EP1644113A1 - Method for applying substances onto nonporous silica gel particles - Google Patents

Abstract

The invention relates to a method for producing nonporous silica gel particles which have an adjustable uniform size of less than 15 ñm and on or onto the surface of which biologically and/or chemically active substances are immobilized or applied. Also disclosed is the use thereof, preferably for separating molecules, enriching samples, or performing chromatography, catalysis, analysis, bonding reactions, or other chemical reactions.

Description

Process for applying substances to non-porous silica particles

description

The invention relates to non-porous silica gel particles with an adjustable uniform particle size of less than 15 μm, on the surface of which biologically and / or chemically active substances are immobilized or applied, a process for their preparation and their use preferably for carrying out molecular separations, chromatography, catalysis , Analytics, sample enrichment, binding or other chemical reactions.

The advantage of immobilizing biomolecules on a carrier material was recognized years ago and lies in the simplified purification of the product e.g. with a molecular separation and above all in the possibility of multiple reuse. In addition, molecules such as enzymes immobilized on a carrier material show greater activity and stability than in solution. Various carrier materials and methods are known for immobilizing biomolecules on carrier surfaces.

Various organic polymers (polysaccharides such as Sepharose, Sephadex, synthetic polymers such as polyacrylamides, phenol-formaldehyde) and inorganic materials (glass, aluminum oxides or silicates) are suitable as carrier materials for this. A major disadvantage of organic carrier materials is their sensitivity to organic solvents and high temperatures, low pressure stability and relatively high production costs. For this reason, several methods for applying biomolecules to inorganic substrates have been developed, which do not have the above-mentioned disadvantages of organic carriers. The known methods for immobilizing biologically active substances on an inorganic support range from simple physical adsorption to covalent binding to the support surface. For proteins such as antibodies and enzymes in particular, fixation by means of covalent bonds has proven to be particularly advantageous. For example, US Pat. No. 3,652,761 describes a method for coupling antibodies or antigens to surfaces via amino groups, the surfaces having previously been treated with 3-aminopropyltriethoxysilanes or N- (2-aminoethyl) -3-aminopropyltrimethoxysilanes. A coupling of enzymes to inorganic carriers by means of bifunctional spacers is known from EP0158909. Immobilization of physiologically active substances on the inorganic carriers pretreated with aminoalkylalkoxysilanes is described in EP0325404.

Compared to organic polymers, the inorganic support materials used in the known processes, such as glass or silicates, have a relatively small reactive surface, so that they can only take up a small amount of molecules and are therefore of limited use for many questions. An improved method is proposed in EP0158909, wherein spherical Si0 ₂ particles are used as the carrier, the mean particle size of which is between 15 and 80 μm and the specific surface area is between 250 and 800 m ² / g. Although this document does not explicitly describe whether these are porous or non-porous particles, porous particles can be inferred from the information on the specific surface.

Several processes for producing porous silica gel particles are known from the literature, such as e.g. from US4,983,369. However, are porous

Particles are usually only of advantage for small analyte molecules. Proteins and

Enzymes usually have diameters that are too large to enter the po- fit the backing material. For these analytes, the effective surface area is reduced by the total area of the pores, so that the advantage over non-porous particles disappears. But also for analytes that are small enough to get into the pores, the use of porous versus non-porous particles can have disadvantages. Since only mass transfer takes place in the pores by diffusion, a much lower flow rate must be set in the separation process in order to avoid peak broadening of the eluents due to the poorer mass transfer (Van-Deemter curve). This can lead to slower separations, which is particularly disadvantageous in routine procedures. In addition, for many questions, such as in environmental analysis, the analyte quantities are so small that the high loading density and thus the use of porous materials is not required.

The present invention is therefore based on the object of circumventing the disadvantages of the known methods for applying substances to inorganic material supports and of providing a method for immobilizing or applying substances onto non-porous silica gel particles with an adjustable uniform particle size of less than 15 μm , This process is intended to enable a significantly higher number of immobilized molecules, a denser loading of the columns and a considerable acceleration of physical, chemical or biochemical processes on the carrier surfaces.

To achieve this object, the invention primarily proposes a method with the features mentioned in claim 1. Further developments of the invention are the subject of the remaining dependent and independent claims 2 to 21, the wording of which, like the wording of the summary, is made the content of the description by reference. The above-mentioned aim is achieved according to the invention in that in a first process step silica gel particles with an average particle size smaller than 15 μm, in particular with the size of 0.2 to 1.2 μm are produced, in the second step the surface - This silica gel particle is pretreated for immobilization of molecules, and finally in the third step the molecules to be immobilized are bound to the particle surface either directly or via previously attached spacers.

