EP1421376A2

EP1421376A2 - Bioanalytical recognition surface with optimised recognition element density

Info

Publication number: EP1421376A2
Application number: EP02797619A
Authority: EP
Inventors: Michael Pawlak; Gert L. Duveneck; Ning-Ping Huang; Susan Margaret De Paul; Marcus Textor; Rolf Hofer; Eveline SCHÜRMANN-MADER
Original assignee: Zeptosens AG
Current assignee: Bayer AG
Priority date: 2001-08-27
Filing date: 2002-08-24
Publication date: 2004-05-26
Also published as: WO2003021253A3; AU2002361223A1; WO2003021253A2; US20040253596A1

Abstract

The invention relates to a recognition surface on a support with an optimal (with regard to the surface) binding capacity for recognition and binding of one or several analytes from samples brought into contact with said surface, characterized in that a) said recognition surface comprises a mixture of specific biological, biochemical or synthetic recognition elements for the recognition and binding of said analytes with components which are neutral to said analytes, in other words, do not bind said analytes and b) said specific recognition elements form less than a complete monolayer, relative to the whole surface area or any partial area thereof. The invention also relates to a method for the qualitative and/or quantitative determination of one or more analytes in one or more samples, characterized in that said samples and optionally further reagents are brought into contact with the above recognition surface and changes resulting from the binding of the analyte or analytical substances added for detection of the analytes are measured by optical or electronic signals.

Description

Bioanalytical detection surface with optimized density of the detection elements

The invention relates to a detection surface on a support with an optimal (in terms of area) binding capacity for the detection and binding of one or more analytes from one or more samples brought into contact with this surface, characterized in that a) said detection surface is a mixture of specific ones biological or biochemical or synthetic recognition elements for the recognition and binding of said analytes with components which are “neutral” with respect to these analytes, ie components that do not bind these analytes, and b) said specific recognition elements, based on the entire recognition surface or any partial area thereof, less than a complete one Take monolayer.

The invention also relates to a method for the qualitative and / or quantitative detection of one or more analytes in one or more samples, characterized in that said samples and optionally further reagents are brought into contact with a detection surface according to the invention and from the binding of the analyte or further to Analyte detection of used detection substances resulting changes in optical or electronic signals can be measured.

For the detection of one or more analytes from a sample with a complex mixture of a large number of substances, methods are common in which one or more so-called recognition elements, which are biological, biochemical or synthetic, are immobilized on a solid support before they are then immobilized Form are brought into contact with said sample and the analytes contained therein bind to the detection elements specific to them. The solid support can be both a macroscopic type with a surface area of square millimeters to square centimeters and also a microscopic type, for example in the form of so-called beads, ie approximately spherical particles with typical diameters in the micrometer range. The surface of such a solid Carrier with recognition elements immobilized thereon will hereinafter be referred to as a "recognition surface".

Compared to methods in which the analytes and their recognition elements come together as reaction or binding partners in homogeneous liquid solution, these methods bound to a solid support offer a number of advantages, for example easier separation or differentiation of bound analyte molecules from the rest of the sample. These advantages are bought with a restriction of the diffusive mixing between analyte molecules and recognition elements.

The nature of these surfaces is of great importance for the production of detection surfaces for the highly efficient and highly selective binding of one or more analytes to be detected in a sample. In order to achieve the lowest possible detection limits, it is desirable to immobilize as many detection elements as possible in a small space in such a way that as many analyte molecules of one type as possible can then be bound in the later detection method. At the same time, it is desirable to maintain the reactivity and biological or biochemical functionality of the recognition elements as high as possible during immobilization, i.e. to minimize any signs of denaturation due to immobilization. Another goal is to largely prevent non-specific binding or adsorption of analyte molecules, which in many cases has a limiting effect on the detection limits that can be achieved.

In WO 84/01031 and in US Pat. Nos. 5,807,755, 5,837,551 and 5,432,099, the immobilization for the analyte of specific recognition elements in the form of small "spots" with partially significantly less than 1 mm ² area on solid supports is proposed. For this purpose, it is postulated as an advantage to be able to determine the concentration of the analyte by binding only a small part of the analyte molecules present, which is dependent only on the incubation time but - in the absence of a continuous flow - essentially independent of the absolute sample volume. At the same time, it is to be expected for such an arrangement that a greater density of bound analyte molecules is achieved in these spots than if these were distributed over an area completely covered with recognition elements. However, here too, just as in the case of large-area immobilization of detection elements on a macroscopic surface, a high density of the immobilized detection elements in the measurement areas thus generated can have a limiting effect on the maximum number of analyte molecules which can be bound to the surface. An important reason for such a limitation of the binding capacity can be steric hindrance.

The present invention solves the problem of already providing a detection surface with a maximum binding capacity and at the same time minimizing the extent of unwanted non-specific binding to the surface. The mixture of specific recognition elements and components which are “neutral” with respect to the analytes, that is to say components which do not bind these analytes, furthermore has a promoting effect on the binding ability of said specific recognition elements by denaturing said recognition elements on the surface of the support (which impair or even cancel the binding ability) In addition, this mixture promotes a uniform distribution of the detection elements within the detection surface, for example by preventing the detection elements from accumulating in clusters after application in liquid solution and evaporation of the solution.

The first object of the invention is a detection surface with an optimal (in terms of area) binding capacity for the detection and binding of one or more analytes from one or more samples brought into contact with this surface, characterized in that a) said detection surface is a mixture of specific biological or biochemical or synthetic recognition elements for recognizing and binding said analytes with components which are “neutral” with respect to these analytes, ie components which do not bind these analytes, and b) said specific recognition elements, based on the entire recognition surface or any partial area thereof, less than a complete monolayer , take in. Said specific recognition elements, based on the entire recognition surface or any partial area thereof, preferably form one tenth to one half of a complete monolayer. In addition, it is preferred that said specific recognition elements and components which are “neutral” to the analytes, based on the entire recognition surface or any partial area thereof, together form at least two thirds of a complete monolayer.

For a large number of applications, it is desirable to determine not only a single analyte, but a large number of analytes simultaneously. A second object of the invention is accordingly a structured detection surface with an optimal (in terms of area) binding capacity for the detection and binding of one or more analytes from one or more samples brought into contact with this surface, characterized in that a) said detection surface in discrete , spatially separated measuring ranges, a mixture of specific biological or biochemical or synthetic recognition elements for the recognition and binding of said analytes with components which are “neutral” with respect to these analytes, ie, those analytes that do not bind, and b) said specific recognition elements, based on the area of the discrete ones Take up measuring ranges less than a complete monolayer.

For this variant, too, it is preferred that said specific recognition elements, based on the entire recognition surface or any partial area thereof, form a tenth to a half of a complete monolayer. In addition, it is also preferred for a structured recognition surface according to the invention that said specific recognition elements and components which are “neutral” with respect to the analytes, based on the entire recognition surface or any partial area thereof, together form at least two thirds of a complete monolayer.

For the purposes of the present invention, spatially separated measuring areas, as part of a detection surface according to the invention, are to be defined by the area, the biological or biochemical or synthetic detection elements immobilized there for the detection of an analyte from a liquid sample and those with the detection elements mixed, "neutral" molecules compared to said analytes. These surfaces can have any geometry, for example the shape of points, circles, rectangles, triangles, ellipses or lines. It is possible that in a two-dimensional arrangement up to 1,000 000 measuring ranges are arranged, whereby a single measuring range can take up an area of 10 ^"4 mm ² - 10 mm ² . Typically, the density of the measurement areas can be more than 10, preferably more than 100, particularly preferably more than 1000 measurement areas per square centimeter.

