EP0784794A1

EP0784794A1 - Bonding matrix and an analysing device for the simultaneous analysis of various analytes

Info

Publication number: EP0784794A1
Application number: EP95934123A
Authority: EP
Inventors: Hans-Georg Batz; Peter Sluka; Wolfgang Mutter
Original assignee: Roche Diagnostics GmbH; Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1994-10-06
Filing date: 1995-09-30
Publication date: 1997-07-23
Also published as: JPH10506996A; DE4435727A1; WO1996011404A1

Abstract

The object of the invention is a bonding matrix containing a flat supporting material covered with a plurality of spatially separated fields horizontally and parallel to its surface which contain stationary binding partners, each of which can attach a corresponding free binding partner of a specific bond pair, in which the regions are formed by: a) separate metal coating spots, b) thin and substantially laterally homogeneous securing layers of a bonding layer of a binding partner B1 secured by anchor groups to the metal spots. The bonding matrix may have a plurality of identical or different binding partners and thus acts as an analysis component for the simultaneous analysis of various analytes in a sample or identical analytes in different samples.

Description

Binding matrix and analysis element for the simultaneous analysis of different analytes

The invention relates to a binding matrix made of a flat carrier material which is covered by a multiplicity of horizontally spatially separated regions which can be covered with different specific binding partners for free reactants, a corresponding analysis element, a method for its production and the use of the analysis element for the simultaneous detection of different free reactants or analytes or for the simultaneous detection of an analyte in different samples.

In order to detect different analytes spatially next to one another due to specific binding reactions, for example in the form of an immunoassay, the most common method is to adsorptively load the wells of microtiter plates with different antibodies or via a fixation layer, such as RSA / streptavidin, and one analyte in each well to determine. The comparatively large dimensions of microtiter plates require large amounts of reagent and samples. Due to the uneven geometry of the wells, only certain detection methods such as UV / VIS absorption or measurement of fluorescence in solution can be used as a measure of the binding of analyte to the solid phase.

An improvement in multiple analyte detection on a common analysis support was achieved by miniaturizing the analysis elements by adsorbing antibodies or antigen spots on nitrocelullose (Walch et al., J. Immunol. Meth. (1984), 66, 99-102; Derer et al., J. Allergy Clin. Immunol. (1984), 74, 85-92). These analysis elements are used in particular for allergy diagnosis. As with microtiter plates, the disadvantage here is that the adsorptive loading of the surface with immunologically active reagents does not result in an orderly and defined covering of the surface, which has an unfavorable effect on the reproducibility of measurement results. The strong dependence of the Coefficients of variation of the measurement results from the nature of the surface with adsorptive loading is described, inter alia, in US Pat. No. 4,980,299. While the statistical fluctuations of the surface coverage with adsorptive loading are less important for large surfaces, they can have a very negative effect on reproducibility with increasing miniaturization of the surfaces, since micro-inhomogeneities in particular are more important here. Quantitative determinations are therefore difficult to make.

Multiple reagent carriers for the parallel synthesis of different polypeptide or polynucleotide strange are described in WO 92/10092, WO 91/07087 and EP 0 138 855. In WO 92/10092, a homogeneous carrier material is uniformly functionalized over the entire surface by introducing amino groups. A reactant with a photochemically cleavable group is bonded to this functionalized surface over a large area. By applying a mask, the photochemically cleavable group can be selectively removed at certain points and an amino acid or a nucleotide base can be selectively coupled chemically. By using different masks, the same procedure can be continued at different sites with different amino acids or nucleotide bases. A large number of different polynucleotide strands, which can be used for binding and determining complementary polynucleotide strands, are thus obtained on the support surface after many working steps. The disadvantage of this method is that it is very complex to produce such a microstructuring with different analyte binding partners. The assignment of binding partners at exposed areas is very uneven and not very reproducible.

