DE102006051482A1 - Arrangement for detecting substances, manufacturing the arrangement and its use - Google Patents

Abstract

The invention relates to an arrangement and its use for the specific detection of substances from samples, in particular a biochip, based on a substrate and a bonding layer located on the substrate. The arrangement has a carrier substrate and a bonding layer formed on the carrier substrate and a test substance coupled to the bonding layer, wherein the carrier substrate is a semiconductor substrate and the attachment layer is at least one inorganic insulator layer formed on the semiconductor substrate and at least one on and / or in the insulator layer Area is acted upon with at least one medium and at least one organic test substance and in the at least one applied area between the insulator layer and the at least one test substance is a coupling and coupled to the insulator layer test substance is adapted to interact with a test substance. The invention further relates to a method for the production of the arrangement in which at least a portion of an inorganic insulator layer formed on a semiconductor substrate is subjected to at least one medium and at least one organic test substance and to it due to interactions between the organic test substance and the inorganic insulator layer a coupling between the organic test substance and the inorganic insulator layer comes and the area is formed, ...

Description

The The invention relates to an arrangement for the specific detection of Substances from samples, in particular a biochip, based on a Substrate and a bonding layer located on the substrate, a procedure for the manufacture of the arrangement and its use, in particular for Capture association events.

application areas of the invention are, inter alia, environmental technology, food technology, the pharmaceutical industry and the life sciences: the Biology, biochemistry, biotechnology, medicine, and the Medical technology.

In Nature finds interactions between molecules or small particles instead of using conventional Microscopes are not observed. Despite their small size, these can Substances affect the health and well-being of humans take, for. In the case of infection with a virus or the mutation of a body's own Gene or the resulting gene product. The presence of such substances can with Help of a biologically active arrangement detected or neutralized become.

biological active arrangements consisting of support materials and applied / immobilized thereon organic compounds, in particular biologically active substances, comes in today's biotechnology and the so-called life sciences an outstanding importance too. Purpose of such carriers, the mostly based on non-water-soluble inorganic or organic polymers are formed is generally the local Fixation or immobilization and the enrichment or concentration biologically active substances, for example of detection molecules or of Pharmaceuticals. This also creates surfaces that are suitable for the most diverse Applications, e.g. as detection systems or as a pharmacon supplier, can be used. The state of the art is in particular biochips and functionalized microparticles (bioparticles) that are specific Identify and bind target structures at the molecular level.

One Biochip is generally a rigid carrier, on its surface biological or chemically active substances, in particular molecules, mostly by electrostatic, hydrophobic, hydrophilic and / or covalent bond immobilized to the surface molecules of the carrier are. The task of immobilized biologically or chemically active substances is it that they are the association of bioactive substances from one Sample, e.g. a test solution, with which they are brought into contact. This allows in particular also bioactive substances can be made visible from a solution, if these with the immobilized biologically active substances, e.g. Peptides, proteins or DNA interact or associate.

• 1. einem Träger bzw. Trägersubstrat mit meist planarer/ebener Oberfläche, oft in der Form eines Mikroskop-Objektträgers,
• 2. einer Anbindungsschicht, die sich auf der Trägeroberfläche befindet und
• 3. biologisch oder chemisch aktiven Substanzen bzw. Prüfsubstanzen die an oder in der Anbindungsschicht immobilisiert sind.

A biochip is usually made up of three components:

• 1. a carrier or carrier substrate with a mostly planar / planar surface, often in the form of a microscope slide,
• 2. a bonding layer, which is located on the support surface and
• 3. biologically or chemically active substances or test substances which are immobilized on or in the attachment layer.

purpose the immobilized biologically active substance or substances is then the specific attraction and / or enrichment of bioactive substances or test substances from a test medium, which then on the Carrier surface, mostly through a physically active label, e.g. with a dye or a radioactive isotope, or with the help of a dye or an enzyme-labeled probe, for example a primer or an antibody, detectable are.

was standing The technology is particularly made up of carriers Glass or plastic covered with a metal or metal bonding layer with a bonding layer of surface-bound organic Coated molecules which are the immobilization of proteins, peptides, or oligonucleotides on the surface or nearby the surface allows. The bonding layer is necessary because the conventional support materials, e.g. Glass or plastic, under normal conditions are chemically hardly reactive or inert and therefore no direct and in solution allow permanent attachment of the biologically active substance. The Coating of the carrier with the connection layer is carried out by a usually consuming chemical process, e.g. a silanization of glass in which the Carrier surface covalent with molecules is occupied, which then in a subsequent step immobilization of biologically active substances.

Carriers for the production of so-called 1D biochips (xD = x-dimensional) generally have a bonding layer of surface molecules with free reactive groups, for example amino, epoxy, aldehyde or maleimide groups. It is also possible, for example, a connection or an application of elements, such as gold, for the subsequent immobilization. If a biologically active substance, for example a protein receptor, is applied to the surface thus functionalized, then the substance can be covalently bound to the functionalized (eg glass) via a chemical reaction. Surface when the receptor has a suitable chemically functional group that reacts with a free reactive group of the molecules of the attachment layer and form a covalent bond. It is also customary that the bonding layer is additionally provided with a crosslinker or a polypeptide is attached in order subsequently to immobilize the desired biologically active substance specifically.

Carrier to Production of 2D or 3D biochips have a bonding layer from covalently bound surface molecules, often lipids, longer Alkane derivatives or organic polymers, e.g. formed as hydrogel, the mostly non-covalent immobilization of biologically active Substances, e.g. from receptors over allow hydrophobic and / or electrostatic interaction.

For an overview of common biochips, see the publication Angenendt P. Drug Discov Today. 2005 Apr 1; 10 (7): 503-11 given.

adversely In the prior art is quite common that the common carrier only metallized or wet-chemical must be functionalized with the binding layer, wherein labor-intensive and time-consuming steps, e.g. Reaction and washing steps, necessary are. Should be about a glass chip or particles for a subsequent connection be silanized by peptides or proteins, it will last for several hours in a jar with one Spent silanization solution, by stirring, shake or ultrasound is moved, then washed and in one Oven dried. The movement of the molecules in solution is u.a. important, a uniform connection layer on the support surface at the chemical reaction between the carrier and the dissolved molecules, e.g. Alkoxysilane or chlorosilanes to achieve. For example, biochips or particles with glass as carrier substrate are produced, so due to the conditions in the production of Bonding layer often the use of special glass substrate, e.g. Quartz glass for high temperature resistance or minor impurities required. But also the connection a polymerized layer or polymer layer, e.g. made of (nitro) cellulose, PVDF or a hydrogel requires professional skill and is expensive. Furthermore The bonding layer thus produced is often chemically sensitive and requires special storage of the carrier, e.g. under vacuum or Protective gas. A disadvantage of the common carrier materials is still that their surface properties for many Applications only limited are suitable. The usual Carrier material glass For example, is a solidified melt with a rough surface, the disadvantageous to a uniform Bonding layer and directional binding, e.g. from microarrays, can affect. As Microarray a carrier material is called, on which is a big one Number of biochemical proofs or tests on the narrowest space, usually only fingernail-sized are located. But also biochips immobilized with only a few locally immobilized Tests are known and used in practice, for example so-called "low density" biochips consist of many locally addressed, mostly in circular "spots" on the surface, bound molecules or larger biological Components (e.g., cell organelles or cells). In this case, for example. DNA molecules or proteins selected, which can associate specifically with substances from a test solution, wherein the bound substances are visible on the surface be made. From the resulting pattern of detected Substances on the biochip, e.g. Serum antibodies or tissue DNA, can be such as pathogens, e.g. Viruses, or tissue changes, tumors, for example, in the examined sample.

Of the Biochip sector has a first significant upswing in time of the human genome project. Powered by its impressive Many start-up companies have accelerated this market and results Enter automation of gene analysis. Now, since the human Genes completely sequenced, the focus is on the development of high-performance biochips, the simple readout of biological signals in diagnostics and enable research.

Next There is a great need for classical DNA analyzes Chips, the gene products (proteins or peptides) or whole cells can prove as biological markers. In particular, the routine examination of patients by doctors and medical personnel will by the use of such biochips be relieved in the future as they have the simultaneous examination allow a variety of parameters.

For one Detection of pathogens in blood serum are in particular biochips suitable, providing a simple and defined attachment of peptides or proteins. These have the advantage that they are in principle all possible Make substances visible, with which they interact according to the key-lock principle. This can, for example also made significantly more pathogens visible or detectable become as possible with today's DNA chips.

In practice, however, in particular the application of proteins / peptides and the reading of the current generation of bio-chips proves to be difficult, as the common carrier for DNA (glass chips) for this often insufficient material characteristics ( Sheridan C. Nature Biotech 23 3-4 (2005) ). If, for example, peptides are to be coupled to the surface in a controlled manner, the methods customary for this purpose require a particularly high outlay with low reproducibility.

functionalized Microparticles, e.g. can be formed as nanoparticles allow the sensitive detection, marking and treatment of target structures a biological or biotechnological system. Under the biological System are essentially organisms, cell tissues, cells, Cell components, DNA, RNA, cDNA, mRNA, cRNA, carbohydrate proteins and / or peptides and structures derived therefrom, under the biotechnological system are all possible investigation orders to understand their properties of biological systems or derived structures are detectable.

was standing the technique is a layered structure of functionalized microparticles, which corresponds to the described 3-component structure of a biochip, and that in the 1D, 2D or 3D embodiment depending on the purpose can vary. A typical microparticle for targeted and delayed release of active substances in an organism includes, for example, immobilized Lipids or hydrogels with incorporated active ingredients as well as covalently, via the Attachment layer, bound antibodies to recognize the target structure in the organism.

There the existing arrangements for detecting substances from a Sample, especially biochips, through the multitude of process steps for their preparation, in particular in the activation of the carrier surfaces, e.g. the elaborate ones Process steps in the silanization, in their reproducibility limited are big Need for arrangements that are easy and reliable to manufacture and accurate reproducible results, also as a peptide or protein chip, in their use enable.

task Therefore, it is the object of the present invention to be efficient and easy indicate an arrangement to be produced with outstanding properties, preferably formed as a biochip or functionalized particles and can be used, based on a substrate and a bonding layer, at the to be tested Substances are simply and directly coupled or immobilized, for detecting target substances or test substances from a sample Method for producing the arrangement and its use, in particular for detecting and / or detecting bioactive substances from a Test medium, preferably of DNA, RNA, proteins, peptides, cells, their components and / or viruses from a liquid.

These The object is achieved by an arrangement according to claim 1, a method for the preparation of the arrangement according to claim 8, as well as its use according to claim 17 solved. The other claims are preferred embodiments the invention. The invention in its different embodiments and technical features is according to the claims and realized the description given here.

crux The invention is the completely surprisingly found manufacturability of arrangements for detecting substances from a sample for which no used in the prior art method for activating the support i.e. chemical or metallic modification of the support material with organic molecules or electrically conductive Elements, wherein an organic or metallic binding layer on the carrier is formed, e.g. Silanization or crosslinking to polymers on glass surfaces or depositing a gold layer, are more necessary but the bioactive substance (test substance) to be immobilized directly on a Fixed phase is connected or is.

The basis of the invention is therefore the unexpected finding that, with a suitable choice of carriers and test substances, no silanization or comparable modification for the subsequent immobilization of the test substances is necessary for the production of devices for detecting substances from a sample, and the use of this finding different way. In principle, the invention thus consists in that, instead of the known metallic or organic bonding layers, an inorganic bonding layer is used for the targeted immobilization of substances to be tested or of capture substances. Silanization in the context of the invention is to be understood in particular as meaning the chemical modification of a substrate, eg of glass, with silanes or silane derivatives, for example as in the printing step Halliwell & Cass, A factorial analysis of silanization conditions for the immobilization of oligonucleotides on glass surfaces, Anal Chem. 2001 Jun 1; 73 (11): 2476-83 described.

The arrangement according to the invention for the specific detection of substances from a sample, in particular for detecting association events, comprises a substrate and at least one bonding layer or coupling layer located on the substrate. According to a solution of the object according to the invention, the substrate comprises a semiconductor or semiconductor material, subsequently also a semiconductor substrate (US Pat. 1 Numbers are given here and, where appropriate, for clarity) and the bonding layer is an inorganic layer on the semiconductor substrate, especially one not in water formed under normal conditions, for example at dryness and room temperature, preferably substantially non-electrically conductive, hereinafter also inorganic insulator layer ( 2 ) called. Sub-insulating materials are to be understood in exceptional cases, also conductive materials that produce or include a spatial isolation of the semiconductor substrate.

When Semiconductors are in accordance with the invention in particular all elemental semiconductors, e.g. Silicon or germanium, and all compound semiconductors, e.g. Gallium arsenide, in their various Modifications, e.g. by doping, to understand. The semiconductor substrate can be in a variety of spatial Dimensions may be formed, for example. As a flat chip, voluminous particles or as a solid phase deposited semiconductor film or thin film.

When inorganic insulator layer is in particular the areal Extension of a directly over the semiconductor substrate lying uniform mass of an inorganic Insulator, i. a substantially non-hydrocarbon compounds containing material that inhibits the flow of electrical charges and its electrical conductivity with changing Temperature does not change substantially, understand. Under insulating layer is, however, in exceptional cases also a conductive Layer, which is a spatial Isolation of the semiconductor substrate generates or comprises to understand.

According to another solution of the object according to the invention, however, glass is also suitable as the substrate and the bonding layer is formed as a non-organically modified, ie covalently coated with organic molecules, and not metallically modified, eg coated with a gold, glass surface. The shape of the arrangement according to the invention in its different embodiments, in particular with the features described below and in the claims, is analogous such that instead of the semiconductor substrate ( 1 ) with the at least one inorganic insulator layer formed thereon ( 2 ) a glass substrate (glass substrate ( 1 )) with at least one glass surface layer formed thereon (glass surface layer ( 2 ) is (are) set, wherein the at least one glass surface layer ( 2 ) is not organically or metallically modified, for example, is non-silanized, in particular untreated or unmodified. The features specified above and below for the semiconductor substrate and the inorganic insulator layer apply correspondingly to the glass substrate and the glass surface layer, as well as to the medium ( 4 ) and the test substance coupled to the glass surface layer.

According to a further solution of the object of the invention is as substrate but also a substantially water-insoluble solid or solid, eg plastic, ceramic, stone, wood, cellulose, metal, etc., particularly preferably plastic, suitable, and the bonding layer is as not organically modified, ie covalently coated with organic molecules, and non-metallized, for example, covered with a gold layer, inorganic insulator layer, in particular silicon oxide layer or silicon dioxide layer formed, in particular as a thin film or film with common thin-layer method, eg by vapor deposition or sputtering, is applied to the solid. The shape of the arrangement according to the invention in its different embodiments, in particular with the features described below and in the claims, is analogous such that instead of the semiconductor substrate ( 1 ) with the at least one inorganic insulator layer formed thereon ( 2 ) a solid (solid ( 1 )) with at least one silicon oxide layer formed thereon (silicon oxide layer ( 2 ) is (are) set, wherein the at least one silicon oxide layer ( 2 ) is not metallized and / or organically modified or is, for example non-silanized, in particular untreated or unmodified. The features specified above and below for the semiconductor substrate and the inorganic insulator layer apply correspondingly to the solid and the glass surface layer, as well as to the medium ( 4 ) and the test substance coupled to the glass surface layer. Untreated in the context of the invention are also previously cleaned surfaces, for example, by conventional in semiconductor manufacturing cleaning method, such as cleaning in Caro's acid, ethanol, ultrapure water, especially in the ultrasonic bath to understand.

