DE10332848B4 - Microarrays of immobilized biomolecules, their preparation and use - Google Patents

Abstract

A microarray of immobilized biomolecules, which has at least two surface areas B which are spatially separated from one another and each comprise several spatially separated places P on which biomolecules are immobilized, in which the mean distance of the boundary line of an area B to its center point is the area of 10 μm to 200 μm.

Description

The present invention relates to microarrays of immobilized biomolecules, their preparation and their use. Likewise, the present invention relates to devices based on such microarrays, in particular chips and dip sticks.

With the increasing practical importance of molecular biology analysis, the standard techniques associated with a very high work and cost, often lasting days or weeks, are reaching their limits. New test formats such as biochips should enable enormous time and cost savings. Since a large number of different substances immobilized in a confined space can be brought into contact simultaneously with a single sample to be analyzed by this technique, the information content obtained per measurement from a chip is very high.

For a review of the most popular biochip systems, see, for example, Bowtell D.D. L. Nature Genetics Supplement 21 (1999) 25-32; Lockhart et al. Nature Biotechnology 14 (1996) 1675. For a review of protein and antibody arrays, see Cahill, D. J. Immunol. Methods, 250 (2001) 81-91.

A typical example of a so-called "high density array" is the GeneChip from Affymetrix, an oligonucleotide array with typically more than 400 different Capture probes that are built up base by base on the glass wafer. The chips are characterized by a very high information density of up to 40,000 oligonucleotides / cm ² . The individual "spots" are rectangular.

Low density arrays are available from Genometrix, for example. A maximum of 250 spots of 50 μm in diameter are printed in the wells of a 96-well microtiter plate using a capillary pin bundle of up to 1000 single capillaries. Conventional techniques are used for coupling to the surface, based in particular on aminosilanizations and NHS-activated haptens, epoxy-activated surfaces, carbodiimide coupling on carboxyl groups and biotin / streptavidin bonds (cf. WO 97/18226 A1 ; EP 0 910 570 A1 ; WO 98/29736 A1 ).

Several methods are currently available for the production of microbioarrays. The most common and also used methods for mass production of biochips are lithography techniques and spotting techniques (see Cheng et al. (Editor), Biochip technology, Harvard Academic Publishers 2001). The lithographic techniques are lithographic processes borrowed from semiconductor technology, which allow molecules to be localized on a surface. For example, describes the US 5,599,695 a method of making oligonucleotide arrays comprising covering a surface occupied by a molecular layer of protected compounds which allow attachment of oligonucleotides with a mask, deprotecting and thus treating the sites uncovered by the mask Contact surface with oligonucleotides. The oligonucleotides are bound to the deprotected sites of the surface and thus form an array corresponding to the mask occupied by oligonucleotides areas / places. Such techniques are particularly suitable for the production of very high density chip oligonucleotide arrays and allow the resolution of a few 100 nm. However, such lithographic processes are generally incompatible with biologically active molecules such as proteins and cells , Furthermore, the equipment costs for such procedures are very high.

The spotting techniques are printing processes. The biomolecules are applied to a surface as solutions with a printing system in the form of a regular dot pattern. At the heart of the printing system is a printhead, which either has a series of needles or is a micro-dosing head. The latter allows parallel printing of different solutions of biomolecules. Namely, when printing with a micro-dosing head, different liquids can be filled into the reservoirs which are connected to the micro-dosing head. You can successively perform several 100 to 1000 printing operations without refilling and thus generate a corresponding number of spots on a surface. However, with the usual spotting techniques it is not possible to produce spots with a diameter below 100 μm. Thus, this technique is not suitable to produce so-called high-density chips, but only low-and medium-density chips. Although various developments of the spotting technique have been described, which make it possible to apply solutions of biomolecules as spots with a resolution in the range <1 micron on a surface. In this context, those of Boland et al. (222nd ACS National Meeting 2001, pp. 26-30), the nanopipette method (Bruckbauer et al., J. Am. Chem. Soc., 2002, 124, 8810), dip-pen nanolithography (DPN, Mirkin et al., Science 1999, 283, p. 661 and Science 1999, 286, p. 523). However, these technologies are very time consuming and also associated with high equipment costs.

More recently, a stamping technique, so-called microcontact printing (μCP), has been variously applied to the preparation of arrays of biomolecules on surfaces (see Martin et al., Langmuir 1998, 14, p. 3971, Bernard et al., Langmuir 1998, 14 , 2224, Chieng et al., Sensors and Actuators 2002, B 83, p 22, Tan et al., Langmuir 2002, 18, p 519, Inerowicz et al., Langmuir 2002, 18, p. 5263 and WO01 / 67104 ). With the μCP technique, which was originally developed for other purposes, the immobilized biomolecules can be localized, ie in defined areas, applied to a surface. In this case, the procedure is generally such that the stamp surface is first brought into contact with a solution of the biomolecule to be immobilized, the stamp is optionally dried and then the stamp surface is brought into contact with the surface to be structured. In this case, the biomolecule is transferred to the surface in those areas in which the stamp surface is in contact with the surface. Surface areas that are not in contact with the stamp surface, however, remain unchanged. In this way you get defined area on the surface, which are covered with biomolecules to represent us as an image of the stamp. With the μCP technique, structures with a resolution up to about 100 nm can be produced. In contrast to the spotting technique, which allows several areas to be simultaneously coated on a single surface with different biomolecules, only identical areas can be created with the μCP technique. For the production of biochips, however, arrays are generally required which have a plurality of regions which differ from one another with regard to the type and / or the coverage of the biomolecules.

Inerowicz et al. (Langmuir 2002, 18, p. 5263) describe a μCP technique in which strip-shaped protein regions are first applied to a surface by means of a polydimethylsiloxane stamp having strip-shaped elevations. In a second stamping process, a stamp having the same structure is then wetted with a solution of a second protein and rotated by 90 ° onto the surface already coated with the first protein. Thus one obtains a surface with two different, orthogonally arranged protein strips. A third protein can be applied to the surface not yet occupied by the first two proteins by rinsing the surface with a solution of the third protein. In this way, however, only three different biomolecules can be applied to the surface. In addition, an overlap between different biomolecules is naturally unavoidable. Therefore, such arrays have only a low selectivity.

Chieng et al. (Sensor and Actuators 2002, B 83, pp 22-29) describe a method for the production of protein arrays, in which one first inserts a protein solution in so-called micro-wells, then with a stamp the protein solution from these micro-wells receives and on imprinted a suitable surface. In this way, protein arrays can be produced which have several regions which differ in the type of protein assignment. With the measures described there, however, only comparatively large areas (edge length> 500 μm) can be produced. A similar procedure is described by Renault et al., Angew. Chem. Int. Ed. 2002, 41, 2320. In this case, a mask with a plurality of through holes is placed on a stamp surface, in which holes solutions of different antigens are filled. As a result, the antigens are applied to the stamp surface. Subsequently, the stamp surface is contacted with a solution of antibody and then printed on a suitable surface. In this way, protein arrays can be produced which have several different regions with respect to their proteins. With both methods, however, only large areas can be produced. Both methods are extremely complicated to carry out and have the disadvantage of low selectivity. This is due to capillary forces of protein fluids.

The WO 02/48676 A2 describes multilayer microarrays for producing bioarrays, comprising a first membrane with very fine through holes arranged on a surface and a second membrane with comparatively large holes arranged above it. Solutions of biomolecules are filled into the bulging holes and penetrate through the fine holes on the surface. After removal of the lower membrane, a pattern of immobilized biomolecules on the surface is thus obtained. In this way, however, only bioarrays with very large areas of different biomolecules can be obtained. In addition, these regions must have relatively large distances from one another in order to achieve sufficient resolution since capillary forces cause the biomolecules to smear beyond the range limits. With the three latter methods, only those biochips can be produced which have a comparatively small number of regions with different biomolecules. In addition, these methods are very expensive in their implementation.

The object of the present invention is to provide arrays of immobilized biomolecules which, on the one hand, have the largest possible number of regions which can be occupied by different biomolecules and which have an increased resolution with respect to the different biomolecules. In addition, these arrays should be easy to manufacture without the need for the costly techniques required for high resolution spotting techniques.

This object is achieved by microarrays of immobilized biomolecules, also referred to below as microbioarrays, which have at least two, preferably at least ten, in particular at least fifty and especially at least one hundred spatially separated surface regions B arranged on a surface, each surface region comprising a plurality of preferably on average at least ten, in particular at least one hundred spatially separated sites P, on which the biomolecules are immobilized and wherein the extent of the areas on average a distance of the boundary line of a respective area B to its area center in the range of 10 .mu.m to 200 .mu.m in particular 25 microns to 200 microns corresponds. Such microbioarrays are the subject of the present invention.

• – mittels Spotting-Techniken Biomoleküle ortsaufgelöst auf eine geeignet strukturierte Stempelfläche aufbringt, so dass jeder Spot mehrere der erhabenen Bereiche der Stempelfläche, beispielsweise säulenförmige Erhebungen, abdeckt und dann die Stempelfläche mit der Oberfläche, auf der die Biomoleküle immobilisiert werden sollen, in Kontakt bringt und gegebenenfalls überschüssige, d. h. nicht immobilisierte Biomoleküle entfernt (Variante 1); oder
• – mittels μCP-Technik ein dem Array entsprechendes Muster an Verbindungen V. weiche eine Immobilisierung von Biomolekülen auf einer Oberfläche bewirken, im Folgenden auch als Kupplungsmittel V bezeichnet, auf die Oberfläche aufbringt, wobei man einen Array erhält, der eine Vielzahl räumlich getrennter Plätze P aufweist, die mit der Verbindung V belegt sind, anschließend eine Lösung der Biomoleküle auf die so bereitgestellte Oberfläche spottet, so dass jeder Spot mehrere Plätze P abdeckt und anschließend überschüssige Biomoleküle entfernt (Variante 2).

Surprisingly, these microbioarrays can be prepared by a hitherto unknown and easy to implement combination of conventional spotting techniques with the μCP technique (see the cited prior art), either

• - By means of spotting techniques biomolecules spatially resolved on a suitably structured stamping surface so that each spot covers several of the raised areas of the stamp surface, such as columnar elevations, and then brings the stamp surface with the surface on which the biomolecules are to be immobilized in contact and optionally excess, ie not immobilized biomolecules removed (variant 1); or
• A pattern corresponding to the array of compounds V. which causes immobilization of biomolecules on a surface, hereinafter also referred to as coupling agent V, is applied to the surface, resulting in an array having a plurality of spatially separated sites P which are occupied by the compound V, then mocks a solution of the biomolecules on the thus provided surface, so that each spot covers several places P and then removes excess biomolecules (variant 2).

Both variants of the method combine the high resolution achieved by the μCP technique and the advantages of the spotting technique, which makes it possible to generate different regions of immobilized biomolecules in a simple manner.

The invention thus relates to a method for the production of microbioarrays, in which one according to variant 1 on a stamp surface, which has a plurality of raised regions (hereinafter surveys), one or more mutually different biomolecules in at least two, preferably at least ten, in particular at least 50, especially at least 100, z. B. 100 to 10,000 spatially separated areas B (= spots) so applied that the biomolecules in the surface areas B respectively a plurality of surveys usually at least 10, preferably at least 50, in particular at least 100, z. B. 100 to 10,000 surveys cover the stamp surface, then the stamp surface with the surface on which the biomolecules are to be immobilized, brings into contact and optionally not immobilized biomolecules, eg. B. by washing, removed.

The number corresponds to this. Arrangement and size of the raised regions of the stamp surface covered by the biomolecules in the surface regions B, in each case the number, arrangement and size of the sites P of a region on which the biomolecules are immobilized. The number, arrangement and size of the surface areas B corresponds in a first approximation to the number, arrangement and size of the spots on the stamp surface.

