DE10035953A1 - Spherical, magnetic silica particles with adjustable particle and pore size and adjustable magnetic content for the purification of nucleic acids and other biomolecules - Google Patents

Abstract

The invention relates to a method for producing magnetic SiO2 particles, comprising the following steps: a) alkoxysilanes are dispersed in water, acid-catalytically hydrolyzed and condensed to form an SiO2 hydrosol; b) a magnetic particle-sol mixture is produced by adding magnetic particles, for example usual magnetic particles, magnetic colloids and/or ferrofluids to the SiO2 hydrosol; c) dispersing the magnetic particle-sol mixture in an organic solvent which is immiscible with water; and d) adding a base to the magnetic particle-sol mixture during or after the dispersion in the organic solvent in order to form a gel.

Description

The present invention relates to magnetic silica particles with adjustable pore and Particle size and adaptable magnetic content, a process for their production and the use of magnetic particles in the area of nucleic acid and biomolecule Separation.

Silica gels of various origins have long been used as chromatography media Known, however, their use specifically for nucleic acid purification was just beginning recognized in the 80s and described in DE 32 11 309. For the particles listed there are pure silica gels that are used exclusively in column chromatography Find use.

Silanized iron oxide particles for the immobilization of enzymes are from the US patent 4,152,210 known. Also for the purpose of enzyme immobilization are in U.S. Patent 4,343,901 ferromagnetic particles by a sol-gel technique are described.

PCT application EP 97/04828 describes 50-1500 nm large monodisperse magnetic particles which consist of an SiO ₂ core which is given magnetic properties by coating with iron oxide. Subsequent silanization of the iron oxide layer enables the particles to bind nucleic acids.

Magnetic silica hybrid particles, consisting of a polystyrene core, on the magnetite and then a silica layer is polymerized on are from PCT / US 95/12988 known. The particles are used for antibody and cell separation.

Iron oxide particles coated with colloidal SiO ₂ are disclosed in US Pat. No. 4,280,918.

V. Goetz et al. (Biotechn & Bioengineering, Vol. 37, 614, 1991) describe 20-100 µm magnetic silica gel particles capable of enzyme immobilization, which are produced by electrostatic coating of nickel powders with silica sols. In a similar way, AM Homola and M. R Lorenz (IEEE Transaction on Magnetics, Vol. 22, 716, 1986) describe electrostatic coating of silica particles on Fe ₂ O ₃ particles for the production of magnetic storage media.

Colloidal metal particles for use in immunoassay are included in the application PCT / US 97/03886.

From US Pat. No. 5,320,944, 0.2-3 μm large magnetic particles are known which pass through Coating a polymer particle with iron oxides obtained magnetic properties. By further coating the particles with silanes, nylon or polystyrene then antibodies are coupled to the particles for use in the immunoassay. The manufacture of an optical sensor in the form of glass or transparent Polymer carriers on which precious metal colloids improve the effector molecule connections for use in immunoassays are derived from the PCT Registration AT 98/00222 forth.  

The synthesis of silica gels for gel permeation chromatography using Cer (IV) - Initiation with vinyl monomers are grafted to the nonspecific protein adsorption suppressing is known from PCT / EP 94/01378.

Organosilanized colloidal silica gel particles as biological separation media are in U.S. Patents 4,927,749 and 4,927,750 and PCT application US 99/00403 discloses, the stability of the colloids and the manner of silanization in Stand in the foreground.

Magnetic particles that contain a magnetic core material and an inorganic one Oxide coated are disclosed in EP 0343 934.

Polymer particles with another containing a magnetic material Polymer layer are coated on a third to interact with biomolecules capable polymer coating is applied, are in PCT application FR 97/00912 described.

Pearlescent color pigments of 10-60 µm in size, coated with magnetite and for separating biological mixtures are provided, go from PCT application DE 97/01300 out.

