Magnetic pigment

The invention relates to magnetic particles with a glass surface, methods for cleaning a biological material, in particular nucleic acids using glass particles in the presence of chaotropic salts, methods for isolating these biological materials and methods for concentrating biological materials and the transfer of biological materials from solutions with high concentration of salts in solutions with low concentration of salts.

Some biological materials, especially nucleic acids, have special requirements with regard to their isolation from the natural environment. On the one hand, they are often present in very low concentrations and, on the other hand, they are often in the vicinity of many other solid and dissolved substances that impair their isolation or determination.

There has been no shortage of attempts recently to propose methods and materials for isolating nucleic acids from their natural environment. From proc. Natl. Acad. USA 76, 615-619 (1979) describes the binding of nucleic acids from agarose gels in the presence of sodium iodide in ground flint glass.

In anal. Biochem. 121, 382-387 (1982) describes the purification of plasmid DNA from bacteria on glass dust in the presence of sodium perchlorate.

DE-A 37 34 442 describes the isolation of single-stranded M13 phage DNA on glass fiber filters by precipitation of the phage particles with the aid of acetic acid and lysis of the phage particles with perchlorate. The nucleic acids bound to the glass fiber filters are eluted after washing with a methanol-containing buffer in Tris / EDTA buffer.

In anal. Biochem. 175, 196-201 (1988) describes a similar method for the purification of DNA from Lambdaphagen.

The previously known methods of the prior art have in common the selective binding of nucleic acids to glass surfaces in chaotropic salt solutions, the nucleic acid being separated from impurities such as agarose, proteins or cell debris. To separate the glass particles from the contaminants, either centrifugation of particles or suction of liquids through glass fiber filters is used according to the prior art. However, this is a limiting step that severely hinders the processing of large numbers of samples.

In anal. Biochem. 201, 166-169 (1992) and PCT GB 91/00212 describe the use of magnetic particles for immobilizing nucleic acids after precipitation by adding salt and ethanol. Here, the nucleic acids are agglutinated, including the magnetic particles. The agglutinate is separated from the original solvent by applying a magnetic field and washing. After a washing step, the nucleic acids are dissolved in a Tris buffer. However, this method has the disadvantage that the precipitation is not selective for nucleic acids, but instead a large number of solid and dissolved substances are also agglutinated. It is therefore not possible with this method to remove any existing inhibitors for certain enzymatic reactions to a sufficient extent.

US-A-4,233,169 describes a porous glass which contains magnetic particles embedded.

There is currently also so-called magnetic, porous glass, which contains magnetic particles in a porous, particulate glass matrix, and the surface of which is coated with a streptavidin-containing layer. This product can be used to isolate biological materials, for example proteins or nucleic acids, if they are modified in a complex preparation step so that they are covalently bound to biotin.

The object of the invention was to provide better materials for the immobilization of biological materials and a simple method for isolating biological materials, in particular nucleic acids, which is suitable for routine diagnostics.

The invention relates to magnetic particles with an outer glass surface which is essentially pore-free or has pores with a diameter of less than 10 nm. Another subject is ferromagnetic particles with a glass surface, methods for isolating biological materials, in particular nucleic acids, and methods for producing magnetic glass particles.

The person skilled in the art refers to solid materials with a small diameter. Sometimes such particles are also called pigments. Particles which have an average grain size of less than 100 μm are particularly suitable for the purposes of the present invention. They particularly preferably have an average grain size of between 10 and 60 μm. The grain size distribution is preferably relatively homogeneous, in particular there are almost no particles <10 μm or> 60 μm.

Materials that can be attracted by a magnet, ie, for example, ferromagnetic or superparamagnetic materials, are referred to as magnetic. Magnetic are also understood to mean materials which are referred to as soft magnetic materials, e.g. B. ferrites. Ferromagnetic materials are particularly preferred in the sense of the invention, in particular if they have not yet been magnetized. In this context, premagnetization is to be understood as contacting a magnet, which increases the remanence. Ferromagnetic materials, such as. B. magnetite (Fe₃O4 or Fe₂O₃.

An outer surface of a particle is understood to mean the coherent surface from which perpendiculars can be formed in the direction of the surroundings of the particle, which do not intersect the same particle again.

A pore is understood to mean a recess in the outer surface of the particle, in which the surface extends so far into the particle that an imaginary perpendicular formed in the recess on the surface in the direction of the immediate vicinity of the particle intersects the particle at least once. Pores also extend deeper than a radius of the pore into the particle.

A glass in the sense of the present invention is understood to mean a silicon-containing amorphous material. The glass may contain other materials, e.g. B.<tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">B₂O₃</entry><entry namest="col2" nameend="col2" align="left">(0 - 30 %),</entry></row><row><entry namest="col1" nameend="col1" align="left">Al₂O3</entry><entry namest="col2" nameend="col2" align="left">(0 - 20 %),</entry></row><row><entry namest="col1" nameend="col1" align="left">CaO</entry><entry namest="col2" nameend="col2" align="left">(0 - 20 %),</entry></row><row><entry namest="col1" nameend="col1" align="left">BaO</entry><entry namest="col2" nameend="col2" align="left">(0 - 10 %),</entry></row><row><entry namest="col1" nameend="col1" align="left">K₂O</entry><entry namest="col2" nameend="col2" align="left">(0- 20 %),</entry></row><row><entry namest="col1" nameend="col1" align="left">N₂O</entry><entry namest="col2" nameend="col2" align="left">(0 - 20 %),</entry></row><row><entry namest="col1" nameend="col1" align="left">MgO</entry><entry namest="col2" nameend="col2" align="left">(0 - 18 %),</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Pb₂O₃</entry><entry namest="col2" nameend="col2" align="left">(0 - 15 %).</entry></row></tbody></tgroup></table></tables>

To a lesser extent, 0-5%, a variety of other oxides, such as. B. Mn₂O₃ TiO₂. As₂O₃, Fe₂O₃, CuO, CoO, etc. may be included. Surfaces with a composition of borosilicate glass, flint glass or silica have proven to be particularly effective. Borosilicate glasses which are particularly preferred from the point of view of the yield of nucleic acids have a boron oxide content of more than 25%; a glass with the composition SiO was found to be particularly valuable₂/ B₂O₃ 70/30 recognized. Glasses which are formed by the so-called gel-sol process and subsequent drying and compacting of the layer formed are particularly preferred in the sense of the invention. The main features of this process are known and have been described e.g. B. in CJ Brinker, GW Scherer "Sol Gel science - The physics and chemistry of Sol Gel Processing", Academic Press Inc. 1990 and Sol-Gel Optics, Processing and Applications Lisa C. Klein Ed. Kluwer Academic Publishers 1994, page 450 ff. as well as in DE-A-1941191, DE-A-3719339, DE-A-4117041 and DE-A-4217432. However, it has not yet been described for magnetic particles. It was not to be expected that magnetic particles could be generated with this, which have very surprising properties when isolating biological materials, in particular nucleic acids. In the gel-sol process, alkoxides of network-forming components, e.g. B. SiO₂ B₂O₃ Al₂O₃, TiO₂ ZrO₂ GeO₂ together with oxides and salts of other components, e.g. B. in alcoholic solution, submitted and hydrolyzed. The equation shows the manufacture of sodium boron aluminum silicate glass.<chemistry id="chem0001" num="0001"><img file="EP1577389A2_D0001.tif" /></chemistry>

The hydrolysis process of the starting components is started by adding water. The reaction proceeds relatively quickly, since the alkali ions have a catalytic effect on the rate of hydrolysis of the silicic acid ester. After the gel formation has ended, the resulting gel can be dried and compressed into a glass by a thermal process.

The quantitative ratio of sol / pigment has a considerable influence on the yield of magnetic pigment according to the invention. There are limits to the fact that the pigment content is so low that a mass that can still be pumped and sprayed is created. If the pigment content is too low, the fine content, e.g. B. of non-magnetic material too large and disturbing. 10 to 25 g pigment / 100 ml sol were found to be appropriate quantitative ratios with regard to the pigment yield.

The slurry is preferably sprayed through a nozzle to form a powder and the aerosol is dried on a falling section. The nozzle is preferably heated to accelerate drying of the slurry. Depending on the geometry of the nozzle, the nozzle temperature is preferably approximately 120 to 200 ° C. A compromise is found by sufficient evaporation rate, but avoiding splashing.

In view of the yield, the compression temperature should be as high as possible. However, if it is too high, the particles stick together and agglomerates form which should be screened out. The post-treatment in air leads to a loss of magnetic properties at too high temperatures, which is why excessive temperatures should be avoided.

