HK1151583B - Device and method for detecting molecular interactions - Google Patents

Description

The invention relates to devices for detecting specific interactions between target and probe molecules.

In modern tests, the probes are placed in the form of a library of substances on carriers, so-called microarrays or microarrays or chips, so that a sample can be analysed simultaneously on several probes in parallel (see D. J. Lock, E. A. Winzhart, Genome, Matrix expression and DNA arrays; 405, 2000, 827-836). For the manufacture of microarrays, the probes are usually produced in a similar way to the one described above, for example, in a synthetic sample of type WO 5,08B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 5,14B, WO 68B, WO 68B, WO 68B, WO 68B, WO 68B, WO 68B, WO 68B 5,85B, WO 68B, WO 68B 5,85B, WO 68B 5,78B, WO 68B 5,78B, WO 68B 5,78B, WO 68B 5,78B, WO 68B 5,78B, WO 68B 5,78B, WO 68B 5,78B, WO 68B 5,78B, WO 68B 5,78B, WO 68B 5,78B, WO 68B 5,8B, WO 68B 5,78B 5, WO 68B 5, WO 68B 5,8B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5, WO 68B 5,

The requirement for binding a target molecule, for example in the form of a DNA or RNA molecule, marked with a fluorescence group to a nucleic acid probe in the microarray is that both the target molecule and the probe molecule are present in the form of a single-stranded nucleic acid. Only between such molecules can an efficient and specific hybridization take place. Single-stranded nucleic acid target and nucleic acid molecules are usually obtained by thermal denaturation and selection of parameters such as temperature, conductivity and concentration of helix-dissolving molecules.

A typical example of the use of microarrays in biological testing is the detection of microorganisms in samples in biomedical diagnostics. This takes advantage of the fact that the genes for ribosomal RNA (rRNA) are ubiquitous and have sequence segments that are characteristic of the species in question. These species-specific sequences are applied to a microarray in the form of single-stranded DNA oligonucleotides. The target DNA molecules to be tested are first isolated from the sample to be tested and marked with markers, such as fluorescent molecular markers.

A number of methods and technical systems are described for detecting molecular interactions using microarrays or probe arrays on solid surfaces, some of which are also commercially available.

Classical systems for detecting molecular interactions are based on comparing the fluorescence intensities of spectrally selectively excited target molecules marked with fluorophores. Fluorescence is the property of certain molecules to emit light when excited with light of a certain wavelength. This results in a characteristic absorption and emission behavior.

US 2004/018523 describes a device for performing biological reactions on a substrate layer with a large number of oligonucleotide binding sites

US 5.863.502 reveals a parallel reaction device to perform reactions in it.

The specific quantitative detection of fluorescence signals is carried out by modified methods of fluorescence microscopy, whereby the absorption wavelength of light is separated from 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.

Technical solutions known to date differ in their optical construction and the components used. Problems and limitations may result from signal noise (the background), which is largely determined by effects such as bleaching and quenching of the dyes used, autofluorescence of the media, assembly elements and optical components, and scattering, reflections and foreign light in the optical construction.

This results in a high technical effort to build highly sensitive fluorescence detectors for qualitative and quantitative comparison of probe arrays, especially for medium and high throughput screening, which require specially adapted detection systems with a certain degree of automation.

To optimize standard epifluorescence designs for reading molecular arrays, CCD-based detectors are known to discriminate between optical effects such as scattering and reflections by stimulating the fluorospheres in the dark field by illumination or illumination (see, e.g., C. E. Hooper et al., Quantitative Photon Imaging in the Life Sciences Using Intensified CCD Cameras, Journal of Bioluminescence and Chemiluminescence (1990), p. 337-344).

Other methods for the quantitative detection of fluorescence signals are based on confocal fluorescence microscopy. For example, confocal scanning systems described in US 5.304.810 are based on the selection of fluorescence signals along the optical axis by means of two pinholes. This results in a high adjustment cost for the samples and the installation of a high-performance autofocus system. Such systems are technically complex. The required components such as lasers, pinholes, possibly shaded detectors such as PMT, avalanche diodes or CCD, are highly complex, high-precision mechanical and optical components that must be integrated and optimized with considerable effort (US 5.459.94, US 5.359.98, US 5.859.98; US 5.859.98, US 5.859.98; and the price of the miniature and the functional components must be highly limited.

Analyses based on probe arrays are currently usually fluorescent optically read (see, e.g., A. Marshall and J. Hodgson, DNA Chips: An array of possibilities, Nature Biotechnology, 16, 1998, 27-31; G. Ramsay, DNA Chips: State of the Art, Nature Biotechnology, 16, Jan. 1998, 40-44). However, the disadvantages of the above-described detection devices and methods are the high signal background, which results in limited accuracy, the sometimes considerable technical effort and the high costs associated with the detection methods.

A number of particularly confocal systems are known to be suitable for detecting low-integrated array-format substance libraries installed in fluidic chambers (see e.g. US 5,324,633, US 6,027,880, US 5,585,639, WO 00/12759).

The methods and systems described above are, however, only very limited in their adaptability to the detection of highly integrated molecular arrays, particularly in fluid systems, especially because of the scattering, reflections and optical aberrations which occur there.

There is therefore a need for highly integrated arrays that can be used to provide high-precision qualitative and/or quantitative evidence of the interaction between probes and targets with relatively little technical effort.

The effect of polarisation axis distortion by polarized excited fluorophores is used for microtiter-format quantification. There are also approaches to build cost-effective high throughput systems (HTS systems) by using appropriately modified polymer films as polarization filters (see I. Gryczcynski et al., Polarisation sensing with visual detection, Chem. 71, 1999, 1241-1251).

Recent developments use the fluorescence of inorganic materials, such as lanthanides (M. Kwiatowski et al., solid-phase synthesis of chelate-labelled oligonucleotides: application in triple-color ligase-mediated gene analysis, Nucleic Acids Research, 1994, 22, 13) and quantendots (M. P. Bruchez et al., semiconductor nanocrystals as fluorescent biological labels, Science 1998, 281, 2013).

The international patent application WO 00/72018 describes optical devices for detecting gold bead-marked samples and making them visible by means of silver reinforcement. However, the devices are only suitable for detection by static measurement. In static measurement, after the interaction of the targets with the probe placed on the probe array and after the start of the reaction leading to precipitation on the interaction elements of the array, an image is taken and the gray values of the concentrations are assigned to the measured concentration, depending on the degree of precipitation formation.

WO 02/02810 provides a method for the qualitative and/or quantitative detection of targets in a sample by molecular interactions between probes and targets on probe arrays, whereby the time course of precipitation at the array elements is detected in the form of signal intensities, i.e. a dynamic measurement is made.

Such dynamic measurement requires the recording of series of images under, for example, certain thermal conditions or at a certain stage of the process, e.g. when certain solutions are present at the time of recording.

The DNA molecules are typically replicated by polymerase chain reaction (PCR). RNA molecules must be converted into complementary DNA (cDNA) by reverse transcription. This cDNA can then also be replicated (amplified) by PCR. PCR is a standard laboratory method (e.g., in Sambro et al. (2001); Cold Spring Laboratory, Cold Spring, N.Y., 3rd edition).

DNA replication by PCR is relatively fast, miniaturised methods allow high sample throughput in small sample volumes and automation makes it labour efficient.

However, it is not possible to characterize nucleic acids by replication alone, and it is necessary to use post-amplification analytical methods such as nucleic acid sequencing, hybridization and/or electrophoretic separation and isolation to characterize the PCR products.

In general, devices and procedures for nucleic acid amplification and detection should be designed to minimise the need for an experimenter to perform the procedure. The advantages of procedures that allow nucleic acid replication and detection and require minimal intervention by an experimenter are obvious. On the one hand, contamination is avoided. On the other hand, the reproducibility of such procedures is significantly increased because they are accessible to automation. This is also extremely important for the authorisation of medicinal products for diagnostic procedures.

A wide variety of methods are currently available for nucleic acid amplification and detection, where the target material is first duplicated by PCR amplification and the identity or genetic status of the target sequences is then determined by hybridization against a probe array.

Both PCR amplification of nucleic acids and their detection by hybridisation are subject to a number of fundamental problems, as are methods combining PCR amplification of nucleic acids and their detection by hybridisation.

In a method combining PCR amplification and detection by hybridisation, where detectable markers, for example in the form of fluorescent-labeled primers, are introduced into the nucleic acids or target molecules to be detected, a wash step is usually performed before actual detection, to remove the excess of the unrealized primer in comparison with the amplification product, and to remove nucleotides with a fluorescent marker that do not participate in the detection reaction or do not specifically hybridise with the nucleic acid probes of the micro-molecule, in order to reduce the high-altitude background caused by these molecules.The detectable signal is also significantly reduced for the nucleic acids to be detected, which specifically hybridise with the nucleic acid probes of the microarray. The latter is mainly due to the fact that the balance between the targets bound by hybridisation and those in solution is no longer present after the wash step. Nucleic acids which have already hybridised with the nucleic acid probes in the array are washed off the binding site by washing and thus washed away with the molecules in solution.

There is therefore a need for highly integrated arrays that can be used to provide high-precision qualitative and/or quantitative evidence of the interaction between probes and targets with relatively little technical effort.

There is also a need for devices that allow PCR and analytical reaction, such as a hybridisation reaction, to be carried out in a reaction chamber.

The present invention is therefore intended to overcome the problems of state of the art mentioned above, which arise in particular from the lack of compatibility of the assay with the test system.

In particular, the present invention is intended to provide devices for the qualitative and/or quantitative detection of molecular interactions between probes and targets on probes arrays with high accuracy and sensitivity and in a simple and cost-effective manner.

It is also the task of this disclosure to provide methods or devices for amplification and for the qualitative and quantitative detection of nucleic acids, which can minimise the interference of the experimenter in the detection process.

A further purpose of the present invention is to provide devices for qualitative and/or quantitative detection of target molecules that provide a high signal-to-noise ratio for detection of interactions on the microarray without affecting the interaction between the target and probe molecules on the array.

A further purpose of the present invention is to provide devices which achieve a high dynamic resolution in detection, i.e. the detection of weak probe/target interactions alongside strong signals.

The purpose of the invention is also to provide devices which allow the near simultaneous multiplication and characterization of nucleic acids at a high throughput.

These and other tasks of the present invention are solved by providing the embodiments described in the claims.

According to the disclosure, methods for the qualitative and/or quantitative demonstration of molecular interactions between probe and target molecules are provided, which do not require the exchange and/or removal of solutions, i.e. in particular washing or rinsing steps.

Such disclosure procedures shall include, in particular, the following steps: (a) introducing a sample containing target molecules into a reaction chamber containing a microarray, the microarray comprising a substrate with probes immobilized on array elements; where no solution exchange and/or removal of solution from the reaction chamber takes place after the sample is introduced and before and during detection.

The present invention also provides devices suitable for the performance of such procedures.

In particular, the present invention provides a device for the qualitative and/or quantitative detection of molecular interactions between probe and target molecules in a sample solution, containing: a microarray of probe molecules immobilized on array elements, the microarray being placed on a first surface of the device; and a reaction chamber formed between the first surface with the microarray placed on it and a second surface, the first surface being elastically deformable at least in the area below the microarray, so that the microarray is conducive relative to the second surface, so that the distance between the microarray and the second surface is variable enough to remove the sample solution between the microarray and the second surface substantially,whereby the reaction chamber is provided with laterally boundary compensating ranges which, when the distance between the microarray and the second surface is reduced to such an extent that the sample solution is essentially removed from the microarray to the second surface, maintain the volume of the sample solution in the reaction chamber constant. In particular, the variability of the distance between the microarray and the second surface, which is usually the detection range of the device of the invention, makes it possible to reduce or completely avoid the signal background caused by the marked target molecules, which does not have specific affinities to the probes' microarray molecules and therefore cannot interact with them.

A method for the qualitative and/or quantitative detection of molecular interactions between probe and target molecules, including the following steps, is also provided, as disclosed: (a) Introducing a sample solution containing target molecules into a reaction chamber of a device of the invention as described above; and (b) Detecting an interaction between the target molecules and the probe molecules immobilized on the substrate.

The devices for detecting target molecules according to the invention are designed in such a way that the detection process and, if necessary, the amplification of the target molecules require as few interventions as possible by an experimenter in the reaction chamber. This has the essential advantage of avoiding contamination. Furthermore, the reproducibility of the detection methods is significantly increased compared to conventional methods, since the detection method is accessible to automation due to the minimisation of external interference. The above mentioned advantages play an important role in the approval of diagnostic procedures.

