WO2005014852A1

WO2005014852A1 - Microarrays of immobilized biomolecules, production thereof, and use thereof

Info

Publication number: WO2005014852A1
Application number: PCT/EP2004/007473
Authority: WO
Inventors: Haitao Rong; Stefano Levi; Jürgen GROLL; Martin Möller
Original assignee: Sustech Gmbh & Co. Kg
Priority date: 2003-07-18
Filing date: 2004-07-08
Publication date: 2005-02-17
Also published as: DE10332848A1; DE10332848B4

Abstract

The invention relates to microarrays of immobilized biomolecules comprising at least two spatially separated sectors B that are located on a surface. Each sector is provided with several spatially separated points P at which the biomolecules are immobilized. On average, the dimension of said sections corresponds to a distance between the boundary line of a respective sector B and the centroid thereof, said distance ranging from 10 µm to 250 µm. The invention also relates to a method for producing micro-bioarrays, according to which biomolecules are applied at a local resolution to an adequately structured matrix surface by means of spotting techniques such that each spot covers several of the raised zones of the matrix surface, e.g. column-shaped elevations, whereupon the matrix surface is contacted with the surface on which the biomolecules are to be immobilized and surplus biomolecules, i.e. biomolecules that are not immobilized, are removed if necessary (variant 1); alternatively, a pattern of compounds V that cause biomolecules to immobilize on a surface and are also called coupling means V in the description, is applied to the surface by means of µCP technology, said pattern corresponding to the array, whereby an array is obtained which is provided with a plurality of spatially separated points P that are occupied by compound V, whereupon a solution of said biomolecules is spotted onto the prepared surface such that one spot covers several points P to which compound V is bound, and surplus biomolecules are then removed (variant 2).

Description

Microarrays of immobilized biomolecules, their production and their use

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

With the increasing practical importance of molecular biological analysis, the established standard techniques, which often involve days and weeks, are very labor intensive and costly. New test formats such as biochips should enable enormous time and cost savings. Since many different substances immobilized in a very small space can be brought into contact with a single sample to be analyzed simultaneously with this technology, the information content obtained from a chip per measurement is very high.

An overview of the best-known biochip systems can be found, for example, in Bowell 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 is given by 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 over 400 different capture samples that are built up base by base on the glass wafer. The chips are characterized by a very high information density of up to 40,000 oligonucleotides / cm ^2. The individual "spots" are rectangular.

Low density arrays are available, for example, from Genometrix. A maximum of 250 spots with a diameter of 50 μm are printed with a capillary pin bundle of up to 1000 individual capillaries into the cavities of a 96-well microtiter plate. Conventional techniques are used for coupling to the surface, which are based in particular on amino silanizations and NHS-activated haptens, epoxy-activated surfaces, carbodiimide couplings on carboxyl groups and biotin / streptavidin bonds (cf. WO 97/18226; EP 0910570 A1; WO 98/29736).

Several methods are currently available for producing microbioarrays. The most common and also used for the mass production of biochips are lithography techniques and spotting techniques (see Cheng et al. (Editor), Biochip technology, Harvard Academic Publishers 2001). The lithography techniques are lithographic processes that are borrowed from semiconductor technology and with which molecules can be applied to a surface in a localized manner. For example, US Pat. No. 5,599,695 describes a method for producing oligonucleotide arrays, in which a surface which is covered with a molecular layer of protected compounds which permit the connection of oligonucleotides is covered with a mask which is not covered by the mask Deprotected areas and brings the surface treated in this way into contact with oligonucleotides. The oligonucleotides are bound to the deprotected areas of the surface and thus form an arrangement of areas / places occupied by oligonucleotides corresponding to the mask. Such techniques are particularly suitable for the production of oligonucleotide arrays with a very high spot density (high density chips) and allow the resolution of a few 100 nm. However, such lithographic processes are generally not compatible with biologically active molecules such as proteins and cells , Furthermore, the equipment costs for such processes are very high.

Spotting techniques are printing processes. The biomolecules are applied as solutions with a printing system in the form of a regular dot pattern on a surface. The heart of the printing system is a printhead that either has a series of needles or is a microdosing head. The latter allows the parallel printing of different solutions of the biomolecules. When printing with a microdosing head, different liquids can be filled into the reservoirs which are connected to the microdosing head. Several 100 to 1000 printing processes can be carried out one after the other without refilling, thus creating a corresponding number of spots on a surface. With the previously common spotting techniques, however, it is not possible to produce spots with a diameter below 100 μm. This technique is therefore not suitable for producing so-called high-density chips, but only low- and medium-density chips. 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 μm to a surface. In this context, those of Boland et al. (222nd ACS National Meeting 2001, pp. 26-30) developed the method, the nanopipette method (Bruckbauer et al. J. Am. Chem. Soc. 2002, 124, 8810), the 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 involve high equipment costs.

Recently, a stamp technique, the so-called micro-contact printing (μCP), has been used variously to produce arrays from 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 WO01 / 67104). With the μCP technology, which was originally developed for other purposes, the biomolecules to be immobilized can be localized, i.e. H. be applied to a surface in defined areas. As a rule, the procedure is such that the stamp surface is first brought into contact with a solution of the biomolecule to be immobilized, the stamp is dried if necessary, and then the stamp surface is brought into contact with the surface to be structured. Here, the biomolecule is transferred to the surface in those areas in which the stamp surface is in contact with the surface. However, surface areas that are not in contact with the stamp surface remain unchanged. In this way, defined areas on the surface are obtained, which are covered with biomolecules and thus represent an image of the stamp. With the μCP technology, structures with a resolution down to about 100 nm can be produced. In contrast to the spotting technique, which allows the simultaneous application of several areas to which different biomolecules are occupied, the μCP technique can only be used to create identical areas. For the production of biochips, however, arrays are generally required which have several regions which differ from one another with regard to the type and / or the occupancy density of the biomolecules.

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

Chieng et al. (Sensor and Actuators 2002, B 83, pp. 22-29) describe a process for the production of protein arrays, in which one first inserts a protein solution into so-called micro-wells, then picks up and absorbs the protein solution from these micro-wells printed a suitable surface. In this way, protein arrays can be produced which have several regions which differ in the type of protein assignment. With the measures described there, however, only comparatively large areas (edge length> 500 μm) can be produced. A similar process is described by Renault et al., Angew. Chem. Int. Ed. 2002, 41, 2320. Here, a mask with a multiplicity of through holes is placed on a stamp surface, solutions of different antigens being filled into the holes. As a result, the antigens are applied to the stamp surface. The stamp surface is then brought into contact with a solution of antibodies and then printed on a suitable surface. In this way it is also possible to produce protein arrays which have several different regions with regard to their proteins. However, only large areas can be produced with both methods. Both methods are extremely complicated to carry out and have the disadvantage of low selectivity. This is due to the capillary forces of the protein liquids.

WO 02/48676 describes multilayer microarrays for the production of bioarrays, which comprises a first membrane arranged on a surface with very fine through holes and a second membrane arranged above it with comparatively large holes. Solutions of biomolecules are filled into the large holes and penetrate the surface through the fine holes. After removing the lower membrane, a pattern of immobilized biomolecules is obtained on the surface. In this way, however, only bioarrays with very large areas of different biomolecules can be obtained. In addition, these areas must be at a comparatively large distance from one another in order to achieve sufficient resolution, since capillary forces cause the biomolecules to smear beyond the area limits. With the last three methods only those biochips can be produced that one have a comparatively small number of regions with different biomolecules. In addition, these procedures are very complex to carry out.

The object of the present invention is to provide arrays of immobilized biomolecules which on the one hand have as large a number of areas as possible which can be occupied by different biomolecules and which have an increased resolution with respect to the different biomolecules. In addition, these arrays should be easy to manufacture without the use of expensive techniques, such as are required for high-resolution spotting techniques.

This object is achieved by microarrays of immobilized biomolecules, hereinafter also called microbioarrays, which have at least two, preferably at least ten, in particular at least fifty and in particular at least one hundred spatially separated surface areas B which are arranged on one surface, each surface area having a plurality of, preferably has on average at least ten, in particular at least one hundred, spatially separated locations P, on which the biomolecules are immobilized, and the extent of the regions, in particular a distance between the boundary line of a respective region B and its center point in the region of 10 μm to 250 μm in particular Corresponds to 25 μm to 200 μm. Such microbioarrays are the subject of the present invention.