EP-A-0 134 215, EP-A-0 304 202 and EP-A-0 271 974 describe miniaturized analysis elements in which antibody spots are applied by conventional coating techniques to, for example, polystyrene surfaces by adsorption. Here, too, the fluctuations in the surface assignments caused by strong heterogeneities of the surfaces occur and cause poor reproducibility of the measurement results. Another problem is the non-specific binding of proteins from serum or plasma to the surface, which has a disadvantageous effect, in particular at low concentrations of the analytes and the solid-phase binding partners, both for the binding of the solid-phase binding partners and for the binding of the analyte to the solid-phase binding partner. The object of the invention was therefore to eliminate the disadvantages of the prior art described above and to provide a binding matrix on which a large number of solid-phase bonding partners for different analytes can be easily reproduced on a very small area in a selectively adjustable surface coverage and at the same time locally can be applied to a limited extent separately from one another, with which, after the application of the various solid phase binding partners, a large number of free analytes from a solution can be bound and detected in a very small space, and with which the multiple analyte determination can thus be reproducibly and qualitatively reduced, while simultaneously reducing non-specific binding of sample components and in particular can be carried out quantitatively

The task is solved by a binding matrix and an analysis element as characterized in the claims

The invention relates to a binding matrix comprising a flat carrier material which, parallel to its surface, is covered with a large number of horizontally spatially separated regions which contain binding partners immobilized, each of which is capable of binding a corresponding binding partner of a specific binding pair, characterized by that the areas are formed from a) metal layer spots not touching each other, b) thinned and essentially laterally homogeneous binding layers of a binding partner B 1 via anchor groups each bound to the surface of the metal spots

A flat binding matrix is understood to mean an essentially flat, preferably a planar, matrix. Although it can in principle have an arbitrarily large surface, the advantages of the invention over the prior art are particularly evident in the case of small surfaces. Analysis elements with a surface of are therefore advantageous less than 100 cm ^, preferably between 1 cm ^ and 25 cm ^

A large number of non-touching metal layer regions (“metal spots”) are applied next to one another in the horizontal direction on the carrier material surface of the binding matrix These areas each preferably form a 10-100 nm thick layer on the carrier material and can take up any surface, that is to say circular or angular. The advantages of the invention are particularly effective when these areas are very small with a diameter between 0.1 and 1 mm, very particularly preferably between 0.3 and 0.6 mm.

The metal layer areas should not touch each other, that is to say the free surfaces of the metal spots facing away from the carrier material should be surrounded by intermediate surfaces, the surfaces of which consist of a material different from the metal spots. In the preferred case, the intermediate surfaces between the metal spots are formed by a non-metallic carrier material itself. Favorable here are carrier materials which have only a very slight non-specific interaction with biological molecules, e.g. Immunreagen¬ have. A distance of 0.1 to 1 mm from the outer boundaries of the metal spots is advantageous in order to facilitate the later separate application of reagents.

Since the spots can have very small areas according to the invention, a very large number of spots can be accommodated on a very small surface of the carrier material.

10-100 spots per cm ^ surface of the carrier material are particularly advantageous

20-50.

Polystyrene, polycarbonate, polyethylene acrylate, polyethylene, polypropylene or glass, for example, can serve as the carrier material.

Precious metals, in particular gold, silver or palladium, are preferably used as metals.

A "thinned" binding layer of a binding partner B 1 which is uniform on all metal spots or on different metal spots of different binding partners B 1 ', B1 ^" etc. is bound to the metal spots. However, only one type of binding partner should be bound in a diluted binding layer on each spot Such thinned binding layers are described in WO 92/10757, to which reference is made here in full. A “thinned” binding layer is understood to mean a monolayer of a specific binding partner of a molecular type that points away from the carrier material into the space, the surface of the metal spot not being completely occupied. The degree of coverage with the binding partner, which is a measure of the dilution, can be expressed as the quotient of the bound thickness of the binding layer divided by the theoretical layer thickness in the case of dense packing.

The degree of coverage of the metal spots with a monolayer of the binding partner B1 is less than 100%, preferably 0.1 to 90%, preferably 0.7 to 70%, particularly preferably 1 to 40%.

The adsorption of the binding layer of the binding partner is mediated via anchor groups. Thiol, disulphide or phosphine groups are suitable as anchor groups. For example, thiol or disulphide groups are particularly suitable as anchor groups for gold or silver surfaces and phosphine groups for a palladium surface.