The arrangement according to the invention is characterized in that on and / or in the inorganic insulator layer ( 2 ) at least one (surface) area or section, subsequently also area ( 3 ), with at least one medium, in particular with a liquid, solid and / or gas, hereinafter also referred to as medium ( 4 ), and with at least one organic, in particular bio-organic, substance to be immobilized, subsequently also test substance ( 5 ) is acted upon or brought into contact, wherein within the at least one acted upon area ( 3 ) between the inorganic insulator layer ( 2 ) and the at least one test substance ( 5 ) there is a connection or coupling without silanized intermediate layer. According to the invention, they are connected to the insulator layer ( 2 ) coupled test substances suitable to interact with one or more test substances from a sample with which the arrangement is possibly brought into contact or is.

Under silanized intermediate layer in the sense In particular, the invention is to be understood as meaning a layer formed on the carrier substrate from silane derivatives, in particular wet-chemical, to which a test substance is bound or coupled, as described, for example, in the document Halliwell & Cass, A factorial analysis of silanization conditions for the immobilization of oligonucleotides on glass surfaces, Anal Chem. 2001 Jun 1; 73 (11): 2476-83 is described.

Under the name "on and / or in the insulator layer " in particular to understand the parts of the inorganic insulator layer, which come into contact with a medium and / or a test substance or interact, ie, for example, the interface the isolate layer to the periphery and / or the medium and / or the test substance accessible molecules near or below the surface the insulator layer. Is the semiconductor substrate, for example, from a porous Material formed, the term "on and / or in" also refers to the spatially within the semiconductor substrate disposed portions of the insulator layer. Under "on and / or in the isolar layer " in certain circumstances but also to understand that the medium and / or the test substance at least partially penetrate into the semiconductor substrate, if it is absorbed by the insulator layer analogously to a sponge.

The arrangement according to the invention has the characteristic property that in the at least one applied area (FIG. 3 ) a stable, direct coupling or attachment without silanized (eg with alkyl silanes) or polymerized or crosslinked (eg polyvinyl, cellulose) intermediate layer between the insulator layer ( 2 ) and the at least one test substance ( 5 ) is present. The arrangement according to the invention is characterized in particular by the fact that the coupling is stable in air and in solution, ie, in particular, that the coupled substance is also after several hours or several days of washing the arrangement or area ( 3 ) with liquid, for example a physiological buffer, still detectable on the insulator layer ( 2 ) is coupled or bound. In particular, it is also advantageous that the test substance remains stable for months and longer in air or vacuum on the inorganic insulator layer. Direct coupled means that the test substance or its molecules are formed by direct contact with the molecules of the inorganic insulator layer in the region (FIG. 3 ) are attached, preferably by interaction of a nucleophilic group of the test substance with the surface molecules of the insulator layer are connected, wherein a covalent or in particular electrostatic compound or interaction is formed or prevails.

The to the insulator layer ( 2 ) coupled test substance ( 5 ) preferably interacts with a test substance, in particular with at least one test substance detectable by radiation and / or ionization, whereby association events are determined by mass spectrometry or by detection of radiation directly on and / or in the insulator layer ( 2 ) preferably in the at least one area ( 3 ) are detectable detectable by means of an external detector. In principle, all possible devices for detecting radiation, eg scanners, imagers, IR ellipsometers, scintigraphs or X-ray devices, in particular for the detection of radiation in the wavelength ranges of visible light, UV light or IR, and all common mass spectrometers, eg MALDI or ESI, but is not limited thereto. An advantage of this is that the arrangement is usually used quickly and easily with existing laboratory equipment and with known measurement methods in diagnostics and research. In particular, all standard methods and methods for the measurement of biochips or other solid-phase assays (for example ELISAs or comparable solid-phase-based assays with dye-labeled molecules) are suitable for measuring the arrangement. For example, all common methods for the detection of enzyme activity are suitable, especially when the enzyme or the enzyme substrate to the applied area ( 3 ) and the assembly is contacted with enzyme substrate or enzyme. If the enzyme substrate is bound to the solid phase or the insulator layer in the applied region, advantageously labeled (eg with a dye or radioactive isotope) enzyme substrate is coupled. But even a detectable change in solution is detectable when the substrate is dissolved and the enzyme is immobilized.

The area ( 3 ), in particular a concentration or amount of test substance suitable for the detection is applied since an advantageous characteristic of the arrangement according to the invention is a concentration-dependent coupling of the test substance to the applied area ( 3 ), ie that with decreasing concentration of the test substance also decreasing detection signals are observed, whereby, for example, also association constants can be determined. At high concentrations, eg> 2 M and 1 M, no concentration-dependent coupling may be observed due to the saturation of the surface with coupled test substance. As a result, concentration-dependent signals or binding data can also be detected if the coupled test substance interacts with the test substance from the solution.

Preferably, for example when the arrangement is designed as a biochip, the on and / or in an area ( 3 ) detectable signals, in particular by radiation and / or by mass spectrometry Characteristics, from on and / or in at least one further area ( 8th ) and / or on and / or in the surrounding area ( 10 ) detectable signals detectable distinguishable, in particular depending on the coupled test substance.

As a semiconductor substrate ( 1 All known solids or materials with semiconductor properties are suitable, for example silicon, titanium oxide or gallium arsenide, in particular in their different, preferably conventional, embodiments, for example various dopings.

Preferably, the semiconductor substrate is ( 1 ) with the insulator layer formed thereon ( 2 ) in the usual dimensions of a biochip or a particle, in particular a bizarre bioparticle, preferably a microparticle or nanoparticle, formed or configured, whereby it is particularly easy to use for common Vefahren for the use of biochips or particles.

In Particularly suitable development of the arrangement according to the invention is the arrangement as a preferably planar biochip and / or as a 1D, 2D or 3D biochip, for the optical or scintigraphic recording of association events by means of an external detector formed.

It is preferred if the semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) is formed in the usual dimensions of a slide or a slide carrier, since it corresponds in size to the standard for biochips. But also the training ien appropriate dimensions with a porous, structured, in particular lithographically structured surface is suitable.

Particularly preferred is the semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) in the dimensions length × width = (7.5 +/- 1) cm × (2.5 cm +/- 0.5 cm, preferably exactly 7.5 cm × 2.5 cm, with the usual deviations in the biochip production, eg. In sawing processes, and / or in a thickness of> 0.3 mm, in particular of 1 mm (+/- 0.7 mm), preferably exactly 1 mm, formed with customary production-related deviations.

Advantageously, the semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) is formed as an oxidized silicon wafer or as a part thereof. Silicon wafers generally have almost perfectly reproducible material properties and also their surface modification, in particular by oxidation, particularly preferably thermal oxidation and / or electrochemical oxidation and / or formation of a natural oxide, is particularly easy and reproducible to produce.

In an advantageous embodiment of the arrangement according to the invention, the semiconductor substrate ( 1 ) is formed as amorphous, polycrystalline silicon or preferably monocrystalline silicon, in particular with a {111} or {100} or {110} preference orientation, particularly preferably with a {111} preference orientation, whereby the carrier material is virtually perfect and reproducible Material is formed. But other crystalline preference orientations of the silicon are suitable.

It is particularly favorable if the semiconductor substrate ( 1 ) is at least partially, in particular continuously and / or uniformly doped, preferably p- and / or n-doped, particularly preferably p-doped, in particular with an electrical resistance of <100 ohm / cm ² , preferably <20 ohm / cm ² , particularly preferably <1 ohm / cm ² , particularly preferably <0.1 ohm / cm ² , since the arrangement thus has particularly favorable properties, for example for the directional coupling of the test substance. As impurities all customary for the doping of semiconductors impurities are suitable for p-doped substrates, in particular boron or phosphorus.

The semiconductor substrate ( 1 ) is preferably formed as a silicon wafer or as a part thereof, in particular as a sawn from a silicon wafer or by other fragmentation, eg by scratching and breaking of the wafer or by means of a liquid jet or a laser chip received. This has, inter alia, the advantageous property that the arrangement can be produced particularly accurately in terms of its material properties or dimensions and thus enables particularly well reproducible detection systems. Arrangements according to the invention which can be used as bioparticles are preferably formed by the mechanical comminution of wafer material, for example by means of a ball mill or tribomechanical methods.

It is particularly suitable if the semiconductor substrate ( 1 ) is formed homogeneously, for example, as a complete, ie within its entire dimensions, uniform silicon material, for example as a consistently not or only with one kind / type of impurities doped silicon chip, which also particularly simple uniform carrier properties of the arrangement, in particular on the entire insulator layer ( 2 ) which is on the homogeneous semiconductor substrate.

Is the semiconductor substrate ( 1 ) formed as a preferably homogeneous semiconductor substrate without doping, the arrangement is particularly favorable for an optical measurement without background or noise used.

For various applications, in particular unpolished semiconductor, in particular silicon um substrates, preferably unpolished silicon wafers or chips formed therefrom, since they allow, for example, a particularly simple and uniform incubation of the assembly surface with a sample, even without the use of a coverslip or other conventional incubation devices.

Is the semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) of an at least partially, preferably formed on at least one side, in particular on the impacted with the test substance page, as a polished silicon wafer or a part thereof, the arrangement is particularly favorable as a biochip with a mirror surface and the associated advantages can be used ,

The inorganic insulator layer ( 2 ) arranged on the semiconductor substrate is preferably formed by chemical reaction from the material of the semiconductor substrate ( 1 ), whereby a particularly ordered and solid bonding layer on the semiconductor substrate ( 1 ) is present.

The inorganic insulator layer ( 2 ) of the erfindunsgemäßen arrangement is preferably as a layer of silicon nitride, silicate, or an inorganic polymer, particularly preferably as silicon dioxide, hereinafter also silicon dioxide layer ( 7 ), in particular with or without inclusions of foreign atoms (doping) formed. This has the advantage that the bonding layer is particularly simple, for example by reaction of a silicon semiconductor substrate ( 1 ) with oxygen or nitrogen, can be produced.

In a particularly favorable development of the arrangement according to the invention, the inorganic insulator layer ( 2 ) as a silicon dioxide layer ( 7 ), in particular in the at least one area ( 3 ; 8th ) is formed as a non-natural, preferably dry and / or thermal oxide or as a dry and / or thermal nitride, whereby particularly advantageous material properties, eg a sponge-like stable structure, of the insulator layer are ensured. But also a Silkat layer ( 7 ), which is produced wet-chemically and preferably subsequently dried and which preferably has a porous structure, is particularly suitable for various applications, or the silicon dioxide layer is advantageously formed as a natural, for example formed in air and / or by contact with distilled water, silicon oxide.

Preferably, the inorganic insulator layer ( 2 ) as a silicon dioxide layer ( 7 ) with a thickness or layer height of> 10 nm, preferably> 30 nm, in particular 100 nm (+/- 70 nm), particularly preferably 100 nm (+/- 10 nm) formed, whereby particularly favorable properties, eg. For the Reflection and amplification of radiation at the boundary layers of the inventive arrangement, are present. Is the semiconductor substrate ( 1 ) are formed from a silicon wafer or from parts thereof, for example with rectangular or square dimensions (chip), so are the oxide thickness and the optical properties of the insulator layer, for example. A blue or red coloration by wavelength-dependent refraction and / or reflection of light in the appropriate wavelength, specifically adjustable, which are advantageous for a variety of applications of the arrangement. For the production of a specific oxide thickness of the insulator layer ( 1 ) on a silicon wafer (semiconductor substrate ( 1 )), in particular electrochemical and preferably thermal oxidation processes are suitable, since by means of which the desired silicon dioxide thickness (oxide thickness) can be produced in a particularly simple and reproducible manner. It is particularly favorable if the semiconductor substrate is formed as a silicon wafer or chip and the insulator layer is produced by conventional thermal oxidation processes.

According to the invention, the region or region of the insulator layer within which the at least one organic test substance (eg catcher molecules) is coupled, for example in different regions, with at least one medium ( 4 ), in particular a liquid, which preferably contains at least one solvent, hereinafter also solvent ( 9 ), or as at least one solvent ( 9 ) is formed. As has been found quite surprisingly, in particular liquid, preferably a combination of an aqueous and an organic solvent, acts as a mediator for the specific and stable coupling of a test substance to the insulator layer according to the invention.

Particularly suitable is the medium ( 4 ) for the application, if there is at least one solvent ( 9 ), which is formed as an aqueous and / or organic solvent, in particular a hydrophilic and / or polar solvent. In particular, water, for example having a neutral pH, is to be understood as aqueous solvent. As an organic solvent ( 9 ) is preferably an alcohol, dialkyl acid, alkyl halide, pyrollidone, furan, DMSO, acetonitrile, acetone or one of their derivatives suitable, in particular ethanol, DMF, dichloromethane, NMP, THF or one of their derivatives.

The medium ( 4 ), which enables the coupling of the test substances according to the invention, preferably comprises water having an acid, neutral or basic pH and / or an organic solvent having an acidic, neutral or basic pH, an electrolyte and / or a buffer or is formed as such. Particularly suitable is a basic, in particular physiological, pH.

The medium is preferably ( 4 ) is formed as a mixture of aqueous and organic solvents, in particular of an aqueous and an organic solvent, in particular as a mixture of an aqueous solution and an organic solvent, preferably with a deficit of organic solvent. Even a percentage only small volume fraction, in particular <10%, preferably <1%, particularly preferably <0.1%, of organic solvents is sufficient, whereby the coupling of particularly sensitive to organic solvents test substances, such as proteins, in a simple manner is guaranteed.

It is particularly favorable if the medium ( 4 ) is at least partially electrically conductive, in particular with a conductance> 0.05 μS / cm, preferably> 20 μS / cm, more preferably> 1 mS / cm, more preferably> 50 mS / cm is formed, in particular if it is a dissolved Solid, preferably the test substance and / or a salt, particularly preferably the test substance and a dissolved salt.

In an advantageous embodiment of the arrangement according to the invention, the at least one medium ( 4 ) and test substance ( 5 ) area ( 3 ) as at least two non-silanized areas / districts ( 3 ; 8th ) is formed on the insulator layer. In particular, at least two regions are to be understood as a plurality of preferably spatially separated regions, which are formed either as a replica, eg several equally acted upon regions, for example two or more at least partially similar arrays, or each with different test substances or with different concentrations of a Prüfsusbtanz acted upon ,

Particularly preferred are the at least two non-silanized regions ( 3 ; 8th ) at least partially from one on and / or in the insulator layer ( 2 ) surrounding area ( 10 ), ie a region of the insulator layer which is preferably not exposed to test substance. This also ensures a simple delineation of the at least two regions as well as a particularly good comparability and distinctness of the radiation emanating from it or its mass spectrometric properties, in particular if the regions ( 3 ; 8th ) are formed as spots, preferably as spatially separated, particularly preferably completely separate, and / or location-addressed spots. In particular, the common definition, as it is used, for example, for spot synthesis or (micro) arrays, is to be understood by "spot".

In a particularly favorable embodiment of the arrangement according to the invention, the at least two regions ( 3 ; 8th ) with at least partially in the at least one medium ( 4 ) dissolved test substance, in particular with a mixture containing aqueous buffer solution, organic solvent and dissolved test substance. This has the advantageous property that the arrangement according to the invention can be produced and used in a particularly simple way, namely in a single process step, eg pipetting or spotting, from a semiconductor substrate and an insulator layer formed on the semiconductor substrate. "Spotting" is to be understood in particular as the location-addressed application of a suitable volume of a liquid or solution to a solid phase, the solution being distributed with a circular or stain-like extent on the surface of the solid phase, in particular also hemispherical or in another form in the Insulator layer penetrates.

