DE10332848A1 - Microarrays of immobilized biomolecules, their preparation and use - Google Patents

Abstract

The present invention relates to microarrays of immobilized biomolecules having at least two spatially separated areas B arranged on a surface, each area having a plurality of spatially separated sites P on which the biomolecules are immobilized, and the extent of the areas on average corresponds to a distance of the boundary line of a respective area B to its area center in the range of 10 mum to 250 mum. DOLLAR A The invention also relates to a method for the production of microbioarrays, in which either DOLLAR A - spatially resolved by spotting techniques biomolecules applied to a suitably structured stamp surface, so that each spot more of the raised areas of the stamp surface, such as columnar elevations covers and then bring the stamp face into contact with the surface on which the biomolecules are to be immobilized and, if necessary, add excess DOLLAR A d. H. not immobilized biomolecules removed (variant 1); or DOLLAR A - by means of muCP technique a pattern corresponding to the array of compounds V, which cause immobilization of biomolecules on a surface, hereinafter also referred to as coupling agent V, applied to the surface, wherein an array is obtained, a plurality of spatially having separate sites P, which are occupied by the compound V, then a solution of the biomolecules on the so-called ...

Description

The The present invention relates to microarrays of immobilized biomolecules whose Production and use thereof. Also relates to the present Invention Devices based on such microarrays, in particular Chips and dip sticks.

With the increasing practical importance of molecular biology Analytics come across those associated with a very high work and cost, often Days or weeks of established standard techniques at borders. New test formats such as biochips are said to save a tremendous amount of time and money enable. Since with this technique a lot of different immobilized in a confined space Substances simultaneously with a single sample to be analyzed can be contacted is the information content obtained per measurement from a chip very high.

An overview of the The best-known biochip systems are found, for example, in Bowtell D.D. L. Nature Genetics Supplement 21 (1999) 25-32; Lockhart et al, Nature Biotechnology 14 (1996) 1675. An Overview of Protein and Antibody Arrays gives 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, which are built 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 (see WO 97/18226; EP 0910570 A1 ; WO 98/29736).

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 of <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. At the. 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.

In younger Time was variously a stamping technique, the so-called Microcontact printing (μCP) used to make arrays of biomolecules on surfaces (see Martin et al., Langmuir 1998, 14, p. 3971, Bernard et al., Langmuir 1998, 14, p. 2225, 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 WO 01/67104). With the μCP technology, the original was developed for other purposes, which can be immobilized biomolecules localized, d. H. in defined areas, applied to a surface become. This is usually done in such a way that you first the stamp surface with a solution brings the biomolecule to be immobilized in contact, the stamp if necessary, dry and then with the stamp surface the surface to be structured brings in contact. Here, the biomolecule is in those areas, in which the stamp area with the surface is in contact, on the surface transferred. Surface areas, not with the stamp area on the other hand, remain unchanged. In this way you get defined Area on the surface, those with biomolecules are occupied us so represent an image of the stamp. With the μCP technology For example, structures with a resolution of up to about 100 nm can be produced. Unlike the spotting technique, which allows it on one surface to apply several areas simultaneously, with different ones biomolecules are occupied, can with the μCP technology only identical areas are generated. For the production of biochips however, typically arrays are needed that have multiple areas which, with regard to the type and / or the occupancy density the biomolecules are different from each other.

Inerowicz et al. (Langmuir 2002, 18, p. 5263) describe a μCP technique, in which by means of a polydimethylsiloxane stamp, the strip-shaped elevations has, first strip Protein areas on a surface applies. In a second stamping process then becomes a stamp with the same structure with a solution of a second protein wetted and rotated 90 ° imprinted on the already coated with the first protein surface. So receives to get a surface with two different, orthogonally arranged protein strips. A third protein may not be on the first two yet Proteins occupied surface be applied by applying a solution of the surface third protein rinses. That way you can however, only three different biomolecules are applied to the surface. Furthermore is an overlap naturally not between different biomolecules preventable. 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 a protein solution into so-called micro-wells, subsequently with a stamp the protein solution from these micro-wells and imprints on a suitable surface. In this way, it is possible to produce protein arrays that contain several Have areas that differ in the type of protein occupancy. With the measures described there However, only comparatively large areas (edge length> 500 μm) can be produced. A similar Method describes Renault et al., Angew. Chem. Int. Ed. 2002 41, 2320. Here, a mask with a plurality of continuous holes on a stamp area put, taking in the holes solutions filled with different antigens become. As a result, the antigens are applied to the stamp surface. Subsequently becomes the stamp area with a solution of antibodies brought into contact and then printed on a suitable surface. That way you can also protein arrays are produced, which in terms of their Proteins have several different areas. With both procedures However, only large areas can be produced. Both methods are extremely complicated and have their implementation the disadvantage of a low selectivity. This is due to capillary forces of protein liquids due.

The WO 02/48676 describes multilayer microarrays for the production of bioarrays having a first membrane disposed on a surface with very fine continuous holes and one about it arranged second membrane with comparatively large holes. In the big ones holes become solutions of biomolecules filled and penetrate through the fine holes on the surface. After removal of the lower membrane is thus obtained a pattern of immobilized Biomolecule on the surface. That way you can but only bioarrays with very large areas of different biomolecules to be obtained. In addition, must these areas have comparatively large distances from one another to a sufficient resolution to achieve because capillary forces smearing the biomolecules over the Area boundaries. With the last three mentioned procedures Only those biochips can be produced that are comparatively have low numbers of areas with different biomolecules. In addition, these methods are very expensive in their implementation.

Of the present invention is based on the object, arrays immobilized biomolecules provide, on the one hand, the largest possible number of areas, which may be occupied by different biomolecules and the one increased resolution in terms of the different biomolecules exhibit. In addition, these arrays should be easy to manufacture, without the need of costly techniques, such as she for high-resolution Spotting techniques are required.

These The task is performed by microarrays of immobilized biomolecules, below also called microbioarrays, solved, the at least two, preferably at least ten, in particular at least fifty and especially at least one hundred spatially separated surface areas B, which are arranged on a surface, wherein every surface area a plurality, preferably on average at least ten, in particular at least a hundred spatially separate places 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 area of 10 μm up to 250 μm 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, welche 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
• - Apply by μCP technique an array corresponding pattern of compounds V, which cause immobilization of biomolecules on a surface, hereinafter also referred to as coupling agent V, applied to the surface, wherein one obtains 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 Process variants combine the high resolution provided by the μCP technique is achieved and the benefits of the spotting technique that allows it different areas of immobilized biomolecules in easier Way to produce.

