EP1610899A2 - Vorrichtung zur vervielf ltigung und zum nachweis von nuklei ns uren - Google Patents
Vorrichtung zur vervielf ltigung und zum nachweis von nuklei ns urenInfo
- Publication number
- EP1610899A2 EP1610899A2 EP04725346A EP04725346A EP1610899A2 EP 1610899 A2 EP1610899 A2 EP 1610899A2 EP 04725346 A EP04725346 A EP 04725346A EP 04725346 A EP04725346 A EP 04725346A EP 1610899 A2 EP1610899 A2 EP 1610899A2
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- EP
- European Patent Office
- Prior art keywords
- detection
- substrate
- reaction
- detected
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/4738—Diffuse reflection, e.g. also for testing fluids, fibrous materials
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- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/59—Transmissivity
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/14—Process control and prevention of errors
- B01L2200/143—Quality control, feedback systems
- B01L2200/147—Employing temperature sensors
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- 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
- B01L2300/0636—Integrated biosensor, microarrays
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- 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
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- 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
- B01L2300/0877—Flow chambers
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- 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
Definitions
- the invention relates to devices and methods for the amplification of nucleic acids and for the detection of specific interactions between molecular target and probe molecules.
- Biomedical tests are often based on the detection of an interaction between a molecule that is present in a known quantity and position (the molecular probe) and an unknown molecule to be detected or unknown molecules (the molecular target or target molecules).
- the probes are stored in the form of a substance library on supports, the so-called microarrays or microarrays or chips, so that a sample can be analyzed simultaneously on several probes (see e.g. DJ Lockhart, EA Winzeler, Genomics, gene expression and DNA arrays; Nature 2000, 405, 827-836).
- the probes are usually immobilized in a predetermined manner on a suitable matrix, for example described in WO 00/12575 (see, for example, US 5,412,087, WO 98/36827) or generated synthetically (see, for example, US 5,143,854).
- An interaction between the probe and the target molecule is usually detected as follows: After the probe or probes have been fixed in a predetermined manner to a specific matrix in the form of a microarray, the targets are brought into contact with the probes in a solution and under incubated defined conditions. As a result of the incubation, a specific interaction takes place between the probe and the target. The binding that occurs is significantly more stable than the binding of target molecules to probes that are not specific for the target molecule. To remove target molecules that have not been specifically bound, the system is washed with appropriate solutions or heated. The detection of the specific interaction between a target and its probe can then be carried out by a large number of methods, which generally depend on the type of marker which was introduced into target molecules before, during or after the interaction of the target molecule with the microarray.
- markers are fluorescent groups, so that specific target-probe interactions with high spatial resolution and compared to other conventional detection methods, especially mass-sensitive methods, can be read with fluorescence optics with little effort (A. Marshall, J. Hodgson , DNA chips: An array of possibilities, Nature Biotechnology 1998, 16, 27-31; G. Ramsay, DNA Chips: State of the art, Nature Biotechnology 1998, 16, 40-44).
- Antibody libraries, receptor libraries, peptide libraries and nucleic acid libraries can be used as substance libraries that can be immobilized on microarrays or chips.
- the nucleic acid libraries play by far the most important role. These are microarrays on which deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules are immobilized. A prerequisite for the binding of a target molecule, for example marked with a fluorescence group, in the form of a DNA or RNA molecule to a nucleic acid probe of the microarray is that both the target molecule and the probe molecule are in the form of a single-stranded nucleic acid. An efficient and specific hybridization can only take place between such molecules. Single-stranded nucleic acid target and nucleic acid probe molecules are usually obtained by heat denaturation and optimal selection of parameters such as temperature, ionic strength and concentration of helix-destabilizing molecules.
- a typical example of the use of microarrays in biological test methods is the detection of microorganisms in samples in biomedical diagnostics.
- rRNA ribosomal RNA
- These species-characteristic sequences are applied to a microarray in the form of single-stranded DNA oligonucleotides.
- the target DNA molecules to be examined are first isolated from the sample to be examined and provided with markers, for example fluorescent markers.
- the labeled target DNA molecules are then incubated in a solution with the probes applied to the microarray, unspecific interactions are removed by appropriate washing steps and specific interactions are detected by fluorescence-optical evaluation.
- the detectable microorganisms theoretically depend only on the number of specific probes that have been applied to the microarray.
- the particularly quantitative detection of fluorescence signals is carried out using modified fluorescence microscopy methods.
- the light of the absorption wavelength is separated from that of the emission wavelength by means of filters or dichroites and the measurement signal is imaged by means of optical elements such as lenses and lenses on suitable detectors such as two-dimensional CCD arrays.
- the analysis is generally carried out using digital image processing.
- the technical solutions known to date differ in terms of their optical structure and the components used. Problems and limitations of such structures result from the signal noise (the background), which is caused by effects such as bleaching and quenching of the dyes used, auto-fluorescence of the media, assembly elements and optical components as well
- CCD-based detectors In order to optimize standard epifluorescence structures for reading out molecular arrays, CCD-based detectors are known which, in order to discriminate against optical effects such as scattering and reflections, implement the excitation of the fluorophores in the dark field by incident light or transmitted light (see for example CE Hooper et al., Quantitative Photon Imaging in the Life Sciences Using Intensified CCD Cameras, Journal of Bioluminescence and Chemiluminescence (1990), pp. 337-344).
- the imaging of the arrays takes place either in an exposure or by rastering using higher-resolution optics.
- the use of multispectral exposure sources enables relatively easy access to different fluorophores through the use of different excitation filters (combinations).
- the advantage of the confocal method lies in this elimination of the disturbing scattered light component outside the focal plane.
- the overall picture arises from the fact that the point of light is guided over the object in a cell shape. This is also known as scanning.
- the image data records recorded during this raster process are subsequently combined to form a 2D or 3D image.
- the task of the scanning unit is to move the light beam in a grid pattern over the object under the microscope. There are basically four options:
- the object is movable and is guided past the fixed laser.
- the laser is also at rest here.
- the movable laser beam is guided past the object.
- the object is at rest.
- the laser beam is moved in one axis, the object in the other axis.
- confocal scanning systems described in US Pat. No. 5,304,810 are based on the selection of the fluorescence signals along the optical axis by means of two pinholes. This results in a high adjustment effort for the samples or the
- a number of particularly confocal systems are known which are suitable for the detection of low-integrated substance libraries in the array format which are mounted in fluidic chambers (see, for example, US Pat. No. 5,324,633, US Pat. No. 6,027,880, US Pat. No. 5,585,639, WO 00/12759).
- the methods and systems described above can only be adapted to a very limited extent, especially because of the scattering, reflections and optical aberrations that occur there.
- high demands are made with regard to spatial resolution, which, however, have so far not been technically feasible.
- Cytogenetics (2000) Bari, Italy has great potential with regard to sensitivity and miniaturization of the construction of the detection technology, especially since light sources from data transmission are used for excitation (980 nm diode laser).
- this technology is currently not commercially available for the detection of target / probe interactions on arrays.
- a detector would contain components for light emission (e.g. laser, LED, high pressure lamps), a system for modulating the excitation and detection light (e.g. chopper discs, electronic shutter) and detection of the time-delayed signal (e.g. CCD, CMOS camera).
- the low compatibility of the particles with biological samples generally proves to be problematic.
- Saturation level precludes quantification after completion of the precipitation reaction. It is not possible to design the experiment parameters in such a way that it is undoubtedly ensured that the saturation level is not reached on any of the array elements, since the reaction rate depends to a large extent on temperature, light, salt concentration, pH and other factors.
- Probe arrays are provided, in which the temporal course of precipitation on the array elements is detected in the form of signal intensities, i.e. a dynamic measurement is carried out. Each array element is then assigned a value based on a curve function that describes the formation of precipitation as a function of time, which quantifies the interaction between the probe and target on an array element and thus the amount of bound targets.
- Admission required. This requires a complex interaction of the individual components of a highly integrated array, particularly in applications in the area of genotyping.
- RNA molecules typically happen through the polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- cDNA complementary DNA
- PCR is a standard laboratory method (such as in Sambrook et al. (2001) Molecular Cloning: A laboratory manual, 3rd edition, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press).
- the duplication of DNA by PCR is relatively fast, enables a high sample throughput in small batch volumes due to miniaturized processes and is work efficient due to automation.
- devices and methods for the amplification of nucleic acids and their detection should be designed in such a way that as few interventions as possible by an experimenter are necessary.
- the advantages of methods that enable nucleic acids to be reproduced and detected and in the course of which an experimenter only has to intervene minimally are obvious.
- contamination is avoided.
- the reproducibility of such processes is significantly increased because they are accessible to automation. This is extremely important with regard to the approval of diagnostic procedures.
- There are currently a large number of methods for the amplification of nucleic acids and their detection in which the target material is first amplified by PCR amplification and the identity or the genetic state of the target sequences is subsequently determined by hybridization against a probe array.
- the amplification of the nucleic acid or target molecules to be detected is generally necessary in order to have sufficient amounts available for qualitative and quantitative detection in the context of the hybridization.
- PCR products that are used as targets for array hybridization reactions generally have a length of at least approximately 60 base pairs. This corresponds to the Sum of the lengths of the forward and reverse primers used for the PCR reaction and the region which is amplified by the PCR and has complementarity with the probe on the array.
- Single-stranded molecules of this length are often not unstructured in solution, that is to say linearly stretched, but rather have more or less stable secondary structures, such as hairpins or other helical structures. If these secondary structures relate to the area of the target which is complementary to the probe, the formation of the secondary structures mentioned prevents efficient hybridization of the target to the probe. The formation of secondary structures can thus also inhibit efficient hybridization and make it difficult, if not impossible, to quantitatively and qualitatively evaluate the process results.
- detectable markers for example in the form of fluorescence-labeled primers, are introduced into the nucleic acids or target molecules to be detected in a method which combines PCR amplification and their detection by hybridization, a procedure is usually carried out before the actual detection
- washing step serves to remove the unreacted primers which are present in large excess compared to the amplification product and those nucleotides which are provided with a fluorescent marker and which do not participate in the detection reaction or do not hybridize specifically with the nucleic acid probes of the microarray. This is intended to reduce the high signal background caused by these molecules. Such an additional process step, however, significantly slows down the detection process. Furthermore, the detectable signal also significantly reduced for the nucleic acids to be detected, which hybridize specifically with the nucleic acid probes of the microarray. The latter is based primarily on the fact that the balance between the targets bound by the hybridization and in solution is no longer present after the washing step.
- Nucleic acids that had already hybridized with the nucleic acid probes on the array are detached from the binding site by washing and thus washed away with the molecules in solution.
- a detectable signal is only left if the washing or rinsing step of the molecules in solution is carried out faster than the detachment of the already hybridized nucleic acids.
- the object of the present invention is therefore to overcome the above-mentioned problems of the prior art, which result in particular from the lack of compatibility of the assay with the test system.
- Another object of the present invention is to provide methods and devices for the amplification and for the qualitative and quantitative detection of nucleic acids, with which a high signal-to-noise ratio is ensured in the detection of interactions on the microarray, without to impair the hybridization between the nucleic acids to be detected and the nucleic acid probes on the array.
- Another object of the present invention is to provide an apparatus for
- the invention is also based on the object of providing a device which enables the almost simultaneous duplication and characterization of nucleic acids with a high throughput.
- Amplification and for the qualitative and quantitative detection of nucleic acids in a sample is provided, which comprises the following steps:
- Detection can preferably be performed during the cyclic amplification reaction, i.e. in the course of one or more cycles of the amplification reaction, and / or after completion of the cyclic amplification reaction.
- the method according to the invention for the amplification of nucleic acids and their detection is designed in such a way that as few interventions as possible by an experimenter are necessary. This offers the main advantage that contamination is avoided. Furthermore, the reproducibility of the The method according to the invention is significantly increased compared to conventional methods, since the method according to the invention is accessible to automation due to the minimization of external interventions. The advantages mentioned above play an important role with regard to the approval of diagnostic methods.
- a device for amplification and for the qualitative and quantitative detection of nucleic acids by means of a method as described above comprising the following elements:
- reaction chamber which is formed between a chamber support and a microarray, the microarray comprising a substrate with nucleic acid probes immobilized thereon on array elements and the temperature in the reaction chamber being controllable and / or regulatable by the temperature control and regulating unit;
- hybridization between the nucleic acids to be detected and the nucleic acid probes immobilized on the substrate being detectable with the device without removing molecules from the reaction chamber that are not hybridized with the nucleic acids immobilized on the substrate.
- the reaction chamber of the device according to the invention is preferably designed as a capillary gap between the chamber support and the microarray.
- the object is achieved according to the invention in that a device for the duplication and detection of nucleic acids is provided which has at least one temperature control and / or regulating unit; a reaction chamber containing a detection area with a probe-substance library immobilized thereon; as well as preferably an optical system with which the temporal course of precipitation formation on the detection surface can be detected.
- a chip located in the reaction chamber which comprises a carrier with a detection area on which a substance library is immobilized, ensures the possibility of providing a very high probe density in the reaction chamber.
- the electrocaloric control or regulation by the temperature control and / or regulating unit allows the setting of defined temperatures both during the processing of the sample to be examined in the reaction chamber and during the detection of the hybridization events. This ensures both improved control and optimization of the detection reaction. Furthermore, the setting of defined temperatures via the temperature control unit allows the implementation of complex reactions such as, for example, amplification reactions by PCR.
- the devices according to the invention are further characterized in that, inter alia, due to the preferably integrated optical system or the reader system, detection of molecular interactions also in the
- the device according to the invention preferably contains an integrated optical system in one aspect of the present invention, with which the time course of precipitation formation on the detection surface can be detected, ensures an exact determination of the relative quantitative amount of nucleic acids bound to the substance library.
- the devices according to the invention allow processing, conditioning reactions and chip-based characterization of nucleic acids to be carried out almost simultaneously, in a time-efficient manner and less prone to failure.
- a processing or conditioning reaction is understood according to the invention to mean a reaction whose reaction products can be characterized by chip-based experiments.
- a device according to the invention for the detection of molecular interactions in closed reaction chambers preferably consists of four main functional elements (see Figure 1).
- the mechanical, electrical and fluidic recording of the reaction chamber takes place in a recording module (1).
- the reaction chamber is also referred to below as a microreactor.
- An optical system (2) is provided for the detection of the reaction results.
- the reaction results can be processed into an analysis result in a controller (3). If necessary, the analysis result is made available for storage and / or further processing by suitable connecting elements (4).
- reaction chamber which is advantageous as a component of the invention
- the reaction chamber which may be identified by a bar code, is installed in a fluidic recording module, where it can be filled with one or more reaction solutions.
- the reaction chamber also optionally has electrical contacts, as a result of which thermal control or regulation of reactions in the reaction chamber is ensured, for example, via integrated sensor and / or heating elements. This is particularly advantageous for carrying out thermally sensitive amplification reactions for DNA or RNA, hybridizations of DNA or RNA or reactions for signal amplification such as, for example, by metal precipitation on appropriately labeled target molecules and bound to the substance library.
- the solutions such as reaction and / or rinsing solutions, which may be required for carrying out the replication and detection reactions, can be introduced into the reaction chamber via suitable connecting elements such as channels. Suitable controllers can be used to monitor the course of the reaction.
- the optical system ensures the imaging of the substance library during or after completion of the duplication and / or detection reactions on a suitable detector, which is designed, for example, as a two-dimensional, electrically readable detection element.
- the sample is illuminated by means of an illumination module or a light source of the optical system and the resulting signals are imaged in a filtered manner in accordance with the markings used.
- the optical system also ensures a kinetic, ie dynamic recording of the reaction results.
- the optical system of the device according to the invention is preferably suitable for recording the chronological course of a silver deposit to amplify hybridization signals between gold-labeled target molecules and the substance library.
- the highly integrated structure of the device according to the invention allows the transfer of several images during the course of the reaction for processing in a suitable data processing module or controller.
- the device according to the invention also ensures the transfer of the raw data or analysis results to external computer or computer networks, for example for storing this data, via electronic interfaces which may be present.
- a microarray comprising a substrate with molecular probes immobilized thereon on predetermined areas.
- the substrate essentially comprises ceramic materials.
- a further aspect relates to the use of a substrate made of essentially ceramic materials for producing a microarray with molecular probes immobilized thereon on predetermined regions of the substrate.
- a probe or a probe molecule or a molecular probe is understood to mean a molecule which is used to detect other molecules by means of a specific, characteristic binding behavior or a specific reactivity. Any kind of molecules that can be coupled to solid surfaces and have a specific affinity are suitable for the probes arranged on the array. In a preferred embodiment, these are biopolymers from the classes of peptides, proteins, nucleic acids and / or their analogs. The probes are particularly preferably nucleic acids and / or nucleic acid analogs.
- nucleic acid molecules of a defined and known sequence are referred to as probes, which are used to detect target molecules in hybridization methods.
- Both DNA and RNA molecules can be used as nucleic acids.
- the nucleic acid probes or oligonucleotide probes can be oligonucleotides with a length of 10 to 100 bases, preferably 15 to 50 bases and particularly preferably 20 to 30 bases in length.
