently, by way of swelling techniques, for example. This brings about possibilities of post-coding after core particle synthesis.

(53) The deliberate adaptation of the densities makes it possible to control the sedimentation behavior of the particles, according to the analytical application, for increased colloidal stability at relatively low densities (e.g. between 1.05 and 1.25) or for more rapid separation of the particles by means of centrifugation, in the case of increased densities (e.g. between 1.1 and 1.5).

(54) The scattered-light intensity in flow-cytometric applications of the hybrid microparticles is equivalent at least to that of pure polystyrene, or even above, depending on the mass fraction of the accreted shell. In comparison to pure silicate microparticles, therefore, the hybrid microparticles yield higher light-scattering intensities for a given size.

(55) At the same time, the hybrid microparticles provide a silicatic surface which can be functionalized by modification in situ or afterward. As a result, it is possible to generate not only individual functional groups but also deliberate mixed surfaces on the hybrid microparticle surface, and these can be optimized advantageously in each case for the analytical issue at hand.

(56) The structure of the silicate shell can be adjusted, finally, in a controlled way via the selection of the polymer cores, stabilized by PVP with different molecular weights. Closed, smooth and closed, hillocky structures here are more accessible by larger molecules in the case of surface area increase of up to 200%. Conversely, open and riven silicate shells are less accessible for large molecules, but do display the greatest increases in surface area, thereby making the adaptation of small molecules advantageous here.

(57) The present invention has been elucidated with reference to working examples. These working examples should in no way be understood as imposing any limitation on the present invention. The claims which follow represent a first, non-binding attempt to define the invention generically.

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