The Invention thus relates to a process for the production of microbioarrays, in which one according to variant 1 on a stamp surface, which has a multiplicity of raised areas (in the following surveys), one or more mutually different biomolecules in at least two, preferably at least ten, in particular at least 50, specifically at least 100, z. B. 100 to 10,000 spatially separated surface areas B (= spots) so that the biomolecules in the surface areas B a plurality of surveys usually at least 10, preferably at least 50, in particular at least 100, z. 100 cover up to 10000 elevations of the stamp surface, then the stamp surface with the surface, on the biomolecules be immobilized, in contact and if necessary non-immobilized biomolecules, z. B. by washing, removed.

there corresponds to the number, arrangement and size of the area areas B through the biomolecules covered, raised areas of the stamp surface respectively the number, arrangement and places P one size Area where the biomolecules are immobilized. The number, arrangement and size of the areas B corresponds to first approximation 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. One hundred to ten thousand of mutually spatially separated 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. B. 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 related to the preparation of the arrays of the invention limited 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, in which however, the area B also larger expansions, e.g. > 500 microns or more. However, it is preferable to use these processes for the preparation of Arrays according to the invention one.

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, unlike conventional spots, are comparatively sharp, ie "coffee stain forms", as occur with 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, in particular 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.

Of the Term "array" denotes a Arrangement of defined places P, in particular a local Assignment of certain substances to defined places P, taking different places P may be assigned the same or different substances. at the assigned substances are according to the invention biomolecules. The spatial Dimensions of the seats P are in the μm range or in the sub-micron range. Therefore, be the arrays of the invention also referred to as microbioarrays or as microarrays of immobilized biomolecules.

The microbioarrays according to the invention have several areas, which in turn are spatially separated from each other separate seats P are subdivided. The squares P within each area is assigned the same substance type, although it is also conceivable mixtures of two or more Substance types the places Assign P to an area. courts P in different areas are usually in terms of composition and / or the occupation density of the respective substance type from one another differ. As a rule, therefore, places P are in different places Regions of different biomolecules be immobilized.

Of the Term "biochip" refers to here an array of biomolecules, the on a solid carrier are immobilized.

• • 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 several interacting molecules.

• Nucleic acids, in particular oligonucleic acids, for example 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, eg Fab, Fab ', F (ab) ₂ , synthetic;
• • proteins, eg allergens, inhibitors, receptors;
• Enzymes, eg peroxidases, alkaline phosphatases, glucose oxidase, nucleases;
• • small molecules (haptens), eg pesticides, hormones, amino acids, antibiotics, drugs, dyes, synthetic receptors, receptor ligands.

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

a In particular, one particular type of biomolecule is as a ligand to call. Ligands interact with and, in particular, bind they - preferably specific - to certain targets.

In the arrays of the invention are the biomolecules in defined places P a surface immobilized. These places P have a certain shape and extent. The shape of the places is P in principle any. Preferably, the shape of the places P is uniform and has, for example, a polygon, z. B. an n-sided with n = 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and among them, preferably one right angular with n = 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and below preferably a rectangular geometry, a circular or a ellipsoid geometry. Also mixed forms of these geometries are in principle suitable, e.g. stretched ovals. Preferably, the places P have a circular or ellipsoidal geometry. The spatial extent of these places P correlates naturally with the Distance of the boundary line of a particular place to his Centroid. This distance is on average usually in the range of 50 nm up to 50 μm, preferably 100 nm to 25 μm, in particular 0.25 μm up to 20 μm and especially 0.5 μm up to 10 μm.

Of the Distance between adjacent places P, defined by the distance between the centers of the surfaces of adjacent ones courts P is usually in the range of in the range of 200 nm to 200 microns, often in Range from 1 μm to 150 μm, especially in the range of 5 microns up to 100 μm and especially in the range of 10 microns up to 90 μm. The mean minimum distance between adjacent places P, defined by the Minimum distance that the boundary lines of two neighboring places P have, is usually in the range of 100 nm to 150 microns, often in the range from 500 nm to 120 μm, especially in the range of 2 microns up to 100 μm, and especially in the range of 5 μm up to 80 μm (Number average).

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

According to the invention the seats P of the array different surface areas Assign B The arranged on the microbioarrays according to the invention surface areas B usually have a uniform geometry, preferably an ellipsoidal or circular Geometry on. Their extent naturally correlates with the Distance of the boundary line of a respective area B to its Centroid. This distance is on average usually in the range of 10 microns to 250 microns, preferably in the range of 25 μm up to 200 μm and in particular in the range of 30 microns to 150 microns. For circular surface areas B 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 Arrays. The mean distance between adjacent areas B, defined by the mean distance of the centers of the area adjacent Areas, is usually a value of 50 microns, especially 100 microns and specifically 200 μm not below. The minimum distance between adjacent areas B, defined by the minimum distance that the boundary lines of two adjacent Have areas, a mean value of 5 microns, in particular 10 μm and especially 50 μm not below.

The microbioarrays according to the invention show in the respective surface areas B in each case a plurality, on average at least 10, preferably at least 50 and in particular at least 100 and up to 10000, preferably to 5000 and especially to 1000 spatially separated courts P on which the biomolecules are immobilized.

According to the invention biomolecules immobilized in a microarray on a surface, d. H. stationary bound. The bond can over covalent interactions, coordinative interactions, ionic or electrostatic interactions, hydrogen bonds, hydrophobic interactions and mixed forms of the aforementioned interactions respectively.

The Connection can be made both directly to the surface as well as via a the attachment of the biomolecule mediating substance V done. These substances V is They are usually compounds that are covalently attached to the surface the biomolecules to be immobilized, bound, and simultaneously to the biomolecule can bind, for example by covalent interaction, ionic Interaction, coordinative interaction, hydrogen bonds, hydrophobic interactions and mixed forms of such interactions, as used, 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 The invention is the connection via covalent interactions. The biomolecules are then over, for example Ester, amide, sulfonamide, imino, amidino, urethane, urea, Thiourethane, thiourea, sulfide, sulfonyl, ether or amino groups to the biomolecules bound.

In another embodiment In the invention, the biomolecules are one bound by a metal atom, coordinated binding. In these cases the attachment of the metal atom to the surface occurs via functional Groups that form a coordinative bond with the metal atom can, for example Carboxyl groups, hydroxyl groups, amino groups, SH groups, oxime groups, Aldehyde groups, keto groups or heterocyclic groups with a Imino nitrogen as in pyridine, quinoline, imidazole or oxazole. Preferably the metal atoms over at least 2, z. B. 2, 3 or 4 such groups bound as a chelate. examples for Suitable metals are especially transition metals, especially transition metals the third period, that in the aqueous Environment can form divalent ions, for example copper (II), Nickel (II), zinc (II), cobalt (II) and iron (II). Bind the biomolecules then to these metals one, preferably over two or three spatially adjacent functional groups of the aforementioned kind.

In a third embodiment the binding of the biomolecules takes place via coordinating hydrogen bonds, as for example in the base pairing of nucleotide sequences occur. In this embodiment are short oligo or polynucleotide sequences with usually at least two, z. B. 2 to 100, especially 2 to 20 bases over preferably covalent bonds bound to the surface. The biomolecule points then an accordingly at least partially complementary oligo- or polynucleotide sequence, wherein at least 2, preferably at least 10 adjacent bases to a corresponding number of adjacent ones Bases complementary are.