In a particularly suitable development of the arrangement according to the invention, the at least two non-silanized regions ( 3 ; 8th ) formed as an array, in particular as a microarray and / or low-density array, whereby a particularly good readability and utilization of the inventive arrangement is possible.

The environment area ( 10 ) comprising at least one and preferably at least two non-silanized regions ( 3 ; 8th ) is particularly preferably formed as silicon dioxide, ie as a substantially untreated region of the insulator layer (FIG. 2 ) or is chemically modified, in particular with organic molecules, except the test substance. Thus, a quick usability or a reduction of unspecific background signals is made particularly easy, preferably when the surrounding area ( 10 ) with blocking reagent and / or a nucleophilic compound, preferably with a thiol, for example mercaptoethanol or cysteine is acted upon.

Especially is suitable for different applications even if the inorganic insulator layer, preferably over their entire areal Expansion, before exposure to medium and test substance is purified, e.g. by bathing in or wetting with an acid or Base, eg Caro'scher Acid (hydrogen peroxide / sulfuric acid mixture), or an organic solvent, e.g. Ethanol, whereby the surrounding area, for example, as a silica with cleaned surface is formed.

In a particularly suitable development of the arrangement according to the invention is the Prüfsub or test substances, in particular as molecules with at least one nitrogen, sulfur, oxygen, phosphorus, halogen, dye or radioactive isotope formed, or includes these, whereby a particularly stable connection or coupling between the insulator layer and the test substance is prepared.

The at least one organic test substance ( 5 ) as at least two different test substances ( 5 ; 11 ), ie that at least two different test substances or at least two different concentrations of a test substance are preferably spatially separated from each other coupled to the insulator layer and / or the test substance or the at least two different test substances each as molecules with at least one, preferably edge or terminally arranged in the molecule, nitrogen, sulfur, oxygen, phosphorus, halogen, dye or radioactive isotope are formed or include these. As a result, the arrangement is particularly advantageous for location-dependent association with and for the detection of several different test substances from a sample suitable or it can be made visible by the test substance, such as an enzyme, changes, eg by enzymatic cleavage, the coupled sample substance, and it is produced a particularly stable and directed coupling of the test substance to the inorganic bonding layer.

In a particularly suitable development of the arrangement according to the invention, the at least one organic test substance ( 5 ), preferably terminal, at least one nucleophilic chemical group or function, in particular a thiol, amino, hydroxyl, carbonyl, cyano, nitrile, Si, Se, He, sulfate, sulfide, alkene or alkyne -, or phosphine group, or a / e by the interaction with the insulator layer resulting r function or residue, whereby the Stabiltät the coupling is particularly well ensured, in particular by a thiol function, for example of a cysteine residue or the thereof By "end-to-end" in the context of the invention, in the case of proteins or peptides, the N-terminus or the C-terminus, preferably the N-terminal or C-terminal amino acid position, is to be understood, in the case of oligonucleotides the 5'- or the 3 "end of the oligonucleotide sequence.

Preferably, the at least one organic test substance ( 5 ) or comprises, as a linker molecule, amino acid, carbohydrate, lipid, dye, drug, biotin, peptide, polypeptide, oligonucleotide, protein, cell compartment, cell or structure derived therefrom, whereby the arrangement is in principle universal for a variety of applications, in particular for detection all possible bioorganic components or structures (test substances) is suitable.

The at least one organic test substance ( 5 ) or DNA, RNA, cDNA, mRNA, cRNA, PNA, receptor, ligand, protein A, protein G, streptavidin, antibody or structure derived therefrom, whereby the specificity of the association or interaction with the bioorganic test substance is particularly good is ensured. Particularly preferred is the test substance ( 5 ) is formed as a synthetic, naturally occurring or recombinantly expressed or obtained by enzymatic, acidic or basic cleavage of polypeptides, preferably as a synthetically produced peptide.

In a particularly advantageous development of the arrangement according to the invention, the at least one organic test substance ( 5 ) as a peptide of preferably L and / or D-amino acid residues, in particular the genetically encoded amino acids and / or their preferably naturally occurring modifications, for example phosphoserine, threonine, or tyrosine, in particular also, for example. Radiolabelled, formed or contains these. Peptides as a test substance generally have the advantage that they are generally easy to produce synthetically and in high purity and, comparable to whole proteins, specific association properties may have, for example, if the peptide is an antibody epitope, preferably N- or C-terminal with a Cysteine residue is formed. But also peptides with at least one non-naturally occurring amino acid residue, for example formed from anthranil or from aminobenzoic acid, are particularly suitable for a wide variety of requirements.

In another advantageous embodiment of the arrangement according to the invention, the at least one organic test substance ( 5 ) are formed as linker molecules with free / unbound chemically reactive groups. As linker molecules are in particular all possible molecules, with the exception of the insulator layer chemically reacting silanes and silane derivatives to understand that have two different, preferably end and / or laterally arranged in the molecule, different chemically reactive groups or functions, wherein the molecules on a Function to the insulator layer ( 2 ) are coupled and possibly functionalized with bioorganic substances on the other function. If the test substance is formed, for example, as cysteine, this is preferably coupled via the thiol or the radical produced from the thiol by interaction with the insulator layer, its side chain to the insulator layer and can be oxidized via a subsequent chemical reaction by means of its amino acid. or carboxyl group to couple another substance or capture molecules, for example, an amino acid residue or a peptide. If the test substance is formed, for example, as cysteine linked to biotin via an amide bond in an N-terminal manner, streptavidin can be coupled to the surface in a subsequent step, or has been coupled, thus allowing, for example, sandwich arrangements (eg insulator layer-biotin-streptavidin biotinylated component) or are formed.

Suitably, the linker molecule or the test substance is at least one crosslinker, which is connected chemically, in particular covalently, with a bioorganic substance, eg an oligonucleotide or a protein, and by means of the other function, in particular a nucleophilic group, preferably a thiol , to the insulator layer ( 2 ) is coupled.

silanes or cross-linked polymers (e.g., PVDF, cellulose) are used as linker molecules in the sense of Invention essentially not suitable because they only with comparatively high effort to produce or usable.

Particularly preferred is the at least one test substance ( 5 ; 8th ) arranged with the majority of their coupled to the surface molecules spatially directed on the insulating layer, that is, that the molecules are uniformly aligned on the surface, or with preferential orientation, in particular depending on the orientation of the semiconductor substrate. If the test substance is formed, for example, as a peptide, protein or oligonucleotide labeled terminally with a nucleophilic group, in particular a thiol, the test substance is in particular via the thiol group to the attachment layer (US Pat. 2 ) and is otherwise freely accessible for interaction with a test substance, preferably perpendicular or at other suitable angles to the insulator surface.

are the test substances as several peptides or several other synthetically produced molecules in particular oligonucleotides formed, these are preferred uniform with a preferably terminal or pendent nucleophilic Group, e.g. designed a thiol, which in particular also for several at least partially similar test substances, e.g. Wild-type sequence and mutant, a particularly uniform and directional connection and ensures good comparability of results is.

One Another aspect of the invention relates to a simple and efficient Method of making a device for specific detection of substances from a sample, in particular for producing a An arrangement which a carrier substrate and one on the carrier substrate formed bonding layer and coupled to the bonding layer test substance comprises, preferably for producing the inventive arrangement. In the work that led to the invention, it was completely surprising found that the impingement on a semiconductor substrate formed inorganic insulator layer with a medium and a test substance for the stable and directed connection of the test substance to the insulator layer leads. This is especially true for oxidized, non-silanized silicon surfaces.

According to the process of the invention, at least one, in particular non-silanized and not covalently coated with organic molecules and not coated with a metal area ( 3 ) one on a semiconductor substrate ( 1 ) formed inorganic insulator layer ( 2 ) with at least one medium ( 4 ), in particular with a gas, solid and / or preferably liquid, and with at least one organic test substance ( 5 ), in particular with a nucleophilic group, for example a thiol, comprising bioorganic substance, or brought into contact, whereby as a result of interactions between the organic test substance ( 5 ) and the inorganic insulator layer ( 2 ), in particular in air and in liquids stable, coupling or connection between the organic test substance ( 5 ) and the inorganic insulator layer ( 2 ), and the organic test substance ( 5 ) within the affected area ( 3 ), in particular without silanized and / or without organic and / or without metallic intermediate layer, to the and / or in the insulator layer ( 2 ) is attached or immobilized, and is useful for a detectable specific association with a test substance.

The Form or expansion of the insulator layer on the surface of the Semiconductor substrate can, as needed, different be, and is, for example, as a closed layer, which in particular enclosing the entire semiconductor substrate or only a part thereof, in particular only one side of it, or is as individual areas, eg. B. by means Lithography, formed.

An air-stable coupling or connection is to be understood in particular as meaning that the coupled test substance can still be detected after minutes, hours and / or more than one week, in particular after more than one month, especially after more than one year after production of the arrangement. for example, by incubation with a sample, is coupled to the insulator layer, when the inventively prepared assembly in this time of ambient air under normal conditions, for example at 23 ° C and atmospheric pressure, or vacuum, or inert gas atmosphere, is stored. A stable coupling or attachment in solution is to be understood in particular that the coupled test substance even after more than one hour, especially after more than 12 hours, especially after more than 24 hours, 36 hours, 48 hours or 72 hours detectable, eg by subsequent incubation with a sample, is coupled to the insulator layer, when the inventively prepared assembly at this time in a physiological buffer solution, eg Tris or phosphate buffer, under normal conditions, for example at 23 ° C and atmospheric pressure, is stored. The connection can also be stable under other than normal conditions.

For the application with medium ( 4 ) and test substance ( 5 ) are in principle all standard methods or other known methods suitable by means of which a rigid surface can be brought into contact with a medium and / or a test substance, bswp. by incubating, pipetting or spotting or printing on the surface. If, for example, an arrangement is to be produced which has uniform properties over the entire surface of its at least one insulator layer, ie is evenly coated with the at least one test substance, then the semiconductor substrate with the insulator layer formed thereon, for example in a vessel which can be filled with liquid, incubated with the medium and / or the test substance such that it is preferably wetted completely with the medium and / or the test substance, for example by bathing the substrate with the insulator layer formed thereon in a liquid in which the test substance is at least partially dissolved. If the medium and / or the test substance is to be applied or functionalized selectively to one or more regions on and / or in the inorganic insulator layer, for example for the production of a low-density chip, then the medium and / or the test substance preferably by means of a nozzle or pipetting device, in particular a pipette or a pipetting, applied to the insulating layer, in particular in spatially separated locations. For the production of microarrays, correspondingly cylindrical arrayers, eg contact or piezo printers, are used. For a particularly uniform and reproducible coating or coupling of the insulator layer with different test substances or different concentrations of a test substance, it is also suitable that first the insulator layer over a large area with the at least one medium, eg by bathing or wetting the surface, for example. With an organic Solvent, preferably with an organic solvent and an aqueous solvent, more preferably with an organic solvent and an aqueous solvent and a dissolved solid, which is preferably formed as a common component for buffering aqueous solutions, such as tris or phosphate, or as a salt is and subsequently applied to or in the large area with medium baufschlagten area preferably location-addressed different test substances and / or different concentrations or amounts of a test substance, so that a plurality of differently functionalized districts / areas are formed within the large area.

advantageously, the admission of the insulator layer with medium and / or test substance at all possible Environmental conditions performed are, and is preferably carried out at room temperature, whereby a particularly simple production of the arrangement according to the invention guaranteed is. But also the use of medium and / or test substance with a temperature over and especially below the usual Room temperature, e.g. 23 ° C, is suitable, e.g. a 4 ° C warm solution the test substance, which also sensitive test substances particularly easy to and / or can be coupled in the insulator layer.

The at least one medium is preferably selected such that it consists of molecules and / or dissolved in it Solids is formed, which under the conditions selected in the process can not react chemically with the insulator layer, i. in particular no covalent bond with the inorganic insulator layer enter, i. that as a medium for the application of a silicon dioxide layer e.g. a mixture with an organic solvent, Water and a dissolved Salt or a dissolved one Buffer component selected becomes.

• 1. Die Isolatorschicht wird aufeinanderfolgend mit einem organischen Lösungsmittel, z.B. DMF, einer wässerigen Flüssigkeit, z.B. einer Salzlösung/Puffer, und der Prüfsubstanz, z.B. einem gelösten Peptid, beaufschlagt, vorzugsweise in der genannten Reihenfolge. Das wenigstens eine Medium (4) umfasst dabei drei Medien bzw. Bestandteile, z.B. DMF und Puffer und Lösungsmittel des Peptids oder umfasst zwei Medien, wenn bspw. der Puffer und das Lösungsmittel des Peptids identisch sind, und das Peptid z.B. in den Konzentrationsbereichen 1 μM bis 1 mM gelöst ist.
• 2. Die Isolatorschicht wird mit einem, vorzugsweise bei Raumtemperatur flüchtigen, organischen Lösungsmittel, z.B. DCM, und nachfolgend mit einer wässerigen, d.h. Wasser beinhaltenden, Flüssigkeit, in der die Prüfsubstanz zumindest teilweise gelöst ist, z.B. mit einer gepufferten Lösung eines Proteins, beaufschlagt.
• 3. Die Isolatorschicht wird mit einer Lösung, in der die Prüfsubstanz zumindest teilweise gelöst ist, beaufschlagt. Die Lösung umfasst dabei insbesondere ein wässeriges Lösungsmittel, bevorzugt ein organisches Lösungmittel und ein wässeriges Lösungsmittel, besonders bevorzugt ein organisches Lösungsmittel und ein wässeriges Lösungsmittel und einen darin gelösten Feststoff, der insbesondere als gängige Pufferkomponente, z.B. Tris, oder als Salz, z.B. Natriumchlorid, gebildet ist. Solch ein Einschritt-Verfahren, d.h. die gleichzeitige Beaufschlagung der Isolatorschicht mit Medium (4) und Prüfsubstanz (5) ermöglicht eine besonders einfache, schnelle und zuverlässige Herstellung der Anordnung zum Erfassen von Testsubstanzen.

Depending on the type of arrangement to be produced, the method is preferably carried out with three, two or one method step (s), for example

• 1. The insulator layer is successively charged with an organic solvent, for example DMF, an aqueous liquid, for example a salt solution / buffer, and the test substance, for example a dissolved peptide, preferably in the order mentioned. The at least one medium ( 4 ) comprises three media or components, for example DMF and buffer and solvent of the peptide or comprises two media, if, for example, the buffer and the solvent of the peptide are identical, and the peptide is dissolved, for example in the concentration ranges 1 uM to 1 mM.
• 2. The insulator layer is treated with a, preferably volatile at room temperature, organic solvent, for example DCM, and subsequently with an aqueous, ie water-containing, liquid in which the test substance is at least partially dissolved, for example, with a buffered solution of a protein acted upon.
• 3. The insulator layer is treated with a solution in which the test substance is at least partially dissolved, applied. The solution comprises in particular an aqueous solvent, preferably an organic solvent and an aqueous solvent, more preferably an organic solvent and an aqueous solvent and a solid dissolved therein, in particular as a common buffer component, for example tris, or as a salt, for example sodium chloride formed is. Such a one-step process, ie the simultaneous exposure of the insulator layer with medium ( 4 ) and test substance ( 5 ) allows a particularly simple, fast and reliable production of the arrangement for detecting test substances.