A detection surface according to the invention is usually generated on a solid support. There are a large number of methods for applying the recognition elements to a carrier surface and different types of interaction of these recognition elements with the surface carrying them, which ensure their immobilization. For example, the application and subsequent adhesion can take place through electrostatic interaction or, more generally, through physical adsorption. The orientation of the recognition elements is then generally statistical. In addition, there is the risk that when the sample containing the analyte and possibly further reagents are added sequentially to the detection surface, some of the detection elements are washed away. It can therefore be advantageous if an adhesion-promoting layer is applied to the carrier surface for the immobilization of biological or biochemical or synthetic recognition elements. For many applications, for example in the case of analyte detection based on optical methods, it is advantageous if this adhesion-promoting layer is transparent at least at one excitation wavelength. The thickness of such an optional adhesion-promoting layer is preferably less than 200 nm, but particularly preferably less than 20 nm. The optional adhesion-promoting layer can, for example, be a chemical compound from the groups of silanes, functionalized silanes, epoxides, functionalized, charged or polar polymers and "self-organized passive or functionalized monolayers or multilayers ".

It is preferred that discrete (spatially separated) measurement areas, as a component of this detection surface, are generated by spatially selective application of biological or biochemical or synthetic detection elements on a surface of a carrier or on an adhesion-promoting layer additionally applied to a carrier surface, preferably using an or several methods from the group of processes involving "inkjet spotting", mechanical spotting using a pen, pen or capillary, "micro contact printing", fluidic contacting of the measuring areas with the biological or biochemical or synthetic recognition elements by their supply in parallel or crossed microchannels, under the influence of pressure differences or electrical or electromagnetic potentials as well as photochemical or photolithographic immobilization processes.

It is also advantageous if "areas" between the spatially separated measurement areas are "passivated" to minimize non-specific binding of analytes or their detection substances, i.e. that "chemically neutral" compounds are applied between the spatially separated measuring ranges relative to the analyte or to one of its detection substances, preferably consisting, for example, of the groups consisting of albumins, in particular bovine serum albumin or human serum albumin, casein, non-specific, polyclonal or monoclonal, foreign or empirically for the analyte (s) to be detected are non-specific antibodies (in particular for immunoassays), detergents - such as, for example, Tween 20 -, fragmented natural or synthetic DNA that does not hybridize with polynucleotides to be analyzed, such as an extract of herring or salmon sperm (in particular for polynucleotide hybridization assays), or also uncharged, but hydrophilic polymers, such as polyethylene glycols or dextrans, are formed.

The biological or biochemical or synthetic recognition elements can be selected from the group consisting of proteins, for example mono- or polyclonal antibodies and antibody fragments, peptides, enzymes, aptamers, synthetic peptide structures, glycopeptides, oligosaccharides, lectins, antigens for antibodies (e.g. Biotin for streptavidin), with additional binding sites functionalized proteins ("tag proteins", such as "histidine tag proteins") and their complex formation partners. Another group of compounds, which are also preferred as recognition elements, include nucleic acids (for example DNA, RNA, oligonucleotides) and nucleic acid analogs (for example PNA) and their derivatives with artificial bases. A third preferred group of compounds as recognition elements comprises soluble, membrane-bound proteins isolated from a membrane, such as, for example, receptors and their ligands. Said “neutral” components, which do not bind the analyte or analytes, can also be selected from the groups consisting of albumin, in particular bovine serum albumin or human serum albumin, casein, nonspecific, polyclonal or monoclonal, foreign species or empirically unspecific antibodies for the analyte (s) to be detected ( especially for immunoassays), detergents - such as Tween 20 -, fragmented natural or synthetic DNA that does not hybridize with polynucleotides to be analyzed, such as an extract of herring or salmon sperm (especially for polynucleotide hybridization assays), or also uncharged but hydrophilic polymers , such as polyethylene glycols or dextrans.

WO 00/65352 describes coatings with graft copolymers (“graft copolymers”) with a polyionic main chain, for example binding to a support (electrostatically) and “non-interactive” (adsorption-resistant) side chains, for coating bioanalytical sensor platforms or Implants for medical applications.

A preferred embodiment of a detection surface according to the invention is characterized in that the detection elements are bound to the free end or near the free end of a fully or partially functionalized, "non-interactive" (adsorption-resistant, uncharged) polymer, said non-interactive "(adsorption-resistant, uncharged) ) Polymer is bound as a side chain to a charged, polyionic polymer as the main chain and forms together with this a polyionic, multifunctional co-polymer.

A large group of embodiments of a detection surface according to the invention is characterized in that the polyionic polymer main chain is cationically (positively) charged at approximately neutral pH. For example, it can be selected from the group of polymers which comprises amino acids with a positive charge at approximately neutral pH, polysaccharides, polyamines, polymers of quaternary amines and charged synthetic polymers. The cationic polymer main chain can also comprise one or more molecular groups from the group consisting of lsysine, histidine, arginine, chitosan, partially deacetylated chitin, amine-containing derivatives of neutral polysaccharides, polyaminostyrene, Polyamine acrylates, polyamine methacrylates, polyethyleneimines, polyaminoethylenes, polyaminostyrenes and their N-alkyl derivatives.

Further suitable molecular groups as part of a polyionic main chain are described in WO 00/65352, which is fully introduced here as part of this description.

Another large group of embodiments is characterized in that the polyionic polymer main chain is anionically (negatively) charged at approximately neutral pH. Within this group, the cationic main chain can be selected from the group of polymers comprising amino acids with attached groups with negative charge at approximately neutral pH, polysaccharides and charged synthetic polymers with negatively charged groups.

The cationic polymer main chain can comprise one or more molecular groups from the group which comprises polyaspartic acid, polyglutamic acid, alginic acid or their derivatives, pectin, hyaluronic acid, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, dextran sulfate, polymethyl methacrylic acid, oxidized cellulose, carboxymic acid, carboxymic acid, maloxymic acid, carboxymethyl acid, maloxymic acid, carboxymethyl acid, maloxic acid, carboxymethyl acid.

The "non-interactive" (uncharged) polymer "as the side chain can be selected from the group comprising poly (alkylene glycols), poly (alkylene oxides), neutral water-soluble polysaccharides, polyvinyl alcohols, poly-N-vinylpyrrolidones, phosphorylcholine derivatives, non-cationic poly ( meth) acrylates and their combinations.

It is preferred that the biological or biochemical or synthetic recognition elements are bound to the “non-interactive” side chain at its free end or close to its free end via reactive groups: it is particularly preferred that said reactive groups are selected from the group comprising hydroxy (-OH), carboxy (-COOH), ester (-COOR), thiols (-SH), N-hydroxysuccinimide, maleimidyl, quinone, vinyl sulfone, nitrilotriacetic acid ("nitrilotriacetic acid", NTA) and combinations thereof. A number of other suitable polymers and preferred embodiments are additionally described in WO 00/65352.

In general, it is preferred that a detection surface according to the invention is applied to an essentially optically transparent carrier.

