EP1230213A1 - Articles having an activated surface for immobilizing macromolecules and method for producing such articles - Google Patents

Abstract

The invention relates to articles having an activated surface for immobilizing bioorganic macromolecules. Said articles comprise: a substrate with a surface; a dendritic skeleton linked to the substrate surface, and; a number of first coupling groups linked to the dendritic skeleton for immobilizing bioorganic macromolecules. The articles can be produced using a method comprising the following steps: providing a substrate with a surface; linking the substrate surface to a dendritic skeleton, and; furnishing the dendritic skeleton with a number of first coupling groups for immobilizing bioorganic macromolecules.

Description

 Articles with activated surface for immobilization of macromolecules and method for producing such articles

The present invention relates to articles, in particular sensors, with an activated surface for immobilizing, in particular, bioorganic macromolecules, and to processes for their production. The methods according to the invention include, in particular, methods for generating activated sensor surfaces for the highly efficient covalent immobilization, in particular of bioorganic macromolecules.

The detection of, for example, bioorganic macromolecules via affinity reactions on biosensors requires substrate surfaces that are coated with a reaction partner of the interacting biomolecules (solid phase method). In order to be able to permanently fix this reaction partner on a sensor matrix (as an example of a substrate surface), this must first be chemically modified so that reactive coupling groups are used Immobilization will be provided. These coupling groups are typically reactive hydroxyl, amino, carboxyl, acyl halide, aldehyde, isothiocyanate or epoxy groups, which are covalently linked to the surface via spacers. It is particularly attractive because it minimizes costs if the sensor can be regenerated and thus used multiple times. The immobilization method should therefore be designed in such a way that the macromolecules are fixed on the surface via covalent, oxidation-stable linker systems.

Previous strategies for immobilization are based on linear linkers. For example, silicon dioxide surfaces can be activated by coating with an N-alkylamino-silane for the coupling of bioorganic macromolecules (Chrisey, L; O ^' Ferrall, CE; Spargo, BJ; Dulcey, CS; Calvert, JM Nucleic Acids Research 24/15 3040-3047 (1996)). Another possibility is to use gold-coated surfaces by coupling the macromolecules via a functionalized alkylthiol SAM (SAM = self-assambled monolayer) (Bardea, A .; Dagan, A .; Willner, I. Anal Chim. Acta 385, 33-43 (1998)). If the macromolecule to be immobilized contains thiol groups, direct coupling through chemisorption to the gold surface is possible, cf. DE 19807339 A1. Glass surfaces can be functionalized by using suitable silanes. Glass surfaces activated with epoxyalkylsilane are frequently used to immobilize hydroxyl- or amino-functionalized biomolecules (see US Pat. No. 5,919,626), while thiolated macromolecules can be coupled to thiolsilane-activated surfaces via disulfide bridges (see US Pat. No. 5,837,860). Another possibility is to use surfaces coated with avidin / streptavidin, which have a high affinity for biotinylated substances (cf. DE 3640412 A1; DE 19724787 A1).

The methods listed, however, need to be optimized, since they generally only lead to a low occupancy density and a lack of regenerability of the surfaces produced in this way. For example, the Au-sulfur bond is oxidatively destroyed in the presence of oxygen, avidin / streptavidin surfaces are denatured during thermal regeneration and silylated glass surfaces are unstable with regard to alkaline media pH> 8 (see Sandoval, JE et. al., Anal. Chem. 63, 2634-2641 (1991)). The glass surfaces obtained by silylation also have only a low loading density, which often leads to an insufficient sensitivity of the sensors (cf. Southern et al. Nature genetics Supplement 21, 5-9 (1999)).

There are now several works aimed at improving the loading density and regenerability of the surfaces, e.g. B. by the use of covalent (see Löfas, S. et al. Pure Appl. Chem. 67, 829-834 (1995)) or chemisorptive on gold (see Johnsson, B .; Löfas, S .; Lindquist, G. Anal. Biochem. 198, 268-277 (1991)) bound dextran intermediate layers with hydrogel properties. However, the immobilization on these surfaces takes place statistically within the hydrogel matrix, which has a disadvantageous effect on the accessibility of the immobilized component, since the heterogeneous interphase reaction is limited by diffusion processes (cf. Southern et al. Nature genetics Supplement 21, 5-9 (1999 )). This problem also occurs with the approach of covalently binding surface-activated microparticles (CPG, magnetic beads, Merrifield resin, etc.) to the sensor surfaces in order to achieve an increase in surface area (see US Pat. No. 5,900,481). The surfaces prepared in this way are microporous and prove problematic in affinity reactions due to limited diffusion.

By Beier et al. describes a method with which it is possible to increase the number of potential binding sites for macromolecules on the sensor surface through the in-situ construction of branched linker systems (see Beier, M .; Hoheisel, JD Vol27 / No9, 1970-1977 (1999 )). However, after prior amination of the support (substrate) (silylation, RFPE = radio frequency plasma discharge in ammonia, etc.), the successive repetition of a 2-step synthesis lasting approx. 1-2 days, followed by a final end group activation, is necessary. Typically, 8 or more reaction steps over several days are required to produce the activated surfaces.

