DE10041809A1 - Arrays of immobilized biomolecules, their manufacture and use - Google Patents

Abstract

The invention relates to arrays of immobilised biomolecules, coupled by means of coupling groups, preferably quinone, to a carbonaceous support surface. The carbonaceous surface comprises at least one polymer based on cycloolefins, or may be obtained whereby a glass, metal or ceramic surface is treated with an aqueous solution of at least one carbon-containing compound which may be hydrolysed and the surface subjected to thermal treatment. Anthraquinone and polycycloolefin surfaces based on norbornene are preferably used, or surfaces silanised with hydrophobic residues according to the sol-gel technique. The advantages of said arrays lie above all in the quality thereof, in particular with relation to the homogeneity and reproducibility with which the biomolecules are immobilised. The invention further relates to methods for the immobilisation of biomolecules and the use of the arrays for diagnostic purposes.

Description

The present invention relates to arrays of immobilized biomolecules on carbon-containing gen surfaces, their manufacture and use. Also relates to the present Invention devices based on arrays, in particular chips and dipsticks as well Carrier with structured surface for the targeted immobilization of biomolecules.

Come across with the increasing practical importance of molecular biological analysis those associated with a very high workload and cost, often days or Weeks of established standard techniques at the limits. New test formats such as Biochips should enable enormous time and cost savings. As with this technique many different substances immobilized in a very small space at the same time can be brought into contact with a single sample to be analyzed is the pro Measurement of information content obtained from a chip is very high.

An overview of the most popular biochip systems is given by Bowtell D. D. L. nature genetics supplement 21 (1999) 25-32.

A typical example of a so-called "high density array" is the GeneChip from Affymetrix, an oligonucleotide array with typically more than 400 different capture probes, which are built 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. However, the spot areas are not homogeneous and poor hybridization can often be observed, especially at the edges (cf. also US Pat. No. 5,744,305; US Pat. No. 5,800,922; US Pat. No. 5,445,934; US Pat. No. 5,795,716).

For example, "Low Density Arrays" are available from Genometrix. Maximum of 250 spots 50 µm in diameter are made up of up to 1000 individual pins with a capillary pin bundle capillaries are printed in the cavities of a 96-well microtiter plate. For coupling to the Conventional surface techniques are used, in particular Aminosila nizations and NHS-activated haptens, epoxy-activated surfaces, carbodiimide coupling lungs to carboxyl groups as well as biotin / streptavidin bonds are based (cf. WO 97118226; EP 0910570 A1; WO 98/29736).

The quality of these biochips is sufficient for satisfactory use, especially in medical diagnostics, however, are not enough. There are various reasons for this.

For example, the carrier materials commonly used pose problems. Glass surfaces first require silanization. These are solutions of silanes applied to the glass surfaces in aprotic solvents. The on the surface Any OH groups that are present hydrolyze the silanes, which then undergo formation condense a network. Such coatings are generally very inhomogeneous and not very reproducible and also depend heavily on the priorities treatment of the glass surface. They are often a disadvantage of membranes or gels occurring diffusion problems and especially with nylon membranes a high rear basic fluorescence. For plastics, there are usually no functional groups that can be coupled. Only Polystyrene is of some importance for the adsorption of proteins.

The coupling of biomolecules to the surface is also critical. Will the Biomole When applied in a structured manner, the evaporation of the tiny amounts of liquid has a positive effect strong influence on the spot quality. The drying process leads to strong Differences in concentration within a spot. The very short response times of often only a few seconds are usually not enough for a completely homogeneous saturation the surface with biomolecules. The inhomogeneities of the spots and the A lack of alignment of the biomolecules leads to high variations in almost all cases coefficient and a low dynamic measuring range.

An independent control of the biochip quality or a comparison of different ones Systems would be desirable. There are usually different control spots in the Integrated in most of the available arrays. However, these only allow a statement whether the sample preparation and incubation were successful and whether all of them are necessary Reagents were added. An evaluation of the results and a plausibility The user is responsible for checking the validity. Uniform standards of quality times are missing from biochip arrays.

The present invention is therefore based on the technical problem, qualitative high-quality biochips with homogeneously and reproducibly applied biomolecules for To make available.

To solve this problem, the invention proposes the biomolecules to a carbon to couple substance-containing surface, which is either based on polycycloolefins or through a certain hydrophobization process is available.

The present invention therefore relates to arrays of immobilized biomolecules, which are coupled to a carbon-containing carrier surface, characterized in that that the carbonaceous surface is at least one polymer based on cycloolefin contains or is obtainable by having a glass or metal surface with a aqueous solution of at least one hydrolyzable carbon-containing compound treated and heated the surface.

The invention has numerous advantages:
The arrays generated are very robust and have good storage properties. The arrays can be encapsulated and / or integrated into automatic analysis devices. Biomolecules immobilized as spots have a uniform coating, ie they are homogeneous, in particular "coffee stain shapes" are avoided. Good discrimination rates are achieved for oligonucleotide arrays and a high dynamic range for hybridization experiments of <10 ⁴ is made available. Intra- and inter-array variations are small. In this way, an intraassay variance of less than 7% and an interassay variance of less than 12% can be achieved. This enables the arrays to be used in the diagnostic / analytical area.

The term "array" denotes an arrangement of defined places, in particular one local assignment of certain substances to defined places, with different The same or different substances can be assigned to places. A defined one Place corresponds to a certain area, the value of which has an intraassay variance of advantageously less than 15%, preferably less than 7% or an interas say variance of advantageously less than 20%, preferably less than 15% and in particular less than 10% when one faces one another compares. A type of substance is usually assigned to a place, although it is also conceivable to add mixtures of two or more types of substances to one place organize. Two-dimensional arrays are preferred. The places form expediently a regular two-dimensional pattern of fields. Within an array, each type of substance or each type of substance mixture has one or more fields be assigned.

The term "biochip" refers to an array of biomolecules that are on a solid Carrier covalently, adsorptively or via other physical / chemical interactions are immobilized.

The term "biomolecules" denotes any biochemical and biological substances, both individual molecules and several molecules that interact with one another. Examples include:

• - Nucleic acids, especially oligonucleic acids, e.g. B. single and / or double strands gige, 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, e.g. B. Fab, Fab ', F (ab) ₂ , synthetic;
• - proteins, e.g. B. allergens, inhibitors, receptors;
• - enzymes, e.g. B. peroxidases, alkaline phosphatases, glucose oxidase, nucleases;
• - small molecules (haptens), e.g. B. pesticides, hormones, antibiotics, pharmaceuticals, dyes fe, synthetic receptors, receptor ligands.

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

According to a special aspect, a certain type of biomolecule is to be used as a ligand describe. Ligands interact with and in particular bind them - preferably specific - to specific targets.

The biomolecules are coupled to a carrier surface for immobilization. This can be done in a number of ways. In principle, the surfaces in be functionalized in a suitable manner, e.g. B. with epoxides, NHS esters or aldehydes for coupling amino-modified biomolecules. A preferred one according to the invention The possibility is to implement the coupling via certain groups (Kopp treatment groups), those with non-functionalized and in particular the following in more detail carbon-containing surfaces described are reactive. Particularly preferred are in this context groups that form a bond with hydrocarbon radicals the can. Structures that can be activated by radiation should be mentioned here in particular, like coumarins, benzofurans, indoles, anglecins, psoralens, groups, the carbenes, Can generate nitrenes or excited ketones, e.g. B. azides, diazo compounds, Diazirines, ketones such as diphenyl ketones, benzophenones and acetophenones, and peroxides. According to a preferred embodiment of the invention, the coupling takes place about quinones. On the basis of this embodiment are now representative for further useful coupling groups special configurations of arrays according to the invention described.

Quinones are usually photochemically reactive and can be removed by means of suitable Coupling radiation to carbonaceous materials. The coupling usually takes place via covalent bonds.

According to the invention, the term “quinone” describes compounds which have at least 2 conjugated carbonyl groups as part of at least one cyclic hydrocarbon structure. The meaning of the term "quinone" is common. The quinones include, for example, anthraquinones, phenanthrenequinones, benzoquinones, naphthoquinones and other quinones, some representatives of which are shown in FIG. 1 of WO 96/31557, which are part of this application by reference. These quinones, like the coupling groups mentioned above, can be substituted. A list of useful substituents, which are part of this application by reference, can be found on pages 11 and 12 of WO 96/31557. Anthraquinones are preferred.

Quinone and biomolecule are linked to one another. In principle, this link can be based on any physical-chemical path, especially covalent and adsorptive, Interactions are based and direct or indirect, for example via spacers or further at least bifunctional linkers take place. So are quinone conveniently derivatives used which are at least monosubstituted. Examples are advantageous se quinonecarboxylic acid derivatives, in particular via ester and preferably amidamine connections to the biomolecule or optionally to a spacer or other linker are bound. In a corresponding manner, a person skilled in the art is able to use quinones to provide other functional groups that can form bonds, which are stable under the conditions of manufacture and use of the arrays. Ether, Amine, sulfide and disulfide bonds are mentioned here by way of example.

In addition, the quinones can carry one or more further substituents. So one can determine the polarity and thus in particular the solubility and affinity of the quinones influence and advantageously to certain modalities such as the application of the Adapt quinones to the carrier surface or to the surface material.

The term "quinone derivative" is used below for substances that at least have a quinone structure. These include B. functionalized quinones, Link fations of quinones with spacers, linkers and / or biomolecules or modified Variants thereof, for example activated, protected or in any other way derivatives prepared for synthetic purposes.

According to one embodiment, quinones are linked directly to biomolecules. These The arrangement of quinone derivatives according to the invention is abbreviated as "Q-B" in the following and is mainly used for biomolecules with a relatively high molecular weight, like antibodies, proteins and enzymes.

According to a further embodiment, quinones are attached to biomolecules via spacers knotted. This arrangement of quinone derivatives according to the invention is described below with "Q-S-B" denotes and is mainly used for biomolecules with a relatively low Molecular weight, such as nucleic acids, in particular oligonucleic acids and haptens.