Preferably, within the at least one applied area ( 3 ), a direct coupling, stable in air and in solutions, in particular without a silanized or polymerized or crosslinked (eg polyvinyl, cellulose) intermediate layer, in particular if a semiconductor substrate ( 1 ), an inorganic insulator layer ( 2 ), a medium ( 4 ) and a preferably bioorganic test substance ( 5 ) with the characteristics characterized below. In the implementation of the method according to the invention is obtained or formed by attachment or accumulation of molecules of the test substance to the rigid attachment layer, in particular a coupling or immobilization of the test substance to the insulator layer, which is stable in air and in solution, ie coupled substance even after one or several hours or one or more days of washing the arrangement formed by the method or the area ( 3 ) by incubation with liquid, for example a physiological buffer, still detectable on the insulator layer ( 2 ) is coupled or bound. In particular, it is also advantageous that the test substance remains stable for months and longer in air or vacuum on the inorganic insulator layer, so that particularly good storage properties are ensured. Direct coupled means that the test substance or its molecules are formed by direct contact with the molecules of the inorganic insulator layer in the region (FIG. 3 ), preferably via interaction of a nucleophilic group of the test substance, in particular a thiol, with the surface molecules of the insulator layer, forming a covalent or especially electrostatic compound or interaction, in particular between the nucleophilic group of the test substance and the surface molecules of the insulator layer such that the test substance is stably and detectably coupled to and / or in the insulator layer.

To another solution the task of the invention is accordingly used as substrate glass or selected and as a bonding layer is a not organically modified, and not metallic modified, e.g. covered with a gold layer on the glass surface formed glass or boundary layer selected. Under organically modified is in particular the covalent Occupancy of the glass surface with organic (linker) molecules, For example, silanes or polymerized or crosslinked polymers (Cellulose, PVDF).

The implementation of the method according to the invention in its different embodiments, in particular with the features described below and in the claims, is carried out analogously such that instead of the semiconductor substrate ( 1 ) with the at least one inorganic insulator layer formed thereon ( 2 ) a glass substrate (glass substrate ( 1 )) with at least one glass surface layer formed thereon (glass surface layer ( 2 )), wherein the at least one glass surface layer ( 2 ) is not organically or metallically modified, for example, is non-silanized, in particular untreated or unmodified. The features of the semiconductor substrate and the inorganic insulator layer which are particularly preferred for the method and which are specified above and below apply correspondingly to the glass substrate and the glass surface layer, as well as to the medium ( 4 ) and the test substance to be coupled to the glass surface layer.

According to another solution of the object according to the invention is as a substrate substantially not soluble in water solid or solid, such as a polymer, glass, ceramic, stone, wood, metal, etc., particularly preferably plastic, used or selected and as a bonding layer is a non-organically modified, and not metallically modified, for example, coated with a gold layer, formed on the solid silicon oxide layer, preferably silicon dioxide layer, in particular as a thin film or film with common thin-layer method, eg by vapor deposition or sputtering on the solid is applied, selected. Organically modified is in particular the covalent occupancy of the silicon oxide surface with organic (linker) molecules, for example silanes, or polymerized or crosslinked polymers (eg cellulose, PVDF). The thickness of the deposited layer is particularly preferably formed to be greater than or equal to the wavelength detected for the detection. The features of the semiconductor substrate and the inorganic insulator layer which are particularly preferably selected or preferred for the method apply to the solid and the silicon oxide layer as well as to the medium. 4 ) and the test substance to be coupled to the silicon oxide layer.

The implementation of the method according to the invention in its different embodiments, in particular with the features described below and in the claims, is carried out analogously such that instead of the semiconductor substrate ( 1 ) with the at least one inorganic insulator layer formed thereon ( 2 ) a solid substrate (solid substrate ( 1 )) with at least one silicon oxide layer formed thereon (silicon oxide layer ( 2 )), wherein the at least one silicon oxide layer ( 2 ) is not metallized or not organically modified, eg non-silanized or, especially untreated or unmodified. The features of the semiconductor substrate and the inorganic insulator layer which are particularly preferred for the method and which are particularly preferred for the method apply correspondingly to the solid substrate and the silicon oxide layer, as well as to the medium ( 4 ) and to be coupled to the glass surface layer test substance ( 5 ).

By the implementation of the method according to the invention, the test substance ( 5 ) in particular to the insulator layer ( 2 ) ( 5 ) that it interacts with at least one preferably by radiation and / or ionization detectable test substance, which is optionally applied to the arrangement formed by the method below, whereby association events by mass spectrometry, by thermal desorption or by detection of radiation directly on and / or in the insulator layer ( 2 ) preferably in the at least one area ( 3 ) are detectable detectable by means of a preferably external detector. The association events are indirectly detectable, for example if the test substance is an enzyme that converts the test substance, or the association events are directly detectable, for example by the presence of the test substance after the association. In principle, all possible devices for detecting radiation, eg scanners, (lean, IR ellipsometers, scintigraphs or X-ray devices, and all conventional mass spectrometers, eg MALDI or ESI) are suitable as detectors for detecting the arrangement formed by the method according to the invention As a result, the arrangement formed by the method can generally be examined quickly and easily with already existing laboratory equipment and with known measuring methods in diagnostics and research All the standard methods are for detecting or measuring the arrangement, in particular after incubation with a test substance for the measurement of biochips or other solid-phase-based assays for measuring particles (eg ELISAs or corresponding or comparable solid-phase-based assays with dye-labeled detection molecules, in particular in microtiter plates), for example also all common methods for the detection of E enzyme activity are suitable when the enzyme or enzyme substrate is applied to the applied area ( 3 ), the assembly being contacted with enzyme substrate or enzyme.

The area ( 3 ) is applied in particular with a suitable concentration for the detection or amount of test substance, since an advantageous characteristic of the method according to the invention the formation of a concentration-dependent coupling of the test substance and / or in the insulator layer ( 2 ) within the affected area ( 3 ), ie that as the concentration of the selected test substance decreases, subsequently decreasing detection signals can be observed. As a result, concentration-dependent signals or binding data can also be detected if the coupled test substance optionally subsequently interacts with a test substance from a sample. Advantageously, even low concentrations or quantities of test substance are suitable for producing an arrangement for detecting test substances by means of the method according to the invention. Suitably, the test substance selected is a dissolved test substance which is present in a concentration of <1 M, preferably <100 mM, more preferably <10 mM in solution. In a particularly suitable manner, a concentration of the test substance of <1 mM, preferably <100 μM, more preferably <10 μM is selected or used for the application. However, a solution of the test substance in a concentration of <1 μM, preferably <100 nM, more preferably <10 nM, in particular <1 nM, is advantageously suitable for the application, in particular if the test substance has a high affinity for the test substance, wherein, for example, the association between the test and test substance is designed over an association constant Ka> 10 ⁶ / M under physiological conditions. If, for example, the test substance is very large or particularly active, for example formed as a cell, a concentration of the test substance which is <100 pM, preferably <10 pM, particularly preferably <1 pM, is also suitable.

If the test substance is applied to the bonding layer in the form of a solution or suspension ( 2 ) are applied, so are in particular conventional or common volumes of solution, as used or selected in standard methods or other methods for the production of biochips or bioparticles. If, for example, a microarray is to be produced by means of the method according to the invention, a volume of test substance solution or suspension in the nanoliter range, for example 1 nl or less, is suitable for the production of low-density chips, a volume in the microliter range (eg 1 μl or less).

Conveniently, in the use of the method according to the invention a test substance or a concentration in several, esp especially at least two, preferably three, more preferably more than three, replicas or replicas preferably applied to the insulator layer location-addressed. For this purpose, for example, several preferably equal volumes of a solution with the at least partially dissolved test substance are applied to the insulator layer at different locations / positions or offset, so that a plurality of identically functionalized regions ( 3 ) on or in the rigid connection layer ( 2 ), which allow a particularly good readability of the arrangement, for example by calculating the average value or the standard deviation of signals, as well as a parallelization of detection systems, for example the parallel measurement of different samples, the local or offset, eg as a pattern or regularly, be applied to the biochip according to the invention.

In a particularly advantageous embodiment of the method according to the invention is preferably by location-addressed admission of the insulating layer ( 2 ) with at least two, preferably at least three, more preferably more than three different concentrations of a test substance a dilution series of the coupled test substance on the solid phase. If, for example, a dilution arrangement of a peptide, a protein or an oligonucleotide is to be prepared on the solid phase, a millimolar or micromolar solution, for example with a concentration of 1 mM to 1000 mM, preferably <100 mM, more preferably <10 mM, or at a concentration of 1 nM to 1000 nM, the test substance is chosen as the starting solution, and diluted, for example by means of conventional dilution series, for example in 1: 1, 1: 5 or 1:10 dilution steps, and both the starting solution and the diluted / n Lösunge / n preferably locally addressed, in particular at different locations or multiply in one place, applied to the bonding layer.

For the method according to the invention, for example for producing a biochip, the material parameters (semiconductor substrate ( 1 ), Insulator layer ( 2 ), Medium ( 4 ) and test substances ( 5 )) is preferably chosen such that, in particular after incubation of the assembly thus produced with a sample, the on and / or in the one area ( 3 ) are distinguishable in particular by radiation and / or signals detectable by mass spectrometric parameters of signals which act on and / or in a second area, preferably not or with another test substance or with a different concentration of the test substance (surrounding area ( 10 ) or further area ( 8th )) are possibly detectable.

As a semiconductor substrate ( 1 In principle, all known solids or materials having semiconductor properties are suitable for the process according to the invention, for example silicon, titanium oxide or gallium arsenide, in particular in their different, preferably conventional, embodiments.

Preferably, for the method according to the invention a semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) in the usual dimensions of a biochip or a particle, in particular a common bioparticle, preferably a microparticle or nanoparticle selected, whereby the assembly to be produced is particularly easy to use for common Vefahren for the application of biochips or particles.

In Particularly suitable development is by means of the method according to the invention a preferably planar biochip and / or a 1D, 2D or 3D biochip usable arrangement made of an optical or scintigraphic Detecting association events by means of an external detector allows.

For the process according to the invention, a semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) are selected in the usual dimensions of a slide or a slide carrier insert, whereby an arrangement corresponding in size to the standard for biochip is produced.

Particularly preferred for the method according to the invention is a semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) in the dimensions length × width = (7.5 +/- 1) cm × (2.5 cm +/- 0.5 cm, preferably exactly 7.5 cm × 2.5 cm with the usual deviations in their production , For example, in sawing processes, and / or in a thickness of> 0.3 mm, in particular of 1 mm (+/- 0.7 mm), preferably exactly 1 mm, with the usual production-related deviations chosen, whereby a particularly good handling the arrangement to be produced is ensured.

For the inventive method is used as a semiconductor substrate ( 1 ) particularly preferably a silicon wafer or a part thereof, in particular a sawn from a silicon wafer or by other fragmentation, eg by scratching and breaking of the wafer or by means of a liquid jet or a laser, received chip selected. This has, inter alia, the advantageous property that the arrangement can be produced particularly accurately in terms of its material properties or dimensions and thus enables particularly good reproducibility of the arrangements or detection systems according to the invention. If by means of the method according to the invention arrangements are to be made which can be used as bioparticles, they are preferably obtained by mechanical comminution of wafer material, for example with By means of a ball mill or tribomechanical method, formed particles are selected as a semiconductor substrate, but the production is also possible by other dicing methods.

Advantageously, for the method according to the invention, a semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ), which is formed as an oxidized silicon wafer or as a part thereof. Silicon wafers generally have almost perfectly reproducible material properties and also their surface modification, in particular by oxidation, is particularly simple, homogeneous and reproducible to produce, whereby a particularly good reproducibility of the process or the arrangement and measurement results produced thereby is ensured.

In an advantageous development of the method according to the invention, a semiconductor substrate ( 1 ), which is formed as amorphous, polycrystalline silicon or preferably monocrystalline silicon, in particular with a {111} or {100} or {110} preferred orientation, particularly preferably with a {111} preference orientation, whereby an arrangement based on a nearly perfect and reproducible carrier material is formed, can be produced. However, other preferred crystalline orientations of the silicon material are also suitable for the process according to the invention.

It is particularly favorable if, for the method according to the invention, a semiconductor substrate ( 1 ), which is at least partially, in particular continuously and / or uniformly doped, preferably p- and / or n-doped, is particularly preferably p-doped, in particular with an electrical resistance of <100 ohm / cm ² , preferably < 20 ohms / cm ² , more preferably <1 ohm / cm ² , particularly preferably <0.1 ohm / cm ² , <15 ohms / cm ² , since then the assembly thus produced particularly favorable properties, eg. A directed coupling of the test substance , having. Suitable impurities of the doped material used are all customary for the doping of semiconductors impurities suitable for p-doped substrates, in particular boron, aluminum, indium and / or gallium, for n-doped substrates phosphorus, arsenic and / or antimony. For the process according to the invention, undoped semiconductor substrate, for example silicon, which is preferably particularly pure, for example impurities in the ppm range or below, may also be used, whereby, for example, in optical measurements a particularly favorable signal-to-noise ratio due to low background signals is reachable.

It is particularly suitable if, for the method according to the invention, a semiconductor substrate ( 1 ), which is preferably completely homogeneously formed, for example, a silicon material which is uniform throughout its entire dimensions, for example a silicon chip which is doped throughout or which is doped only with one kind / type of foreign atoms, thereby also resulting in particularly simple uniform carrier properties of the arrangement , in particular over the entire insulator layer ( 2 ) can be produced.

For the production of particularly favorable arrangements be for the inventive method preferably unpolished semiconductor, in particular silicon substrates, preferably unpolished silicon wafers or chips formed therefrom, chosen or used, since they, for example, a particularly simple and uniform incubation the layout surface with a sample, even without using a coverslip or other common Incubation devices.

Is used for the inventive method as a semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) an at least partially, preferably on at least one side, in particular on the impacted with the test substance, polished silicon wafer or a part thereof, or a polished and oxidized silicon wafer or a part thereof, so is advantageously the arrangement can be produced as a biochip with a reflective surface and the associated advantages.

Especially it is also suitable if for the inventive method an inorganic insulator layer is selected, the surface of which before Exposure to medium and test substance cleaned is, e.g. by means of an acid or base and / or an organic solvent, in particular using an ultrasonic bath or a shaker. If, for example, an arrangement with particularly favorable, e.g. levels, surface properties be prepared so as a semiconductor substrate with the on it formed insulator layer etched with hydrofluoric acid and then by means of Carowscher Acid oxidized Selected silicon substrate, which is preferably dried in an oven.

As a bonding layer, which is arranged or formed on the semiconductor substrate, an inorganic insulator layer is preferably selected, which by chemical reaction from the material of the semiconductor substrate ( 1 ) is formed, whereby a particularly ordered and solid bonding layer on the semiconductor substrate ( 1 ) can be used for the inventive method.

As an inorganic insulator layer ( 2 ) for the process according to the invention is preferably a layer of silicon nitride, silicate or, more preferably, of silicon dioxide, hereinafter also referred to as Si silicon dioxide layer ( 7 ), in particular with or without inclusions of impurities (doping). This has the advantage that the bonding layer is particularly simple, for example by reaction of a silicon semiconductor substrate ( 1 ) with oxygen or nitrogen, can be produced.

In a particularly favorable development of the invention, the inorganic insulator layer ( 2 ) a silicon dioxide layer ( 7 ), in particular in the at least one area ( 3 ; 8th ) is formed as a non-natural, preferably dry and / or thermal oxide or as a dry and / or thermal nitride, whereby particularly advantageous properties of the manufacturable by the method according to the invention arrangement, for example by a spongy formed stable structure of the insulator layer, can be ensured. But even a porous inorganic insulator layer, for example, wet-chemically formed on or in the semiconductor substrate and then preferably dried in the oven, is particularly suitable as a bonding layer.