The following definitions are used to describe the present invention, among others: For the purposes of the present invention, a probe, molecule or molecular probe is a molecule used to detect other molecules by a specific, characteristic bonding behavior or reactivity. For the probes arranged in the array, any type of molecule is eligible that can be attached to solid surfaces and has a specific affinity. A preferred embodiment is biopolymers, in particular biopolymers from the classes of peptides, proteins, antigens, antibodies, carbohydrates, nucleic acids and/or their analogues and/or polymers that are the biopolymers of the above-mentioned mixtures.

A probe is a specifically defined and known sequence of nucleic acid molecules used to detect target molecules in hybridization processes. Nucleic acids can be used as both DNA and RNA molecules. For example, the nucleic acid or oligonucleotide probes may be around oligonucleotides with a length of 10 to 100 bases, preferably 15 to 50 bases, and preferably 20 to 30 bases in length. Typically, according to the invention, the probes are single-stranded nucleic acid molecules or molecules of nucleic acid, pre-stranded molecular analogues or molecules that have at least a single sequence of sequences that can be detected by a single sequence of DNA molecules.

A target or target molecule is defined in the present invention as the molecule to be detected by a molecular probe. A preferred embodiment of the present invention is the nucleic acid targets to be detected. However, the probe array according to the invention may also be used by analogy to detect peptide/probe interactions, protein/probe interactions, carbohydrate/probe interactions, antibody/probe interactions, etc.

If the targets of the present invention are nucleic acids or nucleic acid molecules detected by hybridization against probes arranged in a probe array, these target molecules generally comprise sequences of 40 to 10,000 bases, preferably 60 to 2,000 bases, also preferably 60 to 1,000 bases, in particular 60 to 500 bases, and most preferably 60 to 150 bases. Their sequence may include the sequences of primers as well as those defined by the template's primer. The target molecules may in particular be single-stranded or double-stranded nucleic acid molecules, of one or both strands, or unlabeled radioactive in a way that can be detected by a standard method of detection.

The target sequence is the sequence range of the target identified by hybridization with the probe, and is also referred to as the area addressed by the probe.

A substance library is defined as a large number of different probe molecules, preferably at least two to 1,000,000 different molecules, preferably at least 10 to 10,000 different molecules, and most preferably between 100 and 1,000 different molecules. In special designs, a substance library may also include 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.

For the purposes of the present invention, a probe array is an arrangement of molecular probes or a library of substances on a carrier, the position of each probe being determined separately. Preferably, the array includes defined locations or predetermined areas, so-called array elements, which are particularly preferably arranged in a particular pattern, with each array element usually containing only one species of probes. The arrangement of the molecules or probes on the carrier may be produced by covalent or non-covalent interactions. The probes are arranged on the side of the carrier facing the reaction chamber. A position within the arrangement, i.e., the array, is usually called a spot.

For the purposes of the present invention, an array element or a predetermined range or spot or an array spot is an area on a surface designated for the deposition of a molecular probe, the sum of all array elements occupied being the probe array.

A carrier element or carrier or substance library carrier or substrate is a solid body on which the probe array is constructed for the purpose of the present invention. The carrier, which is also commonly referred to as a substrate or matrix, may be, for example, an object carrier or wafer or ceramic materials. In a special design, the probes may also be immobilized directly on the first surface, preferably on a sub-area of the first surface.

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The detection layer is the second surface of the device according to the present invention, preferably the probes deposited on the microarray are essentially at the detection level when detecting the interaction between probes and targets, in particular by reducing the distance between the microarray and the second surface to about zero.

The term chamber body is used to describe the solids that make up the reaction chamber for the purposes of the present invention.

The reaction chamber or chamber is defined in the present invention as the space formed between the microarray and the second surface or detection plane, preferably as a variable capillary gap. The reaction space is bounded on the side by side walls, which may be e.g. made as elastic seals. The probes immobilized on the microarray are located on the inside of the reaction chamber facing the side. The base surface of the reaction chamber or chamber is defined by the first surface or surface of the array. The thickness of the reaction chamber or chamber is defined as the thickness of the reaction chamber or chamber, for example, between 1 mm and 3 mm, with a maximum width of 1 mm and a maximum width of 3 mm, for example, in the presence of a detection chamber or chamber, and a maximum width of 1 mm and a maximum width of 3 mm, for example, in the presence of a detection chamber or chamber.

The distance between the microarray and the second surface is, for the purposes of the present invention, the distance between the surface of the microarray substrate, i.e. the side of the microarray facing the reaction chamber, and the side of the second surface facing the reaction chamber.

A capillary gap is a reaction chamber which can be filled by capillary forces acting between the microarray and the second surface. Typically, a capillary gap has a small thickness, e.g. not more than 1 mm, preferably not more than 750 μm and preferably not more than 500 μm. The capillary gap is also described as having a thickness in the range of 10 to 300 μm, 15 μm to 200 μm or 25 μm to 150 μm. In special designs of the present invention, the capillary gap has a thickness of 50 μm, 60 μm, 70 μm, 80 μm or 90 μm, and the reaction chamber has a thickness of not more than 2 mm.

For the purposes of the present invention, a cartridge or reaction cartridge is a unit of the reaction chamber with a chamber body and corresponding enclosure.

A confocal fluorescence detection system is a fluorescence detection system in which the object in the focal plane of the lens is illuminated by a point light source, point light source, object and point light detector in precisely optically conjugated 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 which maps the entire volume of the reaction chamber is a nonconfocal fluorescence detection system, i.e. a fluorescence detection system in which the illumination by the point source light is not limited to the object, and thus has no focal limit.

Conventional arrays or microarrays of the present invention comprise about 50 to 10,000, preferably 150 to 2,000 different species of probe molecules on a preferably square surface of e.g. 1 mm to 4 mm x 1 mm to 4 mm, preferably 2 mm x 2 mm. In further designs, microarrays of the present invention comprise about 50 to about 80,000, preferably about 100 to about 65,000, particularly preferably about 1,000 to 10,000 different species of molecules on a surface of several mm.^{2 and}Other, of a width of less than 30 cm^{2 and}, preferably about 1 mm^{2 and}Other, of a width of less than 30 cm^{2 and}, preferably 2 mm^{2 and}Other, of a width of less than 30 cm^{2 and}and preferably about 4 mm^{2 and}Other, of a width of less than 30 cm^{2 and}For example, a conventional microarray has 100 to 65,000 different species of probe molecules on a surface of 2 mm x 2 mm.

A marking or marker for the purpose of the present invention means a detectable unit, such as a fluorophore or an anchor group, to which a detectable unit can be coupled.

A replication reaction or amplification reaction, as defined in this disclosure, usually comprises 10 to 50 or more amplification cycles, preferably about 25 to 45 cycles, preferably about 40 cycles.

For the purposes of this disclosure, an amplification cycle is defined as a single step of amplification of the cyclic amplification response.

For the purposes of this disclosure, an amplification product is a product obtained by amplifying, multiplying or amplifying the nucleic acid molecules to be amplified by the cyclic amplification reaction, preferably by PCR.

For the purposes of this disclosure, denaturation temperature means the temperature at which the double stranded DNA is separated during the amplification cycle, the denaturation temperature being usually above 90°C, preferably around 95°C, especially in the case of PCR.

For the purposes of this disclosure, the annealing temperature is the temperature at which the primers hybridise to the nucleic acid to be detected.

For the purposes of this disclosure, the temperature of chain extension or extension is the temperature at which the nucleic acid is synthesised by incorporation of the monomer building blocks.

For the purposes of this disclosure, an oligonucleotide primer is an oligonucleotide that binds to, or hybridizes with, the DNA to be detected, also known as target DNA, whereby the synthesis of the DNA counterstrand to be detected is initiated at the site of binding in the cyclic amplification reaction. In particular, a primer is usually a short DNA or RNA oligonucleotide with preferably about 12 to 30 bases, which is complementary to a section of a larger DNA or RNA hybrid molecule and has a free 3-OH group at its 3'-end. This free 3'-OH group allows the primer to serve as a substrate for any 5'-OH polymers or 5-3'-OH polymers that are in the range of 15 to 30 nucleotides before the synthesis of the newly synthesised RNA. The primer is located between the 12'-sequence of the nucleotide and the free 3'-nucleotide of the newly synthesized RNA.

A template or template strand is usually a double stranded nucleic acid molecule or a nucleic acid strand that serves as a template for the synthesis of complementary nucleic acid strands.

For the purposes of the present invention, molecular interaction means in particular a specific, covalent or non-covalent bond between a target molecule and an immobilized probe molecule.

Hybridization is the formation of double-stranded nucleic acid molecules or duplex molecules from complementary single-stranded nucleic acid molecules. The association is preferably always in pairs of A and T or G and C. In the context of a hybridization, for example, DNA-DNA duplexes, DNA-RNA or RNA-RNA duplexes can be formed.

For the purposes of the present invention, processing refers in particular to purification, concentration, labelling, amplification, interaction, hybridization and/or washing and rinsing steps, and other steps in the process of detecting or detecting targets by means of substance libraries.

For the purposes of the present invention, a sample or solution or analyte or solution is a liquid to be analyzed, which contains in particular the target molecules to be detected and, if necessary, amplified.

For the purpose of the present invention, the term "reaction chamber replacement" refers in particular to the washing or rinsing steps. For example, the replacement of solutions is used to eliminate molecules with detectable markers that do not specifically interact with probes on the microarray, whereby after interaction the sample solution is replaced by an unlabeled solution. Molecules that do not specifically interact with probes on the microarray are replaced, e.g., by a primary detectable marker that has not been reshaped during the amplification reaction, or by a detectable marking molecule that has no complementary specific action on the target molecule that is replaced with this target molecule.

Removal of solutions from the reaction chamber is understood as the steps for the present invention in which molecules with detectable markers that do not specifically interact with probes on the microarray are removed from the reaction chamber. Molecules that do not specifically interact with probes on the microarray are, for example, primers with a detectable marker that have not been implemented during the amplification reaction or target molecules with a detectable marker that do not have a complementary probe on the target array that specifically interacts with that molecule.

However, if, for the purposes of the present invention, no solution exchange and/or removal of solutions occurs in the reaction chamber between the introduction of the sample containing target molecules into a reaction chamber and the detection of the interaction, it is conceivable that during this period solutions may be added to the reaction chamber without any exchange or removal of solutions already present in the reaction chamber.

The present disclosure thus covers a method for the qualitative and/or quantitative detection of molecular interactions between probe and target molecules, including in particular the following steps: (a) introducing a sample containing target molecules into a reaction chamber containing a microarray, the microarray comprising a substrate with probes immobilized on array elements; where no solution exchange and/or removal of solution from the reaction chamber takes place after the sample has been introduced and before or during detection.

An essential feature of the disclosure method in this aspect of this disclosure is that the detection of an interaction between the target molecules to be detected and the probe molecules immobilized on the substrate of the microarray is done without any exchange of solutions in the reaction chamber or removal of solutions from the reaction chamber, i.e. the detection of the interaction between targets and probes can be done without the need for splicing or washing shells following the interaction and/or without the interaction reaction releasing molecules from the reaction chamber that are not specifically removed by the microarray.

This can be achieved in particular by focus selective detection methods in the exposure-based method, such as confocal techniques or by the application of deep selective lighting due to the evanescent dissociation of excitation light (TIRF) based on total reflection in the sample substrate or methods based on the use of waveguides. Such focus selective methods are particularly desirable when further exclusion of the background signals produced by fluorescence molecules present in the liquid, i.e. non-hybridised, is required to increase confluence. When using fluorescence-labelled molecules, the specific interaction of fluorescence-specific fluorescence can be discriminated by means of total fluorescence or internal fluorescence-reflection methods such as target microscopy (TIRF) or fluorescence-reflection.

Examples are CCD-based detectors that detect optical effects such as scattering and reflections by discriminating between the excitation of the fluorophores in the dark field by illumination or through-light (see, e.g., C. E. Hooper et al., Intentative Photone Imaging in the Life Sciences Usingsified CCD Cameras, Journal of Bioluminescence and Chemoluminescence (1990), 337-344). Alternatives to fluorescence detection systems that can be used in the disclosure process are white-light structures as described in WO 00759, WO 0025113 and WO 96/27025; they are basic analog systems, e.g. USB 99325, USB 99634, USB 996405, USB 987505, USB 986505, USB 987505, USB 986505, USB 987505, USB 986505, and other basic analog systems; they are used in structural imaging, such as in the process of fluorescence, in the process of fluorescence separation, and in the process of microwave emission; they are used in other types of fluorescence systems such as in the process of fluorescence separation, such as in the process of fluorescence separation, and in the process of microwave emission.