The anchor group for adsorption to the solid phase is preferably not attached directly to the binding partner, but is linked via a spacer molecule, preferably via a flexible spacer molecule. The spacer molecule particularly preferably contains at least one alkylene group of the formula (CH2) _n. Where n is a natural number from 1-30, preferably 2-30, particularly preferably 2-15. On one side, the spacer molecule contains the anchor group (for example the thiol or disulphide group), which is suitable for adsorption on the surface of the metal spots. On its other side, that is, away from the carrier material, the spacer molecule contains one or more linkage groups via which the binding partner or a component thereof is linked to the spacer molecule. These linkage groups can be, for example, an amino or hydroxyl function which is linked, for example, to a carboxyl function of the binding partner to form an ester or amide group. However, the spacer molecule can also contain a carboxyl function as a linking group, which in turn is then linked to a reactive amino or hydroxyl function of the binding partner.

The preparation of dilute binding layers is described in WO 92/10757. A first possibility is to use a spacer molecule which is linked to two or more molecules, preferably two molecules of the binding partner. An example of such a spacer molecule is cystamine, which contains a disulphide group as an anchor group and two amino functions as a linking group and can therefore be linked to two molecules of an activated binding partner. When adsorbed onto a gold surface, such molecules form a thinned binding layer with a degree of coverage significantly less than 100% of the binding partner

Another possibility for producing a dilute binding layer is the incorporation of a hydrophilic linker group between the spacer molecule and the binding partner. This linker is in particular a straight-chain molecule with a chain length of 4-15 atoms. A linker group is preferred which contains one or more hydrophilic ethylene oxide units, preferably between 1 and 5. The hydrophilic linker group is particularly preferably formed by an amine- or hydroxyl-terminated polyethylene oxide.

A further spacer molecule, which consists of an alkylene group of the formula (CH2) _n and a linking group, in which n is a natural number from 2-15, can preferably be installed between the hydrophilic linker and the binding partner. 1,8-diamino-3,6-dioxaoctane has proven to be a particularly suitable linker. When adsorbed onto a gold surface, such compounds form a monolayer with an occupancy density of, for example, 19% with biotin as binding partner, which is able to form a free reaction partner (streptavidin) with high affinity and within a very short time to form a densely packed film to tie. Such thinned binding layers are therefore particularly preferred.

Very particularly preferred are the “diluted” binding layers which, in addition to the binding partners bound via anchor groups and spacers, also contain spacer molecules which are connected to an anchor group, but to which no binding partner is bound. Such compounds are also referred to below as dilution molecules. For example, if you have biotinylated and non-biotinylated spacer molecules in a ratio of 1:10 to 1 2, a dilute biotin monolayer is obtained which can bind a free binding partner at high speed and with a large capacity

Suitable dilution molecules contain an anchor group and a spacer component and optionally a linker molecule, the number of CH2 groups of the spacer molecule not more than 1-5, preferably not more than 1-2, of the number of CH2 groups of the spacer molecule distinguishes, which is bound to the binding partner. It has also proven to be expedient for the minimum chain length of the dilution molecule to be 6 atoms (without anchor group and hydrophilic linker group)

Instead of the binding partner, there is preferably a hydrophilic function at the end of the diluent molecule remote from the anchor group, such as, for example, a hydroxyl group, a carboxylic acid group, a carboxylic acid ethyl ester or methyl ester group, a carboxylic acid amide group, a carboxylic acid amide group substituted with one or two methyl or ethyl groups , a sulfonic acid group or a sulfonamide group. It is also preferred to bind a hydrophilic linker (as defined above) or part of a hydrophilic linker to the end of the diluent remote from the anchor group.

It is also possible to link a spacer with a binding partner and a spacer without a binding partner via a covalent bond. When using gold or silver surfaces, this linkage is preferably carried out via a disulphide bridge.

In such mixed binding layers, which consist of dilution molecules (spacer molecules without binding partner) and spacer molecules with binding partner, the proportion of spacer molecules with binding partner is advantageously 0.1-90 mol%, preferably 0.5-50 mol. % and particularly preferably 1-40 mol%.

Any partner of a specific binding pair can be used as binding partner, for example antibodies, antigens, proteins, lectins, biotin, streptavidin or polynucleotides. A diluted binding layer, which is formed by the same binding partner B 1, is preferably applied to all spots. These are preferably biotin or biotin-analogous molecules such as destihobiotin, iminobiotin or HABA (4-hydroxyphenyl-azobenzoic acid), which also react with streptavidin.