The term “essentially optically transparent” is to be understood to mean that a layer characterized thereby is at least 95% transparent, at least at the wavelength of a light irradiated by an external light source, for its optical path perpendicular to said layer, provided that the layer is not reflective In the case of partially reflecting layers, “essentially optically transparent” is understood to mean that the sum of transmitted, reflected and optionally coupled into a layer and guided therein is at least 95% of the incident light at the point of incidence of the incident light.

An from an external light source towards the detection surface, i.e. H. According to the present invention, both light emitted by a carrier in the direction of the detection surface and from the opposite side, if appropriate by a medium radiated above the detection surface in the direction of the detection surface, should generally be referred to as an “excitation light.” This excitation light can be used, for example, for excitation luminescence, or specific fluorescence or phosphorescence.

It is preferred that the essentially optically transparent carrier comprises a material from the group comprising moldable, sprayable or millable plastics, metals, metal oxides, silicates, such as, for. B. glass, quartz or ceramics.

A possible embodiment is characterized in that the detection surface according to the invention is applied to an adhesion-promoting layer applied to the essentially optically transparent carrier, which is also essentially optically transparent.

A special embodiment is characterized in that recesses for the production of sample containers are structured in the surface of said carrier. This Recesses typically have a depth of 20 μm to 500 μm, preferably from 50 μm to 300 μm.

It is preferred that the essentially optically transparent carrier comprises a continuous optical waveguide or divided into individual waveguiding regions. It is particularly advantageous if the optical waveguide is an optical layer waveguide with a first, essentially optically transparent layer (a) facing the detection surface on a second, essentially optically transparent layer (b) with a lower refractive index than layer (a).

It is preferred that said optical layer waveguide is substantially planar. Suitable planar optical layer waveguides and their modifications are, for example, in patent applications WO 95/33197, WO 95/33198, WO 96/35940, WO 98/09156, WO 99/40415, PCT / EP 00/04869 and PCT / EP 01 / 00605. The content of these patent applications is therefore fully introduced as part of this description.

It is preferred that, for coupling excitation light into the optically transparent layer (a), this layer is in optical contact with one or more optical coupling elements from the group consisting of prism couplers, evanescent couplers with matched optical waveguides with overlapping evanescent fields, End face couplers are formed with focusing lenses, preferably cylindrical lenses, arranged in front of one end face of the wave-guiding layer, and grating couplers.

It is advantageous if the excitation light is coupled into the optically transparent layer (a) with the aid of one or more grating structures (c) which are pronounced in the optically transparent layer (a).

It is characteristic of a further variant that the coupling out of light guided in the optically transparent layer (a) takes place with the aid of one or more lattice structures (c ') which are pronounced in the optically transparent layer (a) and have the same or different periods and Have grid depth like grid structures (c). Another component of the invention is a method for the qualitative and / or quantitative detection of one or more analytes in one or more samples, characterized in that said samples and optionally other reagents are brought into contact with a detection surface according to the invention according to one of the aforementioned embodiments and from the Binding of the analyte or other changes in optical or electronic signals resulting from the detection substances used to detect the analyte can be measured.

The invention also relates to a method for the qualitative and / or quantitative detection of one or more analytes in one or more samples, characterized in that said samples and optionally further reagents are brought into contact with and out of a structured detection surface according to one of the above-mentioned embodiments the binding of the analyte or other changes in the detection substances used for the detection of analytes resulting from optical or electronic signals emanating from the discrete measuring ranges are measured in a spatially resolved manner

It is preferred that the one or more samples are preincubated with a mixture of the various detection reagents for determining the analytes to be detected in said samples and these mixtures are then brought into contact with a detection surface according to the invention in a single addition step.

It is also preferred that the detection of the one or more analytes is based on the determination of the change in one or more luminescences.

The excitation light from one or more light sources can be radiated in an incident light excitation arrangement. It can also be irradiated in a transmission light excitation arrangement.

A method is preferred which is characterized in that the detection surface, optionally mediated via an adhesion-promoting layer, is arranged on an optical waveguide, which is preferably essentially planar, that the one or more samples are brought into contact with the one or more analytes to be detected therein and optionally further detection reagents sequentially or after mixing with said samples in a single step with said detection surface and that the excitation light from one or more light sources in the optical waveguide, analogously is coupled in as described above for the optical layer waveguide.

Characteristic of a special embodiment of the method according to the invention is that the detection of the one or more analytes on a detection surface above a grating structure (c) or (c ') formed in the layer (a) of an optical layer waveguide on the basis of the result of the binding of the analyte and / or further detection reagents, on the immobilized biological or biochemical or synthetic recognition elements, resulting changes in the resonance conditions for coupling an excitation light into the layer (a) of a carrier designed as a layer waveguide or for coupling out light carried in the layer (a).

A variant of the method according to the invention is particularly preferred, which is characterized in that said optical waveguide is designed as an optical layer waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower refractive index than layer (a), that excitation light continues to be coupled into the optically transparent layer (a) with the aid of one or more grating structures which are pronounced in the optically transparent layer (a) and is guided as a guided wave to the measuring areas (d) thereon, and that the evanescence continues Field of said guided wave generated luminescence of luminescent molecules is detected with one or more detectors and the concentration of one or more analytes is determined from the intensity of these luminescence signals.

Here, (1) the isotropically emitted luminescence or (2) coupled into the optically transparent layer (a) and coupled out via a grating structure (c) or (c ') coupled luminescence or luminescence of both components (1) and (2) can be measured simultaneously , It is part of the method according to the invention that a luminescent dye or luminescent nanoparticle is used as the luminescent label to generate the luminescence, which can be excited and emitted at a wavelength between 300 nm and 1100 nm.

It is preferred that the luminescence label be bound to the analyte or in a competitive assay to an analog of the analyte or in a multi-stage assay to one of the binding partners of the immobilized biological or biochemical or synthetic recognition elements or to the biological or biochemical or synthetic recognition elements.

Another embodiment of the method is characterized in that a second or even further luminescence label with the same or different excitation wavelength as the first luminescence label and the same or different emission wavelength is used.

It is preferred that the second or even more luminescent label can be excited at the same wavelength as the first luminescent dye, but emit at other wavelengths.

It is particularly advantageous if the excitation spectra and emission spectra of the luminescent dyes used overlap only slightly or not at all.

A variant of the method consists in that charge or optical energy transfer from a first luminescent dye serving as donor to a second luminescent dye serving as acceptor is used to detect the analyte.

Another embodiment of the method is characterized in that, in addition to the determination of one or more luminescences, changes in the effective refractive index on the measurement areas are determined.

A further development of the method is characterized in that the one or more luminescences and / or determinations of light signals are carried out polarization-selectively at the excitation wavelength. It is preferred that the one or more luminescences are measured with a different polarization than that of the excitation light.

Part of the invention is a method according to one of the aforementioned embodiments for the simultaneous or sequential, quantitative or qualitative determination of one or more analytes from the group of proteins, such as antibodies or antigens, receptors or ligands, chelators, with additional binding sites functionalized proteins ("Tag Proteins ", such as" histidine tag proteins ") and their complex formation partners, oligonucleotides, DNA or RNA strands, DNA or RNA analogs, enzymes, enzyme factors or inhibitors, lectins and carbohydrates.

Possible embodiments of the method are characterized in that the samples to be examined are, for example, naturally occurring body fluids such as blood, serum, plasma, lymph or urine or tissue fluids or egg yolk.