In particular, the devices described in WO 2004/087951 in which the reaction chamber is formed by a capillary cleft are suitable for performing a disclosure-compliant detection procedure without exchanging solutions in the reaction chamber and/or removing solutions from the reaction chamber prior to detection.

In a further embodiment of this aspect of this disclosure, the replacement and/or removal of solutions from the reaction chamber is avoided by detection by mass change on the array surface, as described e.g. in WO 03/004699; the relevant content of WO 03/004699 is hereby expressly referred to.

A further embodiment of this aspect of this disclosure avoids the replacement and/or removal of solutions from the reaction chamber by detection by acoustic surface wave detection, as described in Z. Guttenberg et al., Lab Chip, 2005; 5(3):308-17.

In a further embodiment of this aspect of this disclosure, the replacement and/or removal of solutions from the reaction chamber is avoided by detection by electrochemical detection by electrodes on the surface of the array, such as measurement of the change in redox potentials (see e.g. X. Zhu et al., Lab Chip, 2004; 46):581-7) or cyclic voltometry (see e.g. J. Liu et al., Anal Chem, 2005; 779):275766-21; J. Wang, Anal Chem, 2003; 7515):3941-5.

In a further embodiment of this aspect of this disclosure, the replacement and/or removal of solutions from the reaction chamber is avoided by detection by electrical detection by electrodes on the surface of the array, such as impedance measurement (see, inter alia, S.M. Radke et al., Biosens Bioelectron, 2005; 208):1662-7).

In another embodiment of this aspect of this disclosure, the exchange and/or removal of solutions from the reaction chamber is avoided by using a microarray with FRET probes (fluorescence resonance energy transfer). The use of such FRET probes is based on the formation of fluorescence-quick pairs, so that a fluorescence signal is only produced when a target molecule has bound to the complementary probe on the surface. The use of FRET probes is described, for example, in B. Liu et al., PNAS 2005, 102, 3, 589-593; K. Usui et al., 2004; 8:209-13; J.A. Cruz et al., 2004; J.A. Molgu and J.A. Cruz et al., 2002; 76:41-626; J.A. Molgu and J.A. Szosich (2002-82); and Biotechnology (2002-83).

Another particularly preferred embodiment of this aspect of this disclosure avoids the replacement and/or removal of solutions from the reaction chamber by using a device of the invention for the qualitative and/or quantitative detection of molecular interactions between probe and target molecules in a sample solution, as described in detail below, which includes: a microarray of probes, immobilized on array elements, where the microarray is placed on a first surface of the apparatus; a reaction chamber formed between the first surface with the microarray placed on it and a second surface, where the first surface is elastically deformable at least in the area below the microarray, so that the microarray is conducive relative to the surface so that the distance between the microarray and the second surface is variable in such a way that the sample solution can be removed essentially between the microarray and the second surface, where the reaction chambers are provided with comparative areas, which are bounded by the distance between the microarray and the second surface, in such a way that the distance between the microarray and the second surface is constant in the case of the second sample solution, and the volume of the microarray is kept constant in the second sample chamber.

The present disclosure also concerns the use of FRET probe molecules as described above and/or detection methods selected from the group consisting of Total Internal Reflection Fluorescence Microscopy (TIRF) as described above, Confocal Fluorescence Microscopy as described above, Mass Change Detection methods as described above, Acoustic Surface Wave Detection methods as described above, Electrochemical and/or Electrical Detection methods as described above, To prevent exchange of solutions in a reaction chamber and/or Removal of solutions from a reaction chamber or to remove any molecules in the reaction chamber or between a target and a molecule during the detection and detection of quantitatively and qualitatively the following: (a) Introducing a sample containing target molecules into a reaction chamber which has a microarray, the microarray comprising a substrate with probes immobilized on array elements;

The present invention relates in particular to a device for the qualitative and/or quantitative detection of molecular interactions between probe and target molecules in a sample solution, comprising: a microarray of probe molecules immobilized on array elements, the microarray being placed on a first surface of the device; and a reaction chamber formed between the first surface with the microarray placed on it and a second surface, the first surface being elastically deformable at least in the area below the microarray, so that the microarray is conducive relative to the second surface, so that the distance between the microarray and the second surface is variable enough to remove the sample solution between the microarray and the second surface substantially,where there are compensating areas on the side of the reaction chamber which, when the distance between the microarray and the second surface is reduced to such an extent that the sample solution is substantially removed from the microarray and the second surface, maintain the volume of the sample solution in the reaction chamber constant. Following interaction between probe molecules and target molecules, the labelled molecules present in the sample solution which do not interact with the probe molecules will cause an undesirable background, in particular if the probe and/or target molecules are nucleic acids and/or nucleic acid analogues, this background will be caused by the labelled primers and/or labelled nucleic acids present in the sample solution which are not hybridised with the probe molecules.

A known way of removing background interference is to exchange the sample solution after interaction with an unlabeled solution, e.g. a non-fluorescent solution, but this option is generally expensive and prone to interference due to corrosion, aging of the solutions and leakage problems.

An essential feature of the device is that the distance between the microarray and the second surface is variable. A variable distance between the microarray and the second surface means that the reaction chamber of the device is compressible. In particular, the distance between the microarray and the second surface is so variable that the microarray can be bound and/or reversibly with its active side, i.e. the side on which the nucleic acids are immobilized, on the second surface.

A compressible reaction chamber thus allows the displacement of sample solution containing marked molecules which do not interact with the probe molecules and thus constitute an undesirable background by reducing the distance between the microarray and the detection plane before the detection is performed. In this way, the detection of interactions between probe and target molecules is possible with any optical detection system without exchanging the sample solution for an unlabeled solution before detection. For example, a simple fluorescence microscopic image of the DNA chip to detect the interaction signal with the invention of a fluorescence exchange device of the sample signal without marking is a possibility, especially against unlabeled fluids.

Finally, the embodiments of the device according to the invention described below ensure, in particular, that focusing of optical detection systems is no longer necessary, thus, the device according to the invention allows, for example, that, unlike the fluorescent optical detection systems for nucleic acids used so far, a simple fluorescent microscope without autofocus function can be used as a selector for the detection of hybridization between targets and probes, without the need for specific liquid washing steps such as target handling, such as the removal of target-bound molecules such as non-hybridized nucleic acids.

This provides an extremely cost-effective system for detecting and, where appropriate, amplifying target molecules in a sample, despite the multifunctional sample handling and analysis that can be performed with the device of the invention.

The device according to the invention, which can also be used as a disposable cartridge, thus allows a complete genetic analysis with only a small apparatus. The device according to the invention thus allows the performance of detection procedures at the site, e.g. in a blood donation. A result can be obtained within a short time, preferably within 1⁄2 to 2 h. All the steps of the device according to the invention, which can be performed as a disposable cartridge, can be performed by means of detailed steps such as processing, sampling, amplification and hybridization. The operator must be familiar with the task of detection and the specifics of the test.

Preferably, the distance between the microarray and the second surface is variable in a range from about 0 to about 1 mm. Other preferred lower limits for the distance between the microarray and the second surface are about 0.1 μm, about 1 μm and about 10 μm. Other preferred upper limits for the distance between the microarray and the second surface are about 0.01 mm, about 0.5 mm, about 1 mm and most preferably about 0.3 mm. Surprisingly, the interaction between probes and targets on the array surface is not affected even when the distance between substrate surface and second surface is close to zero or approximately 0.3 mm.

Preferably, the device of the invention also includes a detection system. It is preferable that the detection system be an optical system. Examples of optical systems suitable for the present invention are detection systems based on fluorescence, optical absorption, resonance transfer, etc. Preferably, the optical detection system is a fluorescent optical system.

In another embodiment, the detection system is connected to at least one spacer holder which, when applied to the second surface, sets a distance between the detection system and the second surface. If the distance between the microarray and the second surface is approximately zero, the spacer holder also sets the distance between the surface of the chip and the optical system of the detection device. This allows the variance in the distance between the optical detection device and the microarray surface to be kept very small. The variance includes only the thickness variations of the second fan, generally a glass surface, the curvature of the second surface and the thicknesses due to contamination on the surface of the chip between the detection and the pressure plate respectively.The invention provides for laterally delimiting compensation areas for the reaction space formed between the first and second surfaces, which, when the distance between the microarray and the second surface is reduced, essentially keep the volume in the reaction chamber constant. Preferably, the reaction space formed between the first and second surfaces is also laterally delimited by elastic seals. The elastic seals are particularly useful for viscous rubber. To ensure the detection of interactions between probe and target molecules,the second surface is made of an optically transparent material, preferably glass.

In the device according to the invention, the first surface is designed, at least in the area below the microarray, so that the microarray is conductive relative to the second surface, so that the distance between the microarray and the second surface is variable. The first surface may be so designed at least in the area below the microarray that the microarray is conducive towards the second surface so that the distance between the microarray and the second surface is reducible and/or the microarray is conducive in a direction opposite to the second surface so that the distance between the microarray and the second surface is enlargeable.The first surface is preferably made of an elastic plastic material, e.g. an elastic membrane. In addition, it may be preferable that the first surface be formed by means of two overlapping layers, with an outer layer of the two overlapping layers having a gap at least in the area below the microarray. In this design, it is preferable that the inner layer of the two overlapping layers be formed by an elastic seal or a sealing membrane, which usually also limits the reaction space laterally (see Figure 6).The higher temperatures in the PCR chamber create an internal pressure which causes the reaction chamber to be pressure-resistant despite the relatively unstable sealing membrane. This design is therefore similar to a self-closing valve. To ensure the elasticity of the sealing membrane, the membrane is preferably equipped with a compensating fold (see Figure 6).

Furthermore, the device may be provided with at least one means of guiding the microarray relative to the second surface, hereinafter referred to as the means of guiding the first surface, preferably selected from the group consisting of a rod, a pen, a plunger and a screw.

The device may include at least one means of guiding the first surface, which leads the microarray towards the second surface in such a way that the distance between the microarray and the second surface is reduced, and/or which leads the microarray in a direction opposite to the second surface in such a way that the distance between the microarray and the second surface is increased.

The microarray is particularly desirable if it is conductive by pressure and/or pulling of the medium on the first surface relative to the second surface.

The above spacers on the second surface may be used as a counter-support for the means of guiding the first surface.

Furthermore, it may be preferable that the first surface be capable of being moved by the first surface guide to vibration, in particular to a vibration of 10 to 30 Hz, preferably around 20 Hz. This may remove bubbles above the chip which would impede detection and/or increase the rate of interaction, e.g. hybridization rate, by a mixing effect caused by the vibration of the first surface guide.

Similarly, it may be preferable to have the second surface conducive to the first surface so that the distance between the microarray and the second surface is variable.

The second surface may be conducive relative to the first surface in such a way that the distance between the microarray and the second surface is reducible and/or that the distance between the microarray and the second surface is enlargeable.

This can be ensured in particular by making the second surface accessible to the second surface by pressure and/or pull of the spacer relative to the first surface, so that the distance between the microarray and the second surface is changeable.

In another preferred embodiment of the device of the invention, both the first and second surfaces are conductive in such a way that the distance between the microarray and the second surface is variable.

In another embodiment, the device is designed so that the microarray on the first surface is already in its original state on the second surface forming the detection plane, preferably in a closed position. The first surface is conductive so that the distance between the microarray and the second surface can be increased. Preferably, the first surface is made of an elastic material.

In another embodiment of the device according to the invention, the first surface is designed to rotate around a rotating axis. The rotating axis divides the first surface into two thigh-sections. The microarray is arranged on a first thigh-section of the first surface in this design. The rotating axis for the rotation movement preferably runs in the middle of the first surface, i.e. the two thigh-sections are preferably the same size. The first surface is preferably made of an elastic material.

In a first position of the first rotatable surface, the first surface is essentially parallel to the second surface. The surface of the microarray is essentially bounded to the second surface in the first position, i.e. the substrate surface with the probes immobilized on it is essentially not moistened by the sample solution. Between the second rib section of the first surface and the second surface, a space is formed in this first position, hereinafter also referred to as the processing chamber. This processing chamber can be used as a chamber for processing the sample solution.

In a second position of the first rotatable surface, the first surface is arranged at an angle of 180° to the second surface. The surface of the microarray is not attached to the second surface in this second position, i.e. the probe molecules immobilized on the substrate of the microarray are freely accessible to and can interact with the target molecules present in the sample solution. The processing chamber is compressed in the second position.

