WO2008019651A2 - Dispositif de détermination de substances, procédé de réalisation et utilisation de ce dispositif - Google Patents

Dispositif de détermination de substances, procédé de réalisation et utilisation de ce dispositif Download PDF

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Publication number
WO2008019651A2
WO2008019651A2 PCT/DE2007/001362 DE2007001362W WO2008019651A2 WO 2008019651 A2 WO2008019651 A2 WO 2008019651A2 DE 2007001362 W DE2007001362 W DE 2007001362W WO 2008019651 A2 WO2008019651 A2 WO 2008019651A2
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WO
WIPO (PCT)
Prior art keywords
test substance
insulator layer
organic
layer
semiconductor substrate
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PCT/DE2007/001362
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German (de)
English (en)
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WO2008019651A3 (fr
Inventor
Nikolaus Bartels
Bernhard Ay
Prosper Hartig
Michael Portwich
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Nikolaus Bartels
Bernhard Ay
Prosper Hartig
Michael Portwich
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Application filed by Nikolaus Bartels, Bernhard Ay, Prosper Hartig, Michael Portwich filed Critical Nikolaus Bartels
Publication of WO2008019651A2 publication Critical patent/WO2008019651A2/fr
Publication of WO2008019651A3 publication Critical patent/WO2008019651A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00572—Chemical means
    • B01J2219/00576—Chemical means fluorophore
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides

Definitions

  • 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 method for the production of the device and its use, in particular for detecting association events.
  • Fields of application of the invention are, inter alia, the environmental technology, food technology, the pharmaceutical industry and the
  • Substances can be detected or neutralized by means of a biologically active arrangement.
  • Bioly active arrangements consisting of support materials and organic compounds applied thereto / immobilized, in particular biologically active substances, are of outstanding importance in today's biotechnology and so-called life sciences.
  • Purpose of such carriers which are usually formed on the basis of non-water-soluble inorganic or organic polymers, is quite generally the local fixation or immobilization and the accumulation or concentration of biologically active substances, for example.
  • Detection molecules or pharmaceuticals As a result, surfaces are also created that can be used for a wide variety of applications, for example as detection systems or as pharmaceutical suppliers.
  • biochips and functionalized microparticles which can specifically recognize and bind target structures at the molecular level are state of the art.
  • a biochip is generally a rigid support on the surface of which biologically or chemically active substances, in particular molecules, are immobilized, usually by electrostatic, hydrophobic, hydrophilic and / or covalent binding to the surface molecules of the support.
  • the object of the immobilized biologically or chemically active substances is that they allow the association of bioactive substances from a sample, eg a test solution, with which they are brought into contact.
  • bioactive substances can also be made visible from a solution if they interact or associate with the immobilized biologically active substances, for example peptides, proteins or DNA.
  • a biochip is usually made up of three components:
  • Purpose of the immobilized biologically active substance or substances is then the specific attraction and / or accumulation of bioactive substances or test substances from a test medium, which then on the support surface, usually by a physically active label, e.g. with a dye or a radioactive isotope, or with the aid of a dye- or an enzyme-labeled probe, for example a primer or an antibody, are detectable.
  • a physically active label e.g. with a dye or a radioactive isotope
  • a dye- or an enzyme-labeled probe for example a primer or an antibody
  • the prior art is in particular carriers of glass or plastic, which are coated with a bonding layer of metal or with a bonding layer of surface-bound organic molecules, which allows the immobilization of proteins, peptides, or oligonucleotides on the surface or in the vicinity of the surface.
  • the bonding layer is necessary since the conventional support materials, for example glass or plastic, under normal conditions are hardly chemically reactive or inert and therefore do not permit direct and permanent attachment of the biologically active substance in solution.
  • the coating of the carrier with the bonding layer by a in the Usually complex chemical process, such as a silanization of glass, in which the support surface is covalently coated with molecules, which then allow the immobilization of biologically active substances in a subsequent step.
  • Carriers for the production of so-called 1D biochips 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, to attach or apply elements, e.g. of gold, for subsequent immobilization.
  • a biologically active substance for example a protein receptor
  • the substance can covalently bind to the functionalized (eg glass) surface via a chemical reaction if the receptor has a suitable chemically functional group which reacts with a free reactive group of molecules of the attachment layer and can form a covalent bond.
  • 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.
  • Supports for the production of 2D or 3D biochips have an attachment layer of covalently bound surface molecules, often lipids, longer alkane derivatives, or organic polymers, e.g. formed as hydrogel, which is a mostly non-covalent immobilization of biologically active substances, e.g. of receptors via hydrophobic and / or electrostatic interaction.
  • covalently bound surface molecules often lipids, longer alkane derivatives, or organic polymers, e.g. formed as hydrogel, which is a mostly non-covalent immobilization of biologically active substances, e.g. of receptors via hydrophobic and / or electrostatic interaction.
  • a disadvantage of the prior art is quite generally that the common carriers are first metallized or wet-chemically functionalized with the binding layer which requires labor-intensive and time-consuming steps, eg reaction and washing steps. If, for example, a glass chip or particle is to be silanized for subsequent attachment of peptides or proteins, it is placed in a vessel containing a silanization solution for several hours, which is agitated by stirring, shaking or ultrasound, then washed and dried in an oven. The movement of the molecules in solution is important, inter alia, in order to achieve a uniform bonding layer on the support surface in the chemical reaction between the support and the dissolved molecules, for example alkoxysilane or chlorosilanes.
  • the use of special glass substrate eg quartz glass for high temperature resistance or low impurities, is often required due to the conditions in the production of the bonding layer.
  • a polymerized layer or polymer layer for example of (nitro) cellulose, PVDF or a hydrogel requires expert skill and is expensive.
  • the bonding layer thus prepared is often chemically sensitive and requires a special storage of the carrier, for example under vacuum or inert gas.
  • a disadvantage of the common support materials is further that their surface properties are only partially suitable for many applications.
  • the common carrier material glass for example, is a solidified melt with a rough surface, which can have a detrimental effect on a uniform connection layer and directional connection, for example of microarrays.
  • a microarray is a carrier material on which a large number of biochemical detections or tests are located on the narrowest, usually only fingernail-sized, space. But also biochips with only a few spatially fixed immobilized tests are known and used in practice, for example so-called "low density" biochips. The tests consist of many locally addressed, mostly in circular “spots" on the surface, bound molecules or larger biological components (eg cell organelles or cells).
  • DNA molecules or proteins are selected which can associate specifically with substances from a test solution, the bound substances being subsequently made visible on the surface.
  • substances from a test solution eg serum antibodies or tissue DNA
  • pathogens eg viruses, or tissue changes, eg tumors
  • the biochip sector has experienced a first major upswing in the human genome project. Driven by its impressive results, many start-up companies have entered this market to accelerate and automate gene analysis. Now that human genes are fully sequenced, the focus is on the development of high performance biochips that enable easy readout of biological signals in diagnostics and research.
  • biochips are suitable which enable a simple and defined binding of peptides or proteins. These have the advantage that, in principle, they can visualize all possible substances with which they interact according to the key-lock principle. As a result, for example, significantly more pathogens can be made visible or detectable than is possible with today's DNA chips.
  • Functionalized microparticles which may be formed, for example, as nanoparticles, allow the sensitive detection, labeling and treatment of target structures of a biological or biotechnological system.
  • a biological system are essentially organisms, cell tissues, cells, To understand cell components, DNA, RNA, cDNA, mRNA, cRNA, carbohydrates proteins and / or peptides and structures derived therefrom, under the biotechnological system are all possible arrangements to understand, with the help of properties of biological systems or structures derived therefrom are detectable.
  • a typical microparticle for the targeted and delayed release of active substances in an organism comprises, for example, immobilized lipids or hydrogels with incorporated active substances and covalently linked antibodies via the attachment layer for the detection of the target structure in the organism.
  • Object of the present invention is therefore to provide an efficient and easy to manufacture arrangement with outstanding properties, which is preferably formed and used as a biochip or functionalized particles, based on a substrate and a bonding layer on the substances to be tested easily and directly coupled or immobilized, for detecting target substances or test substances from a sample, a method for producing the arrangement and their use, in particular for detecting and / or detecting bioactive substances from a test medium, preferably DNA, RNA, proteins, peptides, cells , their components and / or viruses from a liquid.
  • This object is achieved by an arrangement according to claim 1, a method for producing the arrangement according to claim 8, and their use according to claim 17.
  • the further claims are preferred embodiments of the invention.
  • the invention in its various aspects and technical features is realized according to the claims and the description given here.
  • 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.
  • 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 sense of the invention is in particular the chemical modification of a substrate, e.g. glass, with silanes or silane derivatives, e.g. as described in the reference in 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.
  • 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.
  • the substrate comprises a semiconductor or semiconducting material, hereinafter also semiconductor substrate (1, numbers are given here and possibly for clarity) and the bonding layer is an inorganic layer located on the semiconductor substrate, in particular a non-water-soluble solid which is under normal conditions, for example at dryness and room temperature, preferably substantially non-electrically conductive, hereinafter also called inorganic insulator layer (2).