Other embodiments are characterized in that the sample to be examined is an optically cloudy liquid, surface water, a soil or plant extract, a bio- or synthesis process broth.

It is also possible that the samples to be examined are prepared from biological tissue parts or cell cultures.

The invention furthermore relates to the use of a method according to the invention for quantitative or qualitative analyzes for determining chemical, biochemical or biological analytes in screening methods in pharmaceutical research, combinatorial chemistry, clinical and preclinical development, for real-time binding studies and for determining kinetic parameters in affinity screening and in research, on qualitative and quantitative analyte determinations, in particular for DNA and RNA analysis and the determination of genomic or proteomic differences in the genome, such as single nucleotide polymorphisms, for measuring protein-DNA interactions, for determining control mechanisms for the m-RNA expression and for protein (bio) synthesis, for the preparation of toxicity studies and for the determination of expression profiles, in particular for the determination of biological and chemical marker substances such as mRNA, proteins, peptides or low-molecular organic (messenger) substances, as well as for the detection of antibodies , Antigens, pathogens or bacteria in pharmaceutical product research and development, human and veterinary diagnostics, agrochemical product research and development, symptomatic and presymptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for therapeutic drug selection, for the detection of pathogens, Pollutants and pathogens, especially of salmonella, prions, viruses and bacteria, especially in food and environmental analysis.

Example:

1. Materials

Poly (L-lysine) hydrobromide (molecular weight approx. 20 kDa), streptavidin from Streptomyces avidinii (molecular weight approx. 60 kDa), protein avidin (molecular weight approx. 66 kDa), biotinylated goat anti-rabbit (ie bound to biotin) Immunoglobulin (anti-R-IgG biotin, molecular weight approx. 150 kDa) and biotinylated bovine serum albumin (BSA biotin, molecular weight approx. 66 kDa) were obtained from Sigma-Aldrich (Buchs, Switzerland). The N-hydroxysuccinimidyl ester of methoxy-poly (ethylene glycol) propionic acid (MeO-PEG-SPA, molecular weight 2 kDa) and the α-biotin-ω-N-hydroxysuccinimidyl ester of poly (ethylene glycol) carbonate (biotin-PEG-CO ₂ -NHS , Molecular weight 3.4 kDa) were obtained from Shearwater Polymers Inc. (Huntsville, USA). Rabbit immunoglobulin (anti-human albumin) (R-IgG, molecular weight approx. 150 kDa) and rabbit anti-bovine serum albumin (anti-BSA, molecular weight approx. 150 kDa) were obtained from DAKO (Glostrup, Denmark). All antibody reagents mentioned were polyclonal. Control serum N (human) was obtained from Hoffmann-La Röche (Basel, Switzerland). 4- (2-hydroxyethyl) piperazin-1-ethanesulfonic acid (HEPES) and other chemicals for making buffers were obtained from Fluka (Buchs, Switzerland).

All aqueous solutions were prepared with ultrapure water (18 MΩcm) from an "EasyPure Reverse Osmosis System" (Barnstead Thermolyne, Dubuque, USA).

2nd carrier

A thin-film waveguide designed as a grating coupler (TiO-SiO ₂ solgel as a waveguiding layer on a glass substrate, period of the coupling grating in the waveguiding layer: 417 nm) (micro vacuum Ltd., Budapest, Hungary) with a sputtered thereon, 12 nm thin Nb ₂ O ₅ layer. Before their first use, these supports, with Nb ₂ O ₅ as the top layer, were sonicated in 0.1 M HC1 for 10 minutes, thoroughly rinsed with ultrapure water, blown dry with nitrogen and subsequently with oxygen plasma in a PDC plasma cleaner / sterilizer for 2 hours -32G (Harrick, Ossining, USA). 3. Synthesis of PLL-g-PEG and its derivatives

The synthesis of PLL-g-PEG was described by Sawhney and Hubbell (A. S. Sawhney, J.A. Hubbell, Biomaterials 13 (1992) 863-870). The method used for the studies on which the present application is based is based on a rule developed by Elbert and Hubbell (D.L. Elbert, J.A. Hubbell, J. Biomed. Mater. Res. 42 (1998) 55-65). According to WO 00/65352, a grafting ratio between g = 3 and g = 5 has been found for PEG part chains with a molecular weight of 2 kDa as an optimum for immobilizing as large an amount of polymers as possible on negatively charged metal oxide surfaces while at the same time guaranteeing minimal non-specific binding (adsorption) of proteins on the metal oxide surfaces coated with this polymer, a ratio of g = 3.5 is used for the experiments described in this example, the percentage of the PEG chains being attached to it bound biotin ("biotinylated PLL-g-PEG") varies.

Figure 1 shows schematically the synthesis of PLL-g-PEG. N-Hydroxy-succinimidyl esters of both biotinylated and non-biotinylated poly (ethylene glycol) ("PEG") are reacted with poly (L-lysine) ("PLL") in a stoichiometric ratio to produce the desired product. The details of this synthesis are described below under 3.1 and 3.2. A longer chain length is selected for the biotinylated PEG chains than for methoxy-PEG in order to ensure good accessibility of the polymer-bound biotin.

The nomenclature chosen below for the designation of the various PLL-g-PEG derivatives includes the molecular weights of the polymer partial chains of the copolymers, the grafting ratio and the percentage of biotinylated PEGs. Accordingly, “PLL (20) -g / ^" 5.57-PEG (2) / PEG-Biotin (3.4) 30%" a polymer formed from a main chain made of poly (L-lysine) with a molecular weight of 20 kDa and side chains, 70% consisting of poly (ethylene glycol) with a molecular weight of 2 kDa and 30% of biotinylated poly (ethylene glycol) with a molecular weight of 3.4 kDa. The grafting ratio of 3.5 means that on average two out of every seven lysine groups (lysine units) are biotinylated or non-biotinylated PEG chains are bound. Since all of the polymers mentioned in this example are produced from similar precursors as an alternative to "PLL-g-PEG / PEG-Biotin30%", the abbreviation "PPB30" should also be used. Abbreviations are used for other percentages of biotinylated PLL-g-PEGs.

3.1 Synthesis of PLL (20) -g / 3.57-PEG (2)

Poly (L-lysine) hydrobromide ("PLL-HBr") is dissolved in 25 ml of sodium tetraborate buffer ("STBB", 50 mM, pH 8.5) per gram of PLL-HBr. The solution is stirred and then filtered (0.22 μm Durapore membrane, sterile Millex GV, Sigma-Aldrich, Buchs, Switzerland) and filled into a sterile culture tube. MeO-PEG-SPA powder is then added in a suitable amount according to the stoichiometric ratio while stirring the solution evenly. After stirring the solution for a further six hours at room temperature, the solution is transferred to a dialysis tube (Spectr / Por dialysis tube, molecular weight limitation (cut-off) 6-8 kDa, Sochochim, Lausanne, Switzerland). Dialysis is carried out for 24 hours in one liter of phosphate buffered saline ("PBS", 10 mM, pH 7.0), followed by 24 hours of further dialysis in one liter of deionized water. The product is then kept at a temperature of -50 ° for 48 hours C and a pressure of 0.2 mbar freeze-dried.