The first swivel surface is preferably swivel by pulling on the first thigh section of the first surface and/or by applying pressure on the second thigh section of the first surface.

The chip or substrate or first surface may preferably be made of silicon, ceramic materials such as aluminium oxide ceramics, borophloat glasses, quartz glass, single crystalline CaF_{2 and}The choice of materials should also be based on the future use of the device or chip. For example, if the chip is used for the characterisation of 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 DNA or RNA molecules, but may also be functionalized by peptides and/or proteins, such as antibodies, receptor molecules, pharmaceutically active peptides and/or hormones, carbohydrates and/or mixed polymers of these biopolymers.

In another preferred embodiment, the molecular probes are immobilized on the substrate surface via a polymer linker, e.g. a modified silane layer. Such a polymer linker can be used to derivatize the substrate surface and thus immobilize the molecular probes. In the case of a covalent bonding of the probes, polymers, e.g. silanes, are used that are functionalized or modified with reactive functionalities such as epoxides or aldehydes.

The chamber body of the reaction chamber is preferably made of materials such as glass, plastic and/or metals such as stainless steel, aluminium and brass. For example, injection-molded plastics may be used to manufacture it. Plastics such as macrolone, nylon, PMMA and Teflon are conceivable, among others. In special designs, electrically conductive plastics such as polyamide with 5-30% carbon fibres, polycarbonate with 5-30% carbon fibres, polyamide with 2-20% stainless steel fibres and PPS with 5-40% carbon fibres and especially 20-30% carbon fibres may be preferred. Alternative and/or additional materials may be used to expand the reaction chamber and also to complete the chamber by means of materials such as septic acid, for example, a spray. For example, the choice of materials such as polystyrene and PCM should be made by selecting the appropriate direction of the reaction.

In particular, the chamber body shall be designed to allow the microarray to be pushed into the second surface with its active side, i.e. the side of the array on which the nucleic acid probes are immobilized, in a closed and/or reversible manner.

In a special embodiment, the device of the invention comprises modules selected from the group consisting of a chamber body, preferably of plastic; a septum or seal sealing the reaction chamber; a DNA chip and/or a second optically transparent surface, preferably a glass plate, where the second surface may also serve as a chip (see Figures 2 and 3). In this embodiment, chamber bodies and seal are made elastically so that the DNA chip is pressed against the glass lid with its active side in a closed and rebellious manner, thus completely suppressing the fluorescent fluid analysis between the DNA chip and the detection surface (see Figure 5). This is done by means of a computerised imaging device that cannot be affected by fluorescence.

The second surface of the chamber body is preferably made of transparent materials such as glass and/or optically transparent plastics such as PMMA, polycarbonate, polystyrene or acrylic.

The reaction chamber is preferably designed as a capillary gap of variable thickness between the second surface and the microarray. By forming a capillary gap between the chip and the detection plane, capillary forces can be used to safely fill the reaction chamber. These capillary forces are already present in the uncompressed state of the reaction chamber, but can be increased by compressing the reaction chamber.

The possibility of compressing the reaction chamber and thus reducing the gap between the microarray and the detection plane provides further possibilities for handling the liquid within the reaction chamber. For example, a further embodiment of the present invention provides for several sub-chambers instead of a single chamber, with the separations between the sub-chambers not raised to the second surface, so that a fluidic connection exists between the sub-chambers in the uncompressed state of the reaction chamber. Compression of the reaction chamber allows the chambers to be separated from each other. This allows the interfaces between the chambers to be handled by compression valves.

A special design of these sub-chambers, separated by valves, is the division of the reaction chamber of the device according to the invention into different PCR chambers. In each chamber, individual primers are presented. The sub-chambers are initially filled with the analyte at the same time. The reaction chamber is then compressed. Afterwards, the reaction chamber goes through the temperature cycle for PCR. Since each sub-chamber is filled with different primers, a different amplification reaction takes place in each chamber.

After PCR, hybridisation takes place, whereby each sub-chamber may contain an individual chip area or individual chip, but it is also possible to allow a fluid connection between the sub-chambers by increasing the distance between the microarray and the second surface, so that the different amplifiers mix and thus hybridise on a chip surface.

The advantage of this embodiment with valve-separated sub-chambers is that it increases the multiplexity of PCR, i.e. the number of independent PCRs with a sample that is limited for biochemical reasons in a single-stack reaction, thus allowing the number of PCRs to be adjusted to the possible number of probes on the chip surface.

In a further embodiment of the present invention, the reaction chamber thus comprises at least two sub-chambers, with the first uncompressed state having a fluidic connection between the sub-chambers and the second compressed state having no fluidic connection between the sub-chambers.

In particular, each sub-chamber is preferably assigned to a defined region of the microarray.

The sub-chambers may be formed in particular by providing cavities between the microarray and/or the second surface as walls.

The walls between the sub-chambers are particularly preferably formed by elastic seals.

Of course, this design of the process unit with valve-separated sub-chambers can be combined with all the compression principles described above.

In another embodiment of the device according to the invention, the first surface is made of a partially deformable elastic material, e.g. an elastic membrane. By compressing only part of the reaction chamber, it is possible to create, among other things, sub-chambers in which the chip is directed towards the second surface, sub-chambers which can be separated from each other and sub-chambers which cannot be changed. This allows simple pump systems to be realized in the reaction chamber, which can be used, for example, to pump salts into the hybridization chamber at the end of a multiplication reaction. This can be advantageous, for example, to optimize the chemical conditions of the PCR hybrid, whereby the PCR hybridization is only optimized for the implementation of the PCR hybridization.

When dividing the reaction chamber into several subchambers, it is preferable to use several agitating agents. Usually, the agitating agents are identical to the agents used to guide the first surface. This allows individual chambers to be agitated specifically. This can be useful, for example, to realize separate reproduction rooms and/or hybridization rooms.

Of course, this embodiment of the device of the invention can also be combined with several agitators with all the compression principles described above.

The components or modules of the device of the invention described above, selected from the group consisting of a chamber body, seals bordering the reaction chamber on the side, a microarray and a detection plane, constitute the so-called process unit of the device of the invention.

The process unit of the device of the invention is preferably modular, i.e. the process unit can include any combination of modules, which can also be replaced during analysis.

In another preferred embodiment, the device according to the invention may additionally include a temperature control and/or control unit for controlling and/or regulating the temperature in the reaction chamber. Such a temperature control and/or control unit for controlling and/or regulating the temperature in the reaction chamber may include, in particular, heating and/or cooling elements or temperature blocks. The heating and/or cooling elements or temperature blocks may be arranged so that they contact the first and/or second surface. A particularly effective temperature control and control is ensured by contacting both the first and second surface.

In this embodiment, the microarray substrate, or the first and/or second surface, is connected to heating and/or cooling elements and/or temperature blocks and should preferably be made of materials that are well conductive. Such heat-conductive materials have the essential advantage of ensuring a homogeneous temperature profile over the entire surface of the reaction chamber and thus allowing temperature-dependent reactions such as PCR to be homogeneous, high-efficiency and highly controllable and/or controllable throughout the reaction chamber.

Thus, the substrate of the microarray, or the first surface, or the second surface, in a preferred embodiment, consists of materials with a high thermal conductivity, preferably in the range of 15 to 500 Wm.^{- 1}K^{- 1}, especially preferred in the range 50 to 300 Watt^{- 1}K^{- 1}and most preferably in the range of 100 to 200 Watt^{- 1}K^{- 1}Examples of suitable thermoconductive materials are silicon, ceramic materials such as aluminium oxide ceramics and/or metals such as stainless steel, aluminium, copper or brass.

If the substrate of the microarray or the first or second surface of the device of the invention consists essentially of ceramic materials, aluminium-oxide ceramics are preferably used, such as A-473, A-476 and A-493 ceramics from Kyocera (Neuss, Germany).

Preferably, the microarray substrate or the first or second surface on the back side, i.e. the side opposite the reaction chamber, shall be equipped with miniaturised temperature sensors and/or electrodes, if any, or shall have heating structures, allowing the sample liquid to be tempered and the sample liquid to be mixed by an induced electrosmotic flow.

The temperature sensors may be manufactured, for example, as nickel-chromium-thin film resistance temperature sensors.

The electrodes may be manufactured, for example, as gold-titanium electrodes and in particular as quadrupols.

The heating and/or cooling elements may preferably be selected to allow rapid heating and cooling of the liquid in the reaction chamber. Rapid heating and cooling means that the heating and/or cooling elements can transmit temperature changes in the range of 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. Preferably the heating and/or cooling elements may also transmit temperature changes from 1 K/s to 10 K/s.

The heating and/or cooling elements, e.g. resistance heaters, may be made, for example, as nickel-chromium thin film resistance heaters.

For further details on the specification and dimensions of the temperature sensors, heating and/or cooling elements, temperature chargers and electrodes, see the contents of the international patent application WO 01/02094.

In a preferred embodiment, the reaction chamber is heated by the use of a chamber body made of electrically conductive material. Such an electrically conductive material is preferably an electrically conductive plastic such as polyamide, possibly with 5-30% carbon fibres, polycarbonate, possibly with 5-30% carbon fibres and/or polyamide, possibly with 2-20% stainless steel fibres. Preferably, the electrically conductive plastic PPS (polyphenyl tempensulfide) with 5-40% carbon fibres, preferably with 20-30% carbon fibres is used. It is also preferable that the chamber be equipped with a heating system that provides adequate expansion and/or reduction of the chamber temperature, which is also ensured at the lowest temperature and/or the maximum temperature of the chamber, as appropriate to the specific characteristics of the chamber.

The heat can be brought into and discharged into the reaction chamber by various means, including external microwave radiation, internal or external resistance heating, internal induction loops or surfaces, water cooling and heating, friction, irradiation with light, particularly IR light, air cooling and/or heating, friction, temperature radiators and fur elements.

Temperature measurements in the reaction chamber may be performed in a variety of ways, such as by means of integrated resistance sensors, semiconductor sensors, light wave conductor sensors, pyrochrome dyes, pyrochrome liquid crystals, external pyrometers such as IR radiation and/or temperature sensors of all kinds integrated in the microarray guidance medium.

Temperature measurements in the reaction chamber may also be performed by integrating a temperature sensor into the chamber body, e.g. by injecting during the chamber body manufacturing process, by non-contact measurements using a pyrometer, IR sensor and/or thermopiles, by contact measurements, e.g. by a temperature sensor integrated in the device and contacting an appropriate surface or volume of the chamber body or chamber, by measuring the temperature-dependent change in refractive index at the detection point, by measuring the temperature-dependent change in the colour of specific molecules in the solution, measuring the temperature change of specific colour bonds in the solution, measuring the Sarray or a colour change in the chamber band/seal and by measuring the temperature-dependent change in the pH of the solution.In addition, a spontaneous temperature limitation can be achieved by a jump-up in the resistance of the heater, with the corresponding jump temperature preferably in the range of 95°C to 110°C. At the jump-up temperature, the resistance of the heater changes upwards, which means that almost no current flows and therefore hardly any heat is released.

The temperature control unit may be integrated into the first and/or second surface in a design where the process unit is equipped with a heater (see Figure 4) to allow the temperature change in PCR and hybridisation.

The process unit preferably has a low heat capacity, so that maximum temperature changes of e.g. at least 5 K/s can be achieved with low energy demand.

The cooling of the process unit can preferably also be achieved by permanently cooling the space surrounding the process unit to a lower temperature and thus passively cooling the cartridge, thus eliminating the need for active cooling of the reaction cartridge.

In a further design, the temperature control unit may comprise temperature blocks, each preheated to a defined temperature. In particular, the process unit in this embodiment does not have an integrated heater.

The heat transfer between the temperature blocks of the temperature control and control unit is preferably ensured by the temperature blocks contacting the first surface and/or second surface of the device of the invention. The temperature blocks may preferably be arranged in a linear or rotary arrangement and thus be integrated, for example, in the detection device. Figure 7 shows a rotary unit comprising several temperature blocks, each set to a defined temperature. The temperature blocks below the process unit are brought to a temperature defined by the respective temperature block. The temperature blocks are only prefabricated in such a way that they are clearly capable of producing a high or even a low temperature. The maximum temperature change in the process unit is not measurable at this temperature change.

In a further design, the temperature control unit is integrated into the first surface guide and/or agitator and/or spacer unit; heat transfer is effected by contacting the first surface and/or spacer unit with the first surface and/or the second surface.