In the case of antibodies, antigens, haptens or polynucleotides, it is also possible for different binding partners to form different, dilute binding layers on individual spots. This is particularly preferred for polynucleotides. In extreme cases there is a diluted binding layer of another binding partner on each metal spot. Such a binding matrix can already be used for binding and for detecting different free binding partners of the matrix binding partners. In addition, it is possible that different layers of binding layers of the same or different binding partners are applied, which have a different degree of dilution from spot to spot.

The degree of coverage of the binding partner B 1 of the first binding layer on the metal spots can be determined by measuring the thickness of the binding layer. The measured layer thickness decreases with the degree of coverage of the binding layer. A binding layer with biotin as the binding partner has a thickness of 0.7 nm, this thickness being less than the calculated length of 3.7 nm of the molecule.

The present invention furthermore relates to a method for producing a binding matrix according to the invention, characterized in that a) metal layer spots are applied to the surface of the carrier material in such a way that they do not touch each other b) the free surface of the metal layer spots is in each case incubated with a reaction solution, containing molecules to create a thinned binding layer.

In a first step, discrete metal spots separated from one another in the horizontal direction are applied to the carrier material of the analytical element in a layer parallel to the surface of the carrier material. These spots are preferably applied in a regular pattern. In order to obtain the desired pattern of metal spots on the surface of the carrier material, a mask is preferably used in which the desired pattern is left free and which is placed on the carrier material. The pattern of the metal spots is then applied by vapor deposition or sputtering on the mask. If necessary, the surface of the carrier material can be provided beforehand with an adhesion promoter, for example chrome Analysis element for surface plasmon resonance are used, so a metal layer thickness of 10 - 100 nm is preferred. Otherwise, the thickness can also be greater

In addition to applying metal spots via a mask, it is also possible to cover the analysis element over the entire surface with a metal layer and to subsequently generate a desired spot pattern by etching away metal. It is also possible to cover a spot on a metal surface by covering the metal with a non-metallic one To produce a coating, for example wax, the coating representing the intermediate surfaces between the spots

In a second step, the individual metal spots are covered with a thinned binding layer. If this thinned binding layer is to be the same on all spots, the carrier element is advantageously immersed in a solution which contains the molecules necessary for producing a thinned first binding layer. On the one hand, this can preferably be a mixture of anchor spacer molecules with and without a binding partner in a ratio determining the degree of thinning, on the other hand it can be a solution of those molecules which, as described above, likewise produce dilute binding layers

If the diluted binding layers on different spots are to contain different binding partners, then different solutions are applied to individual spots, each containing a different binding partner in a form which produces a diluted binding layer. Instead of or in addition, different "degrees of dilution" of the binding films on different spots can also be achieved, for example by a different proportion of thinning molecules being present in the individual solutions, which are applied to different spots. The spatially separate application of the reagents for dilute binding layers can be carried out using useful methods such as pipetting, stamping or printing techniques, for example ink-jet. If necessary, excess solution is removed again. These thinned lateral binding layers are microscopically homogeneous, as can be demonstrated, for example, by surface plasmon microscopy (B Rothenhausler and W Knoll, Surface Plasmon Microscopy, Nature, Vol 332, page 615-617 (1988). With a resolution of 5 μ, no thickness differences can be measured

A preferred embodiment is a binding matrix which contains a diluted binding layer of the same binding partner on each spot. Each of these binding layers can then be used for binding identical or different binding partners

If the thinned binding layer has the same binding partner on all of its spots on its surface, this binding matrix with a thinned binding layer, which is applied in the form of spatially separated discrete areas on the carrier material via metal spots, now serves for the reproducible and the degree of thinning of the respective binding layer adjustable or optimizable application of a large number of the same or different binding partners for free analytes to these discrete areas and thus forms the basis for a multianalytical analysis element which enables the simultaneous, precise qualitative but also quantitative determination of a large number of analytes in a sample or an analyte is allowed in various samples in a small space, only very small amounts of reagent being required and these determinations being little disturbed by non-specific binding of blood or serum components such as fibrinogen compared to conventional "Mul" tiparameter "analysis elements such as microtiter plates allow a much higher number of analysis areas. Up to 100,000 spots are possible on an area of 100 cm ^, but the production of the binding matrix is nevertheless very simple

Another object of the invention is therefore an analysis element for the determination of different free reactants in a sample or for the determination of a reactant in different samples, containing a flat support material which covers a plurality of spatially separated areas parallel to its surface is on which different or identical specific binding partners for free reactants are immobilized, characterized in that it contains a binding matrix according to the invention to which one binders B 1 or, in the presence of an additional binding partner B2, one to B2 speci - contains fish-bound further binding layer which contains binding partner B3 on its surface for free reactants or analytes to be determined.