Magnetic hybrid particles that consist of a polymer core, initially with a Coated ferrofluid and then coated with a functional polyacrylate are the subject of US Pat. No. 5,648,124;

The particles known from the prior art have in relation to the separation of Some disadvantages of nucleic acids on the one hand are not a number of carrier media magnetic (US 4,927,750; DE 32 11 309; PCT / US 99/00403; PCT / EP 94/01378), so that a rapid separation of the particles, as required today in routine analyzes, is not possible is, d. that is, one relies on column chromatography methods, on the other hand point Magnetic particles based on silica or polystyrene with a magnetic oxide are coated, a high specific density (PCT / EP 97/04828, US 4,152,210, EP 3211309, 5,320,944). This results in insufficient dispersibility, which leads to leads to rapid settling of the particles. The use of these particles in an immunological or Nucleic acid assays, which are mainly carried out in suspension, are therefore sustainable impaired, one is therefore dependent on additional mechanical mixing. The decisive disadvantage of the coated particles is, however, that the Metal oxides both as a core material and as a coating material despite the subsequent silanization can come into direct contact with the analysis solution. This poses a serious problem in nucleic acid analysis e.g. B. in the context of Polymerase chain reaction ("PCR"), since the polymerases used in the PCR in Contact with iron compounds can be deactivated. In the from the state of the Magnetic particles known in the art are also not special procedural measures specified that lead to a targeted Pore size adjustment, a parameter that has a critical impact on efficiency who has nucleic acid purification. The methods known from the prior art for The production of the magnetic particles is fundamentally very complex and requires one manufacturing process lasting several hours.

The object of the present invention is to overcome the disadvantages of the known silica Bypass separation media and process for making magnetic particles To enable silica base that the previous labor and time consuming Bypass coating techniques entirely and are able to particle and Adjust pore sizes and the magnetic portion in a targeted manner so that the Separation media can be used more efficiently for biomolecule or nucleic acid isolation can.  

The starting point of the invention are prepolymers in the form of silica hydrosols magnetic colloids or magnetic particles are mixed and then in heterogeneous phase can be polycondensed to spherical polymer particles.

The silica brine is produced by the known sol-gel processes Hydrolysis of alkoxysilanes using dilute mineral acids or acids organic basis. The mineral acids are hydrochloric acid or carboxylic acids such as acetic acid, Formic acid or propionic acid are used.

To obtain the silica brine, the alkoxysilanes are dispersed in water and hydrolyzed by adding acid. Silicic acid orthoesters of aliphatic alcohols can be used as alkoxysilanes, preferably methyl, ethyl or propyl esters being used individually or as mixtures. As a result, condensation to low-polymer SiO ₂ hydrosols takes place, which gradually lead to more or less viscous sols or gels through further polycondensation. This sol-gel process is usually carried out at lower temperatures, preferably with ice cooling. In order to better mix the heterogeneous silane-water phase, the reaction is carried out in an ultrasonic bath or with the aid of an ultrasonic sonotrode. Depending on the composition, sonication times of 5 to 30 minutes are sufficient, the sonication times generally decreasing with increasing acid concentration. The mineral acids which are preferably used consistently have a concentration of 0.02 to 1 mol / liter, the volume fraction of the acids in the batch being 15-35%, preferably 20-28%. The monocarboxylic acids are used as pure acids; their volume share is 15-30%.

The stoichiometry of the gel will depend on the type of hydrolysis and Polycondensation determined. Acid catalysis generally leads to higher ones Hydrolysis rates with slowed polycondensation, while conversely the addition of Bases that promote polycondensation.

As is generally known from the prior art, the specificity of the nucleic acid Purification decisively determined by the pore structure, such that Nucleic acid molecules with a molecular weight <50 kDa, preferably on separation supports are separated with a pore size of <30 nm, while for nucleic acids <50 kDa Carriers with a pore size of 5-30 nm are particularly suitable.

Both hydrolysis and polycondensation reaction mechanisms leading to the sol can are used to specifically change or adjust the pore structure of the gels. This can be achieved concretely in that the sol phase in the presence of a defined Acid concentration is produced. Run under otherwise identical test conditions Acid concentrations of <0.15 mol / liter generally to pore sizes of <30 nm, whereas a decrease in the acid concentration to <0.15 mol / liter with a Reduction in pore size below 30 nm goes hand in hand. The mentioned Parameter settings are fundamentally with the respective conditions of the linked base-catalyzed polycondensation. So usually the above-mentioned pore dimensions consistently with the addition of a 1-3% Realize ammonia solutions. The use of more concentrated bases (6-25%) leads in contrast, under otherwise identical acid concentration ratios to pore diameters of <30 nm.