Preferably, the device also includes a treatment unit for purifying and/or concentrating the sample solution and/or controlling the loading and/or discharge of the reaction chamber with fluids. Fluids are understood to be liquids or gases within the scope of the present invention. In addition, the analytical solution can be buffered in the treatment unit. Finally, the treatment unit can also be used to provide the necessary analytical chemicals. The connection of the fluid containers to the reaction chamber can be carried out as in the international patent application WO 01/02094.

In particular, this embodiment prefers to connect the reaction chamber and the treatment unit by means of two cannulas, the cannulas being arranged so that a first cannula ensures the supply of fluids from the treatment unit to the reaction chamber and a second cannula ensures the escape of air displaced from the reaction chamber by the fluids supplied. A sample introduced into the treatment unit can thus be passed through the cannulas to the reaction chamber of the process unit.

The treatment unit may be designed to be detachable from the process unit, so that after filling the reaction chamber with the sample solution and any other reaction fluids, the treatment unit can be separated from the process unit, preferably removed, and, if necessary, disposed of.

The following are embodiments of integrated or unintegrated reaction chamber filling units, also referred to as filling or processing units.

The reaction solution is usually introduced into a specific opening of the filling unit by means of a suitable tool, e.g. a pipette, and the fluids are conveyed to the device by pressure from the pipette or by another pressure-generating tool, e.g. a syringe or an automated unit, e.g. a functional component of a processing machine.

The filling unit is preferably designed in an ergonomically sound manner for manual operation and preferably has easily accessible openings on the outside for the introduction of the reactive substances.

A filling unit shall preferably include a suitable fluid interface to penetrate the chamber body seal. In particular, cannulae, e.g. of stainless steel or polymers, are used, usually between 0.05 mm and 2 mm in diameter. Preferably, at least one or more cannulae are arranged, preferably two, one of which can be used to fill with a reactive liquid and another to dispense the reaction chamber and absorb excess liquids. Such cannulas may be fixed or interchangeable with the filling unit, preferably with a soluble filling unit being achieved by the filling connection for the realization of single-use articles.

The filling unit may also include a unit to cover the cannula, so that after the systems are separated, injury to the user from the cannula or contamination of the environment can be prevented.

The filling unit preferably also includes a suitable mechanical interface for the precise contact of the reaction cartridge, e.g. by means of special snap closures, which allows the chamber body seal to penetrate at preferred points.

When processing the reaction cartridge in appropriate automatic processing machines, appropriate mechanical arrangements shall be made to allow adjustment and positioning in the equipment, in particular for the positioning for the exchange and/or supply of liquids and the positioning of the reaction cartridge for detection of signals after the reaction has been carried out in the reaction chamber.

The device or filling unit may also include an integrated waste receptacle to accommodate excess or displaced gaseous or liquid media, such as protective gas fillings or buffers. The waste receptacle may, for example, be filled with another gaseous, liquid or solid medium which binds the liquid or gaseous substances, such as cellulose, filter materials, silica gel, in a reversible or irreversible manner. The waste receptacle may also have an exhaust vent or be equipped with a pressure relief to improve the overall filling behaviour of the unit.

The waste container can also be constructed as a separate module, in which case the filling unit is equipped with corresponding fluid interfaces leading outwards, which can meet commercial standards such as LuerLock.

In the first special embodiment, the filling is carried out with a removable filling unit with an integrated waste receptacle. The filling unit is used in particular for the single filling of the reaction chamber. The filling unit is designed, for example, to be fixed to the cartridge or temporarily fixed, the samples are introduced into the reaction chamber and, after filling, the filling unit is separated from the cartridge and disposed of. In this first special embodiment, the filling unit is also surrounded by an integrated waste receptacle, which may be designed as described above. An example of this introduction is shown in Figure 22A. The process for filling a modular reaction unit is shown in Figure 23.

In a second special embodiment, the filling unit is used with an integrated filling unit. Here the filling unit is an integral part of the reaction cartridge and is therefore not separated from it, the disposal of the filling unit and the cartridge is carried out simultaneously. The filling unit is preferably used for single filling of the reaction chamber and possibly for further fluid steps within the process. The filling unit also preferably includes a technical device that realizes a preferential arrangement of the cannulas in the system, in particular to prevent an accidental insertion of the cannulas into the seal of the chamber.This technical arrangement may be carried out, for example, by introducing springs, elastic elements or certain recesses and elevations to achieve a grating. The filling unit in this embodiment also includes a filling and waste channel, which includes corresponding outward flow interfaces which may also meet commercial standards such as LuerLock. Such interfaces may have a form or force lock to further systems and are used for the supply and/or discharge of gaseous and/or liquid media. An example of this is shown in Figure 24.

In a third special embodiment, the filling unit is used preferably for single filling of the reaction chamber and possibly for further internal fluid steps. In this embodiment, the filling unit also includes a technical device that provides a pre-positioning of the cannula in the reaction cartridge and is therefore not separated from it, and the disposal of the filling unit and the cartridge is carried out simultaneously. The filling unit is used preferably for single filling of the reaction chamber and possibly for further internal fluid steps. The filling unit also includes a technical device that provides a pre-positioning of the cannula in the reaction cartridge and is therefore not separated from it, in order to prevent the unintended insertion of a single filling in the chamber. For example, the filling unit can be integrated into a specific process by means of a system of elasticity and a single filling in the form of a single filling, in order to prevent the unintended insertion of a single filling in the chamber. However, it may also be used in the case of a pre-positioning of a single filling in the chamber. For example, this can be integrated into a specific case of the filling unit by means of a special system of elasticity and a special effects.

A special embodiment of the arrangement of cannulae for pressure compensation during the crushing process is described below. For example, the cannulae of a cartridge filling tool may be arranged in such a way that both a loose filling and the transfer of excess reaction solutions when the reaction chamber is compressed are possible. This can be achieved preferably by an adapted design of the seal and cannula arrangement, in which the cannulae preferentially penetrate into the compensation areas within the reaction chamber. Such an arrangement is particularly useful when the excess volume cannot be absorbed by a special sealing device.

The device may also include a unit connected to the detection system to control the test procedure and/or to process the signals received by the detection system. The control and/or processing unit may be a microcontroller or an industrial computer. This combination of detection unit and processing unit, which ensures the conversion of the reaction results into the analytical result, allows, inter alia, the use of the device as a handheld device, for example in medical diagnostics.

Furthermore, the device of the invention preferably has an additional interface for external computers, which allows, inter alia, the transfer of data for storage outside the device.

In another preferred embodiment, the device is encoded, preferably with a data matrix and/or a barcode, containing information on the substance library and/or the execution of the replication and/or detection reaction. Such an individual identification number enables the sampling or detection device to automatically identify which test has been performed. To this end, during the manufacture of the device, a data set is stored in a database containing information on the substance library, the execution of the data matrix and/or a barcode. The data set may include, in particular, information on the arrangement of the samples on the array and the number of samples to be evaluated in the data matrix. The data set may then be automatically recorded in a computer, in such a way as to ensure the timeliness of the execution of the test and, where appropriate, the temperature and/or temperature controller and/or the data set in the data matrix.

The coding, like a data matrix, does not necessarily have to contain all the information, but can simply contain an identifier by which the required data is loaded from a computer or a data carrier.

The device according to the invention is extremely easy to manufacture. The geometric tolerances of the dimensions of the individual components can be very large, for example, from 1/10 to 2/10 mm, so that, for example, the process unit can consist of only four individual components that can be placed in a simple way. In Figures 10 and 11, embodiments are shown that, due to the inventive design, are also easy to manufacture, although they consist of several parts. The geometric tolerances of the dimensions of the individual components can be very large, for example, with 1/10 to 2/10 mm, so that, for example, the spray molding of seals and camera bodies can be carried out in a cost-effective way.

In another aspect of this disclosure, a method for the qualitative and/or quantitative detection of molecular interactions between probe and target molecules is provided, which includes the following steps: (a) Introduce a sample, preferably a sample solution containing target molecules, into a reaction chamber of a device as described above; and (b) Detect an interaction between the target molecules and the probes immobilized on the substrate.

The disclosure method shall allow for the qualitative and/or quantitative detection of molecular interactions between probe and target molecules in a reaction chamber without requiring the exchange of sample or reaction fluids after the interaction and before detection to remove a disturbance background.

The usual method of detecting interaction between the probe and the target molecule in this disclosure is as follows: after fixing the probe (s) in a specified way to a specific matrix in the form of a microarray or after providing a microarray, the targets are brought into contact with the probes in a solution and incubated under defined conditions.

Detection of the specific interaction between a target and its probe can then be done by a variety of methods, usually depending on the type of marker inserted into target molecules before, during or after the interaction of the target molecule with the microarray. Typically, such markers are fluorescent groups, so that specific target probe interactions can be detected with high spatial resolution and low fluorescent optical effort compared to other conventional detection methods, especially mass-sensitive methods (see e.g., A. Nature array, J. Hodgson, Marshall, DNA: An array of Nature, Biotechnology possibilities, 1998, Ramsay, G. 16, 27, Chips of the State of DNA, 1998, Biotechnology, 16, 40-44).

Depending on the library of substances immobilized on the microarray and the chemical nature of the target molecules, this test principle can be used to study interactions between nucleic acids and nucleic acids, between proteins and proteins, and between nucleic acids and proteins (for an overview see F. Lottspeich, H. Zorbas, 1998, Bioanalytics, Spectrum Akademischer Verlag, Heidelberg/Berlin).

In particular, antibody libraries, receptor libraries, peptide libraries and nucleic acid libraries are considered as potential drug libraries that can be immobilized on microarrays or chips.

The most important role is played by the nucleic acid libraries, which are microarrays on which deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules are immobilized.

In a preferred embodiment of the disclosure procedure, the microarray and secondary surface are spaced before detection in step (b) in a position that allows for processing of the sample solution and/or interaction between the target molecules and the probe molecules immobilized on the substrate, for example amplification of nucleic acids to be detected and/or hybridization between nucleic acids to be detected and nucleic acids immobilized on the substrate.

Furthermore, it is preferable that in step (b) the distance between the microarray and the second surface is changed, preferably reduced, i.e. the detection is preferably done at a reduced distance between the microarray and the detection plane.

In the case of a design, the distance between the microarray and the second surface of the microarray is directed towards the second surface in order to reduce the distance, preferably by applying pressure to the first surface on at least one means of guiding the first surface, such as a stick, rod, pen and/or screw, the pressure point of the means being particularly below the microarray.

The microarray may be pushed towards the second surface or detection plane by the first surface being elastically deformable at least in the area below the microarray; alternatively, the first surface may be formed by means of two overlapping layers, with an outer layer of the two overlapping layers having a protrusion at least in the area below the microarray and an inner layer of the two overlapping layers being made of an elastic seal.

The means of guiding the first surface, e.g. a pen, a stick, a stick and/or a screw, may not be used only to apply pressure to the first surface. If bubbles arise on the DNA chip which would make detection difficult, they can be removed by agitation through the means of guiding the first surface, e.g. with a vibration frequency of about 20 Hz applied to the first surface, particularly in the form of an elastic membrane.

Furthermore, the problem is often that the interaction, e.g. hybridization, takes a very long time on the chip surface. This is due, among other things, to the fact that the interaction or hybridization rate is diffusion-determined. Preferably, the interaction or hybridization rate can be increased by agitation over the medium to guide the first surface, e.g. with a vibration frequency of about 20 Hz applied to the first surface, especially in the form of an elastic membrane, since the agitation or vibration leads to a mixing in the reaction chamber.

In the case of a further design, the second surface is directed towards the first surface in order to reduce the distance between the microarray and the second surface, which can be ensured in particular by applying pressure from the spacer to the second surface in the direction of the first surface.

In the case of a further design, the first surface is directed towards the second surface and the second surface towards the first surface in order to reduce the distance between the microarray and the second surface.

Further embodiments of the first surface relative to the second surface and the second surface relative to the first surface are described below. These embodiments are not only suitable for positioning the first surface or the probe array relative to the second surface or the detection surface, but can also be used in particular to move the probe array relative to the detection surface.

In one embodiment, the probe array is directed against or moved in the chamber relative to the detection surface by means of a magnetic field; for example, the probe array and/or the second surface contains a magnetic material or contains a component which is a mixture of magnetic materials and/or is contained in a form of a fully or partially magnetic material; furthermore, it may be preferable to move the probe array and/or the second surface passively by means of a magnetic field, by moving a magnetic body which is located below the respective surface and is connected to it, for example.

In another embodiment, the probe array is moved and/or positioned relative to the detection surface by gravity.

