Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
  • G01N21/6454—Individual samples arranged in a regular 2D-array, e.g. multiwell plates using an integrated detector array
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • 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
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  • B01J19/0093—Microreactors, e.g. miniaturised or microfabricated reactors
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
  • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
  • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• • G—PHYSICS
  • G01—MEASURING; TESTING
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  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/251—Colorimeters; Construction thereof
  • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
• • G—PHYSICS
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  • 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
  • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
• • G—PHYSICS
  • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
  • G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
  • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
  • G03F7/70—Microphotolithographic exposure; Apparatus therefor
  • G03F7/70216—Mask projection systems
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  • B01J2219/00306—Reactor vessels in a multiple arrangement
  • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
  • B01J2219/00315—Microtiter plates
  • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
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  • 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
  • B01J2219/00709—Type of synthesis
  • B01J2219/00711—Light-directed synthesis
• • 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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0627—Sensor or part of a sensor is integrated
  • B01L2300/0654—Lenses; Optical fibres
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/069—Absorbents; Gels to retain a fluid
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
  • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/97—Test strip or test slide
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/973—Simultaneous determination of more than one analyte

Definitions

• the invention relates to a method and a measuring device for determining a large number of analytes in a sample.
• the invention further relates to a kit and a measuring substance carrier for use in carrying out the method.
• Macromolecules often polymers such as nucleic acids and proteins, and substances derived from them as well as the products of special enzymatic synthesis services.
• the genetic information is in the form of an enormous variety of different nucleic acid sequences, the DNA.
• IVD procedures have become an indispensable tool in clinical diagnostics. Attempts are increasingly being made to increase the number of determinable parameters. New technologies lead to the so-called multiplex analysis, in which an attempt is made to analyze a plurality or a large number of analytes in one process " ⁇ Integrate. At the same time, attempts are made to downsize or miniaturize the devices. Most IVD methods record only a few parameters, ie well below 100, in one pass. The simultaneous detection of nucleic acids, for example in order to recognize a genetic predisposition , and other factors, for example: Auto-antibodies in the context of an autoimmune disease such as rheumatism have not yet taken place.
• Nucleic acid diagnostics which can detect infections with HIV, have relied on molecular biological methods such as polymerase chain reaction (PCR) or sequencing either provide only limited information or are very complex and therefore expensive. Therefore, they are sometimes unsuitable for the collection of large amounts of data in widespread use for cost reasons.
• PCR polymerase chain reaction
• a new and well-regarded approach is nucleic acid analysis by hybridization to immobilized oligonucleotides (oligos) on a solid Underground, e.g. a silicon
• a signal is generated by the binding of complementary strands to the immobilized oligos, an extremely dense and miniaturized arrangement of the oligos on the chip being achieved, for example by photolithographic synthesis, and thus the number of
• Fluctuations e.g. connected in the execution of experiments and assays, but also corresponding personnel costs per generated information and usually a high expenditure of time.
• HIV HIV, HBV etc.
• This invention should enable the rapid, cost-effective analysis of food e.g. be possible due to the presence of certain genes or proteins.
• BioChips are miniaturized hybrid functional elements with biological and technical components, e.g. Biomaterials immobilized on the surface that can serve as specific interaction partners (e.g. antibodies, DNA oligonucleotides) and a silicon matrix. Most of these are
• BioChip arrays Functional elements arranged in rows and columns, one speaks of BioChip arrays. Since thousands of biochemical functional elements can be arranged on the BioChip, they have to be manufactured using microtechnical methods.
• Known BioChips can be classified according to the following criteria:
• Chromatographic procedures Interaction of analytes with a solid phase, mostly immobilized interaction partners (eg hybridization of nucleic acids to DNA oligonucleotides). Detection method (optical, electrical).
• Marker-based e.g. absorption, fluorescence or luminescence
• marker-free detection methods light generation for reaction detection
• Manufacturing process e.g. synthesize oligonucleotides directly on the BioChip light-activated, spot synthesized oligonucleotides, coat beads or tubes.