In addition to hydrolysis and polycondensation as structure-determining factors, Surprisingly, it can also be shown that using both reaction mechanisms the viscosity of the sol phase can be used to adjust the particle sizes.  The viscosity of the brine is derived directly from the specific reaction of the brine Gel formation stops during the aging process. In the course of this process occurs Formation of oxo bridges water and alcohol from the sol from a parallel Viscosity increase is accompanied. This increase in viscosity results in otherwise the same Process conditions (stirring speed, chemical composition) to a Particle enlargement. In general, particles with a size of <100 µm predominantly with a sol viscosity of <40 cp and particles <200 µm from sols with a viscosity of <40 cp.

The composition of the silane approach according to the invention not only meets the morphological requirements for the separation media, it also surprisingly creates the conditions for the production of spherical magnetic silica particles. These are:

• a) Miscibility of the brine with aqueous magnetic colloids or magnetic particle suspensions
• b) formation of defined polymer droplets when dispersed in suitable organic Solvents.

As soon as the silane suspension is homogenized by hydrolysis in the first production step the sol can be used for further particle production. An instant one However, use of the brine is not absolutely necessary, since the brine, depending on Acid concentration and sol approach, a fluid consistency over several days maintained and can still be processed after days.

In the second process step, a magnetic colloid or is added Ferrofluids to the sol. By definition, "colloids" or "ferrofluids" include all magnetic nanoparticles, either with or without the addition of a stabilizer, Surfactants or emulsifiers form a stable aqueous colloidal dispersion.

Magnetic and transition metal oxides, ferrites or come as magnetic nanoparticles other nanoparticulate compounds in question, which are either ferro, long distance or are superparamagnetic in nature. The production of such colloids or ferrofluids are variously described in: US 4,628,037; Br. 1,439,031; EP 0275 285; US 3,917,538; US 4,827,945; US 4,329,241.

The main criterion for the selection of suitable colloids or ferrofluids is their Compatibility with the silica sol, i. that is, the colloid may be in contact with the sol phase do not flocculate or agglomerate. Ferrofluids, which are a homogeneous dispersion with the Soles are those that either loaded surfactants z. B. in the form of aromatic or contain aliphatic sulfonic acid derivatives or aliphatic carboxylic acids. Such fluids are also commercially available.

Alternatively, magnetic colloids can also be used without the addition of surfactants or emulsifiers or other surface-active substances. For this, the well-known Precipitation process from iron (II) and iron (III) salt solutions, such as u. a. by Khalafalla and Reimers (Br. Patent. 1,439,031), Shinkai et al. (Biocatalysis, Vol. 5, 61, 1991) or Kondo et. al. (Appl. Microbiol. Biotechn., Vol. 41, 99, 1994). Crucial to The synthesis of these colloids is by either carefully removing the foreign ions Magnetopheresis or dialysis. The raw product is used in magnetopheresis through a chromatographic column packed with steel wool (more preferred Diameter 0.4-10 mm), which is between the poles of a strong permanent or Electromagnet is located, passed through. Because of the generated High gradient magnetic field, the excess salt solution runs through the column unhindered through while the colloid or ferrofluid is retained. By washing it several times of the retained colloid, residual salts can largely be removed. To  Removing the column from the magnetic field or switching off the magnetic field can do that Colloid can then be recovered by simple elution.

In addition to the use of the above colloids or ferrofluids, there are basically also such Magnetic particles can be used for encapsulation, which, for. B. in the form of polyvinyl acetate, polyvinyl alcohol, dextran, polyacrolein, polystyrene, albumin or Alginate. Such magnetic particles, which generally have a particle size of 0.05 have up to 3 µm and are used in the field of biomolecule purification known from the prior art: PCT / EP96 / 02398; 3. Appl. Polymer Sci., Vol. 50, 765, 1993; Anal. Biochem., Vol. 128, 342, 1983; US 4,654,267; J. Cell Sci., Vol. 56, 157, 1982; Proc. Natl. Acad. Sci., Vol. 78, 579, 1981; DE 35 08 000. Magnetic particles of this type are also commercially u. a. under the name Dynabeads, BioMag, Estapor, M-PVA, AGOWA, BioBeads or SPHERO are offered. T. to registered Trademarks.

These magnetic particles are used in a similar way to the colloids or ferrofluids Production of the silica particles according to the invention used.