The term “spacer” denotes multi- and, in particular, bifunctional linkers of certain types Length, which in the present case usually consists of quinone on the one hand and biomolecule on the other hand are linked. In principle, spacers can be homo- or hetero (bi) functional be, being a hetero (bi) functionality a variation of in itself by a certain Quinone derivative enables given functionality. So can quinone and biomolecule optimally linked to one another through the choice of appropriate heterobifunctional spacers on the one hand to correspond to the type of binding specified by the quinone derivative and on the other hand to provide functionality that is effective, but also enables the most gentle link possible, especially with proteins. Acts the biomolecule is a ligand, can have its type and, in particular, its length of the spacer, the interaction between biomolecule and target can be influenced. Especially the accessibility of immobilized biomolecules for the interaction with the Optimize target.

Preferred quinone derivatives are anthraquinon-2-yl derivatives of the formula I.

Phenanthrenequinon-3-yl derivatives of the formula II

where in the formulas I and II the radicals R can be identical or different and independently of one another for the abovementioned useful substituents and preferably for C ₁ -C ₄ -alkyl, C ₃ -C ₁₀ -cycloalkyl, C ₆ -C ₁₀ -aryl or C ₇ -C ₁₂ aralkyl, z1 and z2 independently of one another stand for a value of 0, 1, 2, 3 or 4 and A1 stands for CO, CH ₂ , CS, O or S. Preferably z1, z2 are zero and A is CO or CH ₂ . Anthraquinone-2-cabonic acid derivatives are particularly preferred. As immobilized biomolecules, these quinone derivatives are linked to at least one biomolecule, optionally via spacers and / or further linkers.

Suitable spacers are, for example, homobifunctional compounds such as diamines, Diacids, e.g. B. dicarboxylic acids, or ethylene glycol oligomers, or heterobifunctional Compounds such as amino acids, e.g. B. β-alanine, glycine, 6-aminocapric acid or γ-aminobutyric acid, arylacetylenes, biotins, avidins, strepavidins, oligosaccharides, e.g. B. off Riboses or deoxyriboses, nucleic acids, especially oligo- (d) A or - (d) T, fatty acids, Phosphoric acid esters and derivatives and / or combinations thereof.

Spacers can also be made up of several parts (linkers) that go through with each other covalent and / or non-covalent bonds are linked. Examples of covalent linkable parts of a spacer are heterobifunctional elements such as amino acids, for example 6-aminocaproic acid or γ-aminobutyric acid, several of which for Extension of a spacer can be connected to each other. examples for Non-covalently linkable parts of a spacer are molecules between which Can form affinity bonds, such as between biotin and avidin or streptavidin or their analogues. Biotin / avidin spacer, avidin / biotin / avidin spacer, anti-biotin antibodies / biotin / avidin spacers or Dig / anti-Dig spacers are preferred Spacer of this aspect.

Spacers can have one or more binding sites for biomolecules (e.g. Dendrimers). The heterobifunctional elements mentioned above, for example usually have one, two or three such binding sites, while affinity Molecules that form bonds, such as avidin or streptavidin, have multiple binding sites exhibit. The latter can lead to an advantageous increase in immobilized biomolecules lead per unit area.

Spacers can also have variable binding sites that depend on their offer different binding options for each configuration. This is especially true especially for affinity bonds, the binding affinity of which depends on the configuration of the binding partners involved can vary.

Preferred spacers are based on
Diamines of formula III

-NH- (CHR ¹ ) _n1 -NH- (III)

Amino acids of formula IV

-NH- (CHR ¹ ) _n1 -CO- (IV)

Polyethylene glycols of the formula V

- (OCH ₂ CH ₂ ) _n2 - (V)

Oligo sugar phosphates of the formula VI

- (O-PO ₂ -O-5'-Sugar-3 ') _n1 -O-PO ₂ - (VI)

or combinations thereof, in particular

-NH- (CHR ¹ ) _n1 -NH- (OCH ₂ CH ₂ ) _n2 - (VII)

where in the formulas III to VII n1 corresponds to a value from 1 to 50 and in particular from 5-20 and n2 corresponds to a value from 2 to 100 and preferably 5 to 50, R ^{1 has} the meanings given for R, where several radicals R ¹ may be the same or different (with n1 ≠ 1) and the sugar is in particular ribose and deoxyribose, preferably in the D-form. In the combination, n1 corresponds in particular to a value from 2 to 6 and preferably from 3, while n2 in particular corresponds to a value from 2 to 20, preferably 4 to 10, e.g. B. 6 corresponds. ^{R 1} preferably represents α-C substituents of naturally occurring amino acids, in particular hydrogen and C ₁ -C ₄ -alkyl. The link to the quinone can in principle take place at both binding sites (QS- or -SQ); QS derivatives are preferred as shown above.

In principle, the above apply to the binding of quinones or spacers to biomolecules Comments on the binding of quinones to spacers accordingly. Covalent bonds, in particular -O-, -NH- or -S- are preferred.

Biomolecules can specify certain functionalities for this, but they can too appropriately modified. For example, proteins can be via amino, sulfide or Carboxy groups, and nucleic acids bind via 5 'or 3 "-OH groups. Proteins can, for example, be reductively modified in a manner known per se to To convert disulfide bridges into free sulfide groups, and nucleic acids can contribute for example implemented with known and also commercially available reagents in order to convert terminal OH groups into groups containing amino groups ren.

According to a preferred embodiment, nucleic acids are above their respective 5 'end attached to quinone or spacer. This can advantageously be done via polyethylene Lenglycol spacers take place, in particular the spacers of the formula VII.

In addition to immobilized biomolecules, other substances can be found on the carbon-containing material be immobilized gene surfaces that are not a biomolecule in the sense of the invention include. These can be in their own places in the array or together with Biomole kulen be immobilized in a statistical distribution in one place (co-immobilized). Appropriately, such molecules are also attached to the carrier via quinones area coupled. In principle, therefore, quinones and quinone derivatives, which are derive from the respective immobilized biomolecules, but no biomolecule in itself include sense of the invention. These include in particular quinone derivatives, in particular functionalized quinones (Q), optionally with spacers (Q-S) and / or other groups are linked. Suitable further groups (B ') can be in particular derive from immobilized biomolecules, e.g. B. Analogues or parts of a Biomolecule that is not included in the with a biological sample, in particular an analyte analytical interaction are able to occur, to which the corresponding biomolecule in is able to. For example, the part of a biomolecule can be a nucleotide, dinucleotide or a trinucleotide that does not match the nuclei present in the biological sample acids are able to hybridize, while the biomolecule is an oligonucleotide that is responsible for this be able to.

In principle, more than 60, 100, 600, 1000, 5000, 10000, 40,000, 100,000, 400,000 or 1,000,000 defined places can be arranged on an array per cm ^{2 of carrier surface.} As a rule, arrays according to the invention have 500 to 1,000,000 spaces / cm ² . A special embodiment are arrays with 500 to 10000 places / cm ² and in particular 1000 to 5000 places / cm ² (low density). Another particular embodiment are arrays with 10,000 to 1,000,000 places / cm ² and in particular 50,000 to 250,000 places / cm ² (high density). The same or different quinone derivatives, sometimes different biomolecules, can be immobilized in different places.

Purpose of the arrangement described above using co-immobilized Quinone derivatives are to provide places with defined densities of immobilized biomolecules put. According to the invention, densities are realized in this way that are smaller than that Value of the maximum occupancy of immobilizable molecules per unit area corresponds to. Thus, arrangements according to the invention can be attached to a surface at a total of x unit-coupled quinones or quinone derivatives (x-y) immobilized biomolecules have, where y is the number of co-immobilized quinone derivatives. Preferably the value x corresponds to the maximum number that can be coupled to a surface unit Quinones (degree of saturation), d. H. preferred arrays have fields of immobilized biomoles küle on whose surface in relation to the coupling of the quinone derivatives used is saturated. The proportion of immobilized biomolecules (x-y / x) is advantageously at least 0.001, preferably at least 0.1, and in particular at least 0.75. In In certain cases, the proportion can advantageously be less than 1, preferably less be than 0.75 and in particular less than 0.5.

According to a particular embodiment, arrays according to the invention contain spaces with biomolecules of the same kind immobilized in different densities. In shape Internal calibration of standard series can thus be made possible.

According to an advantageous embodiment, a site contains immobilized biomoles Make a label that is proportional to the amount of quinone derivatives coupled. purpose this embodiment is an assessment of the immobilized biomolecules in particular take into account the amount and distribution of the immobilized biomolecules to be able to.

Immobilized biomolecules and / or co-immobilized quinone derivatives can be used for this purpose be marked. The spacers are preferably marked.

In principle, all types of marking are suitable. Preferably the marking is so chosen that it does not, or only minimally, interferes with the subsequent analytics, the Use of the arrays is therefore not or only marginally influenced. These are above all any necessary markings of the sample, ie in particular of nucleic acids re-targets to be considered. On the other hand, the marking can be chosen so that it is with the marking of the sample interacts. That of interactive markings outgoing signal may be distinguishable from that of the corresponding runs out of non-interacting markings. The interaction can, for example way to influence the signal intensity, e.g. B. amplify or quench, or the signal modify, e.g. B. change the wavelength of absorbed or emitted light.

According to the invention, marking systems which can be recognized spectroscopically, photochemically, biochemically, immunochemically, electrically, optically or chemically, are suitable. This includes both direct marking systems, such as radioactive markers (e.g. ³² P, ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C), magnetic markers, chromophores, for example UV, VIS or IR absorbing compounds, fluorophores, chemi- or bioluminescent markers, transition metals, which are usually chelated, or enzymes, e.g. B. horseradish peroxidase or alkaline phosphatase and the detection reactions coupled to them, as well as indirect labeling systems, for example haptens, such as biotin or digoxigenin, which can be recognized via appropriate detection systems.

Advantageous chromophores have an intense color that of the surrounding ones Molecules is only slightly absorbed. Dye classes, such as quinolines, triarylmetha ne, acridines, alizarines, phthaleins, azo compounds, anthraquinones, cyanines, phenaza thionium compounds or phenazoxonium compounds are representative of called 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. According to the invention it is advantageous that one and the same fluorophore, depending on the excitation and detection principle, several can emit distinguishable radiation.