In view of the existing disadvantages of the known methods, a first (partial) object of the present invention was to provide an article, in particular a sensor, with an activated surface that has a high density reactive coupling groups and also has a high physical-chemical stability to thermal and chemical regeneration steps.

From a procedural point of view, it was a second (partial) object of the present invention to specify a method which allows such an activated surface to be built up in a few reaction steps.

A third (partial) object of the present invention was to provide a corresponding method for producing an article (in particular a sensor) with a bioorganic macromolecule immobilized on the surface.

The first (partial) object is achieved by an article with an activated surface for immobilizing, in particular, bioorganic macromolecules, comprising an substrate with a surface, a dendrimeric framework linked to the substrate surface and a number of first linked to the dendrimeric framework

Coupling groups (e.g. NHS or isothiocyanate groups) for

Immobilization of bioorganic macromolecules.

The article can in particular be a (bio) sensor or sensor element, such as a DNA microarray, a protein array of antibodies, receptors and / or enzymes, a test array of peptides, peptoids, or low-molecular compounds such as Act pharmacophores or other active ingredients.

Depending on the requirements of the individual case, the dendrimeric scaffold can comprise a large number of identical or different first coupling groups (e.g. NHS esters, isothiocyanate groups, nucleic acid strands or the like). For example, sensors should often be able to detect a wide variety of analytes, and it then makes sense to offer these different analytes different first coupling groups.

Advantageously, the substrate surface is linked to the dendrimeric scaffold via a linking unit, which (in the manufacture of the article, see below) by reaction of an initiator group assigned to the substrate surface with a complementary functional group assigned to the dendrimeric scaffold. For example, if an initiator group with a free carboxy function was originally assigned to the surface and the complementary group of the dendrimeric skeleton was an amino function, the linking unit is an amide. Further examples of initiator groups and complementary functional groups are given below in connection with the explanation of the production processes according to the invention. Knowing the educt groups, it is clear to the person skilled in the art how the (product) linking unit is structured.

The dendrimeric skeleton can in particular comprise the following dendrimeric basic units, which can be crosslinked in the dendrimeric skeleton: starburst dendrimers and their in particular chemically or biochemically modified derivatives, metallodendrimers, carbosilane dendrimers, polysilane dendrimers, glycosyl-containing saccharide chargers and dendrimers - Dendrimers and their derivatives, peptide and oligopeptide dendrimers and their derivatives as well as nucleotide and oligonucleotide dendrimers and their derivatives.

The dendrimeric framework advantageously carries first coupling groups for immobilizing, in particular, bioorganic macromolecules or other substances which (during the manufacture of the article, see below) can be formed by reacting a functional group assigned to the dendrimeric framework with a homo- or heterobifunctional linker substance (linker molecule) , If the functional group assigned to the dendrimeric skeleton is, for example, an amino function and the linker substance is the homobifunctional substance phenylene-1,4-diisothiocyanate, the dendrimeric skeleton carries an isothiocyanate group as the first coupling group. Further examples of linker substances are given below in connection with the explanation of the production process according to the invention. Knowing the educt groups, it is clear to the person skilled in the art how the first (product) coupling group, which is monofunctional when using bifunctional linker molecules, is structured. The dendrimeric framework advantageously comprises dendrimeric basic units linked to the substrate surface, which are cross-linked to one another, for example with the aid of bifunctional linker substances (linker molecules). For the resulting benefits, see below.

The second (partial) object is achieved by a method for producing an article with an activated surface for immobilizing, in particular, bioorganic macromolecules, with the following steps:

Providing a substrate with a surface

Linking the substrate surface with dendrimeric basic units (in particular the basic units of a dendrimeric framework) and

Equipping the dendrimeric scaffold with a number of first coupling groups for immobilization, in particular bioorganic macromolecules.

The article produced is then usually an article as described in more detail above.

The substrate surface is usually first modified with a reactive initiator group, ie. the initiator group is connected to an (unmodified) substrate surface, before the (modified) substrate surface is then linked (and thus further modified) to the dendrimeric framework in a subsequent step. The substrate surface can be modified using standard methods, cf. Fig. 6.

The reactive, surface-bound initiator group can be selected from one of the following chemically reactive groups: hydroxyl, amino, carboxyl, acyl halide, ester, aldehyde, epoxy or thiol group. It can also be selected from one of the following biologically or chemically reactive groups: disulfides, metal chelates, nucleotide and oligonucleotides, peptides or haptens, such as, for example, biotin, digoxigenin, dinitrophenyl groups or similar groups.

The (unmodified) substrate surface to which the reactive initiator group is coupled will preferably be selected from the following group: consists of: carrier materials based on metals, semimetals, semiconductor materials, metal and non-metal oxides; glasses; plastics; organic and inorganic polymers; organic and inorganic films and gels, in particular as a coating for one of the aforementioned materials.