• Carrier types glass chips, plastic chips, microtiter plates, tubes or beads.
• Optical detection serial in the scanner or parallel with a CCD camera.
• Particles can be examined in a method based on fluorescence labeling of the microparticles by using etched glass fiber microwaves in the back light, after which an analyte determination method is built up.
• US Pat. No. 4,767,205 describes a method for the coded identification of a selected object, wherein uniform microparticles with a size of 2 to 20 ⁇ m are coded with respect to a specific combination of size, shape and color, and inserted into the object to be identified.
• Patent contains no reference to a method for the determination of analytes.
• DE OS 36 288 describes an immunological test method in which a reaction vessel is used, on the surface of which several analyte-specific antibodies are immobilized.
• WO 98/00292 describes a method for the detection of an antigen in a sample, wherein a colored solid phase, for example a microtiter plate, is used. There is no evidence of particulate solid phases.
• the invention represents an important alternative to other parallel methods and multiplex formats, e.g. represented by biochips or "coated tubes”.
• the subject of the invention according to claim 1 is a method for determining a multiplicity of analytes in a sample, comprising the steps:
• the method according to the invention is particularly useful for the diagnosis of clinical parameters, e.g. for the nucleic acid
• Diagnostics for basic biological research, forensics, for food analysis and for the screening of medical products.
• the invention further relates to a kit according to claim 29, in particular for carrying out the method according to one of claims 1-27.
• the kit comprises
• the subject of a measuring device for determining a plurality of analytes in a sample in particular according to the method according to the invention.
• the measuring device comprises an electronic image acquisition sensor for the unexpanded image-wise detection of the optical behavior of an ensemble of microparticles which have been brought into contact with the sample and are applied to a carrier, means for excitation of the signal output signaling
• a data processing device evaluating image data from the electronic image acquisition sensor for providing information about the presence of certain analytes in the sample on the basis of the detected optical behavior of the
• the electronic image capture sensor is preferably a color-capable CCD image sensor.
• the image sensor can be connected directly to the carrier, so that cumbersome optical image transmission lenses, light guides and optical enlarging elements can be omitted.
• the invention further relates to a carrier for carrying out the method according to the invention, the carrier being a
• Has cavity in particular a flat capillary gap for receiving the ensemble of microparticles between two plates with their flat sides opposite one another, at least one of which is transparent.
• the distance between the two plates is chosen so that only one layer of adjacent to the other in the cavity Microparticles fit.
• the distance between the plates can optionally be adjustable.
• the invention further relates to a carrier for carrying out the method according to the invention, the carrier having a large number of capillary channels, in particular running parallel to one another, and being optically transparent at least in the region of the capillary channels.
• the carrier enables the microparticles to be presented in a mobile manner during optical detection in a simple manner.
• Frractal SMART Chip - FSC With the carrier called “Fractal SMART Chip - FSC” here, thousands of different assays (measurements) can be carried out in one measurement process.
• the principle of the fractal BioChip is based on a double coding of the solid phase, which serves as the immobilization partner, from microparticles (beads).
• Catcher molecules e.g. oligonucleotides
• these coded beads e.g. color-coded glass or polymer beads
• the surface of the SMART Beads can be localized and identified at any time.
• the target analyte sought in the sample e.g. a DNA analyte
• the target analyte sought in the sample can also be encoded (e.g. with a fluorescence label).
• the appropriate target analyte molecules are then stored in the analysis system on the capture molecules on the SMART
• This accumulation preferably takes place in a three-dimensional vessel under correspondingly favorable fluidic mixing conditions.
• the SMART beads are placed on a carrier, which preferably forms a planar arrangement.
• the SMART Beads do not have to be placed in a fixed grid or geometric grid. Due to the planar alignment of the SMART beads and the optical, double coding, all SMART beads can be detected in parallel with the help of CCD techniques, both in the stationary and in the moving state of the
• optically imaging lens systems can be dispensed with if light directed in parallel is used and the size of the SMARTS is chosen such that it covers a sufficient number of pixel elements of the color CCD chip.