A sol of primary particles is first produced. A process for the production of spherical SiO ₂ particles by hydrolytic polycondensation of tetraalkoxysilanes is known from the prior art (W. Stöber et al., J. Colloid and Interface Science 26, 62 (1968) and 30, 568 (1969)). While maintaining basic reaction conditions, this method is modified in the first step of the present invention to produce uniform, non-porous silica gel particles as follows.

According to the invention, the tetraalkoxysilane is placed in a preheated aqueous-alcoholic-ammoniacal hydrolysis mixture and mixed well. After an hour of reaction, the synthesis is usually complete and the spherical silica gel material can be separated from the remaining solvent. Depending on the choice of experimental parameters, silica gel particles of different average particle sizes can be obtained. According to the invention, the silica gel particles advantageously have an average particle size of less than 15 μm, but preferably between 0.2 μm and 5 μm, in particular between 0.2 μm and 1.2 μm, with a standard deviation of not more than 10%. The specific surface area is 2 m ² / g to 20 m ² / g. For example, ethanol is used for the production of particles with medium sizes between 0.2 μm and 0.3 μm, and isopropanol for larger particles. For particles from a size of 1 μ, preference is given to as tetraethoxysilane (TEOS) added, especially in two steps at intervals of about 30 min.

In the second step of the method, a modification of the particle surface necessary for the immobilization of molecules takes place according to the invention. Since silica gel naturally does not contain any functional groups for binding molecules, the surface is modified by treatment with suitable substances to such an extent that the molecules to be immobilized can be bound to the support material in a further step either directly or via a spacer. In particular, amino, epoxy, thio, phospho or carboxyl groups are introduced onto the carrier surface in this step.

In a particularly preferred embodiment variant, the particle surface is treated with aminoalkylalkoxysilanes, in particular with 3-aminopropyltriethoxysilane or / and with N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, in order to introduce amino groups to the support surface. In the next step, these can be linked with a carboxyl group e.g. a protein (e.g. in a carbodiimide process) or with the carbonyl group of a spacer such as e.g. Glutaraldehyde react.

In the case of an epoxy-functionalized particle surface, for example, the binding of substances to the surface can e.g. via already existing (as with proteins) or specially introduced amino groups (oxirane method) or also thionyl groups.

In a preferred embodiment, the substances to be applied are bound via bifunctional spacers. For this purpose, the suitable, preferably bifunctional, spacers are covalently bound to the functional groups introduced beforehand. Bifunctional spacers with amino, thio, phospho, carboxyl, hydroxyl or epoxy groups are advantageous End used with which the molecules to be immobilized should react in the next step. The use of spacers which can react with the amino groups located on the surface has proven particularly advantageous. In a particularly preferred variant, glutaraldehyde or glutaraldehyde is used for this. It is also possible to use numerous other spacers, such as, for example, alkyl chains, divinyl sulfone or other spacers known from the prior art.

The use of multivalent building blocks is also conceivable. In this case, a duplication of the surface functionality can be achieved, which can advantageously lead to an increase in the loading density. As a non-limiting example, the trifunctional linker 3,5-dicarbomethoxyphenoxyacetic acid p-nitrophenyl ester can be mentioned, which can almost double the surface functionality after covalent attachment to amino-functionalized support material.

Finally, in the third step, the molecules to be applied are bound to the carrier surface. In a preferred embodiment, the molecules are covalently coupled to the carrier surface. The substances to be applied can either be directly with the functional groups of the surface (eg hydroxyl, carboxyl, thiol or amino group of the molecule on hydroxyl, amino, carboxyl or other activated carrier material) or with the previously applied carrier material Spacers react. In a particularly preferred variant, the molecules are covalently bound to the surface by means of a reaction between an amino group of the molecule and a carbonyl group of the glutaraldehyde spacer. The actual coupling can be preceded by an activation reaction of the corresponding functional groups of one of the components, solid phase or the substance to be applied. In general, it proves to be advantageous to Functional groups introduced surface to activate, since the activation of the dissolved component can lead to inter- or intramolecular crosslinking as a side reaction. The usual methods known to those skilled in the art are used for this.