The same arrangement can also be achieved by variant 2 of the method according to the invention. For this purpose, a compound V is applied to a stamp surface having a multiplicity of elevations and stamped onto the surface on which the biomolecules are to be immobilized. In this way, one obtains a surface having a plurality of spatially separated locations P, wherein the number, arrangement and size of the places P corresponds to the number, arrangement and size of the raised areas of the stamp surface and which are occupied by the connection V. Subsequently, one or more different solutions of the immobilized biomolecules in at least two, preferably at least ten, especially at least fifty and especially at least one hundred, z. B. one hundred to ten thousand spatially separate surface areas B so that the surface areas B each have a plurality, usually at least ten, preferably at least fifty and especially at least one hundred, z. One hundred to ten thousand of the places P, which are occupied by the connection V, cover. The compound V brings about a binding, ie immobilization of the biomolecules on the surface. Subsequently, one will remove the non-immobilized biomolecules. In the arrays thus obtained, the number, arrangement and size of the separated regions B correspond to the number, arrangement and size of the spots.

The inventive method according to the variants 1 and 2 is not limited to the production of the arrays according to the invention but also allows the production of arrays with an array of places P and areas B, which corresponds to the arrays according to the invention, but in which the area B also larger expansions , z. B.> 500 microns or above. However, preference is given to using these processes for the production of the arrays according to the invention.

The invention has a number of advantages:
The microbioarrays according to the invention allow an increase in capacity compared to conventional biochips produced by spotting techniques, since four to ten spots with the same biomolecule occupancy are generally required in the latter. to ensure a sufficiently accurate detection of the analyte. By contrast, in the arrays according to the invention, only a surface area corresponding in size to a spot is required. Due to the division of the areas into several places P, a clear increase in sensitivity, ie a better signal-to-noise ratio, is also achieved in the detection of analytes. In addition, significantly lower amounts of biomolecules are required for the production. Furthermore, the arrays according to the invention are relatively easy to produce. The expense for their production corresponds even at a resolution corresponding to that of a high-density biochip, only the production cost of a medium-density biochip produced by conventional spotting technology. The method according to the invention can also be used in a simple manner for the mass production of high-density arrays, since the μCP technique allows a repeated application of the stamping process without the need for intermittent spotting steps. Due to the attachment of the biomolecules to sites P within a given area, the area boundaries are relatively sharp, unlike conventional spots, ie, "coffee stain forms" such as occur in conventional arrays made by spotting techniques are avoided. This allows a very dense arrangement of the individual surface areas on the surface of the array, without having to worry about smearing these areas. Furthermore, it is surprisingly found that the method according to the invention does not occur in the case of complex biomolecules in which there is the danger of denaturation, especially in the case of proteins, such denaturation. Therefore, a particularly preferred embodiment of the invention relates to protein microbial arrays, ie microarrays of immobilized proteins.

The term "array" denotes an arrangement of defined locations P, in particular a spatial assignment of specific substances to defined locations P, it being possible for different sites P to be assigned the same or different substances. The assigned substances are according to the invention biomolecules. The spatial dimensions of the places P are in the micron range or in the sub-micron range. Therefore, the arrays according to the invention are also referred to as microbioarrays or as microarrays of immobilized biomolecules.

The microbioarrays according to the invention have several areas, which in turn are subdivided into spatially separated locations P. The locations P within each area are assigned the same substance type, although it is also conceivable to associate mixtures of two or more substance types with the sites P of a region. Places P in different areas will generally differ with regard to the composition and / or the coverage of the respective substance type. As a rule, therefore, different biomolecules will be immobilized on sites P in different regions.

The term "biochip" refers to an array of biomolecules immobilized on a solid support.

• – Nukleinsäuren, insbesondere Oligonukleinsäuren, z. B. einzel- und/oder doppelsträngige, lineare, verzweigte oder circuläre DNA, cDNA, RNA, PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid), PSNA (Phosphothioate Nucleic Acid);
• – Antikörper, insbesondere humane, tierische, polyklonale, monoklonale, rekombinante, Antikörperfragmente, z. B. Fab, Fab', F(ab)₂, synthetische;
• – Proteine, z. B. Allergene, Inhibitoren, Rezeptoren;
• – Enzyme, z. B. Peroxidasen, Alkalische-Phosphatasen, Glukose-Oxidase, Nukleasen;
• – kleine Moleküle (Haptene), z. B. Pestizide, Hormone, Aminosäuren, Antibiotika, Pharmaka, Farbstoffe, synthetische Rezeptoren, Rezeptorliganden.

The term "biomolecules" refers to any biochemical and biological substances, both as single molecules and as multiple interacting molecules. For example:

• - Nucleic acids, in particular oligonucleic acids, eg. Single and / or double-stranded, linear, branched or circular DNA, cDNA, RNA, PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid), PSNA (Phosphothioate Nucleic Acid);
• - Antibodies, in particular human, animal, polyclonal, monoclonal, recombinant, antibody fragments, z. Fab, Fab ', F (ab) ₂ , synthetic;
• Proteins, e.g. B. allergens, inhibitors, receptors;
• - enzymes, eg. Peroxidases, alkaline phosphatases, glucose oxidase, nucleases;
• Small molecules (haptens), e.g. Pesticides, hormones, amino acids, antibiotics, drugs, dyes, synthetic receptors, receptor ligands.

According to a further aspect, the term "biomolecule" defines the ability of a substance to be able to enter into a specific analytical interaction with a biological sample or a part thereof, in particular the analyte.

In a particular aspect, a particular type of biomolecule is designated as a ligand. Ligands interact with and, in particular, bind to specific targets, preferably specifically.

In the arrays according to the invention, the biomolecules are immobilized on defined sites P of a surface. These places P have a certain shape and extent. The shape of the places P is basically arbitrary. Preferably, the shape of the places P is uniform and has, for example, a polygonal, z. B. an n-sided with n = 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and preferably a rectangular with n = 3, 4, 5, 6, 7, 8, 9th , 10, 11 or 12, and of these, preferably a rectangular geometry, a circular or an ellipsoidal geometry. Mixed forms of these geometries are fundamentally suitable, for. B. stretched ovals. Preferably, the places P have a circular or ellipsoidal geometry. The spatial extent of these places P naturally correlates with the distance of the boundary line of a respective place to its center of area. This distance is on average usually in the range of 50 nm to 50 .mu.m, preferably 100 nm to 25 .mu.m, in particular 0.25 .mu.m to 20 .mu.m and especially 0.5 .mu.m to 10 .mu.m.

The distance between adjacent places P, defined by the spacing of the surface centers of adjacent places P, is generally in the range of in the range of 200 nm to 200 μm, frequently in the range of 1 μm to 150 μm, in particular in the range of 5 μm to 100 μm and especially in the range of 10 microns to 90 microns. The mean minimum distance between adjacent places P, defined by the minimum distance which the boundary lines of two adjacent places P have, is generally in the range of 100 nm to 150 μm, frequently in the range of 500 nm to 120 μm, in particular in the range of 2 μm to 100 μm, and especially in the range of 5 μm to 80 μm (number average).

The arrangement of the places P on the surface may be regular or irregular. Preference is given to regular arrangements, i. H. Arrangements in which the average distance, the two adjacent places P have, not more than 20% and especially not more than 10% varies, or deviates from the mean.

According to the invention, the positions P of the array can be assigned to different surface areas B. The surface regions B arranged on the microbioarrays according to the invention generally have a uniform geometry, preferably an ellipsoidal or circular geometry. Their extent naturally correlates with the distance of the boundary line of a respective area B to its center of area. This distance is on average usually in the range of 10 .mu.m to 250 .mu.m, preferably in the range of 25 .mu.m to 200 .mu.m and in particular in the range of 30 .mu.m to 150 .mu.m. For circular surface areas B, this corresponds to half the diameter of the respective surface area. The respective surface areas B usually form a uniform arrangement on the surface of the array. The mean distance between adjacent regions B, defined by the mean distance between the surface centers of adjacent regions, will generally not fall below a value of 50 .mu.m, in particular 100 .mu.m and especially 200 .mu.m. The minimum distance between adjacent areas B, defined by the minimum distance that the boundary lines of two adjacent areas have, will not be less than 5 μm on average, in particular 10 μm and especially 50 μm.

The microbioarrays according to the invention have in the respective surface areas B in each case a plurality of at least 10, preferably at least 50 and in particular at least 100 and up to 10,000, preferably up to 5000 and in particular up to 1000 spatially separated sites P, on which the biomolecules are immobilized.

According to the invention, the biomolecules in the microarrays are immobilized on a surface, i. H. fixedly bound. The binding can be via covalent interactions, coordinative interactions, ionic or electrostatic interactions, hydrogen bonds, hydrophobic interactions and mixed forms of the aforementioned interactions.

The binding can take place both directly to the surface as well as via a compound V mediating the binding of the biomolecule. These substances V are usually compounds which are covalently bonded to the surface on which the biomolecules are to be immobilized, and which can simultaneously bind to the biomolecule, for example by covalent interaction, ionic interaction, coordinative interaction, Hydrogen bonds, hydrophobic interactions and mixed forms of such interactions, as for example in the base pairing of oligonucleotides, in the protein-protein interaction, antibody-antigen binding and in the protein-ligand interaction occur (affinitive interactions).

In a preferred embodiment of the invention, the attachment takes place via covalent interactions. The biomolecules are then bound to the biomolecules via ester, amide, sulfonamide, imino, amidino, urethane, urea, thiourethane, thiourea, sulfide, sulfonyl, ether or amino groups, for example.

In another embodiment of the invention, the biomolecules are bound via a metal atom mediated coordinate bond. In these cases, the metal atom is attached to the surface via functional groups which can form a coordinative bond with the metal atom, for example carboxyl groups, hydroxyl groups, amino groups, SH groups, oxime groups, aldehyde groups, keto groups or heterocyclic groups with an imino nitrogen such as in pyridine, quinoline, imidazole or oxazole. Preferably, the metal atoms are over at least 2, e.g. B. 2, 3 or 4 such groups bound as a chelate. Examples of suitable metals are in particular transition metals, especially transition metals of the third period, which can form divalent ions in an aqueous medium, for example copper (II), nickel (II), zinc (II), cobalt (II) and iron (II). The biomolecules then bind to these metals via one, preferably two or three spatially adjacent functional groups of the aforementioned type.

In a third embodiment, the binding of the biomolecules takes place via coordinating hydrogen-bond bonds, as occur, for example, in the base pairing of nucleotide sequences. In this embodiment, short oligo or polynucleotide sequences are generally at least two, e.g. B. 2 to 100, in particular 2 to 20 bases bonded via preferably covalent bonds to the surface. Accordingly, the biomolecule will have an oligonucleotide or polynucleotide sequence at least partially complementary thereto, at least 2, preferably at least 10, adjacent bases being complementary to a corresponding number of adjacent bases.

In a fourth embodiment of the invention, the biomolecules via affinitive interactions, eg. For example, protein-protein interactions, such as occur in the binding of antigens to antibodies, or protein-ligand interactions, for example via a biotin-streptavidin or biotin-avidin system bound.

As surfaces for the arrays according to the invention, basically all surfaces can be considered on which biomolecules can be immobilized without causing deactivation of the biomolecules. These surface materials may be the surface of a support material or a modified surface of a support material. The carrier materials can be rigid or flexible. Typical support materials include oxidic support materials such as glass, quartz, ceramics, semimetals such as silicon and germanium, common semiconductor materials, eg. As doped silicon or doped germanium, metals and metal alloys, especially based on gold, polymers, eg. As polyvinyl chloride, polyolefins such as polyethylene, polypropylene, polymethylpenthene, polyesters, fluoropolymers such as Teflon, polyamides, polyurethanes, polyalkyl (meth) acrylates, polystyrene, blends and composites of the aforementioned materials and the like. Preference is given to materials whose surface has a low roughness. Preferably, the roughness characterizing R _a value is not more than 100 nm and in particular not more than 50 nm. Preferred support materials are glass and silicon wafers.