The magnetic-colloid-sol mixture is dispersed in an organic solvent with stirring in the subsequent step. Suitable dispersants are solvents which are immiscible with water and which form either emulsions or dispersions. It has surprisingly been found that organic solvents in particular lead to a stable dispersion which have a distribution coefficient of <2. According to C. Laane et al. (in "Biocatalysis in Organic Media", Laane et al. Ed., Elsevier, Amsterdam, pp 65, 1987) the logarithm of the distribution coefficient is defined in a standard octanol-water two-phase system. Solvents that meet these properties are e.g. B. chloroform, trichlorethylene, 1.1.1-trichloroethane, hexane, petroleum ether, toluene; Carbon tetrachloride, heptane, octane. Mixtures of the above solvents, which have a density of approximately 1 g / cm ³ , are also suitable for dispersing.

The production of the magnetic particles is basically with the pure ones specified above Solvents possible. With regard to a narrower size distribution of the particles and a better dispersion behavior could surprisingly be shown that the organic phase one or two emulsifiers or surfactants can be added. Additives of this type are e.g. E.g .: polyglycerol esters, polyethylene glycol castor oil derivatives, Polyoxyethylene sorbitan fatty acid esters, alkylphenylpolyethylene glycol derivatives, Block copolymers from castor oil derivatives, propylene oxide-ethylene oxide block copolymers, modified polyesters, polyethylene glycol ether derivatives, polyoxypropylene ethylene diamine Block copolymers sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene, Polyoxyethylene alcohol derivatives, polyhydroxy fatty acid polyethylene glycol Block copolymers, substances of this type are commercially available u. a. under the Trade name: Hypermer®, Renex®, Estol®, Pluronic®, Eumulgin®, Tetronic®, Triton®, Brij®, Lameform®, Pripol®, Arlacel®, Prisorine®, Span®, Tween®, Dehymuls® or Synperonic® known.

The emulsifier concentrations relevant for the production of the magnetic particles lie between 0.1 and 15%, preferably between 0.5 and 6% by volume or weight.

In principle, conventional vegetable oils can also be used for dispersing Use mixtures of vegetable oils with the organic solvents, it did however, the use of organic solvents has shown some advantages over oils Have advantages. These are due to the low viscosity of the organic compounds against the oil. The low viscosity enables the separation of the  synthesized magnetic particles from the reaction mixture within a few seconds Using a commercially available hand magnet. This separation step requires at Use of vegetable oils, however, up to a few hours, followed by extensive Washing processes with organic solvents, which are necessary to remove the residual oil to remove the magnetic particle. In the present method, however, the used solvents can be easily recovered by redistillation. The volume ratio of organic phase to hydrosol is usually 8: 1 to 30: 1 and 2: 1 to 4: 1 in relation to the volume ratio sol to magnet colloid. The The proportion by weight of the magnetic solid in the sol approach is between 10-55%. The possibility of simple and targeted adjustment of the magnetic component by mere Mixing the magnetic colloid using this method over the prior art distinguishes, opens up a wide range of applications, which is simply the separation of Biomolecules and nucleic acids or biomolecule analysis as in PCT / EP 97/04828; DE 32 11 309; US 5,320,944; U.S. 4,927,749; US 4,927,750; PCT / US 99/00403, PCT / FR 97/00912, goes far. This affects e.g. B. the use of Magnetic particles as biocatalysts in biotechnological fermentation processes, as Protein encapsulation matrix, as a "stimulus-response" gel or as an adsorber in the Hemoperfusion. These additional applications result from the fact that with the help of Processes and products according to the invention both biomolecules such as enzymes or proteins encapsulated in the sol-gel matrix by simple admixing to the sol approach as well covalently coupled to the silica support via active groups, as will be explained below can be.

To fix the magnetic silica dispersion to defined magnetic particles, the added a base during the last process step during the mixing process. This Adding the base leads to a solid gel formation of the Polymer droplet. The higher the chosen one, the shorter the gelation process Base concentration is. Ammonia is preferably used as the base, other bases, such as B. NaOH can in principle also be used. The caustic soda is in the Usually as a 0.05 to 0.1 molar solution, ammonia consistently in the form of a 1 to 12% aqueous solution used. The volume ratios of base to sol are usually around 1: 2 to 1: 4.

Since the gelling reaction is very rapid, the manufacturing process requires that Base particles including the synthesis of the sol and the ferrofluid or colloid Time spent less than an hour. That means compared to all conventional ones Procedure a time saving of 60 to 90%.