Alternatively, the substances to be coupled can be fixed to the particle surface via a non-covalent bond. For example, biotinylated molecules can be coupled to a surface previously coated with streptavidin or avidin. A non-covalent bond between a receptor and its ligand or an antibody and its antigen or the like can also be used to couple substances to the particle surface. Such non-covalent couplings belong to the prior art and can be carried out by a person skilled in the art (see, for example, J. Cai and J. Henion, J. Chromatogr., 1997, 691 (2), 357-370; TM Phillips and JM Krum, J. Chromatogr. B, 1998, 715 (1), 55-63.) Such a non-covalent bond is very stable under physiological conditions, but can easily be broken by changes in pH or organic solvents. If the silica gel particles coated with substances are used several times, a covalent bond as described above is therefore preferred.

According to the invention, the molecules to be applied can be any biologically or chemically active substances. Proteins or peptides (such as antibodies, enzymes, receptors or ligands), polymers or oligomers of the nucleus class (DNA, RNA, PNA, thioRNA and their derivatives), carbohydrates or metabolites are examples of, but not restrictive, proteins to call. As chemically active substances, for example, catalysts (for example rhodium-diphosphine diamine complexes) can be immobilized on the particle surface. The method according to the invention has the advantage over the known methods that, on the one hand, by using very small particles (average particle size smaller than 15 μm, advantageously between 0.2 and 1.5 μm, in particular between 0.2 and 1.2 μm) a larger effective surface is available for immobilization, so that a significantly higher loading capacity than when using larger non-porous particles is possible. As a result, many physical (e.g. separation) or chemical (catalysis, binding) or biochemical (enzymatic reactions, receptor-ligand, antibody-antigen-binding reactions) processes taking place on the particle surface can be accelerated many times over. In addition, molecular mixtures such as, for example, slightly purified enzymes can be used in the loading because of the high binding capacity. Furthermore, because of their non-porosity and thus a larger effective surface, the silica gel particles according to the invention, which are loaded with molecules, can be used for many purposes much more effectively than porous carrier materials, since the pores are inaccessible for many larger molecules, such as proteins. Finally, a better packing bed is possible due to the small size dispersion and a small average particle size.

Furthermore, the invention comprises non-porous spherical silica gel particles with a particle size, in particular an average particle size, of less than 15 μm, which are loaded with biologically and / or chemically active substances. The silica gel particles can preferably be produced by the process described above.

Furthermore, the invention comprises the use of the silica gel particles loaded with biologically and / or chemically active molecules when carrying out physical, chemical or biochemical processes on their surface. Thus, the silica gel particles described in the present invention can be used in a molecular separation such as a nucleic acid, protein or other separation, for example on a Hybridization or a ligand-receptor principle is used. Another possible use is to carry out enzymatic or catalytic reactions, for example in different production processes, enzyme or catalyst molecules being applied to the surface of the silica gel particles. Molecular separations, chromatography or sample enrichment can also be carried out on the particles described. Furthermore, all kinds of binding reactions (antibody-antigen, receptor ligand, enzyme substrate, etc.) can be carried out with the described silica gel particles, which can be used, for example, in the field of analysis or diagnostics. By binding organic macromolecules (eg proteins, peptides, enzymes), these can be immobilized and studies on structure-function relationships can be carried out. Such silica gel particles are also suitable for attaching various metal complexes, for example for selective hydrogenation or also for attaching pieces of DNA for nucleic acid synthesis. The chemical industry, medical laboratory diagnostics, drug or crop protection agent development, quality control and research and development are among the sectors in which the silica gel particles according to the invention can be used particularly advantageously.

Further advantages, features and possible applications of the invention will be explained in more detail below with reference to the exemplary embodiments with reference to the drawings. Even without further explanations, it is assumed that a person skilled in the art can use the above description in the broadest scope. The preferred embodiments and examples for the method according to the invention are therefore only to be understood as a descriptive disclosure, in no way as a limitation in any way. The drawings show: Figure 1: An electron micrograph of silica gel particles with an average particle diameter of 0.7 microns;

FIG. 2: Course of the UV absorption of nitrophenolate during the cleavage (saponification) of p-nitrophenyl caprylate for determining the activity of the lipase on various materials;

Figure 3: Diagram of the ²⁹ Si-CP / MAS-NMR spectrum of silica gel particles with an average particle diameter of 0.7 μm. In contrast to porous silica gel, the signal of the surface species Q ^{2 can} hardly be seen (92 ppm) due to the much smaller surface area. In the case of porous materials with a surface that is many times higher, a clearly recognizable signal would have to appear here.

embodiments

Production of silica gel particles with an immobilized enzyme

a) Production of non-porous silica gel particles with a uniformly adjustable particle size

Uniformly spherical silica gel particles with different diameters in the range from 0.2 to 1.2 μm can be produced by the hydrolysis of tetraethoxysilane (TEOS) in the presence of ammonium hydroxide in an alcoholic medium under strictly defined concentration ratios of the starting materials used.