In general, it is recommended that the surfaces of inert support materials, eg. Glass, ceramics, metal, metalloid such as silicon, polyolefin such as polyethylene or polypropylene, d. H. in a way to treat them having a plurality of functional groups R capable of reacting with complementary reactive groups R 'to form a chemical bond, preferably a covalent chemical bond. These functional groups R allow the biomolecules to be directly linked via a chemical bond or indirectly, i. H. by means of one of the above-defined substances V, which is covalently bonded to the surface via such groups to immobilize on the surface.

Reactive groups R in the context of this invention are those which react with nucleophiles in an addition reaction including a Michael reaction or in a substitution reaction, e.g. As isocyanate groups, (meth) acrylic groups, vinylsulfone groups, oxirane groups, aldehyde groups, oxazoline groups, carboxylic acid groups, Carbonsäureester- and carboxylic anhydride groups, carboxylic acid and sulfonic acid halide groups, but also the complementary, reacting as a nucleophile groups such as alcoholic OH groups , primary and secondary amino groups, thiol groups and the like. Examples of carboxylic ester groups are in particular so-called active ester groups of the formula -C (O) OX in which X is pentafluorophenyl, pyrrolidine-2,5-dione-1-yl, benzo-1,2,3-triazol-1-yl or a carboxamidine residue, and NHS esters (N-hydroxy-succinimide ester). Reactive groups within the meaning of the invention are also nucleophilic groups which react with the aforementioned electophilic groups to form bonds, e.g. As NH ₂ , SH, or OH, but especially NH ₂ . It goes without saying that a surface according to the invention either predominantly electrophilic groups or predominantly nucleophilic groups, wherein in the case of NCO / NH ₂ , both groups may be present side by side.

An overview of reactive groups R and functional groups R complementary thereto is given in Table 1, the reactive groups R in the first row and the complementary groups R 'in the first column being given: TABLE 1 Complementary Functional Groups Reactive group ^R Complementary group R ' isocyanate (Meth) acrylic / vinyl sulfone SH -NH ₂ -OH CHO oxirane isocyanate X * X X X (Meth) acrylate X X X vinylsulfone X X X SH X X X X -NH ₂ X X X X -OH X X X COOH X oxirane X X X Aktivester / NHS X X CHO X X * In the presence of water

Also suitable as reactive groups R are free-radically polymerizable C =C double bonds, eg. B. in addition to the above-mentioned (meth) acrylic groups and vinyl ether and vinyl ester groups, further activated C = C double bonds, activated C = C triple bond and N = N double bonds with allyl groups in terms of an ene reaction or with conjugated diolefin Groups react in the sense of a Diels-Alder reaction. Examples of groups which can react with allyl groups in the sense of an ene reaction or with dienes in the sense of a Diels-Alder reaction are maleic acid and fumaric acid groups, maleic acid ester and fumaric acid ester groups, cinnamic acid ester groups, propiolic acid (ester) groups, Maleic acid amide and fumaric acid amide groups, maleimide groups, azodicarboxylic ester groups and 1,3,4-triazoline-2,5-dione groups.

The groups R in a broader sense also include groups acting as ligands, the metal ions, in particular of transition metals and especially of transition metals of the third period, which can form divalent ions in an aqueous medium, such as copper (II), nickel (II), zinc (II), cobalt (II) and iron (II), which in turn mediate coordinate binding to the biomolecule. Examples of groups acting as ligands include carboxyl groups, hydroxyl groups, amino groups, SH groups, oxime groups, aldehyde groups, keto groups and heterocyclic groups having an imino nitrogen, such as in pyridine, quinoline, imidazole or oxazole. Preferably, the group acting as ligands on the surface are arranged so that the metal atoms over at least 2, z. B. 2, 3 or 4 such groups are bound as a chelate.

In a preferred embodiment, the organic surface layer has on its surface such functional groups which are amenable to addition or substitution reaction by nucleophiles. Preferred R groups are isocyanate, isothiocyanate, aldehyde, active ester, acrylic and methacrylic groups such that the surface-active biomolecules have one of the following functional groups selected from amide groups, urethane groups, urea groups, thiourethane groups, ester groups, imino groups, thiourea groups or sulfoethyl groups and optionally via a spacer.

In a specific embodiment of the invention, the binding of the biomolecules or of the substance V takes place via isocyanate groups present on the surface layer to form urethane or urea groups.

In another particularly preferred embodiment of the invention, the surface has a multiplicity of nucleophilic groups, in particular NH ₂ groups, such that the biomolecules used for the immobilization or the substance V is a group which is complementary to this, for example an isocyanate group, an ester group , z. B. have an active or NHS ester group.

To produce surfaces having a plurality of reactive groups R, it is possible, for example, to activate the surfaces of inert support materials by treatment with acids or alkalis. Activation may also be by oxidation (flame-off), by electron beam irradiation, or by plasma treatment with an oxygen-containing plasma as described by P. Chevallier et al. J. Phys. Chem. B 2001, 105 (50), 12490-12497; in JP 09302118 A2 ; in DE 10011275 ; or by D. Klee. et al. Adv. Polym. Sci. 1999, 149, 1-57. The activation of the surface can also be carried out by means of plasma treatment with an NH ₃ -containing plasma, as described in US Pat US 6,017,577 . 6,040,058 and 6,080,488 is described. Plasma-modified, amino-produced PE films are also commercially available.

Frequently, activating the surface also includes applying an organic coating having on its surface a plurality of the above-defined functional groups R. This coating is also referred to below as an adhesive layer. For this purpose, bi- or polyfunctional organic compounds or polymers are applied to the surface, which has at least one functional group which react with the functional groups on the surface of the support material to form bonds (covalent, ionic and / or coordinative) and the at least one additional additional functional group R of the type described above. These substances can be applied to the carrier surface as thin or ultrathin layers, in particular as monomolecular or multimolecular layers, or produced on the surface of the carrier material by means of chemical vapor deposition (CVD). According to the invention, very thin adhesive layers with a thickness of preferably <100 nm, in particular <10 nm and especially monomolecular layers, in particular self-assembling monolayers (SAM), for example of thiol compounds which have at least one further group R, are preferred. especially for the treatment of noble metal surfaces, of silane compounds which have at least one group R, in particular for the production of monolayers on silicon or glass surfaces, furthermore macromolecular monolayers obtained by grafting polymers onto the surface or by graft polymerization on the surface and their polymers have suitable functional groups R.

In a preferred embodiment of the invention, compounds which have silane groups, in particular trialkoxysilane groups, as bonding-promoting groups are used to produce the adhesive layer. Examples of such compounds are trialkoxyaminoalkylsilanes such as triethoxyaminopropylsilane and N [(3-triethoxysilyl) propyl] ethylenediamine, trialkoxyalkyl-3-glycidyl ether silanes such as triethoxypropyl-3-glycidyl ether silane, trialkoxyalkyl mercaptans such as triethoxypropyl mercaptan, trialkoxyallylsilanes such as allyltrimethoxysilane, trialkoxy (isocyanatoalkyl) silanes and trialkoxysilyl (meth) acryloxyalkanes and - (meth) acrylamidoalkanes such as 1-triethoxysilyl-3-acryloxypropane. These compounds are applied in very thin layers, in particular as monolayer on the surface, preferably after previous activation. Methods for this purpose are known from the cited prior art.

Furthermore, for the production of adhesive layers and polyammonium compound with free primary amine groups, as z. By J. Scheerder, J.F. J Engbersen, and D.N. Reinhoudt, Red. Trav. Chim. Pays-Bas 1996, 115 (6), 307-320, and by Decher. Science 1997, 277, 1232-1237 to be described for this purpose. Also suitable are polyfunctional polymers which are absorbed by the chain backbone on the carrier surface and which have further functional groups R. Particular mention should be made here of monomolecular coatings of polylysine and / or polyethyleneimine.

The adhesive layer is preferably applied to the carrier, in particular in the case of inert carrier materials, after activation by treatment with acids or alkalis, by oxidation (flaming), by electron irradiation or by a plasma treatment in the manner described above.

The activation of the surface can also be effected in such a way that, instead of an adhesive layer or preferably on the adhesive layer or on another activated surface, a further coating is applied which is particularly suitable for immobilizing the biomolecules and such groups instead of the reactive groups R. which allows binding of the biomolecules via coordinative bonds, via hydrogen bonds, via hydrophobic interactions or via affinitive bonds. For this purpose, one will generate on the surface of a preferably monomolecular layer of the above compounds V, z. As layers of chelating agents, low molecular weight ligands for protein sequences such as biotin, from proteins such as avidin or streptavidin, or from oligonucleotides. It goes without saying that you will make such an activation only when the production of the arrays according to variant 1 takes place.

Support materials which have such an organic surface layer (adhesive layer) are known in principle, for example from the cited prior art and largely commercially available, for example from the company Bioslide Technologies, Walnut CA, USA (NH ₂ -functionalized support, CHO-functionalized Supports, epoxy-functionalized supports, polylysine-coated supports), from Xenopor Corp., Hawthorne, NJ, USA (NH ₂ -functionalized supports, aldehyde-functionalized supports, epoxide-functionalized supports, maliimide-functionalized supports, nickel chelate Carrier, streptavidin-functionalized carriers, biotinylated carriers, thiol-modified carriers), from Greiner Bio-One GmbH, (amino-functionalized carriers, aldehyde-functionalized carriers, streptavidin carriers), Xan Tec Bioanalytics GmbH (with polysaccharide hydrogels coated supports, streptavidin-functionalized supports, biotin-functionalized supports er, ultrahydrophobic alkyl layers, carboxyl-functionalized supports, hydrazone-functionalized supports, thiol-functionalized supports) Amersham Biosciences UK Ltd., England (polymeric amino-modified surface), Schott Glas, Mainz, Germany (multi-functionalized aminosilane coatings, Nexterion ^® carrier), company Sunergia Group Inc. (amino-modified support, isothiocyanate-modified support).

In a preferred embodiment of the invention, the biomolecules are bound to a hydrogel-forming surface layer. This surface layer should naturally be very thin, preferably in the dry state have a thickness ≤ 50 microns, especially ≤ 10 microns and especially ≤ 1 micron, more preferably ≤ 500 nm and especially ≤ 100 nm, so as not to adversely affect the function of the biochip. As a rule, this hydrogel-forming layer will have an average thickness of at least 0.5 nm, preferably at least 1 nm and especially at least 5 nm. A hydrogel-forming layer is understood to mean polymeric layers which swell on exposure to moisture by intercalation of water.

The hydrogel-forming coatings can be applied directly to the surface of the support, e.g. Example, be activated by treatment with acids or alkalis, by oxidation (flaming), by electron beam irradiation or by a plasma treatment surface, or to the above-described adhesive layer. Hydrogel-forming coatings are naturally preferred which have on their surface a large number of free functional groups R as described above. Carriers with hydrogel coatings are well known in the art and are also commercially available for the purposes of biochip production, for example, from Perkin-Elmer, Life and Analytical Sciences, Boston, MA, USA (polyacrylamide hydrogel coated carriers having hydrogel ^® coated Slides) and XanTec Bioanalytics GmbH (coated with polysaccharide hydrogels carrier, the free at its surface carboxyl, amino, hydrazino, SH, streptavidin or biotin groups.

In a particularly preferred embodiment, the hydrogel layer is composed of mutually crosslinked star-shaped prepolymers, wherein the star-shaped prepolymers have on average at least 4, on the shelf 4 to 12, preferably 5 to 10 and especially 6 to 8 polymer arms A, which by themselves are water soluble. Such coatings are known in principle, for example from the German patent applications 10203937.2 and 10216639.0 , the disclosure of which is hereby incorporated by reference. These surfaces are characterized by a particularly low affinity for non-specific binding of biomolecules, especially proteins and cells. In addition, these surfaces have a plurality of functional groups R, in particular the groups mentioned in Table 1, which can be used in the manner described above for immobilizing the biomolecules. In addition, the hydrogel-forming coatings described therein are extremely stable to aging and mechanical stress.