Preferably, an inorganic insulator layer ( 2 ) selected as the silicon dioxide layer ( 7 ) with a thickness or height of> 10 nm, preferably> 30 nm, in particular 100 nm (+/- 70 nm), particularly preferably 100 nm (+/- 10 nm) is formed, whereby particularly favorable properties, for example the reflection and amplification of radiation at the boundary layers, the producible arrangement are present. Is used as a semiconductor substrate ( 1 a silicon wafer or a part thereof, eg with rectangular or square dimensions (chip), the oxide thickness also determines the optical properties of the insulator layer, for example a blue or red coloration by reflection of light in the corresponding wavelength at the silicon. Silicon oxide interface, specifically used, which are advantageous for a variety of applications of the arrangement. For the use of a specific oxide thickness of the insulator layer ( 1 ) on a silicon wafer (semiconductor substrate ( 1 )) are particularly suitable electrochemically and preferably thermally produced oxides, since by means of which the arrangement is particularly simple and reproducible in their properties, in particular the material properties of the bonding layer, produced or usable. It is particularly advantageous if a silicon wafer or chip with a silicon dioxide layer formed by a conventional thermal oxidation method, for example by heating and contacting the wafer with a gas consisting predominantly of oxygen, is selected as the semiconductor substrate with the insulator layer formed thereon. Preferably, however, an electrochemically formed oxide which is preferably dried in an oven is used as the inorganic insulator layer.

In the method according to the invention, the district of the insulator layer within which the organic test substance (eg catcher molecules), eg in different areas, is coupled, is preferably used with at least one medium ( 4 ), in particular a liquid, which preferably contains at least one solvent, hereinafter also solvent ( 9 ), or as at least one solvent ( 9 ) is formed. As has been found quite surprisingly, in particular liquid, preferably a combination of an aqueous and an organic solvent, acts as a mediator for the specific and stable coupling of the test substance to the insulator layer according to the invention.

Particularly suitable for the process according to the invention is a medium ( 4 ) is selected for the application, the at least one liquid solvent ( 9 ), which is formed as an aqueous and / or organic solvent, in particular as a hydrophilic and / or polar solvent. In particular, water, for example having a neutral pH, is to be understood as aqueous solvent. As an organic solvent ( 9 ) is preferably an alcohol, dialkyl acid, alkyl halide, pyrollidone, furan, DMSO, acetonitrile, acetone or one of their derivatives suitable, for example, ethanol, DMF, dichloromethane, NMP, THF or one of their derivatives.

The medium ( 4 ) selected for the process according to the invention preferably comprises or has an acidic or basic pH water and / or an organic acidic or basic organic solvent, an electrolyte and / or a buffer. Particularly suitable is a medium having a basic, in particular physiological, pH value is selected.

Preference is given as medium ( 4 ) a mixture of aqueous and organic solvents, in particular from an aqueous and an organic solvent, in particular selected as a mixture of an aqueous solution and an organic solvent, preferably with a deficit of organic solvent. The use of only a small percentage by volume of the organic solvent in the mixture, in particular <10%, preferably <1%, more preferably <0.1%, is suitably chosen, whereby the coupling of particularly sensitive to organic solvents test substances, eg Proteins or cells, is guaranteed.

It is particularly favorable if, for the process according to the invention, an at least partially electrically conductive medium ( 4 ), in particular with a conductivity> 0.05 μS / cm, preferably> 20 μS / cm, particularly preferably> 1 mS / cm, particularly preferably> 50 mS / cm is selected, in particular, if there is a dissolved solid, preferably the test substance and / or a buffer component and / or a salt, preferably the test substance and a Buffer component, more preferably the test substance and a dissolved salt includes. But even the use of a non-electrically conductive medium may be suitable.

In an advantageous development of the process according to the invention, at least two non-silanized regions or regions (preferably not covalently occupied by organic molecules) are preferably ( 3 ; 8th ) on the insulator layer with at least one medium ( 4 ) and in each case acted upon with at least one test substance in such a manner, for example by pipetting suitable, in particular non-intermeshing (spot) volumes, that at least two identically or in particular differently functionalized areas are formed. In particular, at least two regions are to be understood as a plurality of preferably spatially separated regions which are formed either as a replica, for example several regions which are subjected to the same effect or are respectively exposed to different test substances or to different concentrations of a test substance, with several differently functionalized regions ( 3 ; 8th ) are formed.

Particularly preferably, the application is carried out in such a way, for example by means of pipetting or automated spotting, that at least two non-silanized regions ( 3 ; 8th ) are formed with coupled test substance which at least partially or completely from one on and / or in the insulator layer ( 2 ) surrounding area ( 10 ), ie a region of the insulator layer which is preferably not exposed to test substance, are surrounded. This also makes possible a simple delineation of the at least two regions as well as a particularly good comparability and distinctness of the radiation emanating from it or its mass-spectrometrically detectable properties, in particular if the regions ( 3 ; 8th ) are formed as spots, preferably as spatially separated and / or location-addressed spots. For various applications, it may also be suitable to use an inorganic insulator layer whose surface has a non-closed silane layer and which in particular is not silanized in the areas in which test substance is coupled.

In a particularly favorable development of the method according to the invention, at least two regions ( 3 ; 8th ) each with at least partially in the at least one medium ( 4 ) dissolved test substance, in particular with a mixture containing aqueous buffer solution, organic solvent and dissolved test substance. This has the advantageous property that the method makes it possible in a particularly simple manner, namely in a single process step, to produce an arrangement for the detectable detection of a plurality of association events, from a semiconductor substrate and an insulator layer formed on the semiconductor substrate. In order to enable particularly uniform coupling conditions, the test substances to be coupled are preferably dissolved in the same medium.

In a particularly advantageous development of the process according to the invention, the at least two non-silanized regions ( 3 ; 8th ) as an array, in particular as a microarray and / or low-density array produced, whereby a particularly good readability and utilization of the inventive arrangement is made possible. For the preparation of the arrays according to the invention, all common application techniques, eg pipetting, and customary volumes are suitable, which are used in the conventional exposure of glass slides with sample substances for producing a biochip array, in particular a low-density or microarray.

The environment area ( 10 ), which preferably has at least one and particularly preferably at least two non-silanized regions ( 3 ; 8th ) is preferably formed such that it is not exposed to test substance, and in particular as only with medium ( 4 ) acted upon or preferably untreated silica of the insulator layer ( 2 ) remains. For various applications, however, it is also suitable that the surrounding area ( 10 ) is chemically modified, in particular with organic molecules, except the test substance. Thus, a quick usability or a reduction of non-specific background signals is particularly easy to produce, preferably when the surrounding area ( 10 ) with blocking reagent and / or a nucleophilic compound, preferably with a thiol, for example mercaptoethanol or cysteine is applied. The passivation of the surrounding area ( 10 ) can also be produced, for example, during the subsequent incubation of the inventive arrangement with a sample containing blocking reagent or serum. Under blocking reagent are essentially all commonly used in biotechnology substances for the passivation of surfaces with organic compounds suitable, in particular dissolved milk powder, albumin or protein fragments, with which preferably the test substance is not specifically associated.

In a particularly suitable development of the method according to the invention is / are as In particular, molecules or substances with at least one nitrogen, sulfur, oxygen, phosphorus, halogen, dye and / or radioactive isotope are selected, or the selected test substance comprises these, whereby a particularly stable connection or coupling between the insulator layer and the test substance is possible.

It is particularly preferred for the process according to the invention if, as the at least one organic test substance ( 5 ) at least two different test substances ( 5 ; 11 ), ie that at least two different test substances or at least two different concentrations of one test substance are preferably spatially-addressed or spatially separated from the isolator layer and / or the test substance or the at least two different test substances are each as molecules with at least one, preferably arranged at the edge or terminal in the molecule, nitrogen, sulfur, oxygen, phosphorus, halogen, dye or radioactive isotope are or include. As a result, an arrangement for location-dependent association with and for the detection of several different test substances from a sample is produced in a particularly simple manner or subsequently modifications effected by the test substance, eg an enzyme, eg by enzymatic cleavage, of the coupled test substance can be visualized. and / or it is thereby produced a particularly stable and directed coupling of the test substance to the inorganic bonding layer.

In a particularly suitable development of the process according to the invention, the at least one selected organic test substance ( 5 ), preferably terminal, at least one nucleophilic chemical group or function, in particular a thiol, amino, hydroxyl, carbonyl, cyano, nitrile, Si, Se, He, sulfate, sulfide, alkene or Alkane, or phosphine group, whereby the Stabiltät the coupling is particularly well ensured, in particular by a thiol function, for example of a cysteine residue. In the case of proteins or peptides, in particular the amino terminus or the carboxyl terminus, preferably the N-terminal or C-terminal amino acid position, is termed terminal, for oligonucleotides the 5 '' or the 3 '' end of the oligonucleotide sequence.

Preferably, the at least one organic test substance ( 5 ) selected a biooorganic test substance, which is preferably formed as a linker molecule, amino acid, carbohydrate, lipid, dye, drug, biotin, peptide, polypeptide, oligonucleotide, protein, cell compartment, cell or structure derived therefrom or contains, whereby the arrangement in principle universal for a variety of applications, in particular for detecting all possible bioorganic components or structures (test substances) can be produced. Especially suitable is the use of peptides and / or proteins as test substance (s) ( 5 ), since the method according to the invention enables, inter alia, the production of peptide and / or protein chips with particularly advantageous properties.

Particularly preferred is as the at least one organic test substance ( 5 ) DNA, RNA, cDNA, mRNA, cRNA, PNA, receptor, ligand, protein A, protein G, streptavidin, antibody or structure derived therefrom or the test substance contains these, whereby the specificity of the association or

Interaction with the bioorganic test substance is particularly well ensured. Particular preference is given as the test substance ( 5 ) a synthetic, naturally occurring or recombinantly expressed or obtained by enzymatic, acidic or basic cleavage of polypeptides peptide, preferably used as a synthetically produced peptide.

In a particularly advantageous embodiment of the method according to the invention is as the at least one organic test substance ( 5 ) One or more peptides of preferably L- and / or D-amino acid residues and / or glycine and / or β-alanine, in particular from the genetically encoded amino acids and / or their preferably naturally occurring modifications, for. As phosphoserine, threonine, or tyrosine, in particular also, for example. With a radioactive or dye marking, selected, or the Profubststanz includes these. Peptides as a test substance generally have the advantage that they are generally easy to produce synthetically and in great purity and, comparable to whole proteins, may have specific association properties, eg. For example, when the antibody selected is an antibody epitope N- or C-terminally formed with a cysteine residue. Synthetic, preferably chromatographically purified, peptides are therefore particularly preferably chosen as the test substance to be coupled in for the method according to the invention. However, peptides with at least one non-naturally occurring amino acid residue, for example formed from anthranil- or from aminobenzoic acid, are particularly preferred for a very wide variety of requirements.

In another advantageous embodiment of the method according to the invention, the at least one organic test substance ( 5 ) or selected as linker molecules with free / unbound chemically reactive groups. As Linkermo In particular, molecules are to be understood as meaning all possible molecules, with the exception of silanes, which have two different chemically reactive groups or functions, preferably end and / or side, arranged in the molecule, the molecules having a function on the insulator layer. 2 ) and possibly connected to bioorganic substances via the other function. If, for example, cysteine is chosen as the test substance, it is preferably coupled to the insulator layer via the thiol function of its side chain and can couple via a subsequent chemical reaction by means of its amino or carboxyl group another substance or capture molecules, for example an amino acid residue or a peptide. If a test substance ZB is an N-terminal cysteine linked to biotin via an amide bond, then in a subsequent step streptavidin can be coupled to the surface, whereby, for example, sandwich arrangements (eg insulator layer-biotin-streptavidin-biotinylated component) are possible or formed , If a lipid is selected as the test substance or linker, which preferably comprises a nucleophilic group, preferably thiol, then the area of the insulator layer acted upon with it is functionalized, for example, for the subsequent immobilization of at least partially hydrophobic substances, in particular a transmembrane protein. However, the immobilization on the test substance or the linker can also be carried out chemically, covalently and / or by electrostatic or hydrophilic interactions.

Suitably, at least one crosslinker is selected as the linker molecule or the test substance, which is already chemically, in particular covalently, connected via one of its at least two linker functions with a bioorganic substance, eg an oligonucleotide or a protein, and by means of the other function, in particular a nucleophilic one Group, preferably a thiol, to the insulator layer ( 2 ) is coupled.

silanes or crosslinking polymers (e.g., PVDF, cellulose) forming substances are as linker molecules or test substance not suitable for the purposes of the invention, since they only with comparatively high Expenditure can be used.

The at least one test substance ( 5 ; 8th ) are arranged with the majority of their coupled to the surface molecules spatially directed on the insulating layer, that is, that the molecules are aligned uniformly, in particular with a preferred orientation, on the surface or are coupled. If, for example, a peptide, protein or oligonucleotide labeled terminally with a nucleophilic group, in particular a thiol, is selected as the test substance, then the test dye substance is in particular bound to the attachment layer via the thiol group (US Pat. 2 ) and is otherwise freely accessible for any subsequent interaction with a test substance.

Become preferably peptides or other synthetically produced molecules, in particular Oligonucleotides, as test substance selected so these are preferably uniform with a nucleophilic group, e.g. an end or side Thiol designed, which in particular even with several at least partially similar Test substances, e.g. Wild-type sequence and mutant, a particularly uniform and directional Connection and good comparability of the results is ensured.

Around one for to make the use of a particularly suitable arrangement is after loading the insulator layer with the at least one Medium and the at least one test substance excess medium and / or excess test substance preferably removed by washing from the surface.

In In practice, volumes are applied when the isolator layer is applied chosen the solution containing the test substance, which leads to a rapid evaporation of the solvent under normal conditions, e.g. at room temperature. The excess, uncoupled substances after evaporation of the solvent remain on the assembly are then subsequently washed from the surface, preferably with an aqueous Liquid, preferably a buffer solution, particularly preferably water, removed.

For the production various arrangements, e.g. of biochips with respect to aggregate changes sensitive proteins, the exposure to the test substance takes place preferably below the usual Room temperature, preferably <23 ° C especially preferably <5 ° C, and optionally with solvent additives, the a rapid evaporation of the solvent at least hinder, e.g. Oil, and the surface becomes after the admission with the or the at least partially dissolved Test substance / s and before the full one Evaporation of the solvent washed accordingly.

Even after extensive exposure of the insulator layer, eg by bathing or wetting, with medium ( 4 ) and / or test substance ( 5 ), the surface is advantageously washed accordingly.

For a special easy handling, the arrangement is dried after washing or if necessary kept moist.

A further aspect of the invention relates to the use of the arrangement according to the invention or of the arrangement produced by the method according to the invention for the specific detection of substances from a sample, in particular for the detection of association events. The use according to the invention is characterized in that the arrangement with at least one sample, subsequently sample ( 13 ), which may be a test substance, hereinafter also referred to as test substance ( 14 ).

For the use according to the invention are in principle all standard methods or other methods for Use of a biochip, for example for an immunoassay or gene expression profiling, as well as all usual Methods for the use of bioparticles, e.g. for investigations of Bioparticles for use as diagnostics, therapeutics or cleansers or for the production of a therapeutic agent.