By changing the quantity of the educts (water, ammonia, TEOS) and the solvent (ethanol or isopropanol), the size of the silica gel obtained can be gradually changed from 0.2 to 1.2 μm (see Table 1):

Table 1: Amounts of the educts used to synthesize the spherical silica gel particles with different diameters: ^a) ethanol, ^b) isopropanol, ^c) two-step reaction

First, the alcohol is mixed with water and ammonia solution and warmed up with gentle stirring. After the reaction temperature is reached, the tetraethoxysilane is added all at once in one step. After about an hour of reaction, the synthesis is complete and the spherical silica gel material can be separated from the remaining solvent. Ethanol is used to produce particles with diameters between 0.2 μm and 0.3 μm. The more viscous isopropanol is used to display larger particles. From a size of 1 μm, the display takes place in 2 stages and the TEOS is added at intervals of approx. 30 min.

The sol-gel process therefore looks like this:

Si (OC ₂ H ₅ ) ₄ + 4 H ₂ 0 hydrolysis ^ _Si ( ₀ H) ₄ + 4 C ₂ H ₅ OH

Cofeondensation n Si (OH) ₄ - - [SiO _x (OH) _4-2x ] + xn H ₂ 0

Dehydration [SiO _x (OH) ₄ . _2x ] * - (Si0 _4/2 ) _n + n (4-2x) / 2 H ₂ 0

The uniform shape and a uniform particle size (average particle diameter 0.7 μm) can already be seen from the SEM images shown in FIG. 1. An average surface area of 2.9 m ² / g results for the particles shown as examples.

b) Functionalization of the particle surface with aminopropyltriethoxysilane

1 g of silica gel particles with a particle diameter of 0.7 μm is activated at 180-200 ° C. After the silica gel has cooled to 70 ° C., it is suspended in 100 ml of toluene. 510 μl of aminopropyltriethoxysilane (triple excess) are added to the suspension, and the reaction mixture is heated under reflux for 20 h. Finally, the silica gel is filtered off with a glass frit and washed twice with toluene, acetone and pentane.

c) Connection of the bifunctional spacer glutardialdehyde

100 mg of the aminopropyl silica gel from step b) are reacted with 1 ml of an aqueous 5% glutardialdehyde solution. The solution is shaken for 4 hours, then the imine bond formed is reduced for 2 hours by 1 ml of NaCNBH ₃ (5 mg / ml). The resulting material is washed with water and loading buffer (150 mM NaCl, 10 mM Phos- phat buffer, pH 7.4) washed several times and stored in loading buffer.

d) Immobilization of an enzyme

1 ml of an enzyme solution (lipase, 1 mg / ml) is mixed with 0.5 ml of a high salt buffer (1, 5 M Na ₂ S0 ₄ in 100 mM phosphate buffer, pH 7.4) and the silica gel from step c) given. The suspension is shaken for 1 minute and then 1 ml of the same buffer is added. After 5 more minutes, 1 ml of the buffer is added and the solution is shaken for 24 hours. The suspension is then centrifuged and the supernatant separated. The precipitate is mixed with capping buffer and shaken for 2 h. At the end, the modified silica gel is washed three times with loading buffer (150 mM NaCl, 10 mM phosphate buffer, pH 7.4), three times with 1 M NaCI solution and three times with loading buffer and stored therein.

e) Checking the activity of the immobilized enzyme

To check the enzymatic activity of the material, the cleavage (saponification) of p-nitrophenyl caprylate is selected. Since nitrophenol has a significant shift in the UV absorption maximum depending on the pH value, the activity of the lipase at the concentration of the nitrophenolate can be monitored by UV spectrometry. The results for the lipase (FIG. 2, NPS) bound to the non-porous silica gel particles show a significantly increased activity compared to the free lipase in glycol (FIG. 2, lipase in glycol) and the bare silica gel (FIG. 2, blank) , The reduced activity of the free lipase can be explained by its agglomeration in solution. A reference material without lipase immobilization is also tested to rule out that the saponification of the ester takes place on the silica gel surface instead of the lipase. For this purpose, all steps of the covalent binding with the exception of lipase addition. There is a curve similar to that for the bare silica gel (FIG. 2, comparative material).