• This direct use (no optics) of highly parallel CCD matrix chips with a large number (currently 8 million color pixels per 1 cm 2 ) of pixels (optical measuring points or sensors) makes it possible to use a large number of SMART beads based on their specific color coding
• CCD chips can currently recognize approximately 1 6 million colors.
• the maximum number of different spectral identities, which can still be detected without errors, is due to the necessary spectral gradation for the optical measurement and the possibility of reproducible coloring of the SMART beads in the
• the identification includes the determination of the SMART Bead class (color) as well as the exact position. This is made possible by the large number of CCD pixels available for detection per SMART bead. This multitude of measured values forms the basis which allows extensive, mathematical multiplexing in the identification of the individual SMART beads in a two-dimensional array. The processing of the data amounts is easily possible due to the development of the performance with a simultaneous price drop of modern computer systems.
• the detection of the detection reaction can make both qualitative and quantitative statements:
• Attachment partner found (evaluation of the fluorescence-labeled labels on the localized beads).
• the SMART beads - relative to other bead processes - allow easy fluidic handling both in production (production and biochemical coating) and in the analysis system including the detection unit, which can be optimized down to the handling of a single microparticle. The same applies to detection.
• identification of the individual SMART beads is possible, particularly for applications in DNA analysis.
• the invention thus covers the following requirements in clinical diagnostics: * Large number of simultaneously determinable parameters (achieved through miniaturized SMART beads and high-resolution optical detection).
• the basic solution in this system goes from the coding (specific identity of the individual microparticles) to the biochemical coating of the surface for the
• Detection method labeling or coating of the SMART beads through the mixing of the SMART beads with the sample to be examined in the analysis system through to presentation and detection.
• each SMART Bead class gets its own identity, resulting from the uniform
• Carrier bodies become identifiable individuals, resulting in a recognition of the individual SMART Bead classes (a class Here, more than one SMART bead with the same identity) is made possible in the detection unit.
• the microparticles (beads or microspheres) only become actual SMART beads through their individual coding.
• the general coding is, for example, the particle size, geometry, weight and physical properties such as magnetizability or the like. applicable. However, these principles have only a very limited number of "gradations" so that only a few different encodings can be generated.
• the SMART beads preferably have an average diameter of at least 10 ⁇ m, preferably at least 20 ⁇ m, particularly preferably from 25 to 200 ⁇ m and most preferably from 25 to 80 ⁇ m.
• Optically transparent or optically non-transparent beads can be used, beads with the highest possible optical transparency being preferred, which enable simple analysis using the transmitted light method. It is also preferred to use SMART beads with the smallest possible size variance, with at least 50%, particularly preferably at least 70% and most preferably at least 90% of the beads having a diameter of up to 20% and preferably up to 10% from average diameter of the respective SMART bead species differs.
• the methods for color coding the SMART beads include, among other things, a translucent coloring of the material, a translucent coloring of the surface, an opaque coloring of the material, an opaque coloring of the surface, embedding of fluorescent dyes or a luminescence process.
• Coloring or electromagnetic codes on the other hand, have a large number of gradations, which enables the identification of a large number of SMART beads.
• the idea of the present invention is based on the use of color coding, that is to say a spectral identity of the SMART beads in combination with the ever-increasing performance of CCD matrix chips for optical detection. These chips can currently identify approximately 70 million colors.
• the binding of the analyte to its specific immobilized interaction partner on the surface of a defined SMART bead should be used.
• SMART Beads each receive the same equipment with an immobilized interaction partner, whereby the interaction partner can be bound covalently, but also non-covalently via adsorptive or specific high-affinity interactions (e.g. biotin / avidin or streptavidin or hapten / anti-apten antibody).
• the presence of the analyte in the test material thus leads to binding and thus signal generation only on this spectrally identifiable class of SMART beads. If the respective class-specific equipment is known, this signal is used to identify the bound analyte and thus to detect it in the test material.