Star-shaped prepolymers are understood as meaning those polymers which have a plurality of polymer chains bonded to a low molecular weight central unit, the low molecular weight central unit generally having from 4 to 100 skeletal atoms such as C atoms, N atoms or O atoms.

The number-average molecular weight of the polymer arms is generally in the range from 200 to 20,000 daltons, preferably in the range from 300 to 10,000 daltons, in particular in the range from 400 to 8,000 daltons and especially in the range from 500 to 5,000 daltons. Accordingly, the star-shaped prepolymer has a number-average molecular weight in the range of generally at least 1500 daltons, preferably 2000 to 100,000 daltons, in particular 2500 to 50,000 daltons and especially 3000 to 30,000 daltons. The hydrogel-forming properties of the surface layer are ensured by the water solubility of the polymer arms. It is generally ensured if the molecular structure, ie at least the type of repeating units, preferably also the molecular weight of the polymer arm, corresponds to a polymer whose solubility in water is at least 1% by weight and preferably at least 5% by weight ( at 25 ° C and 1 bar). Examples of such polymers with sufficient water solubility are poly-C ₂ -C ₄ -alkylene oxides, polyoxazolines, polyvinyl alcohols, homopolymers and copolymers which contain at least 50 wt .-% of N-vinylpyrrolidone in copolymerized form, homopolymers and copolymers containing at least 30 wt. % Hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, acrylamide, methacrylamide, acrylic acid and / or methacrylic acid in copolymerized form, hydroxylated polydienes and the like. The polymer arms A are preferably derived from poly-C ₂ -C ₄ -alkylene oxides and are in particular selected from polyethylene oxide, polypropylene oxide and polyethylene oxide / polypropylene oxide copolymers, which may have a block or a random arrangement of the repeat units. Particular preference is given to star-shaped prepolymers whose polymer arms A are derived from polyethylene oxides or from polyethylene oxide / polypropylene oxide copolymers having a propylene oxide content of not more than 50%.

In order to ensure a crosslinkability of the prepolymers, they have functional groups at the ends of their polymer arms, which react with complementary functional groups to form covalent bonds. Examples of such groups are those in Table 1 under R bwz. R 'mentioned functional groups.

For the preparation of hydrogel-forming coatings, which are composed of interconnected star-shaped prepolymers, in particular those prepolymers have proven that have at the ends of their polymer arms isocyanate groups and particularly preferably isocyanate end groups derived from aliphatic diisocyanates, in particular those such obtained by addition of isophorone diisocyanate (IPDI) to the chain ends of OH group-terminated star prepolymer precursors.

The inventively used for the prepolymers are z. T. known, for. B. from the WO 98/20060 . US 6,162,862 , Gotz et al., Macromol. Mater. Closely. 2002, 287, p. 223, Bartelink et al. J. Polymer Science 2000, 38, p. 2555, DE 10216639.0 and DE 10203937 (star-shaped polyether prepolymers), Chujo Y. et al., Polym. J. 1992, 24 (11), 1301-1306 (star-shaped polyoxazoline prepolymers), WO 01/55360 (star-shaped polyvinyl alcohols, star-shaped, vinylpyrrolidone-containing copolymers) or can be prepared by the methods described therein.

The provision of the organic surface, which is essentially composed of crosslinked star-shaped prepolymers, therefore usually comprises the deposition of the star-shaped prepolymers on the surface of a support and subsequent crosslinking of the reactive groups of the star-shaped prepolymers. If desired, the steps of deposition and crosslinking may be repeated. You get in this way to thicker layers.

• i.a. Abscheiden des sternförmigen Präpolymeren auf der Oberfläche eines Trägers durch Aufbringen einer Lösung wenigstens eines sternförmigen Präpolymeren, das im Mittel wenigstens vier Polymerarme A aufweist, die für sich gesehen in Wasser löslich sind und an ihren freien Enden eine reaktive funktionelle Gruppe tragen, auf die zu Oberfläche des Trägers;
• i.b. anschließend Durchführen einer Verknüpfungsreaktion der reaktiven Gruppen untereinander und Entfernen des Lösungsmittels; und
• i.c. gegegebenenfalls Umwandeln der funktionellen Gruppen auf der Oberfläche der Oberflächenschicht.

In general, the method of depositing the star-shaped prepolymers comprises the following steps:

• ia depositing the star-shaped prepolymer on the surface of a carrier by applying a solution of at least one star-shaped prepolymer having on average at least four polymer arms A, which are individually soluble in water and carry at their free ends a reactive functional group to which Surface of the carrier;
• ib subsequently performing a linking reaction of the reactive groups with each other and removing the solvent; and
• optionally, converting the functional groups on the surface of the surface layer.

In step ia, a coating is obtained which is composed of uncrosslinked prepolymers and which has a multiplicity of functional groups on its surface. In this coating, a linking reaction of the reactive groups is carried out with each other, wherein the linking reaction after application and removal of the solvent can be carried out by subsequent treatment of the coating with a crosslinker or removing the solvent, if the solution already contains a suitable crosslinker and / or the functional groups of the prepolymer undergo a chemical reaction with each other under binding formation.

Examples of deposition methods are the immersion of the surface to be coated in the solution of the prepolymer and the spin coating - in this case the solution of the prepolymer is applied to the rotating at high speed surface to be coated. It goes without saying that the coating measures are usually carried out under dust-free conditions for the production of ultra-thin coatings.

In the immersion process, the substrates are immersed in a solution of the star-shaped prepolymer in a suitable solvent, and the solution is then allowed to run off so that a thin liquid film with as uniform a thickness as possible remains on the substrate. This is then dried. The resulting film thickness depends on the concentration of the prepolymer in the solution. Subsequently, the networking is triggered.

In spin coating, the initially non-rotating substrate is generally completely wetted with the solution of the star-shaped prepolymer. Subsequently, the substrate to be coated with high rotational speeds, usually at least 50 U / min, often at least 500 U / min, z. B. 500 to 30000 U / min, preferably above 1000 U / min, z. B. 1000 to 10000 U / min, and more preferably 3000 to 8000 U / min, set in rotation, wherein the solution is largely thrown off and a thin coating film remains on the surface of the substrate. Subsequently, a networking is triggered here.

The concentration is usually at least 0.001 mg / ml, preferably at least 0.01 mg / ml, in particular at least 0.1 mg / ml, particularly preferably at least 1 mg / ml. The concentration of the prepolymer in the solution will usually not exceed a value of 500 mg / ml, preferably 250 mg / ml and especially 100 mg / ml. Naturally, the concentration of the coating can be controlled via the concentration.

To prepare the solutions of the prepolymers, all solvents which have little or no reactivity with respect to the functional groups of the prepolymer to be dissolved are fundamentally suitable. Of these, those are preferred which have a high vapor pressure and can thus be easily removed. Preference is therefore given to those solvents which have a boiling point below 150.degree. C. and preferably below 120.degree. C. under normal pressure. Examples of suitable solvents are aprotic solvents, e.g. As ethers such as tetrahydrofuran (THF), dioxane, diethyl ether, tert-butyl methyl ether, aromatic hydrocarbons such as xylenes and toluene, acetonitrile, propionitrile and mixtures of these solvents. In the case of prepolymers having OH, SH, carboxyl, (meth) acrylic and oxirane groups and protic solvents such as water or alcohols, eg. For example, methanol, ethanol, n-propanol, isopropanol, n-butanol and tert-butanol, and mixtures thereof with aprotic solvents suitable. In the case of prepolymers with isocyanate groups, water and mixtures of water with aprotic solvents are surprisingly suitable in addition to the abovementioned aprotic solvents, since the degradation of the isocyanate groups of the prepolymers in such solutions takes place comparatively slowly.

The crosslinking of the prepolymers can take place in different ways. For example, the prepolymers may react with a crosslinking agent. As crosslinking agents, basically all compounds having 2 or more functional groups are suitable, which react with the functional groups of the prepolymer to form bonds.

Accordingly, according to a first embodiment of the invention, the provision of the organic surface by a process in which the linkage of the reactive groups of the prepolymer by the addition of a compound Vm1 triggers having at least two complementary reactive groups per molecule with the reactive groups of the star shaped prepolymer to form bonds.

The polyfunctional compounds Vm1 may be low molecular weight compounds, for. B. aliphatic or cycloaliphatic diols, triols and tetraols, z. As ethylene glycol, butanediol, diethylene glycol, triethylene glycol, trimethylolpropane, pentaerythritol and the like, aliphatic or cycloaliphatic diamines, triamines or tetramines, e.g. Ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, 1,8-diamino-3,6-dioxaoctane, diaminocyclohexane, isophoronediamine and the like, aminoalcohols such as ethanolamine, diethanolamine, aliphatic or cycloaliphatic dithiols, dicarboxylic acids or tricarboxylic acids such as sebacic acid, glutaric acid, adipic acid, Phthalic acid, isophthalic acid, or the aforementioned diisocyanates, depending on which type of reactive groups the prepolymer has. In contrast to the prepolymers, the low molecular weight polyfunctional compounds generally have a molecular weight <500 g / mol.

The polyfunctional compound Vm1 may already be contained in the solution of the prepolymer. In the initially formed surface layer of largely uncrosslinked prepolymers then react, for. As drying or heating of the layer, the reactive groups of the crosslinking agent with the reactive groups of the prepolymer and thus form a layer of crosslinked prepolymers, which usually has a plurality of functional groups on its surface.

In principle, prepolymers which have at least four polymer arms A which are inherently soluble in water and have a reactive functional group at their free ends which react with the reactive groups of the prepolymer to form bonds are also suitable as polyfunctional compounds Vm1. In other words, in order to provide the hydrogel coating, it is also possible according to the invention to use solutions of at least two different prepolymers, wherein the one prepolymer has reactive groups which have a complementary reactivity to the reactive groups of the other prepolymer.

In another embodiment of the invention, the linking of the reactive groups in the prepolymer is initiated by adding a sufficient amount of a compound Vm2 which reacts with a portion of the reactive groups of the prepolymer to form functional groups having a reactivity complementary thereto. In the case of prepolymers with isocyanate groups, for example, the crosslinking can be initiated by treating the coated article with water, eg. B. by storage in a humid atmosphere or under water. In this case, a part of the isocyanate groups react to form amino groups, which in turn react with the remaining isocyanate groups to form bonds, resulting in a layer of cross-linked prepolymers. The crosslinking agent Vm2 is thus here water. Thus prepared coatings, when freshly prepared, still have free isocyanate groups. Upon prolonged exposure to moisture, the surface isocyanate groups are converted to amino groups. In a variant of this embodiment, solutions of the prepolymer in water or in a mixture of water with one or more water-miscible solvents. Examples of suitable water-miscible solvents are, in particular, those which do not react with the isocyanate groups or react only much more slowly than water. Examples include cyclic ethers such as tetrahydrofuran and dioxane, furthermore N-alkylamides such as N-methylpyrrolidone, dimethylformamide and dimethylacetamide. The mixing ratio of water: solvent is generally in the range from 1: 100 to 100: 1, preferably in the range from 50: 1 to 1:10 and especially in the range from 20: 1 to 1: 1. This variant is particularly suitable for producing thicker layers. The concentration of prepolymer is then preferably in the range of 0.1 to 500 mg / ml, in particular in the range of 0.5 to 200 mg / ml and especially in the range of 1 to 100 mg / ml.

The hydrogel layer composed of crosslinked, star-shaped prepolymers is preferably provided on a support which is activated and / or provided with an adhesive layer.