Fluorophores can be used alone or in combination with a quencher (e.g. Molecular Beacons) are used.

Preferred fluorophores are 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 and IRD41.

Particularly preferred fluorophores are Cy5, 5-Tamra and Cy3.

The above exemplary list shows that a large number of distinguishable Markie ments is available, so that an appropriate ratio of marks of the immobilized molecules on the one hand and, if necessary, the sample on the other can be realized.

Cycloolefin-based polymers are known per se. One or more of the following are useful:

• - a relatively low total surface energy, which is in particular lower than for example that of polymethyl methacrylate (PMMA) and preferably in one Range from 25 to 35 mN / m and more preferably in a range from 28 to 32 mN / m lies. A relatively low polar component is particularly advantageous here. So amounts the ratio of the dispersive part to the polar part according to the OWRK method advantageously at least 2: 1, preferably 3: 1 and particularly preferably a little at least 4: 1;
• - a relatively low surface roughness. Grid is an advantage force microscopy (AFM) determined rms roughness levels of less than 1.5 nm and preferably less than 1.0 nm. The rms roughness is advantageously in a range from 0.6 to 0.8 nm;
• - a relatively high transmission, advantageously at least 85% and preferably at least 90% transmissions at λ = 400 nm and / or λ = 800 nm (to be determined by means of UV-VIS spectrophotometry);
• - a relatively low reflection, advantageously less than 10% and preferably less than 9% reflection at λ = 400 nm and / or λ = 800 nm (using To determine UV-VIS spectrophotometry);
• - A relatively low water absorption, which is advantageously less than 0.01 wt% (ASTM D570);
• - a relatively low proportion of foreign particles. It is advantageous here if the content of foreign particles with a diameter of 0.5 μm or more, less than 10 ⁵ particles / g and preferably less than 5 × 10 ⁴ particles / g;
• - A relatively low self-fluorescence, suitably similar to that of Pyrex glass is.

For example, reference is made here to those in EP 0 591 892, US 5,008,356, US 5,087,677, WO 97/46617, EP 0 713 893, EP 0 827 975, EP 0 997 482 described polycycloolefins (polymers and copolymers) which are part of this application.

Preferred representatives of this group are based on structural units of the formula VIII

where the radicals R ² , R ³ , R ⁴ , R ^{5 can be} identical or different and, independently of one another, represent hydrogen, halogen, hydroxy, cyano or optionally alkyl substituted once, twice, three times, four times or five times with halogen, cyano or alkyl , Alkenyl, alkynyl, cycloalkyl, cycloalkenyl, alkoxy, alkoxycarbonyl, alkanoyloxy, aryl or aralkyl, or R ² together with R ³ or R ⁴ together with R ^{5 represent} alkylidene optionally substituted in the manner described above, or 2 or several of the radicals R ² , R ³ , R ⁴ , R ^{5 form} a double bond or, with appropriate modification of the aforementioned single-bond radicals, a ring or several rings, in particular cycloaliphatic rings, which in turn form single-bond radicals R ² , R ³ , R ⁴ , R ⁵ can be substituted. The symbol stands for a carbon single or double bond.

Unless stated otherwise, “Alk” or “alk” stands primarily for hydrocarbons radicals with 1 to 15 carbon atoms, and "aryl" especially for aromatics with 6 to 10 Hydrocarbon radicals, especially phenyl and naphthyl.

Preferred polymers of this type are based on structural units of the formula IX

where R ⁶ to R ¹³ independently of one another have the meanings given ^{above for R 1} to R ^4. Preferably, R ⁶ to R ⁹ independently represent hydrogen, C ₁ -C ₆ -alkyl or C ₁ -C ₆ -alkylidene, or 2 or more of the radicals R ⁶ to R ⁹ together form a cyclohexane ring, cyclopentane ring, norbornene ring or benzene ring. These are also the preferred meanings of R ¹⁰ to R ¹³ .

The polymers described above can be referred to as ring opening polymers or copolymers. They can be obtained from monomers of the formula X

where R ² to R ^{5 have} the meanings given above, and optionally further comonomers by ring-opening polymerization or copolymerization and optionally subsequent hydrogenation.

Preferred representatives of these polymers can be obtained from monomers of the formula XI

where R ⁶ to R ^{9 have} the meanings given above, and / or
Monomers of formula XII

where R ¹⁰ to R ^{13 have} the meanings given above,
and optionally further comonomers in a corresponding manner.

Further preferred representatives of polymers based on cycloolefin are based on structural units of the formula XIII

where R ² to R ^{5 have} the meanings given above and R ¹⁴ , R ^{15 can be} identical or different and independently represent hydrogen, alkyl or aryl.

Preferred polymers of this type are based on structural units of the formula XIV

where R ⁶ to R ^{13 have} the meanings given above. R ¹⁴ , R ¹⁵ are preferably hydrogen, C ₁ -C ₆ -alkyl or C ₆ -C ₁₀ -aryl, in particular hydrogen.

These polymers can be referred to as addition polymers or copolymers. They can be obtained by addition polymerization or copolymerization of at least one cycloolefin monomer of the formula X, preferably at least one cycloolefin polymer of the formula XI and / or at least one cycloolefin monomer of the formula XII with a monomer of the formula XV

where R ¹⁴ , R ^{15 have} the meanings given above,
and optionally further comonomers.

Specific examples of monomers of the formula X can be found in EP 0 827 975 A2 Pages 6, line 41 to page 8, line 21, in EP 0 997 482 A1 on page 4, lines 24-49, in EP 0 713 893 A1 in column 3, line 39 to column 5, line 2 and in the No. 5,008,356 in column 6, lines 37-67 and in Tables 1 and 2. To this Reference is made in full to this text, with the monomers being part of this Registration are.

Specific examples of the monomers of the formula XV are given in EP 0 827 975 A2 pages 10, line 54 to page 11, line 3, and in EP 0 997 482 A1 on page 4, Called lines 55-58. Reference is also made to these statements in full taken, whereby these monomers are also part of this application.

Preference is given to polycycloolefins derived from norbornene derivatives, d. H. especially Norbornene and the above information correspondingly substituted norbornene derivatives, are available.

A particular embodiment of cycloolefin-based polymers which can be used according to the invention are obtainable by addition polymerization of vinyl compounds of the formula XV, in particular ethylene, with

• a) Norbornene monomers, in particular norbornene, tetracyclododecene or dicyclo pentadiene;
• b) monocycloolefin monomers, in particular cyclopentene or cyclohexene; or
• c) Cyclic conjugated diene monomers, in particular cyclopentadiene or cyclohexa serve.

Such addition polymers are, for example, according to the US Pat. No. 5,087,677 be procedures are available.

Another particular embodiment of polymers that can be used according to the invention Cycloolefin-based are obtainable by ring-opening polymerization of norbornende derivatives and subsequent hydrogenation of the double remaining after polymerization ties. Are the norbornene derivatives used with aromatic groups substituted? do they indicate or exhibit polycyclic structures with norbornene and aromatic structures Relementen on it, it is advantageous if at least 90% of the unsaturated bonds of the main polymer chain are hydrogenated during at least 80% of the aromatic structure ren are preserved.

Such ring-opening polymers are, for example, according to that in EP 0 713 893 A1 described method available.

For example, an addition polymer of norbornene and ethylene is available from the Company Hoechst sold under the trade name Topas®. More commercially available Cycloolefin-based polymers, for example, have the trade names Zeonex®, in particular Zeonex®480 R and Zeonex®480, and are available from Zeon.

If the carrier surface contains at least one polymer based on cycloolefin, it can additionally contain further polymers, in particular polyolefins. A preferred one Embodiment of carbon-containing surfaces consists essentially of one Polymer based on cycloolefin or a mixture of several polymers Cycloolefin based.

The carrier can be made of a single material. This is especially true for carriers with a surface made of polycycloolefin is preferred. However, carriers are also possible which are composed of different materials, e.g. B. where an inventive Surface is applied to other carrier materials. In principle, the inven carbon-containing surfaces according to the invention on any useful carrier material lien be upset. For example, the following substances can be used as carrier material can be used: glass (standard glass, Pyrex glass, quartz glass), plastics preferred high purity or low intrinsic fluorescence (such as polyolefins, e.g. PE (polyethylene), PP (Polypropylene), polymethylpentene, polystyrene, PMMA (poly (methyl methacrylate)), polycar bonat, Teflon), metals (such as gold, chromium, copper, titanium, silicon), oxidic materials (Ceramics, aluminum-doped zinc oxide (TCO), silica, aluminum oxide).

As an alternative to the carbon-containing surfaces described above on Polycy cloolefin-based surfaces according to the invention can be obtained in that glass, metal or ceramic surfaces with an aqueous solution of at least one Treats hydrolyzable carbonaceous compound and dries the surface.

Appropriate configurations of this procedure are based primarily on the Regulations for sol-gel processes.

Suitable hydrolyzable carbon-containing compounds are based primarily on Elements M of the main groups III to V and the subgroups II to IV of the Periodensy stems of the elements, in particular Si, Al, B, Pb, Sn, Ti, Zr, V and Zn, of which Si, Al, Ti and Zr and particularly Si are preferred. These compounds have at least one hydrolyzable group A and at least one non-hydrolyzable carbonaceous group Group B on. In general, the molar ratio of groups A to groups B in the Connections are 10: 1 to 1: 2.

Hydrolyzable groups A include halogens, in particular Cl and Br, alkoxy groups, in particular C _1-4 alkoxy groups, aryloxy groups, in particular C _6-10 aryloxy groups, acyloxy groups, in particular C ₁ -C ₄ acyloxy groups, and alkyl carbonyl groups, in particular C _1-4 alkyl carbonyl groups. Preferred groups are Cl, methoxy, ethoxy, n-propoxy, i-propoxy, butoxy, phenoxy, acetoxy, propionyloxy and acetyl.