- the probes are single-stranded nucleic acid molecules or molecules of nucleic acid analogs, preferably single-stranded DNA molecules or RNA molecules, which have at least one sequence region which is complementary to a sequence region of the target molecules.
- the probes can be placed on a solid carrier substrate, e.g. in form of a microarray.
- a solid carrier substrate e.g. in form of a microarray.
- they can be radioactive or non-radioactive, so that they can be detected using the detection reaction customary in the prior art.
- a target or a target molecule is understood to mean the molecule to be detected with a molecular probe.
- the targets to be detected are nucleic acids.
- the probe array according to the invention can also be used analogously for the detection of protein / probe interactions, antibody / probe interactions, etc.
- the targets in the context of the present invention are nucleic acids or nucleic acid molecules which are detected by hybridization against probes arranged on a probe array
- these target molecules generally comprise sequences with a length of 40 to 10,000 bases, preferably from 60 to 2,000 bases, also preferably from 60 to 1,000 bases, particularly preferably from 60 to 500 bases and most preferably from 60 to 150 bases.
- Your sequence may contain the sequences of primers, as well as the sequence areas of the template defined by the primers.
- the target molecules can in particular be single-stranded or double-stranded nucleic acid molecules, one strand or both strands of which are radioactive or non-radioactive, so that they can be detected in one of the detection methods customary in the prior art.
- the target sequence is the sequence region of the target which is detected by hybridization with the probe. According to the invention, it is also said that this area is addressed by the probe.
- a substance library is understood to mean a multiplicity of different probe molecules, preferably at least two to 1,000,000 different molecules, particularly preferably at least 10 to 10,000 different molecules and most preferably between 100 and 1,000 different molecules. In special configurations, a substance library can also comprise only at least 50 or fewer or at least 30,000 different molecules.
- the substance library is preferably arranged as an array on a support in the reaction chamber of the device according to the invention.
- a probe array is understood to mean an arrangement of molecular probes or a substance library on a support, the position of each probe being determined separately.
- the array preferably comprises defined locations or predetermined areas, so-called array elements, which are particularly preferably arranged in a specific pattern, each array element usually containing only one species of probe.
- the arrangement of the molecules or probes on the carrier can be generated by covalent or non-covalent interactions.
- a position within the array, i.e. of the array, is commonly referred to as a spot.
- the probe array thus forms the detection area.
- an array element or a predetermined area or a spot or an array spot is understood to mean an area on a surface intended for the deposition of a molecular probe, the sum of all occupied array elements is the probe array.
- a carrier element or carrier or substance library carrier or substrate is understood to be a solid on which the probe array is built.
- the carrier which is usually also referred to as a substrate or matrix, can be, for example, specimen slides or wafers or else ceramic materials.
- the totality of molecules deposited in an array arrangement on the substrate or the detection surface or the substance library deposited in an array arrangement on the substrate or the detection surface and the carrier or substrate is often also referred to as a "chip”, “microarray”, “Microarray”, “DNA chip”, probe array, etc. called.
- a detection area is understood to mean the area of the substrate on which the substance library, preferably in an array arrangement, is deposited.
- a chamber support is understood to mean a solid body which forms a base area for the reaction chamber.
- the chamber carrier is preferably arranged on the side opposite the substrate or substance library carrier.
- the chamber carrier can also serve as a substrate or substance library carrier at the same time.
- a chamber body is understood to mean the solid body forming the reaction chamber.
- the substance library carrier or the chip is usually part of the chamber body, the Substance library support can be formed from a different material than the rest of the chamber body.
- a reaction chamber or a reaction space is the space that is formed between the chamber support and the microarray and is preferably designed as a capillary gap.
- the base area of the reaction chamber or the reaction space is defined by the base area of the array or the chamber support.
- the thickness of the reaction space or the reaction chamber or the capillary gap is, in particular, the distance between the chamber body and the matrix or microarray.
- a reaction space in the context of the present invention usually has a small thickness, for example a thickness of at most 1 cm, preferably of at most 5 mm, particularly preferably of at most 3 mm and most preferably of at most 1 mm.
- a capillary gap denotes a reaction space which can be filled by capillary forces which act between the chamber support and the microarray.
- a capillary gap has a small thickness e.g. of at most 1 mm, preferably at most 750 ⁇ m and particularly preferably at most 500 ⁇ m.
- a thickness in the range from 10 to 300 ⁇ m, from 15 ⁇ m to 200 ⁇ m or from 25 ⁇ m to 150 ⁇ m is preferred as the thickness of the capillary gap.
- the capillary gap has a thickness of 50 ⁇ m, 60 ⁇ m, 70 ⁇ m, 80 ⁇ m or 90 ⁇ m. If the reaction space or the reaction chamber has a thickness of more than 2 mm, the
- a confocal fluorescence detection system is understood in the context of the present invention to be a fluorescence detection system in which the object, ie the miroarray, is illuminated in the focal plane of the objective by a point light source. Point light source, object and point light detector lie in exactly optically conjugate planes. Examples of confocal systems are described in A. Diaspro, Confocal and 2-photon-microscopy: Foundations, Applications and Advances, Wiley-Liss, 2002.
- a fluorescence-optical system that images the entire volume of the reaction chamber is understood to mean a non-confocal fluorescence detection system, that is to say a fluorescence detection system in which the illumination by the point light source is not directed onto the object, i.e. the microarray.
- a fluorescence detection system thus has no focal limitation.
- microarrays within the scope of the present invention comprise approximately 50 to 10,000, preferably 150 to 2,000 different species of probe molecules on a, preferably square, area of e.g. 1 mm to 4 mm x 1 mm to 4 mm, preferably 2 mm x 2 mm.
- microarrays within the scope of the present invention comprise approximately 50 to approximately 80,000, preferably approximately 100 to approximately 65,000, particularly preferably approximately 1,000 to approximately 10,000 different species of probe molecules on one
- a conventional microarray has from 100 to 65,000 different species of probe molecules in an area of 2 mm x 2 mm.
- a microtiter plate is understood to mean an arrangement of reaction vessels in a specific grid, which allows the automated execution of a large number of biological, chemical and laboratory tests.
- a marker or a marker denotes a detectable unit, for example a fluorophore or an anchor group, to which a detectable unit can be coupled.
- the sample to be analyzed is the liquid to be analyzed with the nucleic acid molecules to be amplified or to be detected.
- a amplification reaction or an amplification reaction usually comprises 10 to 50 or more
- Amplification cycles preferably about 20 to 40 cycles, particularly preferably about 30 cycles.
- a cyclic amplification reaction is preferably a polymerase chain reaction (PCR).
- an amplification cycle is a single amplification step of the cyclic amplification reaction.
- a PCR amplification step is also referred to as a PCR cycle.
- the amplification product is a product from the amplification or duplication or amplification of the nucleic acid molecules to be amplified by the cyclic amplification reaction, preferably designated by the PCR.
- a nucleic acid molecule amplified by PCR is also referred to as a PCR product.
- the denaturation temperature is understood to be the temperature at which the double-stranded DNA in the
- the denaturation temperature especially in the case of a PCR, is usually more than 90 ° C., preferably about 95 ° C.
- the annealing temperature is understood to mean the temperature at which the primers hybridize to the nucleic acid to be detected.
- the annealing temperature especially in the case of a PCR, is usually in the range from 55 ° C. to 65 ° C. and is preferably approximately 60 ° C.
- the chain extension or extension temperature is understood to be the temperature at which the nucleic acid is synthesized by incorporating the monomer units.
- the extension temperature in particular in the case of a PCR, is usually in the range from about 70 ° C. to about 75 ° C. and is preferably about 72 ° C.
- an oligonucleotide is referred to as an oligonucleotide primer which binds or hybridizes to the DNA to be detected, also called target DNA, the synthesis of the counter strand of the DNA to be detected starting from the binding site in the cyclic amplification reaction ,
- a short DNA or RNA oligonucleotide with preferably about 12 to 30 bases is usually referred to as a primer, which is complementary to a section of a larger DNA or RNA molecule and has a free 3-OH group on its 3 '- End decreed.
- the primer can be used as a substrate for any DNA or RNA Polymerases are used that synthesize nucleotides on the primer in the 5 '-3' direction.
- the sequence of the newly synthesized nucleotides is predetermined by the sequence of the template hybridized with the primer, which is beyond the free 3'-OH group of the primer.
- Primers of conventional length comprise between 12 and 50 nucleotides, preferably between 15 and 30 nucleotides.
- a double-stranded nucleic acid molecule or a nucleic acid strand, which serves as a template for the synthesis of complementary nucleic acid strands, is usually referred to as a template or template strand.
- Hybridization is the formation of double-stranded nucleic acid molecules or duplex molecules from complementary single-stranded nucleic acid molecules. The association preferably always takes place in pairs of A and T or G and C.
- a hybridization e.g. DNA-DNA duplexes, DNA-RNA or RNA-RNA duplexes are formed.
- Hybridization can also form duplexes with nucleic acid analogs, e.g. DNA-PNA duplexes, RNA-PNA duplexes, DNA-LNA duplexes and RNA-LNA duplexes.
- Hybridization experiments are usually used to demonstrate sequence complementarity and thus the identity between two different nucleic acid molecules.
- processing is understood to mean purification, labeling, amplification, hybridization and / or washing and rinsing steps, and further process steps carried out in the detection or detection of targets with the aid of substance libraries.
- a substrate consisting essentially of ceramic materials or comprising essentially ceramic materials
- a substrate which comprises at least 75%, preferably at least 85% and particularly preferably at least 90% ceramic materials.
- a substrate consisting essentially of aluminum oxide ceramics or essentially comprising aluminum oxide ceramics means a substrate which comprises at least 75%, preferably at least 85% and particularly preferably at least 90% of aluminum oxide ceramics.
- an aluminum ceramic is understood to mean a ceramic material which essentially consists of aluminum oxide.
- a ceramic material consisting essentially of aluminum oxide is understood to mean a ceramic material which comprises at least 75%, preferably at least 85% and particularly preferably at least 90% aluminum oxide.
- a first subject of the present invention is thus in particular a device for the duplication and detection of nucleic acids, which comprises the following elements:
- An essential feature of the device according to the invention in this aspect of the present invention is the integration of a temperature control and / or regulating unit, a temperature-adjustable reaction chamber and an optical system, which ensures dynamic measurement of signal amplification reactions by precipitation, in one device.
- the device according to the invention in this aspect of the present invention is characterized in that, due to the integrated optical system or the reader system, detection of molecular interactions also in the
- the device according to the invention in this embodiment contains an integrated optical system with which the time course of precipitation formation on the detection surface can be detected ensures an exact determination of the relative quantitative amount of nucleic acids bound to the substance library.
- the optical system ensures the imaging of the substance library during or after the completion of the duplication and / or detection reactions on a suitable detector, which is designed, for example, as a two-dimensional, electrically readable detection element.
- a suitable detector which is designed, for example, as a two-dimensional, electrically readable detection element.
- the sample is illuminated by means of an illumination module or a light source of the optical system and the resulting signals are imaged in a filtered manner in accordance with the markings used.
- the optical system also ensures a kinetic, ie dynamic recording of the reaction results.
- the optical system is the
- the device according to the invention is suitable for recording the chronological course of a silver deposit to amplify hybridization signals between gold-labeled target molecules and the substance library.
- the highly integrated structure of the device according to the invention allows the transfer of several images during the course of the reaction for processing in a suitable data processing module or controller.
- the device according to the invention can, for example, be designed such that the chamber body of the reaction chamber, which contains the chip with the detection surface, is sealingly applied to a chamber carrier in such a way that a sample space with a capillary gap is formed between the chamber carrier and the detection surface or the substrate of the chip that is temperature-adjustable and flow-controllable.
- This type of construction makes it possible to carry out reactions which only take place efficiently in certain temperature ranges and to preferably detect the reaction products simultaneously by chip-based experiments.
- the device according to the invention can thus be used, for example, to multiply nucleic acid molecules almost simultaneously by PCR and to detect the PCR products by chip-based experiments.
- the sample liquid for such reactions can be heated and cooled efficiently by appropriate means for temperature regulation.
- the device according to the invention can also be used to carry out a reverse transcription reaction and in this way convert mRNA into cDNA and to characterize the reaction products by hybridization on the chip. In this way, so-called "gene profiling" can be carried out. Since both reverse transcription and hybridization are carried out in one chamber, this process is extremely time-efficient and less prone to failure.
- Restriction digestion can be carried out at desired temperatures in the reaction chamber and the reaction products can be characterized by hybridization on a chip.
- the enzymes can be denatured by heat deactivation.
- the device according to the invention thus enables time-efficient restriction fragment length polymorphism mapping (RFLP mapping).
- ligation can also be carried out, for example.
- nucleic acid target / nucleic acid probe complexes as a function of the temperature can be investigated with the device according to the invention.
- Devices according to the invention can also be used to carry out the binding behavior of proteins as a function of the temperature. For example, it can be used to test whether antibodies are able to retain their antibodies after being heated for a longer period of time bind appropriate antigens. The prerequisite for this is that in this case the chip is not functionalized by nucleic acid molecules, but by the corresponding proteins or peptides.
- the chamber body of the reaction chamber preferably consists of materials such as glass, plastic and / or metals such as stainless steel, aluminum and brass.
- plastics suitable for injection molding can be used for its production.
- plastics like Makrolon, Nylon, PMMA and Teflon are conceivable.
- the reaction space between the substance library support and the chamber support can also be closed off by septa, which, for example, enable the reaction space to be filled by means of syringes.
- the chamber body consists of optically transparent materials such as glass, PMMA, polycarbonate, polystyrene and / or topaz. The choice of materials must be adapted to the intended use of the device. For example, they are
- the chamber support preferably consists of glass, plastics, silicon, metals and / or ceramic materials.
- the chamber support can consist of aluminum oxide ceramics, nylon and / or Teflon.
- the chamber support consists of transparent materials such as glass and / or optically permeable plastics, for example PMMA, polycarbonate, polystyrene or acrylic.
- the chamber carrier and / or the substrate is preferably connected to means for applying temperature which are integrated in the device according to the invention and should then preferably consist of materials which are good heat conductors.
- thermally conductive materials offer the significant advantage that they ensure a homogeneous temperature profile over the entire area of the reaction space and thus temperature-dependent reactions such as a PCR in the entire reaction chamber can be controlled or regulated homogeneously, with high yield and with high accuracy.
- the chamber carrier and / or the substrate thus exist in a preferred one
- Embodiment made of materials with a high thermal conductivity preferably with a thermal conductivity in the range from 15 to 500 Wm ⁇ K “1 , particularly preferably in the range from 50 to 300 Wm ⁇ K “ 1 and most preferably in the range from 100 to 200 Wm ⁇ K “1 "1 , whereby the materials are usually not optically transparent.
- suitable thermally conductive materials are silicon, ceramic materials such as aluminum oxide ceramics and / or metals such as stainless steel, aluminum or brass.
- the substrate consists of materials with a high thermal conductivity, such as ceramic materials.
- the substrate is connected to a means for applying temperature, whereby the opposite side, the chamber support, can be made of a material that does not have a pronounced thermal conductivity, such as e.g. a material that is also used for the rest of the chamber body.
- a material that does not have a pronounced thermal conductivity such as e.g. a material that is also used for the rest of the chamber body.
- the substrate or the carrier of the device according to the invention essentially consists of ceramic materials
- aluminum oxide ceramics are preferably used.
- alumina ceramics are the ceramics A-473, A-476 and A-493 from Kyocera (Neuss, Germany).
- the ceramics differ essentially in the respective proportion of aluminum oxide (A-473: 93%, A-476: 96% and A-493: 99,%) and in their surface roughness.
- Aluminum oxide ceramics which have the lowest possible surface roughness are particularly preferably used.
- the chamber support and / or the substrate is on its back, i.e.
- the side facing away from the reaction chamber is provided with optionally miniaturized temperature sensors and / or electrodes or has heater structures there, so that temperature control of the sample liquid and mixing of the sample liquid by an induced electroosmotic flow is possible.
- the temperature sensors can, for example, be designed as nickel-chromium thin-film resistance temperature sensors.
- the electrodes can be designed, for example, as gold-titanium electrodes and in particular as a quadrupole.
- the means for applying temperature can preferably be selected so that the liquid in the capillary gap can be heated and cooled quickly. Rapid heating and cooling is understood here to mean that the means for subjecting the temperature to temperature changes in a range from 0.2 ° K / s to 30 ° K / s, preferably from 0.5 ° K / s to 15 ° K / s, particularly preferably from 2 ° K / s to 15 ° K / s and most preferably from 8 ° K / s to 12 ° K / s or about 10 ° K / s can be conveyed. Temperature changes of 1 ° K / s to 10 ° K / s can preferably also be conveyed by the means for applying temperature.
- the means for applying temperature for example in the form of heaters, can be embodied, for example, as a nickel-chrome thin-film resistance heater.