• – Der mittlere Abstand der Begrenzungslinie der Stirnfläche einer Säule zum Flächenmittelpunkt der Stirnfläche beträgt im Mittel üblicherweise etwa 50 nm bis 50 μm, vorzugsweise 100 nm bis 25 μm, insbesondere 0,25 μm bis 20 μm und speziell 0,5 μm bis 10 μm. Dies entspricht bei den bevorzugten zylindrischen Säulenformen einen mittleren Durchmesser der Säulen im Bereich von 100 nm bis 100 μm, vorzugsweise 200 nm bis 50 μm, insbesondere 0,5 μm bis 40 μm und speziell 1 μm bis 20 μm.
• – Der mittlere Abstand benachbarter Säulen, definiert durch den mittleren Abstand der Flächenmittelpunkte der Stirnflächen benachbarter Säulen liegt in der Regel im Bereich von 200 nm bis 200 μm, häufig im Bereich von 1 μm bis 150 μm, insbesondere im Bereich von 5 μm bis 100 μm und speziell im Bereich von 10 μm bis 90 μm.
• – Der mittlere minimale Abstand zweier benachbarter Säulen liegt in der Regel im Bereich von 100 nm bis 150 μm, häufig im Bereich von 500 nm bis 120 μm, insbesondere im Bereich von 2 μm bis 100 μm, und speziell im Bereich von 5 μm bis 80 μm (Zahlenmittel). Vorzugsweise ist der mittlere Abstand zweier benachbarter Säulen, definiert durch den Abstand der Flächenmittelpunkte der Stirnmflächen der Säulen, wenigstens doppelt so groß wie der mittlere Abstand der Begrenzungslinie der Stirnfläche einer Säule zu dem Flächenmittelpunkt der Stirnfläche.

As already explained, the microbioarrays according to the invention can be produced by a combination of microcontact printing (μCP) and spotting technique. The arrangement and size of the areas is determined by the spotting technique. The arrangement of the places P within these areas B results from the type of stamp used. Accordingly, the stamp has on its surface a plurality of spatially separated raised areas, z. B. a plurality of band-shaped or in particular columnar elevations, which correspond in terms of their dimension and arrangement of the raised areas of the desired arrangement and the desired dimensions of the places P. In particular, punches with a large number of columnar elevations are preferred. The columns and their arrangement are characterized in particular by the following dimensions:

• The average distance between the boundary line of the end face of a column and the center of area of the end face is on average usually about 50 nm to 50 μm, preferably 100 nm to 25 μm, in particular 0.25 μm to 20 μm and especially 0.5 μm to 10 μm. In the preferred cylindrical column shapes, this corresponds to an average diameter of the columns in the range of 100 nm to 100 μm, preferably 200 nm to 50 μm, in particular 0.5 μm to 40 μm and especially 1 μm to 20 μm.
• The average distance between adjacent columns, defined by the mean distance between the center areas of the faces of adjacent columns, is generally in the range from 200 nm to 200 μm, frequently in the range from 1 μm to 150 μm, in particular in the range from 5 μm to 100 μm and especially in the range of 10 microns to 90 microns.
• The average minimum distance between two adjacent columns is generally in the range of 100 nm to 150 μm, frequently in the range of 500 nm to 120 μm, in particular in the range of 2 μm to 100 μm, and especially in the range of 5 μm to 80 μm μm (number average). Preferably, the average distance between two adjacent columns, defined by the distance between the surface centers of the front surfaces of the columns, at least twice as large as the average distance of the boundary line of the end face of a column to the surface center of the end face.

The end faces of the columns correspond to the geometry of the places P and are in particular circular or ellipsoidal and particularly preferably circular.

According to a preferred embodiment of the invention, the columns are regularly arranged on the stamp surface, i. H. the individual spacing of adjacent columns will on average not deviate more than 20% and preferably not more than 10% from the average spacing of the columns.

In the case of band-shaped elevations, the distances of the bands to one another, which were stated for the distances between adjacent columns, apply to one another. The average width of the band-shaped elevations is generally in the range of 100 nm to 100 .mu.m, preferably 200 nm to 50 .mu.m, in particular 1 .mu.m to 30 .mu.m and especially 2 .mu.m to 20 .mu.m.

The height of the elevations over the stamp base area is generally in the range of 0.5 to 500 .mu.m, in particular in the range of 0.5 to 50 .mu.m and especially in the range of 1 to 10 microns. It is of minor importance for the process according to the invention. In order to ensure a sufficient stability of the columns, the ratio of height H to the distance A between the boundary lines of the end face of a column and its center of area H: A is not more than 10, in particular not more than 5. D. h. a cylindrical column with a diameter of about 1 micron will not exceed a height of 5 microns and especially 2.5 microns. The same applies to the ratio of the width of a band-shaped elevation to its height over the stamp base area.

Preferably, the columnar elevations of a stamp are formed by an elastic material in order to ensure a sufficiently good transmission. As a rule, the elastic material is a polymeric elastomer, for example a crosslinked polydimethylsiloxane. Such punches are known from the cited prior art or can be prepared by the methods specified therein (see in particular Kane et al., Biomaterials 1999, 20, pp. 2363-2376 and references cited therein, Xia et al., Chem. Rev. 1999, 99, 1823-1848 and references cited therein, Michel et al., IBM J. Res. & Dev. 45, 2001, 697-718, and literature cited therein, Tan et al., Langmuir 2002, 18, p 519-523, and literature cited therein). In general, such stamps are made by molding a master having a plurality of wells, which correspond to the later surveys of the stamp. For this purpose, as a rule, a mixture of a suitable elastomer precursor with a crosslinker is applied to a structured casting mold which constitutes a negative of the stamp surface (master, usually a silicon wafer) and cured. After curing, an elastic stamp is obtained whose stamp surface represents the positive image of the master. Such methods are extensively described in the prior art cited herein, to which reference is made for further details. The surface of the stamp may also be provided with a hydrophilic surface layer. Such modified stamps are described, for example, by Donzel et al., Adv. Mater. 2001, 13, p. 1164-1167. The preparation of this master is carried out according to known methods, for example by photolithographic methods, as described in detail in the cited prior art and the literature cited therein.

The thickness of the stamp and its surface dimensions are of minor importance and depends primarily on the dimensions of the array to be produced. If the stamp material is an elastomer, it has proved advantageous if the stamp surface is arranged on a rigid or flexible support, for example on a metal foil. Usually, the elevations and the stamp surface of the same, preferably elastomeric material. However, the thickness of the material forming the stamp surface is preferably at least 1 μm, in particular at least 0.1 mm to about 1 cm.

According to variant 1 of the method according to the invention, one or preferably several different biomolecules are brought into at least 2, preferably at least 10, in particular at least 50 and particularly preferably at least 100, eg. B. 100 to 10,000 and in particular 100 to 10,000 preferably to 5000 and especially to 1000 spatially separated surface areas so that the biomolecules in each area each have a plurality, preferably at least 10, in particular at least 50 and especially at least 100, z. B. cover 100 to 1000 of the elevations of the stamp surface. The application of the biomolecules can be carried out in a conventional manner by means of conventional spotting techniques sequentially or in particular simultaneously. The apparatuses required for this purpose are the dot matrix printers, dispensers, platters, micropipettes and the like customary for the spotting techniques, with which the liquid drops can be applied to a surface by a contact method or a contact-free method (contact mode, non-contact Fashion).

In a first embodiment of variant 1, the biomolecules are applied as a solution directly onto the stamp surface. When applying the solutions of the biomolecules on the stamp surface, it has proven to be advantageous to avoid premature evaporation of the solvent. Suitable methods for this are the cooling of the stamp and / or the saturation of the atmosphere with the respective solvent. When cooling the stamps are usually strongly cooled before spotting and then subjected to the spotting process. In the saturation method, the working chamber is saturated with the solvent in which the biomolecule is dissolved. Preferably, the spotting conditions are adjusted so that the solvents immediately evaporate when applied to the die surface. The specialist can easily determine the conditions required for this with a few routine experiments. Preferably, the spot process is carried out at temperatures in the range of 5 to 50 ° C and in particular at about room temperature in a partially saturated with solvent atmosphere, d. H. an atmosphere containing vaporized solvent, but in an amount below its saturation concentration, preferably in the range of 10 to 80% of the saturation concentration.

For a clean transfer of the biomolecules on the surface, it has proven to be advantageous to dry the stamp surface after wetting with the solution of the biomolecule. Furthermore, it has proven to be advantageous if the concentration of the biomolecules in the solution is in the range of 1 nmol / ml to 1 .mu.mol / ml. The required for a clean transfer of the biomolecules from the stamp contact time is usually a few seconds to 30 min. Longer contact times are usually not required but not disadvantageous.

The amount of liquid dispensed is important because it determines the size of the surface areas B, ie in particular the spot size. Liquid quantities in the femtosecond to nanoliter range are applied to the shelf. Thus, spots are available with a diameter of about 150 to 200 microns with a contactless applied amount of about 10 nl to 2 ul. Spots with diameters in the range of 10 μm can be generated by applying amounts of about 0.01 μl to 0.1 μl using capillary dispensers.

The solvents for preparing the solutions of the biomolecules depend in a manner known per se on the nature of the biomolecule to be dissolved. Often the solvent is water or aqueous mixtures of water with organic solvents that are immiscible with water indefinitely. These include, for example, alcohols such as methanol, ethanol, isopropanol or n-butanol, dimethylformamide, Dimethylfulfoxid, tetrahydrofuran and the like and mixtures thereof with water. Depending on the nature of the biomolecule, only organic solvents, eg. B. the aforementioned solvents and their mixtures are used, especially in the case of low molecular weight compounds. In addition to the biomolecules, the solutions may also contain surface-active substances, for example emulsifiers such as alkyl sulfates, alkyl sulfonates, ethoxylates of long-chain alkanols and their sulfuric acid half esters, ethoxylated hydroxycarboxylic acids, ethoxylated esters of long-chain carboxylic acids with glycerol, quaternary ammonium compounds and the like. Furthermore, in particular aqueous solutions of the biomolecules may contain conventional buffers such as PBS buffer, SSC buffer and the like.

In a second embodiment of variant 1, solutions of the biomolecules are applied in a plurality of spatially separated surface areas onto a smooth, ie unstructured surface on which the biomolecules are not immobilized. This surface can be regarded as a release surface. The application of the solutions is carried out in a conventional manner by means of conventional spotting techniques, as described above. The quantity of liquid dispensed is in this case dimensioned such that the arrangement and size of the spots corresponds to the later surface area B of the array. Subsequently, the stamp surface of the above-described, preferably formed from an elastic material, structured stamp is brought into contact with the release surface. As a result, the biomolecules on the stamp surface of the transferred to a textured stamp. Thereafter, the stamp surface of the stamp is then brought into contact with the surface on which the biomolecules are to be immobilized. Regarding the used solutions of the biomolecules, the concentrations, the structuring of the punches and the contact times the above applies. Suitable release surfaces are the abovementioned inert supports and in particular unstructured elastic stamps.

Subsequent to the application of the biomolecules and the concomitant coupling of the organic surface, a washing process is generally carried out. The purpose of this washing process is, in particular, to remove uncoupled biomolecules. In accordance with the solution previously used for applying the biomolecules, aqueous solvents or solvent mixtures are preferably used for washing. In principle, the above statements apply here accordingly.

According to a particular embodiment, washing is carried out with an aqueous solvent or solvent mixture containing a surfactant, preferably a nonionic surfactant. Particularly preferred are polyalkoxylated, and in particular polyethoxylated Fettsäurester of polyols, in particular glycerol or sorbitol, for example sorbitan fatty acid esters sold under the trade name Tween ^®. In particular, the use of ethoxylated sorbitan hexylaurate has proved to be useful.

The concentration of surfactant in the washing solution is suitably chosen so that on the one hand effective removal of uncoupled biomolecules is ensured, on the other hand, the washing solution with the immobilized biomolecules is compatible. Concentrations ranging from 0.01% to 10%, and preferably from 0.05% to 2%, by weight have been found to be useful.