These microbioarrays can surprisingly be produced by a hitherto unknown and easy-to-carry out combination of conventional spotting techniques with the μCP technique (see the prior art cited in the introduction), with either biomolecules being applied in a spatially resolved manner to a suitably structured stamp surface using spotting techniques, so that each spot covers several of the raised areas of the stamp surface, for example columnar elevations, and then brings the stamp surface into contact with the surface on which the biomolecules are to be immobilized and, if necessary, removes excess, ie non-immobilized, biomolecules (variant 1); or by means of μCP technology, a pattern of compounds V corresponding to the array, which bring about an immobilization of biomolecules on a surface, hereinafter also referred to as coupling agent V, is applied to the surface, an array being obtained which has a plurality of spatially separated places P which are occupied with the compound V, then a solution of the biomo- spot on the surface provided so that each spot covers several places P and then remove excess biomolecules (variant 2).

Both process variants combine the high resolution achieved by the μCP technique and the advantages of the spotting technique, which allows different areas of immobilized biomolecules to be generated in a simple manner.

The invention thus relates to a method for producing microbioarrays, in which, according to variant 1, one or more different biomolecules in at least two, preferably at least ten, in particular at least one, on a stamp surface which has a plurality of raised areas (hereinafter referred to as elevations) 50, especially at least 100, e.g. B. 100 to 10,000 spatially separated surface areas B (= spots) so that the biomolecules in the surface areas B each have a plurality of elevations, as a rule at least 10, preferably at least 50, in particular at least 100, for. B. 100 to 10,000 surveys of the stamp surface, then the stamp surface with the surface on which the biomolecules are to be immobilized, in contact and possibly non-immobilized biomolecules, for. B. removed by washing.

The number, arrangement and size of the raised areas of the stamp area covered by the biomolecules in the surface areas B each correspond to the number, arrangement and size of the places P of an area on which the biomolecules are immobilized. The number, arrangement and size of the surface areas B corresponds in a first approximation to the number, arrangement and size of the spots on the stamp surface.

The same arrangement can also be achieved by variant 2 of the method according to the invention. For this purpose, a compound V is applied to a stamp surface which has a large number of elevations and stamped onto the surface on which the biomolecules are to be immobilized. In this way, a surface is obtained which has a multiplicity of spatially separated places P, the number, arrangement and size of the places P corresponding to the number, arrangement and size of the raised areas of the stamp surface and which are occupied by the connection V. Then one or more different solutions of the biomolecules to be immobilized are brought into at least two, preferably at least ten, in particular at least fifty and especially at least one hundred, e.g. B. one hundred to ten thousand from each other 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, e.g. B. cover a hundred to ten thousand of the places P, which are occupied by the connection V. The compound V thereby causes a binding, ie immobilization of the biomolecules on the surface. Then the immobilized biomolecules will be removed. 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 method according to the invention according to variants 1 and 2 is not limited to the production of the arrays according to the invention but also allows the production of arrays with an arrangement of places P and regions B which corresponds to the arrays according to the invention, but in which the surface region B also has larger dimensions , e.g. > 500 μm or more. However, these methods are preferably used to produce the arrays according to the invention.

The invention has a number of advantages:

The microbioarrays according to the invention allow an increase in capacity compared to conventional biochips produced by spotting techniques, since the latter generally require four to ten spots with the same biomolecule occupancy in order to ensure a sufficiently accurate detection of the analyte. With the arrays according to the invention, on the other hand, only a surface area that corresponds in size to a spot is required. Due to the division of the areas into several places P, a significant increase in sensitivity, ie a better signal / noise ratio in the detection of analytes, is also achieved. Significantly smaller amounts of biomolecules are also required for production. Furthermore, the arrays according to the invention are comparatively simple to manufacture. Even with a resolution that corresponds to that of a high-density biochip, the effort for their production corresponds only to the manufacturing effort 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 technology allows the stamping process to be used repeatedly without spotting steps being necessary in between. Due to the connection of the biomolecules to places P within a respective area, the area boundaries are comparatively sharp, in contrast to conventional spots, ie "coffee stain shapes" as used in conventional Common arrays that are produced by spotting technology are avoided. This allows a very dense arrangement of the individual surface areas on the surface of the array without fear of smearing these areas. Furthermore, it is surprisingly found that the method according to the invention does not occur in the case of more complex biomolecules in which there is a risk of denaturation, in particular in the case of proteins. A particularly preferred embodiment of the invention therefore relates to protein microbioarrays, that is to say microarrays of immobilized proteins.

The term "array" denotes an arrangement of defined places P, in particular a local assignment of certain substances to defined places P, different places P being able to be assigned the same or different substances. According to the invention, the assigned substances are biomolecules. The spatial dimensions of the places P are in the μm range or in the sub-μm range. The arrays according to the invention are therefore also referred to as microbioarrays or as microarrays of immobilized biomolecules.

The microbioarrays according to the invention have a plurality of regions, which in turn are subdivided into spatially separated locations P. The same substance type is assigned to the places P within each area, although it is also conceivable to assign mixtures of two or more types of substance to the places P of an area. Places P in different areas will generally differ from one another with regard to the composition and / or the occupancy density of the respective substance type. As a rule, different biomolecules will be immobilized on places P in different areas.

The term “biochip” here denotes an array of biomolecules that are immobilized on a solid support.

The term “biomolecules” denotes any biochemical and biological substances, both as individual molecules and as several interacting molecules. Examples include:

• 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, for example Fab, Fab ', F (ab) ₂ , synthetic;

Proteins, e.g. Allergens, inhibitors, receptors;

Enzymes, e.g. Peroxidases, alkaline phosphatases, glucose oxidase, nucleases;

Small molecules (haptens), e.g. Pesticides, hormones, amino acids, antibiotics, pharmaceuticals, dyes, synthetic receptors, receptor ligands.

According to a further aspect, the term “biomolecule” defines the ability of a substance to be able to interact with a biological sample or a part thereof, in particular the analyte.

According to a special aspect, a certain type of biomolecule can be called a ligand. Ligands interact with and in particular bind - preferably specifically - to certain targets.

In the arrays according to the invention, the biomolecules are immobilized on defined positions P on a surface. These places P have a certain shape and extension. The shape of the places P is basically arbitrary. Preferably, the shape of the places P is uniform and has, for example, a polygon, e.g. B. an n-square with n = 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and among them preferably a rectangular geometry, a circular or an ellipsoidal geometry. Mixed forms of these geometries are also generally suitable, e.g. straight ovals. The places P preferably have a circular or ellipsoidal geometry. The spatial extent of these places P naturally correlates with the distance between the boundary line of a respective place and its center point. This distance is generally in the range from 50 nm to 50 μm, preferably 100 nm to 25 μm, in particular 0.25 μm to 20 μm and especially 0.5 μm to 10 μm.

The distance between adjacent places P, defined by the distance of the surface center points of adjacent places P, is generally in the range from 200 nm to 200 μm, often 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 from 10 μm to 90 μm. The mean minimum distance between adjacent places P, defined by the minimum distance that the boundary lines of two adjacent places P have, is generally in the range from 100 nm to 150 μm, often in the range from 500 nm to 120 μm, in particular in the range from 2 μm to 100 μm, and especially in the range from 5 μm to 80 μm (number average).

The arrangement of the places P on the surface can be regular or irregular. Regular arrangements are preferred, i. H. Arrangements in which the mean distance, which two adjacent places P have, does not vary by more than 20% and especially not more than 10%, or deviates from the mean.

According to the invention, the locations P of the array can be assigned to different surface areas B. The surface areas B arranged on the microbioarrays according to the invention generally have a uniform geometry, preferably an elipsoidal or circular geometry. Their extension naturally correlates with the distance between the boundary line of a respective area B and its center point. This distance is generally in the range from 10 μm to 250 μm, preferably in the range from 25 μm to 200 μm and in particular in the range from 30 μm to 150 μm. In the case of circular surface areas B, this corresponds to half the diameter of the respective surface area. The respective surface areas B generally form a uniform arrangement on the surface of the array. The average distance between adjacent areas B, defined by the average distance between the surface center points of adjacent areas, will generally not fall below a value of 50 μm, in particular 100 μm and especially 200 μm. The minimum distance between adjacent areas B, defined by the minimum distance that the boundary lines of two adjacent areas have, will on average not fall below a value of 5 μm, in particular 10 μm and especially 50 μm.