3.2 Synthesis of biotinylated PLL-g-PEG

Biotinylated PLL-g-PEG is synthesized in a manner similar to that previously described. Biotin-PEG-CO ₂ -NHS powder is slowly added in an appropriate amount according to the stoichiometric ratio to the filtered solution of PLL-HBr solution and stirred for one hour. MeO-PEG-SPA is then added in an amount appropriate to the stoichiometric ratio, and the resulting solution is stirred for a further five hours. The further steps of dialysis and product recovery are the same as described above. 3.3 Determination of the grafting ratio and the percentage of biotin

The grafting ratio and the percentage of biotin in the biotinylated PEG derivatives are estimated using 1H-NMR. The lyophilized polymers are dissolved in D ₂ O and the spectra recorded with a 300 MHz NMR spectrometer. The values determined from this are summarized in Table 1.

Table 1: Grafting ratio and percentage of biotin in biotinylated PEG derivatives, determined using 1H-NMR.

4. Determination of the amount of surface adsorbed molecules using a lattice coupler sensor as a carrier

The mass of adsorbed polymer on the Nb ₂ θ ₅ surfaces is determined on the basis of the difference in the coupling conditions for light coupling into a grating coupler sensor before and after application of the respective polymer layers. The principle of operation of a grating coupler sensor is described, for example, in US Pat. No. 4952056. A grid coupler structure (BIOS I, ASI AG, Zurich, Switzerland) was used as the measuring instrument.

The values of the mass of surface adsorbed material are determined on the basis of the equation by Feijter (JJ Ramsden, J. Stat. Phys. 73 (1993) 853-877). Using an Raleigh interferometer, an incremental value of dn / dc = 0.158 cm ³ / g was determined for the adsorption of the polymers on a surface and used as a basis for the further calculations. For the protein adsorption on a surface a value of dn / dc = 0.182 cm ³ / g provided (JJ Ramsden, DJ Roush, DS Grill, R. Kurrat, RC Willson, J. Am. Chem. Soc. 117 (1995) 8511-8516).

5. Coating the Nb ₂ θs surfaces with polymers

A carrier pretreated according to section 2 of this example is equilibrated in HEPES-1 buffer (10 mM HEPES, pH 7.4) for at least five hours before an experiment, then inserted into the lattice coupler measuring instrument and there in HEPES-1 for another hour - Buffer equilibrated until a stable baseline, ie a stable resonance angle for coupling the excitation light into the highly refractive waveguiding layer using the coupling grating has been achieved.

Solutions of PLL-g-PEG (15 μM) and PLL-g-PEG / PEG-Biotin in HEPES -1 buffer are filtered through 0.22 μm Durapore membranes and mixed immediately before use. The Nb ₂ O ₅ surfaces are coated in situ in the lattice coupler structure by bringing the metal oxide surface of the support into contact with the solution of the polymer mixture. This is done for 30 minutes under constant flow at a flow rate of 1 ml / h. The coated substrate is then rinsed with HEPES -1 buffer for 30 minutes.

6. Carrying out the protein binding assays 6.1. Standard Assay Protocol

For most of the experiments described below, the polymer-coated supports are sequentially under continuous flow (flow rate: 1 ml / h) with solutions of streptavidin (100 μg / ml), anti-R-IgG-biotin (100 μg / ml) and finally R-IgG (200 μg / ml) incubated. Each of these incubation steps has a duration of 15 minutes, which is sufficient at the selected concentrations to saturate all available binding sites. This is followed by a 30-minute washing step with the respective buffer to remove unbound, previously added molecules. HEPES buffer solutions were used in each case. 6.2. Special assay protocols

For the investigations of the influence of streptavidin surface densities on the binding behavior, which are described further below, the same process steps are carried out as in 6.1. with the exception that a streptavidin concentration of only 2.5 μg / ml is used and the incubation time is 45 minutes.

BSA biotin (100 μg / ml) and rabbit anti-bovine serum albumin (“anti-BSA”, 200 μg / ml) are used to investigate the dependency of the optimal surface density of the recognition elements on the size of the molecules to be bound from a supplied sample. used instead of anti-R-IgG-biotin and R-IgG.

7. Application of polymers with detection elements partially bound to them on a carrier

A thin-film waveguide (TiO ₂ -SiO ₂ sol gel as a waveguiding layer on a glass substrate, period of the coupling grating in the waveguiding layer: 417 nm) with a 12 nm thin Nb ₂ O ₅ layer applied thereon serves as a carrier. At a set pH of 7.4 of a subsequently applied solution, the surface of Nb ₂ O _{5 is} negatively charged (isoelectric point IEP = 3.6), while PLL main chains of PLL-g-PEG and PLL-g-PEG / PEG-biotin are strongly positive are loaded. It is believed that the strong adsorption of polymers, which include PLL as an essential component, on Nb ₂ O ₅ -coated surfaces is based primarily on electrostatic interaction between this metal oxide surface and the polymer as a multi-charged adsorbate.

The aim of applying a mixture of PLL-g-PEG and PLL-g-PEG / PEG-Biotin is to achieve an optimal binding capacity of the polymer-coated surface by adjusting the mixing ratio and at the same time to minimize non-specific binding. Biotin, bound as a recognition element in the polymer PLL-g-PEG / PEG-biotin, serves as a specific recognition element for molecules such as, for example, avidin or streptavidin, to which "biotinylated" molecules (ie with Biotin-linked molecules), such as anti-RIgG-biotin, can be bound, which in turn can serve as recognition elements for an analyte (in this example, R-IgG).

As a result of the application of this polymer mixture with PLL-g-PEG / PEG-biotin on the Nb ₂ θ ₅ surface, the biotin binding sites are surrounded by non-binding PEG chains, so that the protein adsorption is reduced. Such properties are described in a number of patents (e.g., U.S. Patent Nos. 5820882, 5232984, 5380536, 6231892, 5462990, 5627233 and 5849839). WO 00/65352 also describes polymers of this type with attached biotin molecules. From these documents, however, there is no indication that - as in the present invention - the binding capacity can be optimized by adjusting the proportion of bound biotin. This optimization of the binding capacity is adjusted according to the invention, in this example by setting the proportion ratio between PLL-g-PEG and PLL-g-PEG / PEG-biotin.

The mass of adsorbed polymer on the Nb ₂ θ ₅ surfaces is determined on the basis of the difference in the coupling conditions for light coupling into the lattice coupler before and after application of the respective polymer layers. From this, values of 167 +/- 8 ng / cm ² adsorbed polymer for pure PLL-g-PEG and 213 +/- 13 ng / cm ² for pure PPB20 are determined. Taking into account the molecular weights and the grafting ratio determined by means of NMR, the surface concentrations of the adsorbed polymers are determined for each mixing ratio used. Within the experimental accuracy there is a uniform value of 2.5 +/- 0.1 pmol / cm ² , which leads to the conclusion is that the mixing ratio of the polymers on the surface is the same as before in solution.

8. Binding of streptavidin to the recognition surface

With the addition of a solution of streptavidin (100 μg / ml) to surfaces with different mixing ratios (PLL-g-PEG: PPB20), under continuous flow, a Saturation signal for the binding of streptavidin to the surface coated with biotinylated polymers was reached within 15 minutes. After a washing step with HEPES -1 buffer, the amount of bound streptavidin is determined for the surfaces with the different polymer mixture ratios. The amount of surface-bound biotin is determined from NMR measurements and the mixing ratio of the polymers in solution.