An essentially pore-free surface is understood to mean a surface that is permeated with less than 5%, preferably less than 2%, particularly preferably less than 0.1%, with pores as defined above. Types of pores are present, they preferably have a diameter of less than 10, particularly preferably 1 nm.

For the purposes of the invention, particular preference is given to particles which have a core composed of TiO₂ contain coated mica and magnetite particles immobilized thereon, the composite thus formed being enclosed by the glass layer. Both the core and the magnetite particles are crystalline and not porous. The spaces on the surface of the mica, which are not occupied by the magnetite particles, are covered by a thicker glass layer than the tips of the magnetite particles, so that an essentially non-porous glass surface results.

The non-porosity of the magnetic particles refers only to the outer surface, not to the inside of the particle, so that the particle can be porous inside if the surface is only of substantially non-porous glass or a glass surface with pores of less than a diameter 10 nm is enclosed.

Surprisingly, the magnetic particles according to the invention are particularly advantageously suitable for isolating biological materials from samples. In particular, long nucleic acids are destroyed very little or not at all when they are immobilized on them. The material of the core is also a natural resource and therefore ecologically unproblematic. The production of the particles according to the invention is also very inexpensive and inexpensive.

The invention also relates to ferromagnetic particles with a glass surface. Superparamagnetic particles are described in the prior art. It has now been found that ferromagnetic particles, when coated with a glass surface, have considerable advantages in the isolation of biological materials. As long as the ferromagnetic particles were not yet exposed to a magnetic field, they sediment only under the influence of gravity. They can be easily and quickly suspended again by shaking. The process of deposition without the influence of a magnetic field is preferably slower than the immobilization of biological materials on their surface. This applies in particular to nucleic acids. The ferromagnetic particles can be collected in a simple manner by means of a magnet at a specific point in the sample liquid in order to separate the liquid from the particles and thus from the immobilized biological materials.

The glass surface of the ferromagnetic particles according to the invention can be pore-free or contain pores. For the reasons mentioned above for the magnetic particles according to the invention, it is preferred that the outer surface of the ferromagnetic particles is also essentially pore-free or has pores with a diameter of less than 10 nm. The ferromagnetic particles according to the invention also preferably have a grain size between 10 and 60 μm, particularly preferably 20 and 50 μm. Particularly preferred are particles in which pores that may be present in the surface have a diameter of less than 10, particularly preferably 1 nm. An example of a ferromagnetic particle according to the invention is the above-mentioned composite of mica and magnetite particles, enclosed by a glass layer.

The invention also relates to a method for isolating a biological material by<ul id="ul0001" list-style="dash" compact="compact"><li>Contacting a sample which contains the biological material in a liquid with the magnetic particles or the ferromagnetic particles according to the invention under conditions in which the biological material binds to the particle surface, and</li><li>Separation of the biological material from the liquid.</li></ul>

Biological materials are understood to be materials on a particulate or molecular basis. These include in particular cells, e.g. B. viruses and bacteria but also human and animal isolated cells such as leukocytes, and immunologically active low and high molecular weight chemical compounds such as haptens, antigens, antibodies and nucleic acids. Nucleic acids, e.g. B. DNA or RNA.

Samples within the meaning of the invention are, for example, clinical samples such as blood, serum, mouthwash, urine, cerebral fluid, sputum, stool, punctate and bone marrow samples. The sample can also be used in the field of environmental analysis, food analysis or molecular biological research, e.g. B. from bacterial cultures, phage lysates and products of amplification processes, for. B. PCR.

According to the invention, the magnetic particles have an inner core to which the outer glass surface is applied. The core can be a composite, but also simple iron cores. The core can also consist of a crystalline or ceramic or glass-like structure in which iron oxide is embedded.

The described method can be used to isolate native or modified biological material. Native biological material is understood to mean material whose structure has not been irreversibly changed compared to the naturally occurring biological materials. However, this does not exclude the modification of other components of the sample. For example, if cells are to be isolated, the medium surrounding the cells may have been modified, but not the cells as such. If nucleic acids are to be isolated, they should also be in the native form, i.e. not be denatured, cut or modified by coupling reactive groups. The term native biological material therefore does not include, in particular, biotinylated nucleic acids. Examples of native biological materials are phage DNA or cellular nucleic acids from blood.

Modified biological materials include materials that are not found in nature, e.g. B. nucleic acids modified by attachment of reactive, detectable or immobilizing groups, e.g. B. biotinylated nucleic acids.

In certain cases, the sample can be used in the isolation method according to the invention without pretreatment. In many cases, however, the sample should be digested using a suitable method and the biological material contained in the sample released. Methods for the digestion of samples are known to the person skilled in the art and can be chemical, enzymatic or physical in nature. A combination of these methods is also possible. Examples include lysis by ultrasound, high pressure or by shear, by alkali, detergents or chaotropic salt solutions, or by the action of proteinases or lipases. Sambrook et al .: Molecular Cloning, A Laboratory Manual, 2nd Addition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al .: Current Protocols in Molecular Biology 1987, specifically with regard to the digestion methods for obtaining nucleic acids , J. Viley and Sons, NY.

In addition to the biological material to be isolated, the sample can contain further components, e.g. B. cell debris, proteins, salts and other substances not to be isolated in a liquid. This sample, which preferably contains the biological material in native form, is brought into contact with the particles under conditions in which the desired biological material binds to the particle surface. The conditions for this depend on the type of biological material, but are known in principle. They also depend on the type of binding via which the biological material is bound to the surface. If, for example, immunological interactions are to be used for binding, then conditions must be selected which are suitable for the formation of immune complexes. For nucleic acids, in the case of the modified nucleic acids, binding is possible via the groups of nucleic acids which represent the modification, e.g. B. Biotin via binding to surfaces coated with streptavidin. In the case of nucleic acids in particular, however, the case of the direct binding of nucleic acids to glass is preferred, among other things because there is no need to modify the nucleic acids and even native nucleic acids can be bound. The binding of native nucleic acids to glass particles can be carried out analogously to methods of the prior art. It is preferably carried out in the presence of chaotropic salts, the concentration of these salts being between 2 and 8 mol / l, preferably 4 to 6 mol / l. Chaotropic salts are e.g. B. sodium iodite, sodium perchlorate, guanidininium thiocyanate, guanidinium isothiocyanate or guanidinium hydrochlorite, but is not limited to these compounds.

To bring the sample into contact with the particles, the sample is mixed with the particles and incubated for a time sufficient for the binding. The length of the incubation is generally known to the person skilled in the art from treatment with non-magnetic particles. Optimization is possible by carrying out a determination of the amount of immobilized biological material on the surface at different times. Incubation times between 10 seconds and 30 minutes can be expedient for nucleic acids.

Depending on the size and type of magnetic particles, the particles are separated from the liquid during the incubation period or the suspension is retained over a longer period. If the particles have a very small particle size and are superparamagnetic, the suspension is preserved over a longer period. If the particles are larger, the particles are slowly separated from the liquid during the incubation. Such aggregates form in particular when ferromagnetic particles are involved. In the preferred case that the ferromagnetic particles are not premagnetized, a particularly gentle separation is guaranteed.

The immobilization preferably does not take place by precipitation by lowering the solubility of the materials to be immobilized. Instead, the immobilization is based on biospecific interactions (capture molecules) or adsorption. This largely avoids non-specific inclusions of impurities.

After the incubation, the biological material is separated from the liquid. This is generally achieved by separating the material bound to the magnetic particles using a magnetic field. For example, the magnetic particles can be drawn to the wall of the vessel in which the incubation took place. The liquid containing the sample ingredients that were not bound to the magnetic particles can then be removed. This distance depends on the type of vessel in which the incubation took place. Suitable process steps are pipetting off or suctioning off the liquid.

The magnetic particles can then, if desired, be cleaned one or more times with a washing solution. The washing solution is chosen so that the biological material does not detach from the particle surface as far as possible, but contaminants that are not to be isolated are washed away as well as possible. This washing step preferably takes place by incubating the washing solution with the particles, with a resuspension of the particles preferably being carried out, e.g. B. by shaking or applying a magnetic field that is not identical to the first magnetic field. The contaminated washing solution is preferably removed in the same way as the sample in the above-mentioned step for binding the biological material.

After the last washing step, a short drying step of the magnetic particles can be carried out in a vacuum or by evaporating (letting) the liquid, whereby a pretreatment with acetone is also possible.