  • Sub-insulating materials are to be understood in exceptional cases, also conductive materials that produce or include a spatial isolation of the semiconductor substrate.
  • Semiconductors according to the invention are in particular all elemental semiconductors, e.g. Silicon or germanium, and all compound semiconductors, e.g. Gallium arsenide, in its various forms or modifications, e.g. by doping, to understand.
  • the semiconductor substrate can be formed in a wide variety of spatial dimensions, for example as a flat chip, voluminous particles or as a semiconductor film or thin-film applied to a solid phase.
  • the inorganic insulator layer is, in particular, the areal extent of a uniform mass of an inorganic insulator lying directly above the semiconductor substrate, i. a substantially non-hydrocarbon compounds containing material which inhibits the flow of electrical charges and its electrical conductivity does not change substantially with changing temperature to understand.
  • a conductive layer which generates or comprises a spatial isolation of the semiconductor substrate to understand.
  • 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 figure of Arrangement according to the invention in its various embodiments, in particular with the features described below and in the claims, is analogous in that instead of the semiconductor substrate (1) with the at least one inorganic insulator layer (2) formed thereon, a glass substrate (glass substrate (1)) with at least a glass surface layer (glass surface layer (2)) formed thereon (set), wherein the at least one glass surface layer (2) is not organically or metallically modified, eg, non-silanized, especially untreated or unmodified is.
  • the above and below specified features for the semiconductor substrate and the inorganic insulator layer apply respectively 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.
  • 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.
  • a substantially water-insoluble solid or solid eg plastic, ceramic, stone, wood, cellulose, metal, etc., particularly preferably plastic, suitable
  • 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,
  • 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 (2) formed thereon a solid (solid (1) ) with at least one silicon oxide layer formed thereon (silicon oxide layer (2)), said at least one silicon oxide layer (2) being non-metallized and / or organically modified or being, for example, non-silanized , in particular untreated or unmodified.
  • the above and below specified features for the semiconductor substrate and the inorganic insulator layer apply respectively 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 previously cleaned surfaces, for example by in the Semiconductor manufacturing conventional cleaning methods, such as cleaning in Caro ' shear 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, also referred to below as 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, also referred to as test substance (5), acted upon or brought into contact, wherein within the at least one applied area (3 ) between the inorganic insulator layer (2) and the at least one test substance (5) is a connection or coupling without silanized intermediate layer.
  • the test substances coupled to the insulator layer (2) in this way are suitable for interacting with one or more test substances from a sample with which the arrangement is or may be brought into contact.
  • a silanized intermediate layer in the sense of the invention is to be understood in particular as a layer formed on the carrier substrate from silane derivatives, in particular wet-chemical, to which a test substance is bound or coupled, as for example in the publication Halliwell & Cass, A factorial analysis of silanization for the immobilization of oligonucleotides on glass surfaces, Anal Chem. 2001 Jun. 1; 73 (11): 2476-83.
  • the term "on and / or in the insulator layer” is to be understood in particular as meaning the parts of the inorganic insulator layer which can come into contact or interact with a medium and / or a test substance, ie, for example, the interface of the isolate layer to the periphery and / or or the molecules accessible to the medium and / or the test substance in the vicinity of or below the surface of the insulator layer If the semiconductor substrate is formed, for example, from a porous material, the term “on and / or in” also refers to the spatial one Regions of the insulator layer arranged within the semiconductor substrate. Under “on and / or in the isolar layer” is under some circumstances but also to understand that the medium and / or the test substance at least partially can penetrate into the semiconductor substrate when it is absorbed by a similar to a sponge of the insulator layer.
  • the arrangement according to the invention has the characteristic property that in the at least one applied area (3) a stable, direct coupling or connection without interlayer, in particular without silanized (eg with alkylsilanes) or polymerized or crosslinked (eg polyvinyl, cellulose) interlayer, is present between the insulator layer (2) and the at least one test substance (5).
  • the arrangement according to the invention is characterized in particular by the fact that the coupling is stable in air and in solution, i. in particular, that the coupled substance also after several hours or several days of washing the arrangement or the area (3) with liquid, e.g. a physiological buffer, still detectably coupled to the insulator layer (2).
  • 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 attached by direct contact with the molecules of the inorganic insulator layer in region (3), preferably by interaction of a nucleophilic group of the test substance with the surface molecules of the insulator layer are attached, wherein a covalent or electrostatic in particular Compound or interaction is formed or prevails.
  • the test substance (5) coupled to the insulator layer (2) 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 region (3) by means of an external detector detectable detected.
  • 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 region (3) by means of an external detector detectable detected 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.
  • the advantage of this is that the arrangement usually quickly and easily with already existing Laboratory equipment and with known measuring methods in diagnostics and research can be used.
  • 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
  • all common methods for the detection of enzyme activity are suitable, especially when the enzyme or the enzyme substrate is bound to the applied area (3) and the arrangement is brought into contact with enzyme substrate or enzyme.
  • 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) is in particular subjected to a concentration or amount of test substance suitable for the detection, 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), i. that as the concentration of the test substance decreases, decreasing detection signals are also observed, as a result of which, for example, association constants can also be determined.
  • concentration-dependent coupling may be observed due to the saturation of the surface with coupled test substance.
  • concentration-dependent signals or binding data can also be detected if the coupled test substance interacts with the test substance from the solution.
  • the signals detectable on and / or in a region (3), in particular by radiation and / or by mass spectrometric characteristics, from and / or in at least one further region (8) and / or / or detectable detectable signals in the environmental region (10), in particular depending on the coupled test substance.
  • semiconductor substrate (1) all known solids or materials with semiconductor properties are suitable, for example, silicon, titanium oxide or gallium arsenide especially in their different, preferably common, embodiments, such as different dopings.
  • the semiconductor substrate (1) with the insulator layer (2) formed thereon is preferably formed or configured in the usual dimensions of a biochip or a particle, in particular a strange bioparticle, preferably a microparticle or nanoparticle, making it particularly easy to use for conventional methods Biochips or particles can be used.
  • the arrangement is formed as a preferably planar biochip and / or as a 1D, 2D or 3D biochip for the optical or scintigraphic detection of association events by means of an external detector.
  • the semiconductor substrate (1) with the insulator layer (2) formed thereon is formed in the usual dimensions of an object carrier or a slide carrier, since it corresponds to the common standard for biochips in terms of its dimensions.
  • the training in the corresponding dimensions with a porous, structured, in particular lithographically structured surface is suitable.
  • the semiconductor substrate (1) with the insulator layer (2) formed thereon 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.
  • the semiconductor substrate (1) as amorphous, polycrystalline silicon or preferably monocrystalline silicon, in particular with a ⁇ 111 ⁇ - or ⁇ 100 ⁇ - or ⁇ 110 ⁇ - preferred orientation, particularly preferably with a ⁇ 111 ⁇ -Vorzugsorienttechnik formed, whereby the carrier material is formed as a nearly perfect and reproducible material.
  • a ⁇ 100 ⁇ preferred orientation of the semiconductor substrate (1) has proven to be particularly suitable for the arrangement according to the invention, with a particularly favorable arrangement being present due to the technical properties and wide distribution of this material, and the manufacturability being further simplified. But other crystalline preference orientations of the silicon are suitable.
  • 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 ohms / cm 2 , preferably ⁇ 20 ohm / cm 2 , more preferably ⁇ 1 ohm / cm 2 , particularly preferably ⁇ 0.1 ohm / cm 2 , since the arrangement thus particularly favorable properties, eg. For the directed coupling of the test substance has.
  • 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 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, educated.
  • 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.
  • the semiconductor substrate (1) is formed homogeneously, for example as a completely, ie within its entire dimensions, uniform silicon material, for example as a silicon chip that is not doped consistently or doped with only one kind / species of foreign atoms, thereby also particularly simple uniform carrier properties of the arrangement, in particular over the entire insulator layer (2), which is located on the homogeneous semiconductor substrate, are produced.
  • the semiconductor substrate (1) is formed as a preferably homogeneous semiconductor substrate without doping, the arrangement can be used particularly advantageously for an optical measurement without background or noise.
  • unpolished semiconductor substrates in particular silicon substrates, preferably unpolished silicon wafers or chips formed therefrom, are suitable for various applications since, for example, a particularly simple and uniform incubation of the arrangement surface with a sample, even without the use of a coverslip, or other conventional ones Incubation devices.
  • the semiconductor substrate (1) with the insulator layer (2) formed thereon is formed from at least partially, preferably at least one side, in particular on the side exposed to the test substance, as a polished silicon wafer or a part thereof, then the arrangement is particularly suitable as a biochip with a reflective surface and the associated advantages.
  • the inorganic insulator layer (2) disposed on the semiconductor substrate is preferably formed by chemical reaction from the material of the semiconductor substrate (1), whereby a particularly ordered and fixed bonding layer is provided on the semiconductor substrate (1).