• the detection principle thus corresponds to established methods, for example in the development of ELISA tests (enzyme linked immunosorbent assay).
• the coating of beads with an immobilized interaction partner is "state of the art" (eg avidin coating 5 of magnetic beads, Dynal; antibody coating in the ELISA
• the aim is to achieve the fastest possible and at the same time safe and therefore efficient detection since only the precisely measured events contribute to the measurement result.
• One way to achieve a high throughput in the detection is to present the SMART Beads as quickly as possible - iy - on the detector - for example analogous to the flow cytometer principle, where the particles are pressed through a capillary at high speed and measured "online" at the outlet in the "particle beam".
• the possibility preferred according to the invention is a parallelization of the detection, for example by a two-dimensional presentation of the SMART
• CCD chips should preferably be used directly, ie without using a
• Optics can be used. As a result, the dimensions for the area on which the SMART beads are to be provided for detection are predetermined. High-resolution standard chips (no custom-made products) currently have external dimensions of approx. 25 x 37 mm. For example, if you want around 200,000 on this area
• each SMART bead could have a diameter of 60 ⁇ m and each SMART bead would still cover about 40 color pixels, which enables reliable detection. With the densest packing by moving them in rows, even more SMART beads would have to be accommodated or the SMART beads could be even larger. Miniaturization and thus further parallelization are of course conceivable.
• the following techniques are possible as methods for the planar application of SMART beads on a carrier: two-dimensional capillary gap, parallel capillaries or tubes, streaking, stamping
• an excitation light source is necessary which also meets the requirements of the color coding method used is sufficient, as the chosen detection method for a successful reaction on the surface of the SMART beads. Possibly. Two separate light sources must also be provided for the two steps, for example if the identity of the SMART beads is measured by an absorption measurement and the reaction is verified by a
• Fluorescence signal to be detected Possible types of light are parallel, planar light (continuous or as a flash) or local, local excitation, for example by means of light lines or points of light.
• heterogeneous e.g. white light for absorption measurement
• optical recognition of the spectral identity of a single SMART bead in the SMART bead array provides two pieces of information:
• SMART bead class e.g. SMART beads with the same color
• SMART beads with the same color belongs to a particular SMART bead, from which the analyte bound on its surface can be determined
• Modern, high-resolution CCD camera chips are preferably used for the detection of SMART beads. These enable both a very fast and a highly parallel detection of the
• the second information of the position detection is necessary for the presentation method without a precisely specified position (capillary gap, smear etc.) in order to be able to assign the subsequent signal of the detection method exactly to a SMART bead.
• the information can be used for quality assurance for the processes with a given position (spotting etc.).
• Analytes lead directly or indirectly to a detectable signal, preferably a light signal. This can e.g. by an excitation light (fluorescence) or by photon emission (luminescence).
• a detectable signal preferably a light signal. This can e.g. by an excitation light (fluorescence) or by photon emission (luminescence).
• the steps of detecting the analyte signal and recognizing the bead coding can be carried out in any order.
• the light signal is detected on the CCD chip, differentiated according to both wavelength (color) and intensity.
• the recorded spectrum can be evaluated qualitatively or quantitatively.
• the differentiation of wavelengths and intensities also allows differentiation of signal sources.
• the fractal chip (FSC) shifts the information about the equipment of a measuring point (e.g. a DNA oligo of a certain sequence) from the localized synthesis or
• Coating eg photolithography, spotting
• the separation of the synthesis of the actual sensors (i.e. the SMART beads) and the incubation with the test material enables the connection of very different areas of biochemical diagnostics in this format. Specifically, the simultaneous analysis of DNA, proteins and other parameters of interest should be made possible.
• the SMART beads manufactured in a batch process can be made available for a variety of diagnostic and analytical questions. This results in great advantages for production, which is reflected in the costs.