The biological material thus purified can, if desired, be removed from the magnetic particles. This step also depends on the type of binding of the biological material to the magnetic particles. In the event that the biological material is native nucleic acids and the magnetic particles are glass-coated particles, the nucleic acid can be removed from the particles according to the invention by means of an elution buffer with a low salt content. Such buffers are known from DE 3724442 and Analytical Biochemistry 175, 196-201 (1988). Buffers with a content of less than 0.2 mol / l are used in particular as elution buffers with a low salt content. In a particularly preferred embodiment, the elution buffer contains Tris. In another particular embodiment, the elution buffer is demineralized water.

In a further embodiment, the cleaning and isolation method described can be carried out following an immunomagnetic separation of cells (for example viral particles or prokariontic or eucariontic cells) from a body fluid or a tissue. For this purpose, the sample with magnetic particles, to which an antibody against an antigen is immobilized on the cell, e.g. B. incubated with shaking. Such particles can be particles according to the invention, but also commercially available (eg MACS microbeads from Miltenyi Biotec GmbH, Bergisch Gladbach, FRG). After applying a magnetic field, one or more washing steps take place with a saline washing solution. Particles are obtained to which the desired cells are bound. Finally, the bound cells are resuspended in a saline buffer. In a preferred embodiment, this salt-containing buffer is a chaotropic salt solution, so that the nucleic acids present in the cell are released from the cells.

Combining the above-described isolation of cells with the likewise described isolation of nucleic acids, preferably in their native form, on the magnetic particles according to the invention results in a particularly advantageous method for isolating nucleic acids from cell-containing samples. Advantages of this embodiment are the possible simplicity (single-tube method), high sensitivity (particularly important in medical microbiology and oncology) and the ease of automation.

The biological materials isolated as a result of the method according to the invention can now be used in any way. For example, they can be used as a substrate for various enzymatic reactions. In the case of nucleic acids, sequencing, radioactive or non-radioactive labeling, amplification of one or more sequences contained therein, transcription, hybridization with labeled probe nucleic acids, translation or ligation may be mentioned as examples. An advantage of the method according to the invention is that the separation of the biological material from the liquid is very simple. In the prior art, either a centrifugation step was used to separate glass particles from impurities or, in the case of the binding of the biological material to glass fiber filters, the liquid was sucked through this liquid. This is a limiting step that hinders the processing of large numbers of samples.

With the particles according to the invention, a more effective separation of the biological materials from impurities is possible. In particular, inhibitors for certain enzymatic reactions can be removed to a particularly good extent according to the invention. The yield of biological material is comparatively high. Fractionation of long nucleic acids was not observed. The particles according to the invention are preferably magnetizable more quickly.

An isolation of nucleic acids from a cell-containing sample is shown schematically in FIG.

FIG. 2 shows the separation of nucleic acids isolated according to the invention in an agarose gel.

FIG. 3 shows the separation of reaction products after isolation and PCR amplification according to the invention.

FIG. 4 shows a gel of the results from example 4.

In Figure 1, an isolation of nucleic acids from a cell-containing sample is shown schematically. The sample (specimen), which contains cells, is pretreated sample-specifically so that the cells in which the nucleic acids are to be detected are in a suitable form. This includes e.g. B. in samples from which body fluids have been removed, the addition of reagents, for. B. for the liquefaction of viscous samples, for. B. Saliva samples. An antibody bound to a solid phase, preferably to a bead (bead), which can recognize and bind the cell, is added to the sample prepared in this way in a vessel. For example, antigens on the cell surface have proven to be suitable partners for the antibody. The specificity of the antibody can depend on the specificity of the analysis task to be solved. If the solid phase is the wall of the vessel, the cells are bound directly to the wall. In the event that the solid phases are pearls, they are separated from the liquid by suitable separation methods. This can be done, for example, by filtration. In the case of magnetic beads, separation is possible by applying a magnetic field to the outer wall of the vessel. The separated cells are washed with a liquid to remove contaminants which would interfere with the detection with the medium surrounding the cells. Conditions are preferably used in which the cells are neither detached from the solid phase nor destroyed. Then the cells are destroyed, the so-called lysis. One possibility is to treat the cells with chaotropic salts. Other options are the action of proteinases and detergents.

In the preferred embodiment, the particles according to the invention are added to the lysis mixture. After a suitable exposure time, which can be optimized by loading the surface with nucleic acids, the particles are separated from the liquid surrounding them, which contains further and undetectable cell components. Again, this is preferably done by applying a magnetic field to the vessel wall using a magnet.

In order to remove any impurities that may still be adhering, washing is preferably carried out with a liquid which is selected such that the nucleic acids to be determined do not detach from the glass surface. To remove the nucleic acids from the glass surface, a so-called elution buffer is added, which has reagent conditions under which the nucleic acids detach from the glass surface. These are especially low salt conditions. Depending on the intended further treatment of the nucleic acids, the liquid can now be separated from the particles and processed further. It is preferred to carry out this separation with an applied magnetic field so that the particles are present separately.

The following examples illustrate the invention.

example 1

Production of the magnetic particles according to the invention

6 different brines were used. The brine was produced according to the following schemes:<ul id="ul0002" list-style="none" compact="compact"><li>Sol 1 (SiO₂: B₂O₃ = 7:3):</li><li>The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 86.6 ml tetraethyl orthosilicate</li><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+ 14.1 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. Then add dropwise<ul id="ul0003" list-style="none" compact="compact"><li>+ 37.8 ml trimethyl borate.</li><li>The sol is then kept at 50 ° C. for 2 hours. Then add</li><li>+ 14.1 ml 0.15 M HCl</li></ul>

Sol 2 (SiO₂: B₂O₃ = 4:1): <ul id="ul0004" list-style="none" compact="compact"><li>The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 100.5 ml tetraethyl orthosilicate</li><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+16.3 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. This is followed by the dropwise addition of + 25.6 ml trimethyl borate. The sol is then kept at 50 ° C. for 2 hours. Then + 16.3 ml 0.15 M HCl is added<ul id="ul0005" list-style="none" compact="compact"><li>Sol 3 (SiO₂: B₂O₃ = 85:15):</li><li>The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 107.8 ml tetraethyl orthosilicate</li><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+ 17.5 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. Then add 19.4 ml of trimethylborate dropwise. The sol is then kept at 50 ° C. for 2 hours. This is followed by the addition of + 17.5 ml 0.15 M HCl Sol 4 (SiO₂: B₂O₃ = 4: 1; 2 mol% P₂O₅): <ul id="ul0006" list-style="none" compact="compact"><li>The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 100.5 ml tetraethyl orthosilicate</li><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+ 16.3 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. Then add dropwise<ul id="ul0007" list-style="none" compact="compact"><li>+ 25.6 ml trimethyl borate</li><li>The sol is then kept at 50 ° C. for 2 hours. Then + 16.3 ml 0.15 M HCl is added</li><li>+ 1.63 g P₂O₅</li></ul><ul id="ul0008" list-style="none" compact="compact"><li>Sol 5 (SiO₂: B₂ = 4: 1 mol% of Al₂O₃):</li><li>The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 100.5 ml tetraethyl orthosilicate</li><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+ .16.3 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. Then add dropwise<ul id="ul0009" list-style="none" compact="compact"><li>+ 25.6 ml trimethyl borate.</li><li>The sol is then kept at 50 ° C. for 2 hours. Then + 16.3 ml 0.15 M HCl is added</li><li>+ 3.06 g AlCl₃</li></ul><ul id="ul0010" list-style="none" compact="compact"><li>Sol 6 (SiO₂: B₂O₃ = 4: 1 mol% ZrO₂)</li><li>The synthesis was carried out in a 250 ml round bottom flask with constant stirring. 100.5 ml tetraethyl orthosilicate</li><li>+ 7 ml of anhydrous undenatured ethanol</li><li>+ 16.3 ml 0.15 M HCl</li></ul>

A two-phase mixture is formed, which is stirred at room temperature until it becomes single-phase. Then add dropwise<ul id="ul0011" list-style="none" compact="compact"><li>+ 25.6 ml trimethyl borate</li><li>+ 5.15 ml of zirconium (IV) propylate, 70% by weight solution in 1-propanol</li><li>The sol is then kept at 50 ° C. for 2 hours. Then + 16.3 ml 0.15 M HCl is added</li></ul>

After a further 2 hours at 50 ° C., 22.5 g of Iriodin 600 (Black Mica) were stirred into 150 ml of the brine and then coated with a spray dryer (Büchi 190, Mini Spray Dryer). The nozzle temperature of the spray dryer was 134 ° C.