The dendrimeric framework, which is usually linked to it via the reactive initiator group of a modified substrate surface, can comprise several identical or different functional groups per basic dendrimer unit. These are preferably aminoalkyl, hydroxyalkyl or carboxyalkyl groups which are bonded in the periphery of the dendrimeric base unit and can be converted into the first coupling group with the aid of the linker substance.

The dendrimeric basic units can preferably be selected from the following group, which consists of: starburst dendrimers and their chemically and biochemically modified derivatives, metallodendrimers, glycosyl-containing dendrimers, saccharide and oligosaccharide dendrimers and their derivatives, peptide and oligopeptide dendrimers and their derivatives and nucleotide and oligonucleotide dendrimers and their derivatives.

The dendrimers to which the basic dendrimeric units correspond in the finished article according to the invention are generally synthesized in a conventional manner before carrying out the process according to the invention (Zeng, F .; Zimmerman, SC; Chem. Rev. 97, 1681-1712 (1997)) and provided for performing the method according to the invention.

It can be dendrimers that

(a) in situ through covalent or non-covalent interactions through a parallel process, for example through self-assembly based on hydrogen bonding, Van der Waals, Coulomb or metal ligand interaction, or

(b) are built in situ by a successive process via covalent or non-covalent interactions such as self-assembly through hydrogen bonding, Coulomb interaction or the metal-ligand interaction. Advantageously, the dendrimeric basic units (ie the educt dendrimers or the dendrimeric skeleton formed therefrom) are equipped with a number of coupling groups for immobilization, in particular bioorganic macromolecules, by reacting functional groups assigned to the dendrimeric basic unit with a homo- or heterobifunctional linker substance (linker molecule).

The dendrimeric base units can be equipped with the first coupling groups before or after the substrate surface is linked to the dendrimeric base units.

In particular, homobifunctional linker substances that can be used are: photochemically, chemically, biochemically or biologically active compounds such as, for example, dicarboxylic acids and their anhydrides, disuccinimidyl glutarate,

Phenylenediisothiocyanates, ß / s- [ß- (4-azidosalicylamido) ethyl] disulfide, 1, 4-ß / s-

Maleimidoalkanes, 1,6-hexane-b / s-vinyl sulfone.

The following heterobifunctional linker substances can be used in particular: photochemically, chemically, biochemically or biologically active compounds such as, for example, 3 - [(2-aminoethyl) dithio] propionic acid, Λ / -α-

Maleimidoacetoxy] succinimide, 4- [p-azidosalicylamido] butylamine, Λ / - [(ß-malimido-propoyloxyjsuccinimide, Λ / - [κ-malimidoundecanoic acid] hydrazide, succinimidyl-6- [3-

(2-pyridyldithio) propionamido] hexanoate, Λ / -succinimidyl iodoacetate.

Knowing the functional groups assigned to the dendrimeric basic units and the bifunctional linker substances to be used, the person skilled in the art can precisely predict which functionalities the dendrimeric framework of the finished article carries.

In view of a high physicochemical stability, a process design (and accordingly the resulting articles) is particularly preferred in which the surface-bound dendrimeric base units are crosslinked with the aid of homobifunctional linkers, with the exception of carboxylic acid anhydrides, so that a polymeric thin film is formed Basic dendrimeric units are created. The linker substances are preferably but not necessarily the same ones that are used to generate the coupling groups; reference is made to the above explanations regarding preferred linker substances.

In view of a high immobilization capacity, on the other hand, a process design (and accordingly the resulting articles) is particularly preferred in which the first coupling group is generated with the aid of heterobifunctional linkers or with carboxylic acid anhydrides. In this process design, each functional group of the dendrimeric base unit is converted into an active coupling group; reference is made to the above explanations regarding preferred linker substances.

Accordingly, the person skilled in the art will select dendrimers (dendrimeric basic units) and linker substances in particular with a view to the substances to be immobilized and with a view to good crosslinking of the dendrimeric basic units.

In accordance with the preferred process design, a macromolecular surface is built up from the surface-fixed dendrimers by covalent crosslinking. The surfaces generated in this way have two decisive advantages compared to common linear linker systems. Due to the (covalent) cross-linking of the immobilized dendrimers, the surfaces have an increased physicochemical stability and thus enable the loss-free regenerability of the surfaces loaded with bioorganic macromolecules (for example sensor surfaces). Furthermore, the immobilization efficiency is drastically increased by using polyfunctionalized dendrimers. In combination with the high flexibility of the linker system due to the chemical nature of the dendrimers, there is a high sensitivity in heterogeneous affinity reactions, such as, for example, the hybridization of nucleic acids and of antibody-antigen, of protein-protein, of protein-ligand or of receptor -Substrate interactions.