  • the inorganic insulator layer (2) of the arrangement according to the invention is preferably formed as a layer of silicon nitride, silicate, or an inorganic polymer, particularly preferably as silicon dioxide, subsequently also called silicon dioxide layer (7), in particular with or without inclusions of impurities (doping). This has the advantage that the bonding layer can be produced in a particularly simple manner, for example by reaction of a silicon semiconductor substrate (1) with oxygen or nitrogen.
  • the inorganic insulator layer (2) is configured as a silicon dioxide layer (7), especially in the at least one area (3; 8) as a non-natural, preferably dry and / or thermal oxide or as a dry and / or thermal nitride is formed, whereby particularly advantageous material properties, such as a sponge-like formed stable structure, the insulator layer are guaranteed.
  • a silicate layer (7) which is prepared 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 natural, eg in air and / or by contact with distilled water formed, formed silicon oxide.
  • the inorganic insulator layer (2) is preferably in the form of a silicon dioxide layer (7) with a thickness of> 10 nm, preferably> 30 nm, in particular 100 nm (+/- 70 nm), particularly preferably 100 nm (+/- 10 nm), whereby particularly favorable properties, for example, for the reflection and amplification of radiation at the boundary layers of the inventive arrangement, are present.
  • the semiconductor substrate (1) is made of a silicon wafer or parts thereof, e.g. With a rectangular or square dimensions (chip) formed so are by 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 corresponding wavelength, adjustable for a variety of Applications of the arrangement are advantageous.
  • 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.
  • at least one medium (4) in particular a liquid acted upon, preferably at least one solvent, hereinafter also referred to as solvent (9) comprises, or as at least one solvent (9) is formed.
  • 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.
  • the medium (4) for the application if it comprises at least one solvent (9), which is formed as an aqueous and / or organic solvent, in particular a hydrophilic and / or polar solvent.
  • solvent (9) which is formed as an aqueous and / or organic solvent, in particular a hydrophilic and / or polar solvent.
  • water for example having a neutral pH, is to be understood as aqueous solvent.
  • organic solvent (9) an alcohol, dialkyl acid amide, alkyl halide, pyrollidone, furan, DMSO, acetonitrile, acetone or one of their derivatives is preferably suitable, especially ethanol, DMF 1 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 acidic, 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 (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.
  • the medium (4) is at least partially electrically conductive, in particular with a conductivity> 0.05 ⁇ S / cm, preferably> 20 ⁇ S / cm, particularly preferably> 1 mS / cm, particularly preferably> 50 mS / cm, in particular if it contains a dissolved solid, preferably the test substance and / or a salt, particularly preferably the test substance and a dissolved salt.
  • the at least one area (3) acted upon by medium (4) and test substance (5) is formed as at least two non-silanized areas / districts (3, 8) on the insulator layer.
  • the at least two regions are to be understood as meaning a plurality of preferably spatially separated regions, which are formed either as a replica, e.g. a plurality of equally applied areas, e.g. two or more at least partially similar arrays, or in each case with different test substances or with different concentrations of a strigsusbeam are applied.
  • the at least two non-silanized regions (3-8) are at least partially formed by an environmental region (10) formed on and / or in the insulator layer (2), i. a preferably not impacted with test substance district of the insulator layer, surrounded.
  • 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; 8) are spots, preferably spatially separated, particularly preferably complete separate, and / or location-addressed spots are formed.
  • the common definition, as it is used, for example, for spot synthesis or (micro) arrays, is to be understood by "spot".
  • the at least two regions (3; 8) are exposed to test substance dissolved at least partially in the at least one medium (4), in particular with a mixture comprising aqueous buffer solution, organic solvent and dissolved test substance.
  • a mixture comprising aqueous buffer solution, organic solvent and dissolved test substance.
  • Solid phase 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.
  • the at least two non-silanized regions (3, 8) are formed as an array, in particular as a microarray and / or low-density array, whereby a particularly good readability and utilization of the arrangement according to the invention is made possible.
  • the environmental region (10) is particularly preferably formed as silicon dioxide, i. as a substantially untreated region of the insulator layer (2) or is modified chemically, in particular with organic molecules, except the test substance.
  • rapid availability or reduction of nonspecific background signals is also particularly easily achieved, preferably if the surrounding area (10) is provided with blocking reagent and / or a nucleophilic compound, preferably with a thiol, e.g. Mercaptoethanol or cysteine is applied.
  • the inorganic insulator layer preferably over its entire areal extent, is cleaned prior to exposure to medium and test substance, eg by bathing in or wetting with an acid or base, for example Caro 's acid (hydrogen peroxide / Sulfuric acid mixture), or an organic solvent, for example ethanol, whereby the surrounding area is formed, for example, as a silicon dioxide with a purified surface.
  • an acid or base for example Caro 's acid (hydrogen peroxide / Sulfuric acid mixture)
  • an organic solvent for example ethanol
  • the test substance or test substances is formed in particular as molecules with at least one nitrogen, sulfur, oxygen, phosphorus, halogen, dye or radioactive isotope, or comprises these, whereby a particularly stable Connection or coupling between the insulator layer and the test substance is made.
  • the at least one organic test substance (5) is formed as at least two different test substances (5, 11), i. in that at least two different test substances or at least two different concentrations of one test substance are preferably spatially separated from the insulator layer and / or the test substance or the at least two different test substances are each arranged as molecules with at least one, preferably edge or terminal, molecule , Nitrogen, sulfur, oxygen, phosphorus, halogen, dye or radioactive isotope are or comprise these.
  • the arrangement is particularly advantageously suitable for location-dependent association with and for the detection of a plurality of different test substances from a sample, or it can be derived from the test substance, e.g. an enzyme, changes caused, e.g. are made visible by enzymatic cleavage of the coupled sample substance, and it is a particularly stable and directed coupling of the test substance to the inorganic bonding layer prepared.
  • the at least one organic test substance (5) preferably terminal, at least one nucleophilic chemical group or function, in particular a thiol, amino, hydroxyl, carboxyl (carboxy), cyano, Nitrile, Si, Se, He, sulfate, sulfide, alkene or alkyne, or phosphine group, or one / e by the interaction with the insulator layer resulting function or remainder, whereby the Stabiltusch the coupling is particularly well ensured, in particular by a thiol function, eg of a cysteine residue or of the residue resulting therefrom by interaction with the insulator layer.
  • a nucleophilic chemical group or function in particular a thiol, amino, hydroxyl, carboxyl (carboxy), cyano, Nitrile, Si, Se, He, sulfate, sulfide, alkene or alkyne, or phosphine group, or one / e by the interaction with
  • the at least one organic test substance (5) is 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 is universally suitable for a wide variety of applications, in particular for detecting all possible bioorganic components or structures (test substances).
  • the at least one organic test substance (5) is particularly preferably formed or contains or contains 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 well ensured.
  • the test substance (5) is formed as a synthetic, naturally occurring or recombinantly expressed peptide or obtained by enzymatic, acidic or basic cleavage of polypeptides, preferably as a synthetically produced peptide.
  • peptides as test substance have the advantage that they are generally easy to prepare synthetically and in high purity and, similar to whole proteins, may have specific association properties, e.g. when the peptide is an antibody epitope, preferably N- or C-terminal with a cysteine residue.
  • peptides with at least one non-naturally occurring amino acid residue e.g. formed from anthranil or from aminobenzoic acid, are particularly suitable for a wide variety of requirements.
  • the at least one organic test substance (5) is formed as linker molecules with free / unbound chemically reactive groups.
  • linker molecules are In particular, to understand all possible molecules, except with the insulator layer chemically reactive silanes and silane derivatives, which have two different, preferably end and / or laterally arranged in the molecule, different chemically reactive groups or functions, the molecules via a function of the Insulator layer (2) are coupled and functionalized with bioorganic substances if necessary via the other function.
  • test substance is formed, for example, as cysteine
  • this is preferably coupled via the thiol or the radical generated from the thiol by interaction with the insulator layer, its side chain to the insulator layer and can via a subsequent chemical reaction by means of its amino or carboxyl group another Substance or catcher molecules, for example, an amino acid residue or a peptide couple.
  • streptavidin can be coupled to the surface in a subsequent step or has been coupled, for example sandwich arrangements (eg insulator layer-biotin-streptavidin-biotinylated component ) are possible or formed.
  • the linker molecule or substance is at least one crosslinker which, via the one function, is chemically, in particular covalently, conjugated to a bioorganic substance, e.g. an oligonucleotide or a protein, and is coupled by means of the other function, in particular a nucleophilic group, preferably a thiol, to the insulator layer (2).
  • a bioorganic substance e.g. an oligonucleotide or a protein
  • a nucleophilic group preferably a thiol
  • the at least one test substance (5; 8) with the majority of its molecules coupled to the surface is arranged spatially directed on the isolate layer, ie the molecules are uniformly aligned on the surface, or with preferential orientation, in particular depending on the orientation of the the semiconductor substrate.