In addition to the first diluted binding layer on each metal spot, the multi-reactant analysis element according to the invention additionally contains a further binding layer of the same or different binding partners B3 for free reactants on each spot. Only one type of reactant binding partner is bound to each spot. These reactant binding partners can be coupled to the binding partner B1 of the first binding layer via a specific binding site. These specific binding sites can, for example, be an epitope for an antibody of the first binding layer or also a specific binding molecule, such as biotin, which is bound to the analyte binding partner and which binds with the binding partner of the first binding layer, e.g. Streptavidin, specifically couples.

Specific binding partners for free reactants in solution are used as reactant binding partners. These can be antibodies, antigens, haptens or nucleic acid strands.

In one embodiment, a binding matrix, on the spots of which there is a dilute binding layer of a uniform binding partner B1, serves directly for the reproducible application of a further layer from spot to spot of identical or different reactant binding partners B3, which bind to Bl via a specific binding site.

In another embodiment, the diluted binding layers of the individual spots are each covered by a further monomolecular binding layer with a uniform binding partner B2. The binding partner B2 has a specific binding point for Bl. Since this assignment can be reproduced very well according to the invention even on a very small area, surprisingly the binding partners B2 of the individual spots can in turn be very good for the reproducible assignment of an adjustable amount of a serve another binding layer with the same or different reactant binding partner B3 from spot to spot, which then have a specific binding site for B2. It is crucial for the advantages according to the invention in both embodiments that the binding matrices on the metal spots contain a diluted binding layer

It has surprisingly been found that in the presence of a dilute binding layer on each spot of the binding matrix according to the invention, each spot can be separately occupied with a reproducible and defined amount of reactant binding partners B3 - be it directly or via an intermediate binding partner layer B2 - without furthermore being unspecific Binding of sample components disturb this assignment. This reproducible assignment is surprisingly achieved even with very small dimensions of the spots, which indicates a very low micro-inhomogeneity of the binding layers. As a result, analytical elements with a large number of spots with a constant and homogeneous binding partner assignment of each spot are obtained In a very small space, it is also possible, surprisingly, to easily and reproducibly cover the various spots with reactant binding partners via the degree of thinning of the different binding layers Control so that each binding layer spot can be set individually with respect to the reactant to be detected and with regard to the sensitivity required for the detection of this reactant

Another object of the invention is a method for producing an analytical element for the multiple determination of the same or different free reactants, characterized in that the free surfaces of the binding layers of a binding matrix are each incubated with the same or different solutions, the binding partners B3 for free reactants contain, the binding partners B3 have a specific binding site for the binding partners B 1 or B2

The different binding layers with the same or different binding partners for free reactants can be applied to the binding matrix in different ways.

In the simplest case, the same or different binding partners B3 for free reactants are applied as a solution to different spots, each covered with a first diluted binding layer and possibly covered with a further binding layer with the binding partner B2. These binding partners B3 must be applied all one have a common binding site for the uniform binding partner B1 of the first dilute binding layer or, if appropriate, the binding partner B2 of the second binding layer. They are preferably bound to a uniform binding molecule for the binding partner B 1 or B2. An example of binding partner B3 are different antibodies that are bound to biotin. Different biotinylated antibodies are then applied to different spots, which are covered with a dilute biotin binding layer and a streptavidin binding phase bound to it, these binding to the streptavidin phase, since streptavidin has a total of 4 binding sites. An analytical element is thus obtained which has fields for binding many different free reactants in a very small space. These reactant binding partner layers form a reproducible covering of the binding layers of the binding matrix.

If a uniform biotinylated antibody of the binding partner for a free antigen is applied to each spot, the analysis element can simultaneously measure a large number of samples which contain this antigen to the solid phase-bound antibody.

Other options for applying different binding partners to the respective spots are printing techniques or stamping techniques, in particular ink-jet, or using a micromultipin plate analogous to adding different reagents to microtiter plates. If necessary, excess solution is removed again.