The microbioarrays according to the invention each have a plurality of, on average at least 10, preferably at least 50 and in particular at least 100 and up to 10,000, preferably up to 5,000 and in particular up to 1,000, spatially separated places P on the respective surface areas B, on which the biomolecules are immobilized.

According to the invention, the biomolecules in the microarrays are immobilized on a surface, that is to say they are bound in place. The binding can take place via covalent interactions, coordinative interactions, ionic or electrostatic interactions, hydrogen bonds, hydrophobic interactions and mixed forms of the aforementioned interactions. The connection can take place both directly to the surface and also via a substance V mediating the connection of the biomolecule. These substances V are generally compounds which are covalently bound to the surface on which the biomolecules are to be immobilized and which can simultaneously bind to the biomolecule, for example by covalent interaction, ionic interaction, coordinative interaction, Hydrogen bonds, hydrophobic interactions and mixed forms of such interactions, as occur, for example, in the base pairing of oligonucleotides, in the protein-protein interaction, antibody-antigen binding and in the protein-ligand interaction (affinitive interactions).

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

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

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

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

In principle, all surfaces on which biomolecules can be immobilized can be considered as surfaces for the arrays according to the invention, without the biomolecules being deactivated. These surface materials can be the surface of a carrier material or a modified surface of a carrier material. The carrier materials can be rigid or flexible. Typical carrier materials include oxidic carrier materials such as glass, quartz, ceramics, semimetals such as silicon and germanium, common semiconductor materials, e.g. B. doped silicon or doped germanium, metals and metal alloys, in particular based on gold, polymers, for. B. polyvinyl chloride, polyolefins such as polyethylene, polypropylene, polymethylpentene, polyester, fluoropolymers such as Teflon, polyamides, polyurethanes, polyacrylic (meth) acrylates, polystyrene, blends and composites of the aforementioned materials and the like. Materials with a low roughness surface are preferred. The R _a value characterizing the roughness is preferably not more than 100 nm and in particular not more than 50 nm. Preferred carrier materials are glass and silicon wafers.

As a rule, it is advisable to activate the surfaces of inert carrier materials, for example glass, ceramic, metal, semimetal such as silicon, polyolefin such as polyethylene or polypropylene, ie to treat them in such a way that they have a large number of functional groups R which are associated with complementary reactive groups R 'can form a chemical bond, preferably a covalent chemical bond. These functional groups R make it possible to immobilize the biomolecules on the surface directly via a chemical bond or indirectly, ie by means of one of the substances V defined above, which is covalently bound to the surface via such groups. Reactive groups R in the sense of this invention are those which react with nucleophiles in an addition reaction including a Michael reaction or in a substitution reaction, e.g. B. isocyanate groups, (meth) acrylic groups, vinylsulfone groups, oxirane groups, aldehyde groups, oxazoline groups, carboxylic acid groups, carboxylic acid ester and carboxylic acid anhydride groups, carboxylic acid and sulfonic acid halide groups, but also the complementary, nucleophile-reacting groups such as alcoholic OH groups, primary and secondary amino groups, thiol groups and the like. As an example of carboxylic acid ester groups, so-called active ester groups of the formula -C (O) OX, in which X is pentafluorophenyl, pyrrolidin-2,5-dione-1-yl, benzo-1,2,3-triazol-1-yl or is a carboxamidine residue, and NHS ester (N-hydroxy-succinimide ester). Reactive groups in the sense of the invention are also nucleophilic groups which react with the aforementioned electophilic groups to form bonds, eg. B. NH ₂ , SH, or OH, but especially NH ₂ . It goes without saying that a surface according to the invention either has predominantly electrophilic groups or predominantly nucleophilic groups, and in the case of NCO / NH ₂ both groups can also be present side by side.

Table 1 provides an overview of reactive groups R and complementary functional groups R ', the reactive groups R being indicated in the first row and the complementary groups R' in the first column:

Table 1: Complementary functional groups

Reactive group isocyanate (meth) acrylic / -SH -NH ₂ -OH -CHO oxirane R vinyl sulfone Complementary group R 'isocyanate X * XXX (meth) acrylate XXX vinyl sulfone XXX -SH XXXX -NH ₂ XXXX -OH XXX -COOH X oxirane XXX Reactive group isocyanate (meth) acrylic / -SH -NH ₂ -OH -CHO oxirane R vinyl sulfone active ester / NHS XX CHO XX

* In the presence of water

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

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

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

In a special embodiment of the invention, the biomolecules or the substance V are bound via isocyanate groups on the surface layer with the formation of urethane or urea groups.

In another particularly preferred embodiment of the invention, the surface has a large number of nucleophilic groups, in particular NH ₂ groups, so that the biomolecules used for immobilization or the substance V have a complementary reactive group, for example an isocyanate group, an ester group , for example, have an active or NHS ester group.

To generate surfaces which have a large number of reactive groups R, the surfaces of inert carrier materials can be activated, for example, by treatment with acids or alkalis. The activation can also be carried out by oxidation (flaming), by electron radiation or by a 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. Be. 1999, 149, 1-57. The surface can also be activated by means of plasma treatment with a plasma containing NH ₃ , as described in US Pat. Nos. 6,017,577, 6,040,058 and 6,080,488. Plasma modification-made PE films made with amino groups are also commercially available.

Frequently, the activation of the surface also includes the application of an organic coating which has a large number of the functional groups R defined on its surface. This coating is also referred to below as an adhesive layer. For this purpose, bifunctional or polyfunctional organic compounds or polymers are applied to the surface which has at least one functional group which reacts with the functional groups on the surface of the support material to form bonds (covalently, ionically and / or coordinatively) and the at least one additional one have functional group R of the type described above. These substances can be applied to the carrier surface as thin or ultra-thin layers, in particular as monomolecular or multimolecular layers, or can be produced on the surface of the carrier material by means of "Chemical Vapor Deposition" (CVD). inventions According to the invention, very thin adhesive layers with a thickness of preferably <100 nm, in particular <10 nm and especially monomolecular layers, in particular self-assembling monolayers (self-assembled monolayers, SAM), for example of thiol compounds which have at least one further group R, are preferred, especially for the treatment of noble metal surfaces, of silane compounds which have at least one group R, in particular for the production of monolayers on silicon or glass surfaces, furthermore macromolecular monolayers which are obtained by grafting polymers onto the surface or by graft polymerization on the surface and their polymers have suitable functional groups R.

In a preferred embodiment of the invention, compounds which have silane groups, in particular trialkoxysilane groups, as adhesion-promoting groups are used to produce the adhesive layer. Examples of such compounds are trialkoxyaminoalkylsilanes such as triethoxyaminσpropylsilan and N [(3-triethoxysilyl) propyl] - ethylenediamine, trialkoxyalkyl-3-glycidylether silanes such as triethoxypropyl-3-glycidyl ether silane, trialkoxyalkylmercaptane such as triethoximetoxysilkyloxykyloxykyloxykyloxykyloxykyloxysiloxysiloxysiloxysiloxysiloxysiloxysiloxysiloxysiloxysiloxysiloxysiloxysiloxysilane and tri-ethoxysiloxane lyl (meth) acryloxyalkanes and - (meth) acrylamidoalkanes such as 1-triethoxysilyl-3-acryloxypropane. These connections are applied to the surface in very thin layers, in particular as a monolayer, preferably after prior activation. Methods for this are known from the prior art cited at the beginning.

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

The adhesive layer is preferably applied to the carrier, in particular in the case of inert carrier materials, after activation by treatment with acids or alkalis, by oxidation (flaming), by electron radiation or by plasma treatment in the manner described above. The activation of the surface can also take place in such a way that, instead of an adhesive layer or preferably on the adhesive layer or on a surface activated in some other way, a further coating is applied which is particularly suitable for immobilizing the biomolecules and such groups instead of the reactive groups R. which allows the biomolecules to be linked via coordinative bonds, via hydrogen bonds, via hydrophobic interactions or via affinitive bonds. For this purpose, a preferably monomolecular layer of the abovementioned compounds V will be generated, for example layers of chelate formers, low molecular weight ligands for protein sequences such as biotin, of proteins such as avidin or streptavidin, or of oligonucleotides. It goes without saying that such an activation will only be carried out if the arrays are produced according to variant 1.