A process design is particularly preferred in which the construction of the dendrimeric framework and thus an essential step in the production of articles according to the invention, for example the construction of aminodendrimer Sensor surfaces, via a sandwich preparation. As shown schematically in FIG. 5, two substrates activated in each case, for example by aminosilylation and subsequent activation using, for example, disuccinimidyl glutarate, for example two glass slides with an area of approximately 3 × 8 cm (any other sizes can also be used), are put together in pairs that the immobilization of the dendrimer can take place in the space between the two substrates (for example glass substrates). When using glass slides, typically about 100 microliters of a 10% solution of the (amino) dendrimer are applied to the surface of one of the two slides, and then the drops of the (amino) dendrimer solution are spread by placing the second slide , so that a thin film of the (amino) dendrimer solution forms between the two substrates. Of particular advantage with this procedure is that the reaction rate and homogeneity of the surface reaction are improved by the applied thin film, and on the other hand that only small amounts of the expensive reagent solutions are required. The sandwich preparation can also be used to produce the first coupling group by applying a saturated solution of the linker substance, for example glutaric anhydride or 1,4-phenylenediisothiocyanate, to the substrates coated with dendrimeric units

A particularly advantageous embodiment of the method according to the invention consists in initially modifying large planar substrates, the area of which corresponds, for example, to a multiple of a desired chip surface, using the steps described above with dendrimer layers. Typically, this is done in such a way that a grid of the chip formats to be produced is first applied to known glass, for example 100 cm × 100 cm large glass panes (or corresponding panes of other substrates; significantly larger panes can also be used) by breaking edges are generated on which the glass pane (or the other substrate) can later be divided into the final chip format. The chemical modification is then usually carried out by carrying out the steps described above (and in the examples below), for example cleaning, silylation and activation steps, for example in immersion baths. The The dendrimer is advantageously applied using the sandwich technique mentioned above, and the final activation of the dendrimers by means of homo- or heterobifunctional linkers is preferably carried out in sandwich or immersion treatment. The large-area substrate wafers are then separated into the final format. Alternatively, the large-area dendrimer-coated disks can first be cut up and only then activated as required using bifunctional linkers.

The advantage of this process design (including the alternatives mentioned) is that it increases the scale in the manufacture of chips, so that large numbers of the activated chip surfaces can be produced using the advantageous sandwich technology.

With the method according to the invention it is possible to attach a cross-linked dendrimeric framework with free coupling groups as a macromolecular (intermediate) layer on a substrate surface (see FIG. 1), whereby (a) the number of potential binding sites for the immobilization of bioorganic macromolecules or other substances, (b) the physicochemical stability of the surface is improved and (c) the regenerability of the substrate surfaces (carrier) modified with the bioorganic macromolecules is improved.

The present invention also relates to articles which are obtainable by the process according to the invention (including the special process designs specified above).

The articles according to the invention comprise functionalized solid phases and can be used, for example, as sensor elements, reactor elements and elements with electrical, electronic or optical functions. In addition to the above-mentioned biosensors (DNA and protein arrays), the articles functionalized with bioorganic molecules can also be used advantageously as solid phases in enzyme reactors, in which a high level of physical and chemical resistance and high occupancy densities are required. Because of the possibility of functionalizing DNA microarrays of high integration density with inorganic colloids and semiconductor nanocrystals (cf. Niemeyer, Curr. Opin. Chem. Biol., 4, 609 (2000)) resistant surfaces can also be used advantageously to build up electronic and optically active elements, for example for high-resolution displays.

The third (sub) task is finally solved by a method for producing an article with a (macro or other) molecule immobilized on the surface, with the following step:

Contacting a macromolecule comprising at least one second coupling group

(a) with an article according to the invention or

(b) with an article produced by the process according to the invention with an activated surface under conditions in which at least a first coupling group of the article and a second coupling group of the macromolecule react with one another to form a link between the macromolecule and the article.

In addition to bioorganic macromolecules, other substances such as macromolecular colloids and nanoparticles or low-molecular compounds such as pharmacological substances, hormones, antigens or other active substances can also be immobilized on appropriately prepared articles (a or b) according to the invention.

The invention also relates to articles comprising a (macro or other) molecule linked to the dendrimeric backbone. The (macro) molecule can be, for example, one selected from the group consisting of: antibodies, in particular the classes IgG, IgM, IgA, enzymes; receptors; Membrane proteins, glycol proteins; carbohydrates; nucleic acids; such as. DNA, RNA, peptide nucleic acid (PNA), pyranosyl ribonucleic acid (pRNA).

The term “other substances” includes in particular organic-chemical or inorganic-chemical groups with specific or also potentially active pharmacological, catalytic (for example photocatalytically active substances such as porphyrins and their derivatives or catalysts for the stereoselective transformation of organic or inorganic substrates), optical or electrical properties (eg fluorescent, electroluminescent or electrically conductive polymers (polyaniline, polypyrrole) compounds. Also can Substance libraries, which are accessible, for example, by combinatorial solid-phase synthesis, are immobilized on the article in order to screen the functionality of these bound substances, for example their inhibitory action on biological enzymes or receptors.

The basis of the present invention is, among other things, the surprising observation that nucleic acid-functionalized glass surfaces, which were produced by the method according to the invention, not only have a drastically improved detection limit in the detection of complementary nucleic acids, but also show a significantly increased physicochemical stability that the regenerability of the nucleic acid-modified supports could be carried out many times by treatment with alkaline washing solutions without loss of activity on the surface. It was found that the articles produced by the process according to the invention have a significantly improved physicochemical stability and thus the loss-free regenerability of the carriers modified with the bioorganic macromolecules is possible. In addition, the chemical nature of the dendrimeric basic units means that the linker system for the connection of the bioorganic macromolecules is highly flexible, which results in additional advantages over linear linker systems in terms of the efficiency of the heterogeneous affinity reaction.