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

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 As a rule, it is recommended that the surfaces of inert substrates, e.g. Glass, ceramics, metal, semi-metal like silicon, polyolefin such as polyethylene or polypropylene, to activate, d. H. in a Way to handle that they have a variety of functional groups R, with the complementary reactive groups R 'under training a chemical bond, preferably a covalent chemical Binding, can react. These functional groups R allow the biomolecules directly via a chemical bond or indirectly, d. H. by means of one of the above defined Substances V that over such groups is covalently bound to the surface on which surface to immobilize.

Reactive groups R in the context of this invention are those which react with nucleophiles in an additionre 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 an inventive surface 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, wherein the reactive groups R in the first row and the complementary groups R' in the first column are given: TABLE 1 Complementary functional groups

Suitable are reactive groups R and free-radically polymerizable C = C double bonds, z. 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 the sense of an ene reaction or with conjugated diolefin groups in the sense of a Diels-Alder reaction react. examples for Groups containing allyl groups in the sense of an ene reaction or with dienes in the sense of a Diels-Alder reaction can react, are maleic acid and fumaric acid groups, maleic acid ester and fumaric acid ester groups, cinnamic ester groups, Propiolic acid (ester) groups, Maleinsäureamid- and fumaric acid amide groups, Maleimide groups, azodicarboxylic acid 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 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 has the organic surface layer on their surface such functional groups that undergo an addition or substitution reaction are accessible by nucleophiles. Preferred groups R are isocyanate, isothiocyanate, aldehyde, active esters, Acrylic and methacrylic groups, so that the biomolecules with the Surface over one 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 are.

In a special embodiment The invention involves the attachment of biomolecules or the substance V on the surface layer present isocyanate groups with formation of 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 , For example, 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.

Often included activating the interface also applying an organic coating to theirs surface a plurality of the above-defined functional groups R has. This coating is also referred to below as an adhesive layer. These are bi- or polyfunctional organic compounds or polymers on the surface having at least one functional group, the with the functional groups on the surface of the support material under bond formation (covalent, ionic and / or coordinative) and the at least additional have a further functional group R of the type described above. These substances can as a thin one or ultrathin Layers, in particular as monomolecular or multimolecular Layers applied to the carrier surface or by means of "Chemical Vapor Deposition "(CVD) on the surface of the carrier material be generated. According to the invention preferred are very thin Adhesive layers with a thickness preferably <100 nm, in particular <10 nm and especially monomolecular layers, in particular self-assembling monolayers (self-assembled monolayers, SAM), for example of thiol compounds containing at least one have another group R, especially for the treatment of precious metal surfaces, of Silane compounds which have at least one group R, in particular for producing monolayers on silicon or glass surfaces, further macromolecular monolayers formed by grafting polymers on the surface or are obtained by graft polymerization on the surface and their Polymers having suitable functional groups R.

In a preferred embodiment The invention relates to the formation of the adhesive layer compounds used as the adhesion-promoting groups silane groups, in particular Having trialkoxysilane groups. 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, Trialkoxyalkylmercaptans such as triethoxypropylmercaptan, trialkoxyallylsilanes such as allyltrimethoxysilane, trialkoxy (isocyanatoalkyl) silanes and Trialkoxysilyl (meth) acryloxyalkanes and - (meth) acrylamidoalkanes as 1-triethoxysilyl-3-acryloxypropan. These compounds are in very thin layers, in particular as a monolayer on the surface applied, preferably after previous activation. method This is 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, JFJ Engbersen, and DN Reinhoudt, Recl. Trav. Chim. Pays-Bas 1996, 115 (6), 307-320, and Decher, Science 1997, 277, 1232-1237 for this purpose to be called. 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.

Preferably the adhesive layer is 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 done in a way that instead of an adhesive layer or preferably on the adhesive layer or in any other way activated surface applying a further coating which is used to immobilize the biomolecules is particularly suitable and instead of the reactive groups R such Has groups that have a binding of biomolecules via coordinative Bindings, over Hydrogen bonds, via hydrophobic Interactions or over affinitive bonds allowed. For this one becomes on the surface one preferably monomolecular layer of the above compounds Generate V, e.g. Layers of chelated, 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 one Such an activation will only be made if the production 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 Carriers, 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). Hydrogel-coated carriers, streptavidin-functionalized carriers, biotin-functionalized carriers r, 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 The invention relates to the biomolecules bonded to a hydrogel-forming surface layer. These surface layer should be very thin by nature, preferably in the dry state, a thickness ≦ 50 μm, in particular ≦ 10 μm and especially ≦ 1 μm, especially 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 becomes a medium Thickness of at least 0.5 nm, preferably at least 1 nm and especially at least 5 nm. Under a hydrogel-forming layer One understands polymeric layers, which are exposed to moisture swell by intercalation of water.

The hydrogel-forming coatings may be bonded directly to the surface of the support, for example to a surface activated by treatment with acids or alkalis, by oxidation (flaming), by electron beam or plasma treatment, or to the adhesive layer described above. Hydrogel-forming coatings are naturally preferred which have on their surface a large number of free functional groups R as described above. Carriers having hydrogel coatings are 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 coated hydrogel ^® coated Slides) and XanTec Bioanalytics GmbH (with polysaccharide hydrogels carrier, the free at its surface carboxyl, amino, hydrazino, SH, streptavidin or having 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, usually 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 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 variety 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.

Under stellate prepolymers one understands such polymers, which are several to a low molecular weight Have central unit bonded polymer chains, wherein the low molecular weight Central unit usually 4 to 100 skeletal atoms such as C atoms, N atoms or O atoms will exhibit.

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%.

Around a crosslinkability of the prepolymers to ensure have functional groups at the ends of your polymer arms, which is complementary to this reactive functional groups to form covalent bonds react. examples for such groups are those in Table 1 under R bwz. R 'called functional Groups.

For the production Hydrogel-forming coatings, which crosslinked from each other stellate prepolymers are constructed, in particular, such prepolymers proven, which have isocyanate groups at the ends of their polymer arms and particularly preferably isocyanate end groups derived from aliphatic Diisocyanates are derived, in particular those as by Addition of isophorone diisocyanate (IPDI) to the chain ends of OH group-terminated star-shaped Präpolymervorstufen to be obtained.

The inventively used for the prepolymers are z. T. known, for. From 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 Providing the essentially crosslinked star-shaped prepolymers built-up organic surface therefore usually includes the deposition of the star-shaped prepolymers on the surface a carrier and subsequent Crosslinking of the reactive groups of the star-shaped prepolymers. The steps of Deposition and networking can if desired repeatedly performed become. 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 carrying out a linking reaction of the reactive groups with one another and Ent removing the solvent; and
• optionally, converting the functional groups on the surface of the surface layer.

In Step i.a. receives one coating, which is composed of uncrosslinked prepolymers and which has a plurality of functional groups on its surface. In this coating becomes a linking reaction of the reactive Groups carried out among themselves, where the linking reaction after application and removal of the solvent by subsequent Treat the coating with a crosslinker or on removal of the solvent can be done when the solution already contains a suitable crosslinker and / or the functional Groups of the prepolymer undergo a chemical reaction with bond formation.