For this purpose, in particular the at least / at least one area ( 3 ) at which the at least one test substance ( 5 ), with the at least one sample ( 13 ), eg by direct application by means of pipetting the sample onto the at least one region or indirect application of the sample in the vicinity of the region, eg in or on the surrounding region ( 10 ), and subsequent distribution or supply to the at least one area ( 3 ). In particular, all standard methods or other methods are suitable for contacting, by means of which a sample to be tested can be placed on a biochip or fed to a solid-phase-bound test substance, for example by means of customary Lab-on-a-Chip methods.

When Specifically, sample is a gas, solid, liquid, and / or mixture used therefrom, more preferably a sample is used, which contains at least one test substance, e.g. an analyte and / or a control, preferably at a detectable concentration.

Particular preference is given as a sample ( 13 ), a solution, emulsion, and / or suspension, which optionally contains microparticles or other undissolved components uses, preferably, at least one at least partially dissolved test substance including.

In an advantageous embodiment, as a sample ( 13 ) Body fluid, preferably urine, blood or serum, mucus tear fluid, sweat, sperm, cerospinal fluid and / or a stool sample is used or the sample contains these.

Particularly preferred is as the at least one sample ( 13 ) at least two different samples ( 13 ; 16 ), which are preferably placed on the surface of the arrangement in a location-addressed manner, in particular in each case on a replica of the coupled test substance (s), whereby different samples can also be examined simultaneously with only one arrangement.

Particularly suitable according to the invention is the use of a sample containing as test substance an amino acid, a carbohydrate, a lipid, a dye, a drug, biotin, a linker molecule, a peptide, an oligonucleotide, a protein, cell tissue, a cell, a cell constituent, DNA , RNA, cDNA, mRNA, cRNA, PNA, a protein and / or a peptide or structures derived therefrom, or the test substance comprises these, eg a bioparticle. Preferably, a sample is used which is used as test substance ( 14 ), an antibody, an enzyme, a receptor, a ligand, a protein domain or an antibody epitope, and particularly preferably as IgA, IgG, IgE, IgM, kinase, phosphatase or antibody epitope.

In a particularly favorable development, a sample is used which contains a test substance ( 14 ), which corresponds to that in the field ( 3 ) without silanized intermediate layer to the insulator layer ( 2 ) coupled test substance ( 5 ) interacts, especially with the Prüfsusbtanz ( 5 ) or enzymatically altered.

Particularly preferred according to the invention is the use of a sample containing a test substance ( 13 ) with at least one on and / or in the at least one area ( 3 ; 8th ) coupled test substance interacts demonstrably.

In a particularly advantageous development, a sample is used which contains a test substance ( 14 ), which is preferably covalently linked to a radiation-active marker, for example a labeled peptide, or subsequently connected, for example by incubation with a labeled secondary antibody.

In one for the evaluation of the arrangement is particularly suitable use after contacting the sample and incubation time, especially longer or> 1 min, preferably> 30 min, especially preferably> 1 h, excess sample material preferably removed by washing from the surface, preferably by Wash with a watery Liquid, preferably with water, more preferably with one, in particular physiological, buffer. For a particularly simple reading is the arrangement after washing dried or kept moist if necessary.

In a particularly favorable development of the use according to the invention, the arrangement, preferably the at least one area ( 3 ; 8th ), after contacting the sample by means of an externally or peripherally arranged detector for the detection of radiation, in particular for the detection of radiation in the wavelength ranges of visible light, UV light or IR or for the detection of radioactive radiation, or for the detection of mass spectrometry measurable products or parameters, in particular by means of a scanner, imager or mass spectrometer read.

Further Features and advantageous features of the invention are also from the examples given below.

In 1 schematically show A and C an example of the prior art and B and D an example of the arrangement according to the invention (oblique view from above, section of a biochip, for a microparticle, the surface is fully / consistently occupied by a test substance).

1A On the surface of a carrier substrate (a) of a solidified melt (glass) is a silanization deposited two-component layer (b and c) of a silane derivative, which is connected via the molecular silicon (b) to the carrier substrate and the organic part ( c) the silane derivative which has a chemical function or group to which one or more test substances (d) are attached in the form of an array in a subsequent step. The areas (spots) with test substances are surrounded by the organic layer (c), whose reactive groups are subsequently blocked.

1B On an semiconductor substrate (s) having the features according to the invention, eg on a part of a silicon wafer, an inorganic insulator layer (f) according to the invention, eg a silicon dioxide layer, is arranged on or in the surface thereof by direct interaction of the surface molecules of the insulator layer with one another chemical function or group (except silanes and their derivatives) of a test substance, eg with a thiol, one or more test substances (g) according to the invention, eg peptide / s with a preferably terminal cysteine residue, are coupled, eg as an array of different test substances and / or different amounts of a test substance, preferably as a replica. By way of example, on the left side, a replica of two identical spots of a test substance is located, on the right side a spot with a lower concentration of the same test substance (front) and a spot of a second test substance (rear) is arranged. The areas (spots) with test substances are surrounded by the inorganic insulator layer, which is optionally blocked subsequently, for example with a thiol or blocking reagent or during the use according to the invention, for example by bringing it into contact with body fluid.

1C : Schematic cross section through the arrangement ( 1A ) in the area of the bound test substance (s). The arrangement comprises a large-area organic layer of silane derivative (c) to which the test substances (d) are attached in a localized manner. Since the organic layer (c) usually has hydrocarbon compounds, interfering interactions between the organic layer (c) and the test substance (s) may occur.

1D : Schematic cross section through the arrangement according to the invention ( 1B ) in the range of the coupled test substances. The test substances or the different lateral concentration of a test substance adhere directly to the surface molecules of the inorganic insulator layer (f) on or in the inorganic insulator layer (h).

In 2 A and D show an example of the inventive arrangement with glass as the carrier substrate according to the further solution of the object according to the invention, B and E an example of a particular embodiment of the invention, and C and F each an example of the inventive arrangement with a non-water soluble solid as a substrate according to the other solution of the object of the invention.

2A On a glass substrate (a) having the features according to the invention, eg on a glass chip, a glass surface layer (b) according to the invention, which is not organically or metallically modified, is arranged on or in the surface thereof by direct interaction of the surface molecules of the insulator layer with one another chemical function or group (except silanes and their derivatives) of a test substance, eg with a thiol, one or more test substances (c) according to the invention, eg peptide / s with a preferably terminal cysteine residue, are coupled, eg as an array of different test substances and / or different amounts of a test substance, preferably as a replica. By way of example, on the left side, a replica of two identical spots of a test substance is located, on the right side a spot with a lower concentration of the same test substance (front) and a spot of a second test substance (rear) is arranged. The areas (spots) with test substances are surrounded by the glass surface layer (b), which may be subsequently, for example with a thiol or blocking reagent or blocked in the use according to the invention, for example by bringing it into contact with body fluid.

2 B On a solid (d), which is formed as a solid according to the further solution of the object according to the invention, in particular with the inventive features, for example on a plastic, silicon (e), for example by vapor deposition or sputtering of the solid surface with a silane, deposited , which is oxidized in the region of its surface, for example present as a natural oxide, so that on the solid a silicon layer (f) and a silicon dioxide layer (g) located thereon is arranged on or in the surface thereof by direct interaction of the surface molecules of the insulator layer a chemical function or group (except silanes and their derivatives) of a test substance, eg with a thiol, one or more test substance (s) (h) according to the invention, eg peptide / s with a preferably terminal cysteine residue, are coupled, eg as array different Test substances and / or different amounts of a test substance, preferably as a replica. By way of example, on the left side, a replica of two identical spots of a test substance is located, on the right side a spot with a lower concentration of the same test substance (front) and a spot of a second test substance (rear) is arranged. The areas (spots) with test substance (s) are surrounded by the silicon dioxide layer (g), which is optionally blocked subsequently, for example with a thiol or blocking reagent or during the use according to the invention, for example by bringing it into contact with body fluid.

2C On a solid according to the invention (i), for example on a plastic, a silicon oxide layer (j), for example by deposition of silicon dioxide (eg from a plasma), arranged on or in the surface by direct interaction of the surface molecules of the silicon dioxide layer with a chemical function or group (except silanes and their derivatives) of a test substance, for example with a thiol, one or more test substance (s) (k) according to the invention, eg peptide / s with a preferably terminal cysteine residue, are coupled, eg as an array of different test substances and / or different amounts of a test substance, preferably as a replica. By way of example, on the left side, a replica of two identical spots of a test substance is located, on the right side a spot with a lower concentration of the same test substance (front) and a spot of a second test substance (rear) is arranged. The areas (spots) with test substance (s) are surrounded by the silicon dioxide layer (j), which is optionally blocked subsequently, for example with a thiol or blocking reagent or during the use according to the invention, for example by bringing it into contact with body fluid.

2D : Schematic cross section through the arrangement according to the invention ( 2A ) in the area of the coupled test substance (s). The test substances or the different amounts of a test substance adhere directly to the surface molecules of the glass surface layer (b) on or in the glass surface layer (k).

2E : Schematic cross section through the arrangement according to the invention ( 2 B ) in the range of the coupled test substances. The test substances or the different amounts of a test substance adhere directly to the surface molecules of the silicon dioxide layer (g) on or in the silicon dioxide layer (I).

2F : Schematic cross section through the arrangement according to the invention ( 2C ) in the range of the coupled test substances. The test substances or the different amounts of a test substance adhere directly to the surface molecules of the silicon dioxide layer (j) on or in the silicon dioxide layer (m).

3 schematically shows cross sections (corresponding to 1D ) of typical embodiments of the inventive arrangement. "The hatching / pattern of the semiconductor substrate, the inorganic insulator layer and the coupled test substances is corresponding 1D ,

According to the other and the further solution of the object according to the invention (corresponding to the 2A or C) corresponds to the roughly hatched area of the 3A -G the cross-section of the glass substrate or the solid and the finely hatched area (s) of the cross-section (s) of the glass surface layer (s) or the silicon dioxide layer (s).

3A shows an arrangement in which the surface of the semiconductor substrate is completely covered with an inorganic insulator layer, preferably uniform thickness / height.

3B shows an arrangement in which two sides of the semiconductor substrate (top and bottom) with an inorganic insulator layer of thickness / height x, for example, a silicon dioxide layer, occupied and at least two sides with an inorganic insulator layer, such as a silicon dioxide layer, the thickness / height ≤ x occupied are, for example, an arrangement with a chip which is sawn from a thermally oxidized silicon wafer with an oxide thickness> 4 nm and on whose side surfaces a natural silicon oxide ≤ 4 nm is formed.

3C shows an arrangement in which the Semiconductor substrate is not completely covered with the inorganic insulator layer, are coupled to the location addressed at least two test substances or at least two unterschiedlcheche amounts of a test substance.

3D shows an arrangement in which at least two spatially separated inorganic insulator layers are formed in the surface of the semiconductor substrate to which at least two test substances or at least two different amounts of a test substance are respectively coupled.

3E shows an arrangement in which at least two spatially separated inorganic insulator layers are formed in the surface of the semiconductor substrate, to each of which one of at least two test substances or in each case a quantity of at least two different amounts of a test substance is coupled, wherein the surfaces of the inorganic insulator layers larger ( left) and / or identical (right) with the surface exposed to the test substance.

3F shows an arrangement in which at least two spatially separated inorganic insulator layers are formed on the surface of the semiconductor substrate to which at least two test substances or at least two different amounts of a test substance are respectively coupled.

4 schematically shows special embodiments of the inventive arrangement. The hatching / pattern of the semiconductor substrate, the inorganic insulator layer and the coupled test substances is corresponding 1D , According to the other and the further solution of the object according to the invention (corresponding to the 2A or C) corresponds to the roughly hatched area of the 3A -G the cross-section of the glass substrate or the solid and the finely hatched area (s) of the cross-section (s) of the glass surface layer (s) or the silicon dioxide layer (s).

4A shows a cross-section through an arrangement with recesses in the surface, wherein the test substance (s) are coupled to and / or in the depressions, which are formed for example as a hollow cylinder, as a hollow cone, as grooves or as a combination thereof.

4B shows a cross-section through an arrangement with continuous recesses in the semiconductor substrate, which are formed for example as a bore or channels, wherein the test substance (s) are coupled to and / or in the recesses.

4C shows a plan view of an arrangement according to 4A ,

4D shows a plan view of an arrangement according to 4B ,

Various embodiments of the invention have been tested, which are described below on the basis of non-exhaustive examples according to FIGS 5 - 15 be explained.

When Semiconductor substrate was silicon in different embodiments (single crystal silicon with a {111}, {100} or {110} orientation, polycrystalline silicon or amorphous silicon, the p- (with boron) or n- (with phosphorus, antimony or arsenic) or was not doped and had an electrical resistance depending on the doping, the surface the silicium was atomically smooth, rough, polished, not polished, or porous) used, with the silicon either as a wafer or as part of it was formed or on different solids (plastic, wood, Metals (e.g., aluminum) or glass). As a glass substrate according to the other solution the task of the invention were slides of glass and as a solid according to the others solution the task of the invention were plastic carriers or Polymer films used.

When inorganic insulator layer was silicon dioxide in different Embodiments used as a native or thermal oxide different thickness was formed on or from the silicon, as glass surface layer according to the other solution of inventive task were not organically or metallically modified surfaces of Glass slides used as a silicon oxide layer according to the further solution of inventive task For example, silicon oxide films deposited on the solid were used.

The with test substances to be coated surfaces were either sequentially with an organic solvent and a watery one solvent or aqueous solution were contacted, or were mixed with an organic and a watery one solvent contacted or were not treated. For the production Arrays were filled with liquid exposed surfaces dried.

When test substances were in particular peptides, proteins, oligonucleotides or small (Molecular weight> 75 and <2000 g / mol) organic Used molecules the solved in water or in a watery one Solution, each with or without the addition of an organic solvent.

The solutions of the test substances were either by means of a pipette, an automatic pipettor or an arrayer on the inorganic insulator layer or the Glasoberglächenschicht or the silicon oxide layer applied, or the respective surface to be coated was wetted with the solution of the test substance (s) or completely bathed. For the preparation of sandwich arrangements, eg biotin / streptavidin / biotinylated test substance, the surface was successively loaded with the various components of the sandwich arrangement, for example over a large area with thio-biotin and streptavidin, and then localized with biotinylated peptides, proteins or oligonucleotides, eg as an array.

Excess, i. uncoupled, test substance was optionally subsequently removed by washing from the surface and / or not with test substance occupied areas of the surface were blocked by blocking reagent, e.g. Blocking buffer and / or a nucleophile, e.g. Thiol, blocked.

Excess blocking reagent was optionally removed by washing from the surface and / or the surface of Arrangement dried.

following followed by incubation of the assembly with at least one solution, the contained at least one at least partially dissolved test substance, especially peptides, proteins, oligonucleotides, cell compartments, Cells and / or small (molecular weight> 75 and <2000 g / mol) organic molecules, being the solution with the test substance if necessary after incubation removed from the surface was and / or the surface washed and / or dried.

Possibly. the assembly was subsequently probed with a dissolved labeled probe, e.g. a dye-labeled secondary antibody, which optionally following from the surface was removed and / or washed the surface of the assembly and / or dried.

The Evaluation of the arrangements was carried out with the help of an imager, a Scanner or a MALDI mass spectrometer.

5 shows, for example, spotting (pipetting by means of a pipetting automat) biotinylated cysteine (ie biotin linked via its carboxyl group as amide to the amino group of L-cysteine), (1 mM, 0.1 mM and 0.01 mM dissolved in Sodium phosphate buffer pH 7.4, 1% v / v NMP) on sawn Si wafers (p-doped, boron, 111 orientation, size 75mm × 25mm × 1mm + - 0.1mm, polished side, thermal oxide 1000A + -70, Spot diameter approx. 1mm).