- the chip or the substrate can preferably consist of borofloat glasses, quartz glass, single-crystal CaF 2 , sapphire disks, topaz, PMMA, polycarbonate and / or polystyrene. The choice of materials is also later
- the chips are preferably functionalized by nucleic acid molecules, in particular by DNA or RNA molecules. However, they can also be functionalized by peptides and / or proteins, such as antibodies, receptor molecules, pharmaceutically active peptides and / or hormones.
- Preferred materials are the detection of the temporal course of precipitation formation on the detection area in the dark field, ie changes in the scattering properties of the detection area are detected for the substance library support, optical transparent materials such as glass, particularly preferably borosilicate glass, and transparent polymers such as PMMA, polycarbonate and / or acrylic; for the chamber support, optically transparent materials, such as glass and / or plastics, and in particular optically non-transparent materials, such as silicon, ceramic materials; and for them
- Reaction chamber plastics such as Makrolon, PMMA, polycarbonate, Teflon and the like, metals such as stainless steel, aluminum and / or brass and glass.
- the chamber carrier can consist of optically transparent materials, while the substance library carrier consists of optically non-transparent materials.
- the device according to the invention additionally comprises at least one fluid container which is connected to the reaction chamber, and optionally a unit for controlling the loading and unloading of the
- fluids are understood to be liquids or gases.
- connection of the fluid containers to the reaction chamber can be carried out, for example, as in international patent application WO 01/02094.
- the device according to the invention comprises a unit connected to the optical system for processing signals recorded by the optical system.
- This coupling of the detection unit and processing unit which ensures the conversion of the reaction results into the analysis result, allows, among other things, the use of the device according to the invention as a hand-held device, for example in medical diagnostics.
- the device preferably further comprises an interface for external computers. This allows the transfer of data for storage outside the device.
- the optical system with which the time course of precipitation formation on the detection surface of the chip can be detected, preferably comprises a two-dimensionally readable detector.
- the detector is preferably a camera, in particular a CCD or CMOS camera or a similar camera.
- CCD or CMOS complementary metal-oxide-semiconductor
- CMOS complementary metal-oxide-semiconductor
- the cameras used in the optical system of the device according to the invention ensure that the illumination intensity is distributed homogeneously on the surface to be imaged and that the signals to be detected are reflected by reflection, transmission modulation, scattering, polarization modulation and the like with the detection technology used within the scope of the available ones
- a high local resolution of the area to be detected can also be achieved, for example, by imaging on detectors such as mirror arrays or LCD elements and adjusting them according to a pattern to be detected or an area to be defined, such as for
- the detectors usually record the entire area of the probe array.
- scanning detectors can also be used to read the chip.
- the device according to the invention comprises movable optical components for guiding light or movable mechanical components for holding the reaction chambers, so that guiding the respective components over the individual positions to be scanned, that is to say the respective measuring points, is ensured is.
- the image is recorded by means of a mathematical reconstruction of the image from the respective measuring points.
- the camera in this embodiment is a movable line scan camera.
- the optical system preferably additionally comprises a light source, particularly preferably a multispectral or a coherent light source.
- a light source particularly preferably a multispectral or a coherent light source.
- Examples of light sources in the context of the present invention are lasers, light-emitting diodes (LED) and / or high-pressure lamps.
- the light source of the optical system preferably ensures homogeneous illumination of the wearer.
- light sources can be used in the form of lighting arrays in the inventive device.
- homogeneous illumination of the carrier can be achieved can be ensured, for example, that the light source comprises a plurality of diffusely radiating light sources, the superimposition of which results in homogeneous lighting.
- diffusely scattering LEDs which are arranged in a matrix, enable homogeneous illumination at short distances from the sample.
- the device according to the invention can be designed such that the detection surface can be scanned in a cell-like manner by the light source. If a raster-shaped or scanning guidance of the light beam over the detection surface is desired, the following design options are conceivable for the device according to the invention:
- the detection surface or the reaction chamber can be designed to be movable and can be guided past a stationary light source. If the light source is a laser, the laser is at rest. The detection surface can also be in the rest position and a movable laser beam can be guided over the detection surface. Finally, it is also possible for the light source to be moved in one axis and the detection surface in the other axis.
- the device additionally comprises lenses, mirrors and / or filters.
- the use of filters enables on the one hand the spectral limitation of the homogeneous illumination and on the other hand the illumination of the samples with different wavelengths.
- the device according to the invention additionally comprises filter changers. With these filter changers, the optical filters can be changed quickly and possible misinformation, which occurs, for example, from cleaning, can be clearly identified and eliminated.
- the optical system is preferably designed such that the detection area can be illuminated homogeneously, preferably with an illumination intensity homogeneity of at least 50%, particularly preferably of at least 60% and most preferably of at least 70%.
- the optical system is designed such that the time course of the change in transmission properties of the detection surface can be detected. This can be ensured, for example, that the light source and detector are arranged on opposite sides in the reaction chamber and the reaction chamber including the support for the detection surface is optically transparent at least in the region of the beam path from the light source to the detector.
- the optical system is arranged in such a way that the time course of the change in reflection properties of the detection area can be detected.
- the disadvantage of the poor reflection of the sample is supplemented in this embodiment by transmission effects in which the illuminating light is reflected via a mirror layer behind the sample, either as an independent mirror or as a layer applied to the back of the sample carrier.
- an area radiator can be arranged on the opposite side of the carrier element and thus also to the sensor, for example of a CCD camera. A very compact arrangement is made possible in this way.
- the device additionally comprises a semi-transparent mirror between Light source and support element.
- the light from the light source reaches the sample through a semitransparent mirror and the image is imaged in reflection by the semitransparent mirror and possibly a reading optic on a camera.
- the optical system is arranged such that the time course of the change in scattering properties of the detection area can be detected.
- the light source and detector are preferably arranged on the same side of the surface to be detected. The optical system can with this
- Embodiment can be arranged, for example, such that the sample or the chip can be illuminated at a certain angle, which is preferably less than 45 ° C. and particularly preferably less than 30 ° C.
- the angle of illumination is chosen so that the incident light without the presence of local scattering centers, i.e. e.g. before precipitation forms on the detection surface, not directly in the
- Detection beam path is reflected and thus no signal is detectable. If local scattering centers appear on the detection surface, e.g. due to the formation of a precipitate, part of the incident light enters the detection beam path and thus leads to a measurable signal in the optical system of the device according to the invention.
- the chamber support or the substance library support is particularly preferably not optically transparent, at least in the area of the detection area.
- Suitable non-optically transparent materials are, for example, silicon, ceramic materials or metals.
- the use of a non-optically transparent chamber support has the advantage that lighter, more precise and more homogeneous temperature control of the reaction chamber is ensured due to advantageous physical properties of the support materials, so that a successful implementation of temperature-sensitive reactions such as a PCR is guaranteed.
- the excitation beam path of the light source is designed such that areas of parallel light are present and interference filters can thus be introduced into the optical system without shifting their transmission windows.
- the detection beam path is designed in such a way that areas of parallel light are present and interference filters can thus be introduced into the optical system without shifting their transmission windows.
- Interference filters greatly change their spectral selectivity in the case of non-parallel beam paths. If the presence of areas of parallel light allows the introduction or arrangement of interference filters in the device according to the invention without changing their spectral selectivity, the device according to the invention is also suitable for the detection of molecular interactions of substances labeled with fluorochromes. A universal CCD-based reaction and detection device can thus be implemented.
- the optical system can, when using white or multispectral light such as e.g.
- Halogen lighting, xenon, white light LED and the like for example two filters in the illumination and detection beam path or, when using monochrome light sources such as LED or laser, for example a filter in the detection beam path.
- the reaction chamber is individually identified via a data matrix.
- a data record is stored in a database in the manufacture of the device according to the invention, which contains information about the substance library, the implementation of the
- the data record can contain information about the arrangement of the probes on the array and information about how the evaluation is to be carried out most advantageously.
- the data set or the data matrix can also contain information about the temperature-time regime of a PCR to be carried out, if necessary, for the purpose of duplicating the
- the data record created in this way is preferably given a number which is attached to the holder in the form of the data matrix. If necessary, the created data record can then be called up via the number recorded in the data matrix when reading out the substance library. Finally, the data matrix from the temperature control unit and other controllers such as e.g. a controller for the filling and filling of the reaction chamber can be read out via the fluid container and thus an automatic execution of the duplication and detection reaction can be guaranteed.
- a device for the duplication and detection of nucleic acids which also has a temperature control and / or regulation unit as described above; and also contains a reaction chamber as described above, which comprises a carrier with a detection surface on which a substance library is immobilized, the temperature in the reaction chamber being controllable and / or regulatable by the temperature control and regulating unit.
- the device in this aspect of the present invention has electrical contacts at the respective array spots. These electrical contacts can be contacted via electrodes, for example.
- the substance library support of the device according to the invention preferably has a three-dimensional structure which is formed, for example, by elevations, base and / or through bores, as a result of which the effect in the fusion of the growing conductive material with the precipitate that forms is formed electrical contacts and the resulting change in electrical parameters is supported.
- the device according to the invention based on optical detection preferably has a fluidic unit for exchanging solutions in the reaction chamber, a temperature control or regulating unit, and an optical system suitable for dynamic measurements.
- the above-mentioned units can optionally also be implemented separately in terms of device technology by implementing corresponding interfaces.
- Substance libraries that are immobilized on the microarrays or chips are, in particular, protein libraries such as antibody or receptor protein or Membrane protein libraries, peptide libraries such as receptor ligand libraries, libraries of pharmacologically active peptides or libraries of peptide hormones, and nucleic acid libraries such as DNA or RNA molecule libraries. Nucleic acid libraries are particularly preferred.
- the substance library is preferably in the form of a microarray, particularly preferably with a density of 2 to 10,000 array spots per cm 2 , most preferably with a density of 50 to 5000 array spots per cm, on the substance library carrier or the detection area is immobilized.
- the reaction chamber of the device according to the invention is preferably designed as a capillary gap.
- the capillary gap preferably has a thickness in the range from 10 ⁇ m to 200 ⁇ m, particularly preferably in the range from 25 ⁇ m to 150 ⁇ m and most preferably in the range from 50 ⁇ m to 100 ⁇ m.
- the capillary gap has a thickness of 60 ⁇ m, 70 ⁇ m, 80 ⁇ m or 90 ⁇ m.
- the reaction space or the reaction chamber has a thickness of, for example
- the thickness of the reaction space is 1.5 mm.
- Pre-amplification of the material to be analyzed is furthermore not necessary in all of the above-described embodiments of the device according to the invention.
- Targeted sections of the sample material extracted from bacteria, blood or other cells can be analyzed using PCR (Polymerase chain reaction), in particular in the presence of the device according to the invention or the substance library support, as described in DE 102 53 966, and hybridized to the support. This represents a significant simplification of the workload.
- the device according to the invention is thus particularly suitable for use in the parallel implementation of amplification of the target molecules to be analyzed by PCR and detection by hybridization of the target molecules with the substance library carrier.
- the nucleic acid to be detected is initially amplified by a PCR, with at least one competitor preferably being added to the reaction at the beginning, which inhibits the formation of one of the two template strands amplified by the PCR.
- a DNA molecule is added which competes with the primer used for the PCR amplification of the template for binding to the template and which cannot be extended enzymatically.
- the single-stranded nucleic acid molecules amplified by the PCR are then detected by hybridization with a complementary probe.
- the nucleic acid to be detected is first amplified in a single-strand excess by a PCR and detected by a subsequent hybridization with a complementary probe, a competitor being initially added to the PCR reaction, which is a DNA molecule or a molecule of a nucleic acid Analogs act that can hybridize to one of the two strands of the template, but not to the area that is detected by the probe hybridization, and which is not enzymatically extendable.
- any molecule which preferentially amplifies only one of the two template strands present in the PCR reaction can be used as a competitor in the PCR.
- proteins, peptides, DNA ligands, intercalators, nucleic acids or their analogues proteins, peptides, DNA ligands, intercalators, nucleic acids or their analogues. Proteins or peptides which are capable of binding single-stranded nucleic acids with sequence specificity and have the properties defined above are preferably used as competitors.
- Nucleic acid molecules and nucleic acid analog molecules are particularly preferably used as secondary structure breakers.
- the initial addition of the competitor to the PCR during the amplification essentially inhibits the formation of one of the two template strands. “Essentially inhibited” means that a sufficient one in the context of the PCR
- the single-strand excess achieved by the PCR is 1.1 to 1000, preferably 1.1 to 300, preferably 1.1 to 100, particularly preferably 1.5 to 100, compared to the non-amplified strand. likewise particularly preferably the factor 1.5 to 50, particularly preferably the factor 1.5 to 20 and most preferably the factor 1.5 to 10.
- Competitors are therefore single-stranded DNA- or RNA-binding proteins with specificity for one of the two template strands to be amplified in a PCR.
- it can be aptamers that bind sequence-specifically only to certain regions of one of the two template strands to be amplified.
- Nucleic acids or nucleic acid analogs are preferably used as competitors.
- the nucleic acids or nucleic acid analogs will usually act as a competitor of the PCR in that they either compete for the primer binding site with one of the primers used for the PCR or, due to sequence complementarity, can hybridize with a region of a template strand to be detected. This area is not the sequence that is detected by the probe.
- Such nucleic acid competitors cannot be extended enzymatically.
- nucleic acid analogs can e.g. are so-called peptide nucleic acids (PNA).
- PNA peptide nucleic acids
- nucleic acid analogs can also be nucleic acid molecules in which the nucleotides are linked to one another via a phosphothioate bond instead of a phosphate bond. It can also be a nucleic acid analogue in which the naturally occurring sugar building blocks ribose or deoxyribose against alternative sugars such as e.g. Arabinose or trehalose etc. were exchanged.
- the nucleic acid derivative can be “locked nucleic acid” (LNA).
- LNA locked nucleic acid
- DNA or RNA molecules are preferably used as competitors.
- the inhibition of the amplification is based on one of the two template strands as part of the PCR reaction on different mechanisms. This is discussed in the following using an example of a DNA molecule.
- a competitor e.g. a DNA molecule
- this can have a sequence which is at least partially identical to the sequence of one of the primers used for PCR in such a way that a specific hybridization of the DNA competitor molecule with the corresponding template strand is possible under stringent conditions .
- the DNA molecule used for the competition cannot be extended by a DNA polymerase in this case, the DNA molecule competes with the respective primer for the binding to the template during the PCR reaction.
- the amplification of the template strand defined by the primer can be inhibited in such a way that the production of this template strand is significantly reduced.
- the PCR follows exponential kinetics that are higher than would be expected with the amounts of competitor used. In this way, a single strand excess is produced in an amount which is sufficient for efficient detection of the amplified target molecules by hybridization.
- the nucleic acid molecules or nucleic acid analogs used for competition must not be able to be extended enzymatically.
- Enzymatically not extendable means that the DNA or RNA polymerase used for the amplification cannot use the nucleic acid competitor as a primer, ie is not able 3 'to the respective counter strand to the template from the sequence defined by the competitor synthesize.
- the DNA competitor molecule can also have a sequence which is complementary to a region of the template strand to be detected, which is not addressed by one of the primer sequences and which is not enzymatically extendable. In the course of the PCR, the DNA competitor molecule will then hybridize to this template strand and block the amplification of this strand accordingly.
- sequences of DNA competitor molecules or generally nucleic acid competitor molecules can be selected accordingly. If the nucleic acid competitor molecules have a sequence that is not essentially identical to the sequence of one of the primers used for PCR, but is complementary to another region of the template strand to be detected, this sequence should be chosen so that it is not in falls within the range of the template sequence that is detected during hybridization with a probe. This is necessary because there is no need for a work-up reaction between the PCR and the hybridization reaction. If a nucleic acid molecule that fell within the range to be detected were used as a competitor, this would compete with the single-stranded target molecule for binding to the probe.
- Such competitors preferably hybridize in the vicinity of the template sequence which is detected by the probe.
- the position indication “in the vicinity” is to be understood as it is given for secondary structure breakers.
- the competitors according to the invention can also hybridize in the immediate vicinity of the sequence to be detected, ie exactly one nucleotide removed from the target sequence to be detected. If nucleic acids or nucleic acid analogs that cannot be extended enzymatically are used as competitive molecules, their sequence or structure should be selected such that they cannot be extended enzymatically by DNA or RNA polymerases.
- the 3 'end of a nucleic acid competitor is preferably designed such that it has no complementarity with the template and / or has a different substituent instead of the 3-OH group at the 3' end.
- the nucleic acid Competitor due to the lack of base complementarity at the 3 'end cannot be extended by the common DNA polymerases.
- the nucleic acid competitor preferably has no complementarity with its target sequence at its 3 'end with respect to the last 4 bases, particularly preferably with regard to the last 3 bases, particularly preferably with regard to the last 2 bases and most preferably with regard to the last base.
- Such competitors can also have non-natural bases at the positions mentioned, which do not allow hybridization.