• the realization of 10 3 to 10 6 can be distinguished, among other things, by the moderate size of the SMART beads, by the direct detection and by using the highly developed CCD technology
• SMART Bead classes aimed for in one measurement run. This means a significant improvement in the state of the art, since large problems occur, for example, with fluorescence-based coding of beads from 64 different types. This provides the first multiplex method that can compete with the density of the measuring points with the DNA arrays that have just been developed, but without restricting them to DNA analysis and without the complex and expensive overall chip synthesis. The flexibility and user-oriented expandability of the individual components represent significant advantages of the invention. Color-coded SMART beads for absorption measurements
• SMART beads with a diameter of approx. 60 ⁇ m are easy to produce, color-coded, biochemically labein, fluid to handle and they deliver a strong light signal both for SMART bead identification and for subsequent reaction detection.
• the attainable optimum of differently colored SMART beads at the FSC is determined by the following parameters: number of chemically possible colorings for the unique identification of a SMART bead, minimum permissible size for the error-free
• the absolute sample amount is, for example, 3 ml for wetting approx. 200,000 pieces of the 60 ⁇ m SMART beads in a very reasonable volume range.
• DNA, PNA, proteins etc. can be combined in one format.
• the processes can be easily optimized through the flexible multiplexing of starting materials (e.g. G, A, C, T or longer oligo sequences) or the washing liquids and the SMART bead classes (microparticles of one color). That it will be the most efficient
• Feedstock consumption in particular in comparison with the fluidly very unfavorable, two-dimensional processes such as these can be used with planar DNA chips on a carrier. As a result, very good reproducibility and quality assurance are achieved.
• the SMART Beads enable both processes with their individual advantages.
• Arrays are generated, on the one hand, are superior flow processes, so that less sample material is required and better wetting of the surfaces of the SMART beads with the sample material is achieved. This enables detection of rare sample components with a low concentration in the sample.
• the measuring principle of FSC technology uses the constantly growing potential of high-resolution image acquisition sensors and in particular the CCD chips for the parallel detection of a large number of SMART beads. These should preferably be provided as close as possible in a two-dimensional array on the optical CCD chip sensor for detection (presentation on the detector). Due to the use of a CCD chip, the system focuses on parallelizing the detection to increase throughput. At the same time, not only is the measuring speed increased, but also the measuring quality due to a large number of possible parallel reference measurements.
• Microparticles are positioned on the carrier in a predetermined geometric arrangement.
• a geometrical arrangement of the microbeads is impressed on the array by means of the positioning or presentation method step, the identification of the microbeads based on their color being used only to differentiate the microbead classes, but not to determine the position of the microbeads, since this is already predetermined by the lattice structure.
• Conceivable carriers are glass or plastic chips (chips in the actual sense), a moving belt, a film on the carrier, etc.
• a capillary, two-dimensional, planar gap (see Neubauer Chamber) can be used, on the surfaces of which a hydrophobic grid is applied, which assigns the SMART Beads distributed in the gap an exact position in the gap.
• the method according to the invention which is much more elegant in comparison with the aforementioned method variant with a predetermined correct arrangement of the SMART beads, is an undefined, free and always new, ie fractal arrangement of the SMART beads in a two-dimensional array, the "fractal
• Position detection used.
• the presentation of the beads can be done through a capillary, two-dimensional, planar slit (see Neubauer
• This measuring principle creates the array each time anew and in an undefined spatial arrangement on the surface of the detector.
• the identity of the individual binding sites is determined by the
• Bead-immanent code is determined and not by a given localization on a chip.
• the dynamic (moving) presentation of the SMART beads on the CCD chip detector takes place here, for example, in a large number of fluid compartments or subspaces, which are preferably in the form of parallel channels, e.g. are designed as capillaries.
• the preferred direct detection of the transmitted light signals (without optics between the SMART bead carrier and the detector, preferably a CCD image sensor) has the advantage of a significantly lower amount of energy, which the light for an error-free
• Optical elements between the CCD imager and the SMART bead carrier would absorb light and thus reduce the sensitivity of the measuring device.
• the signal detection is based on the colocalization of a spectrally (colored) marked SMART beads and a signal generated by analyte binding.
• the non-specific background can also be minimized.