In this series of measurements with different mixing ratios of surface-immobilized polymers, the proportion of bound streptavidin increases continuously with the proportion of PPB20 (see FIG. 2). No binding or adsorption is observed on the pure PLL-g-PEG surface, while 2.77 pmol / cm ² streptavidin are bound to a pure PPB20 surface (corresponding to 19.2 pmol / cm ² biotin). From the two-dimensional crystal structure of streptavidin it can be concluded that a single streptavidin molecule has a size of approximately 5.5 nm x 4.5 nm (SA Darst, M. Ahlers, PH Meiler, EW Kubalek, R. Blankenburg, HO Ribi, H. Ringsdorf, RD Kornberg, Biophys. J. 59 (1991) 387-396). As a result, a densely packed streptavidin monolayer can be expected to have a surface density of 6.71 pmol / cm. Consequently, the surface densities of streptavidin bound to the surface under the conditions of the experiment in FIG. 2 correspond to between 0% and 41% of a densely packed monolayer.

For all polymer mixing ratios used in this series, the ratio of bound streptavidin to surface-immobilized biotin is 1: 6.5. The relatively high excess of biotin sub-molecules as immobilized recognition elements compared to bound streptavidin, which was added in an excess, which should lead to the saturation of all available binding sites in terms of quantity, can be explained by the fact that a part of the biotin molecule is not accessible on the surface, but rather could be hidden in the PEG sublayer. On the other hand, one streptavidin molecule could also bind to two or more biotin molecules.

In the case of a surface on which pure PPB50 is immobilized (corresponding to 42 pmol / cm biotin on the surface), approximately 6.65 pmol / cm streptavidin is bound, which corresponds approximately to the amount of a monolayer. In this case, the ratio of bound streptavidin to immobilized biotin is about 1: 5. In the next step, biotinylated anti-rabbit immunoglobulin (anti-R-IgG-biotin) is bound to the surface previously modified with streptavidin (to the remaining binding sites for biotin to streptavidin). This is followed by a washing step with HEPES-1 buffer.

Figure 2 shows the amount of anti-R-IgG biotin bound as a function of the concentration of surface bound biotin. With increasing biotin surface concentration, ie at the same time increasing concentration or surface density of bound streptavidin, the amount of bound anti-R-IgG biotin also initially increases. With a surface concentration (density) of approx. 11.2 pmol / cm ² of biotin bound via PEG / biotin, corresponding to a concentration of 1.68 pmol cm bound streptavidin (or x% of a complete monolayer), a maximum of the amount of bound anti-R -IgG-biotin reached about 0.43 pmol / cm. With a further increase in the density of surface-immobilized PEG biotin and thus the streptavidin bound to it, the amount of bound anti-R-IgG biotin decreases again.

The decrease in the binding capacity for anti-R-IgG biotin can be explained by steric hindrance of the available binding sites on streptavidin. It should also be taken into account that the anti-R-IgG-biotin molecule with a size similar to that of anti-R-IgG (from 14.3 nm x 5.9 nm x 13.1 nm (HD Kratzin, W. Plam, M. Stangel, WE Schmidt, J. Friedrich, N. Hilschmann, Biol. Chem. HS 370 (1989) 263 - 272)) occupy an approximately 2.5 times larger footprint than streptavidin if one has a "foot print" of size 14.3 nm x 5.9 nm.

To test the hypothesis of a decrease in binding capacity due to steric hindrance at a high density of relatively large surface-bound recognition elements, the smaller protein BSA-biotin (molecular weight approx. 150,000) is used instead of anti-R-IgG-biotin (molecular weight approx. 150,000). 50,000), followed by the delivery of the anti-BSA antibody in the subsequent step.

3 shows the results for the sequential adsorption or binding of streptavidin, BSA-biotin and anti-BSA to the support surface covered with mixed polymer layers shown: The amount of bound BSA biotin increases continuously with the amount of surface-immobilized biotin, and clearly above the value at which the maximum was achieved for the streptavidin / anti-E-IgG-biotin system. Up to surface concentrations of 2.77 pmol / cm ² streptavidin no maximum value of the bound BSA-biotin (approx. 0.7 pmol / cm ² at 2.77 pmol / cm ² streptavidin or 11.6 pmol / cm ² surface-bound PEG / biotins) is reached.

In another experiment, pure PPB50 is immobilized on the carrier surface. In the further corresponding assay steps, 6.65 pmol / cm ² streptavidin bind to the surface, but only 0.12 pmol / cm ² BSA-biotin, from which it can be concluded that the immobilization density of the recognition elements also has an optimal value for this smaller molecule but shifted is shifted to higher values of surface-bound streptavidins (between 2.77 pmol / cm ² and 6.65 pmol / cm ² ).

In a last assay step, the “chip” previously brought into contact with anti-R-IgG-biotin is brought into contact with R-IgG as analyte, followed by rinsing with buffer. As can be seen from FIG. 4, the binding behavior for R-IgG very closely follows the trend of the binding curve of anti-R-IgG-biotin, as described above for the binding of anti-R-IgG-biotin.

Claims

claims

1. detection surface with an optimal (in terms of area) binding capacity for the detection and binding of one or more analytes from one or more samples brought into contact with this surface, characterized in that a) said detection surface is a mixture of specific biological or biochemical or synthetic Detection elements for the detection and binding of said analytes with components which are "neutral" with respect to these analytes, ie components which do not bind these analytes, and b) contain said specific detection elements, based on the entire detection surface or any partial area thereof, less than a complete monolayer.

2. Detection surface according to claim 1, characterized in that said specific detection elements, based on the entire detection surface or any partial area thereof, form a tenth to half of a complete monolayer.

3. Detection surface according to one of claims 1-2, characterized in that said specific detection elements and components "neutral" with respect to the analytes, based on the entire detection surface or any partial area thereof, together form at least two thirds of a complete monolayer.

4. Structured detection surface with an optimal (in terms of area) binding capacity for the detection and binding of one or more analytes from one or more samples brought into contact with this surface, characterized in that a) said detection surface in discrete, spatially separated measurement areas, a Mixture of specific biological or biochemical or synthetic recognition elements for the recognition and binding of said analytes with components which are “neutral” with respect to these analytes, ie components that do not bind these analytes, and b) said specific recognition elements, based on the area of the discrete measurement areas, less than a complete one Monolayer, ingest.

5. Structured recognition surface according to claim 4, characterized in that said specific recognition elements, based on the entire recognition surface or any partial area thereof, form a tenth to a half of a complete monolayer.

6. Structured recognition surface according to one of claims 4-5, characterized in that said specific recognition elements and components “neutral” with respect to the analytes, based on the entire recognition surface or any partial area thereof, together form at least two thirds of a complete monolayer.

7. Structured detection surface according to one of claims 4 - 6, characterized in that up to 1,000,000 measuring ranges are arranged in a 2-dimensional arrangement and a single measuring range occupies an area of 10 ^"4 mm ² - 10 mm ²

8. Structured detection surface according to one of claims 4-7, characterized in that the measuring areas are arranged in a density of more than 10, preferably more than 100, particularly preferably more than 1000 measuring areas per square centimeter.