- Nucleic acid competitors that cannot be extended enzymatically can also be 100% complementary to their target sequence if they are modified in their backbone or at their 3 'end in such a way that they cannot be extended enzymatically. If the nucleic acid competitor has a group other than the OH group at its 3 'end, these substituents are preferably a phosphate group, a hydrogen atom (dideoxynucleotide), a biotin group or an amino group. These groups cannot be extended by the usual polymerases.
- a DNA molecule is particularly preferably used which competes with one of the two primers used for PCR for binding to the template and which has been provided with an amino link at the 3 'end during chemical synthesis.
- Such competitors can have 100% complementarity to their target sequence.
- Nucleic acid analog competitors such as PNAs, on the other hand, do not have to have a blocked 3 'OH group or a non-complementary base at their 3' end, since they are not recognized by the DNA polymerases due to the backbone changed by the peptide bond, and therefore are not extended become.
- Corresponding other modifications of the phosphate group which are not recognized by the DNA polymerases, are known to the person skilled in the art. These include Nucleic acids with backbone modifications such as 2'-5 'amide bonds (Chan et al. (1999) J. Chem. Soc, Perkin Trans. 1, 315-320), sulfide bonds (Kawai et al.
- Hybridize templates e.g. the primer binding site
- competitors use secondary structure crusher properties, the efficiency of hybridization can be further increased.
- the DNA competitor molecule can have a sequence complementary to one of the primers.
- antisense DNA competitor molecules can then be used to titrate the primer in the PCR reaction, so that it no longer hybridizes with the respective template strand and accordingly only the template strand defined by the other primer is amplified.
- the nucleic acid competitor can, but need not, be enzymatically extendable.
- nucleic acid competitors when nucleic acid competitors are used in the context of this invention, this includes nucleic acid analog competitors, unless the context indicates otherwise.
- the nucleic acid competitor can bind reversibly or irreversibly to the corresponding strand of the template. The binding can take place through covalent or non-covalent interactions.
- the nucleic acid competitor is preferably bound via non-covalent interactions and is reversible.
- the binding to the template is particularly preferably carried out by forming Watson-Crick base pairings.
- sequences of the nucleic acid competitors are generally based on the sequence of the template strand which is to be detected, in the case of antisense primers, on the other hand, on the primer sequences to be titrated, which, however, are in turn defined by the template sequences.
- the PCR amplification of nucleic acids is a standard laboratory method with which the person skilled in the art is familiar with the diverse possibilities of variation and design.
- a PCR is characterized in that the double-stranded nucleic acid template, usually a double-stranded DNA molecule, is first subjected to heat denaturation for 5 minutes at 95 ° C., as a result of which the two strands are separated from one another.
- the "forward” and “reverse” primers present in the reaction solution attach to the positions complementary to their sequence in the respective template Strands on.
- the “annealing” temperature of the primers depends on the length and base composition of the primers. It can be calculated based on theoretical considerations. Information on the calculation of "annealing” temperatures can be found, for example, in Sambrook et al. (vide supra).
- an elongation step follows in which the activity of those present in the reaction solution DNA polymerase deoxyribonucleotides are linked to the 3 'end of the primer.
- the identity of the inserted dNTPs depends on the sequence of the template strand hybridized with the primer. Since thermostable DNA polymerases are generally used, the elongation step usually takes place between 68-72 ° C.
- RNA e.g. mRNA
- reverse transcription e.g. reverse transcriptase
- thermostable DNA-dependent DNA polymerase is used as the polymerase.
- a thermostable DNA-dependent DNA polymerase is selected from the group consisting of Taq DNA polymerase (Eppendorf, Hamburg, Germany and Qiagen, Hilden, Germany), Pfu DNA polymerase (Stratagene, La JoUa, USA), Tth-DNA polymerase (Biozym Epicenter Technol., Madison, USA), Vent-DNA polymerase, DeepVent-DNA polymerase (New England Biolabs, Beverly, USA), Expand-DNA polymerase (Röche, Mannheim, Germany) used.
- polymerases which have been optimized from naturally occurring polymerases by targeted or evolutionary change is also preferred.
- the use of Taq polymerase from Eppendorf (Germany) or the Advantage cDNA polymerase mix from Clontech (Palo Alto, CA, USA) is particularly preferred.
- a method for the detection of nucleic acids comprises the following steps: a) provision of a device according to the invention as described above; b) interaction of the nucleic acids to be detected with the substance library immobilized on the detection surface; and c) detection of the interaction.
- the targets to be examined can be present in any type of sample, preferably in a biological sample.
- the targets are preferably isolated, cleaned, copied and / or amplified by the method according to the invention before they are detected and quantified.
- the amplification is usually carried out by conventional PCR methods or by a method as described above for parallel amplification of the target molecules to be analyzed by PCR and detection by hybridization of the target molecules with the substance library carrier.
- the amplification is carried out as a multiplex PCR in a two-stage process (see also WO 97/45559).
- a multiplex PCR is carried out using fusion primers whose 3 'ends are gene-specific and whose 5' ends represent a universal region. The latter is the same for all forward and reverse primers used in the multiplex reaction.
- the amount of primer is limited. This allows all multiplex products to be amplified to a uniform molar level, provided that the number of cycles is sufficient to achieve primer limitation for all products.
- universal primers are present which are identical to the 5 'regions of the fusion primers. Amplification takes place up to the desired amount of DNA.
- the detection is preferably carried out in that the bound targets are provided with at least one marking which is detected in step c).
- the label which is coupled to the targets or probes is preferably a detectable unit or a detectable unit which is coupled to the targets or probes via an anchor group.
- the method according to the invention is extremely flexible with regard to the possibilities of detection or marking.
- the method according to the invention is thus compatible with a large number of physical, chemical or biochemical detection methods.
- the only requirement is that the unit or structure to be detected can be coupled directly to a probe or a target, for example an oligonucleotide, or linked via an anchor group that can be coupled to the oligonucleotide.
- the detection of the label can be based on fluorescence, magnetism, charge, mass, affinity, enzymatic activity, reactivity, a gold label and the like. based.
- the marking can be based on the use of fluorophore-marked structures or building blocks.
- the label can be any dye that can be coupled to targets or probes during or after their synthesis. Examples include Cy dyes (Amersham Pharmacia Biotech, Uppsala, Sweden), Alexa dyes, Texas red, fluorescein, rhodamine (Molecular Probes, Eugene, Oregon, USA), lanthanides such as samarium, ytterbium and Europium (EG&G, Wallac, Freiburg, Germany).
- luminescence markers, metal markers, enzyme markers, radioactive markers and / or polymeric markers can also be used in the context of the present invention as a marker or as a detection unit which is coupled to the targets or probes.
- a nucleic acid can also be used as a label (tag), which can be detected by hybridization with a labeled reporter (sandwich hybridization).
- a labeled reporter such as primer extension, ligation and RCA are used to detect the tag.
- the detectable unit is coupled to the targets or probes via an anchor group.
- Preferred anchor groups are biotin, digoxygenin and the like. The anchor group is then reacted with specifically binding
- anchor groups for example streptavidin conjugates or antibody conjugates, which are themselves detectable or trigger a detectable reaction.
- the anchor groups can be converted into detectable units before, during or after addition of the sample comprising the targets or, if appropriate, before, during or after the cleavage of the selectively cleavable bond in the probes.
- the labeling can also be carried out by interaction of a labeled molecule with the probe molecules.
- the label can be labeled by hybridizing one as described above
- Oligonucleotide with an oligonucleotide probe or an oligonucleotide target are described, for example, in Lott Industries and Zorbas, Bioanalytik, Spektrum Akademischer Verlag, Heidelberg, Berlin, 1998, chapters 23.3 and 23.4.
- detection methods are used which, as a result, provide an adduct with a specific solubility product which results in precipitation.
- substrates or starting materials are used for the marking, which can be converted into a poorly soluble, usually colored product.
- enzymes which catalyze the conversion of a substrate into a poorly soluble product can be used in this labeling reaction. Reactions that are suitable for leading to a deposit on array elements, as well as possibilities for the detection of the deposit, are described, for example, in the international patent application WO 00/72018 and in the international patent application
- the bound targets are provided with a label which catalyzes the reaction of a soluble substrate or educt to form a sparingly soluble precipitate on the array element on which a probe / target interaction has taken place or which acts as a seed for the conversion of a soluble substrate or educt to a sparingly soluble precipitate on the array element on which a probe / target interaction has taken place.
- a label which catalyzes the reaction of a soluble substrate or educt to form a sparingly soluble precipitate on the array element on which a probe / target interaction has taken place or which acts as a seed for the conversion of a soluble substrate or educt to a sparingly soluble precipitate on the array element on which a probe / target interaction has taken place.
- enzymes catalyze the conversion of a substrate into a poorly soluble, usually colored product.
- the reaction leading to the formation of a precipitate on the array elements is particularly preferably an enzyme-catalyzed conversion of a soluble substrate or starting material into a poorly soluble product.
- the reaction leading to the formation of a precipitate on the array elements is a peroxidase-catalyzed oxidation of 3,3 ', 5,5'-tetramethylbenzidine.
- Tetramethylbenzidine horseradish peroxidase used.
- other peroxidases are known to the person skilled in the art which can be used for the oxidation of 3,3 ', 5,5'-tetramethylbenzidine.
- the methods according to the invention include the following variants are conceivable for the detection of the probe / target interactions via insoluble precipitates.
- the targets are provided with a catalyst in front of an enzyme which catalyzes the conversion of a soluble substrate or starting material into an insoluble product.
- the reaction which leads to the formation of a precipitate on the array elements in this case is the conversion of a soluble substrate or starting material into an insoluble product in the presence of a catalyst, preferably enzyme, coupled to the targets.
- the enzyme is preferably selected from the group consisting of horseradish peroxidase, alkaline phosphatase and glucose oxidase.
- the soluble substrate or starting material is preferably selected from the group consisting of 3,3'-diaminobenzidine, 4-chloro-1-naphthol, 3-amino-9-ethylcarbazole, p-phenylenediamine HCl / pyrocatechol, 3, 3 ' , 5, 5 '-Tetramethylbenzidin, Naphthol / Pyronin, Bromchlorindoylphosphat, Nitrotetraazoliumblau and Phenazinmethosulfat.
- a colorless soluble hydrogen donor e.g. 3,3'-diaminobenzidine
- horseradish peroxidase transfers hydrogen ions from the donors to hydrogen peroxide to form water.
- the reaction which results in the formation of a precipitate on the array elements is the formation of a metallic precipitate.
- the reaction leading to the formation of a precipitate on the array elements is particularly preferably the chemical reduction of a silver compound, preferably silver nitrate, silver lactate, silver acetate or silver tartrate, to elemental silver.
- a silver compound preferably silver nitrate, silver lactate, silver acetate or silver tartrate
- Formaldehyde and / or hydroquinone are preferably used as reducing agents.
- colloidal gold or defined gold clusters are coupled to the targets, possibly via certain mediator molecules such as streptavidin.
- mediator molecules such as streptavidin.
- the coloration resulting from the gold marking is preferably by subsequent reaction with less noble metals such as Silver reinforced, the gold marking coupled to the targets acting as a seed or catalyst, for example for the reduction of silver ions to a silver strike.
- the targets coupled with gold markings are also referred to below as gold conjugates.
- a relative quantification of the probe / target interaction can also take place.
- the relative quantification of the concentration of the bound targets on a probe array by detecting a precipitate or a precipitate is carried out via the concentration of the labels coupled to the targets, which indicate the reaction of a soluble substrate to a poorly soluble precipitate on the array element a probe / target interaction has taken place, catalyze or act as a seed for such reactions.
- this is Ratio of bound target to gold particles 1: 1.
- it can be a multiple or a fraction thereof.
- the detection method is thus carried out by measuring the change in transmission, reflection or scatter, which is caused by the precipitation which is produced on the array elements on which a probe / target interaction has taken place, by the catalytic action of the label coupled to the bound targets ,
- the temporal course of the precipitation formation on the array elements is thus detected in the form of signal intensities in step c).
- an accurate determination of the relative quantitative amount of bound targets can be ensured.
- the procedure described below is not limited to the silver / gold staining described above, but can be applied accordingly to all detection methods in which the bound targets are provided with a label which unites the reaction of a soluble substrate or starting material catalyzes sparingly soluble precipitate on the array element on which a probe / target interaction has taken place or which acts as a seed for the conversion of a soluble substrate to a sparingly soluble precipitate on the array element on which a probe / target Interaction has taken place.
- the target molecule is e.g. biotinylated by PCR.
- the PCR product is hybridized against a substance library, for example a DNA library.
- Streptavidin-functionalized gold beads are then added to the reaction chamber, which react with the biotinylated hybrids, for example DNA hybrids.
- a silver peening can be produced on the gold beads now specifically bound to the surface, for example by reducing silver nitrate with hydroquinone under the catalytic effect of gold (see, inter alia, WO 00/72018, DE 100 33 334.6, MA Hayat, Immunogold-Silver Staining, CRC Press, New York, 1995).
- I is the light intensity after absorption
- I 0 is the light intensity before absorption
- a is an absorption coefficient multiplied by the shading per unit area b by the silver precipitate.
- Intensity I and time t are available as measured variables. These measured variables are obtained by illuminating the carrier element with the substance library and recording the transmitted light with a camera. This recording is repeated at regular intervals while the silver deposition is being carried out. The brightness values of the individual library regions (spots) are for each
- Silver deposition time series can be related in the following way.
- the area F that shadows a silver ball is:
- the shading per unit area b by the silver precipitate is proportional to the number of silver beads per unit area N, the shading area per silver bead F and a constant k.
- the function for the intensity I is thus calculated as a function of the time t
- O is a device-dependent offset value.
- the number of gold beads that are deposited per unit area depends on the one hand on the amount of targets marked with gold beads and on the other hand on the binding strength of the targets, for example the target DNA, with the probes, for example the spot DNA. If there is no target in the sample which interacts with a probe on the corresponding array element, gold balls are not deposited on the surface of this array element or spot. If the bond between the probe and target is weak, very few gold spheres will settle on the surface of this array element.
- a' represents a measure of the concentration of the target D ⁇ A and the binding strength of the target D ⁇ A at the array element or spot i.
- the silver absorption constant a 'can be calculated from the measurement curves obtained using nonlinear regression using equation (VI).
- the calculation of the constant a 'can be used as a significant measure of the binding strength and the concentration of the target D ⁇ A at a spot.
- the time constant ⁇ of the deposition reaction can be determined from the constant a ':
- Another variant of the evaluation consists in using the gray values of the individual spots directly as measured values after a specified time.
- this method has the disadvantages that it cannot be assessed in advance which time is optimal for the evaluation and that the measured values have a lower statistical certainty.
- any lighting inhomogeneity can only be carried out via a flatfield correction.
- Another aspect of the present invention relates to a method for the amplification and for the qualitative and quantitative detection of nucleic acids in a sample, comprising the following steps: a) introducing the sample into a reaction chamber which is formed between a chamber support and a microarray, the microarray comprising a substrate with nucleic acid probes immobilized thereon on array elements;
- An essential feature of a preferred embodiment of the method according to the invention in this aspect of the present invention is that the detection of a hybridization between the nucleic acids to be detected and the nucleic acid probes immobilized on the substrate of the microarray is carried out without removing those molecules that are not included in the reaction chamber the nucleic acids immobilized on the substrate are hybridized.
- Such molecules can be, for example, primers provided with a detectable marker, which are not during the
- Amplification reaction were implemented, or nucleotides or nucleic acid molecules provided with a detectable marker, for which there is no complementary nucleic acid probe on the array that specifically hybridizes with this nucleic acid. This means that the interaction or hybridization between nucleic acid targets and nucleic acid probes can be detected without rinsing or washing steps being necessary after the hybridization.
- the method according to the invention in this aspect of the present invention preferably does not carry out a washing step for removing molecules which are not hybridized with the nucleic acids immobilized on the substrate, it has surprisingly been found that despite the fact that the molecules do not remove them In the background of the solution, an accurate and sensitive detection of the specific hybridization between target nucleic acids and nucleic acid probes immobilized on the substrate is ensured.
- the method according to the invention thus enables the amplification and the qualitative and quantitative detection of nucleic acids in a reaction chamber, wherein the detection of molecular interactions or hybridizations can take place immediately after the completion of a cyclic amplification reaction, preferably without changing the samples - or reaction liquids is required.
- the method according to the invention also ensures cyclic detection of hybridization events during amplification, i.e. proof of hybridization also during the cyclic amplification reaction.
- the amplification products can be quantified during the amplification reaction and after the amplification reaction has ended.
- the detection thus takes place during the cyclic amplification reaction and / or after the cyclical amplification reaction has ended. This is preferably done
- the cyclic amplification reaction is preferably a PCR.
- three temperatures are conventionally passed through for each PCR cycle.
- the hybridized nucleic acids preferably detach from the microarray at the highest temperature, ie the denaturation temperature.
- a preferred value for the denaturation temperature is 95 ° C.
- a measurement value can thus be determined at this denaturation temperature, which serves as a zero value or reference value for the nucleic acids detected in the respective PCR cycle.
- an annealing temperature of, for example, about 60 ° C., hybridization between the nucleic acids to be detected and the nucleic acids immobilized on the substrate of the microarray is made possible.