As already mentioned, it is possible with the method according to the invention to apply a cross-linked dendrimeric framework with free coupling groups as a macromolecular (intermediate) layer on a substrate surface (cf. FIG. 1). The attachment of the macromolecular layer is achieved, for example, by treating a glass surface (for example amino-, epoxy- or carboxyl-modified (as substrate surface) produced by standard processes) first with a dendrimeric macromolecule and then with a homo- or heterobifunctional linker reagent. By connecting the dendrimeric component to the modified glass surface, the number of binding sites for the immobilization of bioorganic macromolecules (eg certain nucleic acids) is increased. By treatment with the homobifunctional linker reagent, preferably (a) the dendrimer units linked to the substrate surface are used for the immobilization of the activated bioorganic macromolecules, ie equipped with free coupling groups and (b) covalently cross-linked the dendrimer units. Bioorganic macromolecules can be immobilized very stably on the surface of an article produced in this way.

The invention is explained in more detail below using examples with reference to the accompanying figures.

Examples

Example 1:

Process for the production of a glass support with a cross-linked dendrimer structure with free isothiocyanate coupling groups attached to the glass support surface, and immobilization of a bioorganic macromolecule by coupling to the coupling groups

1.1 Aminosilylation of a glass surface (surface activation):

A glass substrate with a surface based on silicon dioxide was provided. The glass surface was cleaned thoroughly (CH2CI2 - H2O2 / H2SO4 + ultrasound → Bidest). The glass surface was then silylated (cf. FIG. 1; step 1) with 3-aminopropyltriethoxysilane in ethanol / water (95: 5) using a method described in the literature (Maskos, U .; Southern EM; Nucleic Acids Res ., 20 (7), 1679-1684 (1992) (other silylation processes are also applicable).

1.2 Carboxy functionalization of the surface

The aminosilylated glass surface prepared according to 1.1 was carboxy-functionalized with a 10 mM solution of disuccinimidyl glutarate in CH2Cl2 / n-ethyldiisopropylamine (100: 1) for 2 h at RT under argon (cf. Fig. 1, step 2). The surface was then thoroughly washed several times with CH2CI2. The carboxy group introduced in this way could then be used as an initiator group

1.3 Linking the surface with an amino-terminated dendrimer (amino-dendrimer immobilization)

The surface was covered with a 10% solution of an aminodendrimer (Fa. Aldrich Chem. Co, sold under the trade name Starburst (PAMAM), see also Yamakawa, Y et al. J. of Polymer Science: Part A 37 3638-3645 (1999) ) wetted in methanol (see Fig. 1, step 3). This step can be carried out, for example, using the advantageous sandwich method already described. After a reaction time of 30 minutes (reaction of the carboxy groups with the amino groups of the dendrimer), the excess of dendrimer was removed by washing and the (glass carrier) surface now provided with dendrimer was dried in a stream of nitrogen.

1.4 Implementation with linker substance (generation of free coupling groups and crosslinking of the dendrimer units)

The glass supports equipped with dendrimer in accordance with 1.3 were transferred into a 20 mM solution of a homobifunctional linker molecule, for example phenylene-1,4-diisothiocyanate, in CH2Cl2 / pyridine (100: 1) (cf. FIG. 1, step 4). After a reaction time of 30 minutes, the mixture was washed thoroughly with CH2Cl2. The glass slide with a dendrimer surface could now be used directly for the immobilization of bioorganic macromolecules described in more detail below, or alternatively it could be kept under argon until use.

1.5. Linking (immobilization) of bioorganic macromolecules - general procedure:

The surface prepared according to 1.4 was wetted with drops of an aqueous solution which contained a bio-organic macromolecule to be immobilized in a typical concentration range of 1 - 100 μM. The bio-organic component was, for example, 5'-amino modified oligonucleotides, such as those available from a variety of commercial suppliers.

The wetted surfaces were incubated in a humidity chamber for several hours. The optimal incubation time was dependent on the type and concentration of the component to be immobilized.

To avoid unspecific binding in the event of intended affinity reactions, the coupling groups which had not reacted during the incubation period were subsequently deactivated. For this purpose, the glass carrier element was, for example, transferred to a 6-aminohexanol solution (100 mM in dimethylformamide (DMF)) in order to inactivate still active isothiocyanate groups of a phenylene diisothiocyanate linker (see 1.4 above). The glass carrier surface was then freed of non-covalently bound bio-organic macromolecules with detergent-containing solutions, for example sodium dodecyl sulfate, then rinsed several times in bidistilled water and dried in a stream of N2. The loaded sensors were stored at - 20 ° C until use.