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

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

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

In a particularly preferred embodiment, the hydrogel layer is composed of interlinked star-shaped prepolymers, the star-shaped prepolymers having on average at least 4, generally 4 to 12, preferably 5 to 10 and especially 6 to 8 polymer arms A which are in themselves seen 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, this surface has a large number of functional groups R, in particular the groups mentioned in Table 1, which can be used in the manner described above for immobilizing the biomolecules. In addition, the hydrogel-forming coatings described there are extremely stable to aging and mechanical stress.

Star-shaped prepolymers are understood to mean those polymers which have a plurality of polymer chains bonded to a low-molecular central unit, the low-molecular central unit generally having 4 to 100 skeletal atoms, such as C atoms, N atoms or O atoms. The number average molecular weight of the polymer arms is generally in the range from 200 to 20,000 daltons, preferably in the range from 300 to 10,000 daltons, in particular in the range from 400 to 8000 daltons and especially in the range from 500 to 5000 daltons. Accordingly, the star-shaped prepolymer has a number average molecular weight in the range of at least 1500 daltons, preferably 2000 to 100000 daltons, in particular 2500 to 50,000 daltons and especially 3000 to 30,000 daltons. The hydrogel-forming properties of the surface layer are guaranteed by the water solubility of the polymer arms. It is generally guaranteed 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, homo- and copolymers which contain at least 50% by weight of copolymerized N-vinylpyrrolidone, homo- and copolymers which have at least 30% by weight. % Copolymerized hydroxyethyl (meth) acrylic! Hydroxypropyl (meth) acrylate, acrylamide, methacrylamide, acrylic acid and / or methacrylic acid, hydroxylated polydienes and the like. The polymer arms A are preferably derived from poly-C ₂ -C ₄ -alkylene oxides and are selected in particular from polyethylene oxide, polypropylene oxide and polyethylene oxide / polypropylene oxide copolymers, which can have a block or a statistical arrangement of the repeating units. Star-shaped prepolymers whose polymer arms A are derived from polyethylene oxides or from polyethylene oxide / polypropylene oxide copolymers with a propylene oxide content of not more than 50% are particularly preferred.

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

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

The prepolymers used according to the invention are e.g. T. known, e.g. B. from WO 98/20060, US 6,162,862, Götz 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 copolymers containing vinylpyrrolidone) or can be prepared by the methods described therein.

The provision of the organic surface essentially composed of crosslinked star-shaped prepolymers therefore usually comprises the deposition of the star-shaped prepolymers on the surface of a support and subsequent crosslinking of the reactive groups of the star-shaped prepolymers. The deposition and crosslinking steps can be repeated if desired. This leads to thicker layers.

As a rule, the process of depositing the star-shaped prepolymers comprises the following steps: i.a. Deposition of the star-shaped prepolymer on the surface of a support by applying a solution of at least one star-shaped prepolymer, which on average has at least four polymer arms A, which are in themselves soluble in water and carry a reactive functional group at their free ends, to the surface the carrier; i.b. then performing a linking reaction of the reactive groups with one another and removing the solvent; and i.e. optionally converting the functional groups on the surface of the surface layer.

In step ia, a coating is obtained which is composed of uncrosslinked prepolymers and which has a large number of functional groups on its surface. In this coating, a linking reaction of the reactive groups to one another is carried out, the linking reaction taking place after the application and removal of the solvent by subsequent treatment of the coating with a crosslinking agent or when removing the solvent if the solution already contains a suitable crosslinking agent and / or functional groups of the prepolymer and enter into a chemical reaction with one another, forming bonds.

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

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

In spin coating, the initially non-rotating substrate is generally completely wetted with the solution of the star-shaped prepolymer. Then the substrate to be coated is rotated at high speeds, usually at least 50 rpm, often at least 500 rpm, e.g. 500 to 30,000 rpm, preferably above 1000 rpm, e.g. B. 1000 to 10000 U / min, and particularly preferably 3000 to 6000 U / min, set in rotation, wherein the solution is largely spun off and a thin coating film remains on the surface of the substrate. Then networking is also triggered here.

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

In principle, all solvents which have no or only a low reactivity towards the functional groups of the prepolymer to be dissolved are suitable for preparing the solutions of the prepolymers. Among these, those are preferred which have a high vapor pressure and are therefore easy to remove. Preferred solvents are therefore those which have a boiling temperature below 150 ° C. under normal pressure and preferably have below 120 ° C. Examples of suitable solvents are aprotic solvents, e.g. B. ethers such as tetrahydrofuran (THF), dioxane, diethyl ether, tert-butyl methyl ether, aromatic hydrocarbons such as xylenes and toluene, acetonitrile, propionitrile and mixtures of these solvents. In the case of prepolymers with OH, SH, carboxyl, (meth) acrylic and oxirane groups, protic solvents such as water or alcohols, e.g. B. methanol, ethanol, n-propanol, isopropanol, n-butanol and tert-butanol, and mixtures thereof with aprotic solvents. In the case of prepolymers with isocyanate groups, water and mixtures of water with aprotic solvents are surprisingly suitable in addition to the abovementioned aprotic solvents, since the isocyanate groups of the prepolymers are degraded comparatively slowly in such solutions.

The prepolymers can be crosslinked in different ways. For example, the prepolymers can be reacted with a crosslinking agent. In principle, all compounds with 2 or more functional groups which react with the functional groups of the prepolymer to form bonds are suitable as crosslinking agents.

Accordingly, according to a first embodiment of the invention, the organic surface is provided by a process in which the linking of the reactive groups of the prepolymer is initiated by adding a compound Vm1 which has at least two complementary reactive groups per molecule which corresponds to the reactive groups of the star-shaped prepolymer react to form bonds.

The polyfunctional compounds Vm1 can be low molecular weight compounds, e.g. B. aliphatic or cycloaliphatic diols, triols and tetraols, e.g. B. ethylene glycol, butanediol, diethylene glycol, triethylene glycol, trimethylolpropane, pentaerythritol and the like, aliphatic or cycloaliphatic diamines, triamines or tetramines, eg. B. ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, 1, 8 ~ diamino-3,6-dioxaoctane, diaminocyclohexane, isophoronediamine and the like, amino alcohols such as ethanolamine, diethanolamine, aliphatic or cycloaliphatic dithiols to dicarboxylic acids such as glic acid, tricarboxylic acids such as sebic acid, tricarboxylic acids, Adipic acid, phthalic acid, isophthalic acid, or around the aforementioned diisocyanates, depending on the type of reactive groups the prepolymer has. In contrast to the prepolymers, the low molecular weight polyfunctional compounds generally have a molecular weight of <500 g / mol. The polyfunctional compound Vm1 can already be contained in the solution of the prepolymer. Then, in the surface layer of largely non-crosslinked prepolymers that initially develops, e.g. B. during drying or when heating the layer, the reactive groups of the crosslinking agent with the reactive groups of the prepolymer and in this way form a layer of crosslinked prepolymers, which generally still has a large number of functional groups on its surface.

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

In another embodiment of the invention, the linking of the reactive groups in the prepolymer is triggered by adding a sufficient amount of a compound Vm2 which reacts with a part of the reactive groups of the prepolymer to form functional groups with a complementary reactivity. In the case of prepolymers with isocyanate groups, crosslinking can be triggered, for example, by treating the coated article with water, e.g. B. by storage in a damp atmosphere or under water. Here, some of the isocyanate groups react to form amino groups, which in turn react with the remaining isocyanate groups to form bonds, forming a layer of crosslinked prepolymers. The cross-linking agent Vm2 is therefore water here. Coatings produced in this way, when freshly produced, still have free isocyanate groups. In the event of prolonged exposure to moisture, the isocyanate groups arranged on the surface are converted into amino groups. In a variant of this embodiment, solutions of the prepolymer in water or in a mixture of water with one or more water-miscible solvents are used. Examples of suitable water-miscible solvents are, in particular, those which do not react with the isocyanate groups or react very slowly than water. Examples include cyclic ethers such as tetrahydrofu- ran and dioxane, also N-alkylamides such as N-methylpyrrolidone, dimethylformamide and dimethylacetamide. The mixing ratio of water: solvent is generally in the range from 1: 100 to 100: 1, preferably in the range from 50: 1 to 1:10 and especially in the range from 20: 1 to 1: 1. This variant is particularly suitable for the production of thicker layers. The concentration of prepolymer is then preferably in the range from 0.1 to 500 mg / ml, in particular in the range from 0.5 to 200 mg / ml and especially in the range from 1 to 100 mg / ml.