9. Structured detection surface according to one of claims 4-8, characterized in that discrete (spatially separated) measurement areas, as part of this detection surface, by spatially selective application of biological or biochemical or synthetic detection elements on a surface of a carrier or on an additional one Carrier surface applied adhesion promoting layer are generated, preferably using one or more methods from the group of methods by "inkjet spotting", mechanical spotting by means of pen, spring or capillary, "micro contact printing", fluidic contacting of the measurement areas with the biological or biochemical or synthetic recognition elements by supplying them in parallel or crossed microchannels, under the influence of pressure differences or electrical or electromagnetic potentials as well as photochemical or photolithographic immobilization gsverfahren is formed.

10. Structured detection surface according to one of claims 4-9, characterized in that areas between the spatially separated measuring areas to minimize non-specific binding of analytes or their detection substances "passivate" ", ie" chemically neutral "compounds are applied between the spatially separated measuring areas with respect to the analyte or one of its detection substances, preferably consisting, for example, of the groups consisting of albumins, in particular bovine serum albumin or human serum albumin, casein, non-specific, polyclonal or monoclonal, Antibodies of a different kind or empirically unspecific for the analyte (s) to be detected (in particular for immunoassays), detergents - such as Tween 20 - that do not hybridize with the polynucleotides to be analyzed, fragmented natural or synthetic DNA, such as an extract of herring or salmon sperm (especially for polynucleotide Hybridization assays), or also uncharged, but hydrophilic polymers, such as polyethylene glycols or dextrans, are formed.

11. Detection surface according to one of claims 1-10, characterized in that said biological or biochemical or synthetic recognition elements are selected from the group consisting of proteins, for example mono- or polyclonal antibodies and antibody fragments, peptides, enzymes, aptamers, synthetic peptide structures, Glycopeptides, oligosaccharides, lectins, antigens for antibodies (eg biotin for streptavidin), with additional binding sites functionalized proteins ("tag proteins", such as "histidine tag proteins") "and their complexing partners is formed.

12. Detection surface according to one of claims 1-10, characterized in that said biological or biochemical or synthetic recognition elements are selected from the group consisting of nucleic acids (for example DNA, RNA, oligonucleotides) and nucleic acid analogs (for example PNA) and their Derivatives with artificial bases is formed.

13. Detection surface according to one of claims 1-10, characterized in that said biological or biochemical or synthetic recognition elements are selected from the group consisting of soluble, membrane-bound proteins and isolated from a membrane, such as receptors and their ligands.

14. Detection surface according to one of claims 1-13, characterized in that said “neutral” components which do not bind the analyte or analytes are selected from the groups which contain albumin, in particular bovine serum albumin or human serum albumin, casein, non-specific, polyclonal or monoclonal, non-specific or empirically unspecific antibodies (in particular for immunoassays) for the analyte or analytes to be detected, detergents such as Tween 20 hybridizing, fragmented natural or synthetic DNA, such as an extract of herring or salmon sperm (in particular for polynucleotide hybridization assays), or also uncharged but hydrophilic polymers, such as polyethylene glycols or dextrans.

15. Detection surface according to one of claims 1-13, characterized in that said recognition elements are bound to the free end or near the free end of a fully or partially functionalized, "non-interactive" polymer, said "non-interactive" polymer as a side chain a charged, polyionic polymer is bonded as the main chain and together forms a polyionic, multifunctional co-polymer.

16. Detection surface according to claim 15, characterized in that the polyionic polymer main chain is cationically (positively) charged at approximately neutral pH.

17. Detection surface according to claim 16, characterized in that the cationic main chain selected from the group of polymers comprising amino acids with positive charge at approximately neutral pH, polysaccharides, polyamines, polymers of quaternary amines and charged synthetic polymers.

18. Detection surface according to claim 17, characterized in that the cationic polymer main chain comprises one or more molecular groups from the group comprising lsysine, histidine, arginine, chitosan, partially deacetylated chitin, amine-containing derivatives of neutral polysaccharides, polyaminostyrene, polyamine acrylates, polyamine methacrylates, polyethyleneimines , Polyaminoethylene, polyaminostyrenes and their N-alkyl derivatives.

19. Detection surface according to claim 15, characterized in that the polyionic polymer main chain is anionically (negatively) charged at approximately neutral pH.

20. Detection surface according to claim 19, characterized in that the cationic main chain selected from the group of polymers comprising amino acids with attached groups with negative charge at approximately neutral pH, polysaccharides and charged synthetic polymers with negatively charged groups.

21. Detection surface according to claim 20, characterized in that the cationic polymer main chain comprises one or more molecular groups from the group comprising polyaspartic acid, polyglutamic acid, alginic acid or its derivatives, pectin, hyaluronic acid, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, dextran sulfate, polymethyl methacrylic acid Cellulose, carboxymethylated cellulose, maleic acid and fumaric acid.

22. Detection surface according to one of claims 15-21, characterized in that the “non-interactive” polymer is selected as the side chain from the group comprising poly (alkylene glycols), poly (alkylene oxides), neutral water-soluble polysaccharides, polyvinyl alcohols, poly-N-vinylpyrrolidone , Phosphorylcholine derivatives, non-cationic poly (meth) acrylates and their combinations.

23. Detection surface according to one of claims 15-21, characterized in that the biological or biochemical or synthetic recognition elements are bound to the “non-interactive” side chain at its free end or close to its free end via reactive groups.

24. Detection surface according to claim 23, characterized in that said reactive groups are selected from the group comprising hydroxy (-OH), carboxy (-COOH), ester (-COOR), thiols (-SH), N-hydroxysuccinimide, maleimidyl, Quinone, vinyl sulfone, nitrilotriacetic acid ("nitrilo triacetic acid", NTA) and their combinations.

25. Detection surface according to one of claims 1-24, characterized in that it is applied to an essentially optically transparent carrier.

26. Detection surface according to claim 25, characterized in that the essentially optically transparent support comprises a material from the group comprising form-, sprayable or millable plastics, metals, metal oxides, silicates, such as, for. B. glass, quartz or ceramics.

27. Detection surface according to one of claims 25-26, characterized in that it is applied to an adhesion-promoting layer applied to the essentially optically transparent carrier, which is also essentially optically transparent.

28. Detection surface according to claim 27, characterized in that the adhesion-promoting layer has a thickness of less than 200 nm, preferably less than 20 nm.

29. Detection surface according to one of claims 27-28, characterized in that the adhesion-promoting layer comprises a chemical compound from the group consisting of silanes, functionalized silanes, epoxies, functionalized, charged or polar polymers and "self-organized passive or functionalized mono- or multilayers".

30. Detection surface according to one of claims 25-29, characterized in that recesses for the production of sample containers are structured in the surface of said carrier.

31. Detection surface according to claim 30, characterized in that said recesses have a depth of 20 μm to 500 μm, particularly preferably from 50 μm to 300 μm.

32. Detection surface according to one of claims 25 to 31, characterized in that the essentially optically transparent support comprises a continuous optical waveguide or divided into individual waveguiding regions.

33. Detection surface according to claim 32, characterized in that the optical waveguide is an optical layer waveguide with a first essentially optically transparent layer (a) facing the detection surface on a second, essentially optically transparent layer (b) with a lower refractive index than Layer (a).

34. Detection surface according to claim 33, characterized in that said optical layer waveguide is substantially planar.

35. Detection surface according to one of claims 32 - 34, characterized in that, for coupling excitation light into the optically transparent layer (a), this layer is in optical contact with one or more optical coupling elements from the group consisting of prism couplers, evanescent Couplers with matched optical waveguides with overlapping evanescent fields, end face couplers with focusing lenses, preferably cylindrical lenses, arranged in front of an end face of the waveguiding layer, and grating couplers are formed.