At least one non-hydrolyzable group B is expediently able to couple to quinones. Groups B of this type are mainly derived from aliphatic, heteroaliphatic, cycloaliphatic, cycloheteroaliphatic or aromatic radicals. These include above all alkyl groups, in particular C ₁ -C ₃₀ -alkyl groups, cycloalkyl groups, in particular C ₃ -C ₁₀ -cycloalkyl groups, alkenyl groups, in particular C ₂ -C ₃₀ -alkenyl groups, aryl groups, in particular C ₆ -C ₁₂ -aryl groups, aralkyl groups pen, in particular C ₇ -C ₁₀ aralkyl groups, it being possible for these groups to be substituted one or more times by alkyl, in particular C ₁ -C ₄ alkyl, alkoxy, in particular C ₁ -C ₄ alkoxy and halogen. Hydrolyzable carbon-containing compounds with at least one aliphatic or cycloaliphatic group B with 3 to 20 carbon atoms, preferably 3 to 16 carbon atoms, are preferred.

A particularly preferred class of hydrolyzable carbon-containing compounds are the silanes of the formula XVI

(R ¹⁷ ) _z Si (R ¹⁶ ) _4-z (XVI)

where the radicals R ¹⁶ , which can be identical or different, independently represent halogen, in particular Cl or Br, C ₁ -C ₄ alkoxy, in particular methoxy and ethoxy, C ₁ -C ₄ acyloxy, in particular acetyloxy, and the radicals R ¹⁷ , which can be the same or different, are independently C ₁ -C ₃₀ -alkyl or C ₃ -C ₁₀ -cycloalkyl, in particular cyclohexyl, and z corresponds to a value of 1, 2 or 3.

Particular mention may be Dichlormethylcyclohexylsilan, phenyltriethoxysilane, (2-phenylethyl) trichlorosilane, phenyldimethylchlorosilane, Dimethoxymethylcyclohexylsilan and (C ₃ -C ₁₆ alkyl) _n - (C ₁ -C ₄ alkoxy) _m - (chloro) silane _l, wherein n , m and l independently of one another stand for a value of 0, 1, 2 or 3 and the sum of n, m and l is 4.

These compounds can be produced in a manner known per se, and in some cases also be obtained commercially.

These compounds are in the aqueous solution to be used according to the invention at least partially hydrolyzed. They preferably form a colloidal solution. Lyosols and especially hydrosols are used in particular. It is in the Usually about disperse systems.

Usable aqueous solutions are based on water and can include additional ones Solvents, especially lower alcohols such as methanol, ethanol and isopropanol, Ketones like acetone, ethers like diethyl ether, sulfoxides like DMSO and others with water contain miscible solvents. The water content, based on the total weight of solvent, is at least 30%, preferably at least 50% and in particular whose at least 80%.

According to a particular embodiment, aqueous solutions are used that have so much Water contain that more than 10, preferably more than 20 and especially more than 40%, advantageously 10 to 99%, preferably 25 to 75%, in particular about 50% of all hydrolyzable groups are hydrolyzed. The pH of the solution is in the Usually below 7, preferably 2 to 5 and especially 3 to 4.

The pH of the aqueous solution can be adjusted with suitable acids, e.g. B. chlorinated water chemical acid or acetic acid, also as an aqueous solution, can be adjusted.

Before the glass or metal surface to be treated with the aqueous solution of the hydrolyzable carbonaceous compound is brought into contact, it is of Advantage of cleaning the surface. This can be done in a manner known per se, e.g. B. with degreasing agents, e.g. B. isopropanol and / or diethyl ether, and / or ultrasound.

The surface is preferably coated with the aqueous solution. To this Conventional coating processes can be used for this purpose, for example dipping, Flooding, pulling, pouring, centrifuging, spraying or brushing. That is preferred Diving. This process is carried out and optimized by the Professional.

The surface and the aqueous solution in contact therewith is then the heated hydrolyzable carbonaceous compound. Solvent evaporates and the coating hardens.

In this context, drying means the removal of solvents, in particular those of water. At least some of the solvent is removed. That is an advantage essentially complete removal of water. Drying can be done by heating men are supported. Usually the heating takes place at temperatures in one Range of 20-500 ° C and preferably 50 ° C to 200 ° C.

The hardened layers usually have thicknesses in the range between 2 μm and 30 µm. Layer thicknesses in a range from 2 to 10 μm and in particular are preferred others from 2 to 5 µm.

The carbon-containing surfaces according to the invention are notable for their low roughness. The R _a value is advantageously less than 0.5 μm, preferably less than 0.2 μm and in particular less than 0.1 μm.

The carbonaceous surfaces of the invention also show a pronounced one water repellent behavior. The contact angle is advantageously more than 60 °, preferably more than 70 ° and in particular more than 80 °.

According to a particular embodiment, parts of the carrier surface have inventions according to arrays on further coatings to which no quinones are coupled.

Metals are particularly suitable, e.g. B. copper, titanium, chromium, gold and especially platinum, or polymers. Such coatings expediently give a surface surface structuring, according to which the places where biomolecules are immobilized, do not have such a coating, while the parts of the surface with a such a coating do not have any immobilized biomolecules. Such arrays can advantageously achieve a more precise coupling of quinones on defined areas exhibit.

The present invention also relates to carriers in the sense of a preliminary product, the surfaces of which are partially covered with suitable materials. Are preferred Carrier with a structured coating (pre-structuring), with recesses in predefine the coating defined places (array), on which then biomolecules can be immobilized.

Carriers in the sense of the invention are usually rigid or semi-rigid materials rer surface and in this sense as solid. Planar surfaces are common chen. In certain cases, however, it can be advantageous to use profiled supports. Elevations and depressions, such as steps, grooves, channels, notches, for example V-notches or mesa structures are possible. These structures can be on the surface surface be arranged that it expediently influence the immobilization of the biomolecules sen. In the case of light-controlled synthesis, for example, incident light can pass through such structures of the carrier surface are specifically influenced, for example mirrored and even be focused, channels can be used to guide fluid.

At least parts of the surface of such supports correspond to those according to the invention Requirements.

The shape of a carrier can be varied and depends primarily on the type the use of the carrier to be described. It can be, for example Slides, microtiter plates, tubes, chips, dipsticks, stamps, caps, particles, Strands, especially fiber bundles, spherical bodies, such as spheres or spheres, Precipitation products, membranes, gels, sheets, containers, capillaries, discs, foils or trade records. Wafer formats can also serve as carriers, the desired ones if are separable. The carrier can also be equipped with other useful components be provided, for example one can encapsulate chips. According to a particular Embodiment is a body, in particular in the form of a dipstick, with a Closure means, e.g. B. provided a snap or screw cap that is placed on a suitable container, especially an incubation vessel, that is placed on the body immobilized biomolecules in contact with a sample fluid located in the container can bring. The body is preferably in the form of a pin, on one of which End of the closure means and expediently at the other end of the Biomolecules are immobilized. At least part of the surface corresponds to the Body the specifications according to the invention. Practically, the body can get out of the Polycycloolefin-based polymer or a suitable composite material with corre corresponding surface parts be shaped. Furthermore, several dipsticks - also according to the embodiment explained above - be individually connected to one another.

The places where biomolecules are immobilized, in particular fields of immobilized biomolecules, can have different geometries. In principle, the shape can be any, e.g. B. circular, rectangular, square or elliptical. The area is preferably less than 1 mm ² , in particular less than 10,000 μm ² , and very particularly preferably less than 1000 μm ² .

According to a particular embodiment of the invention, biomolecules are as Spots immobilized, d. H. as substantially circular fields. Have such spots preferably diameter in a range from 1 μm to 1000 μm, in particular 5 μm up to 20 µm or 50 µm to 500 µm. In particular spots with diameters below 200 µm and especially below 20 µm can be achieved with the one described above Realize pre-structuring in an advantageous manner.

According to a preferred embodiment of arrays according to the invention, these are in the case of biomolecules, around nucleic acids, in particular oligonucleotides with a length from 2 to 500 bases. As a rule, the immobilized oligonucleotides are able to to bind a target nucleic acid. In this sense, the immobilized Oligonu Kleotide also known as probes.

The probes are usually single-stranded oligomers. DNA, RNA as well Nucleic acid analogs and derivatives, such as optionally modified PNA, LNA and PSNA as well as modified DNA or RNA can be used. The minimum length of the probes depends on the complexity of the sample, in particular the number of bases to be detected sender nucleic acids, but also on the type of nucleic acid used as a probe type and the achievable thermodynamic stability between sample and probe.

Depending on the nucleic acid type, probes usually have a length of 8 to 60, preferably from 13 to 25 and in particular from 13 bases for DNA, from 8 to 60, preferably from 13 to 25 and especially from 13 bases for DNA-LNA hybrids, from 8 to 60, preferably from 13 to 25 and especially from 13 bases for PSNA, from 6 to 30, preferably from 8 to 18 and especially from 9 bases for LNA, and from 6 to 18, preferably from 8 to 18 and especially from 9 bases for PNA.

With relatively low complexity of the nucleic acids to be detected, e.g. B. amplificates, in particular PCR amplificates, viruses, plasmids and microorganisms are relative short probes, for example in the range of 8-25 mers and in particular 8-15 mers, are appropriate thirty. With relatively high complexity of the nucleic acids to be detected, e.g. B. non-amplifi decorated RNA; Totally amplified RNA via cDNA, unamplified total genomic mixer DNA, and amplified whole genomic DNA, are longer probes, roughly in the range from 16-60mers for synthetic probes, expedient or the probes consist of PCR products, cDNA, plasmids or DNA from lysed bacteria that also contain a can have a base number in excess of this.

The base sequence of the probes depends primarily on their function. A test probe usually has a sequence of bases that is complementary to a specific target sequence of a nucleic acid to be detected or, according to another aspect, can specifically hybridize with the target sequence. A control probe can in particular have the function of a normalization, mismatch, housekeeping, sample preparation, hybridization or amplification control and accordingly have a base sequence that is complementary to a base sequence of a reference nucleic acid, a nucleic acid to be detected with a similar base sequence, a constitutively expressed nucleic acid, e.g. B. a _{nucleic acid derived from β 2} -microglobulin, β-actin, GAPDH or transferrin receptor gene, a species-specific nucleic acid or an amplificate.