Dendrimer-based, isothiocyanate-functionalized surfaces, which are loaded, for example, with amino-functionalized oligonucleotides, are of particular interest as sensor surfaces for DNA chip technology, cf. Niemeyer, CM .; Blohm, D. Angew. Chem. 111/19 3039-3043 (1999). See also Fig.2 and the associated explanations in this context.

Example 2:

1. Variation: Modification of glass support surfaces using carboxy-terminated dendrimers

The amination of a glass-based carrier was carried out as described in Example 1 under 1.1. To build up a carboxydendrimer surface, a carboxy-terminated dendrimer (trade name Starburst (PAMAM) dendrimer; Aldrich Chem. Co) was first used by known methods, for example as described by Johnsson, B .; Löfas, S .; Lindquist, G. Anal. Biochem. 198 268-277 (1991), esterified in the presence of dicyclohexylcarbodiimide / N-hydroxysuccinimd. The activated dendrimer was placed in DMF on the aminosilylated surface and after a reaction time of 30 minutes the excess dendrimer was washed off with DMF. The surfaces could then be used directly, as described in Example 1, for the immobilization of bioorganic macromolecules. Alternatively, the activated surfaces could be stored under argon.

Example 3:

2. Variation: Construction of macromolecular sensor surfaces based on amino reactive silane surfaces

The glass surfaces are cleaned as described in Example 1, 1.1. In the case of hydrolysis-stable silanes such as 3-carboxypropyltrialkoxysilane, the silanization is carried out as indicated in Example 1, 1.1. If it is a hydrolysis-sensitive silane, such as, for example, 3-glycidoxypryopyltrimethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltriethoxysilane, 3-iodopropyltrimethoxysilane, 3-isothiocyanatotrialkoxysilane and the corresponding trihalosilanes, the silanization of the literature is preferably described under dry conditions such as in the literature and dry conditions (Southern, EM et al .; Nucleic Acids Res., 22 (8), 1368-1373 (1994); other methods are also conceivable. Epoxy, isothiocyanato and iodo-terminated silane surfaces are used directly to bind the amino-functionalized dendrimer while carboxy-functionalized silane surfaces are first activated by reaction with DCC / NHS, as described in Example 7, 7.2 The further reaction of the dendrimer surface with a homo- or heterobifunctional spacer to produce the first coupling group and the macromolecules are attached to it as follows under Example 1, 1.4 - 1.5, see above as described in Example 7, 7.4. Example 4:

3. Variation: Use of plastic carriers

Surfaces of plastic carriers made of, for example, polyethylene, polypropylene, polystyrene, polycarbonate, polyacrylonitrile or their copolymers were produced by known methods, for example by the method of Hartwig, A. et al. Advances in Colloid and Interface Science 52, 65-78 (1994) aminated by radio frequency plasma discharge in an ammonia atmosphere.

The construction of dendrimer-based surfaces was then carried out as described in Example 1, 2 or 7.

Example 5:

4. Variation: Use of gold-coated substrates

Gold layers were applied in a conventional manner to various supports and aminated by known methods, for example by producing an amino-terminated SAM on gold, as described by Glodde, M .; Hartwig, A .; Hennemann, O.-D .; Stohrer, W.-D. Intern. J. of Ahesion & Adhesivs 18 359-364 (1998).

The construction of dendrimer-based surfaces and the connection of bioorganic macromolecules was carried out as described in Examples 1-3.

Example 6:

5. Variation: Use of surfaces based on silicon and other metal and semiconductor carrier materials

Metal and semiconductor surfaces were aminated using known methods, for example by silylation with amino, epoxy, carboxy or thiolsilanes (see Chrisey, L .; O'Ferrall, CE; Spargo, BJ; Duicey, CS; Calvert, JM Nucleic Acids Research 24/15 3040-3047 (1996) and Bhatia, SK et al. Anal. Biochem. 178, 408-413 (1989)) or by hydrosilylation with ω-undecenecarboxylic acid (Sieval, AB et al .; Langmuir 14 (7) 1759-1768 (1998).

The respective activated surfaces were then modified according to Examples 1-3 with a dendrimeric component.

Biorganic macromolecules were immobilized as described in Example 1 under 1.5.

Example 7:

6.Variation: Process for the production of a glass support with a non-crosslinked dendrimer structure with free NHS ester coupling groups attached to the glass support surface

7.1 Aminosilylation of a glass surface (surface activation): The silylation was carried out as described in Example 1 under 1.1.

7.2 Carboxy functionalization of the surface

The aminosilylated glass surface prepared according to 1.1 was carboxy-functionalized with a saturated solution of glutaric anhydride in DMF for 4 h at RT under argon. The surface was then thoroughly washed several times with DMF and water. The free carboxyl groups were then activated with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS). For this purpose, the surface was wetted with a solution of 1 M DCC and 1 M NHS in DMF. After a reaction time of 4 hours, the supports were washed thoroughly with DMF and acetone.

The NHS ester-activated carboxy group introduced in this way could then be used as an initiator group

7.3 Linking the surface with an amino-terminated dendrimer (amino-dendrimer immobilization) The dendrimeric basic unit with the substrate was carried out as in Example 1 below

1.3 executed.