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

As already explained, the microbioarrays according to the invention can be produced by a combination of micro-contact 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 a plurality of spatially separated raised surface areas on its surface, e.g. a multiplicity of band-shaped or, in particular, columnar elevations which correspond to the desired arrangement and the desired dimensions of the places P with regard to their dimension and arrangement of the raised surface areas. Stamps with a large number of columnar elevations are particularly preferred. The columns and their arrangement are characterized in particular by the following dimensions:

The average distance between the boundary line of the end face of a column and the center of the face of the end face is usually on average about 50 nm to 50 μm, preferably 100 nm to 25 μm, in particular 0.25 μm to 20 μm and especially 0.5 μm to 10 μm. With the preferred cylindrical column shapes, this corresponds to an average diameter of the columns in the range from 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 average distance between the center points of the faces of the 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 from 10 μm to 90 μm. The average minimum distance between two adjacent columns is generally in the range from 100 nm to 150 μm, often in the range from 500 nm to 120 μm, in particular in the range from 2 μm to 100 μm, and especially in the range from 5 μm to 80 μm (number average). The average distance between two adjacent columns, defined by the distance of the surface center points of the end faces of the columns, is at least twice as large as the average distance between the boundary line of the end face of a column and the surface center point of the end surface.

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

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

In the case of band-shaped elevations, the same applies to the spacing of the bands from one another as has been said for the spacing between adjacent columns. The average width of the band-shaped elevations is generally in the range from 100 nm to 100 μm, preferably 200 nm to 50 μm, in particular 1 μm to 30 μm and especially 2 μm to 20 μm.

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

The columnar elevations of a stamp are preferably formed from an elastic material in order to ensure a sufficiently good transmission. As a rule, the elastic material is a polymeric elastomer, for example a cross-linked polydimethylsiloxane. Such stamps are known from the prior art cited at the outset or can be produced by the methods specified therein. (see in particular Kane et al., Biomaterials 1999, 20, pp. 2363-2376 and the literature cited therein, Xia et al., Chem. Rev. 1999, 99, 1823-1848 and the literature cited there, 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 literature cited there). As a rule, such stamps are produced by molding a master which has a large number of depressions which correspond to the later elevations of the stamp. For this purpose, a mixture of a suitable elastomer precursor with a crosslinking agent is applied to a structured casting mold which is a negative of the stamp surface (master, usually a silicon wafer) and cured. After curing, an elastic stamp is obtained, the stamp surface of which represents the positive image of the master. Such methods are extensively described in the prior art cited here, to which reference is made for further details. The surface of the stamp can also be provided with a hydrophilic surface layer. Such modified stamps are described, for example, by Donzel et al., Adv. Mater. 2001, 13, pp. 1164-1167. This master is produced by methods known per se, for example by photolithographic methods as described in detail in the prior art cited here and in the literature cited therein.

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

According to variant 1 of the method according to the invention, one or preferably several mutually different biomolecules are brought into at least 2, preferably at least 10, in particular at least 50 and particularly preferably at least 100, for. B. 100 to 10000 and in particular 100 to 10000 preferably up to 5000 and especially up to 1000 spatially separated surface areas so that the biomolecules in each surface area each have a plurality, preferably at least 10, in particular at least 50 and especially at least 100, z. B. cover 100 to 1000 of the elevations of the stamp surface. The biomolecules can be applied in a manner known per se using conventional spotting techniques, one after the other or in particular simultaneously respectively. The devices required for this are the dot matrix printers, dispensers, plotters, micropipettes and the like that are customary for spotting techniques, with which the liquid drops can be applied to a surface using a contact method or a contact-free method (contact mode, non-contact Fashion).

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

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

The amount of liquid dispensed is important because it determines the size of the surface areas B, in particular the spot size. As a rule, amounts of liquid in the femto to nanoliter range are applied. For example, spots with a diameter of approximately 150 to 200 μm are available with a contact-free amount of approximately 10 nl to 2 μl. Spots with diameters in the range of 10 μm can be created by applying amounts of approximately 0.01 nl to 0.1 μl using capillary dispensers. The solvents for producing the solutions of the biomolecules depend in a manner known per se on the type of the biomolecule to be dissolved. The solvent is often water or aqueous mixtures of water with organic solvents which are infinitely miscible with water. 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 solvents, for example the abovementioned solvents and mixtures thereof, can also be used, in particular in the case of low molecular weight compounds. In addition to the biomolecules, the solutions can also contain surface-active substances, for example emulsifiers such as alkyl sulfates, alkyl sulfonates, ethoxylates of long-chain alkanols and their sulfuric acid half-esters, ethoxylated hydroxycarboxylic acids, ethoxylated esters of long-chain carboxylic acids with glycerol, furthermore quaternary ammonium compounds and the like. Furthermore, in particular aqueous solutions of the biomolecules can contain customary buffers such as PBS buffers, SSC buffers and the like.

In a second embodiment of variant 1, solutions of the biomolecules in several spatially separated surface areas are applied to a smooth, ie. H. unstructured surface on which the biomolecules are not immobilized. This surface can be seen as a release area. The solutions are applied in a manner known per se by means of customary spotting techniques, as described above. The amount of liquid dispensed is dimensioned such that the arrangement and size of the spots corresponds to the later surface area B of the array. The stamp surface of the structured stamp described above, which is preferably formed from an elastic material, is then brought into contact with the release surface. As a result, the biomolecules are transferred to the stamp surface of the structured stamp. The stamp surface of the stamp is then brought into contact with the surface on which the biomolecules are to be immobilized. With regard to the solutions used for the biomolecules, the concentrations, the structuring of the stamp and the contact times, the above applies. The inert carriers mentioned above and in particular non-structured elastic stamps are suitable as release surfaces.

Following the application of the biomolecules and the associated coupling of the organic surface, a washing process is generally carried out. Purpose of this The washing process is particularly to remove uncoupled biomolecules. Based on the solution previously used to apply the biomolecules, aqueous solvents or solvent mixtures are preferably used for washing. In principle, the above statements apply accordingly.

According to a particular embodiment, washing is carried out with an aqueous solvent or solvent mixture which contains a surfactant, preferably a nonionic surfactant. Polyalkoxylated, in particular polyethoxylated, fatty acid esters of polyols, in particular of glycerol or sorbitol, for example the sorbitan fatty acid esters sold under the trade name Tween®, are particularly preferred. In particular, the use of ethoxylated sorbitan hexylaurate has proven to be expedient.

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

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

The biomolecules are then applied to the surface pretreated in this way in a plurality of spatially separated surface areas B. This leads to a chemical binding of the biomolecules and thus to an immobilization at those positions P which are occupied by the compound V or in which the compound V is bound. Unbound biomolecules are then rinsed off the surface in a manner known per se, for example by washing with the abovementioned washing liquids. In this way, the array according to the invention is obtained which has a plurality of spatially separated surface areas, each of which comprises a plurality of spatially separated places P, which are occupied by biomolecules, the places P having an identical occupancy within one area and the places P different areas have a similar occupancy or a different occupancy with regard to the occupancy density and / or the type of immobilized biomolecule.

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

Variant 2 can basically be carried out on any surface, the explanations given above apply accordingly. However, it has proven 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 when proteins are immobilized. However, the above-mentioned commercial products, such as those sold by XanTec Bioanalytics GmBH and Perkin-Elmer, are also suitable as carriers with a hydrogel-forming surface.

Accordingly, a particularly preferred embodiment of variant 2 is a process in which: in a first step, a hydrogel-forming surface layer is provided on a support which has a large number of functional groups on its surface which react with complementary functional groups to form bonds, applies a compound V, which has one or more functional groups, to a stamp surface which has a multiplicity of raised surfaces can react with the functional groups of the surface to form a covalent bond, and which furthermore has one or more different functional groups which can form a bond to the biomolecule to be immobilized; the stamp surface thus treated is brought into contact with the hydrogel-forming surface layer, a surface being obtained which has a multiplicity of spatially separated places P which are occupied by the compound V, then one or more different solutions of the biomolecules to be immobilized in at least two spatially separated surface areas B in such a way that the surface areas each cover a large number of the places which are occupied by the compound V, the biomolecules being immobilized in the places and subsequently removing non-immobilized biomolecules.