• SMART beads without a reaction on their surface could differ by 10 from currently 4096 intensity levels in one color channel, and the SMART beads that have found their binding partner are 2-3 intensity levels below or above due to the biochemical reaction.
• the scope of the application ranges from the search for a rare component of a sample, the search for the "needle in a haystack" with, in extreme cases, only a single SMART Bead class (one
• color coding e.g. translucent coloring of the material (e.g. glass, polysaccharide or plastic) from which the SMART beads (microparticles) are made, translucent coloring of the surface of the SMART beads and incubation of the beads with suitable dyes, embedding of fluorescent dyes (or markers) in the SMART Beads, an embedding of absorption dyes in the SMART Beads or the chemical coating of the particle surface with a luminescence marker.
• translucent coloring of the material e.g. glass, polysaccharide or plastic
• translucent coloring of the surface of the SMART beads and incubation of the beads with suitable dyes e.g. translucent coloring of the material (e.g. glass, polysaccharide or plastic) from which the SMART beads (microparticles) are made
• translucent coloring of the surface of the SMART beads and incubation of the beads with suitable dyes e.g. translucent coloring of the surface of the SMART beads and incubation of the beads
• the method according to the invention is arbitrary for determination
• nucleic acids DNA, RNA, in special cases also PNA
• proteins polypeptides and peptides in all forms, e.g. Hormones, growth factors, enzymes, tumor antigens, serum factors, antibodies, carbohydrates in various combinations, e.g. different sugars in
• Cells, subcellular particles or viral particles can be determined.
• the analyte is bound to the microparticles via a specific binding with an immobilized interaction partner.
• the method allows the use of different types of analyte binding reactions, e.g. a hybridization of complementary nucleic acids, where longer molecules such as cDNA, synthetic oligonucleotides, PNA, RNA can be used as interaction partners.
• the analyte can also be determined by a receptor-ligand or antibody-antigen reaction, e.g. natural antibodies, recombinant
• Antibodies and their subunits, antigens, generally proteins and polypeptides of interest as immobilized interaction partners be used.
• Peptides for example synthetic peptides, natural peptides, can also be used as immobilized interaction partners.
• combinatorial chemistry products can be synthesized directly on the beads.
• Two principles for signal generation are primarily used: the direct detection of an analyte previously or in the reaction and the indirect detection by competition of the analyte with a labeled standard of the same substance.
• the first variant is well established for some applications, e.g. for diagnostics of serum components but rather unsuitable.
• the second variant is therefore preferable for these applications, and in principle it also allows easier sample preparation by the
• Direct detection can be carried out by labeling the analytes with a fluorescent dye, a reporter enzyme and subsequent reaction (e.g. chemo- and bioluminescence) by selective
• Labeling of the bound analyte e.g. for nucleic acids by intercalating (fluorescent) dyes, double-stranded proteins or antibodies.
• a secondary detection is by detection of the bound
• Marked standards for competitive detection reactions can be dye, fluorescence or enzyme-coupled (e.g. chemo- and
• Protein standards can be considered Fusion protein with reporter enzyme (see above) or autofluorescent protein (eg GFP), for example for recombinant antibodies, protein hormones, growth factors etc. can be produced.
• Fusion protein with reporter enzyme see above
• autofluorescent protein eg GFP
• Gap entry side and / or negative pressure on the gap exit side are conveyed into the gap. Furthermore, the capillary forces, which can theoretically reach up to 40 g, can also be reinforced by a correspondingly absorbent material (fleece) on the gap exit side.
• the material for the carrier with 2D capillary is e.g. Glass or plastic is possible, whereby carriers with capillaries can be designed inexpensively as disposables, especially in an injection-molded version.
• the necessary "Single Microparticle Handling" can also be used for the sorting processes when putting together the individual SMART Bead Kits.
• the information from a SMART Bead Array should advantageously be read out in a combined excitation and detection unit, with the preferred system being an arrangement of the excitation light source directly above the SMART Bead Array and the CCD chip directly below (or vice versa) the array (Sandwich construction).