  • the test substance is designed, for example, as a peptide, protein or oligonucleotide labeled terminally with a nucleophilic group, in particular a thiol
  • the test substance layer is coupled in particular via the thiol group to the attachment layer (2) and is otherwise exposed accessible for interaction with a test substance before, preferably perpendicular or at other suitable angles to the insulator surface.
  • test substances are formed as a plurality of peptides or a plurality of other synthetically produced molecules, in particular oligonucleotides, they are preferably designed uniformly with a preferably terminal or pendant nucleophilic group, eg a thiol, whereby in particular also with several at least partially similar test substances, eg wild-type sequence Mutant, a particularly uniform and directional connection and a good comparability of the results is guaranteed.
  • Another aspect of the invention relates to a simple and efficient method for producing an arrangement for the specific detection of substances from a sample, in particular for producing an arrangement which comprises a carrier substrate and a bonding layer formed on the carrier substrate and a test substance coupled to the bonding layer, preferably for producing the arrangement according to the invention.
  • an inorganic insulator layer formed on a semiconductor substrate with a medium and a test substance leads to the stable and directed binding of the test substance to the insulator layer. This is especially true for oxidized, non-silanized silicon surfaces.
  • At least one, in particular non-silanized, area not covalently coated with organic molecules and not coated with a metal (3) of an inorganic insulator layer (2) formed on a semiconductor substrate (1) is provided with at least one medium (4).
  • the shape or extent of the insulator layer on the surface of the semiconductor substrate may be different as required, and is, for example, as a closed layer, which in particular encloses the entire semiconductor substrate or only a part thereof, in particular only one side thereof as individual areas, eg by 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. eg by incubation with a sample coupled to the insulator layer, when the assembly made in accordance with the invention is exposed to room air under normal conditions, e.g. at 23 ° C and atmospheric pressure, or vacuum, or inert gas atmosphere is stored.
  • solution-stable coupling or binding is meant, in particular, that the coupled test substance is detectable even after more than one hour, in particular after more than 12 hours, especially after more than 24 hours, 36 hours, 48 hours or 72 hours, e.g.
  • connection can also be stable under other than normal conditions.
  • a physiological buffer solution e.g. Tris or phosphate buffer
  • medium (4) and test substance (5) are in principle all standard methods or other known methods suitable by means of which a rigid surface with a medium and / or a test substance can be brought into contact, 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 uniformly with the at least one test substance is occupied, the semiconductor substrate with the insulator layer formed thereon, for example.
  • the medium and / or the test substance is incubated such that it is preferably completely wetted 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.
  • 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.
  • correspondingly cylindrical arrayers eg contact or piezo printers
  • first the insulator layer over a large area with the at least one medium eg by bathing or wetting the surface, for example.
  • 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 bauftschten 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.
  • aqueous 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 bauftschten area preferably location-addressed different test substances and / or different concentrations or amounts
  • the exposure of the insulator layer with medium and / or test substance can be carried out under all possible ambient conditions, and is preferably carried out at room temperature, whereby a particularly simple production of the arrangement according to the invention is ensured.
  • medium and / or test substance at a temperature which is above and in particular below the usual room temperature, for example 23 ° C is suitable, for example, a 4 ° C warm solution of the test substance, whereby even sensitive test substances are particularly easy to and / or in the insulator layer can be coupled.
  • the at least one medium is preferably selected to be formed from molecules and / or solids dissolved therein which, under the conditions chosen in the process, can not chemically react with the insulator layer, i. especially no covalent bond with the inorganic insulator layer, i. 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 buffer component is selected.
  • a silicon dioxide layer e.g. a mixture with an organic solvent, water and a dissolved salt or a dissolved buffer component is selected.
  • the method is preferably carried out with three, two or one method step (s), depending on the type of arrangement to be produced.
  • the insulator layer is successively treated with an organic solvent, e.g. DMF, an aqueous liquid, e.g. saline / buffer, and the test substance, e.g. a dissolved peptide, preferably in the order mentioned.
  • the at least one medium (4) comprises three media or components, e.g. 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 e.g. is dissolved in the concentration ranges 1 ⁇ M to 1 mM.
  • the insulator layer is treated with an organic solvent, preferably volatile at room temperature, e.g. DCM, and subsequently with an aqueous, i. Water-containing, liquid in which the test substance is at least partially dissolved, e.g. with a buffered solution of a protein, applied.
  • an organic solvent preferably volatile at room temperature, e.g. DCM
  • an aqueous, i. Water-containing, liquid in which the test substance is at least partially dissolved e.g. with a buffered solution of a protein, applied.
  • the insulator layer is charged with a solution in which the test substance is at least partially dissolved.
  • 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.
  • a direct coupling which is stable in air and in solutions, in particular without silanized or polymerized or cross-linked (eg polyvinyl, cellulose) interlayer, is formed within the at least one exposed area (3), in particular if a semiconductor substrate (1) is used for the method according to the invention.
  • a coupling or immobilization of the test substance to the insulator layer which is stable in air and in solution, i.
  • the coupled substance is also washed by incubation with liquid, e.g. after one or several hours or one or more days of washing of the arrangement formed by the method or the area (3).
  • a physiological buffer still detectably coupled to the insulator layer (2).
  • 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 coupling means that the test substance or its molecules are bound by direct contact with the molecules of the inorganic insulator layer in region (3), preferably by interaction of a nucleophilic group of the test substance, in particular a thiol, with the surface molecules of the insulator layer are connected a covalent or, in particular, electrostatic compound or interaction, in particular between the nucleophilic group of the test substance and the surface molecules of the insulator layer, is formed so that the test substance is stably and detectably coupled to and / or in the insulator layer.
  • a nucleophilic group of the test substance in particular a thiol
  • glass is used or selected accordingly as a substrate and as a bonding layer is a non-organically modified, and not metallically modified, eg coated with a gold layer, formed on the glass surface or boundary layer selected.
  • 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) to understand.
  • 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.
  • 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 preferred for the method and which are particularly preferred for the method apply correspondingly to the solid and the Silicon oxide layer, and for the medium (4) and to be coupled to the silicon oxide layer test substance.
  • a solid substrate (solid substrate (1) ) is set or selected with at least one silicon oxide layer (silicon oxide layer (2)) formed thereon, wherein the at least one silicon oxide layer (2) is not metallized or not organically modified, eg non-silanized or, in particular, untreated or unmodified.
  • the above and below specified features of the semiconductor substrate and the inorganic insulator layer particularly preferred for the method apply correspondingly to the solid substrate and the silicon oxide layer, as well as to the medium (4) and the test substance (5) to be coupled to the glass surface layer.
  • the test substance (5) is in particular coupled to the insulator layer (2) in such a way that it can be probed with at least one test substance preferably detectable by radiation and / or ionization, with which the arrangement formed by the method is determined.
  • association events are detectable detectable 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 region (3) 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.
  • all possible devices for detecting radiation eg scanners, imagers, IR ellipsometers, scintigraphs or x-ray devices, and all common mass spectrometers, eg MALDI or ESI, are suitable as detectors for detecting the arrangement formed by the method according to the invention.
  • the advantage of this is that the arrangement formed by the method usually fast and easy with already existing laboratory equipment and can be examined with known measuring methods in diagnostics and research.
  • the range (3) is applied in particular with a concentration or amount of test substance suitable for the detection, since an advantageous characteristic of the method according to the invention is the formation of a concentration-dependent coupling of the test substance to and / or in the insulator layer (2) within the applied range ( 3), ie that with decreasing concentration of the selected test substance also subsequently decreasing detection signals are observable.
  • 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.
  • 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.
  • 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.
  • a concentration of the test substance of ⁇ 1 mM, preferably ⁇ 100 ⁇ M, more preferably ⁇ 10 ⁇ M is selected or used for the application.
  • a solution of the test substance in a concentration of ⁇ 1 ⁇ M, 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.
  • the test substance for example, very large or particularly active, for example formed as a cell,
  • a concentration of the test substance is suitable which is ⁇ 100 pM, preferably ⁇ 10 pM, more preferably ⁇ 1 pM.
  • test substance is applied to the bonding layer (2) in the form of a solution or suspension
  • conventional or conventional volumes of solution are suitable, in particular, as in standard processes or others
  • a volume of test substance solution or suspension in the nanoliter range e.g. 1 nl or less suitable for the production of low
  • Density chips may be capable of microliter volume (e.g., 1 ⁇ l or less).
  • test substance or a concentration in several, in particular at least two, preferably three, more preferably more than three, replicas or replicates on the
  • Insulator layer preferably applied in a localized manner.
  • Detection systems allow, e.g. the parallel measurement of different samples which are locally addressed, e.g. as a pattern or regularly applied to the biochip according to the invention.
  • a dilution series of the coupled test substance on the solid phase is produced by preferably spatially addressed loading of the isolate layer (2) with at least two, preferably at least three, more preferably more than three different concentrations of a test substance.