Another object of the invention is a method for the simultaneous determination of at least two reactants or analytes in a sample solution by means of specific binding reactions of the analytes with solid-phase-bound analyte binding partners on a flat analysis element, characterized in that the sample solution is contacted with an analysis element according to the invention and the presence or amount of solid-phase-bound analytes in different areas of the analysis element is determined separately.

The analyte and the solid-phase-bound reactant binding partner form a specific binding pair. Analytes can be: antibodies, antigens, haptens, nucleic acids, at least are partially complementary to a solid phase-bound nucleic acid, and other specific binding partners.

The detection of the binding of the analyte to the solid phase binding partner is made possible, for example, by the analyte carrying a labeling group or being labeled by binding with another free binding partner. The markings which are customary in immunoassays or in DNA diagnostics come into question as the markings. In particular, the markings with a direct marking, in particular with a fluorescent or luminescent constituent, are advantageous Spots allows an exact qualitative or quantitative detection of this analyte.

In order to be able to detect the binding of the analytes on individual spots next to one another and in particular also quantitatively, location-resolving detection methods can preferably be used. A preferred method is e.g. confocal scanning fluorescence microscopy. In this method, the fluorescent dye used for marking is excited individually for fluorescence on each spot with the aid of a laser of suitable wavelength (scanning). The emitted light is detected by means of sensitive detectors, e.g. integrating CCD cameras recorded, and quantified with suitable image evaluation software. Other non-scanning fluorescence microscopic methods are also well suited for evaluation. In this case, several spots are viewed simultaneously in one image section. The individual spots are then evaluated, e.g. in succession with the help of suitable image analysis software.

Another method of measuring the binding of an analyte to the solid phase binding partner is optical, in particular reflection-optical techniques, in which the increase in the layer thickness of an extremely thin layer with the carrier-fixed binding partner can be observed by binding the free analyte. An overview of these techniques is given in Sadowsky: "Review of optical methods in immunosensing", SPIE, Vol. 1954, Optical Testing and Methology II (1988), 413-419. No special labeling of the analytes is necessary here. A particularly preferred reflection-optical method is the detection of the binding of different analytes in different areas of the analysis element by means of surface plasmon resonance. In these methods, the analytical element consists of a transparent, dielectric material, on which a very small layer of a metallic conductive layer in spot form is applied, which carries the solid phase binding partner. Such analysis elements are also often referred to as optical immune sensors. Examples of such optical immune sensors are described in EP-A 0 1 12 721, EP-A-0 276 142 and in EP-A-0 254 557

Other location-resolving detection methods such as electrochemical measurements, changes in conductivity or measurement of radioactive labeling are also possible. To analyze a sample for certain analytes, the sample is either applied to the analysis element over a large area or separately to individual spots. If necessary, additionally marked binding partners for the analytes to be determined are added. In this case, the analysis element is advantageously washed free of free labeling molecules before measuring the labeled analytes bound to the solid phase.

The analysis element can also be used to determine one or more analytes in various samples.

The invention therefore also relates to a method for the simultaneous determination of an analyte in different samples by means of specific binding reactions of the analyte with solid-phase-bound analyte binding partners on a surface-shaped analysis element, characterized in that different sample solutions are contacted with different areas of an analysis element according to the invention, the same analyte binding partners in the areas are bound and the presence or amount of the solid phase-bound analyte is determined separately in different areas.

The various sample solutions can be applied using standard application techniques such as pipetting, printing and stamping techniques or a micromultipin plate. Another object of the invention is the use of the binding matrix or analysis elements according to the invention for the simultaneous detection of a large number of analytes, in particular in immunoassays, for example for allergy diagnostics or for DNA diagnostics, for example for screening a sample for polynucleotides with certain sequences.

example 1

1. Production of the spots by vapor deposition

"Lexan" polycarbonate films (manufacturer: General Electric, thickness 0.75 mm) with the dimensions 8x8 cm were placed over a metal mask (d = 0.5 mm, material. Aluminum) in a Leybold high vacuum coating system (Univex 450) 36x36 round gold spots (total of 1296 spots) evaporated at equal intervals. The layer thickness of the evaporated gold is 300 nm. The diameter of the spots is 1 mm, the distance from spot to spot is also 1 mm on all sides

2. Coating the multi-gold spot plate with "diluted" biotinthiol binding layers

2.1 "Hydrophilic" layer

2.8 mg (5 x \ 0 ^~ ~ - m) of the biotin thiol compound I and 8.7 mg (4.5 x 10 ~ 4 m) of the diluent II were in 100 ml of 0.05 m potassium phosphate buffer pH 7.25 solved. The freshly coated polycarbonate films were immersed in this solution. After one hour the plates were removed, washed twice with water (bidistilled) and air-dried.