The powder obtained by the spray drying process was then subjected to a temperature treatment under a nitrogen atmosphere (90 l / h). The heating rate was 1 k / min and the holding time was 2 hours at the compression temperature. This temperature was 750 ° C for coating with Sol 1, 860 ° C for coating with Sol 2 and 800 ° C for the other coatings. After the sintering process, the furnace was switched off and the powder was cooled to room temperature. Any agglomerates formed were screened with a 50 µm sieve.

Example 2

Manufacture of GMP1, GMP2, GMP3 and GMP4

GMP1, GMP2, GMP3 and GMP4 are pigments from different production batches, which were obtained from sol 1 from example 1 in a process according to example 1 under the following conditions: <tables id="tabl0002" num="0002"><table frame="all"><tgroup cols="5" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="31.50mm" /><colspec colnum="2" colname="col2" colwidth="31.50mm" /><colspec colnum="3" colname="col3" colwidth="31.50mm" /><colspec colnum="4" colname="col4" colwidth="31.50mm" /><colspec colnum="5" colname="col5" colwidth="31.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center"><b>parameter</b></entry><entry namest="col2" nameend="col2" align="center"><b>GMP1</b></entry><entry namest="col3" nameend="col3" align="center"><b>GMP2</b></entry><entry namest="col4" nameend="col4" align="center"><b>GMP3</b></entry><entry namest="col5" nameend="col5" align="center"><b>GMP4</b></entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Aging of the sol (h) (30 ° C)</entry><entry namest="col2" nameend="col2" align="left">36</entry><entry namest="col3" nameend="col3" align="left">36</entry><entry namest="col4" nameend="col4" align="left">36</entry><entry namest="col5" nameend="col5" align="left">36</entry></row><row><entry namest="col1" nameend="col1" align="left">Pigment fraction of the sol (g / 100 ml)</entry><entry namest="col2" nameend="col2" align="left">5</entry><entry namest="col3" nameend="col3" align="left">15</entry><entry namest="col4" nameend="col4" align="left">8</entry><entry namest="col5" nameend="col5" align="left">20</entry></row><row><entry namest="col1" nameend="col1" align="left">Airflow from the nozzle (%)</entry><entry namest="col2" nameend="col2" align="left">100</entry><entry namest="col3" nameend="col3" align="left">100</entry><entry namest="col4" nameend="col4" align="left">100</entry><entry namest="col5" nameend="col5" align="left">100</entry></row><row><entry namest="col1" nameend="col1" align="left">Air pressure (bar)</entry><entry namest="col2" nameend="col2" align="left">6</entry><entry namest="col3" nameend="col3" align="left">6</entry><entry namest="col4" nameend="col4" align="left">6</entry><entry namest="col5" nameend="col5" align="left">3</entry></row><row><entry namest="col1" nameend="col1" align="left">Nozzle temperature (° C)</entry><entry namest="col2" nameend="col2" align="left">135</entry><entry namest="col3" nameend="col3" align="left">120</entry><entry namest="col4" nameend="col4" align="left">130</entry><entry namest="col5" nameend="col5" align="left">143</entry></row><row><entry namest="col1" nameend="col1" align="left">Compression temperature (° C)</entry><entry namest="col2" nameend="col2" align="left">534</entry><entry namest="col3" nameend="col3" align="left">534</entry><entry namest="col4" nameend="col4" align="left">534</entry><entry namest="col5" nameend="col5" align="left">615</entry></row><row><entry namest="col1" nameend="col1" align="left">02 post-treatment (1 hour)</entry><entry namest="col2" nameend="col2" align="left">(300 ° C)</entry><entry namest="col3" nameend="col3" align="left">(300 ° C)</entry><entry namest="col4" nameend="col4" align="left">300 ° C)</entry><entry namest="col5" nameend="col5" align="left">(400 ° C)</entry></row><row><entry namest="col1" nameend="col1" align="left">Yield of pigment</entry><entry namest="col2" nameend="col2" align="left">low</entry><entry namest="col3" nameend="col3" align="left">high</entry><entry namest="col4" nameend="col4" align="left">medium</entry><entry namest="col5" nameend="col5" align="left">high</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">DNA yield</entry><entry namest="col2" nameend="col2" align="left">low</entry><entry namest="col3" nameend="col3" align="left">high</entry><entry namest="col4" nameend="col4" align="left">high</entry><entry namest="col5" nameend="col5" align="left">high</entry></row></tbody></tgroup></table></tables>

Example 3

PCR sample preparation from whole human blood with magnetic glass particles

Isolation of the nucleic acid

Approx. 10 mg of three glass magnetic particle batches (GMP2 - 4) were placed in Eppendorf reaction vessels. The exact weights are given in Table 1, triplicate determinations were carried out.

40 μl of proteinase K (20 mg / ml, made from lyophilisate) were pipetted into 200 μl of thawed whole blood and mixed immediately. Then 200 μl binding buffer (6 M guanidine-HCl, 10 mM Tris-HCl, 10 mM urea, 30% Triton X-100, pH 4.4) were added, mixed and incubated at 70 ° C. for 10 minutes. After adding 200 μl of i-propanol, the vortex mixer was mixed for 10 seconds, the sample was incubated for 20 minutes at room temperature and mixed again for 10 seconds as before. The magnetic separation was carried out for at least 30 seconds in the Boehringer Mannheim magnetic particle separator (ID No. 1 641 794). The supernatant was removed and analyzed as described below.

The magnetic particles were washed with 500 ul washing buffer (20 mM NaCl, 10 mM Tris-HCl, pH 7.5 (25 ° C), 80% ethanol) by mixing for 10 seconds, incubation for 1 minute at room temperature and mixing for 10 seconds and drawn to the vessel wall with the magnetic particle separator. The supernatant was removed and discarded. The washing procedure was repeated until the washing supernatant was colorless (4 x washing in total). The nucleic acids were then eluted 3 times with elution buffer (10 mM Tris-HCl, pH 8.5) preheated to 70 ° C. in each case by mixing for 10 seconds, incubating for 10 minutes at room temperature and mixing for 10 minutes.

Processing of the supernatant

The supernatant after the first binding to the magnetic glass particles was checked for the content of nucleic acids as follows: The supernatant was placed in a filter tube (Boehringer Mannheim, Id.No. 1744003, e.g. contained in the High Pure PCR Product Purification Kit ) and centrifuged for 1 minute at 8000 rpm in an Eppendorf table centrifuge. The run is discarded and the filter tube is washed twice with 500 μl washing buffer (centrifugation as before). The filter tube is briefly centrifuged dry and then eluted with 2 x 200 µl of 1 x elution buffer preheated to 70 ° C by renewed centrifugation.

Analysis of the eluate and the sample supernatant

50 10 μl of sample buffer were added to μl of the eluates or the supernatants worked up using a filter tube, and 45 μl of this was separated electrophoretically in a 0.8% agarose gel at 120 V for 90 minutes.

Different dilutions of the eluates or the processed supernatants were measured spectroscopically at 260 and 280 nm in a Uvikon 710 (Kontron).

Two 5 ul aliquots of the eluates were checked in duplicate by Expand ™ Long Template PCR (Boehringer Mannheim, Id. No. 1681834) with specific primers for the human tPA gene (expected product length 15 kb). <tables id="tabl0003" num="0003"><table frame="all"><tgroup cols="4" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="39.37mm" /><colspec colnum="2" colname="col2" colwidth="39.37mm" /><colspec colnum="3" colname="col3" colwidth="39.37mm" /><colspec colnum="4" colname="col4" colwidth="39.37mm" /><thead valign="top"><row rowsep="1"><entry namest="col1" nameend="col1" align="left">Mix I</entry><entry namest="col2" nameend="col2" align="left">per approach</entry><entry namest="col3" nameend="col3" align="left">Mix II</entry><entry namest="col4" nameend="col4" align="left">per approach</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">dNTP, 100 mM each</entry><entry namest="col2" nameend="col2" align="center">1 µl</entry><entry namest="col3" nameend="col3" align="left">Expand ™ buffer, 10 x</entry><entry namest="col4" nameend="col4" align="center">5 µl</entry></row><row><entry namest="col1" nameend="col1" align="left">Primer 1, 200 ng / µl</entry><entry namest="col2" nameend="col2" align="center">1 µl</entry><entry namest="col3" nameend="col3" align="left">Expand ™ polymerase</entry><entry namest="col4" nameend="col4" align="center">0.75 µl</entry></row><row><entry namest="col1" nameend="col1" align="left">Primer 2, 225 ng / µl</entry><entry namest="col2" nameend="col2" align="center">1 µl</entry><entry namest="col3" nameend="col3" align="left">H₂O_bidest.</entry><entry namest="col4" nameend="col4" align="center">9.25 µl</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">H₂O_bidest.</entry><entry namest="col2" nameend="col2" align="center">17th µl</entry><entry namest="col3" nameend="col3" /><entry namest="col4" nameend="col4" /></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">20 µl</entry><entry namest="col3" nameend="col3" /><entry namest="col4" nameend="col4" align="center">25th µl</entry></row></tbody></tgroup></table></tables>