  • a millimolar or micromolar solution eg with a concentration of 1 mM to 1000 mM, preferably ⁇ 100 mM, more preferably ⁇ 10 mM, or with a concentration of 1 nM to 1000 nM
  • the test substance is selected as the starting solution, and diluted, for example by means of conventional dilution series, eg : 1, 1: 5 or 1:10 dilution steps, and preferably both the starting solution and the diluted solutions / n are local addressed, in particular at different locations or multiply in one place, applied to the attachment layer.
  • the material parameters are preferably selected such that, in particular after incubation of the assembly thus produced with a sample, the on and / or in which one area (3) is distinguishable in particular by signals detectable by radiation and / or 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 (8)) can be detected if necessary.
  • semiconductor substrate (1) in principle all known solids or materials with 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.
  • an arrangement which can be used as a preferably planar biochip and / or as a 1D, 2D or 3D biochip is made by means of the method according to the invention, which enables an optical or scintigraphic detection of association events by means of an external detector.
  • a semiconductor substrate (1) with the insulator layer (2) formed thereon is preferably selected in the usual dimensions of an object carrier or an object carrier insert, whereby an arrangement corresponding in size to the common standard for biochips is produced.
  • a semiconductor substrate (1) with the insulator layer (2) formed thereon in the dimensions length x width (7.5 +/- 1) cm x (2.5 cm +/- 0.5 cm , preferably exactly 7.5 cm x 2.5 cm with the usual deviations in their production, for example.
  • length x width (7.5 +/- 1) cm x (2.5 cm +/- 0.5 cm , preferably exactly 7.5 cm x 2.5 cm with the usual deviations in their production, for example.
  • a thickness of> 0.3 mm, in particular of 1 mm (+/- 0.7 mm ) preferably exactly 1 mm, selected with the usual production-related deviations, whereby a particularly good handling of the device to be produced is ensured.
  • the semiconductor substrate (1) is particularly preferably a silicon wafer or a part thereof, in particular a saw made of a silicon wafer or by other division, e.g. by scratching and breaking the wafer or by means of a liquid jet or a laser, chip obtained selected.
  • This has u.a. the advantageous property that the arrangement is particularly accurate in their material properties or dimensions produced and thus allows a particularly good reproducibility of the arrangements or detection systems 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, e.g. chosen by means of a ball mill or tribomechanical process, particles formed as a semiconductor substrate, but the production is also possible by other dicing methods.
  • a semiconductor substrate (1) with the insulator layer (2) formed thereon, which is formed as an oxidized silicon wafer or as a part thereof, is selected for the method according to the invention.
  • Silicon wafers usually have nearly 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 and the arrangement and measurement results produced thereby is ensured.
  • a semiconductor substrate (1) is selected which is in the form of amorphous, polycrystalline silicon or preferably monocrystalline silicon, in particular with a ⁇ 111 ⁇ or ⁇ 100 ⁇ or ⁇ 110 ⁇ preferred orientation, particularly preferably with ⁇ 111 ⁇ .
  • -Vorzugsorienttechnik is formed, whereby an arrangement which is formed on a nearly perfect and reproducible support material can be produced.
  • a ⁇ 100 ⁇ preferred orientation of the semiconductor substrate (1) has proven to be particularly suitable for the method according to the invention, wherein the arrangement is particularly simple and inexpensive to produce by the technical characteristics and wide distribution of this material.
  • other preferred crystalline orientations of the silicon material are also suitable for the process according to the invention.
  • a directed coupling of the test substance has.
  • 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.
  • 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 the case of optical measurements, a particularly favorable signal-to-noise ratio due to low background signals is reachable.
  • a semiconductor substrate (1) is selected for the method according to the invention, which is preferably completely homogeneously formed, for example a silicon material which is uniform throughout its entire dimensions, for example a silicon which is doped throughout and which is doped with only one species / species of foreign atoms Chip, is used, whereby particularly simple uniform carrier properties of the arrangement, in particular over the entire insulator layer (2) can be produced.
  • Silicon substrates preferably unpolished silicon wafers or formed therefrom
  • Chips chosen or used, since they, for example, a particularly simple and uniform incubation of the assembly surface with a sample, even without
  • an inorganic insulator layer is selected, the surface of which is cleaned prior to exposure to medium and test substance, e.g. by means of an acid or base and / or an organic solvent, in particular using a
  • Ultrasonic bath or a shaker If, for example, an arrangement with particularly favorable, eg flat, surface properties are to be produced, a silicon substrate etched with hydrofluoric acid and subsequently oxidized by Carow's acid is selected as the semiconductor substrate with the insulator layer formed thereon, which is preferably dried in an oven.
  • an inorganic insulator layer which is formed by chemical reaction from the material of the semiconductor substrate (1) is preferably selected, thereby forming a particularly ordered and strong bonding layer on the semiconductor substrate (1 ) can be used for the inventive method.
  • 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 silicon dioxide layer (7) selected, in particular with or without inclusions of impurities (doping).
  • silicon dioxide layer (7) selected, in particular with or without inclusions of impurities (doping).
  • a silicon dioxide layer (7) is selected as the inorganic insulator layer (2), which is in particular in the at least one region (3; 8) as a non-natural, preferably dry and / or thermal oxide or as a dry and / or thermal nitride is formed, whereby particularly advantageous properties of the manufacturable by the method according to the invention arrangement, eg be ensured by a spongy formed stable structure of the insulator layer.
  • a porous inorganic insulator layer e.g. is formed wet-chemically on or in the semiconductor substrate and then preferably dried in the oven, is particularly suitable as a bonding layer.
  • an inorganic insulator layer (2) which is in the form of a 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, for the reflection and amplification of radiation at the boundary layers, the manufacturable arrangement are present.
  • the oxide thickness also includes the optical properties of the insulator layer, for example a blue or red coloration due to reflection of light in the corresponding one Wavelength at the silicon / silicon oxide interface, can be used specifically for various applications of Arrangement of advantage.
  • the oxide thickness of the insulator layer (1) on a silicon wafer (semiconductor substrate (1)) in particular electrochemically and preferably thermally produced oxides are suitable, since by means of which the arrangement is particularly simple and reproducible in their properties, in particular the material properties of the bonding layer , can be produced or used.
  • 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.
  • a gas consisting predominantly of oxygen is selected as the semiconductor substrate with the insulator layer formed thereon.
  • an electrochemically formed oxide which is preferably dried in an oven is used as the inorganic insulator layer.
  • a liquid which preferably comprises at least one solvent, also referred to below as solvent (9)
  • solvent (9) preferably comprises at least one solvent, also referred to below as solvent (9)
  • solvent (9) 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.
  • a medium (4) is selected for the loading, which comprises 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.
  • a liquid solvent (9) which is formed as an aqueous and / or organic solvent, in particular as a hydrophilic and / or polar solvent.
  • water for example having a neutral pH, is to be understood as aqueous solvent.
  • an alcohol, dialkyl acid amide, alkyl halide, pyrollidone, furan, DMSO, acetonitrile, acetone or one of their derivatives is preferably suitable, for example, ethanol, DMF, dichloromethane, NMP, THF or one of their derivatives.
  • the medium (4) chosen for the process according to the invention preferably comprises or is formed as such water having an acidic or basic pH and / or an organic solvent having an acidic or basic pH, an electrolyte and / or a buffer , Particularly suitable is a medium having a basic, in particular physiological, pH value is selected.
  • the medium (4) used is preferably 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 deficiency of organic solvent.
  • the use of a percentage only small volume fraction of the organic solvent in the mixture, in particular ⁇ 10%, preferably ⁇ 1%, particularly preferably ⁇ 0.1%, is suitably selected, whereby also the coupling of particularly sensitive to organic solvents test substances, e.g. of proteins or cells.
  • an at least partially electrically conductive medium (4) in particular with a conductivity> 0.05 ⁇ S / cm, preferably> 20 ⁇ S / cm, particularly preferably> 1 mS / cm, particularly preferably> 50 mS / cm, especially if it contains a dissolved solid, preferably the test substance and / or a buffer component and / or a salt, preferably the test substance and a buffer component, particularly preferably the test substance and a dissolved salt.
  • a non-electrically conductive medium may be suitable.
  • At least two non-silanized regions or regions (3; 8) preferably not covertly coated with organic molecules are subjected to at least one medium (4) and in each case at least one test substance on the isolator layer, for example by pipetting suitable, in particular not running into each other (spot) volumes that at least two similar or in particular differently functionalized areas are formed.
  • the at least two areas are in particular a plurality of preferably spatially separated districts to understand that are formed either as a replica, eg several areas, which are acted upon similarly, or in each case with different test substances or with different concentrations of a test substance are applied, wherein a plurality of differently functionalized areas (3; 8) are formed.