- the detection or the detection of oligonucleotides present in a PCR cycle is therefore carried out at the annealing temperature.
- the detection is preferably carried out at a temperature below the annealing temperature of an amplification cycle.
- the detection can take place at a temperature in the range from 25 ° C. to 50 ° C. and preferably in the range from 30 ° C. to 40 ° C.
- the hybridization between the nucleic acids to be detected and the nucleic acids immobilized on the substrate of the microarray is first carried out at a low temperature in order to subsequently increase the hybridization temperature increase.
- Such an embodiment offers the advantage that the hybridization time is reduced compared to hybridizations at temperatures below 50 ° C. without losing specificity in the interactions.
- the detection of an interaction between the probe and the target molecule is usually carried out in the context of the present invention as follows: after the probe or the probes have been fixed in a predetermined manner to a specific matrix in the form of a microarray, the targets are in a solution with the probes brought into contact and incubated under defined conditions. As a result of the incubation, a specific one is found between the probe and the target
- the binding that occurs is significantly more stable than the binding of target molecules to probes that are not specific for the target molecule.
- the detection or detection of the specific interaction between a target and its probe can then be carried out by a large number of methods, which generally depend on the type of marker which, before, during or after the interaction of the target molecule with the microarray in target molecules has been introduced.
- markers are fluorescent groups, so that specific target-probe interactions with high spatial resolution and compared to other conventional detection methods, especially mass-sensitive methods, can be read fluorescence-optically with little effort (see, for example, A. Marshall, J Hodgson, DNA chips: An array of possibilities, Nature Biotechnology 1998, 16, 27-31; G. Ramsay, DNA Chips: State of the art, Nature Biotechnology 1998, 16, 40-44).
- Antibody libraries, receptor libraries, peptide libraries and nucleic acid libraries can be used as substance libraries that can be immobilized on microarrays or chips.
- the nucleic acid libraries play by far the most important role. These are microarrays on which deoxyribonucleic acid (DNA)
- RNA molecules are immobilized.
- the nucleic acids to be detected are preferably provided with a detectable marker.
- the detectable marker is particularly preferred
- Fluorescent markers The signal of the molecules in solution, which are not hybridized with the nucleic acid probes of the array, i.e. the background, can be kept low compared to the signal of the nucleic acids hybridized with the nucleic acid probes, in particular, in that, in the method according to the invention, particularly thin reaction chambers, in particular in Form of a
- Capillary gap can be used.
- the accumulation of the target molecules on the array surface due to the specific binding of the probe and target makes it possible, with a correspondingly thin design of the reaction chamber, preferably as a capillary gap, the signals on the microarray can also be represented, for example, with a fluorescence-optical system that images the entire volume of the reaction chamber.
- the sample is thus introduced into a reaction chamber in the form of a capillary gap between the chamber carrier and the microarray.
- the capillary gap preferably has a thickness in the range from 10 ⁇ m to 200 ⁇ m, particularly preferably in the range from 25 ⁇ m to 150 ⁇ m and most preferably in the range from 50 ⁇ m to 100 ⁇ m.
- the capillary gap has a thickness of 60 ⁇ m, 70 ⁇ m, 80 ⁇ m or 90 ⁇ m.
- the process according to the invention is carried out in a reaction chamber or a reaction chamber with a thickness of, for example, from 0.7 mm to 2.5 mm, preferably from 1.0 mm to 2.0 mm and particularly preferably from 0.8 mm to 1.8 mm performed.
- the thickness of the reaction space is 1.1 mm.
- epifluorescence-technical structures with imaging detection technology are required for the detection of a hybridization.
- CMOS, JFIT or scanning PMT as well as wavelength-selected areal or point-scanning illumination, e.g. by means of white light sources, LED, organic LED (OLED) and the like, particularly suitable.
- focus-selective detection methods can of course also be used in the method according to the invention, such as confocal techniques or on the application of depth selective lighting due to e.g. methods based on total reflection, evanescent coupling of ambient light (TIRF) in the sample substrate or the use of waveguides.
- TIRF ambient light
- CCD-based detectors which, in order to discriminate against optical effects such as scattering and reflections, implement the fluorophores in the dark field by incident light or transmitted light (see e.g. CE Hooper et al., Quantitative Photone Imaging in the Life Sciences Using Intensified CCD Cameras, Journal of Bioluminescence and Chemoluminescence (1990), 337-344).
- Other alternatives for fluorescence detection systems that can be used in the method according to the invention are white light structures such as those e.g. are described in WO 00/12759, WO 00/25113 and WO 96/27025; confocal systems, e.g. in US 5,324,633, US 6,027,880, US 5,585,639 and WO 00/12759; Confocal excitation systems based on Nipkow discs with imaging confocal imaging, e.g. in US
- the detection of the hybridization between target nucleic acids and the nucleic acids immobilized on the substrate during the cyclic amplification reaction enables a continuous detection of the signal increase on the probe array or microarray.
- the method according to the invention determines the initial concentration of the nucleic acids to be detected in the sample by correlation with the number of amplification cycles that are required for the hybridization between the nucleic acids to be detected and the nucleic acid probes immobilized on the substrate to be detectable.
- Figure 9 shows the course of the exponential amplification of a target with different initial concentrations of target molecules in the sample.
- a typical detection limit when carrying out the method according to the invention using conventional fluorescence detection is at a target concentration in the range from 1 pM to 10 pM.
- Figure 9 shows that this range is reached depending on the initial concentration of nucleic acids to be detected in the sample after a different number of amplification cycles. Consequently, the starting concentration of the detecting nucleic acids in the sample can be concluded from the number of amplification cycles required to reach this detection limit.
- the sample contains a nucleic acid in a known concentration which interacts or hybridizes with a nucleic acid probe of the microarray.
- a nucleic acid in a known concentration is also referred to in the context of the present invention as a control nucleic acid or control.
- Figure 10 shows the development of a hybridization signal as a function of the number of amplification cycles and of the initial concentration of the target nucleic acids in solution as a result of the exponential amplification.
- Figure 10 also shows that simply determining the When the detection limit is reached, it is possible to quantify the target amount, in particular if a corresponding control nucleic acid is carried in the sample in a known concentration and a corresponding calibration is carried out.
- the targets to be examined can be present in any type of sample, preferably in a biological sample.
- the targets are preferably isolated, cleaned, copied and / or amplified by the method according to the invention before they are detected and quantified.
- the amplification is usually carried out by conventional PCR methods or by a method as described above for parallel amplification of the target molecules to be analyzed by PCR and detection by hybridization of the target molecules with the substance library carrier.
- the amplification is carried out as a multiplex PCR in a two-stage process (see also WO 97/45559).
- a multiplex PCR is carried out using fusion primers whose 3 'ends are gene-specific and whose 5' ends represent a universal region. The latter is the same for all forward and reverse primers used in the multiplex reaction.
- the amount of primer is limited. This allows all multiplex products to be amplified to a uniform molar level, provided that the number of cycles is sufficient to be sufficient for all products
- the detection is preferably carried out in that the bound targets are provided with at least one marking which is detected in step c).
- the label which is coupled to the targets or probes is preferably a detectable unit or a detectable unit which is coupled to the targets or probes via an anchor group.
- the method according to the invention is extremely flexible with regard to the possibilities of detection or marking.
- the method according to the invention is thus compatible with a large number of physical, chemical or biochemical detection methods.
- the only requirement is that the unit or structure to be detected can be coupled directly to a probe or a target, for example an oligonucleotide, or linked via an anchor group that can be coupled to the oligonucleotide.
- the detection of the label can be based on fluorescence, magnetism, charge, mass, affinity, enzymatic activity, reactivity, a gold label, and the like. based.
- the marking can be based on the use of fluorophore-marked structures or building blocks.
- the label can be any dye that can be coupled to targets or probes during or after amplification or synthesis.
- cy dyes (Amersham Pharmacia Biotech, Uppsala, Sweden), alexa dyes, Texas red, fluorescein, rhodamine (Molecular Probes, Eugene, Oregon, USA), lanthanides such as samarium, ytterbium and europium (EG&G, Wallac, Freiburg, Germany).
- luminescence markers luminescence markers, metal markers, enzyme markers, radioactive markers and / or polymeric markers can also be used in the context of the present invention as a marker or as a detection unit which is coupled to the targets or probes.
- a nucleic acid can also be used as a label (tag), which can be detected by hybridization with a labeled reporter. This is also known as sandwich hybridization. Various molecular biological detection reactions such as primer extension, ligation and RC A are used to detect the tag.
- the detectable unit is coupled to the targets or probes via an anchor group.
- Preferred anchor groups are biotin, digoxygenin and the like.
- the anchor groups are reacted in a subsequent reaction with specifically binding components, for example streptavidin conjugates or antibody conjugates, which are themselves detectable or trigger a detectable reaction.
- the anchor groups can be converted into detectable units before, during or after addition of the sample comprising the targets or, if appropriate, before, during or after the cleavage of the selectively cleavable bond in the probes.
- the labeling can also be carried out by interaction of a labeled molecule with the probe molecules.
- the label can be labeled by hybridizing one as described above
- Oligonucleotide with an oligonucleotide probe or an oligonucleotide target are described, for example, in Lott Industries and Zorbas, Bioanalytik, Spektrum Akademischer Verlag, Heidelberg, Berlin, 1998, chapters 23.3 and 23.4.
- detection methods are used which, as a result, provide an adduct with a specific solubility product which results in precipitation.
- substrates or starting materials are used for the marking, which can be converted into a poorly soluble, usually colored product.
- enzymes can be used in this labeling reaction, which catalyze the conversion of a substrate or educt into a poorly soluble product. Reactions that are suitable for leading to a deposit on array elements, as well as possibilities for the detection of the deposit, are described, for example, in the international patent application WO 00/72018 and in the international one
- Patent application WO 02/02810 described, the content of which is hereby expressly incorporated by reference.
- the bound targets are provided with a label which catalyzes the reaction of a soluble substrate or educt to form a sparingly soluble precipitate on the array element on which a probe / target interaction has taken place or which acts as a seed for the conversion of a soluble substrate to a sparingly soluble precipitate on the array element on which a probe-target interaction has taken place.
- a label which catalyzes the reaction of a soluble substrate or educt to form a sparingly soluble precipitate on the array element on which a probe / target interaction has taken place or which acts as a seed for the conversion of a soluble substrate to a sparingly soluble precipitate on the array element on which a probe-target interaction has taken place.
- enzymes catalyze the conversion of a substrate or educt into a poorly soluble, usually colored product.
- the reaction leading to the formation of a precipitate on the array elements is particularly preferably an enzyme-catalyzed conversion of a soluble substrate or starting material into a poorly soluble product.
- the reaction leading to the formation of a precipitate on the array elements is a peroxidase-catalyzed oxidation of 3,3 ', 5,5'-tetramethylbenzidine.
- Tetramethylbenzidine horseradish peroxidase used.
- other peroxidases are known to the person skilled in the art which can be used for the oxidation of 3,3 ', 5,5'-tetramethylbenzidine.
- Table 1 above gives an overview of a number of possible reactions which are suitable for leading to a precipitation of array elements on which there is an interaction between the target and the probe is done.
- the methods according to the invention include the following variants are conceivable for the detection of the probe / target interactions via insoluble precipitates.
- the targets are provided with a catalyst, preferably an enzyme, which catalyzes the conversion of a soluble substrate into an insoluble product.
- the reaction which leads to the formation of a precipitate on the array elements in this case is the conversion of a soluble substrate or starting material into an insoluble product in the presence of a catalyst, preferably enzyme, coupled to the targets.
- the enzyme is preferably selected from the group consisting of horseradish peroxidase, alkaline phosphatase and glucose oxidase.
- the soluble substrate or starting material is preferably selected from the group consisting of 3,3'-diaminobenzidine, 4-chloro-1-naphthol, 3-amino-9-ethylcarbazole, p-phenylenediamine HCl / pyrocatechol, 3,3 ' , 5,5'-tetramethylbenzidine, naphthol / pyronine, Bromochloroindoyl phosphate, nitrotetraazolium blue and phenazine methosulfate.
- a colorless, soluble hydrogen donor for example 3,3'-diaminobenzidine, is converted into an insoluble colored product in the presence of hydrogen peroxide.
- the enzyme horseradish peroxidase transfers hydrogen ions from the donors to hydrogen peroxide to form water.
- the reaction which results in the formation of a precipitate on the array elements is the formation of a metallic precipitate.
- the reaction leading to the formation of a precipitate on the array elements is particularly preferably the chemical reduction of a silver compound, preferably silver nitrate, silver lactate, silver acetate or silver tartrate, to elemental silver.
- a silver compound preferably silver nitrate, silver lactate, silver acetate or silver tartrate
- Formaldehyde and / or hydroquinone are preferably used as reducing agents.
- colloidal gold or defined gold clusters are coupled to the targets, possibly via certain mediator molecules such as streptavidin.
- mediator molecules such as streptavidin.
- the gold marking range coupled to the targets acting as a crystallization seed or catalyst for example for the reduction of silver ions to a silver deposit.
- the targets coupled with gold markings are also referred to below as gold conjugates.
- a relative quantification of the probe / target interaction can also take place.
- the relative quantification of the concentration of the bound targets on a probe array by detecting a precipitate or a precipitate is carried out via the
- Oligonucleotide probes the ratio of bound target to gold particles 1: 1. In other embodiments of the present invention, it can be a multiple or a fraction thereof.
- the detection in this embodiment of the detection method according to the invention is thus carried out by measuring the change in transmission, reflection or scatter, which is caused by the precipitation, which occurs on the array elements on which a probe / target interaction has taken place, by the catalytic effect of the the linked target is generated.
- a non-transparent precipitate is preferably deposited catalytically by means of such interaction hybrids, ie the targets provided with a label such as colloidal gold or defined gold clusters.
- silver as a precipitate has proven particularly advantageous in the case of gold conjugates.
- the temporal course of the precipitation formation on the array elements is thus detected in the form of signal intensities in step c).
- an accurate determination of the relative quantitative amount of bound targets can be ensured.
- the qualitative and / or quantitative detection of the probe / target interaction by measuring the transmitted light absorption can be carried out analogously to the example described above for a method according to the invention using a device according to the invention which comprises an optical system with which the temporal The course of precipitation formation on the detection surface is detectable.
- a prerequisite for the binding of a target molecule, for example marked with a fluorescence group, in the form of a DNA or RNA molecule to a nucleic acid probe of the microarray is that both the target molecule and the probe molecule are in the form of a single-stranded nucleic acid.
- An efficient and specific hybridization can only take place between such molecules.
- Single-stranded nucleic acid target and nucleic acid probe molecules are usually obtained by heat denaturation and optimal selection of parameters such as temperature, ionic strength and concentration of helix-destabilizing molecules. This ensures that only probes with almost perfectly complementary, ie corresponding sequences with the target sequence remain paired (see e.g. AA Leitch, T. Schwarzacher, D. Jackson, IJ Leitch, 1994, in vitro hybridization, Spektrum Akademischer Verlag, Heidelberg / Berlin / Oxford).
- the nucleic acid to be detected is initially amplified by a PCR
- at least one competitor is initially added to the reaction in a further preferred embodiment, which inhibits the formation of one of the two template strands amplified by the PCR.
- a DNA molecule is added which competes with the primer used for the PCR amplification of the template for binding to the template and which cannot be extended enzymatically.
- the single-stranded nucleic acid molecules amplified by the PCR are then detected by hybridization with a complementary probe.
- the nucleic acid to be detected is first amplified in a single-strand excess by a PCR and detected by a subsequent hybridization with a complementary probe, a competitor being initially added to the PCR reaction, which is a DNA molecule or a molecule of a nucleic acid Analogs act that can hybridize to one of the two strands of the template, but not to the area that is detected by the probe hybridization, and which is not enzymatically extendable.
- Competitors can therefore, according to the invention, be proteins, peptides, DNA ligands, intercalators, nucleic acids or their analogs. Proteins or peptides which are capable of binding single-stranded nucleic acids with sequence specificity and have the properties defined above are preferably used as competitors. Nucleic acid molecules and nucleic acid analog molecules are particularly preferably used as secondary structure breakers.
- the initial addition of the competitor to the PCR during the amplification essentially inhibits the formation of one of the two template strands.
- “Essentially inhibited” means that a sufficient single-strand excess and a sufficient amount of the other template strand are produced in the course of the PCR in order to ensure efficient detection of the amplified strand by the hybridization.
- the single-strand excess achieved by the PCR is 1.1 to 1000, preferably 1.1 to 300, preferably 1.1 to 100, particularly preferably 1.5 to 100, compared to the non-amplified strand. likewise particularly preferably the factor 1.5 to 50, particularly preferably the factor 1.5 to 20 and most preferably the factor 1.5 to 10.
- Competitors are therefore single-stranded DNA- or RNA-binding proteins with specificity for one of the two template strands to be amplified in a PCR.
- it can be aptamers that bind sequence-specifically only to certain regions of one of the two template strands to be amplified.
- nucleic acids or nucleic acid analogs are preferably used as competitors.