In another embodiment, the probe array is moved and/or positioned relative to the detection surface by a current generated in the reaction chamber, for example by means of a device designed so that when the probe array is flooded with a liquid, a downward pressure is created on one side of the reaction chamber and an overpressure on the opposite side, causing the probe array to move in the reaction chamber.

In another embodiment, the probe array is moved and/or positioned relative to the detection surface by the action of an electric field.

In another embodiment, local overheating under the probe array creates a gas bubble that causes the chip to move in the chamber or be directed against the detection surface.

By reducing the distance between the microarray and the second surface before detection, the sample solution is preferably essentially removed from the range between the microarray and the detection plane, thus reducing the background signals caused by marked molecules not bound to the array surface, e.g. marked primers and/or marked target nucleic acids not bound to the array surface.

In particular, in the detection of step (b), the distance between the microarray and the second surface is changed so that the sample solution is essentially removed from the microarray and the second surface, the microarray is then essentially in the detection plane and a disturbance background is almost completely avoided.

In another alternative embodiment, the microarray is already in the original state of the device, bound to the second surface forming the detection plane, and is not first brought into the detection plane by guiding the first surface towards the second surface and/or guiding the second surface towards the first surface. During the processing steps, the microarray in this embodiment is not soaked by the sample solution. To perform the interaction reaction, e.g. hybridization, the first surface, which is formed retractively from an elastic material, e.g. an elastic pressure membrane, is brought back from the detection surface. This leads to the chip surface being removed from the detection surface and released by the detection solution.

A further embodiment of the method described in the present disclosure uses a device of the invention as described above, the first surface of which is designed to rotate about a pivot axis.

In a first position, also known as the starting position, the surface of the microarray on the first rib is essentially bounded to the second surface, i.e. the substrate surface with the probes immobilized on it is essentially not moistened by the sample solution.

The first rotatable surface is then moved to a second position, where the first surface is positioned at an angle other than 180°, preferably 45°, to the second surface. This is done preferably by pulling on the first thigh section of the first surface and/or by pressing on the second thigh section of the first surface with a means to guide the first surface as described above. By moving the first surface to the second position, the microarray is carried out from the second surface and the sample is inserted into the cavity or microarray and the second surface. The action on the substrate of the microarray is carried out by immobilized molecules in such a way that the action of the molecules is so rapid that an interaction between the target and the first test solution can be freely achieved. The action of the microarray and the first test solution is such that the molecules can be moved between the target and the test solution in such a way that the reaction between the target and the test molecule can be accelerated.

To perform the detection and, if necessary, further processing, the first flexible surface is brought back to the first position, for example by pressure on the first thigh section of the first surface and/or by pulling on the second thigh section of the first surface or, in the case of elastic design of the first surface, by releasing the first thigh section.

The targets to be tested may be present in any type of sample, preferably a biological sample.

Preferably, targets shall be isolated, purified, copied and/or amplified prior to detection and quantification by the disclosure process.

The DMP also allows for the amplification and qualitative and/or quantitative detection of nucleic acids in a reaction chamber, whereby the detection of molecular interactions or hybridizations can be carried out after the completion of a cyclic amplification reaction without the need to exchange the samples or reaction fluids. The DMP also ensures the cyclical detection of hybridization events at amplification, i.e. the detection of hybridization also during the cyclic amplification reaction. Finally, the DMP allows the quantification of the amplification products during the amplification reaction and after the completion of the amplification.

Amplification is usually performed by conventional PCR methods or by a procedure as described above to perform parallel amplification of the target molecules to be analysed by PCR and detection by hybridisation of the target molecules with the substance library carrier.

In another embodiment, amplification is performed as multiplex PCR in a two-stage process (see also WO 97/45559). In a first stage, multiplex PCR is performed by using fusion primers, whose 3' ends are gene-specific and whose 5' ends are a universal region. The latter is the same for all forward and reverse primers used in the multiplex reaction. In this first stage, the amount of DNA primers is limiting. This allows all multiplex products to be amplified to a uniform molar level, provided that the number of cycles is sufficient to achieve limitation for all products. In a second stage, universal primers are added, which are identical to the 5' primers.

In another preferred embodiment of the disclosure method, the detection is performed during the cyclic amplification reaction and/or after the completion of the cyclic amplification reaction; preferably, the detection is performed during the amplification reaction at each amplification cycle; alternatively, the detection may be determined at every second cycle or every third cycle or at any other interval.

In the case of a linear replication reaction in which the target quantity increases by a certain amount with each step, or an exponential replication reaction, e.g. PCR, in which the target quantity of DNA multiplies with each step, the process unit can thus push the chip to the detection level after each replication step and thus perform the detection. This makes it possible to perform online monitoring of the replication reaction. In particular, in non-linear replication reactions, this makes it possible to determine the output concentration of the target quantity of DNA.

The number of steps can be optimized online. Once the target DNA amount reaches a certain concentration, the replication is stopped. If the target starting concentration is low, the number of steps is increased to perform a safe analysis of the products. With reduced reaction time of positive controls, the analysis process can be stopped very early.

The chemicals required to perform an amplification reaction, such as polymerase, buffer, magnesium chloride, primer, marked, especially fluorescent-marked primer, dNTPs, etc., may be submitted to the reaction chamber, for example, freeze-dried.

Preferably, the cyclic amplification reaction is a PCR. In PCR, three temperatures are usually run for each PCR cycle. Preferably, the hybridised nucleic acids are separated from the microarray at the highest temperature, i.e. the denaturation temperature. A preferred denaturation temperature is 95°C. Thus, at this denaturation temperature, a hybridisation signal can be determined to serve as a zero or reference value for the nucleic acids detected in the respective PCR cycle.

The temperature following the PCR cycle, e.g. an annealing temperature of about 60°C, allows for hybridisation between nucleic acids to be detected and nucleic acids immobilized on the microarray substrate, so that a manifested method is used to detect or detect target nucleic acids present in a PCR cycle at the annealing temperature.

To increase the sensitivity of the exposure method, it may also be advantageous to lower the temperature below the annealing temperature, so that detection is preferably performed at a temperature below the annealing temperature of an amplification cycle, for example, detection may be performed at a temperature in the range of 25°C to 50°C and preferably in the range of 30°C to 45°C.

Another alternative embodiment of the disclosure method involves hybridisation between nucleic acids to be detected and nucleic acids immobilized on the microarray substrate at a low temperature, followed by an increase in the hybridisation temperature, which has the advantage of reducing the hybridisation time compared to hybridisation at temperatures above 50 °C without compromising specificity of the interactions.

Subtracting the zero value or reference value at the denaturing temperature from the value measured at or below the annealing temperature gives a noise-free result in which fluctuations and drift are eliminated.

Normally, the target molecules to be detected are identified by a detectable marker, so in the disclosure method, detection is preferably by having the bound targets be marked with at least one marker detected in step (b).

As mentioned above, the marking coupled to the targets or probes is preferably a detectable unit or a detectable unit coupled to the targets or probes via an anchor group. The detection or marking procedure is extremely flexible in terms of detection possibilities. The detection procedure is therefore compatible with a variety of physical, chemical or biochemical detection methods. The only condition is that the unit or structure to be detected can be directly coupled to a probe or a target, such as an oligonucleotide or an oligonucleotide-coupled anchor group.

The detection of the marking may be based on fluorescence, magnetism, charge, mass, affinity, enzymatic activity, reactivity, a gold marking, etc. Thus, the marking is preferably based on the use of fluorophore-labeled structures or building blocks. In conjunction with the fluorescence detection, the marking may 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), Lanthanide, Samarium, and Europium (EGG, Wallac & Y, Freiburg, Germany).

As already mentioned, the use of the device of the invention in the disclosure procedure ensures the detection of the fluorescence markers by means of a fluorescence microscope without autofocus, e.g. a fluorescence microscope with fixed focus.

In addition to fluorescence markers, luminescence markers, metal markers, enzyme markers, radioactive markers and/or polymer markers may be used as a marker or detection unit coupled to the targets or probes for the purposes of this disclosure.

Similarly, a nucleic acid can be used as a marker (tag) which can be detected by hybridization with a tagged reporter (sandwich hybridization).

In an alternative embodiment of the disclosure process, the detectable unit is coupled to the targets or probes via an anchor group. The preferred anchor groups used are biotin, digoxygenin, etc. The anchor group is implemented in a subsequent reaction with specific binding components, such as streptavidin conjugates or antibody conjugates, which are self-detectable or trigger a detectable response. When using anchor groups, the implementation of the anchor groups into detectable units may take place before, during or after the addition of the samples covering the targets or probes, or, if applicable, during or after the selective spindle in the probes. The selective spindle in the probes is specifically described in the international patent application WO 0383/B88, with respect to the content of which this is expressly described.

The labelling may also be revealed by interaction of a labelled molecule with the probe molecules, for example by hybridisation of a labelled oligonucleotide with an oligonucleotide probe or oligonucleotide target as described above.

Other labelling methods and detection systems suitable for the present invention are described, for example, in Lottspeich and Zorbas, Bioanalytics, Spectrum Akademischer Verlag, Heidelberg, Berlin, 1998, chapters 23.3 and 23.4.

A preferred embodiment of the disclosure process uses detection methods that result in an adduct with a specific solubility product resulting in precipitation. In particular, the labelling process uses substrates or educts that can be converted into a hard soluble, usually coloured product. For example, this labelling reaction may use enzymes that catalyze the conversion of a product substrate into a hard soluble product. Reactions that are capable of precipitating array elements and possibilities for precipitation detection are described, for example, in international patent application WO 00/72018 and international patent application WO 02/28100 whose contents are specifically referred to herein.

In a particularly preferred embodiment of the disclosure method, the bound targets are marked to catalyze the reaction of a soluble substrate or educts to a hard precipitate on the array element where a probe/target interaction has occurred or to act as a crystallization germ for the conversion of a soluble substrate or educts to a hard precipitate on the array element where a probe/target interaction has occurred.

The use of the disclosure method thus allows simultaneous qualitative and quantitative analysis of a wide variety of probe/target interactions, with individual array elements of ≤ 1000 μm, preferably ≤ 100 μm and preferably ≤ 50 μm.

In immunocytochemistry and immunological microtiter plate-based assays, the use of enzymatic markers is known (see E. Lidell and I. Weeks, Antibody Technology, BIOS Scientific Publishers Limited, 1995).

The reaction leading to precipitation on the array elements is particularly preferred, where the enzyme catalyzes the transformation of a soluble substrate or educt into a poorly soluble product.

The preferred peroxidase for the oxidation of 3,3', 5,5'-tetramethylbenzide is sea urchin peroxidase, but other peroxidases are known to be used for the oxidation of 3,3',5,5'-tetramethylbenzide.

It is assumed that 3,3',5,5'-tetramethylbenzide is oxidized to a blue radical reaction under the catalytic action of a peroxidase in a first step (see, e.g., Gallati and Pracht, J. Clin. Chem. Clin. Biochem. 1985, 23, 8, 454).

The following Table 1 gives, without claiming to be complete, an overview of a number of possible reactions that are likely to result in a precipitation of array elements that have been interacted with by the target and the probe: Tabelle 1

Katalysator bzw. Kristallisationskeim	Substrat bzw. Edukt
Meerrettichperoxidase	DAB (3,3'-Diaminobenzidin)
4-CN (4-Chlor-1-Napthol)
AEC (3-Amino-9-Ethylcarbazol)
HYR (p-Phenylendiamin-HCl und Pyrocatechol)
TMB (3,3',5,5'-Tetramethylbenzidin)
Naphtol/Pyronin
Alkalische Phosphatase	Bromchlorindoylphosphat (BCIP) und Nitrotetrazoliumblau (NBT)
Glucoseoxidase	t-NBT und m-PMS (Nitrotetrazoliumblauchlorid und Phenazinmethosulfat)
Goldpartikel	Silbernitrat
Silbertartrat

The detection and/or detection of probe/target interactions via insoluble precipitates is described in particular in WO 02/02810.

The following are embodiments of this disclosure which can be used to overcome problems that may occur in general in the detection of molecular interactions on solid media, such as preventing the formation of Newtonian rings between the detection plane and the probe array. The appearance of Newtonian rings is essentially determined by the type of illumination, the wavelength of the light used for detection, the distance between the detection plane and the probe array and the refractive index of the solution in the chamber.

In addition, Newton rings can be prevented by the application of spacers on the chip and/or the chip facing side of the detection surface.

Furthermore, Newton rings can be prevented by applying the probe array to a rough carrier surface.