6 shows, for example, spotted biotinylated cysteine (1 mM, 0.1 mM, 0.01 mM) on sawn Si wafer (p-doped, boron, 111 orientation, size 75mm x 25mm x 1mm + - 0.1mm, unpolished side , thermal oxide 1000A + -70, spot diameter approx. 1mm).

7 shows, for example, spotted biotinylated cysteine (1 mM, 0.1 mM, 0.01 mM) on sawn Si wafer (p-doped, boron, 111 orientation, size 75mm x 25mm x 1mm + -0.1mm), unpolished side 1mm, after incubation with HRP-labeled streptavidin and visualization with luminol on a Lumi imager shows signals which are dependent on the concentration of the applied test substance.

8th shows, for example, spotted biotinylated cysteine (1 mM, 0.1 mM, 0.01 mM) on sawn Si wafers (p-doped, boron, 111 orientation, size 75mm x 25mm x 1mm + -0.1mm), unpolished 1mm, after long-term test, incubated after 3 months with HRP-labeled streptavidin and visualized with luminol on lumi- (lean, storage at 25 ° C, 4 ° C and -20 ° C (side), thermal oxide 1000A + -70. from top to bottom).

9 For example, spotted Tab2 peptides (in 0.1 M TBS buffer containing concentrations of IM, 0.1 M, 0.01 M) are N-terminally extended with cysteine (columns 1-6, columns 7-9 without extension) with antibody sandwich after 30 days, on sawn Si wafer (p-doped, boron, 111 orientation, size 75mm × 25mm × 1mm + - 0.1mm), unpolished side, thermal oxide 1000 A + -70, tapping diameter about 1mm Upper image detected on the Lumi imager with HRP-labeled antibody, lower image detected on the Affymetrix scanner with fluorescence-dye-labeled antibody.

10 schematically shows examples of test substances (cysteine-biotin, protein (streptavidin), peptide (antibody epitope)), the large surface coupled to the surface of square silicon chips and by means of which subsequently test substances (streptavidin, biotin and monoclonal antibody) from a test solution by means of chemiluminescence be detected (above, the non-luminous chip is the negative control).

11 schematically shows the location-addressed binding of biotin on an inorganic insulator layer on a semiconductor substrate (triangular). For this purpose, a biotin-cysteine solution is pipetted onto the insulator layer and subsequently detected with HRP-labeled streptavidin. The chemiluminescent signals are identical to the pipetting sites. This makes it clear how simple and targeted the functionalization of the surface can be done and the further advantage of the chip, näm that is readily available for use in sandwich immobilizations (eg, biotin-streptavidin-biotinylated antibodies).

12 Figure 3 shows schematically the site-addressed coupling of peptides on an inorganic insulator layer on a semiconductor substrate and the comparison between a cysteine-labeled epitope (Tab2) pipetted in solution and the same unlabeled peptide. Subsequently, it is incubated with primary antibody and screened with HRP-labeled secondary antibody, and the specific binding of molecules and the suitability for immunoassays becomes clear.

13 shows schematically the location-addressed connection of connection to an inorganic insulator layer formed on a semiconductor substrate. Solutions of HRP-labeled streptavidin in different concentrations are pipetted onto the insulator layer and, after wetting the surface with peroxidase substrate, the chemiluminescence measured, whereby signals become visible, which are dependent on the concentration / amount of the coupled protein. Thus, the simple production of the arrangement according to the invention as a protein chip by functionalization with proteins becomes clear and the associated advantages, namely that a wide variety of parameters can be measured and affinities can be determined.

• A) Dazu werden farbstoffmarkierte Oligonukleotide mit verschieden Linkern auf oxidierten Si-Wafern (p-dotiert, Bor, 111-Orientierung, thermisches Oxid 1000 A+–70), gekoppelt und mittels eines Affymetrix-Scanners ausgelesen
• 4ergruppe 1: Hydrazinmodifiziertes Oligo SES, Cy3 gelabelt + C6-SFB (macht aidehydende)
• 4ergruppe 2: Hydrazinmodifiziertes Oligo SES, Cy3 gelabelt
• 4ergruppe 3: Hydrazinmodifiziertes Oligo SMS, Cy3 gelabelt + C6-SFB (macht aidehydende)
• 4ergruppe 4: Hydrazinmodifiziertes Oligo SMS, Cy3 gelabelt
• B) Farbstoffmarkierte Oligonukleotide mit verschieden Linkern werden auf oxidierten Si-Wafern (p-dotiert, Bor, 111-Orientierung, Termisches Oxid 1000 A+–70, gekoppelt und mittels Affymetrix-Scanner ausgelesen
• 4ergruppe 1: Aminomodifiziertes Oligo SES, Cy5 gelabelt + C6-SFB (macht aldehydende).
• 4ergruppe 2: Aminomodifiziertes Oligo SES, Cy5 gelabelt + C6-SANH (macht Hydrazinende)
• 4ergruppe 3: Aminomodifiziertes Oligo SES, Cy5 gelabelt
• 4ergruppe 4: Aminomodifiziertes Oligo SMS, Cy5 gelabelt + C6-SFB (macht aldehydende)
• 4ergruppe 5: Aminomodifiziertes Oligo SMS, Cy5 gelabelt + C6-SANH (macht Hydrazinende)
• 4ergruppe 6: Aminomodifiziertes Oligo SMS, Cy5 gelabelt
• 4ergruppe 7: Aminomodifiziertes Oligo ESE, Cy5 gelabelt + C6-SFB (macht aldehydende)
• 4ergruppe 8: Aminomodifiziertes Oligo ESE, Cy5 gelabelt + C6-SANH (macht Hydrazinende)
• 4ergruppe 9: Aminomodifiziertes Oligo ESE, Cy5 gelabelt
• 4ergruppe 10: Aminomodifiziertes Oligo MEM, Cy5 gelabelt + C6-SFB (macht aldehydende)
• 4ergruppe 11: Aminomodifiziertes Oligo MEM, Cy5 gelabelt + C6-SANH (macht Hydrazinende)
• 4ergruppe 12: Aminomodifiziertes Oligo MEM, Cy5 gelabelt
• 4ergruppe 13: Thiolmodifiziertes Oligo SES, Cy5 gelabelt
• 4ergruppe 14: Thiolmodifiziertes Oligo SMS, Cy5 gelabelt
• 4ergruppe 15: Biotinmodifiziertes Oligo SES, Cy5
• 4ergruppe 16: Biotinmodifiziertes Oligo SMS, Cy5
• 4ergruppe 17: Hazwo-O
• 4ergruppe 18: Wasser

14 shows arrangements according to the invention, which are produced as oligonucleotide microarrays and are useful for the detection of oligonucleotides.

• A) For this purpose, dye-labeled oligonucleotides with different linkers on oxidized Si wafers (p-doped, boron, 111 orientation, thermal oxide 1000 A + -70) coupled and read using an Affymetrix scanner
• Group 4: Hydrazine-modified oligo SES, Cy3 labeled + C6-SFB (makes aidydehyde)
• Group 4: Hydrazine-modified oligo SES, Cy3 labeled
• Group 3: Hydrazine-modified oligo SMS, Cy3 labeled + C6-SFB (makes aidydehyde)
• Group 4: Hydrazine-modified oligo SMS, Cy3 labeled
• B) Dye-labeled oligonucleotides with different linkers are coupled to oxidized Si wafers (p-doped, boron, 111 orientation, termic oxide 1000 A + -70, and read out using the Affymetrix scanner
• Group 1: amino-modified oligo SES, Cy5 labeled + C6-SFB (aldehyde-terminated).
• Group 4: amino-modified oligo SES, Cy5 labeled + C6-SANH (makes hydrazine end)
• Group 3: amino-modified oligo SES, Cy5 labeled
• Group 4: Amino Modified Oligo SMS, Cy5 Label + C6-SFB (makes aldehydes)
• Group 4: Amino Modified Oligo SMS, Cy5 Label + C6-SANH (makes Hydrazine End)
• Group 4: Amino Modified Oligo SMS, Cy5 Labeled
• Group 4: amino-modified oligo ESE, Cy5 labeled + C6-SFB (makes aldehydes)
• Group 4: Amino Modified Oligo ESE, Cy5 labeled + C6-SANH (makes hydrazine end)
• Group 4: amino-modified oligo ESE, Cy5 labeled
• Group 4: Amino Modified Oligo MEM, Cy5 Labeled + C6-SFB (Aldehyde Ending)
• Group 4: Amino-modified oligo MEM, Cy5 labeled + C6-SANH (makes hydrazine end)
• Group 12: Amino-modified oligo MEM, Cy5 labeled
• Group 13: Thiol-modified oligo SES, Cy5 labeled
• Group 14: Thiol-modified oligo SMS, Cy5 labeled
• Group 4: Biotin-modified oligo SES, Cy5
• Group 16: Biotin Modified Oligo SMS, Cy5
• Group 17: Hazwo-O
• Group of four 18: Water

It shows a specific binding of the oligonucleotides and below a specific binding and detection of dissolved oligonucleotides as test substances.

All in the foregoing description, the following claims and The features shown in the illustrations can be used individually as well in any combination for the realization of the invention in its various embodiments be significant.

Claims (20)