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

The biomolecules can be applied in spatially separated surface areas in a manner known per se by means of the usual spotting techniques, as described in detail in variant 1. With regard to the exact process conditions, in particular the temperature and the choice of solvents, the above statements apply accordingly.

The washing step usually required is carried out by rinsing with a suitable washing solution. The explanations given for variant 1 apply accordingly.

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

The binding to the biomolecule can be covalent, ionic, coordinative, hydrophobic interaction, an interaction via hydrogen bonds or a mixed form of the aforementioned bonds (affinitive binding, as occurs, for example, in protein-ligand or protein-protein interactions) , In a first preferred embodiment of the invention, this binding center is a center which can form a coordinative bond to the biomolecule, which is mediated via a transition metal atom. With regard to the type of transition metal atoms, what has been said above applies. In particular, these 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). Such compounds V preferably bind the metal atom as a chelate, ie the compounds V have at least 2, preferably at least 3, e.g. B. 3, 4 or 5 functional groups that can form a coordinative bond to the above metal ions. The spatial arrangement of the functional groups is preferably selected so that the connection is made in the form of a chelate over all functional groups of the connection. Examples of suitable compounds V which are capable of coordinatively binding metal atoms and which simultaneously have a reactive group R 'include, for example, aromatic orthohydroxy aldehydes such as salicylaldehyde, functionalized pyridine and quinoline compounds, such as 8-hydroxyquinoline, dipicolylamine, N-2-pyridylmethylaminoacetate, furthermore polycarboxylic acids with preferably at least 2, for example 2, 3, 4, 5 or 6, carboxyl groups, in particular compounds which contain at least 2, for. B. 2, 3, 4, 5 or 6 carboxymethyl groups and optionally have a further functional group R ', as in iminodiacetic acid, nitrilotriacetic acid and their derivatives, as described by Liu et al., Synthesis, 2002, 10 , 1398, carboxymethylated aspartic acid, N, N, N-tris (carboxymethyl) ethylenediamine, N, N, N, N-tetra (carboxymethyl) ethylenediamine (EDTA) and the like, further ortho-phosphoserine and the like. In a preferred embodiment of the invention, an amino-functionalized derivative of a compound bond, which has at least two carboxymethyl groups, in particular an amino-functionalized derivative of nitrilotriacetic acid such as lysine-NTA (= 6-amino-2- (N, N-bis (carboxymethyl) amino) hexanoic acid). The compounds V, which can form complexes with transition metal ions, can be applied to the surface in complexed form or preferably in uncomplicated form. If uncomplexed compounds V are used, a complexation is then carried out by rinsing with a solution containing metal ions. If appropriate, after the application of the compound V or after the complexation of the compound V bound to the surface, a washing step will be introduced in order to remove excess compound V and / or excess metal ions.

In the case of carboxylic acids as compounds V, they can also be in carboxyl-protected form, ie. H. use as ester. Suitable protective groups for carboxyl groups are known to the person skilled in the art from the relevant literature, preference being given to those groups which can be split off under conditions which affect the surface, eg. B. do not impair or even destroy the hydrogel layer. An example of this is the tert-butyl group, which is thermally, for. B. can be removed by heating to 150-220 ° C, preferably under reduced pressure.

The solutions used for complexation are usually aqueous solutions of water-soluble salts of the metal ions, e.g. Solutions of chlorides, nitrates, sulfates, acetates etc. The concentration of the metal ions in the solution is generally in the range from 1 mM to 1 M. Depending on the type of ligand and the nature of the metal ion, the pH of the solution is in the Rule 5 to 12, in particular 6 to 11. The solutions may optionally contain weak complexing agents, for example Ammonia to ensure the solubility of the metal ions at higher pH values.

In another embodiment of method 2, the compounds V have, in addition to the reactive group R ′ required for the connection of the compound V, a group which can bind to the biomolecule via at least 2 and preferably via at least 3 hydrogen bonds. Such groups are, for example, oligonucleotide groups with at least 2, preferably at least 3, e.g. B. 3 to 100 and in particular 3 to 20 nucleotide bases. Biomolecules which have an oligonucleotide sequence complementary thereto can bind to these groups, the proportion of complementary bases being at least 50%. In a further embodiment of the invention, compounds V are used which are known to have an affinitive interaction with biomolecules. Examples of suitable compounds V are ligands, for example haptens and antibodies, which are functionalized, ie have a group R 'such that they can react with the surface to form a covalent bond. This also includes chemical compounds V which, in addition to at least one reactive group R ', have a further molecular group or amino acid sequence which can bind to the biomolecule in the sense of an affinitive interaction, for example a protein ligand or protein-protein interaction. Examples include biotin derivatives which are modified with a group R 'and thus form an anchor for biomolecules which have a streptavidin or avidin sequence, and streptavidin and avidin derivatives which are modified with a group R', so that they are an anchor for biotinylated biomolecules.

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

Biomolecules that bind to substance V with the formation of a covalent bond have, for example, a group that is complementarily reactive to this (see above). These biomolecules which bind to an oligonucleotide sequence naturally have a sequence which is at least partially complementary to this, preferably at least two adjacent base pairs in the oligonucleotide sequence of the biomolecule being complementary to two adjacent base pairs of the oligonucleotide sequence of substance V. The agreement is preferably at least 50%.

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

In a preferred embodiment of the invention, substance V is a substance which coordinates a transition metal ion of the type described above. In this case, the biomolecule has a group of molecules, which in turn can form a coordinating bond with the transition metal ion as a ligand, preferably as a chelating ligand. However, the gelating effect of this group is preferably not so strong that the gelating part of compound V is completely displaced. Chelating ligands in the biomolecules are in particular those which have at least two, in particular 2 or 3, groups which are 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 are in particular groups which have at least one or preferably 2 or 3, spatially adjacent imidazole groups. Preferred among these are in particular those groups which are derived from histidine or oligohistidine sequences. Such groups are also referred to as histidine tag or His tag.

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

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

In the particularly preferred embodiment of variant 2 with a hydrogel-forming surface layer, compounds V have proven particularly effective which bring about an immobilization of the biomolecule by means of a coordinative bond mediated by a transition metal. Accordingly, the compounds V are preferably selected from the above-mentioned chelating compounds, in particular from the above-mentioned polycarboxylic acids with at least 2 carboxyl groups, for example 2, 3, 4, 5 o- of the 6 carboxyl groups which still have a functional group which reacts with the functional groups of the hydrogel-forming surface layer to form a covalent bond. Examples of these are in particular the compounds already mentioned with at least two, for example 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 combination with a hydrogel-forming surface layer has proven particularly useful for the immobilization of complex biomolecules that easily denature, especially for the immobilization of proteins. A very particularly preferred embodiment of variant 2 with a hydrogel-forming surface layer thus relates to the immobilization of proteins, of proteins which bind to the surface by means of a coordinative bond, and in particular of proteins with histidine tags or comparable tags, as described by Stiborova et al ., Biotechnology and Bioengineering, 2003, 82, 605-611 and in the literature cited there.

With regard to the application of the areas of the biomolecules to the surface structured with the compound V, what has been said above applies to the application of biomolecules to stamp surfaces, with direct spotting (contactless or in contact) being preferred. The biomolecule can be used in pure form or as a raw product of a functionalization, since the biomolecule provided with the binding site selectively binds to the positions P occupied by the substance V.

The application of the biomolecule is usually followed by a washing step. This washing step serves to remove unbound biomolecule from the surface of the array. Basically, such liquids are liquids which dissolve the biomolecule and at the same time do not damage the surface. Particularly suitable are aqueous solvents which, in addition to water, can also contain small proportions, preferably not more than 40 and in particular not more than 10%, of organic, water-miscible solvents, for example alcohols such as methanol, ethanol and the like. These washing liquids can also contain surface-active substances as mentioned above. The proportion of surface-active substances will generally not be more than 5% by weight, based on the washing liquid. Furthermore, the washing liquids can also contain the buffers customary in biotechnology, in particular PBS buffers, SSC buffers and similar, in the concentrations customary for this. The type of additional components is aimed at known manner according to the type of biomolecule immobilized on the surface.

If the immobilized biomolecules are marked in one or more areas, quality control of the array can now follow. For this purpose, the array can be measured with a detection system corresponding to the marking. In particular, the coupling efficiency and thus the immobilization of the biomolecules can be assessed in terms of area, homogeneity and density via the measured amount of immobilized marking.