2.2. "Hydrophobic" layer

2.94 mg (5xl0 ^-5 m) of the biotin thiol compound III and 9.2 mg (4.5 x 10 " ⁴ m) of the thinner IV were dissolved in 100 ml of ethanol pa. The freshly coated polycarbonate films were immersed in this solution After 4 hours the plates were removed, washed twice with ethanol pa and air dried.

IV.

3. Application of streptavidin

The foils coated with gold spots and SAM were immersed in a streptavidin solution for one hour (concentration streptavidin: 0.5 mg / ml in 0.05 m K-phosphate buffer pH 7.2). The chips were then washed with a solution of 50 mM K-phosphate buffer pH 7.2, 2% sucrose, 0.9% NaCl and 0.3% RSA II. The chips were then dried at 25 ° C and 40% humidity for 20 hours.

4. Perform a TSH test

A TSH test was carried out on the spots coated with streptavidin according to the following scheme:

1. Incubation with <TSH> MAK 1.20-Bi (1: 4) for 45 minutes, concentration of the MAK 20 μg / ml, buffer 20 mM K-phosphate pH 7.4.

2. Wash with bidist. Water.

3. 45 minutes incubation with TSH standard "Enzymun", from Boehringer GmbH.

4. Wash with bidist. Water.

5. 30 minutes incubation with fluorescence-latex-antibody (A8) conjugate, 0.05% solids content in 20 mM K-phosphate buffer pH 7.4.

6. Wash with bidist. Water.

7. Measurement on a confocal fluorescence microscope (from Biorad). Results

Two standards (10 μU and 2 μU) were measured. The following relative intensities were obtained:

Graphical evaluation of the measurement results (number of pixels against fluorescence intensity) for the hydrophobic thinned binding layer, see FIG. 1.2

Example 2

Checking the non-specific binding

The streptavidin-coated spots of the diluted hydrophobic binding layer from Example 1 were examined for non-specific binding of the fluorescent latex conjugate mediated by serum components.

1. Wash with bidist. water

2. 45 minutes incubation with sample (standards or serum)

3. Wash with bidist. water

4. 30 minutes incubation with fluorescent latex antibody (A8) conjugate

5. Wash with bidist. water 6. Measurement on the confocal fluorescence microscope

Results

It can be seen that serum components do not interfere with the measurement

Claims

claims

1 binding matrix containing a flat carrier material which is covered parallel to its surface in the horizontal direction with a large number of spatially separated areas which contain binding partners immobilized, each of which is capable of binding a corresponding free binding partner of a specific binding pair, characterized in that that the areas are formed from a) metal layer spots not touching each other, b) thinned and essentially laterally homogeneous binding layers of a binding partner B 1 bound to the metal spots via anchor groups

2 binding matrix according to claim 1, characterized in that the occupancy amounts to 0.1 to 90% of the maximum occupancy.

3. Binding matrix according to claim 2, characterized in that the occupancy is 0.5 to 70% of the maximum occupancy.

4. Binding matrix according to claim 3, characterized in that the occupancy is 1 to 40% of the maximum occupancy

5. Binding matrix according to one of claims 1-4, characterized in that the Metall¬ layer spots consist of gold, silver or palladium and the anchor groups are thiol, disulfide or phosphine groups.

6. Binding matrix according to one of claims 1-5, characterized in that the Anker¬ group is linked to the binding partner B1 via a flexible spacer molecule.

7. Binding matrix according to claim 6, characterized in that the flexible spacer molecule contains at least one alkylene group of the formula (CH2) _n , where n is a natural number between 1 and 30.

8. binding matrix according to Anspmch 6 or 7, characterized in that a spacer molecule is linked to two or more molecules of the binding partner.