Mix 1 is placed in a thin-walled PCR tube with 5 μl of eluate and Mix II is added. The mixture is mixed briefly and covered with 30 µl mineral oil. The approaches are amplified in a Perkin-Elmer Thermocycler 9600 with the following program:<tables id="tabl0004" num="0004"><table frame="all"><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">2nd Minutes</entry><entry namest="col2" nameend="col2" align="left">92 ° C</entry><entry namest="col3" nameend="col3" /></row><row><entry namest="col1" nameend="col1" align="left">10th Seconds</entry><entry namest="col2" nameend="col2" align="left">92 ° C</entry><entry namest="col3" nameend="col3" /></row><row><entry namest="col1" nameend="col1" align="left">30th Seconds</entry><entry namest="col2" nameend="col2" align="left">65 ° C</entry><entry namest="col3" nameend="col3" align="left">10th Cycles</entry></row><row><entry namest="col1" nameend="col1" align="left">12th Minutes</entry><entry namest="col2" nameend="col2" align="left">68 ° C</entry><entry namest="col3" nameend="col3" /></row><row><entry namest="col1" nameend="col1" align="left">10th Seconds</entry><entry namest="col2" nameend="col2" align="left">92 ° C</entry><entry namest="col3" nameend="col3" /></row><row><entry namest="col1" nameend="col1" align="left">30th Seconds</entry><entry namest="col2" nameend="col2" align="left">65 ° C</entry><entry namest="col3" nameend="col3" align="left">20 Cycles</entry></row><row><entry namest="col1" nameend="col1" align="left">12th Minutes + 20 seconds per cycle</entry><entry namest="col2" nameend="col2" align="left">68 ° C</entry><entry namest="col3" nameend="col3" /></row><row><entry namest="col1" nameend="col1" align="left">7 Minutes</entry><entry namest="col2" nameend="col2" align="left">68 ° C</entry><entry namest="col3" nameend="col3" /></row><row rowsep="1"><entry namest="col1" nameend="col1" align="left">subsequently</entry><entry namest="col2" nameend="col2" align="left">7 ° C</entry><entry namest="col3" nameend="col3" /></row></tbody></tgroup></table></tables>

The 50 μl PCR batches were mixed with 10 μl sample buffer and 45 μl thereof were separated electrophoretically in a 0.8% agarose gel at 120 V for 90 minutes.