Furthermore, Newtonian rings can be prevented by applying the probe array to a light-absorbing surface.

The pressure applied to the chip relative to the detection surface can also be permanently varied during the detection process, changing the thickness of the gap between the chip and the detection surface and thus the position of the Newtonian rings.

Another particularly preferred method of preventing Newtonian rings is the use of several light sources from different directions to illuminate and thus stimulate the fluorophores of bound targets.

Background fluorescence produced by the fluorophores of unbound targets in the displaced liquid may cause distortion of the detected signal, preferably by the use of a lens, e.g. applied to the detection surface or chip and/or arranged around the chip or in the imaging optics, designed to illuminate or image only the surface of the probe array.

When using suitable light sources such as lasers, due to the coherence of the light, inhomogeneities in the lighting can occur. Such inhomogeneities can be reduced or avoided by using waveguides and/or mixing filters and/or light of different wavelengths.

The application of an organic or inorganic light-absorbing layer on the probe array carrier, which is non-fluorescent in the selected wavelength range, can reduce or prevent the fluorescence background signal produced by the probe carrier and/or elements behind it.

In all the embodiments of the disclosure procedure described above, preamplification of the material to be analysed is not required. Targeted sub-areas of the sample material extracted from bacteria, blood or other cells can be amplified and hybridized on the medium by means of a polymerase chain reaction (PCR), in particular in the presence of the device of the invention or the substance library carrier as described in DE 102 53 966.

The disclosure-compliant method is therefore particularly suitable for performing parallel amplification of the target molecules to be analysed by PCR and detection by hybridisation of the target molecules with the substance library carrier. The nucleic acid to be detected is first amplified by PCR, preferably by adding at least one competitor to the reaction initially, which inhibits the formation of one of the two template strands amplified by PCR. In particular, a DNA primer is added to the PCR molecule, which is amplified by one of the template strands used for PCR amplification of the template, and cannot be extended enzymatically, but the nucleic acid amplified by PCR is then added initially by a single competitor, which is then amplified by a single DNA template, or by a single hybrid molecule, which is not able to be amplified by the enzyme, and which is initially referred to as a hybrid, and which is not capable of being extended by an enzyme.

Any molecule that produces preferred amplification of only one of the two template strands present in the PCR reaction can be used as a competitor in PCR. Competitors may therefore be proteins, peptides, DNA ligands, intercalators, nucleic acids or their analogues. Competitors are preferred to be proteins or peptides that are capable of binding single-stranded nucleic acids with sequence specificity and have the properties defined above.

Err1:Expecting ',' delimiter: line 1 column 196 (char 195)ⁿ(with n = number of cycles), but a damped amplification kinetics of the form < 2ⁿ- I 'm not .

The single strand surplus obtained by PCR is a factor of 1.1 to 1000 over the non-amplified strand, preferred factor of 1.1 to 300, also preferred factor of 1.1 to 100, particularly preferred factor of 1.5 to 100, also particularly preferred factor of 1.5 to 50, particularly preferred factor of 1.5 to 20 and most preferred factor of 1.5 to 10.

Typically, the function of a competitor will be to selectively bind to one of the two template strands, thus impeding amplification of the corresponding complementary strand. 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. Similarly, they may be aptamers that bind sequence-specifically to specific regions of one of the two amplifying template strands only.

Preferably, nucleic acids or nucleic acid analogues are used as competitors. Usually, the nucleic acids or nucleic acid analogues will act as a PCR competitor by either competing with one of the primers used for PCR for the primer binding site or, due to sequence complementarity, being able to hybridise with a region of a template strand to be detected. This region is not the sequence detected by the probe. Such nucleic acid competitors are not enzymatically extensible.

Err1:Expecting ',' delimiter: line 1 column 630 (char 629)

The preferred competitors are DNA or RNA molecules, in particular DNA or RNA oligonucleotides or their analogues.

Depending on the sequence of the nucleic acid molecules used as competitors or nucleic acid analogues, the inhibition of amplification of one of the two template strands in the PCR reaction is based on different mechanisms.

For example, if a DNA molecule is used as a competitor, it may have a sequence that is at least partially identical to the sequence of a primer used in PCR to such an extent that specific hybridization of the DNA competitor molecule with the corresponding template strand is possible under strict conditions. Since the DNA molecule used in competition is apparently not extensible by a DNA polymerase in this case, the DNA molecule competes with the respective primer during the PCR reaction to bind to the template. Depending on the quantity ratio of the DNA competitor molecule used, this may inhibit the amplification of the primer strand defined by the template strand, so that this way of preparing the primer would be significantly more efficient. This would reduce the kinetic efficiency of the primary strand by using a higher amount of the target material than would be expected by using a single template.

Err1:Expecting ',' delimiter: line 1 column 192 (char 191)

Alternatively to the above, the DNA competitor molecule may have a sequence that is complementary to a region of the template strand to be detected that is not addressed by any of the primer sequences and is not extensible enzymatically.

The expert is aware that the sequences of DNA competitor molecules or more 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 a PCR primer but is complementary to another region of the template strand to be detected, this sequence shall be selected so that it does not fall within the range of template sequence competence to be detected by means of a probe hybridisation. This is necessary because no processing reaction needs to take place between the PCR and the hybridisation reaction. A nucleic acid molecule that falls within the range to be detected would be considered as a single nucleic acid to be used to complement this template sequence to the target.

Err1:Expecting ',' delimiter: line 1 column 180 (char 179)

When using non-enzymatically extensible nucleic acids or nucleic acid analogues as competing molecules, these should be selected in terms of sequence or structure so that they cannot be enzymatically extended by DNA or RNA polymers. Preferably, the 3' end of a nucleic acid competitor should be designed to have no complementarity to the template and/or to replace the 3' end with another substituent.

If the 3' end of the nucleic acid competitor does not have template complementarity, whether the nucleic acid competitor binds to one of the template's primer binding sites or to one of the template's sequences to be amplified by PCR, the nucleic acid competitor cannot be extended by the common DNA polymers due to the lack of base complementarity at the 3' end. This type of non-extensibility of nucleic acid competitors by DNA polymers is not known to the expert. In particular, the nucleic acid competitor does not have 3' end with respect to the last 4 bases, especially with respect to the last 3 bases, especially with respect to the last 2 bases, and most of the above-mentioned bases cannot be preferentially hybridized with respect to its competitors.

Nucleic acid competitors that are not enzymatically extensible may also have 100% complementarity to their target sequence if they are modified in their spine or 3' end to be enzymatically non- extensible.

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.

In particular, such a method prefers to use a DNA molecule competing with one of the two PCR primers for binding to the template and which has been provided with an amino link at the 3' end during chemical synthesis as a competitor.

Other competing phosphate group modifications that are not recognized by the DNA polymerases are known to the professional, including nucleic acids with backbone modifications such as 2'-5' amide bonds (Chankin et al. (1999) J. Chem., Trans 1, 315-320), sulfide bonds (Kawai et al. (1993)), Nucleic Acids, 13-147 (2009), L. (647), T. Amensen et al. (2002) and T. T. Perroning, 213-174 (2000), and Acid Science, 290 (134-164), T. T. (2004) and T. T. (2004).

Multiple competitors that hybridise at different parts of the template (e.g. the primer binding site) can also be used simultaneously in a PCR. If the competitors have secondary structure breaker properties, this can further increase the efficiency of hybridisation.

In an alternative embodiment, the DNA competitor molecule may have a complementary sequence to one of the primers. Such, for example, antisense DNA competitor molecules can then be used, depending on the ratio of antisense DNA competitor molecule to primer, to titrate the primer in the PCR reaction so that it is no longer hybridized with the respective template strand and only amplified accordingly by the template strand defined by the other primer.

When the term "nucleic acid competitor" is used in this disclosure, it includes nucleic acid analogue competitors, unless the context otherwise indicates. The nucleic acid competitor may bind to the relevant strand of the template in a reversible or irreversible manner. The binding may be by covalent or non-covalent interactions.

Preferably, the nucleic acid competitor is bound by non-covalent interactions and is reversible, in particular, preferably, the template is bound by formation of Watson-Crick base pairs.

The sequences of nucleic acid competitors are usually based on the sequence of the template strand to be demonstrated, whereas for antisense primers, they are based on the primer sequences to be titrated, but which are in turn defined by the template sequences.

Err1:Expecting ',' delimiter: line 1 column 545 (char 544)

After annealing the primer, which is typically carried out in a temperature range of 40-75 °C, preferably 45-72 °C and preferably 50-72 °C, an elongation step is followed, whereby the DNA polymerase deoxyribonucleotides present in the reaction solution are linked to the 3' end of the primer by the activity of the DNA polymerase. The identity of the inserted dNTPs depends on the sequence of the template DNA strand hybridized with the primer. Since thermally stable polymerases are usually used, the elongation step usually starts between 68-72 °C.

In symmetric PCR, by repeating this described cycle of denaturation, annealing of the primer and elongation of the primer, an exponential increase in the nucleic acid segment of the target defined by the primer sequences is achieved.

The expert is aware that single-stranded RNA, such as mRNA, can also be used as a template.

In a particularly preferred embodiment, 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 Biolapand, Beverly, USA), Exbs DNA polymerase (Roche, Mannheim, Germany).

The use of polymerases optimized from naturally occurring polymerases by targeted or evolutionary modification is also preferred, in particular the use of Taq polymerase from Eppendorf (Hamburg, Germany) or the Advantage cDNA polymerase mix from Clontech (Palo Alto, CA, USA) is preferred when performing PCR in the presence of the substance library carrier.

A further aspect of the present invention concerns the use of the apparatus of the invention for performing microarray-based tests.

The following are specific designs of the device or process of the invention.

Figure 5 shows that the first surface, here an elastic membrane, preferably with a heating device integrated, is deformed by a pin or a stick, pushing the chip towards the detection plane. Furthermore, a spacer holder on the second surface pushes the detection plane into the reaction chamber, approaching the DNA chip from above until the liquid between the DNA chip and the detection plane is almost completely displaced. By moving the detection surface towards the chip, the elastic seals sealing the reaction chamber are compressed. The displaced liquid deforms the air pressure dilation, causing the air pressure to be compressed in the chambers.

The process unit can also be designed to deform only the first surface, e.g. in the form of an elastic membrane, or to push only the detection plane into the chamber.

The reaction chamber is enclosed on its side and on the side opposite to the detection plane by a sealing membrane to which the DNA chip is attached. The sealing membrane closes a hole at the bottom of the chamber body at the height of the DNA chip. The hole is slightly smaller than the DNA chip. When conducting a PCR in the reaction chamber, the pressure inside the chamber due to the higher temperatures associated with the PCR closes the chamber. The chamber is therefore fixed despite the unstable sealing membrane (principle of the sealing membrane itself).

Examples Example 1: Construction of a reaction cartridge without integrated heater

Figures 8 and 9 show an example of a processing unit without integrated heating and a device to guide the DNA chip against the detection plane, the DNA chip in the device shown can be read by a conventional fluorescence microscope (e.g. axioscope, Zeiss, Jena, Germany).

Example 2: Construction of a reaction cartridge with silicon heating substrate

Err1:Expecting ',' delimiter: line 1 column 333 (char 332)

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The next component is an elastic seal that limits the reaction chamber laterally.

In the centre of the reaction chamber, the DNA chip is fixed so that the probe array faces the detection plane. After the detection plane is installed in the form of a glass surface, it protrudes from the lower half-shell by a further 0,2 mm. The subsequent addition of the upper half-shell, guided by pencils, presses the glass surface against the seal, thus ensuring optimal sealing of the reaction chamber.

The reaction chamber can then be filled with solution, with the exception of the outer chambers, where only the inner chamber is filled with the chip, and the required fluids are injected into the reaction chamber via the cannula.

Biochemical reactions controlled by the silicon heating substrate, such as PCR and/or hybridisation in the reaction chamber, can then be performed.

To detect the intermediate results or the final result, the detection plane is pressed against the DNA chip from above by means of the detection unit spacer holder until the distance between the detection plane and the probe array is approximately zero. The surrounding liquid is then pushed into the outer chambers where it compresses the air there. This process is reversible and can be done, for example, after each PCR cycle.

The compact design, the internal circuit board with EPROM and the integrated heating substrate make this variant of the device particularly suitable for mobile use.

Example 3: Evidence of decrease in background signal by displacement of analyte

All the fluorescence measurements described in this example were made with a fluorescence microscope (Zeiss, Jena, Germany) in the light of a white light source and a cyanide 3 filter set. The signals were recorded with a CCD camera (PCO-Sensicam, Kehlheim, Germany).