The present invention also relates to a method for immobilizing biomolecules,

• a) being at least one derivative of a coupling group, preferably a quinone derivative, applied to a carrier with a carbonaceous surface, exposed and if necessary, the immobilized derivatives are transferred into the biomolecules, the The method is characterized in that the carbon-containing surface at least one polycycloolefin is used; or
• b) wherein having a support surface with at least one hydrolyzable carbon containing compound treated, at least one derivative of a coupling group, preferably wise a quinone derivative, applied to the carbonaceous support surface, exposed and, if necessary, converting the immobilized derivatives into the biomolecules, wherein the method is characterized in that an aqueous solution of the hydrolyte convertible carbonaceous compound is used.

Developments according to the invention of this subject are also based on the preferred embodiment directed to quinones as representative of others suitable coupling groups described.

Exposure takes place to couple the quinone to the carbon-containing surface. exposure here means the radiation of electromagnetic radiation for the purpose of quinone activation. It should be a non-ionizing radiation whose wavelength is in the Usually in the UV-VIS range. The radiation should interfere with other functions as little as possible nell groups of the quinone derivatives interact. For practical reasons, commonly used incoherent continuous light. Monochrome can also be used matic, polarized, pulsed or coherent light can be used. Suitable Light sources are known and can usually be obtained commercially.

The exposure time is measured by an effective coupling of the quinones to the carbon Substance-containing surface with minimal decomposition of the quinone derivative. Usually amounts to the exposure time less than 200 minutes. Exposure times of less than 60 minutes, especially less than 10 minutes and especially less than 5 minutes.

The quinone derivative to be applied is based on at least one functionalized Quinone structure and can be spacers and / or biomolecules or derivatives or parts thereof exhibit. For example, functionalities on spacers and / or biomolecules have suitable protecting or activating groups.

The resulting procedures for immobilizing biomolecules can basically be differentiated into a variant in which the biomolecule while the quinone coupling is present, and another variant in which the Biomolecule only after the quinone has been coupled to an already immobilized quinone vat is added. The variant to be chosen in each individual case depends on the type of biomole küls and its synthesis.

For example, it can be useful to first use the functionalized quinone optionally with the interposition of a spacer to bind to the biomolecule and this prefabricated construct is then attached to the carbonaceous surface couple. This variant offers the advantage that the prefabricated constructs as a substance - If necessary after appropriate purification and advantageously characteristic sated - can be essentially homogeneous, so that the quality of the resulting Arrays are mainly determined by the coupling reaction. This is where the present one offers Invention, in turn, advantageous embodiments that on the one hand result in an effective and Contribute reproducible coupling, on the other hand a control of the coupling result enable nisses and thus the array.

According to a preferred embodiment of the present method are therefore prefabricated, quinone and biomolecule or derivatives thereof having constructs (possibly with the use of spacers and other linkers) on the carbon material-containing surface applied and exposed. If necessary, a Then transfer the immobilized constructs into the desired biomolecules, as a rule, however, this is not necessary. This embodiment takes place above all Application to nucleic acids and especially oligonucleotides.

For this purpose, the nucleic acids are first made available in a manner known per se. Oligonucleotides can be conveniently used with solid phase synthesis methods, such as the phosphate the ester coupling or phosphoramidite method can be synthesized. Come for PNAs suitable peptide synthesis systems for use. Used another option Nucleic acids of natural origin as a template for generation according to the invention suitable nucleic acids, for example in the form of cDNA, ccDNA, RNA or cRNA. Above all, amplicons, particularly PCR products, should be mentioned here.

The nucleic acid is required for binding to functionalized quinone or spacers if necessary to be modified at a suitable point, for example by implementing with Amino modifiers or biotin analogs, such as. B. activatable aryl azide derivatives of Biotin. Binding at the 5 'end of the nucleic acid is preferred.

The quinone is preferably used as a carboxylic acid or reactive acyl derivative, for example se as a carboxylic acid halide, with suitable spacers, biomolecules or Implemented spacer-biomolecule constructs or appropriate derivatives thereof. This implementation tion takes place in a manner known per se, depending on the type of bond to be realized. The Spacers can also be built up step by step, for example by successively starting from the functionalized quinone first the individual linkers and finally adding the biomolecule or molecules, or on the one hand the functionalized quinone with one or more linkers and on the other hand the biomolecule with one or several linkers implemented and then the resulting constructs together adds. If amplificates, in particular PCR products, are to be immobilized, one can first of all the primers to be used with the quinone, optionally with intermediate switching of spacers, derivatizing. The amplificate then already has the quinone and can usually be coupled directly to the surface.

In certain cases, however, it may be necessary and also appropriate that the Biomolecule is not present during the quinone coupling. This is especially true useful if the coupling conditions affect the biomolecule. A another case concerns the possibility of the biomolecules following the coupling to be synthesized successively starting from the immobilized quinone derivatives.

In principle, the known in the solid phase synthesis of Nucleic acids and peptides are used to apply methods.

Shall different biomolecules, especially oligonucleotides and peptides with different union sequences are synthesized in parallel, can be activated by means of photoactivatable Protective groups and selective irradiation only those oligomers are deprotected which are then to be provided with a certain additional module. The selective one Irradiation is usually carried out using suitable lithographic masks. To further Explanation is given here to the basic explanations in this regard in the US patent 5,744,305.

As a rule, the quinone derivative is applied in solution to the carbon-containing carrier surface upset. Suitable solvents should be with the quinone derivative and in particular be compatible with the photoactivated quinone derivative. In addition, it is advantageous that the even distribution of the quinone derivatives on the surface before the Ver the solvent has been evaporated. The result is preferably a homogeneous one Distribution of the quinone derivatives on the treated surface, being in particular it is preferred that the treated surface is saturated with quinone derivatives.

In principle, aqueous and organic solvents can be used. Prefers are aqueous solvents. These can contain organic solvents. Preference wisely, the proportion of organic solvents is less than 10% by volume and in particular less than 5% by volume. Suitable organic solvents include lower alcohols such as methanol, ethanol and 1-propanol, ketones such as acetone, ethers such as Diethyl ether, sulfoxides such as DMSO and other water-miscible solvents. Aqueous solutions with a propanol content of about 1 to 10% by volume, in particular about 5% have proven to be useful.

According to a preferred embodiment of the method according to the invention, sets a hygroscopic salt is added to the solution to be applied. Calcium is preferred chloride. It has proven to be useful to use the hygroscopic salt in the mM range add, preferably about 0.1 mM to 200 mM, preferably 1 mM to 50 mM and especially about 50 mM.

In principle, all techniques are suitable for applying the quinone derivatives that include a allow defined and reproducible delivery of a certain amount of liquid. For example, techniques such as pipetting, dispensing, printing, stamping can be used Application.

For example, non-contact methods such as piezo dispensers and pressure pulses can be used dispenser. Here is the control of temperature and relative humidity Avoidance of satellite spots proved useful. Especially with plastic carriers The grounding of the carrier material to dissipate electrostatic charge is popular makes sense.

Contacting methods, such as capillary dispensers, which use the Liquid with a thin capillary, for example with an inner diameter of 1.5 to 5 µm, and the so-called pin and ring technology. About the The polarity of the medium to be dispensed can guarantee optimal tear-off be e.g. B. by adding salt or solvents such as ethanol or 1-propanol.

The amount of liquid dispensed is important as it determines the area on which the Biomolecules are immobilized, so in particular the spot size is determined. In the As a rule, amounts of liquid in the femto to nanoliter range are applied. So are Spots with a diameter of about 150 to 200 µm with a non-contact applied th quantity available from about 0.5 nl to 1.5 nl. Spots with diameters in the range of 10 µm can be produced by using capillary dispensers to dispense amounts of approx 1 pl applies.

According to a particular embodiment of the method according to the invention the quinone derivatives applied to a pre-structured surface. These are parts the surface treated so that quinone derivatives to the treated areas in the essential not couple. For example, you can parts of the surface with materials coat which, under the coupling conditions, are essentially not with the Quinone derivatives react. Above all, this includes metals such as chromium, copper, titanium, Platinum, gold and the like, other inorganic materials such as glasses or ceramics ken, as well as organic materials, including polymers. There is also one Plasma treatment of the surface, for example with argon plasma, into consideration.

According to a preferred embodiment of this embodiment, one brings first a metal layer, preferably a platinum or chrome layer, on the carbon-containing ge surface. This can be done in a manner known per se, for example can the metal can be sputtered or vapor deposited. Then the parts of the applied layer removed again and thus the parts of the carbonaceous Surface exposed again, on which biomolecules are to be immobilized. Also parts of the metal layer can be removed in a manner known per se. For example, one can be conventionally used for this purpose Apply photoresist, this over a suitable mask, usually a photolithogra phische mask, expose in the places where the metal then, for example wisely by etching, should be removed. This way you can by choosing a certain mask with recesses of a defined size and shape the carbon containing surface as an image of this mask in couplable and non-couplable Areas are divided.

Alternatively, photoresist can also first be applied to the carbon-containing surface be genes. This is then exposed through an inverse mask and developed so that then metal can be applied to those parts of the surface which biomolecules should not be immobilized. Those places where Biomolecules to be immobilized are initially coated with photoresist, before coupling the biomolecules in a suitable manner, for example with appropriate Solvents such as acetone is removed.

In addition to the application of defined and reproducible amounts of liquid this embodiment in particular has the advantage of the shape and the maximum area to be able to choose a place to immobilize biomolecules.

After the quinone derivatives have been applied, the solution is generally left first evaporate medium. This is usually done at room temperature, but it can also can be influenced by choosing suitable higher or lower temperatures.

Following the exposure and the associated coupling of quinones on a washing process is usually carried out on the carbon-containing surface. The purpose of this washing process is, in particular, to add uncoupled quinone derivatives remove. Based on the one previously used to apply the quinone derivative Aqueous solvents or solvent mixtures are also used for washing used. In principle, the above statements apply here accordingly.