  • the application is carried out in such a way, e.g. by pipetting or automated spotting to form at least two non-silanized coupons (3; 8) coupled at least partially or completely to an environmental region (10) formed on and / or in the insulator layer (2), i. a preferably not impacted with test substance district of the insulator layer are surrounded.
  • This also makes possible a simple delimitation 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; 8) act as spots, preferably spatially separated from each other and / or Locally addressed spots are formed.
  • 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.
  • At least two regions (3; 8) are each exposed to a test substance dissolved at least partially in the at least one medium (4), in particular with a mixture comprising aqueous buffer solution, organic solvent and dissolved test substance.
  • a test substance dissolved at least partially in the at least one medium (4), in particular with a mixture comprising aqueous buffer solution, organic solvent and dissolved test substance.
  • 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.
  • the test substances to be coupled are preferably dissolved in the same medium.
  • the at least two non-silanized regions (3; 8) are produced as an array, in particular as a microarray and / or low-density array, thereby enabling a particularly good readability and utilization of the arrangement according to the invention.
  • arrays according to the invention all common application techniques, eg pipetting, and customary volumes are suitable, which are used in the conventional application of glass carriers with sample substances for producing a biochip array, in particular a low-density or microarray.
  • the surrounding area (10), which preferably completely surrounds at least one and particularly preferably at least two non-silanized areas (3; 8), is preferably formed in such a way that it is not exposed to test substance, and in particular as only medium (4) acted upon or preferably untreated silicon dioxide of the insulator layer (2) remains.
  • the surrounding area (10) is chemically modified, in particular with organic molecules, with the exception of the test substance.
  • rapid usability or reduction of nonspecific background signals is particularly easy to produce, preferably if the surrounding area (10) is provided with blocking reagent and / or a nucleophilic compound, preferably with a thiol, e.g. Mercaptoethanol or cysteine is applied.
  • the passivation of the surrounding area (10) can, however, also be produced, for example, during the subsequent incubation of the arrangement according to the invention with a sample containing blocking reagent or serum.
  • 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.
  • molecules with at least one nitrogen, sulfur, oxygen, phosphorus, halogen, dye and / or radioactive isotope are selected as the test substance or test substances, or the selected test substance comprises these, whereby a particularly stable connection or coupling between the insulator layer and the test substance is made possible.
  • At least two different test substances (5, 11) are selected as the at least one organic test substance (5), i. that at least two different test substances or at least two different concentrations of a test substance are preferably spatially separated or 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 terminal arranged in the molecule, nitrogen, sulfur, oxygen, phosphorus, halogen, dye or radioactive isotope are or include.
  • a device for location-dependent association with and for the detection of a plurality of different test substances from a sample is produced in a particularly simple manner or it can subsequently be prepared from the test substance, e.g. an enzyme, modifications effected, e.g. are made visible by enzymatic cleavage of the coupled sample substance, and / or it is thereby produced a particularly stable and directed coupling of the test substance to the inorganic bonding layer.
  • the at least one selected organic test substance (5) comprises at least one nucleophilic chemical group or function, in particular a thiol, amino, hydroxyl, carboxyl, cyano, nitrile, Si, Se, Te, sulfate, sulfide, alkene or alkyne, or phosphine group, whereby the Stabiltusch the coupling is particularly well ensured, in particular by a thiol function, eg from a cysteine residue.
  • a nucleophilic chemical group or function in particular a thiol, amino, hydroxyl, carboxyl, cyano, nitrile, Si, Se, Te, sulfate, sulfide, alkene or alkyne, or phosphine group
  • oligonucleotides the 5 'or the 3' end of the oligonucleotide sequence.
  • the bioorganic test substance selected as the at least one organic test substance (5) is preferably a linker molecule, amino acid, Carbohydrate, lipid, dye, drug, biotin, peptide, polypeptide, oligonucleotide, protein, cell compartment, cell, or structure derived therefrom or comprising, whereby the arrangement is in principle universal for a variety of applications, in particular for detecting all possible bioorganic components or structures (test substances) can be produced.
  • test substance (s) (5) is particularly suitable, since the process according to the invention is useful i.a. allows the production of peptide and / or protein chips with particularly advantageous properties.
  • the at least one organic test substance (5) is particularly preferably chosen or the test substance contains the DNA, RNA, cDNA, mRNA, cRNA, PNA, receptor, ligand, protein A, protein G, streptavidin, antibody or structure thereof, whereby the Specificity of the association or interaction with the bioorganic test substance is particularly well ensured.
  • Particularly preferred as test substance (5) is 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.
  • the at least one organic test substance (5) one or more peptides from preferably L and / or D-amino acid residues and / or glycine and / or ß-alanine, in particular from the genetically encoded amino acids and or their preferably naturally occurring modifications, for example phosphoserine, threonine, or tyrosine, in particular also, for example, with a radioactive or dye marking, chosen, or the Profubststanz includes these.
  • Peptides as a test substance generally have the advantage that they are generally easy to prepare synthetically and in high purity and can have comparable association properties with whole proteins, for example when the peptide is preferably N- or C-terminal with a cysteine residue formed antibody epitope is selected.
  • 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.
  • 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 variety of requirements.
  • the at least one organic test substance (5) is selected as linker molecules with free / unbound chemically reactive groups or comprises these.
  • linker molecules in particular all possible molecules, with the exception of silanes, are to be understood which have two different, preferably end and / or side, molecules arranged in the molecule, different chemically reactive groups or functions, the molecules via a function of the insulator layer (2) be coupled and possibly connected via the other function with bio-organic substances.
  • cysteine is chosen as the test substance, it is preferably coupled to the insulator layer via the thiol function of its side chain, and a further substance or capture molecules, e.g. attach an amino acid residue or a peptide.
  • streptavidin may be coupled to the surface, thereby, for example, sandwiching arrangements (e.g., insulator layer-biotin-streptavidin-biotinylated component).
  • sandwiching arrangements e.g., insulator layer-biotin-streptavidin-biotinylated component.
  • the immobilization on the test substance or the linker can also be carried out chemically, covalently and / or by electrostatic or hydrophilic interactions.
  • 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, is coupled to the insulator layer (2).
  • a bioorganic substance eg an oligonucleotide or a protein
  • a nucleophilic one Group preferably a thiol
  • the method according to the invention particularly preferably arranges the at least one test substance (5; 8) with the majority of its molecules coupled to the surface spatially directed on the isolate layer, i. that the molecules are uniformly aligned, 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 layer is coupled in particular via the thiol group to the attachment layer (2) and is otherwise freely accessible for the optionally subsequent interaction with a test substance.
  • peptides or other synthetically produced molecules are preferably selected as the test substance, these are preferably uniform with a nucleophilic group, e.g. a terminal or lateral thiol, whereby in particular even with several at least partially similar test substances, e.g. Wild-type sequence and mutant, a particularly uniform and directed binding and a good comparability of the results is ensured.
  • a nucleophilic group e.g. a terminal or lateral thiol
  • excess medium and / or excess test substance is preferably removed from the surface by washing.
  • volume of the solution containing the test substance are selected which lead to a rapid evaporation of the solvent under normal conditions, for example at room temperature.
  • the excess, uncoupled substances that remain on the assembly after evaporation of the solvent are then subsequently passed through Washing from the surface, preferably with an aqueous liquid, preferably a buffer solution, more preferably water, removed.
  • the exposure to the test substance is preferably below the usual room temperature, preferably ⁇ 23 0 C particularly preferably ⁇ 5 ° C, and optionally with solvent additives, a rapid evaporation of the At least hinder solvent, eg oil, and the surface is washed after exposure to the or at least partially dissolved test substance (s) and before the complete evaporation of the solvent accordingly.
  • the At least hinder solvent eg oil
  • the surface is advantageously washed accordingly.
  • the arrangement is dried after washing or possibly 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 is brought into contact with at least one sample, also referred to below as sample (13), which optionally contains a test substance, also referred to below as test substance (14).
  • the at least / at least one region (3), to which the at least one test substance (5) is coupled is brought into contact with the at least one sample (13), for example by direct application by means of pipetting the sample the at least one region or indirect application of the sample in the vicinity of the region, for example in or on the surrounding region (10), and subsequent distribution or supply to the at least one region (3).
  • 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.
  • a gas, solid, liquid, and / or a mixture thereof is used as a sample, more preferably a sample containing at least one test substance, e.g. an analyte and / or a control, preferably at a detectable concentration.
  • test substance e.g. an analyte and / or a control, preferably at a detectable concentration.
  • sample (13) a solution, emulsion, and / or suspension which optionally contains microparticles or other undissolved components, preferably containing at least one at least partially dissolved test substance.
  • the sample (13) used is body fluid, preferably urine, blood or serum, mucous tear fluid, sweat, semen, cerospinal fluid and / or a stool sample or the sample contains same.
  • At least two different samples (13; 16) are used as the at least one sample (13), which are preferably placed on the surface of the arrangement in a location-addressed manner, in particular each on a replica of the coupled test substance (s), thereby also having only one arrangement different samples can be examined simultaneously.