Examples for deposition processes are the immersion of the surface to be coated in the solution of prepolymers as well as the spincoating - here becomes the solution of the prepolymer applied to the rotating at high speed surface to be coated. It goes without saying that you can make ultra-thin coatings the coating measures usually carried out under dust-free conditions.

at the immersion process immersed the substrates in a solution of stellate prepolymer in a suitable solvent and lets subsequently the solution drain, leaving a thinner liquid film with as possible uniform thickness remains on the substrate. This is then dried. The resulting film thickness depends from the concentration of the prepolymer in the solution from. Subsequently, will the networking triggered.

At the Spincoating usually becomes the initially non-rotating substrate with the solution of the star-shaped prepolymers Completely wetted. Subsequently is the substrate to be coated with high numbers of revolutions, in usually at least 50 rpm, often at least 500 rpm, e.g. 500 to 30,000 rpm, preferably above 1000 rpm, z. From 1000 to 10,000 rpm, and more preferably 3000 to 6000 rev / min, set in rotation, the solution largely is thrown off and a thinner Coating film on the surface of the substrate remains. Then, here is a networking triggered.

Usually is the concentration is at least 0.001 mg / ml, preferably at least 0.01 mg / ml, especially at least 0.1 mg / ml, more preferably at least 1 mg / ml. The concentration of the prepolymer in the solution is usually a value of 500 mg / ml, preferably 250 mg / ml and especially 100 mg / ml. About the Concentration lets naturally the thickness control the coating.

to Production of the solutions the prepolymer are basically all solvents suitable, which no or only a low reactivity with respect to functional groups of the to be solved Have prepolymers. Of these, preferred are those which have a high vapor pressure and thus easy to remove. Preference is therefore given to such solvent at normal pressure, a boiling temperature below 150 ° C and preferably below 120 ° C exhibit. examples for suitable solvent are aprotic solvents, z. As ethers such as tetrahydrofuran (THF), dioxane, diethyl ether, tert-butyl methyl ether, aromatic hydrocarbons such as xylenes and toluene, further Acetonitrile, propionitrile and mixtures of these solvents. In the case of prepolymers with OH, SH, carboxyl, (meth) acrylic and oxirane groups are also protic solvent such as water or alcohols, eg. Methanol, ethanol, n-propanol, isopropanol, n-butanol and tert-butanol, as well as their mixtures with aprotic solvents suitable. In the case of prepolymers with isocyanate groups are in addition to the aforementioned aprotic solvents surprisingly also water and mixtures of water with aprotic solvents suitable because the degradation of the isocyanate groups of the prepolymers in such solutions comparatively slowly.

The Crosslinking of the prepolymers can be done in different ways. For example, the prepolymers with a crosslinking agent. As crosslinking agents are in principle all compounds with 2 or more functional groups are suitable, which with the functional groups of the prepolymer under bond formation react.

Therefore takes place according to a first embodiment the invention provides the provision of the organic surface a process that involves linking the reactive groups of the prepolymer by the addition of a compound Vm1 triggers the at least two complementary reactive Groups per molecule having, with the reactive groups of the star-shaped prepolymer react under bond formation.

at the polyfunctional compounds Vm1 may be low molecular weight Act connections, z. B. to aliphatic or cycloaliphatic Diols, triols and tetraols, e.g. As ethylene glycol, butanediol, diethylene glycol, Triethylene glycol, trimethylolpropane, pentaerythritol and the like, aliphatic or cycloaliphatic diamines, triamines or tetramines, z. Ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, 1,8-diamino-3,6-dioxaoctane, diaminocyclohexane, isophoronediamine and like, amino alcohols such as ethanolamine, diethanolamine, aliphatic or cycloaliphatic dithiols to dicarboxylic acids or tricarboxylic acids such as sebacic, glutaric, adipic acid, phthalic acid, isophthalic acid, or the aforementioned diisocyanates, depending on which type of reactive groups having the prepolymer. The low molecular weight polyfunctional compounds have the difference to the prepolymers usually a molecular weight <500 g / mol.

The polyfunctional compound Vm1 can already be found in the solution of prepolymers be included. In the first resulting surface layer from largely uncrosslinked prepolymers then react, for. B. when drying or when heating the Layer the reactive groups of the crosslinking agent with the reactive ones Groups of the prepolymer and thus form a layer of interconnected ones prepolymers which usually still have a variety of functional groups on their surface having.

When In principle, polyfunctional compounds Vm1 are also prepolymers suitable, which have at least four polymer arms A, which in itself soluble in water are and at their free ends a reactive functional group which react with the reactive groups of the prepolymer to form bonds react. In other words, to provide the hydrogel coating can According to the invention also solutions of at least two different prepolymers are used, wherein the one prepolymer has reactive groups which are resistant to the reactive groups of other prepolymers a complementary one Reactivity exhibit.

In another embodiment The invention is the link the reactive groups in the prepolymer triggered by that one admits a sufficient amount of a compound Vm2, the with a portion of the reactive groups of the prepolymer to form functional groups react with a complementary reactivity. In the case of prepolymers With isocyanate groups, for example, the crosslinking can be initiated by one treats the coated article with water, for. B. by Store in a humid atmosphere or under water. Here, a part of the isocyanate groups reacts to form amino groups, which in turn with the remaining Isocyanate groups react to form bonds, with a layer from cross-linked prepolymers arises. The crosslinking agent Vm2 is thus here water. Made like that Coatings have, when freshly prepared, still free isocyanate groups. For prolonged exposure to moisture become the surface arranged isocyanate groups converted into amino groups. In a Variant of this embodiment sets one solutions of the prepolymer in water or in a mixture of water with one or more water-miscible solvents one. examples for suitable, water-miscible solvents are in particular those which are not or only very much slower with the isocyanate groups as water enter into a reaction. Examples are cyclic ones Ethers such as tetrahydrofuran and dioxane, furthermore N-alkylamides such as N-methylpyrrolidone, Dimethylformamide and dimethylacetamide. The mixing ratio of water: solvent is usually in the range of 1: 100 to 100: 1, preferably in the range of 50: 1 to 1:10 and especially in the range 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.

Prefers becomes the networked, star-shaped prepolymers constructed hydrogel layer on a carrier provided that activates and / or provided with an adhesive layer is.

• – 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 Stirnflächen der Säulen, wenigstens doppelt so groß wie der mittlere Abstand der Begrenzungslinie der Stirnfläche einer Säule zu dem Flächenmittelpunkt der Stirnfläche.

The microbioarrays according to the invention can be produced, as already explained, 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, for example 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 mean distance between the boundary line of the end face of a column to the center of area of the end face is on average usually about 50 nm to 50 .mu.m, preferably 100 nm to 25 .mu.m, 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 end faces 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 faces the columns correspond to the geometry of the places P and are in particular circular or ellipsoidal, and more preferably circular.

Corresponding a preferred embodiment The invention relates to the columns on the stamp surface arranged regularly, i.e. the individual distance between adjacent columns is on average no more than 20% and preferably not more than 10% of the mean distance of the columns diverge from one another.

at band-shaped Surveys apply with regard to the distance of the bands to each other, that for the distances adjacent columns said. The average width of the band-shaped elevations is in the rule in the range of 100 nm to 100 microns, preferably 200 nm to 50 microns, in particular 1 μm to 30 μm and especially 2 μm to 20 μm.