According to a particular embodiment, washing is carried out with a solvent or Solvent mixture containing a surfactant, preferably a nonionic surfactant. Polyalkoxylated, in particular polyethoxylated, fatty acid esters are particularly preferred of polyols, in particular of glycerol or sorbitol, for example those under the Sorbitan fatty acid esters sold under the trade name Tween®. In particular, the Use of ethoxylated sorbitan monolaurate proved to be expedient.

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

For example, the support surface after exposure to about 0.5 wt .-% to about 2 wt .-% Tween 20 containing solution washed, especially in this Solution to be immersed. The purpose of this first wash is in particular to provide a Avoid coupling the reagents to be removed. Then the Support surface then with about 0.01 wt .-% to about 0.05 wt .-% Tween 20 containing solution are rinsed.

The concentration of quinone derivatives in the solution to be applied is variable and depends in particular on the desired density of immobilized biomolecules. Concentrations in the range from 0.1 µM to 100 µM can be used, with im lower concentration range in certain cases a saturation of the surface with Quinones is not reached, while this is the case in the upper concentration range and excess quinone derivatives that have not coupled due to saturation, can then be removed. Concentrations above at least 5 µM have to saturate the surface with satisfactory homogeneity of the immobilized biomolecules proved to be useful.

After washing with a surfactant-containing solvent or solvent mixture, you can further washing steps follow. For this it may be useful to use organic Solvents, especially lower alcohols, such as isopropanol, to use which can then be removed again by rinsing with water.

Finally, the surface is dried under normal conditions, conveniently at Ambient temperature and, if desired, by blowing dry through air or Inert gas.

Are the immobilized biomolecules according to the above special embodiment with provided with a marking, a quality control of the array can then follow eat. For this purpose, the array can be equipped with a detection system corresponding to the marking be measured. Let over the measured amount of immobilized marker in particular the coupling efficiency and thus the immobilization of the biomolecules assess in terms of area, homogeneity and density.

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

The following applications are mentioned as examples:
Diagnostics, therapy selection and therapy control of tumor and metabolic diseases; Investigation of genetic predisposition (SNPs), in particular the detection of mutations, also in the forensic area; Gene expression control; Sequencing, in particular sequencing by hybridization; Microbiological applications such as in bacteriology and virology, for example for differentiating different strains; ELISA applications with immobilized antibodies, antigens, receptors and ligands in clinical chemistry through to food chemistry and environmental analysis; Allergy diagnostics with immobilized allergens; High throughput screening of substance libraries.

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

In non-competitive assays, the substance to be analyzed (analyte) occurs with the on the Surface immobilized biomolecules in interaction. Usually the analyte previously marked, but marking is also possible after the analyte has already been marked the immobilized biomolecules has interacted, e.g. B. by means of primers Extension or rolling cycle PCR. In these cases, a measurement signal is obtained that is so is larger, the more analyte is present. It is also possible that through the interaction The effect of the sample with the substances immobilized on the surface increases the fluorescence of the immobilized substances is changed (weakening, amplification, e.g. Molecular Beacons) or the activity of an enzyme is changed and this change is registered as a measuring signal. Examples of non-competitive assays are hybridization reactions of PCR products or labeled DNA / RNA to immobi on the surface lized nucleic acids, in particular oligonucleotides or cDNA, and sandwichimmu noassays.

In competitive procedures, a marked substance (marker) is added to the sample, the similar binding properties to the biomoles immobilized on the surface like the analyte itself. There is a competitive reaction between analyte and Markers held around the limited number of binding sites on the surface. Man receives a signal that is lower the more analyte is present. examples for competitive assays are immunoassays (ELISA) and receptor assays.

The arrays according to the invention are suitable for examining any samples biologically of origin that may contain a particular analyte. One embodiment concerns body samples of human and animal origin. If necessary, an preparatory work-up of the analyte present in the sample or a fraction which may contain this analyte. This work-up usually corresponds to standard practice. Samples such as blood and blood components or isolates thereof, tissue, native, frozen, fixed, with and without dissection, cells from body fluids, e.g. B. sputum, lavages, punctures, Exudates and urine, or stool, can advantageously be taken with arrays according to the invention be sought. Such analytical methods using the arrays are in vitro Procedure.

According to a preferred embodiment, arrays according to the invention are for hybridization determination experiments. Expediently, there are nuclei on the carrier surface acids immobilized, which can hybridize as probes with certain targets. A oligonucleotide arrays are a special type of such arrays.

The design of the probes takes place in particular with a view to the target or targets that are of analytical interest (analyte). This is how test probes are usually designed tet that they work with a defined target, for example a specific base sequence, hybridize specifically. Good discrimination is particularly important between perfect match and mismatch. This can be done using the melting points of the hybrids Probe and target as well as corresponding melting points for defined mismatches calculated using the nearest-neighbor model and thus making statements about the discrimination of Perfect match and mismatch are made (cf. e.g. J. J. SantaLucia: Proc. Natl. Acad. Sci. USA 95 (1998) 1460 and Peyret, e. al .: Biochemistry 38 (1999) 3468-3477).

For hybridization, the immobilized probe is brought into contact with a hybridization rgemisch that one of the sample to be examined after appropriate samples preparation derived part, and, if necessary, further expedient additives. It goes without saying that parts of the mixture are initially separated from one another with the probe can be brought into contact. The hybridization conditions are appropriate Usually chosen so that the probe and the target complementary to it are stable hybrids can form. For this purpose, conditions are usually relatively lower at first Stringency chosen, e.g. B. Temperatures of about 20-50 ° C and in particular of about 30-40 ° C and ionic strengths of about 6 × SSPE or lower. Then can then at similar or higher stringency, e.g. B. about 1 × SSPE at about 30-40 ° C to about 0.25 x SSPE can be washed at about 30-50 ° C. It can also be under stringent Conditions are hybridized and the unbound labeled sample portion to be washed away. Furthermore, known agents, e.g. B. detergents, block reagents, denaturing agents, agents accelerating renaturation and Tm-leveling reagents can be used. The optimization of the Hybridisie the log is a matter for the specialist.

Amplified or non-amplified nucleic acids can be used for the hybridization step can be used. The known amplification methods to which the Polymerase chain reaction (PCR), also called nested PCR, asymmetric PCR or Multiplex PCR performed, or alternative methods such as the ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated Amplification (TMA) and the like. Certain versions of this Techniques such as mutation-enriching PCR and / or combinations with others molecular biological methods, such as reverse transcription (RT), can be appropriate. In particular, the arrays according to the invention enable hybridization tion without prior amplification, in particular for the detection of mRNA or thereof derived nucleic acids.

The proof in the sense of the invention includes the determination of whether a certain Target (analyte) is or is not present in a sample (presence or absence). The determination can be made qualitatively or quantitatively.

As a rule, the detection requires a quantification of those nucleic acids that are hybridize an immobilized probe. The quantification can be absolute or relative respectively. Suitable detection systems are sufficiently known to the person skilled in the art. A A widely used option is the introduction of markings, e.g. B. radioacti ver, colorimetric, fluorescent or luminescent type. These are used in the Usually in the nucleic acids present in the sample and in particular in those after Pointing nucleic acids with a certain base sequence or nucleic acids with it similar base sequence introduced, e.g. B. in the course of a hybridization upstream Amplification or in another manner known per se.

The invention is illustrated by the following examples.

Examples

It shows

1 shows the fluorescence of various carrier materials relative to standard glass slide A (Menzel); B: Pyrex glass; C: polymethylpentene (Permanox; Nunc); D: polycycloolefin (Zeonex 480R; Zeon); E: Polycycloolefin (Zeonex 480; Zeon); F: polycycloolefin (Topas; Hoechst); G: polycarbonate (Nunc); H: polystyrene (Nunc); I: polystyrene on Pyrex; J: CxHy on silicon (soft deposited carbon; plasma; the H / C ratio can be adjusted via the hydrogen content, technology IMM Mainz); K: Teflon on Pyrex; L: gold on pyrex;

Figure 2 shows the fluorescence after hybridization of a Cy5-labeled 16-mer oligonucleotide probe immobilized 16mer oligonucleotide for different ver carrier materials. A: polycycloolefin (Zeonex 480R; Zeon); B: polystyrene on pyrex glass; C: Teflon on Pyrex glass; D: CxHy on silicon. The ratio of signal (white bars) to background fluorescence (black bars) can be seen;

. Figure 3 shows the fluorescence after hybridization of a Cy5-labeled 55mer-oligonucleotide sample with polycycloolefin immobilized 18-mer oligonucleotide were being used for the immobilization of solutions with different concentrations of the oligonucleotide (A: 1 uM; B: 3 uM; C: 5 uM ), each of which was applied 5 times in a grid of 400 µm as a spot;

. Figure 4 shows the fluorescence after hybridization of a Cy5-labeled 55mer-oligonucleotide sample with polycycloolefin immobilized 18-mer oligonucleotide as a function of the concentration [.mu.M] of Oligonu kleotids as used for immobilising solution;

Figure 5 shows the fluorescence after hybridization of a Cy5-labeled 55mer-oligonucleotide sample with polycycloolefin immobilized 18 mer oligonucleotide, wherein 5-micron was applied as a spot to immobilize a 10 uM solution of the Oligonu kleotids in a grid of 400.