  • 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.
  • a sample which contains 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 1 IgE, IgM, kinase, phosphatase or antibody epitope.
  • a sample which contains a test substance (14) which interacts with the test substance (5) coupled in the region (3) without silanized intermediate layer to the insulator layer (2), in particular associated with the test surface (5) or enzymatically altered.
  • Particularly preferred according to the invention is the use of a sample which contains a test substance (13) which interacts verifiably with at least one test substance coupled to and / or in at least one area (3; 8).
  • a sample which contains a test substance (14) which is preferably covalently linked to a radiation-active marker, e.g. a labeled peptide, or subsequently linked, e.g. by incubation with a labeled secondary antibody.
  • a radiation-active marker e.g. a labeled peptide
  • the arrangement preferably the at least one region (3; 8), 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, I lean or mass spectrometer read.
  • FIG. 1 shows schematically A and C an example of the state of the art and B and D an example of the arrangement according to the invention (oblique view from above, detail from a biochip, in the case of a microparticle the surface is correspondingly completely / consistently coated with a test substance) ,
  • Figure 1 A On the surface of a carrier substrate (a) of a solidified melt (glass) is a deposited by silanization 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) of the silane derivative, which has a chemical function or group to which in a subsequent step, one or more test substances (d) are attached in the form of an array.
  • the areas (spots) with test substance (s) are surrounded by the organic layer (c), whose reactive groups are subsequently blocked.
  • FIG. 1B on a semiconductor substrate (s) with the features according to the invention, e.g. on a part of a silicon wafer, an inorganic insulator layer (f) of the present invention, e.g. a silicon dioxide layer, disposed on or in the surface thereof by direct interaction of the surface molecules of the
  • 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 inorganic insulator layer, which is optionally subsequently, for example, blocked with a thiol or blocking reagent or in the inventive use, for example by contacting with body fluid.
  • FIG. 1C Schematic cross section through the arrangement (FIG. 1A) in the region of the connected test substance (s).
  • the arrangement comprises a large-area organic layer of silane derivative (c) to which the test substance (s) are attached in a localized manner. Since the organic layer (c) usually has hydrocarbon compounds, there may be troublesome interactions between the organic layer (c) and the test substance (s).
  • FIG. 1 D Schematic cross section through the arrangement according to the invention (FIG. 1B) in the region of the coupled test substance (s).
  • 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).
  • Figure 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 of the invention
  • B and E an example of a particular embodiment of the invention
  • 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.
  • FIG. 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 Insulator layer having a chemical function or group (except silanes and derivatives thereof) of a test substance, for example with a thiol, one or more test substance (s) (c) according to the invention, eg peptide / e with a preferably terminal cysteine residue, are coupled, eg as Array of different test substances and / or different amounts of a test substance, preferably as a replica.
  • a glass substrate 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 Insulator layer having a chemical function or group (except silanes and derivatives thereof
  • 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 glass surface layer (b), 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.
  • Figure 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, e.g. on a plastic, silicon (e), e.g. by sputtering or sputtering the solid surface with a silane which has been oxidized in the region of its surface, e.g.
  • test substance e.g. Peptide / s with a preferably terminal cysteine residue, e.g. as an array of different test substances and / or different amounts of a test substance, preferably as a replica.
  • test substance 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) of test substance (s) are surrounded by the silicon dioxide layer (g), which may be subsequently, e.g. with a thiol or blocking reagent or in the use according to the invention, e.g. by contacting with body fluid, is blocked.
  • FIG. 2C On a solid (i) according to the invention, eg on a plastic, a silicon oxide layer 0), for example by deposition of silicon dioxide (eg from a plasma), is arranged on or in the surface thereof by direct interaction of the surface molecules of the silicon dioxide layer with a chemical function or group (excluding silanes and their derivatives) of a test substance, eg with a thiol, one or more test substance (s) (k) according to of the invention, for example peptide / s with a preferably terminal cysteine residue are coupled, for example as an array of different test substances and / or different amounts of a test substance, preferably as a replica.
  • a test substance (s) (k) for example peptide / s with a preferably terminal cysteine residue are coupled, for example as an array of different test substances and / or different amounts of a test substance, preferably as a replica.
  • 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.
  • FIG. 2 D Schematic cross-section through the arrangement according to the invention (FIG. 2 A) in the region 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).
  • FIG. 2E Schematic cross section through the arrangement according to the invention (FIG. 2B) in the region 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 silicon dioxide layer (g) on or in the silicon dioxide layer (I).
  • FIG. 2F Schematic cross section through the arrangement according to the invention (FIG. 2C) in the region 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 silicon dioxide layer (j) on or in the silicon dioxide layer (m).
  • FIG. 3 schematically shows cross sections (corresponding to FIG. 1D) of typical
  • FIG. 1 D The hatching / pattern of the semiconductor substrate, the inorganic insulator layer and the coupled test substance (s) is shown in FIG. 1 D.
  • the roughly hatched area of FIG. 3 AG corresponds to the cross section of the glass substrate or the solid and the finely hatched area / s to the cross section / the glass surface layer (s) or the silicon dioxide layer (s).
  • FIG. 3A shows an arrangement in which the surface of the semiconductor substrate is completely covered with an inorganic insulator layer, preferably uniform thickness / height.
  • Figure 3B shows an arrangement at the two sides of the semiconductor substrate (top and bottom) with an inorganic insulator layer of thickness / height x, e.g. a silicon dioxide layer, are covered and at least two sides with an inorganic insulator layer, e.g. a silicon dioxide layer occupied thickness / height ⁇ x, e.g. an arrangement with a chip that 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.
  • an inorganic insulator layer of thickness / height x e.g. a silicon dioxide layer
  • an inorganic insulator layer e.g. a silicon dioxide layer occupied thickness / height ⁇ x
  • FIG. 3C shows an arrangement in which the semiconductor substrate is not completely covered by the inorganic insulator layer, to which at least two test substances or at least two different amounts of a test substance are coupled in a location-addressed manner.
  • FIG. 3 D 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 quantities of a test substance are respectively coupled.
  • FIG. 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 Isolator layers are larger (left) and / or identical (right) with the surface exposed to test substance.
  • FIG. 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 quantities of a test substance are respectively coupled.
  • FIG. 4 schematically shows special embodiments of the arrangement according to the invention.
  • the hatching / pattern of the semiconductor substrate, the inorganic insulator layer and the coupled test substance (s) is according to FIG. 1 D.
  • Figure 4 A shows a cross-section through an arrangement with recesses in the surface, wherein the test substance (s) on and / or in the depressions, e.g. are formed as hollow cylinders, as hollow cones, as grooves or as a combination thereof.
  • Figure 4B shows a cross-section through an array with continuous recesses in the semiconductor substrate, e.g. are formed as a bore or channels, wherein the test substance (s) are coupled to and / or in the recesses.
  • FIG. 4 C shows a plan view of an arrangement according to FIG. 4 A.
  • FIG. 4D shows a plan view of an arrangement according to FIG. 4B.
  • silicon in various embodiments (monocrystalline silicon with a ⁇ 111 ⁇ , ⁇ 100 ⁇ or ⁇ 110 ⁇ orientation, polycrystalline Silicon or amorphous silicon which was p- (boron) or n- (with phosphorus, antimony or arsenic) or undoped and exhibited electrical resistance depending on the doping, the surface of the silicon atom being atomically smooth, rough, polished, not polished or porous), with either the silicon formed as a wafer or as a part thereof or deposited on different solids (plastic, wood, metals (eg aluminum) or glass).
  • glass substrate according to the other solution of the object of the invention were glass slides and as a solid according to the further solution of the object of the invention were plastic or polymer films used.
  • silicon dioxide was used in various embodiments, which was formed as a native or thermal oxide of different thickness on or from the silicon, as the glass surface layer according to the other solution of the object according to the invention non-organically or metallically modified surfaces of glass slides were used, and As the silicon oxide layer according to the further solution of the object according to the invention, silicon oxide layers deposited on the solid were used.
  • a layer of silicon dioxide with a thickness of> 4 nm, which was formed in particular as a thermally generated oxide has surprisingly proven to be particularly advantageous.
  • the surfaces to be coated with test substances were either contacted successively with an organic solvent and an aqueous solvent or aqueous solution, or were contacted with a mixture with an organic and an aqueous solvent or were untreated.
  • the surfaces treated with liquid were dried.
  • test substances used were in particular peptides, proteins, oligonucleotides or small (molar mass> 75 and ⁇ 2000 g / mol) of organic molecules which were dissolved in water or an aqueous solution, with or without addition of an organic solvent.
  • the solutions of the test substances were applied to the inorganic insulator layer or the glass surface layer or the silicon oxide layer either by means of a pipette, an automatic pipetter or an arrayer, or the respective surface to be coated was wetted or completely bathed with the test substance solution.
  • 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 subsequently localized with biotinylated peptides, proteins or oligonucleotides, eg as an array.