<u>Results</u>

<tables id="tabl0005" num="0005"><table frame="all"><title>Table 1:</title><tgroup cols="12" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="13.12mm" /><colspec colnum="2" colname="col2" colwidth="13.12mm" /><colspec colnum="3" colname="col3" colwidth="13.12mm" /><colspec colnum="4" colname="col4" colwidth="13.12mm" /><colspec colnum="5" colname="col5" colwidth="13.12mm" /><colspec colnum="6" colname="col6" colwidth="13.12mm" /><colspec colnum="7" colname="col7" colwidth="13.12mm" /><colspec colnum="8" colname="col8" colwidth="13.12mm" /><colspec colnum="9" colname="col9" colwidth="13.12mm" /><colspec colnum="10" colname="col10" colwidth="13.12mm" /><colspec colnum="11" colname="col11" colwidth="13.12mm" /><colspec colnum="12" colname="col12" colwidth="13.12mm" /><thead valign="top"><row rowsep="1"><entry namest="col1" nameend="col12" align="left">Yield of nucleic acids with magnetic glass particles from 200 µl blood</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col2" rowsep="0" /><entry namest="col3" nameend="col3" rowsep="0" /><entry namest="col4" nameend="col7" align="center">Overlap 1: 8</entry><entry namest="col8" nameend="col11" align="center">1. Eluate 1: 8</entry><entry namest="col12" nameend="col12" rowsep="0" /></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" /><entry namest="col4" nameend="col4" align="center">260 nm</entry><entry namest="col5" nameend="col5" align="center">280 in the</entry><entry namest="col6" nameend="col6" align="center">yield</entry><entry namest="col7" nameend="col7" align="center">260/28 0</entry><entry namest="col8" nameend="col8" align="center">260 nm</entry><entry namest="col9" nameend="col9" align="center">280 nm</entry><entry namest="col10" nameend="col10" align="center">yield</entry><entry namest="col11" nameend="col11" align="center">260/28 0</entry><entry namest="col12" nameend="col12" /></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">GMP / 2</entry><entry namest="col2" nameend="col2" align="center">12th mg</entry><entry namest="col3" nameend="col3" align="center">1</entry><entry namest="col4" nameend="col4" align="center">0,021</entry><entry namest="col5" nameend="col5" align="center">0,013</entry><entry namest="col6" nameend="col6" align="center">1.7 µg</entry><entry namest="col7" nameend="col7" align="center">1,6</entry><entry namest="col8" nameend="col8" align="center">0,171</entry><entry namest="col9" nameend="col9" align="center">0,164</entry><entry namest="col10" nameend="col10" align="center">13.7 µg</entry><entry namest="col11" nameend="col11" align="center">1,0</entry><entry namest="col12" nameend="col12" /></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">10th mg</entry><entry namest="col3" nameend="col3" align="center">2</entry><entry namest="col4" nameend="col4" align="center">0,045</entry><entry namest="col5" nameend="col5" align="center">0,035</entry><entry namest="col6" nameend="col6" align="center">3.7 µg</entry><entry namest="col7" nameend="col7" align="center">1,3</entry><entry namest="col8" nameend="col8" align="center">0,137</entry><entry namest="col9" nameend="col9" align="center">0,138</entry><entry namest="col10" nameend="col10" align="center">11.0 µg</entry><entry namest="col11" nameend="col11" align="center">1,0</entry><entry namest="col12" nameend="col12" /></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">9 mg</entry><entry namest="col3" nameend="col3" align="center">3</entry><entry namest="col4" nameend="col4" align="center">0,036</entry><entry namest="col5" nameend="col5" align="center">0,027</entry><entry namest="col6" nameend="col6" align="center">2.9 µg</entry><entry namest="col7" nameend="col7" align="center">1,3</entry><entry namest="col8" nameend="col8" align="center">0,153</entry><entry namest="col9" nameend="col9" align="center">0,164</entry><entry namest="col10" nameend="col10" align="center">12.2 µg</entry><entry namest="col11" nameend="col11" align="center">0,9</entry><entry namest="col12" nameend="col12" /></row><row><entry namest="col1" nameend="col1" align="left">GMP / 3</entry><entry namest="col2" nameend="col2" align="center">10th mg</entry><entry namest="col3" nameend="col3" align="center">1</entry><entry namest="col4" nameend="col4" align="center">0,050</entry><entry namest="col5" nameend="col5" align="center">0,042</entry><entry namest="col6" nameend="col6" align="center">4.0 µg</entry><entry namest="col7" nameend="col7" align="center">1,2</entry><entry namest="col8" nameend="col8" align="center">0,245</entry><entry namest="col9" nameend="col9" align="center">0,246</entry><entry namest="col10" nameend="col10" align="center">19.6 µg</entry><entry namest="col11" nameend="col11" align="center">0,9</entry><entry namest="col12" nameend="col12" /></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">10th mg</entry><entry namest="col3" nameend="col3" align="center">2</entry><entry namest="col4" nameend="col4" align="center">0,033</entry><entry namest="col5" nameend="col5" align="center">0,022</entry><entry namest="col6" nameend="col6" align="center">2.6 µg</entry><entry namest="col7" nameend="col7" align="center">1,5</entry><entry namest="col8" nameend="col8" align="center">0,397</entry><entry namest="col9" nameend="col9" align="center">0,398</entry><entry namest="col10" nameend="col10" align="center">31.8 µg</entry><entry namest="col11" nameend="col11" align="center">1,0</entry><entry namest="col12" nameend="col12" /></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">10th mg</entry><entry namest="col3" nameend="col3" align="center">3</entry><entry namest="col4" nameend="col4" align="center">0,042</entry><entry namest="col5" nameend="col5" align="center">0,030</entry><entry namest="col6" nameend="col6" align="center">3.4 µg</entry><entry namest="col7" nameend="col7" align="center">1,4</entry><entry namest="col8" nameend="col8" align="center">0,278</entry><entry namest="col9" nameend="col9" align="center">0,282</entry><entry namest="col10" nameend="col10" align="center">22.2 µg</entry><entry namest="col11" nameend="col11" align="center">0,9</entry><entry namest="col12" nameend="col12" /></row><row><entry namest="col1" nameend="col1" align="left">GMP / 4</entry><entry namest="col2" nameend="col2" align="center">10th mg</entry><entry namest="col3" nameend="col3" align="center">1</entry><entry namest="col4" nameend="col4" align="center">0,065</entry><entry namest="col5" nameend="col5" align="center">0,056</entry><entry namest="col6" nameend="col6" align="center">0.7 µg</entry><entry namest="col7" nameend="col7" align="center">1,2</entry><entry namest="col8" nameend="col8" align="center">0,135</entry><entry namest="col9" nameend="col9" align="center">0,142</entry><entry namest="col10" nameend="col10" align="center">11.0 µg</entry><entry namest="col11" nameend="col11" align="center">1,0</entry><entry namest="col12" nameend="col12" /></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">11 mg</entry><entry namest="col3" nameend="col3" align="center">2</entry><entry namest="col4" nameend="col4" align="center">0,071</entry><entry namest="col5" nameend="col5" align="center">0,142</entry><entry namest="col6" nameend="col6" align="center">2.4 µg</entry><entry namest="col7" nameend="col7" align="center">0,5</entry><entry namest="col8" nameend="col8" align="center">0,140</entry><entry namest="col9" nameend="col9" align="center">0,142</entry><entry namest="col10" nameend="col10" align="center">11.2 µg</entry><entry namest="col11" nameend="col11" align="center">1,0</entry><entry namest="col12" nameend="col12" /></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">10th mg</entry><entry namest="col3" nameend="col3" align="center">3</entry><entry namest="col4" nameend="col4" align="center">0,066</entry><entry namest="col5" nameend="col5" align="center">0,051</entry><entry namest="col6" nameend="col6" align="center">1.7 µg</entry><entry namest="col7" nameend="col7" align="center">1,3</entry><entry namest="col8" nameend="col8" align="center">0,130</entry><entry namest="col9" nameend="col9" align="center">0,130</entry><entry namest="col10" nameend="col10" align="center">10.4 µg</entry><entry namest="col11" nameend="col11" align="center">1,0</entry><entry namest="col12" nameend="col12" /></row></tbody></tgroup><tgroup cols="12" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="13.12mm" /><colspec colnum="2" colname="col2" colwidth="13.12mm" /><colspec colnum="3" colname="col3" colwidth="13.12mm" /><colspec colnum="4" colname="col4" colwidth="13.12mm" /><colspec colnum="5" colname="col5" colwidth="13.12mm" /><colspec colnum="6" colname="col6" colwidth="13.12mm" /><colspec colnum="7" colname="col7" colwidth="13.12mm" /><colspec colnum="8" colname="col8" colwidth="13.12mm" /><colspec colnum="9" colname="col9" colwidth="13.12mm" /><colspec colnum="10" colname="col10" colwidth="13.12mm" /><colspec colnum="11" colname="col11" colwidth="13.12mm" /><colspec colnum="12" colname="col12" colwidth="13.12mm" /><thead valign="top"><row rowsep="1"><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col2" rowsep="0" /><entry namest="col3" nameend="col3" rowsep="0" /><entry namest="col4" nameend="col7" align="center">2nd Eluate 1: 8</entry><entry namest="col8" nameend="col11" align="center">3rd 1: 4 eluate</entry><entry namest="col12" nameend="col12" /></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" /><entry namest="col4" nameend="col4" align="center">260 nm</entry><entry namest="col5" nameend="col5" align="center">280 nm</entry><entry namest="col6" nameend="col6" align="center">yield</entry><entry namest="col7" nameend="col7" align="center">260/28 0</entry><entry namest="col8" nameend="col8" align="center">260 nm</entry><entry namest="col9" nameend="col9" align="center">280 nm</entry><entry namest="col10" nameend="col10" align="center">yield</entry><entry namest="col11" nameend="col11" align="center">260/28 0</entry><entry namest="col12" nameend="col12" align="center">Σ Eluate</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">GMP / 2</entry><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="center">1</entry><entry namest="col4" nameend="col4" align="center">0,099</entry><entry namest="col5" nameend="col5" align="center">0,101</entry><entry namest="col6" nameend="col6" align="center">7.9 µg</entry><entry namest="col7" nameend="col7" align="center">1,0</entry><entry namest="col8" nameend="col8" align="center">0,057</entry><entry namest="col9" nameend="col9" align="center">0,062</entry><entry namest="col10" nameend="col10" align="center">2.3 µg</entry><entry namest="col11" nameend="col11" align="center">0,9</entry><entry namest="col12" nameend="col12" align="center">23.9 µg</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="center">2</entry><entry namest="col4" nameend="col4" align="center">0,078</entry><entry namest="col5" nameend="col5" align="center">0,076</entry><entry namest="col6" nameend="col6" align="center">6.2µg</entry><entry namest="col7" nameend="col7" align="center">1,0</entry><entry namest="col8" nameend="col8" align="center">0,041</entry><entry namest="col9" nameend="col9" align="center">0,049</entry><entry namest="col10" nameend="col10" align="center">1.6 µg</entry><entry namest="col11" nameend="col11" align="center">0,8</entry><entry namest="col12" nameend="col12" align="center">18.8µg</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="center">3</entry><entry namest="col4" nameend="col4" align="center">0,103</entry><entry namest="col5" nameend="col5" align="center">0,112</entry><entry namest="col6" nameend="col6" align="center">8.2 µg</entry><entry namest="col7" nameend="col7" align="center">0,9</entry><entry namest="col8" nameend="col8" align="center">is missing</entry><entry namest="col9" nameend="col9" /><entry namest="col10" nameend="col10" /><entry namest="col11" nameend="col11" /><entry namest="col12" nameend="col12" /></row><row><entry namest="col1" nameend="col1" align="left">GMP / 3</entry><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="center">1</entry><entry namest="col4" nameend="col4" align="center">0,147</entry><entry namest="col5" nameend="col5" align="center">0,147</entry><entry namest="col6" nameend="col6" align="center">11.8 µg</entry><entry namest="col7" nameend="col7" align="center">1,0</entry><entry namest="col8" nameend="col8" align="center">0,084</entry><entry namest="col9" nameend="col9" align="center">0,098</entry><entry namest="col10" nameend="col10" align="center">3.4 µg</entry><entry namest="col11" nameend="col11" align="center">0,9</entry><entry namest="col12" nameend="col12" align="center">34.8 µg</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="center">2</entry><entry namest="col4" nameend="col4" align="center">0,256</entry><entry namest="col5" nameend="col5" align="center">0,252</entry><entry namest="col6" nameend="col6" align="center">20.5 µg</entry><entry namest="col7" nameend="col7" align="center">1,0</entry><entry namest="col8" nameend="col8" align="center">0,042</entry><entry namest="col9" nameend="col9" align="center">0,043</entry><entry namest="col10" nameend="col10" align="center">1.7 µg</entry><entry namest="col11" nameend="col11" align="center">1,0</entry><entry namest="col12" nameend="col12" align="center">54.0 µg</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="center">3</entry><entry namest="col4" nameend="col4" align="center">0,147</entry><entry namest="col5" nameend="col5" align="center">0,143</entry><entry namest="col6" nameend="col6" align="center">11.8 µg</entry><entry namest="col7" nameend="col7" align="center">1,0</entry><entry namest="col8" nameend="col8" align="center">0,073</entry><entry namest="col9" nameend="col9" align="center">0,093</entry><entry namest="col10" nameend="col10" align="center">2.9 µg</entry><entry namest="col11" nameend="col11" align="center">0,8</entry><entry namest="col12" nameend="col12" align="center">36.9 µg</entry></row><row><entry namest="col1" nameend="col1" align="left">GMP / 4</entry><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="center">1</entry><entry namest="col4" nameend="col4" align="center">0,106</entry><entry namest="col5" nameend="col5" align="center">0,108</entry><entry namest="col6" nameend="col6" align="center">8.5 µg</entry><entry namest="col7" nameend="col7" align="center">1,0</entry><entry namest="col8" nameend="col8" align="center">0,083</entry><entry namest="col9" nameend="col9" align="center">0,098</entry><entry namest="col10" nameend="col10" align="center">3.3 µg</entry><entry namest="col11" nameend="col11" align="center">0,8</entry><entry namest="col12" nameend="col12" align="center">22.8 µg</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="center">2</entry><entry namest="col4" nameend="col4" align="center">0,111</entry><entry namest="col5" nameend="col5" align="center">0,114</entry><entry namest="col6" nameend="col6" align="center">8.9 µg</entry><entry namest="col7" nameend="col7" align="center">1,0</entry><entry namest="col8" nameend="col8" align="center">0,054</entry><entry namest="col9" nameend="col9" align="center">0,063</entry><entry namest="col10" nameend="col10" align="center">2.2 µg</entry><entry namest="col11" nameend="col11" align="center">0,9</entry><entry namest="col12" nameend="col12" align="center">22.3 µg</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col3" align="center">3</entry><entry namest="col4" nameend="col4" align="center">0,135</entry><entry namest="col5" nameend="col5" align="center">0,141</entry><entry namest="col6" nameend="col6" align="center">10.8 µg</entry><entry namest="col7" nameend="col7" align="center">1,0</entry><entry namest="col8" nameend="col8" align="center">0,077</entry><entry namest="col9" nameend="col9" align="center">0,095</entry><entry namest="col10" nameend="col10" align="center">3.1 µg</entry><entry namest="col11" nameend="col11" align="center">0,8</entry><entry namest="col12" nameend="col12" align="center">24.3 µg</entry></row></tbody></tgroup></table></tables>