Fig. 6 shows the fluorescence after hybridization of a Cy5-labeled 24-mer oligonucleotide probe with different 13-mer oligonucleotides is immobilized on a cyclohexyl-silanized glass slide, wherein an oligonucleotide complementary to the sample (left, 2 spots, Perfect Match) and a another oligonucleotide has a base mismatch with the sample (right, 2 spots, mismatch);

Fig. 7 shows the fluorescence after hybridization of a Cy5-labeled 24-mer oligonucleotide probe immobilized polycycloolefin with AQ-coupled PCR amplicon (A, 0.1-0.5 uM), AQ-coupled PCR amplicon (B, 0:01 to 0:05 uM) non-AQ-coupled PCR amplicon (C, 0.1-0.5 μM);

Fig. 8 shows the fluorescence after hybridization of a Cy5-labeled 55mer-oligonucleotide sample with polycycloolefin immobilized 18-mer oligonucleotide that quinone derivative for immobilization in admixture with a Anthra AQ-HEG-5'-T was applied as a spot, wherein the a Total anthraquinone concentration of 10 µM related AQ-HEG-5'-T proportion [%] 0; 10; 25; 50; 75; 90 and 99 mol%;

Fig. 9, the fluorescence after hybridization of a Cy5-labeled 55mer-oligonucleotide sample with polycycloolefin immobilized 18 mer oligonucleotide, whereby immobilization to a 10 uM solution of the Oligonu kleotids 5-fold with a Kapillardispenser (capillary diameter in the range of 1.5-5 uM) was applied as a spot in a grid of 50 μm;

FIG. 10 is a mask for photolithographic pre-structuring the substrate surface with recesses 100 microns in diameter in a grid of 400 microns and positioning marks;

Fig. 11, the fluorescence after hybridization of different concentrations of a Cy5-labeled 55mer oligonucleotide-probe (10 ^-11; 10 ^-10; 10 ^-9 10 ^-8 10 ^-7 10 ^-6 10 ^-5 M) with polycycloolefin -immobilized 18-mer oligonucleotide (200 μm spots), the values marked with O being recorded without a filter, the values marked with Δ with a first filter and the values marked with a second filter;

Fig. 12, the fluorescence after hybridization of different concentrations of a Cy5-labeled 55mer-oligonucleotide probe (2 x 10 ^-13; 2 x 10 ^-12; 2 x 10 ^-11; 2 x 10 ^-10; 2 x 10 ^-9 M) ; with IMNT-S1-immobilized 18-mer oligonucleotide (10 µm spots);

Fig. 13, the fluorescence of polycycloolefin immobilized, Cy5-labeled (B) and non-labeled (A) 18mer-oligonucleotide prior to hybridization and chemiluminescence by hybridization with biotinylated 18-mer oligonucleotide probe and subsequent incubation with streptavidin-HRP (B ' or A ');

Fig. 14 is a schematic exploded view of a dipstick (A) and a mating receptacle (B).

In FIGS. 2 to 9 and 11 to 13, the fluorescence is plotted as an intensity in arbitrarily selected units [AU].

1. Background fluorescence of the support material

The background fluorescence of various carrier materials at 635 nm excitation and 670 nm emission was examined with a high-resolution confocal laser scanner ( FIG. 1). Polycycloolefins, polymethylpentene and polystyrene on Pyrex glass show a lower fluorescence than a standard slide.

2. Manufacture of the probes a) oligonucleotide (biomolecule B)

Oligonucleotides were produced in a manner known per se via a solid phase synthesis using the phosphoramidite method. Oligonucleotides coupled to the solid phase with DMTr-protected 5'-OH and the following sequence were synthesized via the 3'-OH:
SEQ ID NO: 1: 5'-AACAGCTATGACCATG-3 '
SEQ ID NO: 2: 5'TATTCAGGCTGGGGGCTG-3 '
SEQ ID NO: 3: 5'-AGCTGGTGGCGTA-3 '
SEQ ID NO: 4: 5'-TGGTGACGTAGGC-3 '
SEQ ID NO: 5: 5'GTACTGGTGGAGTATTTGATAGTG-3 '.

b) quinone spacer construct (Q-S)

This was in a manner known per se from anthraquinone-2-carboxylic acid, Synthesized mono-Boc-1,3-propanediamine and hexaethylene glycol.

c) quinone spacer oligonucleotide construct (Q-S-B) or quinone spacer T construct

After setting up the above sequences, the DMTr protective group at the 5 'end was again removed under acidic conditions with ZnBr ₂ and the free 5'-OH was reacted with the 2-cyanoethyl-N, N-diisopropyl-chlorophosphoramidite-activated construct QS.

A construct of thymidine and the Q-S construct was obtained in an analogous manner.

The following table shows an overview of the synthesized quinone derivatives:

3. Array fabrication 3.1 Arrays with a plastic surface

Unless expressly stated, this was done under the trade name Zeonex 480® Polycycloolefin available at the time of filing was used.

3.1.1 Oligonucleotide arrays 3.1.1.1 Arrays with spots of 16mers on different surface materials

Aqueous solutions (0.2 M NaCl) of AQ-1 (0.05 µM) were added by hand as 0.5 µL drops pipetted in 4-fold values onto different surfaces (A-D) (pitch 2-4 mm): A: Polycycloolefin (Zeonex 480R; Zeon); B: polystyrene on pyrex glass; C: Teflon on Pyrex Glass; D: CxHy on silicon. After the spots had dried, the carriers were 10 min irradiated with UV light (340 nm) and washed 3 × 5 min with bidest water to over remove excess probes.

3.1.1.2 Arrays with different sizes of spots from 18mers

An aqueous solution (2 mM calcium chloride; 1 vol .-% 1-propanol) of AQ-2 (10 µM) were with a piezo dispenser (1-channel; Genesis NPS 100/4 with Active Tip M, TECAN AG, Hombrechtikon, CH) in a grid of 500 µm on polycycloolefin (Zeonex 480R; Zeon) applied. The drop size was about 1.5 nL. After the The carrier was irradiated with UV light for 1 min. Subsequently, the carrier first immersed in an aqueous solution with 0.5-1% Tween 20 and swirled briefly. It was then rinsed thoroughly with 0.1% Tween 20 solution and the carrier 2 × 10 min Immersed in fresh 0.5-1% Tween 20 solution, shaken to do so. Final The support was then immersed in 2-propanol for 10 minutes with shaking, with water rinsed and air dried at room temperature. The diameter of the result The resulting spots were around 150 to 200 µm. In a corresponding way, spots could with a diameter of about 12 µm can be obtained by using a capillary penser drop sizes of about 1 pL (internal capillary diameter in the range of 1.5-5 µM) in a grid of 50 µm.

3.1.1.3 Array with spots from 18mers in different amounts

5-fold replicas of the solutions used in 3.1.1.2 with different concentrations tions (0.5; 1; 3; 5; 8; 10; 20 µM) of AQ-2 were as described under 3.1.1.2 with the Piezodispensor with a drop size of 1.5 nL immobilized on polycycloolefin.

3.1.1.4 Arrays with spots from 18mers in different densities

Based on the procedure described in Example 3.1.1.2, solutions were found (2 mM calcium chloride; 1% by volume 1-propanol) with the piezo dispenser for a Drop size of 1.5 nL polycycloolefin applied, which in addition to the AQ-2 Contained anthraquinone derivative AQ-5 in different concentrations. The to be applied lowing solutions were adjusted so that the anthraquinone concentration of AQ-2 + AQ-5 was always 10 µM.

3.1.1.5 Arrays with spots of fluorescence-labeled 18-mers

Based on the procedure described in Example 3.1.1.2, solutions were found (2 mM calcium chloride; 1% by volume 1-propanol), on the one hand AQ-2 (10 µM), on the other hand AQ-6 (10 µM) contained, in triplicate with the piezo dispenser with a drop size of 1.5 nL applied to the same polycycloolefin carrier.

3.1.1.6 Arrays with 150 µm or 200 µm spots from 13mers using a piezo or pressure pulse dispenser

Furthermore, based on the procedure described in example 3.1.1.2 se aqueous solutions (2 mM calcium chloride; 1% by weight 1-propanol) of AQ-3 (10 µM) on the one hand with a piezo dispenser (1-channel; Genesis NPS 100/4 with Active Tip M, TECAN AG, Hombrechtikon, CH), on the other hand with a pressure pulse dispenser (24-channel; TopSpot, HSG-IMIT, Villingen-Schwenningen) in a grid of 300 µm or 500 µm spotted on polycycloolefin. The droplet size was about 0.5 nl or 1.5 nl.

3.1.2 Array with spots from PCR amplificate

DNA from the Colo320 cell line (k-ras wild type) was amplified using the primers RasUS1 and RasDS135 specified below. The length of the PCR amplificate was 157 bp. Furthermore, a corresponding amplicon was generated using RasDS135 and an AQ-coupled primer (AQ-7) corresponding to RasUS1.

PCR conditions per batch: 100 ng DNA, 1.5 mM MgCl ₂ ; 100 µM dNTPs; 250 nM primer; 1 U Taq polymerase (Qiagen; HotStar); 7.5% glycerol; in 50 µl of 1 × PCR buffer. Thermocycling: 1 × 95 ° C 15 min; 35x (94 ° C 60 sec, 70 ° C 50 sec, 58 ° C 50 sec, 72 ° C 60 sec); 1 x 72 ° C 7 min.

The PCR products with and without AQ primer were diluted 1: 2 and 1:20 (AQ primer only) with spotting solution (2 mM CaCl ₂ , 1 vol% 1-propanol) and each 1.5 nL in 5-fold Replicates immobilized on polycycloolefin based on the procedure described in Example 3.1.1.2.

3.2 Array with glass surface

Glass slides were precoated with cyclohexyldichloromethylsilane (Fluka) using a sol-gel process. For this purpose, the silane was prehydrolyzed with water and the mixture was slightly acidified with dilute hydrochloric acid. Glass slides (Menzl) previously cleaned with isopropanol (10% diethyl ether) and ultrasound (15 min) were immersed in the hydrolyzate and slowly withdrawn again. The glass slides were dried at 80-150 ° C. AQ-3 and AQ-4 (10 μM each) solutions (2 mM CaCl ₂ ) with a pressure pulse dispenser (24-channel; TopSpot, HSG-IMIT, Villingen-Schwenningen) were added in quadruple replicates to the resulting surface a drop size of 1.5 nL in a grid of 500 µm on the same polycycloolefin support.

3.3 Arrays with a structured surface

First, a chrome layer is sputtered onto the polycycloolefin or glass surface. When using titanium, this is vapor deposited. The subsequently applied photoresist (resist system AZ 1512, Clariant) is then exposed via the mask shown schematically in FIG. 10. The exposed areas of the lacquer are removed during development. Finally, the exposed areas of the metal layer are etched away. The recessed areas have a diameter of 100 µm and the grid is 400 µm. The spot diameters of 12 recessed areas were 102.07 µm for polycycloolefin and 100.74 µm for glass with coefficients of variation of 0.033% and 0.042%, respectively.