  • Uncoupled test substance was optionally subsequently removed from the surface by washing and / or the non-test substance areas of the surface were removed by blocking reagent, e.g. Blocking buffer and / or a nucleophile, e.g. Thiol, blocked.
  • blocking reagent e.g. Blocking buffer and / or a nucleophile, e.g. Thiol, blocked.
  • Excess blocking reagent was optionally removed from the surface by washing and / or the surface of the assembly was dried.
  • the assembly was incubated with at least one solution containing at least one at least partially dissolved test substance, in particular peptides, proteins, oligonucleotides, cell compartments, cells and / or small (molar mass> 75 and ⁇ 2000 g / mol) organic molecules, wherein the Solution with the test substance was optionally removed from the surface after incubation and / or the surface was washed and / or dried.
  • test substance in particular peptides, proteins, oligonucleotides, cell compartments, cells and / or small (molar mass> 75 and ⁇ 2000 g / mol) organic molecules
  • the assembly was subsequently probed with a dissolved labeled probe, e.g. a dye-labeled secondary antibody, which was optionally subsequently removed from the surface and / or the surface of the arrangement is washed and / or dried.
  • a dissolved labeled probe e.g. a dye-labeled secondary antibody
  • Figure 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 wafer (p-doped, boron, 111 orientation, size 75mm x 25mm x 1mm + - 0.1mm, polished side, thermal oxide 1000 A + - 70, spot diameter approx. 1mm).
  • biotinylated cysteine ie biotin linked via its carboxyl group as amide to the amino group of L-cysteine
  • Figure 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 1000 A + -70, spot diameter approx. 1mm).
  • Figure 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, thermal Oxid 1000 A + -70, spot diameter approx. 1 mm, after incubation with HRP-labeled streptavidin and visualization with luminol on a Lumi imager shows signals that are dependent on the concentration of the applied test substance.
  • FIG. 8 shows, for example, spotted biotinylated cysteine (1 mM, 0.1 mM, 0.01 mM) on sawn Si wafers (p-doped, boron, 111 orientation, size 75 mm ⁇ 25 mm ⁇ 1 mm + -0.1 mm), unpolished side, 1mm, after long term test, incubated after 3 months with HRP-labeled streptavidin and visualized with luminol on Lumi-Imager, storage at 25 ° C, 4 ° C and -2O 0 C (from above downward).
  • Figure 9 shows, for example, spotted Tab2 peptides (in 0.1 M TBS buffer, the concentrations 1M, 0.1M, 0.01M) N-terminally extended with cysteine (columns 1-6, columns 7-9 without extension). incubated with antibody sandwich after 30 days, on sawn Si wafer (p-doped, boron, 111 orientation, size 75mm x 25mm x 1mm + - 0.1mm), unpolished side, thermal oxide 1000 A + -70, tapping diameter ca. 1mm, upper image detected on the Lumi imager with HRP-labeled antibody, lower image detected on the Affymetrix scanner with fluorescence-dye-labeled antibody.
  • test substances cyste-biotin, protein (streptavidin), peptide (antibody epitope)
  • test substances cyste-biotin, protein (streptavidin), peptide (antibody epitope)
  • test substances streptavidin, biotin and monoclonal antibody
  • FIG. 11 shows schematically the location-addressed binding of biotin on an inorganic insulator layer on a semiconductor substrate (triangular).
  • a biotin-cysteine solution is pipetted onto the insulator layer and then mixed with
  • Biotin-streptavidin-biotinylated antibody Biotin-streptavidin-biotinylated antibody
  • Figure 12 shows schematically the location-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.
  • ab2 cysteine-labeled epitope
  • FIG. 13 schematically shows 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.
  • FIG. 14 shows arrangements according to the invention, which are produced as oligonucleotide microarrays and can be used for the detection of oligonucleotides.
  • dye-labeled oligonucleotides with different linkers on oxidized Si wafers p-doped, boron, 111 orientation, thermal oxide 1000 A + -.
  • Si wafers p-doped, boron, 111 orientation, quantitative oxide 1000 A + -70, coupled and read by Affymetrix scanner
  • Group 1 Amino-modified oligo SES, Cy5 labeled + C6-SFB (aldehyde-forming) group of 4: Amino-modified oligo SES, Cy5 labeled + C6-SANH (makes
  • Group 4 amino-modified oligo SMS, Cy5 labeled + C6-SFB (aldehyde-forming) group of 4 5: amino-modified oligo SMS, Cy5 labeled + C6-SANH (makes
  • Group 4 amino-modified oligo ESE, Cy5 labeled + C6-SFB (aldehyde-forming) group of 4 8: amino-modified oligo ESE, Cy5 labeled + C6-SANH (makes
  • Group 4 Amino Modified Oligo MEM 1 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 of four 13 thiol-modified oligo SES
  • Cy5 labeled group of four 14 thiol-modified oligo SMS
  • Cy5 labeled group of four 15 biotin-modified oligo SES
  • Group 18 Water A specific binding of the oligonucleotides and subsequently a specific binding and detection of dissolved oligonucleotides as test substances are shown.

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Abstract

L'invention concerne un dispositif et son utilisation pour déterminer de manière spécifique des substances d'échantillons, notamment une biopuce comprenant un support sur lequel se trouve une couche de liaison. Le dispositif comporte un support sur lequel est formée une couche de liaison à laquelle est couplée une substance de contrôle. Le support est semi-conducteur et la couche de liaison est au moins une couche d'isolation inorganique formée sur le support. Sur et/ou dans la couche d'isolation, au moins une zone est soumise à l'action d'au moins un milieu et d'au moins une substance de contrôle organique. Dans cette zone, la couche d'isolation est couplée à la substance de contrôle, laquelle peut interagir avec une substance d'essai. L'invention porte également sur un procédé de réalisation de ce dispositif, selon lequel au moins une zone d'une couche d'isolation inorganique formée sur un support est soumise à l'action d'au moins un milieu et d'au moins une substance de contrôle organique, et des interactions entre la substance de contrôle organique et la couche d'isolation inorganique provoque un couplage entre la substance de contrôle organique et la couche d'isolation inorganique, ladite zone étant formée pour interagir avec une substance d'essai et/ou interagissant avec cette substance d'essai lorsqu'elle est appliquée.
PCT/DE2007/001362 2006-07-31 2007-07-31 Dispositif de détermination de substances, procédé de réalisation et utilisation de ce dispositif WO2008019651A2 (fr)

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EP1215186A1 (fr) * 2000-12-14 2002-06-19 Commissariat A L'energie Atomique Support solide pour l'immobilisation d'oligonucléotides
WO2002048691A1 (fr) * 2000-12-14 2002-06-20 Commissariat A L'energie Atomique Dispositif de renforcement de fluorescence large bande a faibles pertes et capteur optique biologique ou chimique l'utilisant.
US20030082658A1 (en) * 1998-04-10 2003-05-01 Francois Mallet Method for fixing a biological molecule on a support surface
EP1364702A2 (fr) * 2002-05-15 2003-11-26 Samsung Electronics Co., Ltd. Procédé de fabrication d'une plaque matrice de biomolecules avec des zones hydrophiles et hydrophobes
EP1542009A1 (fr) * 2002-08-12 2005-06-15 Hitachi High-Technologies Corporation Procede de detection d'acide nucleique par utilisation de microreseaux d'adn, et appareil de detection d'acide nucleique
US20060121501A1 (en) * 2004-10-22 2006-06-08 Stanislaw Burzynski Method for immobilizing molecular probes to a semiconductor oxide surface

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US6707038B2 (en) * 2001-02-14 2004-03-16 Picoliter Inc. Method and system using acoustic ejection for selective fluid deposition on a nonuniform sample surface
CA2440146A1 (fr) * 2001-03-30 2002-10-24 Amersham Biosciences Ab Analyse par biopuces de polymorphismes a simple nucleotide
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US20030082658A1 (en) * 1998-04-10 2003-05-01 Francois Mallet Method for fixing a biological molecule on a support surface
EP1215186A1 (fr) * 2000-12-14 2002-06-19 Commissariat A L'energie Atomique Support solide pour l'immobilisation d'oligonucléotides
WO2002048691A1 (fr) * 2000-12-14 2002-06-20 Commissariat A L'energie Atomique Dispositif de renforcement de fluorescence large bande a faibles pertes et capteur optique biologique ou chimique l'utilisant.
EP1364702A2 (fr) * 2002-05-15 2003-11-26 Samsung Electronics Co., Ltd. Procédé de fabrication d'une plaque matrice de biomolecules avec des zones hydrophiles et hydrophobes
EP1542009A1 (fr) * 2002-08-12 2005-06-15 Hitachi High-Technologies Corporation Procede de detection d'acide nucleique par utilisation de microreseaux d'adn, et appareil de detection d'acide nucleique
US20060121501A1 (en) * 2004-10-22 2006-06-08 Stanislaw Burzynski Method for immobilizing molecular probes to a semiconductor oxide surface

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