7.4 Implementation with linker substance (creation of free coupling groups)

The glass supports equipped with dendrimeric basic units according to 1.3 were transferred into a saturated solution of glutaric anhydride in DMF. After a reaction time of 4 hours, the mixture was washed thoroughly with DMF and water. The free carboxyl groups were then activated with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS). For this purpose, the surface was wetted with a solution of 1 M DCC and 1 M NHS in DMF. After a reaction time of 4 hours, the supports were washed thoroughly with DMF and acetone. The glass slide with a dendrimer surface could now be used directly for the immobilization of bioorganic macromolecules described in more detail below, or alternatively it could be kept under argon until use.

Examples 1 to 7 above include the attached FIGS. 1 and 2, which are explained in more detail below. They represent:

Figure 1: Surface modification to produce dendrimer-based, macromolecular sensor surfaces.

FIG. 2: attachment of biomolecules to the surfaces according to FIG. 1

FIG. 1 shows the surface modification of a glass carrier, plastic carrier or gold-coated carrier (as a substrate), cf. especially Examples 1, 4, 5 and 7.

Step 1 shows the amino activation of the surface by silylation, RFPD or ω-aminoalkylthiol-SAM, cf. Example 1, 1.1. Step 2 leads to a carboxylated surface by binding a dicarboxylic acid, cf. Example 1, 1.2.

According to step 3, a polyfunctionalized amino dendrimer is fixed on this surface. A 4th generation dendrimer with 64 amino groups is typically used for this, cf. Example 1, 1.3.

The final end group activation and crosslinking of the fixed dendrimers can be seen in step 4. For reasons of clarity, the intramolecular crosslinking of amino end groups is not shown, cf. Example 1, 1.4.

FIG. 2 shows a dendrimer-based, isothiocyanate-functionalized surface which is loaded with bioorganic macromolecules. The macromolecules are amino-functionalized oligonucleotides. Such surfaces are of particular interest as sensor surfaces for DNA chip technology, cf. Niemeyer, CM .; Blohm, D. Angew. Chem. 111/19 3039-3043 (1999). The oligonucleotides are bound as described in Example 1 under 1.5; regeneration can take place, for example, in an alkaline medium (e.g. under the following conditions: use of 50 mM NaOH, room temperature, 2 x 3 min).

Example 8:

Comparative consideration of homogeneity and loading density in DNA arrays which were obtained (a) with a conventional linear linker and (b) by surface modification according to the invention

The examination carried out is explained in more detail with reference to FIG. 3 (with FIGS. 3-1 and 3-2).

Figure 3-1 shows a conventional DNA array carrying 5 ^' amino modified capture oligonucleotides which are surface-fixed via a conventional linear linker. The surface was activated as follows: After silylation with 3-aminopropyltriethoxysilane as described in Example 1 under 1.1, 1,4-phenylenediisothiocyanate was bound. The individual spots were applied to the conventional surface with a piezoceramic pipette to form the oligonucleotide array. The spots of a vertical row were each generated with the same volume of capture oligonucleotide solution: rows 1-4, 9-12 = 2 nl; 5-8, 13-16 = 4 nl. The concentration of the spotted oligomer solution was 10 μmol / l in bidest.

For comparison, FIG. 3-2 shows a DNA array according to the invention with a (sensor) surface according to the invention.

The modification route is described in Example 1.

The same capture oligonucleotide was immobilized as in the case of the array from Fig. 3-1.

To produce the array according to the invention, the spot volume was varied within the respective rows 1-16: upper 7 spots: 2 nl; lower 8 spots: 4 nl. The concentration of the spotted oligomer solution was 10 μmol / l in bidest.

Hybridization and comparative array evaluation:

In both cases, a 1 nM solution of the 3 ^' - Cy5-labeled, complementary nucleic acid was hybridized for 1 h at room temperature. The array was evaluated by fluorescence excitation and reading with a standard CCD camera.

The array from FIG. 3-1 produced by a conventional method, namely by isothiocyanate activation of amino-silylated glass surfaces, shows clear inhomogeneities in the signal intensity within the vertical rows 1-16, although the same intensity was expected due to identical conditions. This effect is due to an inhomogeneous surface modification and can only be achieved in arrays without surface modification according to the invention difficult to prevent. Such effects would severely hinder a quantitative evaluation of the array.

In contrast, the individual signals of the array with surface activation according to the invention from FIG. 3-2 have almost the same intensity within a lower right row. The homogeneity of the surface modification is therefore significantly improved by the method according to the invention.

The different loading density with capture oligonucleotides is evident from the different exposure times during the exposure. 3-1 an exposure time of 120 sec was necessary to be able to detect signals; for recording Fig. 3-2 only 10 sec.

Example 9:

Comparative consideration of the regeneration stability of a DNA array with a conventional linear linker and a DNA array obtained by surface modification according to the invention

The test carried out is explained in more detail with reference to FIG. 4 (with FIGS. 4-1 to 4-8).