There are no special requirements for mixing the sol-solvent mixture restrictions; In general, the usual mixers can be used. For the production of particularly fine particle fractions (<10 µm) are commercially available Dispersing tools that work according to the rotor-stator principle (e.g. Ultra-Turrax®) a rotational speed of up to 20,000 rpm is required. Between stirring speed and particle size there is an inverse proportionality such that particle sizes between 20 and 500 microns generally by stirring speeds in the range of 800-1800 rpm generated and particles in the range of 0.5-10 microns stirring speeds Require 5000-20,000 RPM. The stirring process usually takes 2 to 5 seconds. The Magnetic particles generated can then be removed from the suspension using a hand magnet be separated. This process is followed by several washes with alcohol and Water. The magnetic carriers obtained are usually stored in water. The silica particles obtained can then be used directly for the purification of nucleic acids or proteins can be used according to the known methods.  

In practice, there are not only carrier media for the separation of nucleic acids pure silica base has been found to be useful, it is also known that especially media with cationic groups (anion exchangers) for nucleic acid separation excellent are suitable. Carriers of this type are known from DE 32 11 309 and DE 37 17 209. This type of carrier can be achieved by chemical reaction of the silica particles with epoxy substituted alkoxysilane such. B. 3-glycidyloxypropyltrimethoxysilane or 3- Glycidyloxypropylmethyldiäthoxysilan and subsequent nucleophilic opening of the Produce oxirane ring using tertiary or secondary alkylamines. The synthesis too strong and weakly acidic ion exchangers and metal chelate carriers are through Implementation of the described epoxy-substituted silica particles with the help of Carboxylic acids, sulfites, thiosulfates or amino-substituted carboxylic acids, e.g. B. Nitrilotriacetic acid or iminodiacetic acid possible.

The chemical modification of the silica particles is not limited to the production of ion exchangers. In a particular embodiment, the SiO ₂ supports can be substituted with substituted alkylalkoxysilanes of the general formula X- (CH ₂ ) _n -Si- (OR) ₃ , where X is a halogen, cyano, NH ₂ or mercapto radical, n = 1 -6, preferably 3, R is an alkyl, trialkysilyl radical or H, are reacted. Ligands in the form of peptides, proteins or enzymes can be covalently coupled to the supports modified in this way for the separation according to the affinity principle or for use as biocatalysts. There are several options for this:

• 1. Protein and other ligands can be easily incubated with the halogen substituted carriers can be coupled directly.
• 2. Protein ligands can be used with dialdehydes Covalently bind amino group-substituted magnetic particles.
• 3. Amino-substituted silica supports are first made using Succin anhydride implemented and the carboxyl groups then z. B. with Carbodiimides activated. Protein ligands can be directly attached to the activated groups be coupled.
• 4. Binding of protein and other ligands via the oxirane group of the above described epoxy-substituted silica particles.

Amino-functionalized nucleic acids can be processed in an analogous manner to the proteins couple any type to the silica support using the methods described.

Without further detailed explanations of these couplings and modifications to deal with the u. a. in "Scientific and Clinical Applications of Magnetic Carriers ", Haefeli et al. (Ed.), Plenum Press, New York, 1997, and L.C. Shriver-Lake in "Immobilized Biomolecules in Analysis", T. Cass and F. S. Ligler ed., Oxford University Press, 1998, it is believed that one skilled in the art can Area knows the special reaction methods well and therefore the description can basically use. The execution farms described are therefore in none Way to be interpreted as limiting revelations.

In addition to the possibility of producing magnetic particles from pure silica gel, Surprisingly, it can be shown that the magnetic particles can also be mixed by Silica sols with synthetic, semi-synthetic or natural organic polymers have it made. Regarding the changed morphology and the chemical Reaction behavior compared to pure silica gels results in a modified one Range of properties that opens up an expansion of the application framework. This relates  focus in particular on the application in the purification of high molecular weight Nucleic acids and the separation of proteins. Passes through the polymer mixture one to hybrid polymers that partially have both the chemical-physical properties of organic polymers as well as those of silica gels. Are specific in this regard, the adsorption behavior of the hybrid particles against proteins that mechanical strength and base stability.

Those polymers which are water-soluble and are compatible with the silica sols, d. H. homogeneous mixtures without phase separation can form. The products of the invention and their targeted application in selected biochemical and biotechnological areas are such Polymers preferred, which both a targeted modification of the morphological structure in the With regard to an increase in surface area and also with regard to the pore structure enable.