Basically, all types of marking are suitable. The marking is preferably selected so that it does not interfere with the subsequent analysis, or only minimally, so that it does not influence the use of the arrays, or only to an insignificant extent. For this purpose, any markings on the sample that may be necessary, in particular nucleic acid targets, must be observed. On the other hand, the marking can be chosen so that it interacts with the marking of the sample. The signal emanating from interacting markings can be distinguishable from that emanating from the corresponding non-interacting markings. The interaction can influence the signal intensity, e.g. amplify or quench, or modify the signal, e.g. change the wavelength of absorbed or emitted light.

Marking systems which can be recognized, for example, spectroscopically, photochemically, biochemically, immunochemically, electrically, optically or chemically are suitable according to the invention. These include direct labeling systems such as radioactive markers (e.g. ³² P, ³ H, ¹²⁵ l, ³⁵ S, ¹⁴ C), magnetic markers, chromophores, for example UV, VIS or IR absorbing compounds, fluorophores, chemical or bioluminescent markers, transition metals, which are generally chelate-bound, or enzymes, for example horseradish peroxidase or alkaline phosphatase and the detection reactions linked to them, as well as indirect labeling systems, for example haptens, such as biotin or digoxigenin, which can be recognized by corresponding detection systems ,

Favorable chromophores have an intense color that is only slightly absorbed by the surrounding molecules. Dye classes such as quinolines, triarylmethanes, acridines, alizarines, phthaleins, azo compounds, anthraquinones, cyanines, phenazathionium compounds or phenazoxonium compounds are representative of the broad spectrum of chromophores suitable according to the invention. Fluorescent labels are preferred. You get strong signals with little background, high resolution and high sensitivity. It is advantageous according to the invention that one and the same fluorophore, depending on the excitation and detection principle, can emit several distinguishable radiations. Fluorophores can be used alone or in combination with a quencher (e.g. molecular beacons). Suitable fluorophores are, for example, aminomethylcoumarin acetic 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 fluorocarboxylic acids, in particular of the types 647 and 532, for example as succinimidyl esters, and IRD41.

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

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

The following applications are mentioned as examples:

Diagnostics, choice of therapy and therapy control of tumor and metabolic diseases; Investigation of the genetic predisposition (SNPs), in particular the detection of mutations, also in the forensic field; Detection of promoter methylations (Epigenetics); Gene expression control; Sequencing, in particular sequencing by hybridization; Microbiological applications such as in bacteriology and virology, for example for 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 substance libraries; Toxicogenomics.

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

In non-competitive assays, the substance to be analyzed (analyte) coincides with that on the Interacting surface immobilized biomolecules. As a rule, the analyte is marked beforehand, but marking is also possible after the analyte has already interacted with the immobilized biomolecules, for example by means of primer extension or rolling-cycle PCR. In these cases, a measurement signal is obtained which is greater the more analyte is present. It is also possible that the interaction of the sample with the substances immobilized on the surface changes the fluorescence of the immobilized substances (attenuation, amplification, for example molecular beacons) or changes the activity of an enzyme and this change is registered as a measurement signal. Examples of non-competitive assays are hybridization reactions of PCR products or labeled DNA / RNA to nucleic acids immobilized on the surface, in particular oligonucleotides or cDNA, and sandwich immunoassays.

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

The invention is illustrated by the following examples and figures.

Characters:

Figure 1: A schematic representation of variant 1 of the method according to the invention is shown. FIG. 1a shows the top view of a conventional polydimethylsiloxane stamp, which has a large number of columnar elevations. Figure 1 b shows a cross section through the polydimethylsiloxane stamp shown in Figure 1 a in the supervision. FIG. 1 c shows schematically the application of different solutions of the biomolecules to different areas of the stamp surface and a cross section through the stamp treated in this way. Figure 1 d shows a plan view of the stamp surface thus obtained. FIG. 1e is a schematic representation of the contacting of the treated stamp surface with the substrate surface. Figure 1 f shows a schematic plan view of the stamped surface and a schematic side view of the stamped surface Figure 2: The variant 2 of the method according to the invention is shown in a schematic manner. Figure 2 a shows a cross section of the stamp used. FIG. 2 b shows schematically the printing of substance V onto a substrate surface coated with a hydrogel layer (1). Figure 2 c shows the surface stamped with the substance V (2) in the top view and Figure 2d as a cross section. FIG. 2e schematically shows the top view of a surface and FIG. 2f the cross section thereof, to which several spots (3) of different protein solutions were applied after substance V had been applied.

Figure 3: Figures 3a and 3b show a fluorescence microscope image of an array of immobilized fluorescein prepared according to variant 1 in different magnifications.

FIG. 4: partial view of a fluorescence microscope image of an area produced according to variant 2 with a large number of places on which fluorescent-labeled streptavidin was immobilized via biotin.

Figure 5: Partial view of a fluorescence microscope image of an array produced according to variant 2 with a large number of places on which green fluorescent protein with histidine tag was immobilized using lysine-NTA-nickel (II).

Examples

I. Amino functionalization of the carrier:

The substrate surfaces (glass plates, microscope carriers, silicon carriers) were first pretreated. For this purpose, the substrates were stored for 1 hour 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. Then they were rinsed several times with water. The substrates treated in this way were then stored in deionized water. Alternatively, the substrates pre-cleaned by water and acetone can be treated in a plasma system of the type TePla 100-E from the company Plasma Systems in oxygen plasma for 2 minutes (pressure: 1 mbar, 50W) and then washed with water and acetone. Alternatively, the pre-cleaned substrates can also be treated with oxygen under a 40 W UV lamp with a wavelength of 185 nm for 10 min (distance between substrate surface and light source 2 mm), and then washed with water and acetone become.

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

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

Production example 1:

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

Production example 2:

A 6-arm statistical poly (ethylene / propylene oxide) with an EO / PO ratio of 80/20 with a molecular weight of 18000 g / mol, which had been modified at the ends of the polymer arms with isophorone diisocyanate (produced analogously to Example 11 of DE 10216639.0 ), was dissolved in a concentration of 10 mg / ml in dry tetrahydrofuran. Then an amino-functionalized silicon carrier, produced according to I, was coated by means of spin coating. For this purpose, the non-rotating, dried substrate was first completely wetted with the prepolymer solution before the solution was removed at a final speed of 4000 rpm for 40 seconds. was hurled. The substrate thus coated was then kept overnight in an atmosphere saturated with water vapor, whereby a surface with NH ₂ groups was obtained.

III. Production of bioarrays

Example 1: Preparation of a fluorescein array:

A solution of an isothiocyanate-modified fluorescein derivative (FITC from Aldrich) was dissolved in a mixture of ethanol / dimethyl sulfoxide 50: 1 v / v in a concentration of 100 μM. On a rectangular polydimethyldisiloxane stamp, manufactured according to Tan et al., Langmuir 2002, 18, pp. 519-523 with an area of 15 mm x 15 mm with a regular arrangement of cylindrical columns (diameter 10 μm, height 2 μm, average distance 20 μm ), a very small amount of liquid (<0.5 μl) of this solution was applied with a microliter pipette and then dried, giving a spot with a diameter of <500 μm. The stamp prepared in this way was brought into contact with the amino-functionalized glass carrier produced in I for 2 minutes. The surface was then examined by fluorescence microscopy. In this way, the pattern shown in Figures 3 and 3a is obtained.

Protein arrays and nucleotide arrays can also be produced in an analogous manner:

Example 2: Preparation of an oligonucleotide array

A rectangular polydimethyldisiloxane stamp, produced for example according to Tan et al., Langmuir 2002, 18, pp. 519-523, with an area of 15 mm x 15 mm with a regular arrangement of columns (diameter 5 μm, height 2 μm, average distance 20 μm), is carried out according to the specification by Donzel et al., Adv. Mater. 2001, 13, 1164, modified hydrophilically in the oxygen plasma. 2 spots of 0.1 .mu.l of a solution of 3'-amino-terminated-5'-fluorescence-labeled oligonucleotide in PBS buffer with a concentration of 100 pmol / .mu.l are applied to this stamp using a micropipette. After drying in an inert gas stream, the stamp prepared in this way is brought into contact with a surface freshly produced according to II Example 1 for 2 minutes. It is then washed with PBS. In this way, an almost error-free image of the stamp is obtained, which has 2 separate areas in which the nucleotides occupy a very large number of spatially separated places, as can be demonstrated by fluorescence microscopy. Example 3 Preparation of a Biotin Streptavidin Array:

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

2 spots of 1-2 μl of a solution of fluorescence-labeled streptavidin (Molecular Probes) in PBS buffer (0.04 mg / ml) are applied to the biotin array produced in this way using a micropipette and stored for 4 hours at room temperature. The array is then washed with PBS buffer and dried in a stream of argon. In this way, an array is obtained which has two spatially separated areas which have a large number of places occupied by streptavidin, as can be demonstrated by fluorescence microscopy (see Figure 4).