• This design which is as compact as possible, minimizes the light path and thus also the required light intensity, as well as the overlay effects of neighboring SMART beads.
• the use of complex, space-intensive, light-absorbing and expensive optics should preferably be dispensed with on both the excitation and the detection side.
• Another variant is a vertical alignment of the chip, so that gravitational forces for loading and unloading the chip with the
• SMART Beads can be used.
• the SMART beads come as light sources
• Corresponding optical grating and / or corresponding optics can be located between the excitation light source and the SMART bead chip.
• the detection unit comprises a CCD chip. These currently have about 2000 x 3000 color pixels on an area of approx. 25 x 37 mm, which corresponds to 6 million color pixels or 18 million individual pixels (RGB principle thanks to miniaturized color filters in front of the pixels) (Cannon company). Arrange microparticles on such an area of 25 x 37 mm
• SMART Beads with a diameter of 60 ⁇ m for direct detection, so you get min. 200,000 microparticles (SMART Beads). Each microparticle covers approximately 40 square color pixels with an edge length of 9 to 10 ⁇ m. This results in 40 color or 120 black and white signals per SMART bead with a digital light intensity gradation of 4096 discrete brightness levels per black and white pixel. In any case, there is therefore a sufficient amount of data available for statistical signal verification.
• a particularly interesting aspect in the context of the present invention is the use of a "parallel inspection unit".
• Light source matrix facing-opposite light sensor matrix namely the CCD image sensor, the carrier with the on it or SMART beads located therein must be arranged between the light source matrix and the CCD sensor.
• the LCD unit as a two-dimensional light source matrix can be used both as a light source for absorption measurements for the respective location determination and for fluorescence excitation in the analyte determination (possibly with a different backlight).
• the method according to claim 1 can be modified in the following way:
• the step of applying the microparticles to a support according to feature b) can take place before step a) of bringing the sample into contact with a large number of species of
• Microparticles are made.
• a planar carrier or a chip, preferably used with a capillary gap, such a flat (possibly hollow) plate carrier should be coupled directly to the CCD image sensor in order to be able to carry out an unenlarged image-wise detection of what happens when the microparticles come into contact with the sample.
• Particles from chromatographic matrices e.g. DEAEI-Sepharose, Sephacryl, Sephadex or Sephacel were stained by 5 min incubation with the dyes eosin red, Evans blue and light green. Colored beads were obtained which could be clearly classified using the transmitted light method.
• Xenon and halogen lamps and cold light sources have proven to be suitable as light sources.
• Suitable optics were, for example, 80 to 200 nm
• Telephoto Tokina photo lens
• an achromatic telephoto 300 mm Achromat telephoto lens
• a collimetor Melis-Criot
• the LCD matrix LCX017AL Nony with a chip area of 60 mm 2 (diagonal 46 mm, aspect ratio 5: 4), a number of pixels of 786 432 (corresponding to a pixel area of 830 ⁇ m 2 ) was successfully tested.
• the CCD chip ICX085AK (Sony) with a diagonal of 16.933 mm, an aspect ratio of 5: 4 (10.57 mm x 8.5 mm) and a number of effective pixels of 1300 x 3030 (calculated
• Fig. 1 shows a diagram for explaining an embodiment of the
• FIG. 2 shows a simplified perspective illustration of a detection unit made up of a CCD image sensor and a measurement substance carrier.
• FIG. 3 shows a top view of the measuring substance carrier according to FIG. 2.
• Fig. 4 shows a section through the measuring substance carrier along the in
• the sample material 1 is mixed with the prepared microparticles (SMART beads) in a mixing device 5.
• the ensemble of microparticles 3 introduced for this purpose comprises a multiplicity of differently colored, transparent microparticles 3, microparticles 3 of the same color belonging to a species which has the same capture molecules (eg oligonucleotides) immobilized on the microparticles 3.
• the presence of the target analytes in the sample is detected by using analyte-specific labeled detection reagents in soluble form, with the aid of which binding of the target analytes to the beads can be detected.