According to variant 2 of the method according to the invention, it is possible to produce microarrays of immobilized biomolecules which have on their surface at least two spatially separated surface areas, each comprising a plurality of spatially separated sites P on which biomolecules are immobilized, by forming a compound V can react with the biomolecules to form bonds, in spatially separated locations P on the surface on which the array is to be produced, and then applying the biomolecules in a plurality of spatially separated surface areas so that each surface area a plurality, preferably on average at least 10th , in particular at least 50 and especially at least 100, z. B. 100 to 10,000 preferably to 5000 and in particular to 1000 places P covers. In other words, first, on the surface on which the array is to be generated, the connections V are immobilized at defined locations P.

Subsequently, the biomolecules are applied in several spatially separated surface areas B on the pretreated surface. This leads to a chemical binding of the biomolecules and thus to an immobilization at those sites P, which are occupied by the compound V, or in which the compound V is bound. Subsequently, unbound biomolecules are rinsed off from the surface in a manner known per se, for example by washing with the 0.g. Washing liquids. In this way, one obtains the array according to the invention, which has a plurality of spatially separate surface areas, each comprising a plurality of spatially separated sites P, which are occupied by biomolecules, wherein the places P within a range have a similar occupancy and the places P different surface areas have a similar occupancy or a different occupancy with respect to the occupation density and / or the type of immobilized biomolecule occupancy.

The application of the compounds V localized in the sites P on the surface on which the biomolecules are to be immobilized succeeds, for example, in the μCP technique described above. For this purpose, the compound V is applied uniformly to the stamp surface, for example, by contacting the stamp surface with a solution of compound V. Then, any solvent is removed and then the thus treated stamp surface, which is now occupied by the compound V, with the surface , on which the biomolecules are to be immobilized, brought into contact. In principle, however, the compound V can also be applied to the surface by other methods for producing high-density arrays.

Variant 2 can in principle be carried out on any surfaces, the statements made above apply accordingly. However, it has proved to be particularly advantageous to carry out variant 2 on one of the hydrogel surfaces described above, especially on a hydrogel layer composed of star-shaped prepolymers, in particular if proteins are immobilized. However, as carrier with hydrogel-forming surface are also the above-mentioned commercial products, as sold by XanTec Bioanalytics GmBH and Perlon-Elmer.

• – in einem ersten Schritt auf einem Träger eine Hydrogel-bildende Oberflächenschicht bereitstellt, die an ihrer Oberfläche eine Vielzahl funktioneller Gruppen aufweist, die mit hierzu komplementären funktionellen Gruppen unter Bindungsbildung reagieren,
• – auf eine Stempelfläche, die eine Vielzahl von erhabenen Flächen aufweist, eine Verbindung V aufbringt, die eine oder mehrere funktionelle Gruppen aufweist, die mit den funktionellen Gruppen der Oberfläche unter Ausbildung einer kovalenten Bindung reagieren können, und die ausserdem eine oder mehrere davon verschiedene funktionelle Gruppen aufweist, die eine Bindung zu dem zu immobilisierenden Biomolekül ausbilden können;
• – die so behandelte Stempelfläche mit der Hydrogel-bildenden Oberflächenschicht in Kontakt bringt, wobei man eine Oberfläche erhält, die eine Vielzahl von räumlich getrennten Plätzen P aufweist, die mit der Verbindung V belegt sind,
• – anschließend eine oder mehrere, voneinander verschiedene Lösungen der zu immobilisierenden Biomoleküle in wenigstens zwei voneinander räumlich getrennten Flächenbereichen B so aufbringt, dass die Flächenbereiche jeweils eine Vielzahl der Plätze, welche mit der Verbindung V belegt sind, abdecken, wobei die Biomoleküle an den Plätzen immobilisiert werden, und
• – anschließend nicht immobilisierte Biomoleküle entfernt.

Accordingly, a particularly preferred embodiment of variant 2 is a process in which:

• In a first step, providing on a support a hydrogel-forming surface layer having on its surface a plurality of functional groups which react with complementary functional groups to form bonds,
• On a stamp surface having a plurality of raised surfaces, applying a compound V having one or more functional groups capable of reacting with the functional groups of the surface to form a covalent bond, and which also has one or more different functional groups Has groups that can form a bond to the biomolecule to be immobilized;
• Bringing the thus treated stamp face into contact with the hydrogel-forming surface layer to obtain a surface comprising a plurality of spatially separated sites P occupied by compound V,
• - Subsequently, one or more different solutions of the biomolecules to be immobilized in at least two spatially separated surface areas B so applied that the surface areas each covering a plurality of places, which are occupied by the compound V, wherein the biomolecules immobilized at the squares be, and
• - subsequently removed non-immobilized biomolecules.

The solvent and the concentration of compound V in the solution depend on the nature of compound V. The optimum solvents and concentrations can be determined by the skilled person in a simple manner with a few routine experiments. Preference is given in principle to those solvents which on the one hand dissolve the compound V in the desired concentration and at the same time can be easily removed, in particular solvents having a boiling point below 120 ° C. Examples of solvents are water, aqueous solvent mixtures and organic solvents, eg. As the solvents mentioned in the variant 1 and aqueous solvent mixtures.

The application of the biomolecules in spatially separate areas can be done in a conventional manner by means of the usual spotting techniques, as described in detail in variant 1. With regard to the exact process conditions, in particular the temperature and the choice of solvents, the above statements apply accordingly.

The usually required washing step is carried out by rinsing with a suitable washing solution. Here, the statements made to variant 1 apply accordingly.

The compounds V used in variant 2 are generally those compounds which on the one hand form a bond to the surface on which the biomolecules are to be immobilized and at the same time can form a bond to specific binding centers in the biomolecules. The attachment of the compound V to the surface is usually carried out by covalent bonds, for example via ester, amide, amino, sulfonamide, urethane, urea or thiourethane groups. Accordingly, the compounds V generally have at least one of the abovementioned groups R which can react with the reactive groups R of the surface, in particular the hydrogel-forming surface, to form a covalent bond. Furthermore, the compounds V at least one further binding center, which can form a bond with the biomolecule.

Binding to the biomolecule may involve covalent, ionic, coordinative, hydrophobic interactions, hydrogen bonding interactions, or a hybrid of the aforementioned bonds (affinity binding, such as occurs in protein ligand or protein-protein interactions). , In a first preferred embodiment of the invention, this binding center is a center that can form a coordinate bond to the biomolecule, which is mediated via a transition metal atom. With respect to the nature of the transition metal atoms, the above applies. In particular, they are transition metal atoms of the third period and especially those which can form divalent ions, such as in copper (II), nickel (II), zinc (II), cobalt (II) and iron (II). Preferably, such compounds V bind the metal atom as a chelate, ie the compounds V have at least 2, preferably at least 3, z. B. 3, 4 or 5 functional groups that can form a coordinate bond to the above-mentioned metal ions. The spatial arrangement of the functional groups is preferably chosen so that the connection takes place in the form of a chelate over all functional groups of the compound. Examples of suitable compounds V which are capable of coordinating metal atoms and which simultaneously have a reactive group R 'include, for example, aromatic orthohydroxy aldehydes such as salicylaldehyde, functionalized pyridine and quinoline compounds such as 8-hydroxyquinoline, dipicolylamine, N-2 Pyridylmethylaminoacetat, furthermore polycarboxylic acids with preferably at least 2, z. B. 2, 3, 4, 5 or 6 carboxyl groups, especially compounds containing at least 2, e.g. B. 2, 3, 4, 5 or 6 carboxymethyl groups and optionally a further functional group R ', as in iminodiacetic acid, nitrilotriacetic acid and their derivatives, as described by Liu et al. Synthesis, 2002, 10, 1398, carboxymethylated aspartic acid, N, N, N-tris (carboxymethyl) ethylenediamine, N, N, N, N-tetra (carboxymethyl) ethylenediamine (EDTA) and the like, further orthophosphoserine and Likewise, in a preferred embodiment of the invention, an amino-functionalized derivative of a compound having at least two carboxymethyl groups, in particular an amino-functionalized derivative of nitrilotriacetic acid such as lysine NTA (= 6-amino-2- (N, N-bis (carboxymethyl) amino) hexanoic acid). The compounds V, which can form complexes with transition metal ions, can be applied to the surface in complexed form or preferably in uncomplicated form. When uncomplexed compounds V are used, complexation is then carried out by rinsing with a metal ion-containing solution. Optionally, following the application of Compound V, or subsequent to the complexing of Surface-bound Compound V, a washing step will be introduced to remove excess Compound V and / or excess metal ions.

In the case of carboxylic acids as compounds V you can also in carboxyl-protected form, d. H. as an ester. Suitable protecting groups for carboxyl groups are known to the person skilled in the art from the relevant literature, preference being given to those groups which can be split off under conditions which affect the surface, eg. B. does not affect the hydrogel layer or even destroy it. Example of this is the tert-butyl group, which is thermally, for. B. by heating to 150-220 ° C, preferably at reduced pressure, remove.

The solutions used for complexing are usually aqueous solutions of water-soluble salts of the metal ions, eg. B. Solutions of chlorides, nitrates, sulfates, acetates, etc. The concentration of metal ions in the solution is usually in the range of 1 mM to 1 M. Depending on the nature of the ligand and the nature of the metal ion, the pH of the solution usually 5 to 12, in particular 6 to 11. The solutions may optionally contain weak complexing agent, for. Ammonia to ensure solubility of the metal ions at higher pHs.

In another embodiment of the method 2, the compounds V, in addition to the reactive group R 'required for the connection of the compound V, have a group which can bind to the biomolecule via at least 2 and preferably via at least 3 hydrogen bonds. Such groups are, for example, oligonucleotide groups having at least 2, preferably at least 3, z. B. 3 to 100 and in particular 3 to 20 nucleotide bases. Such biomolecules can bind to these groups, which have a complementary oligonucleotide sequence, wherein the proportion of complementary bases should be at least 50%.

In a further embodiment of the invention, compounds V are used as compounds which, as is known, enter into an affine interaction with biomolecules. Examples of suitable compounds V are ligands, e.g. Haptens and antibodies that are so functionalized, d. H. have a group R 'that can react with the surface to form a covalent bond. These also include chemical compounds V which, in addition to at least one reactive group R ', have a further molecule group or amino acid sequence which can bind with the biomolecule in the sense of an affinitive interaction, for example a protein ligand or protein-protein interaction. Examples of these are biotin derivatives which are modified with a group R 'and thus form an anchor for biomolecules which have a streptavidin or avidin sequence, and streptavidin and avidin derivatives modified with a group R', so that they represent an anchor for biotinylated biomolecules.

The biomolecules to be immobilized naturally have at least one binding site which can form a bond, for example a covalent bond, a coordinate bond, a hydrogen bond or an affinity bond, to the substance V immobilized on the surface. Examples of such groups have in part already been dealt with above in compounds V.

For example, biomolecules that bind to substance V to form a covalent bond have a complementary reactive group (see above). Naturally, these biomolecules which bind to an oligonucleotide sequence have a sequence which is at least partially complementary thereto, wherein preferably at least two adjacent base pairs in the oligonucleotide sequence of the biomolecule are complementary to two adjacent base pairs of the substance V oligonucleotide sequence. Preferably, the match is at least 50%.

When the biomolecule is attached via affinitive interactions, the biomolecule naturally has at least one group which can bind with the substance V in the sense of a protein ligand or protein-protein interaction, for example in a streptavidin-modified compound V, a biotin unit or in the case of a biotin-modified substance as compound V, a streptavidin unit.