- nucleic acids or nucleic acid analogues act as a competitor of the PCR in that they either compete for the primer binding site with one of the primers used for the PCR or, because of a sequence complementarity, can hybridize with a region of a template strand to be detected. This area is not the sequence that is detected by the probe.
- nucleic acid competitors cannot be extended enzymatically.
- nucleic acid analogs can e.g. are so-called peptide nucleic acids (PNA).
- PNA peptide nucleic acids
- nucleic acid analogs can also be nucleic acid molecules in which the nucleotides are linked to one another via a phosphothioate bond instead of a phosphate bond. It can also be a nucleic acid analogue in which the naturally occurring sugar building blocks ribose or deoxyribose against alternative sugars such as e.g. Arabinose or trehalose etc. were exchanged.
- the nucleic acid derivative can be “locked nucleic acid” (LNA).
- LNA locked nucleic acid
- DNA or RNA molecules are preferably used as competitors.
- the inhibition of the amplification of one of the two template strands in the context of the PCR reaction is based on different mechanisms. This is discussed in the following using an example of a DNA molecule. If, for example, a DNA molecule is used as a competitor, this can have a sequence which is at least partially identical to the sequence of one of the primers used for PCR in such a way that a specific hybridization of the DNA competitor molecule with the corresponding template strand under stringent conditions is possible.
- the DNA molecule used for the competition cannot be extended by a DNA polymerase in this case, the DNA molecule competes with the respective primer for the binding to the template during the PCR reaction.
- the amplification of the template strand defined by the primer can be inhibited in such a way that the production of this template strand is significantly reduced.
- the PCR follows exponential kinetics that are higher than would be expected with the amounts of competitor used. In this way, a single strand excess is produced in an amount which is sufficient for efficient detection of the amplified target molecules by hybridization.
- the nucleic acid molecules or nucleic acid analogs used for competition must not be able to be extended enzymatically.
- "Enzymatically non-renewable” means that the DNA or RNA polymerase used for amplification cannot use the nucleic acid competitor as a primer, i.e. is not able to synthesize 3 'of the sequence defined by the competitor the respective counter strand to the template.
- the DNA competitor molecule can also have a sequence which is complementary to a region of the template strand to be detected, which is not addressed by one of the primer sequences and which cannot be extended enzymatically. in the As part of the PCR, the DNA competitor molecule will then hybridize to this template strand and block the amplification of this strand accordingly.
- sequences of DNA competitor molecules or generally nucleic acid competitor molecules can be selected accordingly. If the nucleic acid competitor molecules have a sequence that is not essentially identical to the sequence of one of the primers used for PCR, but is complementary to another region of the template strand to be detected, this sequence should be chosen so that it is not in falls within the range of the template sequence that is detected during hybridization with a probe. This is necessary because there is no need for a work-up reaction between the PCR and the hybridization reaction. If a nucleic acid molecule that fell within the range to be detected were used as a competitor, this would compete with the single-stranded target molecule for binding to the probe.
- Such competitors preferably hybridize in the vicinity of the template sequence which is detected by the probe.
- the position indication “in the vicinity” is to be understood as it is given for secondary structure breakers.
- the competitors according to the invention can also be used directly
- nucleic acids or nucleic acid analogs that cannot be extended enzymatically are used as competitive molecules, their sequence or structure should be selected such that they cannot be extended enzymatically by DNA or RNA polymerases.
- the 3 'end of a nucleic acid competitor is preferably designed such that there is no complementarity with the Has template and / or instead of the 3-OH group at the 3 'end carries another substituent.
- the nucleic acid Competitor due to the lack of base complementarity at the 3 'end cannot be extended by the common DNA polymerases.
- the nucleic acid competitor preferably has no complementarity with its target sequence at its 3 'end with respect to the last 4 bases, particularly preferably with regard to the last 3 bases, particularly preferably with regard to the last 2 bases and most preferably with regard to the last base.
- Such competitors can also have non-natural bases at the common positions, which do not allow hybridization.
- Nucleic acid competitors that cannot be extended enzymatically can also have a 100% complementarity to their target sequence if they are modified in their backbone or at their 3 'end in such a way that they cannot be extended enzymatically.
- nucleic acid competitor has a group other than the OH group at its 3 'end, these substituents are preferably a phosphate group, a hydrogen atom (dideoxynucleotide), a biotin group or an amino group. These groups cannot be extended by the usual polymerases.
- a DNA molecule is particularly preferably used which competes with one of the two primers used for PCR for binding to the template and which has been provided with an amino link at the 3 'end during chemical synthesis.
- Such competitors can have 100% complementarity to their target sequence.
- Nucleic acid analog competitors such as PNAs, on the other hand, do not have to have a blocked 3'-OH grappe or a non-complementary base at their 3 'end, since they are not recognized by the DNA polymerases due to the backbone altered by the peptide bond and are therefore not extended become.
- Corresponding other modifications of the phosphate group which are not recognized by the DNA polymerases, are known to the person skilled in the art. These include Nucleic acids with backbone modifications such as 2'-5 'amide bonds (Chan et al. (1999) J. Chem. Soc, Perkin Trans. 1, 315-320), sulfide bonds (Kawai et al.
- competitors that hybridize to different areas of the template (e.g. the primer binding site) can also be used simultaneously in one PCR. If the competitors over
- the DNA competitor molecule can have a sequence complementary to one of the primers.
- antisense DNA competitor molecules for example, can then be used, depending on the quantitative ratio between the antisense DNA competitor molecule and the primer, for the primer in the Titrate the PCR reaction so that it no longer hybridizes with the respective template strand and accordingly only the template strand defined by the other primer is amplified.
- the nucleic acid competitor can, but need not, be enzymatically extendable.
- nucleic acid competitors when nucleic acid competitors are used in the context of this invention, this includes nucleic acid analog competitors, unless the context indicates otherwise.
- the nucleic acid competitor can bind reversibly or irreversibly to the corresponding strand of the template.
- Binding can take place through covalent or non-covalent interactions.
- the nucleic acid competitor is preferably bound via non-covalent interactions and is reversible.
- the binding to the template is particularly preferably carried out by forming Watson-Crick base pair pairs.
- sequences of the nucleic acid competitors are generally based on the sequence of the template strand which is to be detected, in the case of antisense primers, on the other hand, on the primer sequences to be titrated, which, however, are in turn defined by the template sequences.
- PCR amplification of nucleic acids is a standard laboratory method with its diverse variation and
- a PCR is characterized in that the double-stranded nucleic acid template, usually a double-stranded DNA molecule, is first subjected to heat denaturation for 5 minutes at 95 ° C., as a result of which the two strands are separated from one another. After cooling to the so-called “annealing" temperature (defined by the primer with the lower melting temperature), the “forward” and “reverse” primers present in the reaction solution attach to the positions in the respective template strands which are complementary to their sequence.
- the “annealing” temperature of the primers depends on the length and base composition of the primers. It can be calculated based on theoretical considerations. Information on the calculation of "annealing" temperatures can be found, for example, in Sambrook et al. (vide supra).
- an elongation step follows in which the activity of those present in the reaction solution DNA polymerase deoxyribonucleotides are linked to the 3 'end of the primer.
- the identity of the inserted dNTPs depends on the sequence of the template strand hybridized with the primer. Since thermostable DNA polymerases are generally used, the elongation step usually takes place between 68-72 ° C.
- DNA polymerases that can be used the production of double-stranded DNA templates, the design of primers, the choice of the annealing temperature and variations of the classic PCR, numerous literature are available to the person skilled in the art.
- the person skilled in the art is familiar with the fact that, for example, single-stranded RNA, such as, for example, mRNA, can also be used as a template. This is usually converted into a double-stranded cDNA beforehand by reverse transcription.
- thermostable DNA-dependent DNA polymerase is used as the polymerase.
- a thermostable DNA-dependent DNA polymerase is selected from the group consisting of Taq DNA polymerase (Eppendorf, Hamburg, Germany and Qiagen, Hilden, Germany), Pfu DNA polymerase (Stratagene, La Jolla, USA), Tth-DNA polymerase (Biozym Epicenter Technol., Madison, USA), Vent-DNA polymerase, DeepVent-DNA polymerase (New England Biolabs, Beverly, USA), Expand-DNA polymerase (Röche, Mannheim, Germany) used.
- polymerases which have been optimized from naturally occurring polymerases by targeted or evolutionary change is also preferred.
- DMH mg mg of the PCR in the presence of the substance library carrier, the use of Taq polymerase from Eppendorf (Germany) or the Advantage-cDNA polymerase mix from Clontech (Palo Alto, CA, USA) is particularly preferred.
- a device which is particularly suitable for the amplification and for the qualitative and quantitative detection of nucleic acids by means of a method according to the invention as described above.
- the device according to the invention comprises a temperature control and / or regulating unit; and a Reaction chamber which is formed between a chamber support and a microarray, the microarray comprising a substrate with nucleic acid probes immobilized thereon on array elements and the temperature in the reaction chamber being controllable and / or regulatable by the temperature control and regulating unit.
- the device according to the invention is further configured such that the hybridization between nucleic acids to be detected and nucleic acid probes immobilized on the substrate can be detected without removing molecules from the reaction chamber which are not hybridized with the nucleic acids immobilized on the substrate.
- a chip or microarray located in the reaction chamber which comprises a carrier with a detection surface on which a substance library is immobilized, ensures the possibility of providing a very high probe density in the reaction chamber.
- the reaction chamber of the device according to the invention is preferably designed as a capillary gap.
- the capillary gap preferably has a thickness in the range from 10 ⁇ m to 200 ⁇ m, particularly preferably in the range from 25 ⁇ m to 150 ⁇ m and most preferably in the range from 50 ⁇ m to 100 ⁇ m.
- the capillary gap has a thickness of 60 ⁇ m, 70 ⁇ m, 80 ⁇ m or 90 ⁇ m.
- the reaction space or the reaction chamber has, for example, a thickness of 0.7 mm to 2.5 mm, preferably 1.0 mm to 2.0 mm and particularly preferably 0.8 mm to 1, 8 mm on. In a special embodiment, the thickness of the reaction space is 1.1 mm.
- the electrocaloric control or regulation by the temperature control and / or regulating unit allows the setting of defined temperatures both during the processing of the sample to be examined in the reaction chamber and during the detection of the hybridization events. This ensures both improved control and optimization of the detection reaction. Furthermore, the setting of defined temperatures via the temperature control or regulating unit allows complex reactions to be carried out, such as, for example, amplification reactions by PCR.
- the device according to the invention permits processing, condilioning reactions and chip-based characterization of nucleic acids to be carried out almost simultaneously, in a time-efficient manner and less prone to failure.
- a processing or conditioning reaction is understood according to the invention to mean a reaction whose reaction products can be characterized by chip-based experiments.
- a device according to the invention for the detection of molecular interactions in closed reaction chambers preferably consists of four main functional elements (see Figure 1).
- the mechanical, electrical and fluidic recording of the reaction chamber takes place in a recording module (1).
- the reaction chamber is also referred to below as a microreactor.
- An optical system (2) is provided for the detection of the reaction results.
- the reaction results can be processed into an analysis result in a controller (3). If necessary, the analysis result is made available for storage and / or further processing by suitable connecting elements (4).
- a reaction chamber which can advantageously be used as a component of the device according to the invention is described in detail in international patent application WO 01/02094, the content of which is hereby expressly incorporated by reference.
- the reaction chamber which may be identified by a barcode, is installed in a fluidic receiving module, where it can be filled with one or more reaction solutions.
- the reaction chamber also optionally has electrical contacts, as a result of which thermal control or regulation of reactions in the reaction chamber is ensured, for example, via integrated sensor and / or heating elements. This is particularly advantageous for carrying out thermally sensitive amplification reactions for DNA or RNA, hybridizations of DNA or RNA or reactions for signal amplification such as, for example, by metal precipitation on appropriately labeled target molecules and bound to the substance library.
- the solutions required for carrying out the replication and detection reactions can be introduced into the reaction chamber via suitable connecting elements, such as channels.
- suitable connecting elements such as channels.
- Suitable controllers can be used to monitor the course of the reaction.
- the devices according to the invention also ensure the transfer of the raw data or analysis results to external computer or computer networks, for example for the storage of this data, via electronic interfaces which may be present.
- the device preferably comprises a detection system, in particular an optical system, particularly preferably a fluorescence-optical system.
- the fluorescence-optical system is a system that images the entire volume of the reaction chamber.
- Such systems have the advantage over confocal fluorescence systems or systems for detection by means of evanescent field excitation (see e.g. Biosensors and Bioelectronics, 18 (2003) 489-497) that they are easier to handle and considerably less expensive.
- Examples of the entire volume of the fluorescence optical systems imaging the reaction chamber are epifluorescence structures with imaging detection technology, e.g. on the basis of CCD, CMOS, JFIT or scanning PMT, as well as on the basis of wavelength-selected flat or point-scanning illumination, e.g. by means of white light sources, LED, organic LED (OLED) and the like, particularly suitable.
- imaging detection technology e.g. on the basis of CCD, CMOS, JFIT or scanning PMT
- wavelength-selected flat or point-scanning illumination e.g. by means of white light sources, LED, organic LED (OLED) and the like, particularly suitable.
- focus-selective detection methods can of course also be used in the method according to the invention, such as confocal techniques or on the application of depth selective lighting due to e.g. on total
- Reflection-based evanescent coupling of excitation light (TIRF) in the sample substrate or the use of waveguide-based methods Such foci-selective methods are to be preferred in particular if a further exclusion of the background signals caused by the fluorescent molecules present in the liquid, ie not hybridized, is necessary in order to increase the sensitivity.
- TIRF excitation light
- a device for the duplication and detection of nucleic acids comprises an optical system, preferably a fluorescence-optical system or an optical system with which the time course of
- Precipitation formation on the detection surface is detectable.
- the device according to the invention in this preferred embodiment is characterized in that, due to the integrated optical system or the reader system, detection of molecular interactions is also possible
- the device according to the invention in this preferred embodiment contains an integrated optical system with which the time course of precipitation formation on the detection surface can be detected ensures an exact determination of the relative quantitative amount of nucleic acids bound to the substance library.
- the optical system ensures the imaging of the substance library during or after the completion of the duplication and / or detection reactions on a suitable detector, which is designed, for example, as a two-dimensional, electrically readable detection element.
- a suitable detector which is designed, for example, as a two-dimensional, electrically readable detection element.
- the sample is illuminated by means of an illumination module or a light source of the optical system the resulting signals are shown filtered according to the markings used.
- the optical system also ensures a kinetic, i.e. dynamic recording of the reaction results.
- the optical system of the device according to the invention is suitable for recording the chronological course of a silver deposit to amplify hybridization signals between gold-labeled target molecules and the substance library.
- the highly integrated structure of the device according to the invention allows the transfer of several images during the course of the reaction for processing in a suitable data processing module or controller.
- the optical system with which the time course of precipitation formation on the detection surface of the chip can be detected, preferably comprises a two-dimensionally readable detector.
- the detector is preferably a camera, in particular a CCD or CMOS camera or a similar camera.
- CCD or CMOS complementary metal-oxide-semiconductor
- CMOS complementary metal-oxide-semiconductor
- the cameras used in the optical system of the device according to the invention ensure that the illumination intensity is homogeneous on the image to be imaged Surface is distributed and the signals to be detected by reflection, transmission modulation, scattering, polarization modulation and the like can be mapped with the detection technology used within the available dynamics.
- Such lighting methods are described, for example, in international patent application WO 00/72018 and are also commercially available (for example from Vision & Control GmbH (Suhl, Germany) for dark field lighting and from Edmund Industrieoptik GmbH (Karlsruhe, Germany) for LED ring light).
- a high local resolution of the area to be detected can also be achieved, for example, by imaging on detectors such as mirror arrays or LCD elements and adjusting them according to a pattern to be detected or an area to be defined, as is the case, for example, for fluorescence applications in the German patent application DE 199 14 279 is described.
- detectors such as mirror arrays or LCD elements and adjusting them according to a pattern to be detected or an area to be defined, as is the case, for example, for fluorescence applications in the German patent application DE 199 14 279 is described.
- the advantage of such a detector when measuring reflection or transmission modulations is the integration of the thermal, electrical and fluidic control or regulation in the possibility of optical signal processing and thus in lower technical requirements for the computer technology involved.
- the detectors usually record the entire area of the probe array.
- scanning detectors can also be used to read the chip.
- the device according to the invention comprises movable optical components for guiding light or movable mechanical components for holding the reaction chambers, so that the guiding of the the respective components via the individual positions to be scanned, that is to say the respective measuring points.
- the image is captured by a mathematical reconstruction of the image from the respective measuring points.
- the camera in this embodiment is a movable line scan camera.
- the optical system preferably additionally comprises a light source, particularly preferably a multispectral or a coherent light source.
- a light source particularly preferably a multispectral or a coherent light source.
- Examples of light sources in the context of the present invention are lasers, light-emitting diodes (LED) and / or high-pressure lamps.
- the light source of the optical system preferably ensures homogeneous illumination of the wearer.