In Fig. 14 an exploded view of a dipstick (A) with closure means 1 and attached body 2 , which has an array 3 , and a container 4 with sample fluid 5 is shown. The array 3 can be an array of immobilized biomolecules with or without pre-structuring.

4. Array quality control

The array from Example 3.1.1.5 was measured with a confocal fluorescence scanner. Fig. 13 (left half) shows the scan. Immobilized AQ-2 cannot be seen (section A), while immobilized AQ-6 (section B) supplies a fluorescence signal, on the basis of which the quality of the spots can be assessed.

5. Array hybridization 5.1 16mer probes

The chips (surface samples with array) are placed in 20 mL of a 0.05 µM for 1 h at room temperature Solution of a 5'-Cy5-labeled 16mer sample (SEQ ID NO: 7: 5'-CATGGTCATAGCTGTT-3 ') in 5 × SSC-T (sodium chloride / sodium citrate buffer with 0.1% Tween 20) inserted. The excess sample was then rinsed off with 5 × SSC-T and the fluorescence measured.

5.2 18mer probes

10 μl of a 5'-Cy5-labeled 55mer sample, dissolved in PerfectHyb (Sigma, Deisenhofen), SEQ ID NO: 8 [5'-CTACGTTGCCCCCCTGACCTGCAGCCCCCAGCCTGAATATGTGAACCAGCCAGAT-3 '] was pipetted onto the array. The sample concentration was 10 ^-11 ; 10 ^-10 ; 10 ^-9 ; 10 ^-8 ; 10 ^-7 ; 10 ^-6 ; 10 ^-5 M and 0.05 nM, respectively. It was incubated for 1 h at RT. The excess sample was then briefly rinsed off with 5 × SSPE and the fluorescence was measured.

5.3 Cy5-labeled 18mer probes

20 μl of a 5 nM in 6 × SSPE-T (= 6 × SSPE with 0.1 vol% Tween 20) dissolved at the 5 'end 18mer biotinylated probe (SEQ ID NO: 9: 5'-CAGCCCCCAGCCTGAATA-3 ') was added to pipetted the array. After a washing step with 6 × SSPE-T it was washed with Steptavidin-POD (Sigma; dilution 1: 100 in 6 × SSPE-T) incubated (30 min, RT). It was again with Washed 6 × SSPE-T. 10 µL chemiluminescent substrate (SuperSignal, ELISA, Pierce) was added, the light was exposed for 2 seconds and the chemiluminescence was determined.

5.4 13mer probes; PCR amplificates

30 µl of a Cy5-labeled 24mer sample dissolved in 6 × SSPE with 0.1% Tween 20 (SEQ ID NO: 10: 5'-CTTGCCTACGCCACCAGCTCCAAC-3 ') were pipetted onto the array. The Sample concentration was 5 nM. It was incubated at 37 ° C. for 1 hour. After that, the Excess sample was briefly rinsed off with 6 × SSPE-T and the fluorescence was measured.

Using the dipstick shown in Fig. 14, one proceeds as follows. The hybridization mixture containing the sample is placed in the container 4 . The dipstick is inserted into the container 4 so that the array 3 comes into contact with the hybridization mixture. The container 4 is closed with the closure means 1 . If necessary, the sample, e.g. B. a PCR amplicon, denatured, usually at least 2 min at 94 ° C. Then it can be cooled down to 0 to 4 ° C. The subsequent de hybridization takes place as usual, for. B. 60 min at 37 ° C. The dipstick is then removed, washed and the array is evaluated.

6. Evaluation

The arrays were scanned with a confocal fluorescence scanner (fluorescence) or with a chemiluminescence detector (chemiluminescence) measured.

6.1 Non-specific binding

The investigation of the arrays produced in Example 3.1.1.1 shows that polycycloolefin and polystyrene on Pyrex glass, due to the lower unspecific binding of the sample, show a significantly better ratio of signal to background fluorescence than CxHy on silicon and Teflon on Pyrex glass, which in these disadvantageous carrier materials in one Range is below 10 ( Fig. 2).

6.2 Spot homogeneity

To produce the arrays from Example 3.1.1.3, different amounts of probes were applied. As the probe concentration increases, the homogeneity of the spots increases ( FIG. 3) until the surface is finally saturated ( FIG. 4). With a probe concentration of 10-20 μM, very homogeneous spots about 150-200 μm in diameter are obtained ( FIG. 5).

The spots applied to glass surfaces according to Example 3.2 are also homogeneous. A good discrimination between perfect match and mismatch is achieved ( FIG. 6).

FIG. 7 shows that good immobilization yields can also be achieved for PCR amplificates (cf. Example 3.1.2).

6.3 Probe density

The investigation of an array produced according to Example 3.1.1.4 shows that the amount of hybridized sample and thus the density of immobilized probe AQ-1 steadily decreases with increasing proportion of the non-hybridizing AQ-HEG-5'-T (AQ-6) ( Fig . 8). In this way, the surface density of the immobilized probes can be adjusted in a reproducible manner. In contrast to a simple reduction in the probe concentration, the homogeneity of the spots is retained when diluting with AQ-HEQ-5'-T.

6.4 Precision

The measurement data for the 1-channel piezodispenser according to example 3.1.1.6 The corresponding spots are shown in Table 1.

Table 1

The coefficient of variation (VC) over 64 spots is single for the spot areas drop release (approx. 0.5 nL) at 5%. The coefficient of variation for the spot volumes (Volume) and the spot diameter (Diameter) is below 7% after hybridization. All 64 Spots together formed a regular grid.

The measurement data for the 24-channel pressure pulse dispenser according to example 3.1.1.6 applied spots are shown in Table 2.

Table 2

The coefficients of variation for the spot areas are below 6% and the integrated ones Signals below 7%.

The interassay variances for the spot diameters are in the case of the arbitrary selection of 4 chips out of 400 after hybridization at 7.0%.

The measurement data for the spots applied with the capillary dispenser according to Example 3.1.1.2 are plotted in FIG. 9 and shown in Table 3.

Table 3

As can be seen in the cross section, the approximately 12 µm wide spots are also very uniform. The intraassay variance (n = 20) for the spot areas is below 10% and the Intraassay variance for the volumes is only 11%.

6.5 Dynamic hybridization area

From FIG. 11 it can be seen that the dynamic range of the 200 μm spots obtained according to Example 3.1.1.2 is greater than the dynamic measuring range of the confocal fluorescence scanner used. Attenuating filters therefore had to be introduced into the beam path in the upper concentration range. The dynamic hybridization range for the 200 µm spots is <10 ⁵ . For the 10 μm spots obtained according to Example 3.1.1.2, a dynamic range of <10 ^{4 is achieved} with a detection limit which is one order of magnitude lower ( FIG. 12).

Claims (19)

1. array of immobilized biomolecules which are coupled to a carbonaceous support surface, characterized in that the carbonaceous surface contains at least one cycloolefin-based polymer or is obtainable by having a glass, metal or ceramic surface with an aqueous solution little treated at least a hydrolyzable carbonaceous compound and the surface dries. 2. Array according to claim 1, characterized in that the biomolecules over at least one quinone, preferably an anthraquinone, are coupled. 3. Array according to claim 1 or 2, characterized in that the coupling group and biomolecule are linked via a spacer. 4. Array according to claim 3, characterized in that the marked the spacer is. 5. Array according to one of the preceding claims, characterized in that the polymer is based on norbornene structural units. 6. Array according to one of claims 1 to 4, characterized in that the hydrolyzable carbon-containing compound is a silane of the formula XVI
(R ¹⁷ ) _z Si (R ¹⁶ ) _4-z (XVI)
where the radicals R ¹⁶ independently of one another represent halogen, C ₁ -C ₄ alkoxy or C ₁ -C ₄ acyloxy, the radicals R ¹⁷ independently of one another represent C ₁ -C ₃₀ alkyl or C ₃ -C ₁₀ - Are cycloalkyl and z corresponds to a value of 1, 2 or 3. 7. Array according to one of the preceding claims, characterized in that Parts of the surface have a metal layer, preferably a chrome or platinum layer layer. 8. Carrier with carbon-containing surface for immobilizing biomolecules via coupling groups, characterized in that parts of the surface have a Have coating to which the coupling groups do not couple. 9. Device based on at least one array according to one of claims 1 to 7 in Shape of a chip or dipstick. 10. Device based on at least one carrier according to claim 8 in the form of a Chips or Dipsticks. 11. Dipstick according to claim 9 or 10, comprising a closure means 1 and a body 2 attached thereto, which has at least one array 3 . 12. A method for immobilizing biomolecules, wherein one at least one derivative of a coupling group on a carbon-containing support tiger surface, exposed and if necessary, the immobilized derivatives are converted into the biomolecules, characterized in that there is at least one carbon-containing surface Polycycloolefin used. 13. A method for immobilizing biomolecules, wherein one a support surface with at least one hydrolyzable carbonaceous Connection handled, at least one derivative of a coupling group on the carbonaceous substrate applies the surface, exposed and if necessary, the immobilized derivatives are converted into the biomolecules, characterized in that an aqueous solution of the hydrolyzable carbonaceous compound used. 14. The method according to claim 12 or 13, characterized in that one quinonde derivatives used. 15. The method according to any one of claims 12 to 14, characterized in that one the derivatives of the coupling groups and a hygroscopic salt, preferably Calcium chloride. 16. The method according to any one of claims 12 to 15, characterized in that one Parts of the surface treated so that the derivatives of the coupling groups to the essentially do not couple treated surface. 17. The method according to claim 16, characterized in that one is on the surface applies a metal layer and removes parts of this layer again. 18. The method according to any one of claims 12 to 17, characterized in that one after exposure to a solvent or solvent mixture that washes 0.1 to 10, preferably 0.5 to 2 and in particular 1 wt .-% of a surfactant ent holds. 19. Use of an array according to one of claims 1 to 7 or a device according to claim 9 or 11 for diagnostic / analytical purposes.