The height of Surveys over the stamp base is usually in the range of 0.5 to 500 .mu.m, in particular in the range of 0.5 to 50 μm and especially in the range of 1 to 10 μm. It is for the process according to the invention of secondary importance. To ensure adequate stability of the columns guarantee, is The relationship of height H to the distance A between the boundary lines of the end face of a Pillar and their center of area H: A not more than 10, especially not more than 5. D. h. a cylindrical column with a diameter of about 1 micron becomes a height of 5 μm and in particular 2.5 microns do not exceed. The same applies to The relationship the width of a band-shaped Survey of their height over the Stamp footprint.

Prefers become the columnar elevations a stamp formed by an elastic material to one sufficiently good transmission to ensure. As a rule, the elastic material is a polymeric elastomer, for example, a crosslinked polydimethylsiloxane. Such punches are from the cited prior art known or can be prepared according to 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, pp. 519-523, and cited literature). In general, such stamps are through Impression of a master made using a variety of wells which has the later Surveys of the stamp correspond. This is usually a Mixture of a suitable elastomer precursor with a crosslinker on a textured casting mold that is a negative of the stamp surface (Master, usually a silicon wafer) applied and cured. After curing you get one elastic stamp, whose stamp face the positive image of the master represents. Such methods are extensive in the cited herein State of the art described on the basis of further details is taken. The surface The stamp may also have a hydrophilic surface layer be provided. Such modified stamp, for example by Donzel et al., Adv. Mater. 2001, 13, p. 1164-1167. The production This master is carried out according to methods known per se, for example by photolithographic methods such as those cited herein State of the art and the literature cited therein described in detail are.

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 you bring one or preferably several different from each other biomolecules in at least 2, preferably at least 10, in particular at least 50 and more preferably at least 100, z. B. 100 to 10,000 and in particular 100 to 10,000, preferably up to 5000 and especially up 1000 spatially separate surface areas so on that the biomolecules in every area each a plurality, preferably at least 10, in particular at least 50 and especially at least 100, e.g. B. 100 to 1000 of Elevations of the stamp surface cover. The application of the biomolecules can be known per se Way with the help of usual Spotting techniques take place successively or in particular simultaneously. The equipment required for this purpose are the usual for the spotting techniques Dot matrix printers, dispensers, plotters, micropipettes and the like, with those after a contact procedure or a contact-free Process the liquid drops on a surface can be applied (contact mode, non-contact mode).

In a first embodiment In variant 1, the biomolecules are applied as a solution directly onto the stamp surface. When applying the solutions the biomolecules on the stamp area it has proven to be advantageous to premature evaporation of the solvent to avoid. Suitable methods for this are the cooling of the Stamp and / or saturation the atmosphere with the respective solvent. At the Cool The stamps are usually strongly cooled before spotting and subsequently subjected to the spotting process. In the saturation method, the working chamber with the solvent saturated, in which the biomolecule solved is. Preferably, the spotting conditions are adjusted that the solvents evaporate immediately when applied to the stamp surface have been. The one for this Required conditions can be the expert with few routine experiments easy to detect. The spot process preferably takes place at temperatures in the range of 5 to 50 ° C and especially at about room temperature in a partially solvent saturated The atmosphere, d. H. an atmosphere, the evaporated solvent contains but in an amount below its saturation concentration, preferably in the range of 10 to 80% of the saturation concentration lies.

For a clean transfer the biomolecules on the surface It has proved to be advantageous to the stamp surface after Wetting with the solution of the biomolecule to dry. Furthermore, it has proven to be beneficial when the concentration the biomolecules in the solution in the range of 1 nmol / ml to 1 .mu.mol / ml. The for a clean transfer the biomolecules The contact time required by the stamp is usually a few seconds up to 30 min. longer Contact times are usually not required but not by Disadvantage.

The delivered amount of liquid is significant as it is the size of the area areas B, ie in particular determines the spot size. As a rule, amounts of fluid Applied in the femto to nanoliter range. So are spots with one Diameter of about 150 to 200 microns Available with a contactless applied amount of about 10 nl to 2 ul. Spots with diameters in the range of 10 microns can be generated by Apply by Kapillardispensem amounts of about 0.01 nl to 0.1 ul.

The solvent for the preparation of the solutions the biomolecules depend in a manner known per se according to the nature of the biomolecule to be dissolved. Often it acts it is the solvent around water or around water Mixtures of water with organic solvents with water are infinitely miscible. These include, for example, alcohols such as methanol, ethanol, isopropanol or n-butanol, dimethylformamide, Dimethylfulfoxide, tetrahydrofuran and the like and mixtures thereof with water. Depending on the type of biomolecule, only organic Solvent, e.g. the aforementioned solvents and their mixtures are used, in particular in the case of low molecular weight Links. In addition to the biomolecules can the solutions also 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, furthermore Quaternary ammonium compounds and the like. Furthermore, in particular aqueous solutions of Biomolecules usual 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 Stamping surface of the above-described, preferably formed from an elastic material, structured stamp brought into contact with the release surface. As a result, the biomolecules are transferred to the stamp surface of the structured 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.

in the Connection to the application of biomolecules and the concomitant coupling becomes organic surface usually a washing process performed. Purpose of this washing process In particular, it is to remove uncoupled biomolecules. On the basis to the solution previously used to apply the biomolecules for washing, preferably aqueous solvents or solvent mixtures. 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 expediently chosen so that on the one hand a effective removal of uncoupled biomolecules is ensured, on the other hand the washing solution with the immobilized biomolecules is compatible. Concentrations in the range of 0.01% by weight to 10 Wt .-% and preferably 0.05 wt .-% to 2 wt .-% have as proved appropriate.

According to variant 2 of the method according to the invention succeeds in the production of microarrays immobilized biomolecules, the on its surface at least two, spatially separate areas each having a plurality of spatial separate seats Include P on which biomolecules are also immobilized by a compound V, which with the biomolecules under bond formation can react in spatially separated seats P on the surface, on which the array is to be created, apply and then the biomolecules in several spatial separate surface areas so that applies every area a plurality, preferably on average at least 10, 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. With others Words, first be on the surface, on which the array is to be generated, the connections V at defined places P immobilized.

Then be the biomolecules in several spatial separate surface areas B applied to the thus pretreated surface. Here comes it leads to a chemical binding of the biomolecules and thus to an immobilization in those places P, which are occupied by the compound V, or in which the connection V is bound. Subsequently you rinse unbound biomolecules in a conventional manner from the surface for example by Washing with the o. G. Washing liquids. In this way receives one the array according to the invention, the more, spatially separate areas each having a plurality, spatially separate places P include those with biomolecules are occupied, the places P within a range have a similar occupancy and the places P different surface areas a similar occupancy or occupancy and / or the type of immobilized biomolecule have different occupancy.