- light sources in the form of lighting arrays can also be used in the device according to the invention.
- homogeneous illumination of the carrier can be ensured, for example, by the light source comprising a plurality of diffusely radiating light sources, the superimposition of which results in homogeneous illumination.
- the light source comprising a plurality of diffusely radiating light sources, the superimposition of which results in homogeneous illumination.
- diffusely scattering LEDs which are arranged in a matrix, enable homogeneous illumination at short distances from the sample.
- the device according to the invention can be designed such that the detection surface can be scanned in a cell-like manner by the light source. If a raster-shaped or scanning guidance of the light beam over the detection surface is desired, the following design options are conceivable for the device according to the invention:
- the detection surface or the reaction chamber can be designed to be movable and can be guided past a stationary light source. If the light source is a laser, the laser is at rest. The detection surface can also be in the rest position and a movable laser beam can be guided over the detection surface.
- the light source it is also possible for the light source to be moved in one axis and the detection surface in the other axis.
- the device additionally comprises lenses, mirrors and / or filters.
- the use of filters enables on the one hand the spectral limitation of the homogeneous illumination and on the other hand the illumination of the samples with different wavelengths.
- the device according to the invention additionally comprises filter changers. With these filter changers, the optical filters can be changed quickly and possible misinformation, for example due to contamination, can be clearly identified and eliminated.
- the optical system is preferably designed such that the detection area can be illuminated homogeneously, preferably with an illumination intensity homogeneity of at least 50%, particularly preferably of at least 60% and most preferably of at least 70%).
- the optical system is designed in such a way that the time course of the change in transmission properties of the detection area can be detected. This can be ensured, for example, by the light source and
- Detector are arranged on opposite sides in the reaction chamber and the reaction chamber including the support for the detection area at least is optically transparent in the region of the beam path from the light source to the detector.
- the optical system is arranged such that the time course of the change in reflection properties of the
- Detection area is detectable.
- the disadvantage of the poor reflection of the sample is supplemented in this embodiment by transmission effects in which the illuminating light is reflected via a mirror layer behind the sample, either as an independent mirror or as a layer applied to the back of the sample carrier.
- an area radiator can be arranged on the opposite side of the carrier element and thus also to the sensor, for example of a CCD camera. A very compact arrangement is made possible in this way.
- the device additionally comprises a semi-transparent mirror between the light source and carrier element.
- the light from the light source reaches the sample through a semitransparent mirror and the image is imaged in reflection by the semitransparent mirror and, if appropriate, a reading optic on a camera.
- the optical system is arranged such that the time course of the change in the scattering properties of the detection area can be detected.
- the light source and detector are preferably arranged on the same side of the surface to be detected.
- the optical system can, for example, be arranged such that the sample or the Chip can be illuminated at a certain angle, which is preferably less than 45 ° C. and particularly preferably less than 30 ° C.
- the angle of illumination is chosen so that the incident light is not reflected directly into the detection beam path without the presence of local scattering centers, ie, for example before precipitation forms on the detection surface, and therefore no signal can be detected. If local scattering centers appear on the detection surface, for example due to the formation of a precipitate, part of the incident light reaches the detection beam path and thus leads to a measurable signal in the optical system of the device according to the invention.
- the chamber support or the substance library support is particularly preferably not optically transparent, at least in the area of the detection area.
- Suitable non-optically transparent materials are, for example, silicon, ceramic materials or metals.
- the use of a non-optically transparent chamber carrier has the advantage that lighter, more precise and more homogeneous temperature control of the reaction chamber is ensured due to advantageous physical properties of the carrier materials, so that a successful implementation of temperature-sensitive reactions such as a PCR is ensured.
- the beam path of the light source is designed in such a way that areas of parallel light are present and interference filters can thus be introduced into the optical system without shifting their transmission windows.
- the detection beam path is designed in such a way that regions of parallel light are present and interference filters can thus be introduced into the optical system without shifting their transmission windows. Interference filters greatly change their spectral selectivity in the case of non-parallel beam paths. If the presence of areas of parallel light enables the introduction or arrangement of interference filters in the device according to the invention without changing their spectral selectivity, the device according to the invention is also suitable for the detection of molecular interactions of substances labeled with fluorochromes. A universal CCD-based reaction and detection device can thus be implemented.
- the optical system can, when using white or multispectral light such as e.g. Halogen lighting, xenon, white light LED and the like, e.g. two filters in the illumination and detection beam path or, when using monochrome light sources such as LED or laser, e.g. have a filter in the detection beam path.
- white or multispectral light such as e.g. Halogen lighting, xenon, white light LED and the like, e.g. two filters in the illumination and detection beam path or, when using monochrome light sources such as LED or laser, e.g. have a filter in the detection beam path.
- the device according to the invention can, for example, be designed such that the chamber body of the
- Reaction chamber which contains the chip with the detection surface, is sealingly applied to a chamber carrier in such a way that a sample space is formed with a capillary gap between the chamber carrier and the detection surface or the substrate of the chip, which is temperature-adjustable and flow-controllable.
- Temperature ranges run, carry out and the reaction products preferably to be detected simultaneously by chip-based experiments.
- the device according to the invention can thus be used, for example, to multiply nucleic acid molecules almost simultaneously by PCR and to detect the PCR products by chip-based experiments.
- the sample liquid for such reactions can be heated and cooled efficiently by appropriate means for temperature regulation.
- the device according to the invention can also be used to carry out a reverse transcription reaction and in this way to convert mRNA into cDNA and to characterize the reaction products by hybridization on the chip. In this way, so-called “gene profiling” can be carried out. Since both reverse transcription and hybridization are carried out in one chamber, this process is extremely time-efficient and less prone to failure.
- Restriction digestion can be carried out at desired temperatures in the reaction chamber and the reaction products can be characterized by hybridization on a chip.
- the enzymes can be denatured by heat deactivation.
- the device according to the invention thus enables time-efficient restriction fragment length polymorphism mapping (RFLP mapping).
- ligation can also be carried out, for example.
- the melting behavior of nucleic acid target / nucleic acid probe complexes as a function of the temperature can be investigated with the device according to the invention.
- Devices according to the invention can also be used to carry out the binding behavior of proteins as a function of the temperature. For example, it can be used to test whether antibodies are still able to bind their corresponding antigens after heating for a longer period of time. The prerequisite for this is that in this case the chip is not functionalized by nucleic acid molecules, but by the corresponding proteins or peptides.
- the chamber body of the reaction chamber of the device according to the invention in this aspect of the present invention preferably consists of materials such as glass, plastic and / or metals such as stainless steel, aluminum and brass.
- plastics suitable for injection molding can be used for its production.
- plastics like Makrolon, Nylon, PMMA and Teflon are conceivable.
- Substance library supports and chamber supports can also be closed by septa, which, for example, allow the reaction space to be filled by means of syringes.
- the chamber body consists of optically transparent materials such as glass, PMMA, polycarbonate, polystyrene and / or topaz.
- the choice of materials must be adapted to the intended use of the device. For example, the temperatures to which the device will be exposed should be considered when choosing materials. For example, if the device is to be used for carrying out a PCR, only those plastics may be used which are stable over long periods of time at temperatures such as 95 ° C.
- the chamber support preferably consists of glass, plastics, silicon, metals and / or ceramic materials.
- the chamber support can consist of aluminum oxide ceramics, nylon and / or Teflon.
- the chamber support consists of transparent materials such as glass and / or optically permeable plastics, e.g. PMMA, polycarbonate, polystyrene or acrylic.
- the chamber carrier and / or the substrate is preferably connected to means for applying temperature which are integrated in the device according to the invention and should then preferably consist of materials which are good heat conductors.
- Such thermally conductive materials offer the significant advantage that they ensure a homogeneous temperature profile over the entire area of the reaction space and thus temperature-dependent reactions such as a PCR in the entire reaction chamber can be controlled or regulated homogeneously, with high yield and with high accuracy.
- the chamber support and / or the substrate consist of materials with a high thermal conductivity, preferably with a thermal conductivity in the range from 15 to 500 Wm ⁇ K “1 , particularly preferably in the range from 50 to 300 Wm “ “K “ “ and most preferably in the range of 100 to 200 Wm ⁇ K “1 , the materials usually not being optically transparent.
- suitable thermally conductive materials are silicon, ceramic materials such as aluminum oxide ceramics and / or metals such as stainless steel, aluminum or brass.
- the substrate consists of materials with a high thermal conductivity, such as ceramic Materials.
- the substrate is connected to a means for applying temperature, whereby the opposite side, the chamber support, can be made of a material that does not have a pronounced thermal conductivity, such as a material that is also used for the other chamber body.
- the substrate or the carrier of the device according to the invention is essentially made of ceramic materials
- aluminum oxide ceramics are preferably used.
- alumina ceramics are the ceramics A-473, A-476 and A-493 from Kyocera (Neuss, Germany).
- the ceramics differ essentially in the respective proportion of aluminum oxide (A-473: 93%, A-476: 96% and A-493: 99,%) and in their surface roughness.
- Aluminum oxide ceramics which have the lowest possible surface roughness are particularly preferably used.
- the chamber support and / or the substrate is on its back, i.e. the side facing away from the reaction chamber with possibly miniaturized
- Temperature sensors and / or electrodes provide or have heater fractures there, so that temperature control of the sample liquid and mixing of the sample liquid by an induced electroosmotic flow is possible.
- the temperature sensors can, for example, be designed as nickel-chromium thin-film resistance temperature sensors.
- the electrodes can be designed, for example, as gold-titanium electrodes and in particular as a quadrapole.
- the means for applying temperature can preferably be selected so that the liquid in the capillary gap can be heated and cooled quickly. Rapid heating and cooling is understood here to mean that the means for subjecting the temperature to temperature changes in a range from 0.2 ° K / s to 30 ° K / s, preferably from 0.3 ° K / s to 15 ° K / s, particularly preferably from 0.5 ° K / s to 12 ° K / s and most preferably from 2 ° K / s to 10 ° K / s.
- the means for applying temperature can be designed, for example, as a nickel-chromium thin-film resistance heater.
- the chip or the substrate can preferably consist of borofloat glasses, quartz glass, single-crystal CaF, sapphire disks, topaz, PMMA, polycarbonate and / or polystyrene.
- the choice of materials should also be based on the later intended use of the device or chip. If the chip is used, for example, to characterize PCR products, only materials that can withstand a temperature of 95 ° C may be used.
- the chips are preferably functionalized by nucleic acid molecules, in particular by DNA or RNA molecules. However, they can also be functionalized by peptides and / or proteins, such as antibodies, receptor molecules, pharmaceutically active peptides and / or hormones.
- preferred materials for the substance library support are optical transparent materials such as glass, particularly preferably borosilicate glass, and transparent polymers such as e.g. PMMA, polycarbonate and / or acrylic; for the chamber support, optically transparent materials, such as glass and / or plastics, and in particular optically non-transparent materials, such as silicon, ceramic materials; and for the reaction chamber plastics such as Makrolon, PMMA, polycarbonate, Teflon and the like, metals such as stainless steel, aluminum and / or brass and glass.
- the chamber carrier can consist of optically transparent materials, while the substance library carrier consists of non-transparent materials.
- the device according to the invention additionally comprises at least one fluid container, which is connected to the reaction chamber, and optionally a unit for controlling the loading and unloading of the reaction chamber with fluids.
- fluids are understood to be liquids or gases.
- the connection of the fluid containers to the reaction chamber can be carried out, for example, as in international patent application WO 01/02094.
- the device according to the invention comprises a unit connected to the detection system for processing signals recorded by the optical system. This coupling of the detection unit and processing unit, which ensures the conversion of the reaction results into the analysis result, allows, among other things, the use of the device according to the invention as a hand-held device, for example in medical diagnostics.
- the device preferably further comprises an interface for external computers. This allows the transfer of data for storage outside the device.
- the reaction chamber is individually identified via a data matrix.
- a data record is stored in the manufacture of the device according to the invention in a database, which contains information about the substance library, the implementation of the detection reaction and the like. So the data set in particular
- the data record or the data matrix can also contain information about the temperature-time regime of a PCR to be carried out, if necessary, for the purpose of duplicating the target molecules.
- the data record created in this way is preferably given a number which is attached to the holder in the form of the data matrix. If necessary, the created data record can then be called up via the number recorded in the data matrix when reading out the substance library.
- the data matrix can be read out by the temperature control or regulating unit and other controllers such as, for example, a control for filling and filling the reaction chamber via the fluid containers, and thus an automatic execution of the replication and detection reaction can be guaranteed.
- a device for the duplication and detection of nucleic acids which also has a temperature control and / or regulating unit as described above; and also contains a reaction chamber as described above, which comprises a carrier with a detection surface on which a substance library is immobilized, the temperature in the reaction chamber being controllable and / or regulatable by the temperature control and regulating unit.
- the device in this aspect of the present invention has electrical contacts at the respective array spots. These electrical contacts can be contacted via electrodes, for example. Due to the formation of a metallic deposit on the array elements for signal amplification of the targets bound to the substance library, for example gold-labeled targets, a conductive material grows on the array spots to which such binding has taken place, which leads to a
- the substance library carrier preferably has that of the invention
- Device in this aspect of the present invention has a three-dimensional structure, which is formed, for example, by bumps, base and / or through bores, whereby the effect in the fusion of the growing conductive material by the precipitate which forms and the resultant electrical contacts Modification of electrical parameters is supported.
- the device according to the invention based on optical detection preferably has a fluidic unit for exchanging solutions in the reaction chamber, a temperature control or regulating unit, and an optical system suitable for dynamic measurements.
- the above-mentioned units can optionally also be implemented separately in terms of device technology by implementing corresponding interfaces.
- Substance libraries that are immobilized on the microarrays or chips are in particular protein libraries such as antibody, receptor protein or membrane protein libraries, peptide libraries such as receptor ligand libraries, libraries of pharmacologically active peptides or libraries of peptide hormones, and nucleic acid libraries such as DNA or RNA molecule libraries. Nucleic acid libraries are particularly preferred.
- the substance library is preferably in the form of a microarray, particularly preferably with a density of 2 to 10,000 array spots per cm 2 , most preferably with a density of 50 to 5000 array spots per cm, on the substance library carrier or the detection area is immobilized.
- Pre-amplification of the material to be analyzed is furthermore not necessary in all of the above-described embodiments of the device according to the invention.
- Targeted sections of the sample material extracted from bacteria, blood or other cells can be amplified with the aid of a PCR (polymerase chain reaction), in particular in the presence of the device according to the invention or the substance library support, as described in DE 102 53 966 and hybridized to the support. This represents a significant simplification of the workload.
- the devices according to the invention are therefore particularly suitable for use in the parallel implementation of amplification of the target molecules to be analyzed by PCR and detection by hybridization of the target molecules with the substance library carrier.
- a microarray comprising a substrate or a carrier with molecular probes immobilized thereon on predetermined areas, the substrate or the carrier essentially comprising ceramic materials.
- a carrier element or carrier or substance library carrier or substrate is understood to be a solid on which the probe array is built.
- the totality of molecules deposited in an array arrangement on the substrate or the detection surface or the substance library deposited in an array arrangement on the substrate or the detection surface and the support or substrate is also referred to as a microarray or probe array.
- Substance libraries that are immobilized on the substrates according to the invention are, in particular, protein libraries such as antibody, receptor protein or membrane protein libraries, peptide libraries such as receptor ligand libraries, libraries of pharmacologically active peptides or libraries of peptide hormones, and nucleic acid libraries such as DNA or RNA
- the substance library is preferably in the form of a microarray, particularly preferably with a density of 2 to 10,000 array spots or array elements per cm 2 , most preferably with a density of 50 to 5000 array spots or Array elements per cm, immobilized on the substrate or the substance library support or the detection area.
- a substrate which preferably consists essentially of ceramic materials, has the significant advantage that such substrates have good thermal conductivity and thus, when performing temperature-dependent reactions such as a PCR, a homogeneous temperature profile over the entire area of the reaction space, which is usually on a Side is limited by the substrate with the substance library arranged on it. Temperature-dependent reactions can thus be carried out in a controllable or regulatable manner with high yield and high accuracy. It is preferred that the substrate of the microarray with means for
- the substrate of the microarray according to the invention preferably consists essentially of ceramic materials with a high thermal conductivity, preferably a thermal conductivity in the range from 15 to 500 Wm “ K " , particularly preferably in the range from 50 to 300 Wm ⁇ K “1 and most preferably in Range from 100 to 200 Wm ⁇ K "1 .
- Ceramic materials in the context of the present invention are understood to mean, in particular, materials which have been produced by annealing or firing finely divided, usually moist, shaped clays at temperatures of, for example, 1000 to 1500 ° C.
- the ceramic materials preferably comprise a constituent, ie a ceramic material.
- mixtures of ceramic materials are also conceivable, which can be used, for example, as laminates.
- the substrate preferably essentially comprises one or more aluminum oxide ceramics.
- the substrate consists of at least 90%, preferably at least 95% and particularly preferably at least 99.5% of one or more
- Alumina ceramics examples include the ceramics A-473, A-476 and A-493 from Kyocera (Neuss, Germany).