In a preferred embodiment of the invention, the substance V is a substance which coordinates a transition metal ion of the type described above. In this case, the biomolecule has a molecular group, which in turn can form a coordinating bond as a ligand, preferably as a chelating ligand with the transition metal ion. Preferably, however, the gelatizing effect of this group is not so strong that it comes to a complete displacement of the gelatizing part of the compound V. Chelating ligands in the biomolecules are in particular those which have at least two, in particular 2 or 3, groups suitable for coordination to a metal atom, for example 1, 2 or 3 carboxylate groups and / or 1, 2 or 3 amino groups and / or 1, 2 or 3 imino groups. Of these, preference is given in particular to groups which have at least one or preferably 2 or 3, spatially adjacent imidazole groups. Particularly preferred among these are those derived from histidine or oligohistidine sequences. Such groups are also referred to as histidine tag or His tag.

Such functionalized biomolecules and methods for functionalizing non-functionalized biomolecules are known from the literature, for example from J. Chromate. 411 (1987) 177-184 (histidine tag), Casey et al., J. Immunol. Methods, 1995, 179, 105-116 (histidine tag), Stiborova et al., Biotechnology and Bioengineering, 2003, 82, 605 -611 and references cited therein (metal complexing-mediating tags), Christian Jakob, doctoral thesis of the University of Göttingen, 2001 and literature cited therein (recent methods and overview of biotinylation).

According to a particularly preferred embodiment of variant 2 of the process according to the invention, the hydrogel-forming surface layer is composed of the star-shaped prepolymers described above, which have on average at least 4 polymer arms A, which in themselves are water-soluble. The application of the substance V can take place before, but preferably during or after the crosslinking of the prepolymers. The hydrogel-forming surface layer preferably has a multiplicity of the functional groups R mentioned above in Table 1 and in particular isocyanate groups and / or amino groups.

In the particularly preferred embodiment of variant 2 with a hydrogel-forming surface layer, in particular those compounds V have proved to be effective which bring about immobilization of the biomolecule by a coordinative bond mediated via a transition metal. Accordingly, the compounds V are preferably selected from the above-mentioned chelating compounds, especially among the above-mentioned polycarboxylic acids having at least 2 carboxyl groups, e.g. B. 2, 3, 4, 5 or 6 carboxyl groups, which still have a functional group which reacts with the functional groups of the hydrogel-forming surface layer to form a covalent bond. Examples of these are in particular the compounds already mentioned with at least two, z. B. 2, 3, 4, 5 or 6 carboxymethyl groups, in particular amino-functionalized derivatives of nitrilotriacetic acid such as lysine NTA (= 6-amino-2- (biscarboxymethylamino) hexanoic acid).

Variant 2 in conjunction with a hydrogel-forming surface layer has proven particularly useful for the immobilization of complex biomolecules that readily denature, especially for the immobilization of proteins. A very particularly preferred embodiment of variant 2 with hydrogel-forming surface layer therefore relates to the immobilization of proteins, of proteins which bind to the surface by means of a coordinate bond and especially of proteins with histidine tags or comparable tags, as described by Stiborova et al ., Biotechnology and Bioengineering, 2003, 82, 605-611 and in the literature cited therein.

With regard to the application of the regions of the biomolecules to the surface structured with the compound V, the principle stated above for the application of biomolecules to stamp surfaces applies in principle, with direct spotting (contact-free or with contact) being preferred. In this case, the biomolecule can be used in pure form or as a crude product of functionalization, since the biomolecule provided with the binding site selectively binds to the sites P occupied by the substance V.

In general, the application of the biomolecule is followed by a washing step. This washing step serves to remove unbound biomolecule from the surface of the array. When Wash liquids are basically those liquids into consideration, which solve the biomolecule and at the same time do not damage the surface. Particularly suitable are aqueous solvents which, in addition to water, may also contain small amounts, preferably not more than 40 and in particular not more than 10% of organic, water-miscible solvents, for example alcohols, such as methanol, ethanol and the like. These washing liquids may also contain surfactants as mentioned above. The proportion of surface-active substances will generally amount to no more than 5% by weight, based on the washing liquid. Furthermore, the washing liquids can also contain the buffers customary in biotechnology, in particular PBS buffer SSC buffers and the like, in the concentrations customary for this purpose. The type of additional constituents depends in a manner known per se on the type of biomolecule immobilized on the surface.

If the immobilized biomolecules are provided with a marking in one or more areas, quality control of the array can now follow. For this purpose, the array can be measured with a detection system corresponding to the marking. The measured amount of immobilized label makes it possible in particular to assess the coupling efficiency and thus the immobilization of the biomolecules in terms of area, homogeneity and density.

In principle, all types of marking are suitable. Preferably, the marker is chosen so that it does not interfere or only minimally with the subsequent analysis, so the use of the arrays not or only insignificantly influenced. For this purpose, any necessary markings of the sample, ie in particular of nucleic acid targets, should be taken into account. On the other hand, the label may be chosen to interact with the label of the sample. The signal originating from interactive markers may be distinguishable from that originating from the corresponding non-interacting markers. For example, the interaction may affect signal intensity, e.g. B. amplify or quench, or modify the signal, for. B. change the wavelength absorbed or emitted light.

Suitable according to the invention are marking systems which are suitable for. B. detect spectroscopically, photochemically, biochemically, immunochemically, electrically, optically or chemically. These include both direct labeling systems, such as radioactive labels (eg ³² P, ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C), magnetic markers, chromophores, for example UV, VIS, or IR-absorbing compounds, fluorophores, chemical or bioluminescent markers, transition metals which are usually chelated, or enzymes, e.g. As horseradish peroxidase or alkaline phosphatase and the detection reactions coupled thereto, as well as indirect labeling systems, such as haptens, such as biotin or digoxigenin, which can be detected by appropriate detection systems.

Advantageous chromophores have an intense color, which is only slightly absorbed by the surrounding molecules. Dye classes, such as quinolines, triarylmethanes, acridines, alizarines, phthaleins, azo compounds, anthraquinones, cyanines, phenazathionium compounds or phenazoxonium compounds, are mentioned here as representative of the broad spectrum of chromophores which are suitable according to the invention.

Fluorescent labels are preferred. It gives strong signals with little background, high resolution and high sensitivity. According to the invention, it is advantageous that one and the same fluorophore can emit several distinguishable radiations depending on the excitation and detection principle. Fluorophores may be used alone or in combination with a quencher (eg, molecular beacons). Suitable fluorophores include, for example, aminomethyl caffeine acetic acid (AMCA, blue), EDANS, BODIPY 493/503; FL; FL8r2; R6G; 530/550; 558/568; TMR 542/574; TR 589/617; 630/650; 650/665, 6-FAM fluorescein (green), 6-OREGON green 488, TET, Cy3 (red), Rhodamine (red), 6-JOE, HEX, 5-TAMRA, NED, 6-ROX, TEXAS Red7 (red ), Cy5, Cy5.5, LaJolla Blue, Cy7, Alexa fluorocarboxylic acids, especially type 647 and 532, e.g. As succinimidyl ester, and IRD41.

The invention also relates to a device based on an array according to the invention, which may be designed, for example, as a chip, microfluid device or as a dipstick. Devices based on bioarrays are generally known to the person skilled in the art from the cited prior art, and can be equipped in a manner known per se with the microbioarrays according to the invention.

The present invention also relates to the use of arrays according to the invention for analytical and in particular diagnostic purposes.

By way of example, the following applications may be mentioned:
Diagnosis, treatment choice and therapy control of tumor and metabolic diseases; Examination of genetic predisposition (SNPs), in particular the detection of mutations, also in the forensic area; Detection of promoter methylation (epigenetics); Gene expression control; Sequencing, in particular sequencing by hybridization; Microbiological applications such as in bacteriology and virology, for example for the differentiation of different strains; ELISA applications with immobilized antibodies, antigens, receptors and ligands in clinical chemistry to food chemistry and environmental analysis; Allergy diagnostics with immobilized allergens; High throughput screening of compound libraries; Toxicogenamics.

In principle, arrays according to the invention are suitable both for noncompetitive and for competitive analytical methods (assays).

In noncompetitive assays, the analyte (analyte) interacts with the biomolecules immobilized on the surface. In general, the analyte is previously marked, but it is also possible a marker after the analyte has already interacted with the immobilized biomolecules, z. B. by primer extension or rolling-cycle PCR. In these cases, a measurement signal is obtained which is the greater, the more analyte is present. It is also possible that the fluorescence of the immobilized substances is changed by the interaction of the sample with the substances immobilized on the surface (attenuation, amplification, eg molecular beacons) or the activity of an enzyme is changed and this change is registered as a measurement signal becomes. Examples of noncompetitive assays are hybridization reactions of PCR products or labeled DNA / RNA to nucleic acids immobilized on the surface, in particular oligonucleotides or cDNA, as well as sandwich immunoassays.

In competitive methods, a labeled substance (marker) is added to the sample which has similar binding properties to the biomolecules immobilized on the surface as the analyte itself. There is a competing reaction between analyte and label for the limited number of binding sites on the surface. A signal is obtained which is lower the more analyte is present. Examples of competitive assays are immunoassays (ELISA) and receptor assays).

The invention will be illustrated by the following examples and figures.

Characters:

1 A schematic representation of variant 1 of the method according to the invention is shown. in 1a For example, the top view is shown of a conventional polydimethylsiloxane stamper having a plurality of serrated elevations. 1b shows a cross section through the in 1a shown in the plan polydimethylsiloxane stamp. In 1c schematically shows the application of different solutions of biomolecules on different areas of the stamp surface and a cross section through the thus treated stamp shown. 1d shows a plan view of the stamp surface thus obtained. 1e is a schematic representation of the contacting of the treated stamp surface with the substrate surface. 1f shows a schematic plan view of the stamped surface and a schematic side view of the stamped surface

2 : Shown schematically is the variant 2 of the method according to the invention. 2a shows a cross section of the stamp used. 2 B schematically shows the printing of the substance V on one with a hydrogel layer ( 1 ) coated substrate surface. 2c shows the substance V ( 2 ) stamped surface in the supervision and 2d as a cross section. 2e schematically shows the top view of a surface and 2f whose cross-section represents, on the after application of the substance V several spots ( 3 ) of different protein solutions were applied.

3 : 3a and 3b show a fluorescence micrograph of a prepared according to variant 1 array of immobilized fluorescein in different magnifications.

4 FIG. 2: Partial view of a fluorescence micrograph of a region prepared according to Variant 2 with a multiplicity of sites on which streptavidin fluorescently labeled via biotin has been immobilized.

5 FIG. 2: Partial view of a fluorescence microscope image of an array prepared according to Variant 2 with a multiplicity of sites on which green histidine-tagged protein was immobilized by means of lysine-NTA-nickel (II).

Examples

I. Aminofunctionalization of the carrier:

The substrate surfaces (glass slides, microscope slides, silicon supports) were first pretreated. For this purpose, the substrates were stored for 1 h at 60 ° C. in a mixture of concentrated aqueous ammonia, hydrogen peroxide (25% strength by weight) and water in a volume ratio of 1: 1: 5. Afterwards, they were rinsed several times with water. The treated substrates were then stored in deionized water. Alternatively, the pre-cleaned by water and acetone substrates in a plasma system of the type TePla 100-E from the company Plasma Systems for 2 minutes in oxygen plasma treated (pressure: 1 mbar, 50 W) and then washed with water and acetone. Alternatively, the pre-cleaned substrates can also be treated with oxygen under a 40 W UV lamp with wavelength of 185 nm for 10 min (distance between substrate surface and light source 2 mm), and then washed with water and acetone.

For the amino functionalization, the pretreated substrates were coated with an aminosilyl monolayer. For this purpose, the sample taken from the water and blown dry with nitrogen was transferred to a glovebox. There, the substrates were stored in a 0.5% (v / v) solution of (3-aminopropyl) trimethoxylsilane in dry toluene for 16 h, then thoroughly washed with toluene and stored therein and filtered using a nitrogen atmosphere in a glovebox before use Dried nitrogen stream.