The Applying the compounds V localized in the places P on the surface, on which the biomolecules to be immobilized succeed, for example, in the above described μCP technology. For this is the connection V evenly on the stamp surface applied, for example by contacting the stamp surface with a solution compound V. Subsequently usually becomes any solvent removed and then the thus treated stamp area, which now with the compound V is occupied, with the surface, on which the biomolecules be immobilized, brought into contact. Basically However, the compound V also according to other methods of preparation be applied to the surface of 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. As a carrier but with hydrogel-forming surface are also the above-mentioned commercial products, as sold by XanTec Bioanalytics GmBH and Perkin-Eimer.

• – 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 ge trennten 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 immobilized biomolecules in at least two spatially ge separated surface areas B such that the surface areas in each case cover a plurality of places, which are occupied by the compound V, the biomolecules in the squares be immobilized, and
• - subsequently removed non-immobilized biomolecules.

The solvent and the concentration of compound V in the solution depends on the Type of compound V. The optimal solvents and concentrations can by the skilled person in a simple way with few routine experiments be determined. Preference is given in principle to those solvents which on the one hand solve the compound V in the desired concentration and easy to remove at the same time, especially solvents with a boiling temperature below 120 ° C. Examples of solvents are Water, watery Solvent mixtures and organic solvents, e.g. the solvents mentioned in variant 1 and aqueous Solvent mixtures.

The Applying the biomolecules in spatial separate surface areas can in a conventional manner by means of the usual spotting techniques, as in variant 1 in detail described, done. In terms of the exact process conditions, in particular the temperature and the choice of solvents the above statements apply corresponding.

Of the usually required washing step is done by rinsing with a suitable washing solution. Here apply the statements made to variant 1 accordingly.

The used in variant 2 compounds V are usually those Compounds that on the one hand form a bond to the surface, on the biomolecules be immobilized, and at the same time a bond to special Binding sites in the biomolecules can train. The connection 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 As a rule, the compounds V have at least one of the abovementioned Groups R 'up, which with the reactive groups R of the surface, in particular the hydrogel-forming surface react to form a covalent bond. Farther the compounds V have at least one white binding center which can bind to 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 coordinatively 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-pyridylmethylaminoacetate, furthermore polycarboxylic acids preferably having at least 2, eg 2, 3, 4, 5 or 6 carboxyl groups, especially compounds containing at least 2, z. 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 described carboxymethylated aspartic acid, N, N, N-tris (carboxymethyl) ethylenediamine, N, N, N, N-tetra (carboxymethyl) ethylenediamine (EDTA) and the like, further orthophosphoserine and the like. 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 in complexed form or preferably in uncomplicated form on the surface who the. 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 complexing the surface-bound Compound V, a washing step will be introduced to remove excess Compound V and / or excess metal ions.

in the Trap of carboxylic acids as compounds V, these can also be prepared in carboxyl-protected form, i. H. as an ester. Suitable protecting groups for carboxyl groups are known in the art from the relevant literature, wherein preference is given to those groups which can be split off under conditions the surface, z. B. does not affect the hydrogel layer or even destroy it. example this is the tert-butyl group thermally, e.g. B. by heating at 150-220 ° C, preferably at reduced pressure, remove.

at the solutions used for complexing are usually around watery solutions water Salts of the metal ions, e.g. solutions chlorides, nitrates, sulfates, acetates etc. Concentration of Metal ions in the solution is usually in the range of 1 mM to 1 M. Depending on the nature of the Ligands and the nature of the metal ion is the pH of the solution in Rule 5 to 12, in particular 6 to 11. The solutions may possibly still weak Complexing agents, e.g. Ammonia to the solubility the metal ions at higher to ensure pH values.

In another embodiment of the method 2, the compounds V in addition to the connection the reactive group R 'required for compound V, form a group, the above at least 2 and preferably over at least 3 hydrogen bonds to the biomolecule can bind. Such groups are, for example, oligonucleotide groups with at least 2, preferably at least 3, e.g. B. 3 to 100 and especially 3 to 20 nucleotide bases. To these groups can such biomolecules bind, which have a complementary oligonucleotide sequence, the proportion of complementary Bases should be at least 50%.

In a further embodiment the invention is used as compounds V sol che substances, the known way with biomolecules enter into an affinitive interaction. Examples of suitable compounds V are ligands, e.g. Haptens and antibodies that are so functionalized are, i. have a group R ', that with the surface react to form a covalent bond. This includes as well chemical compounds V, in addition to at least one reactive group R 'another one molecular group or amino acid sequence exhibit that with the biomolecule in the sense of an affine interaction, for example one Protein ligand or protein-protein interaction can bind. Examples are biotin derivatives, modified with a group R ' are and thus an anchor for biomolecules form, which have a streptavidin or avidin sequence, as well as Streptavidin and avidin derivatives modified with a group R ', so that they have an anchor for biotinylated biomolecules represent.

The to be immobilized biomolecules naturally have at least a binding site fixed to those on the surface Substance V is a bond, for example a covalent bond, a coordinative bond, a hydrogen bond or an affinity Can form bond. Examples of such groups were partially already discussed above in the 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 Bio molecule are complementary to two adjacent base pairs of the substance V oligonucleotide sequence. Preferably, the match is at least 50%.

at a binding of the biomolecule via affinitive Interactions, the biomolecule naturally has at least one group, those with the substance V in the sense of a protein ligand or protein-protein interaction can bind, for example, in a streptavidin-modified Compound V, a biotin unit or a biotin-modified Substance as compound V is a streptavidin unit.

In a preferred embodiment of the invention, the substance V is a substance, which is a transition metal ion coordinated the type described above. In this case, that indicates biomolecule a molecular group which, in turn, acts as a ligand, preferably as a chelating agent Ligand with the transition metal ion can make a coordinating bond. However, it is preferable the gelatizing effect of this group is not so strong that it to a complete displacement the gelating part of the compound V comes. chelating Ligands in the biomolecules are in particular those which at least two, in particular 2 or 3 have 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. Preferred among these In particular, groups which are at least one or preferably 2 or 3, spatially have adjacent imidazole groups. Preferred among these are in particular, such groups as histidine or oligohistidine sequences are derived. Such groups are also called histidine tag or His tag designated.

Such functionalized biomolecules and methods of functionalizing non-functionalized biomolecules 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 as well literature quoted there (metal complexing intermediary tags), Christian Jakob, Dissertation of the University of Göttingen, 2001 and quoted therein Literature (Newer Methods and Overview to biotinylation).

According to one particularly preferred embodiment variant 2 of the method according to the invention is the hydrogel-forming surface layer from the above-described crosslinked star-shaped prepolymers constructed, which have on average at least 4 polymer arms A, the for Viewed water soluble are. The application of the substance V can before, preferably however during or followed by the crosslinking of the prepolymers. Preferably The hydrogel-forming surface layer has a plurality of Functional groups R mentioned above in Table 1 and in particular isocyanate groups and / or amino groups.

at the particularly preferred embodiment of variant 2 with hydrogel-forming surface layer have proven in particular those compounds V, the one Immobilization of the biomolecule by one over a transition metal mediate coordinated bonding. Accordingly are the compounds V are preferably among the above-mentioned chelating Compounds selected, especially among the ones mentioned above polycarboxylic with at least 2 carboxyl groups, e.g. 2, 3, 4, 5 or 6 carboxyl groups, which still have a functional group, which with the functional Groups of hydrogel-forming surface layer under formation a covalent bond reacts. Examples are in particular the ones already mentioned Compounds with at least two, e.g. 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 been proven in particular for the immobilization of complex biomolecules, the easily denature, especially for the immobilization of proteins. A very particularly preferred embodiment of variant 2 with Hydrogel-forming surface layer thus relates to the immobilization of proteins, of proteins, which bind to the surface via a coordinative bond and especially of proteins with histidine tags or similar tags, as reported by Stiborova et al., Biotechnology and Bioengineering, 2003, 82, 605-611 and in the literature cited therein.