Arrangement for the specific detection of substances from a sample, in particular for detecting association events, which has a carrier substrate and a bonding layer formed on the carrier substrate and a test substance coupled to the bonding layer, characterized in that the carrier substrate is a semiconductor substrate ( 1 ) and the bonding layer at least one inorganic insulator layer formed on the semiconductor substrate ( 2 ), wherein on and / or in the insulator layer ( 2 ) at least one area ( 3 ) with at least one medium ( 4 ) and at least one organic test substance ( 5 ) is applied, and in the at least one applied area ( 3 ) between the insulator layer ( 2 ) and the at least a test substance ( 5 ) is a coupling, and to the insulator layer ( 2 ) coupled test substance ( 5 ) is capable of interacting with a test substance. Arrangement according to claim 1, characterized in that - in the at least one applied area ( 3 ) between the insulator layer ( 2 ) and the at least one test substance ( 5 ) a direct coupling, stable in air and in liquids, without silanized or polymerized or crosslinked (eg polyvinyl, cellulose) intermediate layer; and / or - to the insulator layer ( 2 ) coupled test substance ( 5 ) interacts with at least one test substance detectable by radiation and / or ionization, whereby association events are determined by mass spectrometry or by detection of radiation on and / or in the insulator layer ( 2 ), preferably in the at least one area ( 3 ), are detectable by means of a detector. Arrangement according to one of the preceding claims, characterized in that - the semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) is formed in the usual dimensions of a biochip or a particle, in particular a microparticle or nanoparticle; and / or the semiconductor substrate ( 1 ) is formed as amorphous polycrystalline silicon or monocrystalline silicon, in particular with a {111} or {100} or {110} preference orientation; and / or - the inorganic insulator layer ( 2 ) as a silicon nitride or silicon dioxide layer ( 7 ) is formed; and / or - the at least one area ( 3 ) as at least two non-silanized areas / districts ( 3 ; 8th ) is formed on the insulator layer, which with the at least one medium ( 4 ) and the at least one test substance ( 5 ) are charged; and / or - the at least one medium ( 4 ) at least one solvent ( 9 ) or as at least one solvent ( 9 ) is formed; and / or - the at least one organic test substance ( 5 ) as at least two different test substances ( 5 ; 11 In particular, the test substance or test substances are formed or comprise molecules comprising at least one of nitrogen, sulfur, oxygen, phosphorus, halogen, dye or radioactive isotope; and / or - to the insulator layer ( 2 ) coupled test substance ( 5 ) interacts with at least one test substance detectable by radiation and / or ionization, whereby association events are determined by mass spectrometry or by detection of radiation on and / or in the insulator layer ( 2 ) preferably in the at least one area ( 3 ) are detectable detectable by means of an externally disposed detector; and / or - on and / or in an area ( 3 ) detectable signals, preferably by radiation and / or by mass spectrometric characteristics of on and / or in at least one further area ( 8th ) and / or on and / or in the surrounding area ( 10 ) detectable signals are distinguishable. Arrangement according to one of the preceding claims, characterized in that - the arrangement is formed as a preferably planar biochip for the optical or scintigraphic detection of association events by means of an external detector; and / or the semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) is formed in the usual dimensions of a slide or a slide carrier insert; and / or the semiconductor substrate ( 1 ) is at least partially p- and / or n-doped, in particular with an electrical resistance of <15 ohms / cm ² ; and / or the silicon dioxide layer ( 7 ) at least partially, in particular in the at least one area ( 3 ; 8th ) is formed as a dry and / or thermal oxide or as a dry and / or thermal nitride; and / or - the at least two non-silanized regions ( 3 ; 8th ) at least partially from one on and / or in the insulator layer ( 2 ) surrounding area ( 10 ) are surrounded; and / or - the medium ( 4 ) is electrically conductive, preferably with a conductance of> 0.05 μS / cm; and / or - the at least one solvent ( 9 ) is formed as an aqueous and / or organic solvent, in particular as a hydrophilic and / or polar solvent; and / or - the at least one organic test substance ( 5 ) at least one nucleophilic group or function, in particular a thiol, amino, hydroxyl, carbonyl, cyano, nitrile, Si, Se, He, sulfate, sulfide, terminal alkene or alkyne, or Phosphane group, or a reaction product formed from it. Arrangement according to one of the preceding claims, characterized in that - the semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) in the dimensions length × width = (7.5 +/- 1) cm × (2.5 cm +/- 0.5 cm); and / or the semiconductor substrate ( 1 ) is formed as a silicon wafer or as part thereof; and / or - the inorganic insulator layer ( 2 ) as a silicon dioxide layer ( 7 ) having a thickness of> 4 nm or> 5 nm, preferably> 10 nm, more preferably> 30 nm, in particular 100 nm (+/- 70 nm) is formed; and / or - the at least two non-silanized regions ( 3 ; 8th ) each with a different concentration of min at least one test substance ( 5 ) or in each case with another of the at least two test substances ( 5 ; 11 ) are charged; and / or - the medium ( 4 ) Water or an organic solvent having an acidic or basic pH, an electrolyte and / or a buffer; and / or - the at least one solvent ( 9 ) comprises or is formed as an organic solvent of molecules having at least one nitrogen, oxygen, halogen and / or ring structure; and / or - the surrounding area ( 10 ) the at least two non-silanized regions ( 3 ; 8th completely surrounds; and / or - the at least one organic test substance ( 5 ) is or contains a linker molecule, amino acid, carbohydrate, lipid, dye, drug, biotin, peptide, polypeptide, oligonucleotide, protein, cell compartment, cell, or structural or reaction product derived therefrom. Arrangement according to one of the preceding claims, characterized in that - the semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) is formed in a thickness of> 0.3 mm, in particular of 1 mm (+/- 0.7 mm); and / or the semiconductor substrate ( 1 ) is formed homogeneously; and / or the silicon dioxide layer ( 7 ) has a blue and / or red color due to production; and / or - the at least two non-silanized regions ( 3 ; 8th ) are formed as spots, in particular spatially separated and / or location-addressed spots; and / or - the medium ( 4 ) is formed as a mixture of an aqueous and organic solvent; and / or - the at least one solvent ( 9 ) is formed as or contains alcohol, Dimethylalkylsäureamid, alkyl halide, pyrollidone, furan or one of their derivatives; and / or - the surrounding area ( 10 ) is formed as silicon dioxide and / or chemically modified, in particular with organic molecules; and / or - the at least one organic test substance ( 5 ) is or comprises DNA, RNA, cDNA, mRNA, cRNA, PNA, receptor, ligand, protein A, protein G, streptavidin, antibody or structure derived therefrom. Arrangement according to one of the preceding claims, characterized in that - the semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) is formed as an oxidized silicon wafer or as a part thereof; and / or - the at least two non-silanized regions ( 3 ; 8th ) are formed as an array, in particular as a microarray and / or low-density array; and / or - the at least one solvent ( 9 ) is formed as or contains ethanol, DMF, dichloromethane, NMP, THF or one of their derivatives; and / or - the surrounding area ( 10 ) is modified by the addition of blocking reagent or a nucleophilic compound; and / or - the at least one organic test substance ( 5 ) is formed as a peptide from L- and / or D-amino acids, in particular gene-coded amino acids and / or whose modifications, or contains these. Arrangement according to one of the preceding claims, characterized in that - the semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) is formed from an at least partially polished silicon wafer or a part thereof; and / or - the at least one area ( 3 ; 8th ) with the at least one test substance ( 5 ; 11 ) contained in the at least one medium ( 4 ) is at least partially dissolved, is applied, in particular with a mixture comprising aqueous buffer solution, organic solvent and dissolved test substance; and / or - the surrounding area ( 10 ) is modified by the action of a thiol, in particular mercaptoethanol or cysteine; and / or - the at least one organic test substance ( 5 ) is coupled as linker molecules with free / unbound chemically reactive groups; and / or - the at least one test substance ( 5 ; 8th ) substantially spatially directed to the lolatorschicht ( 2 ) is coupled. Method for producing an arrangement for the specific detection of substances from a sample, characterized in that at least one area ( 3 ) one on a semiconductor substrate ( 1 ) formed inorganic insulator layer ( 2 ) with at least one medium ( 4 ) and at least one organic test substance ( 5 ) as a result of interactions between the organic test substance ( 5 ) and the inorganic insulator layer ( 2 ) to a coupling between the organic test substance ( 5 ) and the inorganic insulator layer ( 2 ), and the area ( 3 ) is adapted to interact with a test substance and / or interacts with the application of a test substance with this. Process according to claim 9, characterized in that at least one non-silanized region ( 3 ) with the at least one medium ( 4 ) and with at least one organic test substance ( 5 ), so that the organic test substance in the range ( 3 ) without silanized intermediate layer on and / or in the insulator layer ( 2 ) and can be used for interaction with a test substance and / or in the at least one applied area ( 3 ) between the insulator layer ( 2 ) and the at least one test substance ( 5 ) a direct coupling in air and in solutions without silanized or polymerized / cross-linked (eg polyvinyl, cellulose) intermediate layer is formed and / or to the insulator layer ( 2 ) coupled test substance ( 5 ) with at least one further test substance ( 12 ) and due to interactions between the test substance ( 5 ) and the further test substance ( 12 ) to a connection of the further test substance ( 12 ) to the test substance ( 5 ), so that the test substance ( 5 ) by the connection with the further test substance ( 12 ) is modified and / or to the insulator layer ( 2 ) coupled test substance ( 5 ) interacts with at least one test substance detectable by radiation and / or ionization, whereby association events are determined by mass spectrometry or by detection of radiation on and / or in the insulator layer ( 2 ) preferably in the at least one area ( 3 ) can be detected by means of a detector. Method according to one of claims 9-10, characterized in that - the at least one area ( 3 ) with at least one medium ( 4 ) and at least one organic test substance ( 5 ) is applied directly; and / or a semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) is used in the usual dimensions of a biochip or as particles, in particular in the dimensions of a microparticle or nanoparticle; and / or - as a semiconductor substrate ( 1 ) crystalline silicon, preferably amorphous or polycrystalline silicon or monocrystalline silicon, in particular having a [111] or [100] or [110] preference orientation is used; and / or - as an inorganic insulator layer ( 2 ) a silicon nitride or silicon dioxide layer ( 7 ) is used; and / or - at least two non-silanized regions ( 3 ; 8th ) on the insulator layer with the at least one medium ( 4 ) and the at least one test substance ( 5 ) be charged; and / or - as a medium ( 4 ) at least one solvent ( 9 ) or a medium comprising a solvent is used; and / or - as an organic test substance ( 5 ) at least two test substances ( 5 ; 11 ) and / or molecules having at least one nitrogen, sulfur, oxygen, phosphorus or halogen; and / or - to the insulator layer ( 2 ) coupled test substance ( 5 ) interacts with at least one test substance detectable by radiation and / or ionization, whereby association events are determined by mass spectrometry or by detection of radiation on and / or in the insulator layer ( 2 ) preferably in the at least one area ( 3 ) are detected detectable by means of an externally arranged detector. Method according to one of claims 9-11, characterized in that - a semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) is used in the usual dimensions of a slide or a slide carrier insert; and / or a semiconductor substrate ( 1 ), which is undoped or at least partially p- and / or n-doped, in particular with an electrical resistance of <15 ohm / cm ² , is used; and / or a silicon dioxide layer ( 7 ), at least partially, in particular in the at least one area ( 3 ; 8th ) is formed as a dry and / or thermal oxide or as a dry and / or thermal nitride; and / or - the at least two non-silanized regions ( 3 ; 8th ) in such a way, in particular oraddressed, with test substance ( 5 ; 11 . 12 ) are applied, preferably by means of a pipetting automanager, that they are at least partially from one on and / or in the insulator layer ( 2 ) surrounding area ( 10 ) are surrounded; and / or - as a medium ( 4 ) an electrically conductive medium, preferably with a conductance of> 0.05 μS / cm is used; and / or - as the at least one solvent ( 9 ) an aqueous and / or organic solvent, in particular a hydrophilic and / or polar solvent is used, or a solvent which comprises an aqueous and / or organic solvent, in particular a hydrophilic and / or polar solvent; and / or - an organic test substance ( 5 ) having at least one nucleophilic group or function, in particular a thiol, amino, hydroxyl, carbonyl, cyano, nitrile, Si, Se, He, sulfate, sulfide, terminal alkene or alkyne, or Phosphane group is used; and / or - the area ( 3 ) first with the at least one medium ( 4 ) and subsequently with the at least one test substance ( 5 ) is applied. Method according to one of claims 9-12, characterized in that - a semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) in the dimensions length × width = (7.5 +/- 1) cm × (2.5 cm +/- 0.5 cm) is used; and / or - as a semiconductor substrate ( 1 ) a silicon wafer or a part thereof is used; and / or a silicon dioxide layer ( 7 ), in particular silicon dioxide, with a thickness of> 30 nm, in particular 100 nm (+/- 70 nm) is used; and / or - the at least two non-silanized regions ( 3 ; 8th ) each with a different concentration of the at least one test substance ( 5 ) or in each case with another of the at least two test substances ( 5 ; 11 ) be charged; and / or - as a medium ( 4 ) Or an organic solvent, in particular having an acidic or basic pH, an electrolyte and / or a buffer is used or a medium, the water or an organic solvent having an acidic or basic pH, an electrolyte and / or comprises a buffer; As solvent ( 9 ) an organic solvent of molecules having at least one nitrogen, oxygen, halogen and / or ring structure or a solvent comprising an organic solvent of molecules having at least one nitrogen, oxygen, halogen and / or ring structure; and / or the insulator layer ( 2 ) spatially / flatly with the medium ( 4 ) and at least one test substance ( 5 ; 11 ; 12 ), that the at least two regions formed by the application ( 3 ; 8th ) completely of the preferably not contaminated with test substance environment area ( 10 ) are surrounded; and / or - as the at least one organic test substance ( 5 ) a linker molecule, amino acid, carbohydrate, lipid, dye, drug, biotin, peptide, polypeptide, oligonucleotide, protein, cell compartment, cell or a structure derived therefrom is used or an organic test substance ( 5 ) comprising at least one linker molecule, amino acid, carbohydrate, lipid, dye, drug, biotin, peptide, polypeptide, oligonucleotide, protein, cell compartment, cell and / or a structure derived therefrom. Method according to one of claims 9-13, characterized in that - a semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) is used in a thickness of> 0.3 mm, in particular of 1 mm (+/- 0.7 mm); and / or - a homogeneous semiconductor substrate ( 1 ), preferably amorphous or uniformly formed monocrystalline or polycrystalline silicon; and / or a silicon dioxide layer ( 7 ) is used, which has a blue and / or red coloring due to production; and / or the insulator layer ( 2 ), in particular location-addressed, preferably with the aid of a machine with the medium ( 4 ) and / or with the at least one test substance ( 5 . 11 ; 12 ), that the at least two regions ( 3 ; 8th ) are formed as spots, in particular spatially separated and / or location-addressed spots; and / or the insulator layer ( 2 ) in such a way with the medium ( 4 ) and at least one test substance ( 5 ; 11 ; 12 ), that the at least two regions formed by the application ( 3 ; 8th ) are formed as spots, in particular spatially separated and / or location-addressed spots; and / or - as a medium ( 4 ) using a mixture of an aqueous and organic solvent; and / or as solvent ( 9 ) an alcohol, dimethyl alkyl amide, alkyl halide, pyrollidone, furan or one of their derivatives is used or the solvent ( 9 ) contains an alcohol, a dimethylalkylamide, an alkyl halide, a pyrollidone, a furan or one of their derivatives; and / or - the surrounding area ( 10 ), which is preferably formed as Sillikat, not with the medium ( 4 ) and / or the organic test substance ( 5 ; 11 ; 12 ) and / or the surrounding area ( 10 ) is modified chemically, in particular with organic molecules; and / or - as the at least one organic test substance ( 5 ; 11 ; 12 ) DNA, RNA, cDNA, mRNA, cRNA, PNA, receptor, ligand, protein A, protein G, streptavidin, antibody or any structure derived therefrom or the organic test substance is DNA, RNA, cDNA, mRNA, cRNA, PNA, receptor , Ligand, protein A, protein G, streptavidin, antibody or any structure derived therefrom. Method according to one of claims 9-14, characterized in that - as a semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) an oxidized or nitrided silicon wafer or a part thereof is used; and / or the insulator layer ( 2 ) in such a way with the medium ( 4 ) and the at least one test substance ( 5 ; 11 ; 12 ), that the at least two regions ( 3 ; 8th ) are formed as an array, in particular as a microarray and / or low-density array; and / or - as the solvent ( 9 ) Ethanol, DMF, dichloromethane, NMP, THF or one of their derivatives is used or the solvent ( 9 ) Ethanol, DMF, dichloromethane, NMP, THF or one of their derivatives; and / or - the surrounding area ( 10 ) is modified by the addition of blocking reagent or a nucleophilic compound; and / or - as the at least one organic test substance ( 5 ; 11 ; 12 ) Peptides with L and / or D-amino acid residues, which are formed in particular from gene-coded amino acids and / or their modifications, preferably non-natural amino acids, or the at least one organic test substance ( 5 ; 11 ; 12 ) Contains peptides with L and / or D-amino acid residues, in particular from gene-encoded amino acids and / or their Modifi cations, preferably non-natural amino acids are formed. Method according to one of claims 9-15, characterized in that - a semiconductor substrate ( 1 ) with the insulator layer formed thereon ( 2 ) is used from an at least partially polished silicon wafer or a part thereof; and / or - the at least one area ( 3 ; 8th ) with the at least one test substance ( 5 ; 11 ) contained in the at least one medium ( 4 ) is at least partially dissolved, is applied, in particular with a mixture comprising aqueous buffer solution, organic solvent and dissolved test substance; and / or - the surrounding area ( 10 ) is modified by the action of a thiol, in particular mercaptoethanol or cysteine; and / or - as the at least one organic test substance ( 5 ) Linker molecules with after coupling to the insulator layer free / unbound chemically reactive groups, (eg cysteine, cysteine biotin) or peptides with a, preferably terminal, thiol, in particular a cysteine residue, are used or the organic test substance ( 5 ) Contains linker molecules with chemically reactive groups or peptides which are free / unbound after coupling to the insulator layer and having a preferably terminal thiol, in particular a cysteine residue; and / or - the at least one applied area ( 3 ; 8th ) and / or the surrounding area ( 10 ) is washed with at least one liquid, in particular a solvent and / or a wash buffer; and / or - a lithographically structured silicon dioxide layer is used. Use of an arrangement according to one of claims 1-8, in particular produced according to one of claims 9-16, for the specific detection of substances from a sample, in particular for the detection of association events, characterized in that the arrangement with at least one sample ( 13 ), which may be a test substance ( 14 ) contains. Use according to claim 17, characterized in that - conventional methods for the use of a biochip are used or conventional methods for the use of particles, in particular microparticles or nanoparticles, are used; and / or - the at least one area ( 3 ; 8th ) of the arrangement with the at least one sample ( 13 ) is brought into contact; and / or - the sample ( 13 ) the test substance ( 14 ), preferably at a detectable concentration; and / or the test substance ( 14 ) as amino acid, carbohydrate, lipid, dye, drug, biotin, linker molecule, peptide, oligonucleotide, protein, cell tissue, cell, cell constituent, DNA, RNA, cDNA, mRNA, cRNA, PNA, protein and / or peptide or structures derived therefrom is or contains this; and / or the test substance ( 14 ) in the area ( 3 ) to the insulator layer ( 2 ) coupled test substance ( 5 ) interacts, in particular with the test substance ( 5 ) or enzymatically altered. Use according to any one of claims 17-18, characterized in that - an immunoassay or gene expression profiling is performed; and / or contacting the sample ( 13 ) with the arrangement, in particular with the areas ( 3 ; 8th ), by pipetting or supplying the sample ( 13 ) to the order, in particular the areas ( 3 ; 8th ) and / or the surrounding area ( 10 ) he follows; and / or - the at least one sample ( 13 ) than at least two different samples ( 13 ; 16 ) is formed; and / or - the sample is formed as a solution or suspension of the test substance; and / or the test substance ( 14 ) is formed as or contains an antibody, enzyme, receptor, ligand, protein domain or epitope, and / or - at least one test substance ( 13 ) from the sample with at least one on and / or in the at least one area ( 3 ; 8th ) coupled test substance interacts demonstrably; and / or the test substance ( 14 ) with a radiation-active marker ( 17 ) or is connected, and / or - the arrangement, preferably the at least one area ( 3 ; 8th ), with a light source, preferably a lamp or a laser, is irradiated and / or - the arrangement, preferably the at least one area ( 3 ; 8th ), is acted upon by a enzymatic conversion chemilumineszierenden enzyme substrate, and / or - the arrangement, preferably the at least one area ( 3 ; 8th ), by means of an externally arranged detector for the detection of radiation, in particular by means of a detector for the detection of radiation in the wavelength ranges of visible light, UV light or IR or for the detection of radioactive radiation, or by means of a detector for the detection of mass spectrometry measurable products or Parameters, in particular by means of a scanner, imager, counter or mass spectrometer, is read out. Use according to one of claims 17-19, characterized in that - the sample ( 13 ) is formed as or contains body fluid, preferably urine, blood or serum, or as a stool sample; and / or the test substance as IgA, IgG, IgE, IgM, kinase, Phosphatase or Antiköperepitop is formed, or contains, and / or - the test substance ( 14 ) via a labeled probe which is preferably a synthetic peptide or polypeptide, a protein domain, a protein fragment, a protein, in particular a secondary antibody or streptavidin, an oligonucleotide or a substance intercalating into double-stranded oligonucleotides, with the marker ( 17 ), and / or the marker ( 17 ) is a dye, a radioactive isotope, a fluorescent protein, a peroxidase or a phosphatase.