Example 4: Production of a protein array via complex formation

The polydimethylsiloxane stamp used in Example 1, with an area of approximately 15 mm × 15 mm, with a regular arrangement of columns (diameter 10 μm, height 2 μm, average distance 20 μm) is mixed with a solution of lysino-NTA tert-butyl ester, produced 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 coating freshly prepared according to II Example 1. The support thus obtained is then stored overnight in an atmosphere saturated with water vapor and washed with toluene. The carrier obtained is 10 min. stored at 200 ° C under vacuum (0.02 mbar) and after cooling for 5 min. immersed in a solution of NiCI ₂ in water (5 mg / ml) and washed with water. A solution of a His fluorescent green fluorescent protein in PBS buffer (0.03 mg / ml) is then applied to the carrier treated in this way, 10 min. stored and then washed with PBS buffer. The array obtained is examined by fluorescence microscopy. The pattern shown in Figure 5 is obtained.

Claims

claims

1. Microarray of immobilized biomolecules, which has at least two surface areas B arranged spatially separated from one another, each comprising a plurality of spatially separated places P, on which biomolecules are immobilized, wherein the mean distance of the boundary line of a region B from its center point is the Range is from 10 microns to 250 microns.

2. A microarray according to claim 1, wherein the average distance between adjacent places P within the areas B is at least twice as large as the average distance between the boundary line of a place P and its center point.

3. Microarray according to claim 1 or 2, wherein the average distance of the boundary line of a place P to its center point is on average 50 nm to 50 microns.

4. Microarray according to one of the preceding claims, wherein the average distance between adjacent sites is in the range from 200 nm to 200 μm.

5. Microarray according to one of the preceding claims, wherein the places P are described by a circular, ellipsoidal or rectangular area.

6. Microarray according to one of the preceding claims, wherein the places P are arranged regularly within the areas B.

7. Microarray according to one of the preceding claims, wherein the regions B comprise at least 10 spatially separated, flat spaces P.

8. Microarray according to one of the preceding claims, wherein at least two of the surface areas B differ in the type and / or the surface density of the immobilized biomolecules.

9. Microarray according to one of the preceding claims, wherein the regions B have a circular geometry.

10. Microarray according to one of the preceding claims, wherein the average distance between adjacent areas is at least 50 microns.

11. Microarray according to one of the preceding claims, comprising an organic surface layer arranged on a carrier, on which the biomolecules are immobilized.

12. The microarray of claim 11, wherein the surface layer comprises at least one hydrogel-forming layer.

13. The microarray according to claim 12, wherein the hydrogel-forming surface layer is essentially composed of crosslinked star-shaped prepolymers which on average have at least 4 polymer arms A, which are water-soluble per se.

14. Microarray according to claim 12 or 13, wherein the hydrogel-forming surface layer has an average thickness of 1 nm to 100 microns in the dry state.

15. A method for producing a microarray of immobilized biomolecules which has at least two surface areas B which are spatially separated from one another and which each comprise a plurality of spatially separated places P on which biomolecules are immobilized by placing on a stamp surface which a multiplicity of raised areas, one or more biomolecules different from one another in at least two, preferably at least ten and in particular at least one hundred spatially separated area areas B so that the biomolecules in area areas B each cover a plurality of the raised areas of the stamp area and then the stamp area brings into contact with the surface on which the biomolecules are to be immobilized and, if appropriate, removes non-immobilized biomolecules.

16. A method for producing a microarray of immobilized biomolecules, which has at least two surface areas B which are spatially separated from one another and which each comprise a plurality of spatially separated locations P, on which biomolecules are immobilized, by pressing on a stamp surface which has a multiplicity of raised surfaces has a connection V, which brings about immobilization of the biomolecules, brings the thus treated stamp surface into contact with the surface on which the biomolecules are to be immobilized, thereby obtaining a surface which has a multiplicity of spatially separated places P which are connected to the compound V are occupied, and then one or more solutions of the biomolecules to be immobilized which are different from one another are applied in at least two spatially separated surface areas B in such a way that the surface areas each cover a large number of the places which are occupied by the compound V, the biomolecules being attached the places P are immobilized, and then non-immobilized biomolecules are removed.

17. The method according to claim 16, wherein the compound which brings about the immobilization of the biomolecules is selected from compounds which form a covalent or ionic bond with the surface and which have a, covalent, ionic, coordinative or affinitive bond with the biomolecule to be immobilized form.

18. The method according to any one of claims 15 to 17, wherein the average distance between adjacent elevations on the stamp surface is at least twice as large as the average distance of the boundary line of the end face of an elevation to the surface center of the end face.

19. The method according to any one of claims 15 to 18, wherein the average distance of the boundary line of the end face of the elevations to the center of the face of the end face is on average 50 nm to 50 microns.

20. The method according to any one of claims 15 to 19, wherein the average distance between adjacent surveys on the stamp surface is in the range of 200 nm to 200 microns.

21. The method according to any one of claims 15 to 20, wherein the end faces of the elevations on the stamp surface are circular, ellipsoidal or rectangular.

22. The method according to any one of claims 15 to 21, wherein elevations are regularly arranged on the stamp surface.

23. The method according to any one of claims 15 to 22, wherein elevations on the stamp surface have an average height in the range of 0.5 to 500 microns and the ratio H: A of average height H to the average distance A between the boundary lines of the end faces of the Elevations and the surface centers of the end faces is not more than 10.

24. The method according to any one of claims 15 to 23, wherein the elevations of the stamp are formed by an elastomeric material.

25. The method of claim 24, wherein the elastomeric material is polydimethylsiloxane.

26. The method according to any one of claims 15 to 25, wherein the concentration of the biomolecules in the solution is in the range of 0.1 nM to 10 mM.

27. The method according to any one of claims 15 to 26, wherein the surface on which the biomolecules or the compound which brings about the immobilization of the biomolecules to be immobilized is formed by a hydrogel-forming surface layer.

28. The method according to claim 27, wherein in a first step, a hydrogel-forming surface layer is provided on a support, which surface has a multiplicity of functional groups which react with complementary functional groups to form a bond on a stamp surface which has a multiplicity of raised areas, applies a compound V which has one or more functional groups which can react with the functional groups of the surface to form a covalent bond and which also has one or more different functional groups which can form a bond to the biomolecule to be immobilized; the stamp surface thus treated is brought into contact with the hydrogel-forming surface layer, a surface being obtained which has a multiplicity of spatially separated places P which are occupied by the compound V, then one or more different solutions of the immobilizing biomolecules in at least two spatially separated surface areas B in such a way that the surface areas each cover a multiplicity of the places which are occupied by the compound V, the biomolecules being immobilized in the places and subsequently removing non-immobilized biomolecules.

29. The method according to claim 28, wherein the hydrogel-forming surface layer is composed of crosslinked star-shaped prepolymers which on average have at least 4 polymer arms A which are water-soluble in themselves.

30. The method according to claim 28 or 29, wherein the functional groups on the surface layer are selected from isocyanate groups and amino groups.

31. The method according to any one of claims 28 to 30, wherein the binding of the compound V to the biomolecule is a coordinative bond mediated via a transition metal.

32. The method according to claim 31, wherein the compound V is a derivative of polycarboxylic acid having at least 2 carboxyl groups, which has a functional group which reacts with the functional groups of the hydrogel-forming surface layer to form a covalent bond.

33. The method according to claim 31 or 32, wherein the biomolecule to be immobilized is a protein with a His tag.

34. Device based on a microarray according to one of claims 1 to 14 in the form of a chip, a microfluid device or a dipstick.

35. Use of a microarray according to one of claims 1 to 14 or a device according to claim 34 for diagnostic and analytical purposes.