36. Detection surface according to claim 35, characterized in that the coupling of excitation light into the optically transparent layer (a) takes place with the aid of one or more grating structures (c) which are pronounced in the optically transparent layer (a).

37. Detection surface according to claim 35, characterized in that the coupling out of light guided in the optically transparent layer (a) takes place with the aid of one or more grating structures (c ') which are pronounced in the optically transparent layer (a) and are the same or have different period and lattice depth like lattice structures (c).

38. Method for the qualitative and / or quantitative detection of one or more analytes in one or more samples, characterized in that said samples and optionally further reagents are brought into contact with a detection surface according to one of claims 1-37 and from the binding of the analyte or other changes in optical or electronic signals resulting from the detection substances used for analyte detection.

39. Method for the qualitative and / or quantitative detection of one or more analytes in one or more samples, characterized in that said samples and optionally further reagents with a structured detection surface according to a of claims 4 - 37 are brought into contact and changes resulting from the binding of the analyte or other detection substances used for the detection of analytes of optical or electronic signals emanating from the discrete measuring ranges are measured in a spatially resolved manner.

40. The method according to any one of claims 38-39, characterized in that the one or more samples are preincubated with a mixture of the various detection reagents for determining the analytes to be detected in said samples and these mixtures are then each in a single addition step with a detection surface one of claims 1-37 are brought into contact.

41. The method according to any one of claims 38-40, characterized in that the detection of the one or more analytes is based on the determination of the change in one or more luminescences.

42. The method according to any one of claims 38-41, characterized in that the excitation light is irradiated by one or more light sources in an incident light excitation arrangement.

43. The method according to any one of claims 38-41, characterized in that the excitation light is irradiated by one or more light sources in a transmission light excitation arrangement.

44. The method according to any one of claims 38-41, characterized in that the detection surface, optionally mediated via an adhesion promoting layer, is arranged on an optical waveguide, which is preferably essentially planar, that the one or more samples with the one or more analytes to be detected therein and, if appropriate, further detection reagents are brought into contact with said detection surface sequentially or after mixing with said samples in a single step and that the excitation light is coupled into the optical waveguide from one or more light sources using one or more optical coupling elements from the group, those of prism couplers, evanescent couplers with matched optical waveguides with overlapping evanescent fields, end face couplers with one in front The end face of the waveguiding layer arranged focusing lenses, preferably cylindrical lenses, and grating couplers is formed.

45. The method according to claim 44, characterized in that the detection of the one or more analytes on a detection surface above a grating structure (c) or (c ') formed in the layer (a) of an optical layer waveguide on the basis of the binding of the analyte and / or further detection reagents, on the immobilized biological or biochemical or synthetic recognition elements, resulting changes in the resonance conditions for coupling an excitation light into the layer (a) of a carrier designed as a layer waveguide or for coupling out light carried in the layer (a).

46. The method according to claim 44, characterized in that said optical waveguide is designed as an optical layer waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower refractive index than layer (a), that further using excitation light one or more lattice structures, which are pronounced in the optically transparent layer (a), are coupled into the optically transparent layer (a) and guided to the measuring areas (d) located thereon as a guided wave, and that the guided wave in the evanescent field continues generated luminescence of luminescent molecules is detected with one or more detectors and the concentration of one or more analytes is determined from the intensity of these luminescence signals.

47. The method according to claim 46, characterized in that (1) the isotropically emitted luminescence or (2) coupled into the optically transparent layer (a) and via a lattice structure (c) or (c ') coupled luminescence or luminescence of both components ( 1) and (2) can be measured simultaneously.

48. The method according to any one of claims 46-47, characterized in that a luminescent dye or luminescent nanoparticle is used as luminescent label to generate the luminescence, which can be excited and emitted at a wavelength between 300 nm and 1100 nm.

49. The method according to claim 48, characterized in that the luminescence label on the analyte or in a competitive assay on an analog of the analyte or in a multi-stage assay on one of the binding partners of the immobilized biological or biochemical or synthetic recognition elements or on the biological or biochemical or synthetic recognition elements is bound.

50. The method according to any one of claims 48-49, characterized in that a second or further luminescence label with the same or different excitation wavelength as the first luminescence label and the same or different emission wavelength is used.

51. The method according to claim 50, characterized in that the second or still further luminescent label can be excited at the same wavelength as the first luminescent dye, but emit at other wavelengths.

52. The method according to claim 51, characterized in that the excitation spectra and emission spectra of the luminescent dyes used overlap only slightly or not at all.

53. The method according to claim 52, characterized in that charge or optical energy transfer from a first luminescent dye serving as a donor to a second luminescent dye serving as an acceptor is used to detect the analyte.

54. The method according to any one of claims 46-53, characterized in that in addition to the determination of one or more luminescences, changes in the effective refractive index on the measurement areas are determined.

55. The method according to any one of claims 46-54, characterized in that the one or more luminescences and / or determinations of light signals are carried out polarization-selectively at the excitation wavelength.

56. The method according to any one of claims 46-55, characterized in that the one or more luminescences are measured at a different polarization than that of the excitation light.

57. The method according to any one of claims 38 - 56 for the simultaneous or sequential, quantitative or qualitative determination of one or more analytes from the group of proteins, such as antibodies or antigens, receptors or ligands, chelators, with additional binding sites functionalized proteins ("Tag Proteins ", such as" histidine tag proteins ") and their complex formation partners, oligonucleotides, DNA or RNA strands, DNA or RNA analogs, enzymes, enzyme cofactors or inhibitors, lectins and carbohydrates.

58. The method according to any one of claims 38-57, characterized in that the samples to be examined are aqueous solutions, in particular buffer solutions or naturally occurring body fluids such as blood, serum, plasma, lymph or urine or tissue fluids or egg yolk.

59. The method according to any one of claims 38-57, characterized in that the sample to be examined is an optically cloudy liquid, surface water, a soil or plant extract, a bio- or synthesis process broth.

60. The method according to any one of claims 38-57, characterized in that the samples to be examined are prepared from biological tissue parts or cell cultures.

61. Use of a detection surface according to one of claims 1-37 or a method according to one of claims 38-60 for quantitative or qualitative analyzes for the determination of chemical, biochemical or biological analytes in screening methods in pharmaceutical research, combinatorial chemistry, clinical and preclinical development , for real-time binding studies and for determining kinetic parameters in affinity screening and in research, for qualitative and quantitative analyte determinations, in particular for DNA and RNA analysis and for determining genomic or proteomic differences in the genome, such as single nucleotide polymorphisms, for measuring Protein-DNA interactions, for Determination of control mechanisms for m-RNA expression and for protein (bio) synthesis, for the preparation of toxicity studies and for the determination of expression profiles, in particular for the determination of biological and chemical marker substances, such as mRNA, proteins, peptides or low-molecular organic ( Messenger) substances, as well as for the detection of antibodies, antigens, pathogens or bacteria in pharmaceutical product research and development, human and veterinary diagnostics, agrochemical product research and development, symptomatic and presymptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for therapeutic drug selection, for the detection of pathogens, pollutants and pathogens, in particular salmonella, prions, viruses and bacteria, especially in food and environmental analysis.