9. binding matrix according to Anspmch 6 or 7, characterized in that there is a hydrophilic linker group between the spacer molecule and the binding partner.

10. Binding matrix according to one of claims 6 or 7, characterized in that the Binde¬ layers of the individual regions in addition to the spacer molecules linked to binding partners still contain spacer molecules which are provided with anchor groups but are not linked to binding partners.

1 1. binding matrix according to Anspmch 1- 10, characterized in that the metal spots have a diameter of 0.1-1 mm.

12. Binding matrix according to Anspmch 1-1 1, characterized in that between 10 and 100 metal spots are applied per cm ^ of Binde¬ matrix.

13. Binding matrix according to one of claims 1-12, characterized in that differently diluted binding layers are applied to individual spots.

14. Binding matrix according to Anspmch 1-13, characterized in that binding layers with different binding partners are applied to individual or all metal spots.

15. Binding matrix according to Anspmch 1-12, characterized in that binding layers with the same binding partners are applied to the metal spots.

16. Binding matrix according to Anspmch 15, characterized in that the binding partner B 1 is biotin or a biotin analog.

17 binding matrix according to one of claims 14 or 15, characterized in that the binding partner B 1 represents one or more different antigens or haptens capable of binding with antibodies

18 Bindematπx according to one of claims 14 or 15, characterized in that the binding partner B l represents one or more different polynucleotides

19 binding matrix according to Claim 15 or 16, characterized in that a further binding layer of a binding partner B2 which is the same in all areas and which is capable of binding free binding partners of a specific binding pair and via is bound to the binding layers of the binding partner B 1 a specific binding site is bound to Bl

20 binding matrix according to Anspmch 19, characterized in that the binding partner Bl is biotin and the further binding partner B2 is streptavidin

21 analytical element for determining different free reactants in a sample or for determining a reactant in different samples, containing a flat support material which, parallel to its surface, is covered with a plurality of horizontally spatially separated areas on which different or the same specific ones Binding partners for free reactants are immobilized, characterized in that there is a binding matrix according to one of claims 15, 16, 19 or 20, to which c) one to the binding partners B 1 or, in the presence of an additional binding partner B2, one to B2 contains specifically bound further binding layer which contains binding partner B3 on its surface for free reactants to be determined

22. Analysis element according to claim 20 or 21, characterized in that the binding partner Bl is biotin, which forms a dilute layer on the metal spots, the binding partner B2 is streptavidin and the binding partners B3 are biotinylated reactant binding partners

23 Method for producing a binding matrix according to claims 1 -18, characterized in that a) metal layer spots are applied to the surface of the carrier material in such a way that they do not struggle b) the free surface of the metal layer spots is incubated in each case with a reaction solution which contains molecules for producing a dilute binding layer.

24 Process for the preparation of a binding matrix according to Claim 19 or 20, characterized in that the diluted binding layers of a binding matrix according to Claim 15 or 16 are each incubated with a reaction solution which contains a further binding partner B2 for a free binding partner with a specific binding site contains for binding partner B 1

25 Method for producing an analytical element according to Anspmch 21-22, characterized ge indicates that the free surfaces of the binding layers of a binding matrix according to one of claims 15, 16, 19 or 20 are each incubated with the same or different solutions, the binding partners B3 for free reactants, the binding partners B3 having a specific binding site for the binding partners B1 or B2.

26. Method for the simultaneous determination of different free reactants in a sample solution or a free reactant in different sample solutions by means of specific binding reactions of the reactants with solid-phase-bound reactant binding partners on a flat analysis element, by contacting and binding the reactants to discrete, spatially separated binding agents. Areas of the analysis element and determination of the solid phase-bound reactants in the different areas separately from one another, characterized in that an analysis element according to Claim 21 or 22 is used.

27. The method according to Anspmch 26, characterized in that the determination is carried out by means of confocal fluorescence microscopy.

28. The method according to Anspmch 26, characterized in that the determination is carried out by means of surface plasmon resonance

29. Use of a binding matrix according to one of claims 1-20 or an analytical element according to one of claims 21-22 for multiple analyte detection.

30 Use of a binding matrix according to one of claims 1-20 or an analytical element according to one of claims 21-22 for immunoassays

31 Use of a binding matrix according to one of claims 1-20 or an analytical element according to one of claims 21-22 for DNA diagnostics