The DNA array shown in FIGS. 4-1 to 4-3 comprises a conventional linear linker, while the array shown in FIGS. 4-4 to 4-8 has a (sensor) surface according to the invention. The surface modification and hybridization of the arrays was carried out as described in Example 7. Quadrupoles with varying volumes of 1.4 - 5.6 nl were scoffed at. Regeneration was carried out in each case 2 × 3 min with 50 mM NaOH at RT.

Fig. 4-1 shows the array with linear linker after the first hybridization, Fig. 4- 2 shows the array after the regeneration and Fig. 4-3 shows the array after the second hybridization. It can be clearly seen that after the first regeneration step, the signal intensity is greatly reduced. The cause is Detachment of the capture oligonucleotide during the regeneration process due to the inherent instability of the conventional linker.

4-4 to 4-8 show the regeneration properties improved by the method according to the invention. Even after a repeated regeneration process, no decrease in the signal intensity can advantageously be observed.

Other figures:

Figure 5 is a highly schematic illustration of the preferred sandwich preparation technique, see above.

6 is a highly schematic illustration of a preferred method according to the invention for producing an article with an activated surface for immobilizing macromolecules or other compounds.

Claims

claims

1. Article with activated surface for immobilization of macromolecules, comprising a substrate with a surface, a dendrimeric framework linked to the substrate surface and a number of first coupling groups linked with the dendrimeric framework for immobilization of macromolecules

2. Article according to claim 1, characterized in that the article is a sensor or sensor element, an array such as a DNA (micro) array, a protein (micro) array, a peptide, peptoid or low molecular weight compounds such as a pharmacophore-carrying test array, a component of a (micro) reaction vessel or an optically or electronically active element.

3. Article according to one of the preceding claims, wherein the dendrimeric framework comprises a number of dendrimeric basic units, which are selected from the group consisting of: organic dendrimers, starburst dendrimers, metallodendrimers, semi-metallodendrimers, carbosilane dendrimers, polysilane dendrimers, Glycosyl-containing dendrimers, saccharide and oligosaccharide dendrimers, peptide and oligopeptide dendrimers, nucleotide and oligonucleotide dendrimers and derivatives of the aforementioned dendrimers.

4. Article according to one of the preceding claims, wherein the dendrimeric framework comprises dendrimeric basic units which are networked with one another.

5. A method for producing an article with an activated surface for immobilizing macromolecules, comprising the following steps:

Providing a substrate with a surface

Linking the substrate surface with dendrimeric basic units and Equipping the dendrimeric basic units with a number of first coupling groups for the immobilization of macromolecules.

6. The method according to claim 5, wherein the substrate surface is equipped with a number of initiator groups for linking to the dendrimeric base units.

7. The method according to claim 6, wherein the initiator groups are selected from the group consisting of: hydroxyl, amino, carboxyl, ester, acyl halide, aldehyde, epoxy and thiol groups as well as disulfides, metal chelates, nucleotides and Oligonucleotides, peptides and haptens, such as biotin, digoxigenin, dinitrophenyl groups.

8. The method according to any one of claims 5-7, wherein the substrate comprises a carrier material which is selected from the group consisting of: carrier materials based on metals, semi-metals, semiconductor materials, metal, semi-metal and non-metal oxides; glasses; plastics; organic and inorganic polymers; organic and inorganic films, gels and monolayers, in particular as a coating for one of the aforementioned materials.

9. The method according to any one of claims 5-8, wherein the dendrimeric basic units are selected from the group consisting of: starburst dendrimers, metallodendrimers, glycosyl-containing dendrimers, saccharide and oligosaccharide dendrimers, peptide and oligopeptide dendrimers, Nucleotide and oligonucleotide dendrimers and derivatives of the aforementioned dendrimers.

10. The method according to any one of claims 5-9, wherein the dendrimeric basic units are equipped with a number of first coupling groups for the immobilization of macromolecules, by reacting functional groups of the dendrimeric basic units with a bifunctional linker substance.

11. The method according to claim 10, wherein the dendrimeric basic units are equipped with the first coupling groups before the substrate surface is linked to the dendrimeric basic units.

12. The method according to claim 10, wherein the dendrimeric basic units are equipped with the first coupling groups only after the substrate surface has been linked to the dendrimeric basic units.

13. The method according to any one of claims 5-12, wherein the dendrimeric basic units are crosslinked to form a dendrimeric framework.

14. A method for producing an article with a macromolecule immobilized on the surface, with the following step:

Contacting a macromolecule comprising at least one second coupling group

(a) with an article according to any one of claims 1-4 or

(b) with an article produced according to one of claims 5-13 with an activated surface under conditions in which at least a first coupling group of the article and a second coupling group of the macromolecule react with one another to form a link between the macromolecule and the article.

15. The method according to any one of claims 5-14, wherein the macromolecule or the macromolecules are selected from the group consisting of: bioorganic macromolecules such as nucleic acids, antibodies, enzymes, receptors, membrane proteins, glycoproteins, carbohydrates; macromolecular and colloidal nanoparticles; low molecular weight compounds such as pharmacologically active substances, hormones, antigens.

16. Article, characterized in that it is obtainable according to a method according to any one of claims 6-15.