Regarding the Applying the areas of the biomolecules to those with the compound V structured surface applies in principle the above for the application of biomolecules on stamp surfaces said, whereby direct spotting (non-contact or with contact) is preferred. In this case, the biomolecule in pure form or as a crude product a functionalization, since that with the binding site provided biomolecule selectively binds to the occupied with the substance V places P.

In usually closes the application of the biomolecule a washing step. This washing step serves to unbound biomolecule from the surface of the array. As washing liquids are basically such liquids consider which the biomolecule solve and at the same time the surface do not damage. Particularly suitable are aqueous Solvent, in addition to water even small proportions, preferably not more than 40 and in particular not more than 10% organic, water-miscible Solvent, For example, alcohols such as methanol, ethanol and the like can. These washings can also surface-active Contain substances as mentioned above. The proportion of surface-active Substances is usually not more than 5 wt .-%, based on the washing liquid be. Furthermore you can the washing liquids also common in biotechnology Buffer, especially PBS buffer SSC buffer and comparable contain, in the usual for this Concentrations. The type of additional Ingredients depends in a conventional manner on the type of the on the surface immobilized biomolecule.

are in one or more areas the immobilized biomolecules with a Marked, so now can a quality control of the array. For this purpose, the array with a detection system corresponding to the marking be measured. about the measured amount of immobilized label can be particularly the coupling efficiency and thus the immobilization of biomolecules in relation on area, homogeneity and assess density.

Basically all marking types are suitable. Preferably, the label chosen so that she not or only minimally interferes with the subsequent analysis, the use of the arrays so not or only slightly influenced. To Above all, if necessary, necessary markings of the sample, ie in particular of nucleic acid targets to be observed. On the other hand, the mark can be chosen that she interacts with the labeling of the sample. That of interactive Marks outgoing signal may be distinguishable from the one that emanates from the corresponding non-interacting markers. For example, the interaction may affect signal intensity, e.g. strengthen or quenching, or modifying the signal, e.g. absorbed the wavelength or emitted light.

Marking systems suitable for the purposes of the invention are those which can be detected, for example, 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, chemi- or bioluminescent markers, transition metals, which are usually chelated, or enzymes, for example horseradish peroxidase or alkaline phosphatase and the detection reactions coupled thereto, as well as indirect labeling systems, for example haptens, such as biotin or digoxigenin, which can be recognized by corresponding detection systems.

advantageous Chromophores have an intense color, that of the surrounding ones molecules only slightly is absorbed. Dye classes, such as quinolines, triarylmethanes, Acridines, alizarines, phthaleins, azo compounds, anthraquinones, Cyanines, phenazathionium compounds or phenazoxonium compounds here representative for the broad spectrum according to the invention suitable chromophores called.

fluorescent Markers are preferred. You get strong signals with little Background, high resolution and high sensitivity. According to the invention of advantage is that one and the same fluorophore depending on the excitation and detection principle several can emit distinguishable radiations. Fluorophores can be alone or in combination with a quencher (e.g., molecular beacons) be used. Suitable fluorophores are, for example, aminomethylcoumariness acid (AMCA, blue), EDANS, BODIPY 493/503; FL; FL Br2; 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 Fluorine-carboxylic acids, in particular of the type 647 and 532, e.g. as succinimidyl ester, and IRD41.

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

object 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; Toxicogenomics.

in principle Arrays of the invention are suitable as well as noncompetitive as well as for competitive analytical procedures (assays).

at noncompetitive assays, the analyte (analyte) with those immobilized on the surface biomolecules in interaction. As a rule, the analyte is marked in advance, possible but is also a marker after the analyte already with the immobilized biomolecules has interacted, e.g. using primer extension or Rolling cycle PCR. In these cases receives one measurement signal, the the bigger, the more analyte is present. It is also possible that through the interaction the sample with the on the surface immobilized substances, the fluorescence of the immobilized substances is changed (Weakening, gain e.g. Molecular beacons) or the activity of an enzyme is changed and this change as a measuring signal is registered. examples for Non-competitive assays are hybridization reactions of PCR products or labeled DNA / RNA on nucleic acids immobilized on the surface, in particular Oligonucleotides or cDNA, as well as sandwich immunoassays.

at Competitive method, the sample is a labeled substance (marker) added, the similar binding properties to those on the surface immobilized biomolecules has like the analyte itself. There is a competitive reaction between Analyte and marker around the limited number of binding sites on the surface instead of. You get a signal, the lower the more analyte is present. Examples of competivie Assays are immunoassays (ELISA) and receptor assays).

The 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 of a conventional polydimethylsiloxane stamper having a plurality of columnar protrusions is shown. 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 fluorescence-labeled streptavidin was immobilized via biotin.

5 FIG. 2: Partial view of a fluorescence micrograph of an array prepared according to Variant 2 with a multiplicity of sites on which green fluorescent protein with histidine tag is assayed by means of Ly sin-NTA-nickel (II) was immobilized.

1. Aminofunctionalization of the carrier:

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

to Aminofunktionalisierung were the pretreated substrates with coated with an aminosilian monolayer. For this purpose, the from the Water sampled and nitrogen-blown sample into a glove box transferred. There, the substrates were stored in a 0.5% (v / v) solution of (3-aminopropyl) trimethoxylsilane stored in dry toluene for 16 h, then washed thoroughly with toluene and stored therein and in a nitrogen atmosphere before use Glove box dried by means of a filtered stream of nitrogen.

II. Production of a carrier with a hydrogel layer containing on its surface functional groups R having.

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 modified at the ends of the polymer arms with isophorone diisocyanate (prepared analogously to Example 11 of US Pat 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 carrier prepared in 1 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 FIG. 1, 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 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 2 min with the amino-thin prepared in 1 Tionalized glass carrier brought into contact. Subsequently, the surface was examined by fluorescence microscopy. In this way you get that in the 3 and 3a shown pattern.

In analogous way also protein arrays and nucleotide arrays are made:

Example 2: Preparation an oligonucleotide array

One rectangular polydimethyldisiloxane stamp, made e.g. to Tan et al., 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, medium Distance 20 μm), will be in accordance with the requirement by Donzel et al., Adv. Mater. 2001, 13, 1164, in the oxygen plasma modified hydrophilic. On this stamp are by means of micropipette 2 spots of 0.1 μl of a solution of 3'-5'-fluorescence-labeled aminoterminiertem Oligonucleotide in PBS buffer at a concentration of 100 pmol / ul applied. After drying in an inert gas stream, the thus prepared stamp will be with a surface freshly prepared according to II Example 1 2 min brought into contact. Subsequently is washed with PBS. You get in this way, an almost error-free image of the stamp, the 2 separate regions in which the nucleotides a very size Number of spatial separate places prove how fluorescence microscopy can be detected.