• step A the appropriate target analyte molecules can then attach to the capture molecules on the surface of the microparticles in question (SMART beads) under hydriding conditions.
• SMART beads microparticles in question
• the mixing step A can be followed by an amplification step (e.g. PCR amplification etc.).
• an amplification step e.g. PCR amplification etc.
• the measuring substance from the microparticles 3 brought into contact with the sample 1 is placed on a measuring substance carrier 7 in a planar arrangement with an arbitrary distribution of the microparticles 3.
• the measuring substance carrier 7 can be a glass plate or transparent plastic plate, on which the microparticle measuring substance 3 e.g. is spread out, rolled out or stamped if necessary.
• step C in FIG. 1 an image-wise absorption measurement of the ensemble of microparticles 3 arranged on the measurement substance carrier 7 is carried out in a first measurement pass, a CCD image sensor 9 being used as the image detection sensor.
• the measuring substance carrier 7 is located between the CCD image sensor and a spectrally broadband emitting light source 1 1, which is indicated in Fig. 1 with a light bulb symbol.
• the measurement substance carrier 7 and the measurement substance layer provided thereon are preferably illuminated with parallel light from the light source 11.
• the CCD image sensor 9 captures an image of the ensemble of microparticles 3 on the measuring substance carrier 7, a data processing and control device 13 evaluating the image data of the CCD image sensor in order to store which microparticles 3 determine which pixel locations of the CCD image sensor 9 Staining were registered.
• the size of the microparticles 3 is selected so that each microparticle 3 covers several pixels (image point sensors) 15 of the CCD image sensor 9 in the top view projection.
• step D detects the detection reaction.
• the reaction is detected by fluorescence signals which emanate from microparticles 3 to which analyte-specific detection reagents have accumulated.
• a UV light source 17 which is represented in FIG. 1 by an incandescent lamp symbol and which emits parallel light, serves as the fluorescence excitation light source.
• the fluorescence radiation is registered by means of the image sensor 9 in association with the position of the respective fluorescent microparticles 3. From the results of steps C and D, the data processing system can then determine which species of microparticles 3 the microparticle (s) 3 that emitted a fluorescence signal in question belong to.
• FIG. 2 shows a measuring substance carrier chip 7a in the measuring arrangement on a CCD image sensor 9.
• the measuring substance carrier chip 7a (also referred to as a fractal SMART chip or FSC) is also shown in plan view in FIG. 3.
• the chip 7a has a disk-shaped capillary gap or cavity 19 which is delimited at the top and bottom in the illustration according to FIG. 2 by transparent plate sections 21, 23.
• the gap height h (cf. FIG.
• the measuring substance carrier chip 7a has at its right end according to FIGS. 3 and 4 a sample input 25 for the supply of the measuring substance (microparticles 3 after mixing with the sample).
• the sample input 25 is connected to the capillary gap 19.
• the capillary gap 19 extends across a detection area 27 which, according to FIG. 2, is aligned with the active image detection field of the CCD image sensor 9 indicated at 29 when the image sensor 9 and carrier chip 7a correspond to that in FIG. 2 shown detection unit are assembled.
• an absorbent material 30 is provided in a chamber of the chip 7a on the outlet side of the capillary gap 19 on the left in FIGS. 2 and 3, which increases the capillary forces for filling the gap 19 with measuring substance by its suction effect.
• the measuring substance carrier 7a is preferably made of plastic in an injection-molded version, so that it can be used inexpensively as a disposable item.
• the size of the detection area 27 of the chip 7a essentially corresponds to the size of the active image detection field 29 of the CCD sensor corresponds.
• the detection area has the dimensions 2.5 cm ⁇ 3.7 cm.
• Fig. 5 shows schematically the arrangement of Fig. 2 with an LCD matrix 35 as a two-dimensional measuring or excitation light source, the light source elements can be controlled depending on the intensity and color of the light depending on location and time, the control device in question common control device for the LCD matrix and the CCD matrix is.

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