Polymers that are preferred as additives are, for. B. polyvinyl alcohol, Polyacrylic acid, polyamino acids, polysaccharides, proteins or polyvinyl pyrrolidone. According to the invention, 0.5-5% aqueous polymer solutions are usually used, which are mixed with the silica sols before the dispersing process. The volume share The organic polymer solutions in the sol phase are between 5 and 25%. Usually polymers with a molecular weight of 15,000 to 250,000 are used.

The use of the predominantly very hydrophilic polymers also offers with regard to the separation of proteins the possibility that the strongly pronounced with pure silica gels largely suppress non-specific adsorption behavior.

The addition of the organic polymers to the sol approach changes that described above Procedure for the production of pure silica particles basically nothing.

The processes and products according to the invention are described in the examples below described in more detail without limiting them.

example 1

A mixture of 55 ml of tetraethoxylsilane and 15 ml of 0.05 M HCl are mixed in one Ultrasonic bath for 25 min. sonicated under ice cooling. 20 ml of the silica sol at 20 ° C has a viscosity of 36 cp, with 5 ml of a magnetic colloid that according to the Shinkai et al. (Biocatalysis, Vol. 5, 61, 1991) by oxidation of a 0.6 molar iron (II) salt solution was prepared using 0.3 M Na nitrite, mixed. The suspension obtained is dissolved in 280 ml of 1.1.1-trichloroethane in which 0.5% by volume Tween 80 and 0.8 vol .-% Prisorine are dissolved, entered. The batch is stirred (1800 rpm) dispersed for a few seconds; then 10 ml of 1% Ammonia solution added; the dispersion is stirred for a further 3 seconds. After 5 Minutes, the magnetic particles are separated from the suspension using a hand magnet and Washed three times each with approx. 50 ml ethanol and water. There are magnetic particles with a particle size of 20-60 microns. After 12 hours of incubation in water the particles are washed several times with water and then several Dried in vacuo to constant weight for hours. The particles have a medium Pore size of 24 nm. The magnetic particles can be obtained according to the known Methods for the purification of nucleic acids are used.  

Example 2

A mixture consisting of 25 ml of the sol prepared according to Example 1 and 8 ml of one Magnetite colloids, according to the instructions of Khalafalla and Reimers (Br-Pat. 1 439 031) from an iron (III) - / iron (II) salt solution (molar ratio 2: 1) by ammonia Precipitation was obtained in 300 ml of toluene, in each of which 2% by volume Pripol 1009 and Tween 20 are solved using an Ultra-Turrax 'at 12,000 rpm. dispersed. To The addition of 12 ml of 3% ammonia solution is further dispersed for 2 seconds. It separation and processing follows as in Example 1. Magnetic particles with a Particle size of 1-4 microns and a magnetite content of 38 wt .-% obtained.

Example 3

A suspension consisting of 50 ml tetramethoxylsilane and 20 ml 0.3 M HCl are used for 5 min. sonicated in an ultrasonic bath. 20 ml of the sol thus obtained, which has a viscosity of 48 cp, with 8 ml of the magnetite colloid prepared according to Example 2 and 2 ml of a 2.5% polyvinyl alcohol solution (molecular weight = 224,000) are mixed. The The suspension is then stirred (1600 rpm) in 320 ml of hexane, the 0.5 vol .-% Contain Estol SMO 3685 dissolved, dispersed. During the dispersing process, 10 ml 1% ammonia solution is added and stirring is continued for 4 seconds. Separation and The magnetic particles obtained are worked up analogously to Example 1. Carriers are formed with a size of 80-150 microns and an average pore size of 58 nm separated magnetic particle fraction is 5 times with dried toluene after each magnetic separation and then 12 hours after adding 50 ml Toluene and 0.85 g of 3-aminopropyltriethoxysilane boiled under reflux. The magnetic particles are separated magnetically again and 3 times each with toluene and chloroform rewashed. This is followed by drying in vacuo for several hours. The amino modified The product is then treated with 6% glutaraldehyde solution in 25 ml 0.1 M Na carbonate Buffer, pH 9.2, reacted for 3 hours at 35 ° C. It then becomes intense with 0.1 M. Wash phosphate buffer, pH 7.2. The aldehyde-functionalized obtained Magnetic particles are suspended in 15 ml 0.1 M phosphate buffer. 2 ml of the suspension are separated magnetically and in 2 ml of 0.1 M phosphate buffer, pH 7.2, in which 1.2 mg Streptavidin are dissolved, incubated. After 6 hours of reaction at 35 ° C the product washed five times with phosphate buffer. To remaining aldehyde groups to saturate, the magnetically separated product in 5 ml of 0.2 M ethanolamine Incubated room temperature over a period of 5 hours. The then several times Magnetic particles washed with phosphate buffer can be used directly after the known ones Methods for binding biotinylated nucleic acids or biotinylated proteins are used become.