The first eluates were still slightly yellow and partially contaminated with fine magnetic particles.

The analysis of the eluates in the agarose gel (FIG. 2) shows a good reproducibility of the yield. The magnetic particles GMP / 2 - 4 show no significant differences. Eluates 1 (top) and 2 (bottom) have approximately the same nucleic acid concentration (estimated by the gel). Eluate 3 shows only a low nucleic acid concentration. Only a low nucleic acid concentration can also be observed in the supernatants.

The Expand ™ PCR delivers consistently good and specific amplification products with all samples except a few outliers (Table 2). The magnetic glass beads were used to isolate nucleic acids from human blood samples, which in a subsequent PCR provided specific amplicons.<tables id="tabl0006" num="0006"><table frame="all"><title>Table 2.</title><tgroup cols="6" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="26.25mm" /><colspec colnum="2" colname="col2" colwidth="26.25mm" /><colspec colnum="3" colname="col3" colwidth="26.25mm" /><colspec colnum="4" colname="col4" colwidth="26.25mm" /><colspec colnum="5" colname="col5" colwidth="26.25mm" /><colspec colnum="6" colname="col6" colwidth="26.25mm" /><thead valign="top"><row rowsep="1"><entry namest="col1" nameend="col6" align="left">Expand ™ PCR results</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" rowsep="0" /><entry namest="col2" nameend="col6" align="center">15 kb Expand ™ PCR human tPA gene</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" /><entry namest="col3" nameend="col4" align="center">1. Eluate</entry><entry namest="col5" nameend="col6" align="center">2nd Eluate</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">GMP / 2</entry><entry namest="col2" nameend="col2" align="center">1</entry><entry namest="col3" nameend="col4" align="center">is missing</entry><entry namest="col5" nameend="col5" align="center">+</entry><entry namest="col6" nameend="col6" align="center">+</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">2</entry><entry namest="col3" nameend="col3" align="center">+</entry><entry namest="col4" nameend="col4" align="center">+</entry><entry namest="col5" nameend="col5" align="center">+</entry><entry namest="col6" nameend="col6" align="center">+</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">3</entry><entry namest="col3" nameend="col3" align="center">+</entry><entry namest="col4" nameend="col4" align="center">+</entry><entry namest="col5" nameend="col6" align="center">is missing</entry></row><row><entry namest="col1" nameend="col1" align="left">GMP / 3</entry><entry namest="col2" nameend="col2" align="center">1</entry><entry namest="col3" nameend="col3" align="center">+</entry><entry namest="col4" nameend="col4" align="center">+</entry><entry namest="col5" nameend="col5" align="center">+</entry><entry namest="col6" nameend="col6" align="center">+</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">2</entry><entry namest="col3" nameend="col3" align="center">(+)</entry><entry namest="col4" nameend="col4" align="center">+</entry><entry namest="col5" nameend="col5" align="center">+</entry><entry namest="col6" nameend="col6" align="center">+</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">3</entry><entry namest="col3" nameend="col3" align="center">-</entry><entry namest="col4" nameend="col4" align="center">(+)</entry><entry namest="col5" nameend="col5" align="center">+</entry><entry namest="col6" nameend="col6" align="center">+</entry></row><row><entry namest="col1" nameend="col1" align="left">GMP / 4</entry><entry namest="col2" nameend="col2" align="center">1</entry><entry namest="col3" nameend="col3" align="center">+</entry><entry namest="col4" nameend="col4" align="center">+</entry><entry namest="col5" nameend="col5" align="center">+</entry><entry namest="col6" nameend="col6" align="center">+</entry></row><row><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">2</entry><entry namest="col3" nameend="col3" align="center">+</entry><entry namest="col4" nameend="col4" align="center">+</entry><entry namest="col5" nameend="col5" align="center">+</entry><entry namest="col6" nameend="col6" align="center">(+)*</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" /><entry namest="col2" nameend="col2" align="center">3</entry><entry namest="col3" nameend="col3" align="center">+</entry><entry namest="col4" nameend="col4" align="center">+</entry><entry namest="col5" nameend="col6" align="center">is missing</entry></row><row rowsep="1"><entry namest="col1" nameend="col6" align="left">K, BM control DNA</entry></row></tbody></tgroup><tgroup cols="6" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="26.25mm" /><colspec colnum="2" colname="col2" colwidth="26.25mm" /><colspec colnum="3" colname="col3" colwidth="26.25mm" /><colspec colnum="4" colname="col4" colwidth="26.25mm" /><colspec colnum="5" colname="col5" colwidth="26.25mm" /><colspec colnum="6" colname="col6" colwidth="26.25mm" /><tbody valign="top"><row><entry namest="col1" nameend="col6" align="justify">* 3. Eluate</entry></row></tbody></tgroup></table></tables>

In FIG. 3 shows a gel with the reaction products after PCR amplification. MWM III is a molecular weight marker (eluate 1, top; eluate 2, bottom).

Example 4

Binding of DNA length standard to magnetic glass particles

1. Preparation of the magnetic glass particles

12 mg of the glass magnet batch GMP4 were placed in Eppendorf reaction vessels.

2nd Lysis and binding

In a 1.5 ml Eppendorf tube with 12 mg magnetic glass particles, 900 µl lysis buffer (4.6 M GuSCN, 45 mM Tris, 20 mM EDTA, pH 7.3) and 100 µl DNA sample, in the model DNA Length standard III from Boehringer Mannheim (catalog no. 528552) was used, mixed for 2 to 10 seconds until a homogeneous suspension is formed. The solution is incubated for 20 minutes at room temperature, with mixing every 5 minutes.

Magnetic separation takes place in a magnetic particle separator for at least 15 seconds. The supernatant is pipetted off.

3rd Washing and drying

The magnetic glass particles are washed twice with washing buffer (5.2 M GuSCN, 50 mM Tris, pH 6.5) twice with 70% pre-cooled ethanol and once with acetone by removing the magnetic field, pipetting in 800 μl solution, mixing for 2 seconds, incubating for 1 min at RT , the magnetic field is applied and the supernatant is finally pipetted off.

After removal of the acetone, the particles are dried for 10 min at 56 ° C. in a heating block with the lid open.

4th Elution of the DNA

The DNA is eluted with 4x 50 μl elution buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) by incubating for 10 min with repeated shaking at 56 ° C. and finally transferring the supernatant containing DNA into a new Eppendorf tube .

5. Analysis of the eluate

A fifth of the eluate volume was mixed with sample buffer and the DNA was separated on a 1% agarose gel at 90 V. To determine recovery, a dilution series of DNA length standard III was applied to the same gel, which contains the DNA amounts to be expected in the samples.

The quantitative evaluation was carried out by scanning a polaroid photo of the agarose gel. The standard dilution series was used as the calibrator.