The present invention is not restricted to the exemplary embodiments described. Rather, many modifications are possible that can be achieved by those skilled in the art and are thus included in the scope of this invention.

Claims

claims 1. Process for the production of non-porous spherical silica gel particles loaded with biologically and / or chemically active substances and having a particle size of less than 15 μm, characterized in that a) the silica gel particles are produced by hydrolytic polycondensation of tetraalkoxysilanes, b) the surface the silica gel particles are functionalized by treatment with suitable substances before the application of biologically and / or chemically active substances, c) the binding of biologically and / or chemically active substances to the surface of the silica gel particles either directly or via suitable ones molecular spacers. 2. The method according to claim 1, characterized in that the silica gel particles have an approximately uniform size with a standard deviation of not more than 10%. 3. The method according to claim 1 or claim 2, characterized in that the silica gel particles have an average particle size between 0.2 microns and 5 microns, in particular between 0.2 microns and 1.2 microns. 4. The method according to any one of the preceding claims, characterized in that the silica gel particles preferably have a specific surface area between 1 m ² / g and 100 m ² / g, in particular between 2 m ² / g and 20 m ² / g , 5. The method according to any one of the preceding claims, characterized in that ethanol is used to produce silica gel particles with average particle sizes between 0.2 microns and 0.3 microns. 6. The method according to any one of the preceding claims, characterized in that isopropanol is used to produce silica gel particles with average particle sizes between 0.3 microns and 1 micron. 7. The method according to any one of the preceding claims, characterized in that for the production of silica gel particles with average particle sizes between 1 micron and 15 microns tetraethoxysilane is added in two stages at different times. 8. The method according to any one of the preceding claims, characterized in that amino, epoxy, thio, phospho and / or carboxyl groups are introduced onto the surface of the silica gel particles before loading with biologically and / or chemically active substances become. 9. The method according to any one of the preceding claims, characterized in that the silica gel particles before loading with biologically and / or chemically active substances with aminoalkylalkoxysilanes, in particular with 3-aminopropyltriethoxysilane and / or with N- (2-aminoethyl) - 3-aminopropyltrimethoxysilane for the introduction of amino groups are treated. 10. The method according to any one of the preceding claims, characterized in that the spacer is bifunctional and has an amino, thio, phospho, carboxyl, hydroxyl or epoxy group at the end. 11. The method according to any one of the preceding claims, characterized in that the spacer is glutaraldehyde, glutardialdehyde and / or divinyl sulfone. 12. The method according to any one of the preceding claims, characterized in that the spacer is multifunctional. 13. The method according to any one of the preceding claims, characterized in that the binding of the substances to be loaded takes place directly on the functionalized surface of the silica gel particles. 14. The method according to any one of the preceding claims, characterized in that the substances to be loaded are bound to the functionalized surface of the silica gel particles or to the spacers applied thereon via a covalent coupling. 15. The method according to any one of the preceding claims, characterized in that the binding of the substances to be loaded to the functionalized surface of the silica gel particles is preceded by an activation of the functional groups on the surface of the silica gel particles and / or the substances to be loaded. 16. The method according to any one of the preceding claims, characterized in that the binding of the substances to be loaded to the functionalized surface of the silica gel particles or to the spacers applied thereon is a non-covalent bond. 17. The method according to any one of the preceding claims, characterized in that the biological and / or to be loaded chemically active substances are proteins, in particular antibodies, enzymes, receptors or ligands, peptides, polymers or oligomers of the nucleus class, in particular DNA, RNA, PNA, thioRNA and their derivatives, carbohydrates, metabolic metabolites and / or other biochemical or chemical molecules , 18. Non-porous spherical silica gel particles with a particle size of less than 15 μm, which are loaded with biologically and / or chemically active substances. 19. Non-porous spherical silica gel particles according to claim 18, characterized in that they can be produced by a method according to one of claims 1 to 17. 20. Use of non-porous spherical silica gel particles according to claim 18 or claim 19 for carrying out molecular separations, chromatography, catalysis, analysis, sample enrichment, binding or other chemical reactions. 21. Use of non-porous spherical silica gel particles according to claim 18 or claim 19 in the chemical industry, medical laboratory diagnostics, drug or crop protection agent development, quality control and / or research and development.