- the substrate has a surface roughness of 0.04 ⁇ m to 0.12 ⁇ m, preferably 0.06 ⁇ m to 0.1 ⁇ m and particularly preferably approximately 0.08 ⁇ m.
- the substrate is optically transparent.
- the optical transparency of the substrate can be ensured, regardless of the material, in that the substrate has a correspondingly small thickness.
- optically transparent materials such as glass ceramics can be used. Glass ceramics are preferably used which do not have large differences in refractive index between glass and crystal phase.
- An example for an optically transparent material is Ceran® (Schott; Germany). It is also possible to use lithium aluminosilicate glass ceramics.
- the molecular probes are immobilized on the substrate surface via a polymer linker, for example a modified silane layer.
- a polymer linker for example a modified silane layer.
- a polymeric linker can serve to derivatize the substrate surface and thus to immobilize the molecular probes.
- polymers e.g. Silanes
- the activation of a surface by isothiocyanate, succinimide ester and imido ester is known to the person skilled in the art.
- amino-functionalized surfaces are frequently derivatized accordingly.
- coupling reagents e.g. Dicyclohexylcarbodiimide
- the molecular probes are selected in particular from antibodies, protein receptors, peptides and nucleic acids.
- a method for producing a microarray according to the invention as described above which comprises immobilizing molecular probes on predetermined regions of the substrate surface of a substrate which essentially consists of ceramic materials.
- the substrate preferably consists essentially of aluminum oxide ceramics.
- the substrate surface is coated with a polymer linker and the molecular probes are immobilized on the substrate surface via the polymer linker.
- the polymer linker is particularly preferably a modified silane layer.
- the microarray according to the invention can be used in any method for the qualitative and / or quantitative detection of target molecules in a sample by molecular interaction between target molecules and the molecular probes on the microarray.
- the array according to the invention is used to examine the genotypic and / or physiological state of cells.
- microarray according to the invention can be used in particular in the methods according to the invention described above for the reproduction and / or detection of nucleic acids.
- Another aspect of the present invention relates to the use of a
- Substrate for producing a microarray with molecular probes immobilized on predetermined areas of the substrate the substrate essentially comprising ceramic materials.
- the ceramic materials can be embodied as described above in the description of the microarray according to the invention.
- FIG. 1 A preferred configuration of a device according to the invention is shown schematically in FIG.
- a reaction chamber (6) or a microreactor (6) with a substance library (5) arranged on a detection surface is held in a suitable manner in the device.
- Chamber (6) is electrically and fluidically connected to a temperature processing unit (1.1) and a fluid processing unit (1.2), so that temperature control or regulation and / or the exchange of liquids or gases between the chamber (6) and the Containers of the fluid processing unit (1.2) can be realized.
- an identification system e.g. on
- the reaction chamber (6) is assigned to a process sequence defined in a process controller (3).
- the corresponding parameters can thus be transferred to the process control units (1.1) and (1.2).
- the optical system can be controlled by the process controller (3) during or after the fluidic and / or thermal processing, so that a, preferably dynamic, optical detection process can take place.
- the detection is carried out by detecting the change in the transmission properties of the sample areas in a transmitted light device by means of a light source (2.4), which can be illuminated by means of illumination optics (2.3) on the sample with an intensity distribution homogeneity of preferably at least 30% and by means of a detection optics (2.2) can be imaged on a suitable detector (2.1), such as a camera.
- a suitable detector such as a camera.
- the images generated in this way can be digitized in the detection system or in the process controller (3) and can also be analyzed in the latter. Alternatively, the images generated can also be forwarded to external computers or computer networks via a data interface (4).
- the modular structure of these components allows the processing of various tests with different temperature, fluid control and detection parameters.
- new process control parameter protocols can be implemented at any time. If certain tests, particularly in the field of medical diagnostics, are to be carried out without a data connection to the outside, the interface (4) is preferably not accessible to the user, so that all the required process control protocols and the necessary analyzes are implemented in the process controller (3).
- the process control can be carried out via an external computer.
- the distribution of the data flows to the individual technical units or modules of the device according to the invention can be implemented via the interface (4).
- various lighting systems (2.3) and (2.4) are also optionally shown, with which the detection area can be illuminated from below and diagonally from above. It is thus possible in this embodiment to activate different detection methods depending on the type of chip determined by a detector for identification (2.5), for example a data matrix reader.
- a detector for identification for example a data matrix reader.
- the time course of the change in optical transmission properties and the Change in scattering properties that can be induced in the incident light dark field is detectable.
- the device according to the invention has no fluid processing unit (see
- control of thermal parameters and the exchange of liquids can be implemented technically using modules known to the person skilled in the art and also commercially available.
- control and data transmission is of particular interest, which can take place in particular through low-current connections such as TTL level and by means of the described transfer protocols.
- optical detection of kinetically extending signal amplification reactions is carried out by recording modulations of certain optical parameters, in particular transmission, reflection, scattering, diffraction and interference.
- Target molecules marked with gold particles after a specific hybridization with sample molecules immobilized or synthesized on solid surfaces grow layers which significantly change the optical properties of the overall system.
- the signal amplification of gold-labeled target molecules by silver is e.g. described in detail in the international patent application WO 02/02810, the content of which is hereby expressly incorporated by reference.
- the change in transmission properties is detected in a transmitted light arrangement as shown in FIG. 1.
- the light source (2.4) is designed as a white light source such as a halogen lamp, white light LED or as a narrow band light source such as LED, laser diode, organic LED.
- the optical system (2.3) consisting of lenses, mirrors and filters is designed in such a way that the substrate surface to be detected is uniformly illuminated with at least 70%) illumination intensity homogeneity.
- the substrate surface is imaged on a detector by a further optical system (2.2) consisting of lenses, mirrors and filters.
- This can be a two-dimensional CCD or CMOS camera or a moving line camera.
- the change in the transmission properties is recorded as a sequence and the curves of the decrease in transmission at different points are compared.
- the target concentrations can be determined quantitatively, as described in the international patent application WO 02/02810.
- the reaction chamber is made of a substrate that is transparent to the wavelengths used in the detection.
- the reaction chamber is made of glass when using light in the visible wavelength range and made of silicon when using light in the infrared range.
- the change in reflection properties is detected in an incident light arrangement as shown in FIG. 4.
- the optics (2.3) are designed in such a way that uniform illumination of the substrate surface to be detected is guaranteed with at least 70%) illumination intensity homogeneity.
- the substrate surface is covered by the optical system
- the change in reflection properties is recorded as a sequence and the curves of the increase in reflection at different points are compared.
- the target concentrations can be determined quantitatively, as described in international patent application WO 02/02810. The calculation is carried out inversely to that in the detection of the transmission change, since the local reflectivity increases when enriched with silver particles on target molecules.
- the reaction chamber is made of a substrate that is transparent to the wavelengths used in the detection.
- the reaction chamber is made of glass when using light in the visible wavelength range and made of silicon when using light in the infrared range.
- the change in scattering properties is detected in a dark field arrangement as shown in FIG. 5.
- the substrate surface to be detected is uniformly illuminated with at least 60%, preferably at least 70%, illumination intensity homogeneity.
- the substrate surface is imaged on a detector by the optical system (2.2).
- This can be a two-dimensional camera (eg CCD, CMOS) or a moving line camera.
- the change in scatter properties is recorded as a sequence and the curves of the increase in scatter at different points are compared.
- the target concentrations can be determined quantitatively, as described in international patent application WO 02/02810. The calculation is carried out inversely to that in the detection of the transmission change, since the number of local scattering centers and thus the scattering increases when silver particles are enriched on target molecules.
- an exact qualitative and quantitative detection of the hybridized target molecules can be carried out with a preferably homogeneous temperature distribution in the reaction chamber.
- Figure 6 shows a typical image sequence for the detection of the silver growth reaction after the hybridization of specific gold-labeled target molecules on a DNA-based substance library.
- the reaction chamber is made of a substrate that is transparent to the wavelengths used in the detection.
- the reaction chamber is made of glass when using light in the visible wavelength range and made of silicon when using light in the infrared range.
- electrical detection of kinetically extending signal amplification reactions takes place by recording modulations of certain electrical parameters, in particular conductivity, changes in resistance and permeability.
- Target molecules marked with gold particles after a specific hybridization with sample molecules immobilized or synthesized on solid surfaces grow layers which significantly change the optical properties of the overall system.
- the signal amplification of gold-labeled target molecules by silver is e.g. described in detail in the international patent application WO 02/02810, the content of which is hereby expressly incorporated by reference.
- the individual array spots can be electrically contacted and the signals generated there can be discharged parallel to a measuring device.
- the array spots are electrically contacted via electrodes as shown in Figure 7.
- a change in the local resistance can be measured by the fusion with growing conductive material, such as silver.
- the effect of the fusion and the resulting change in resistance is supported by three-dimensional structures on the detection surface, such as elevations, grand and through holes.
- Figure 8 schematically shows an arrangement for measuring these changes in resistance on a single three-dimensional array spot.
- One way of keeping the signal in the solution small compared to the signal on or in the surface is to use particularly thin reaction chambers.
- the enrichment of the target molecules on the array surface due to the specific binding of the probe and target makes it possible, with a correspondingly thin design of the reaction chamber, to display the signals on the probe array with conventional fluorescent optics illuminating or imaging the entire volume.
- Figure 11 shows the relationship between layer thickness or chamber thickness and the number of molecules in the supernatant immediately above the spot, which are provided with a fluorescence marker.
- microarrays used in the context of the present invention when using a detector with 8-bit resolution, such as an epifluorescence microscope (Zeiss, Jena, Germany), show the characteristics of the signal development shown in the following table.
- the number of bound molecules present on a spot or array element of typical size with saturation of the surface can be assumed to be approximately 10 9 , this corresponds to a space requirement of 10 nm per molecule.
- a solution containing a fluorescence-labeled target (Cy3) was introduced into a device according to the invention with a reaction chamber thickness of 100 ⁇ m and a suitable probe array.
- the concentration of the target is 10 nM
- 2x SSPE with 0.1% SDS was used as a suitable buffer.
- a fluorescence image was taken.
- An epifluorescence microscope (Zeiss, Jena, Germany) was used for this purpose, namely an axioscope that is equipped with a PCO-CCD camera and has a mercury vapor lamp to excite the fluorescence.
- the temperature was gradually increased from 5 ° C to 95 ° C. After each increment, incubation was also carried out for five minutes and an image was taken.
- the image data were evaluated with the Iconoclust® software (Clondiag).
- Figure 12 shows a characteristic melting curve for the different probes. The specific course of the duplex dissociation depending on the temperature and the respective sequence is also clear.
- the amplification in the device according to the invention is preferably carried out using exponential amplification techniques such as PCR, in order to quantify the target amount contained in the sample, it is necessary to enable continuous detection of the signal increase on the probe array.
- Genomic DNA from E. coli was used in two different dilutions as the target material to be amplified. After a certain number of cycles, images were taken in the manner described in Example 3. These were subjected to an analysis using the Iconoclust ® software (Clondiag, Jena).
- Hybridization and subsequent detection of hybridization by means of enzymatic precipitation of an organic molecule on a ceramic surface The literature describes mutations in at least 6 genes in the human genome that have been identified as risk factors for the development of thrombosis.
- the company Ogham (Münster) has developed a diagnostic assay for the detection of thrombosis-relevant mutations in human DNA. Using an oligonucleotide probe model system used for this test, the proof of principle for hybridization and the subsequent detection should be provided by enzymatic precipitation of an organic dye on a ceramic surface.
- the probes were placed on the slides from a 10 ⁇ M solution of the oligonucleotides in 0.1 M phosphate buffer / 2.2% sodium sulfate. The probes were then covalently linked to the epoxy grapples on the ceramic surface by baking at 60 ° C. for 30 minutes. A multi-part washing process followed in the following order:
- the oligonucleotides provided by Ogham for the hybridization in the form of a mixture of six different oligonucleotides were in a final concentration of 100 pm and in a final volume of 50 ⁇ l in 6 ⁇ SSPE buffer (52.59 g NaCl, 8.28 g NaH 2 PO 4 ⁇ H 2 O, 2.22 g EDTA x 2H 2 O in 1 1 H 2 O bidest, adjusted to pH 7.4 with NaOH) / 0.005% Triton.
- the probe array was covered with a Hybri-slip (Z36.590-4, Sigma, Taufkirchen, Germany), so that a reaction space with a volume of approx. 20 ⁇ l was created over the probe array.
- the hybridization solution had been denatured (5 min at 95 ° C.)
- it was added to the reaction space above the probe array and the probe array was then incubated at 50 ° C. for 60 min with gentle shaking.
- Hybri-slip was then removed from the ceramic surface. This was followed by two washing steps of 5 min each at room temperature in 2xSSC / 0.01% Triton and 2xSSC. Then 200 ⁇ l of a prepared blocking solution (milk powder, Ogham, Weg, in 6xSSPE / 0.005% Triton) were added to the probe array, covered with a cover glass and then incubated for 10 min at room temperature. The cover glass was then removed and the blocking solution was rinsed with 500 ⁇ l of a prepared conjugation solution (streptavidin-poly HRP, N200, Pierce, dilution 1: 10,000 in 6xSSPE / 0.005% Triton).
- a prepared conjugation solution streptavidin-poly HRP, N200, Pierce, dilution 1: 10,000 in 6xSSPE / 0.005% Triton.
- Figure 1 Schematic representation of a processing and detection device according to the invention for the duplication and detection of nucleic acids.
- Figure 2 Schematic representation of a processing and detection device according to the invention for the duplication and detection of nucleic acids with an external process controller and depending on the individualization of the chip selectable detection systems.
- Figure 3 Schematic representation of a processing and detection device according to the invention for the duplication and detection of nucleic acids without a fluid processing unit.
- Figure 4 reflected light arrangement of the device according to the invention.
- Figure 5 Dark field arrangement of the device according to the invention.
- Figure 6 Representation of an image sequence to document the growth of silver particles during the hybridization of gold-labeled target molecules with probe molecules immobilized on the detection surface in the form of a substance library.
- Figure 7 Schematic representation of a processing and detection device according to the invention for the duplication and detection of nucleic acids by electrical detection of the kinetics of the silver precipitate on a single planar array spot covered with probe molecules.
- Figure 8 Schematic representation of a processing and detection device according to the invention for the duplication and detection of nucleic acids by electrical detection of the kinetics of the silver precipitate on a single array spot covered with through holes provided with probe molecules.
- Figure 9 Course of the exponential amplification of a target at different initial concentrations of nucleic acids to be detected in the sample.
- FIG. 11 Representation of the relationship between chamber thickness and the number of molecules in the supernatant immediately above the spot, which are provided with a fluorescent marker.
- the lines represent different concentrations of target molecules in solution (in pM), calculated for a volume of 10,000 ⁇ m 2 (corresponding to the area of a spot or array element) multiplied by the thickness of the chamber.
- Figure 12 Characteristic melting curve for different probes and specific course of the duplex dissociation depending on the temperature and the respective sequence (see example 3).
- Figure 13 Schematic representation of the arrangement of the probes on the array used in Example 5. One box represents four redundant probes.
- Figure 14 Probe array on a ceramic surface after hybridization and detection by enzymatic precipitation of an organic molecule (see example 5). The layout of the array is shown in Fig. 13.
- Figure 15 Results of the hybridization of an oligonucleotide mixture against a probe array on a ceramic surface with subsequent detection by enzymatic precipitation of an organic molecule (see Example 5).
- Ceramic substrates for defined temperature control in a device according to the invention a so-called assay processor.
- the ceramic substrate and the assay processor have the following specification:
- Carrier material Ceramic carrier Sensor / heater structures: Pt thin film structures
- Figure 17 Schematic representation of the assay processor from Figure 16.
- Figures 18 to 24 Schematic representations of preferred embodiments of the devices according to the invention.
- reaction space 301 enclosed recess in 300, defines reaction space
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Abstract
Description
Claims
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DE10315074A DE10315074A1 (de) | 2003-04-02 | 2003-04-02 | Vorrichtung zur Vervielfältigung und zum Nachweis von Nukleinsäuren |
PCT/EP2004/003532 WO2004087951A2 (de) | 2003-04-02 | 2004-04-02 | Vorrichtung zur vervielfältigung und zum nachweis von nukleinsäuren |
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EP04725346A Withdrawn EP1610899A2 (de) | 2003-04-02 | 2004-04-02 | Vorrichtung zur vervielf ltigung und zum nachweis von nuklei ns uren |
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EP (2) | EP2266699B1 (de) |
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Also Published As
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WO2004087951A2 (de) | 2004-10-14 |
WO2004087951A3 (de) | 2005-01-27 |
EP2266699B1 (de) | 2014-06-04 |
JP4917883B2 (ja) | 2012-04-18 |
JP2006523095A (ja) | 2006-10-12 |
EP2266699A1 (de) | 2010-12-29 |
US20060078929A1 (en) | 2006-04-13 |
DE10315074A1 (de) | 2004-10-14 |
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