The yield of DNA with magnetic glass particles is shown in Table 1 <tables id="tabl0007" num="0007"><table frame="all"><title>Table 1.</title><tgroup cols="9" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="17.50mm" /><colspec colnum="2" colname="col2" colwidth="17.50mm" /><colspec colnum="3" colname="col3" colwidth="17.50mm" /><colspec colnum="4" colname="col4" colwidth="17.50mm" /><colspec colnum="5" colname="col5" colwidth="17.50mm" /><colspec colnum="6" colname="col6" colwidth="17.50mm" /><colspec colnum="7" colname="col7" colwidth="17.50mm" /><colspec colnum="8" colname="col8" colwidth="17.50mm" /><colspec colnum="9" colname="col9" colwidth="17.50mm" /><thead valign="top"><row rowsep="1"><entry namest="col1" nameend="col9" align="left">Yield of DNA length standard III with magnetic glass particles</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="center">Standard No.</entry><entry namest="col2" nameend="col2" align="center">DNA amount in standard [ng]</entry><entry namest="col3" nameend="col3" align="center">Brightness-intensive. Standard (measured value) [rel. Units]</entry><entry namest="col4" nameend="col4" align="center">Sample No.</entry><entry namest="col5" nameend="col5" align="center">Pigment / bead type</entry><entry namest="col6" nameend="col6" align="center">Brightness-intensive. Sample (measured value) [rel. Unitsl</entry><entry namest="col7" nameend="col7" align="center">calculated amount of DNA on gel [ng]</entry><entry namest="col8" nameend="col8" align="center">calculated amount of DNA in sample [ng]</entry><entry namest="col9" nameend="col9" align="center">Recovery [%]</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="center">1</entry><entry namest="col2" nameend="col2" align="center">200</entry><entry namest="col3" nameend="col3" align="center">65</entry><entry namest="col4" nameend="col4" align="center">1</entry><entry namest="col5" nameend="col5" align="center">GMP4</entry><entry namest="col6" nameend="col6" align="center">45</entry><entry namest="col7" nameend="col7" align="center">139</entry><entry namest="col8" nameend="col8" align="center">695</entry><entry namest="col9" nameend="col9" align="center">69,5</entry></row><row><entry namest="col1" nameend="col1" align="center">2</entry><entry namest="col2" nameend="col2" align="center">175</entry><entry namest="col3" nameend="col3" align="center">56</entry><entry namest="col4" nameend="col4" align="center">2</entry><entry namest="col5" nameend="col5" align="center">GMP4</entry><entry namest="col6" nameend="col6" align="center">39</entry><entry namest="col7" nameend="col7" align="center">120</entry><entry namest="col8" nameend="col8" align="center">600</entry><entry namest="col9" nameend="col9" align="center">60,0</entry></row><row><entry namest="col1" nameend="col1" align="center">3</entry><entry namest="col2" nameend="col2" align="center">150</entry><entry namest="col3" nameend="col3" align="center">51</entry><entry namest="col4" nameend="col4" /><entry namest="col5" nameend="col5" /><entry namest="col6" nameend="col6" /><entry namest="col7" nameend="col7" /><entry namest="col8" nameend="col8" /><entry namest="col9" nameend="col9" /></row><row><entry namest="col1" nameend="col1" align="center">4</entry><entry namest="col2" nameend="col2" align="center">125</entry><entry namest="col3" nameend="col3" align="center">44</entry><entry namest="col4" nameend="col4" /><entry namest="col5" nameend="col5" /><entry namest="col6" nameend="col6" /><entry namest="col7" nameend="col7" /><entry namest="col8" nameend="col8" /><entry namest="col9" nameend="col9" /></row><row><entry namest="col1" nameend="col1" align="center">5</entry><entry namest="col2" nameend="col2" align="center">100</entry><entry namest="col3" nameend="col3" align="center">37</entry><entry namest="col4" nameend="col4" /><entry namest="col5" nameend="col5" /><entry namest="col6" nameend="col6" /><entry namest="col7" nameend="col7" /><entry namest="col8" nameend="col8" /><entry namest="col9" nameend="col9" /></row><row><entry namest="col1" nameend="col1" align="center">6</entry><entry namest="col2" nameend="col2" align="center">75</entry><entry namest="col3" nameend="col3" align="center">25</entry><entry namest="col4" nameend="col4" /><entry namest="col5" nameend="col5" /><entry namest="col6" nameend="col6" /><entry namest="col7" nameend="col7" /><entry namest="col8" nameend="col8" /><entry namest="col9" nameend="col9" /></row><row><entry namest="col1" nameend="col1" align="center">7</entry><entry namest="col2" nameend="col2" align="center">50</entry><entry namest="col3" nameend="col3" align="center">17</entry><entry namest="col4" nameend="col4" /><entry namest="col5" nameend="col5" /><entry namest="col6" nameend="col6" /><entry namest="col7" nameend="col7" /><entry namest="col8" nameend="col8" /><entry namest="col9" nameend="col9" /></row><row><entry namest="col1" nameend="col1" align="center">8</entry><entry namest="col2" nameend="col2" align="center">25</entry><entry namest="col3" nameend="col3" align="center">9</entry><entry namest="col4" nameend="col4" /><entry namest="col5" nameend="col5" /><entry namest="col6" nameend="col6" /><entry namest="col7" nameend="col7" /><entry namest="col8" nameend="col8" /><entry namest="col9" nameend="col9" /></row><row rowsep="1"><entry namest="col1" nameend="col1" align="center">9</entry><entry namest="col2" nameend="col2" align="center">10</entry><entry namest="col3" nameend="col3" align="center">4</entry><entry namest="col4" nameend="col4" /><entry namest="col5" nameend="col5" /><entry namest="col6" nameend="col6" /><entry namest="col7" nameend="col7" /><entry namest="col8" nameend="col8" /><entry namest="col9" nameend="col9" /></row></tbody></tgroup></table></tables>

The agarose gel, which served as the basis for the quantitative evaluation, is shown in FIG. 4 shown. It is a 1% ethidium bromide stained agarose gel. Lanes 1 through 10 correspond to a dilution series of DNA length standard III. 1: 1 µg DNA, 2: 200 ng DNA, 3: 175 ng DNA, 4: 150 ng DNA, 5: 125 ng DNA, 6: 100 ng DNA, 7: 75 ng DNA, 8: 50 ng NDA, 9: 25 ng DNA, 10:10 ng DNA.

Lanes 11 and 12 correspond to the DNA eluted from the magnetic glass particles when using 200 ng DNA length standard.

Preferred embodiments of the invention are shown below as part of the description:<ul id="ul0012" list-style="none"><li>1. Magnetic particles with an outer glass surface that is essentially non-porous or has pores with a diameter of less than 10 nm.</li><li>2nd Ferromagnetic particles with a glass surface.</li><li>3rd Particles according to embodiment 1 or 2, characterized in that they have a grain size of between 10 and 60 microns.</li><li>4th Particles according to embodiment 1 or 2, characterized in that any pores present in the surface have a diameter of less than 1 nm.</li><li>5. Particles according to embodiment 1 or 2, characterized in that the particles contain a composite of a core of mica and magnetite particles immobilized thereon, this composite being enclosed by a glass layer.</li><li>6. Process for isolating a biological material<ul id="ul0013" list-style="dash" compact="compact"><li>Contacting a sample containing the biological material in a liquid with particles according to one of claims 1 to 5 under conditions in which the biological material binds to the particle surface, and</li><li>Separation of the biological material from the liquid.</li></ul></li><li>7. Method according to embodiment 6, characterized in that the biological material is nucleic acids.</li><li>8th. Process for the isolation of nucleic acids by<ul id="ul0014" list-style="dash" compact="compact"><li>Contacting a sample which contains the nucleic acid in native form in a liquid with magnetic particles with glass surfaces under conditions in which the nucleic acids in native form can bind to the glass surface and</li><li>Separation of the bound nucleic acids from the liquid.</li></ul></li><li>9. Method according to one of the embodiments 6 to 8, characterized in that the separation takes place with the aid of a magnet.</li><li>10th Method according to one of the embodiments 6 to 9, characterized in that the magnetic particles are not premagnetized when brought into contact with the sample.</li><li>11. Process for the production of magnetic glass particles<ul id="ul0015" list-style="dash" compact="compact"><li>Provision of a magnetic core and</li><li>Enclosing the magnetic core with a substantially non-porous glass surface.</li></ul></li><li>12th Method according to embodiment 11, characterized in that the enclosing includes the deposition of a sol on the surface and the subsequent densification of the sol.</li><li>13. Use of ferromagnetic glass particles for the isolation of nucleic acids in native form.</li><li>14. Use of magnetic glass particles to isolate nucleic acids.<img file="EP1577389A2_D0002.tif" /></li></ul>