Claims (17)

1. A process for producing spherical, magnetic silica particles, characterized in that a silica hydrosol formed by acid-catalytic hydrolysis of an aqueous silane dispersion, which is mixed with a magnetic colloid or ferrofluid or with magnetic particles, in one with water not dispersible miscible organic phase and is crosslinked during the dispersing process by adding a base. 2. A method for producing magnetic silica particles according to claim 1, characterized characterized in that the hydrosol has a viscosity of 5-500 cp. 3. A method for producing magnetic silica particles according to claim 1, characterized characterized in that the water-immiscible organic phase 0.1 to 25 vol .-% contains one or more surface-active substance (s) dissolved. 4. A method for producing magnetic silica particles according to claim 1, characterized characterized in that an organic solvent with an Distribution coefficient <2 is used. 5. A method for producing magnetic silica particles according to claim 1, characterized characterized in that the volume ratio of sol to organic phase is 1: 5-1: 30. 6. A method for producing magnetic silica particles according to claim 1, characterized characterized in that the aqueous silane dispersion 15-35 vol .-% of a 0.02 to 1 molar Contains acid. 7. A method for producing magnetic silica particles according to claim 1, characterized characterized in that the volume ratio of silica sol to magnetic colloid or Ferrofluid or magnetic particles 2: 1-4: 1. 8. A method for producing magnetic silica particles according to claim 1, characterized characterized in that the dispersion crosslinking process takes 1-5 seconds. 9. A method for producing magnetic silica particles according to claim 1, characterized characterized in that the sol before the dispersion process 1-30 vol .-% of an aqueous Solution of an organic polymer, polysaccharide or protein can be added. 10. A method for producing magnetic silica particles according to claim 9, characterized characterized in that as organic polymer polyvinyl alcohol, dextran, starch, Chitosan, alginate, ficoll, polyethyleneimine, polyvinylpyrrolidone, agarose, Polyacrylic acid, hyaluronic acid, pectinate or alginate is used. 11. A method for producing magnetic silica particles according to claim 1, characterized characterized in that the silica particles with the dispersion crosslinking process Alkoxysilanes or substituted alkylalkoxysilanes are implemented. 12. Spherical, magnetic silica particles with a particle size of 0.5-2000 µm and adjustable pore size and adjustable magnetic content for the nucleic acid or Biomolecule separation, obtainable by a dispersion crosslinking process aqueous silica sols, characterized in that in the silica particle  magnetic colloid or ferrofluid or magnetic particles homogeneous over the whole Particles are distributed and encapsulated, the magnetic solids content being 10-55% by weight based on the silica content. 13. Spherical, magnetic silica particles according to claim 12, characterized in that that the magnetic particles formed from the silica sol 2-25 wt .-% of an organic Contain polymers, polysaccharides or proteins. 14. Spherical, magnetic silica particles according to claims 12 and 13, characterized in that the silica particles with an alkoxysilane or a substituted alkylalkoxysilane of the general formula X- (CH ₂ ) _n -Si- (OR) ₃ , where X is a Halogen, epoxy, cyano, NH ₂ or mercapto radical, n = 1-6, R is an alkyl, trialkysilyl radical or H, are implemented. 15. Spherical, magnetic silica particles according to claim 12 to 14, characterized characterized in that the silica particles with oligopeptides, oligosaccharides, Oligonucleotides, polypeptides, polynucleotides, polysaccharides, antibodies, Have antibody fragments or enzyme-coupling groups. 16. Spherical, magnetic silica particles according to claim 14, characterized in that that the epoxy-substituted silica magnetic particles with secondary or tertiary Alkylainines are converted to anion exchangers. 17. Use of the spherical, magnetic silica particles according to one of the claims 12 to 16 for the isolation or analysis of nucleic acids, proteins, antibodies, or antibody fragments and biotinylated nucleic acids, proteins, antibodies or antibody fragments.