II. Preparation of a carrier with a hydrogel layer which has functional groups R on its surface.

Production Example 1

A 6-arm random poly (ethylene / propylene oxide) having an EO / PO ratio of 80/20 with a molecular weight of 3100 g / mol which had been modified at the ends of the polymer arms with isophorone diisocyanate (prepared analogously to Example 11 of DE 10216639.0 ) was dissolved at a concentration of 1 mg / ml in a mixture of tetrahydrofuran and water (1: 9 v / v). Then, an amino-functionalized glass slide prepared according to I was immersed in this solution and withdrawn at a rate of 5 mm / sec perpendicular to the liquid surface. In this way, one obtained a nearly monomolecular coating of the glass carrier with the prepolymer, which has production reasons on its surface isocyanate groups.

Production Example 2:

A 6-arm random poly (ethylene / propylene oxide) having an EO / PO ratio of 80/20 with a molecular weight of 18,000 g / mol which had been modified at the ends of the polymer arms with isophorone diisocyanate (prepared analogously to Example 11 of DE 10216639.0 ) was dissolved in dry tetrahydrofuran at a concentration of 10 mg / ml. Then, an amino-functionalized silicon support prepared according to I was spin-coated. For this purpose, first the non-rotating, dried substrate was completely wetted with the prepolymer solution before the solution was spun off for 40 seconds at a final speed of 4000 rpm. The thus coated substrate was then stored overnight in an atmosphere saturated with water vapor to give a surface having NH ₂ groups.

III. Production of bioarrays

Example 1: Preparation of a Fluorescein Array

A solution of an isothiocyanate-modified fluorescein derivative (FITC from Aldrich) was dissolved in a mixture of ethanol / dimethyl sulfoxide 50: 1 v / v in a concentration of 100 uM. On a rectangular Polydimethyldisiloxan stamp, manufactured by Tan et al., Langmuir 2002, 18, pp 519-523 with an area of 15 mm × 15 mm with a regular array of cylindrical columns (diameter 10 .mu.m, height 2 .mu.m, average distance 20 microns ), a very small amount of liquid (<0.5 .mu.l) of this solution was applied with a microliter pipette and then dried to obtain a spot with a diameter <500 .mu.m. The thus prepared stamp was brought into contact with the amino-functionalized glass carrier prepared in I for 2 min. Subsequently, the surface was examined by fluorescence microscopy. In this way one obtains the pattern shown in Figures 3 and 3a.

Analogously, protein arrays and nucleotide arrays can also be prepared:

Example 2: Preparation of an oligonucleotide array

A rectangular Polydimethyldisiloxanstampel prepared z. Langmuir 2002, 18, pp. 519-523, with an area of 15 mm × 15 mm with a regular arrangement of columns (diameter 5 μm, height 2 μm, mean distance 20 μm) according to the instructions of Donzel et al., Adv. Mater. 2001, 13, 1164, hydrophilically modified in oxygen plasma. 0.1 μl of a solution of 3'-amino-5'-fluorescence-labeled oligonucleotide in PBS buffer at a concentration of 100 pmol / μl are applied to this stamp by means of a micropipette 2 spots. After drying in an inert gas stream, the stamp prepared in this way is brought into contact with a freshly prepared surface according to Example 11, 2 mm. It is then washed with PBS. This gives a nearly defect-free image of the stamp, which has two separate regions in which the nucleotides occupy a very large number of spatially separated sites, as can be detected by fluorescence microscopy.

Example 3 Preparation of a biotin-streptavidin-array

The polydimethyldisiloxane stamp used in Example 2 is wetted with a solution of biotinamidocaproic acid N-hydroxysuccinimide ester in dimethylformamide (10 μg / ml) and then dried. The stamp thus obtained becomes 2 mm. brought into contact with the coating produced by II Example 2. Subsequently, the coating thus obtained is washed successively with dimethylformamide and water to remove unbound ester and dried in an argon stream. In this way, an array of circular regions of immobilized biotin was obtained.

2 spots of a solution of fluorescently labeled streptavidin (Molecular Probes) in PBS buffer (0.04 mg / ml) are applied by micropipette to the biotin array prepared in this way and stored for 4 hours at room temperature. Subsequently, the array is washed with PBS buffer, dried in an argon stream. In this way, one obtains an array of two spatially separated regions having a plurality of occupied streptavidin sites, as can be detected by fluorescence microscopy (see ).

Example 4: Production of a Protein Array via Complexing

The polydimethylsiloxane stamp used in Example 1, with an area of about 15 mm × 15 mm, with a regular array of columns (diameter 10 μm, height 2 μm mean distance 20 μm) is mixed with a solution of lysine NTA-tert-butyl ester, made according to J. Groll, diploma thesis of the University of Ulm, 2001, wetted in toluene (20 mg / ml) and then dried. The stamp thus obtained is 15 min. brought into contact with the freshly prepared according to II Example 1 coating. Subsequently, the support thus obtained is stored overnight in an atmosphere saturated with water vapor and washed with toluene. The resulting support is 10 min. stored at 200 ° C under vacuum (0.02 mbar) and after cooling for 5 min. dipped in a solution of NiCl ₂ in water (5 mg / ml) and washed with water. Subsequently, a solution of His-tagged Green-Fluorescent protein in PBS buffer (0.03 mg / ml) is applied to the thus-treated carrier for 10 min. stored and then washed with PBS buffer. The resulting array is examined by fluorescence microscopy. You get that in the shown pattern.

Claims (35)

A microarray of immobilized biomolecules comprising at least two surface areas B arranged spatially separated on a surface, each comprising a plurality of spatially separated sites P on which biomolecules are immobilized, wherein the mean distance of the boundary line of a region B to its center of area is the region of 10 microns to 200 microns. Microarray according to claim 1, wherein the mean distance of adjacent places P within the areas B is at least twice as large as the mean distance of the boundary line of a place P to its center of area. Microarray according to claim 1 or 2, wherein the average distance of the boundary line of a place P to its center of area is on average 50 nm to 50 μm. A microarray according to any one of the preceding claims, wherein the mean spacing of adjacent sites is in the range of 200 nm to 200 μm. A microarray according to any one of the preceding claims, wherein the locations P are described by a circular, ellipsoidal or rectangular area. Microarray according to one of the preceding claims, wherein the locations P are arranged regularly within the regions B. Microarray according to one of the preceding claims, wherein the regions B comprise at least 10 spatially separated, planar spaces P. Microarray according to one of the preceding claims, wherein at least two of the surface areas B differ in the type and / or areal density of the mobilized biomolecules. Microarray according to one of the preceding claims, wherein the regions B have a circular geometry. A microarray according to any one of the preceding claims wherein the mean spacing of adjacent regions is at least 50μm. Microarray according to one of the preceding claims, comprising an organic surface layer arranged on a carrier, on which the biomolecules are immobilized. A microarray according to claim 11, wherein the surface layer comprises at least one hydrogel-forming layer. A microarray according to claim 12, wherein the hydrogel-forming surface layer is composed essentially of star crosslinked prepolymers interlinked with each other, which comprise on average at least 4 polymer arms A, which in themselves are water-soluble. A microarray according to claim 12 or 13, wherein the hydrogel-forming surface layer has on average a thickness of 1 nm to 100 μm in the dry state. A method of producing a microarray of immobilized biomolecules comprising at least two surface areas B arranged spatially separated from each other on a surface, each comprising a plurality of spatially separated sites P on which biomolecules are immobilized by placing on a plunger surface containing a plurality of raised surfaces Has surfaces, one or more mutually different biomolecules in at least two, preferably at least ten and in particular at least one hundred spatially separated surface areas B so applying that cover the biomolecules in the areas B each have a plurality of the raised areas of the stamp surface and then the stamp surface with the Surface on which the biomolecules are to be immobilized, brings into contact and optionally not immobilized biomolecules removed. A method of producing a microarray of immobilized biomolecules comprising at least two surface areas B arranged spatially separated on a surface, each comprising a plurality of spatially separated sites P on which biomolecules are immobilized by applying to a stamp surface comprising a plurality of raised Has surfaces, a compound V, which causes immobilization of the biomolecules, applies, the thus treated stamp surface with surface on which the biomolecules are to be immobilized, in contact, to obtain a surface having a plurality of spatially separated sites P. , which are occupied by the compound V, and then one or more mutually different solutions of the biomolecules to be immobilized in at least two spatially separated surface areas B so applied that the surface areas each have a plurality of places, which with d cover compound V, immobilizing the biomolecules at sites P, and subsequently removing non-immobilized biomolecules. The method of claim 16, wherein the compound which effects the immobilization of the biomolecules is selected from compounds which form a covalent or ionic bond with the surface and which form a, covalent, ionic, coordinative or affinitive bond with the biomolecule to be immobilized. Method according to one of claims 15 to 17, wherein the mean distance between adjacent elevations on the stamp surface is at least twice as large as the average distance of the boundary line of the end face of a survey to the area center of the end face. Method according to one of claims 15 to 18, wherein the average distance between the boundary line of the end face of the elevations to the surface center of the end face is on average 50 nm to 50 microns. Method according to one of claims 15 to 19, wherein the mean distance between adjacent elevations on the stamp surface in the range of 200 nm to 200 microns. Method according to one of claims 15 to 20, wherein the end faces of the elevations on the stamp face are circular, ellipsoidal or rectangular. Method according to one of claims 15 to 21, wherein elevations are regularly arranged on the stamp surface. Method according to one of claims 15 to 22, wherein elevations on the stamp surface on average have a height in the range of 0.5 to 500 microns and the ratio H: A of average height H to the average distance A between the boundary lines of the end faces of the elevations and the surface centers of the faces is not more than 10. A method according to any one of claims 15 to 23, wherein the protuberances of the stamper are formed by an elastomeric material. The method of claim 24, wherein the elastomeric material is polydimethylsiloxane. The method of any of claims 15 to 25, wherein the concentration of biomolecules in the solution is in the range of 0.1 nM to 10 mM. Method according to one of claims 15 to 26, wherein the surface on which the biomolecules or the compound which causes the immobilization of the biomolecules to be immobilized, is formed by a hydrogel-forming surface layer. A method according to claim 27, wherein In a first step, providing on a support a hydrogel-forming surface layer having on its surface a plurality of functional groups which react with complementary functional groups to form bonds, On a stamp surface having a plurality of raised surfaces, applying a compound V having one or more functional groups capable of reacting with the functional groups of the surface to form a covalent bond, and which also has one or more different functional groups Has groups that can form a bond to the biomolecule to be immobilized; Bringing the thus treated stamp face into contact with the hydrogel-forming surface layer to obtain a surface comprising a plurality of spatially separated sites P occupied by compound V, - Subsequently, one or more different solutions of the biomolecules to be immobilized in at least two spatially separated surface areas B so applying that the surface areas each cover a plurality of places, which are occupied by the compound V, wherein the biomolecules immobilized at the squares be, and - subsequently removed non-immobilized biomolecules. A method according to claim 28, wherein the hydrogel-forming surface layer is composed of star crosslinked prepolymers interlinked with each other, which comprise on average at least 4 polymer arms A, which in themselves are water-soluble. The method of claim 28 or 29, wherein the functional groups on the surface layer are selected from isocyanate groups and amino groups. The method of any one of claims 28 to 30, wherein the binding of compound V to the biomolecule is a transition metal-mediated coordinate bond. The method of claim 31, wherein the compound V is a derivative of the polycarboxylic acid having at least 2 carboxyl groups having a functional group that reacts with the functional groups of the hydrogel-forming surface layer to form a covalent bond. The method of claim 31 or 32, wherein the biomolecule to be immobilized is a protein having a His tag. Device based on a microarray according to one of claims 1 to 14 in the form of a chip, a microfluid device or a dipstick. Use of a microarray according to one of claims 1 to 14 or a device according to claim 34 for diagnostic and analytical purposes.

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