Example 3 Preparation a biotin-streptavidin-array:

Of the Polydimethyldisiloxan stamp used in Example 2 is with a solution of biotinamidocaproic acid N-hydroxysuccinimide ester in dimethylformamide (10 μg / ml) wetted and then dried. The stamp thus obtained is 2 min. with the II example 2 prepared coating brought into contact. Subsequently, will the resulting coating for removing unbound Ester washed successively with dimethylformamide and water and dried in an argon stream. That's how you got an array with circular Areas of immobilized biotin.

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 4 ).

Example 4: Preparation a protein array over complexation

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 support, for 10 min. stored and then washed with PBS buffer. The resulting array is examined by fluorescence microscopy. You get that in the 5 shown pattern.

Claims (35)

Microarray of immobilized biomolecules, the at least two on a surface spatial has separated areas B, the each several, spatially separate places P include on which biomolecules are immobilized, wherein the mean distance of the boundary line a region B to its centroid the range of 10 microns up to 250 μm lies. A microarray according to claim 1, wherein the mean distance neighboring squares 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 centroid. 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 microns. Microarray according to one of the preceding claims, wherein the mean distance between adjacent places in the range from 200 nm to 200 microns. Microarray according to one of the preceding claims, wherein the places P by a circular, ellipsoidal or rectangular surface to be discribed. Microarray according to one of the preceding claims, wherein the places P are arranged regularly within the areas B. Microarray according to one of the preceding claims, wherein the areas B comprise at least 10 spatially separated areas P spatially separated from one another. Microarray according to one of the preceding claims, wherein at least two of the surface areas B in the nature and / or the surface density of the immobilized biomolecules differ. Microarray according to one of the preceding claims, wherein the areas B a circular Have geometry. Microarray according to one of the preceding claims, wherein the mean distance between adjacent areas is at least 50 μm. Microarray according to one of the preceding claims, comprising one on a carrier arranged organic surface layer, on which the biomolecules are immobilized. A microarray according to claim 11, wherein the surface layer comprises at least one hydrogel-forming layer. Microarray according to claim 12, wherein the hydrogel-forming surface layer essentially of interconnected star-shaped prepolymers is constructed, which have on average at least 4 polymer arms A, the for Viewed water soluble are. Microarray according to claim 12 or 13, wherein the hydrogel-forming surface layer has on average a thickness of 1 nm to 100 microns in the dry state. Method for producing a microarray immobilized biomolecules the at least two spatially separated on a surface arranged areas B, each having a plurality of spatially separated courts Include P on which biomolecules are immobilized by placing on a stamp surface, which a variety of raised areas has one or more mutually different biomolecules in at least two, preferably at least ten and in particular at least one hundred spatially separate surface areas B so that the biomolecules in the surface areas B each cover a plurality of the raised areas of the stamp surface and then the stamp surface with the surface, on the biomolecules be immobilized, in contact and if necessary non-immobilized biomolecules away. Method for producing a microarray immobilized biomolecules the at least two spatially separated on a surface arranged areas B, each having a plurality of spatially separated courts Include P on which biomolecules are immobilized by placing on a stamp surface, which is a variety of raised surfaces a compound V which causes immobilization of the biomolecules, Apply, the thus treated stamp surface with surface on the biomolecules To be immobilized, brings into contact, whereby to obtain a surface, the a variety of spatial separate places P, which are occupied by the compound V, and then a or several mutually different solutions of the immobilized biomolecules in at least two from each other spatially separate surface areas B so that the surface areas each a variety of places, which are occupied by the compound V cover, wherein the biomolecules to the seats P are immobilized, and then removed non-immobilized biomolecules. The method of claim 16, wherein the compound which effects immobilization of the biomolecules is selected from compounds that form a covalent or ionic bond with the surface and form the one, covalent, ionic, coordinative or affinitive bond with the biomolecule to be immobilized. A method according to any one of claims 15 to 17, wherein the middle one Distance of adjacent elevations on the stamp surface at least twice as big like the mean distance of the boundary line of the face of a Survey of the center of area the face. A method according to any one of claims 15 to 18, wherein the middle one Distance between the boundary line of the frontal area of the elevations and the center of area the face on average 50 nm to 50 μm is. A method according to any one of claims 15 to 19, wherein the middle one Distance of adjacent elevations on the stamp surface in the area from 200 nm to 200 μm lies. Method according to one of claims 15 to 20, wherein the end faces of the Elevations on the stamp surface circular, ellipsoidal or rectangular. Method according to one of claims 15 to 21, wherein surveys on the stamp surface arranged regularly are. Method according to one of claims 15 to 22, wherein surveys on the stamp surface on average a height in the range of 0.5 to 500 μm exhibit and the ratio H: A of medium height N to the average distance A between the boundary lines of the end faces of Elevations and the center of area of the faces not more than 10. Method according to one of claims 15 to 23, wherein the elevations of the stamp are formed by an elastomeric material. The method of claim 24, wherein the elastomeric Material is polydimethylsiloxane. Method according to one of claims 15 to 25, wherein the concentration the biomolecules in the solution 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 immobilizes the immoblisierenden biomolecules caused by a hydrogel-forming surface layer is formed. A method according to claim 27, wherein you - in a hydrogel-forming first step on a carrier surface layer provides a variety of functional on its surface Has groups with complementary thereto functional groups Bond formation react, - on a stamp surface, the a variety of raised areas has a compound V which has one or more functional Has groups with the functional groups of the surface below Formation of a covalent bond can react, and also one or several of which have different functional groups, the one Can form binding to the biomolecule to be immobilized; - the like treated stamp surface brings into contact with the hydrogel-forming surface layer, where you have a surface gets the a variety of spatial separate places P, which are occupied by the connection V, - then one or several different solutions of the immobilized from one another biomolecules in at least two from each other spatially separate surface areas B so that the surface areas each a variety of places, which are occupied by the compound V cover, wherein the biomolecules to the seats be immobilized, and - not afterwards immobilized biomolecules away. The method of claim 28, wherein the hydrogel-forming surface layer is composed of interconnected star-shaped prepolymers, which have on average at least 4 polymer arms A, seen in isolation water soluble are. The method of claim 28 or 29, wherein the functional Groups on the surface layer are selected from isocyanate groups and amino groups. A method according to any one of claims 28 to 30, wherein the binding the compound V to the biomolecule one over a transition metal mediated coordinative binding. The method of claim 31, wherein the compound V is a derivative of the polycarboxylic acid with at least 2 carboxyl groups, which is a functional group having the functional groups of the hydrogel-forming ends surface layer reacting to form a covalent bond. A method according to claim 31 or 32, wherein the immobilizing biomolecule is a protein with a His tag. Device based on a microarray after a the 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.