(a) Measurement of the fluorescence signal of the analyte according to the gap width

The channels were cast with defined channel depths (5 μm, 10 μm, 28 μm) from Sylgard, with a width of 125 μm, a glass chip was placed over the channels of varying depths, the channels were then filled with a 200 nM solution of a Cy3-labeled oligonucleotide 2 x SSC + 0.2% SDS and the signal measured at an exposure time of 1.5 s.

The measurement results are shown in Figure 12. As the channel depth increases, the signal increases linearly. A regression line was calculated (Equation 1).

The resulting regression equation (Equation 1) now allows the thickness of the layer between the DNA chip and the detection surface to be determined by the background fluorescence signal.

This was verified by stacking two glass plates (chips) with structured markings on their top (crosses, numbers and data matrix in Figure 14) on which focusing was possible. The chips were stacked so that the structured markings were oriented to each other and separated only by a thin liquid layer. A 200 nM solution of a cy3-labeled oligonucleotide in 2 x SSC + 0.2 % SDS was used as liquid. The focusing unit of the microscope, which was equipped with a scaling linearity, allowed the distance between the markings and the layer thickness of the liquid film to be determined in real time. The intensity of the background light is 158 μm below 0.75 μm.

(b) Experiments to reduce or eliminate background fluorescence by compressing the process unit

In these experiments, the hybridization signal was measured in relation to the displacement of the fluorescent analyte by pressing a plunger. The experimental structure is outlined in Figure 15. By pressing the plunger, the silicon chip (3.15 x 3.15 mm) was pressed against a probe chip (DNA chip) and the liquid between the two surfaces was displaced.

To perform the experiment, the chamber was filled with a hybridisation solution, which is a model system for the conditions of a PCR hybridisation. The hybridisation solution contained a Cy3 labelled oligonucleotide (2 nM final concentration in 2 x SSC + 0.2% SDS) which was complementary to the probe array. In addition, the hybridisation solution contained a Cy3 labelled oligonucleotide which also does not hybridise with the probe array and only contributes to the fluorescence background signal in the solution, but not to the specific signals at the spots.

The hybridisation was carried out for 10 min. The hybridisation signals were then read out using a fixed exposure time of 1.5 s. In the test setup, the plunger was pressed further to the probe array (detection surface) between each take-off, reducing the gap between the array and the second surface filled with the hybridisation solution.

Figure 16 shows a recording of the hybridization signal at a gap width of 10 μm. The measurement results for the background signal and the hybridization signal at the spots are shown in Figure 17. Both signals behave linearly to the gap width as expected.

When a grey value of 255 is reached, the measuring instrument is over-controlled, i.e. with a gap width of about 17 μm, the spot intensity can only be measured by reducing the exposure time, which reduces the sensitivity of the measurement.

The difference between the two is very small, but the difference between the two is very small, and the difference between the two is very small.

(c) Amplification, hybridisation and detection as a single-stage reaction

Two process units were assembled and numbered, with the structure shown in Figure 15.

Two identical reaction approaches were developed with the following composition: Other

20mM dNTPs	0,5 µl
1 M Kaliumacetat (Kaac)	3 µl
25mM Mg-acetat Eppendorf	5 µl
Clontech C-DNA PCR Puffer	5 µl
Eppendorf Taq-Polymerase	3 µl
10µM Primer CMV_DP_Cy3 Cy3_5'TGAGGCTGGGAARCTGACA3'	1 µl
10µM Primer CMV_UP_NH2 5'GGGYGAGGAYAACGAAATC3'_NH2	0,66 µl
10µM Primer CMV_UP 5'GGGYGAGGAYAACGAAATC3'	0,33 µl
10µM Primer Entero_DP_Cy3 Cy3_5'CCCTGAATGCGGCTAAT3'	1 µl
10µM Primer Entero_UP_NH2 5'ATTGTCACCATAAGCAGCC3'_NH2	0,66 µl
10µM Primer Entero_UP 5'ATTGTCACCATAAGCAGCC3'	0,33 µl
10µM Prime HSV1_DP_Cy3 Cy3_5'CTCGTAAAATGGCCCCTCC3'	1 µl
10µM Primer HSV1_UP_NH2 5'CGGCCGTGTGACACTATCG3'_NH2	0,66 µl
10µM Primer HSV1_UP 5 'CGGCCGTGTGACACTATCG	0,33 µl
10µM Primer HSV2_UP_Cy3 Cy3_5'CGCTCTCGTAAATGCTTCCCT3'	1 µl
10µM Primer HSV2_DP_NH2 5'TCTACCCACAACAGACCCACG3'_ NH2	0,66 µl
10µM Primer HSV2_DP 5'TCTACCCACAACAGACCCACG3'	0,33 µl
10µM Primer VZV_DP_Cy3 Cy3_5'TCGCGTGCTGCGGC	1 µl
10µM Primer VZV_UP_NH2 5'CGGCATGGCCCGTCTAT3'_NH2	0,66 µl
10µM Primer VZV_UP 5 'CGGCATGGCCCGTCTAT	0,33 µl
Template CMV	1 µl
PCR-Grade Water	22,5 µl
total	50 µl

The process units were filled with 50 μl of reaction solution each and processed according to the following temperature-time regime. Other

1 Denaturieren	95 °C
Dauer	300 s
2 Denaturieren	95 °C
Dauer	10 s
3 Annealing/Extension	60 °C
Dauer	20 s

Repeat steps 2 to 3 35 times Other

4 Denaturierung	95 °C
Dauer	300 s
5 Hybridisierung	40 °C
Dauer	3600 s

The two process units were then treated differently; in the first case (process unit 1) the background fluorescence was reduced by displacing the analyte, this was ensured by pushing the flask upwards towards the detection surface, so that the gap filled with the reaction solution was reduced to the maximum.

In the second case (process unit 2) the analyte was replaced with a non-fluorescent solution, which was replaced with 2 x SSC buffers at a flow rate of 300 μl/min and a flush volume of 900 μl. This procedure is in accordance with the state of the art.

The two strategies for reducing the background fluorescence were then compared and the hybridization signals in both process units were detected using the fluorescence microscope camera setup described.

The exposure time was 5 s (see Figure 18 and Figure 19). Spot intensities were compared with CMV_S_21-3(5'-NH_{2 and}The location of the probes is shown in Figures 18 and 19.

The result of the experiment is summarized in Figure 20. The flushing of the reaction chamber in process unit 2 reduces the hybridization signal compared to the displacement in process unit 1. The method of analyte displacement by the disclosure method is therefore preferable to the exchange of solutions.

To obtain evidence of the quantity and integrity of the amplification product, an additional 5 μl of each reaction solution was analysed on a 2% agarose gel.

Example 4: Device for processing and detecting reaction cartridges of the invention

An apparatus for processing and detecting reaction cartridges according to the present embodiment is shown in Figure 28. The apparatus for performing microarray-based tests with reaction cartridges according to the invention is usually composed of several components which are combined in one apparatus, but may also be modularly assembled from several sub-apparatus. The apparatus may be controlled either via an integrated computer or an interface to an external computer. The structure of the apparatus is illustrated in Figure 28.

A typical procedure is as follows: The fluid interface of the reaction cartridge is manually brought into the filling position by the user, where the cannulas pierce the chamber body seal. The user then fills the reaction mixture into the reaction chamber using a standard laboratory pipette. Both steps can also be performed by a device that is designed accordingly. The fluid interface is then moved back to the output, which can also be done by a device that is designed accordingly.

The reaction cartridge is then inserted into the device. A data matrix reader located in the device detects the unique data matrix on the reaction cartridge and, on the basis of a user-submitted data set, loads the characteristic data for the cartridge and for the test to be performed into the control calculator. This then controls the individual process steps, which may include, for example, amplification and hybridization. The integrated pressure mechanism is then reduced according to the invention to detect the capillary gap in the reaction chamber.

The detection can be done by conventional fluorescent optical imaging or non-imaging systems, and the data obtained is then transmitted to a control computer, which evaluates it and presents or stores it on an internal or external interface.

The user may then remove the test cartridge from the device and dispose of it.

Example 5: Reaction cartridge made of electrically conductive plastic

A reaction cartridge is prepared as shown in Figure 29.

The bottom half-shell (1) of the reaction cartridge consists of electrically conductive plastic as the bottom of the reaction chamber (Conduct 2, RKT, Germany). On the bottom of the chamber floor a foil Pt100 temperature sensor is fixed by means of a suitable adhesive, e.g. Loctite 401 (Loctite, Germany). The bottom half-shell together with the seal (3) and the cover glass (4) forms the reaction chamber of the cartridge of the invention.

The cartridge also has a threaded hole (2) for inserting screws for electrical contact, an upper half-shell (5) of the reaction cartridge, e.g. made of acrylic, a hole (6) for attaching the upper half-shell and a detection window (7) in the upper half-shell.

A standard PCR reaction mixture is prepared: The test chemical is a mixture of a mixture of the following compounds: a mixture of a mixture of a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the following compounds: a mixture of two or more of the two compounds: a mixture of two or more of the two or more of the two compounds; a mixture of two or more of the two or more of the two compounds: a mixture of two or two or more of the two or more of the two compounds.

The reaction chamber is filled with the reaction mixture by means of an insulin syringe (Becton Dickinson, Germany) and a second cannula is inserted through the seal of the chamber body to vent during the filling process.

The chamber is then connected to a control unit by means of the two screws provided for this purpose (CLONDIAG chip technologies GmbH, Germany) and the temperature sensor at the bottom of the lower half-shell is connected to this control unit, which is able to control certain temperatures in the lower half-shell according to a given program.

The following PCR programme is then performed: 5 min 95°C, 30 x (30 s 95°C, 30 s 62°C, 50 s 72°C).

Figure 30 shows a picture of the reaction cartridge with a thermal imaging camera at 95°C.

After completion of the programme, the reaction product is removed from the reaction chamber by means of an insulin injection, and a cannula is inserted through the seal of the chamber body to allow ventilation during the reaction chamber emptying.

The reaction product is now analysed by agarose gel electrophoresis by placing 5 μl of the reaction solution with a suitable buffer (e.g. 5 μl 250 mM in 50% glycerin, bromphenol blue) in a bag of 2% agarose gel and electrophoresing, as shown in Figure 31.

As can be clearly seen, in all cases an amplification product of the correct size and in quantities comparable to the positive control was obtained.

The following The following table shows the data:

An overview of the device in accordance with the invention, including a reading device and the process unit.

The following table shows the figures:

Description of the process unit according to the invention.

Figure 3: The number of

Explosion drawing of the process unit according to the invention, comprising the detection surface, seal, DNA chip and chamber body.

Figure 4: The number of

The chamber body is shown with a plastic-sprayed heating medium in the elastic membrane.

The following table shows the figures:

Description of the state of the process unit according to the invention in the sampling apparatus A) during PCR, B) before detection, and C) during detection.

The following table shows the figures:

The function of the process unit according to the invention with membrane coating, compensating fold and underside hole is shown in A. (a) the process unit is shown in the normal position. (b) the process unit is shown in the compressed form in which the fluorescent solution is displaced between the DNA chip and the detection surface.

Figure 7: The number of

The temperature units are thermostatically set to a temperature, and the temperature in the reaction chamber can be changed by rotation of the plate and/or process unit.

Figure 8: The number of

Figure of an example of a milling and screwing process unit.

Figure 9: The number of

An example of a compression or compression apparatus for the process unit of the invention for detecting hybridization signals in a conventional fluorescence microscope is shown.

The following table shows the figures:

A representation of a process unit according to the invention with a plate as an electrical connection for a heater and temperature sensor.

The following table shows the figures:

Explosion drawing of the process unit shown in Figure 10.

The following table shows the figures:

A representation of the regression lines to determine the width of a fluoro-phore-filled slit.

The following table shows the data:

A representation of the linear progression of the fluorescence signal with increasing exposure time over the measured range.

The following table shows the figures:

Fluorescence recording of two chips superimposed on each other, filled with 200 nM Cy3 fluorophor in the spacing. The intensity of the background is 158 grays at an exposure time of 0.75 s. The gap width measured by the fluorescence microscope is 40.00 μm. Assuming that the measured grays behave linearly at the exposure time (see Figure 13), the gap width obtained from Equation 1 is 42.6 μm. The layer thickness values thus obtained are in good agreement.

The following table shows the figures:

Describe the test design for the wash-free detection of DNA arrays.

The following table shows the figures:

Fluorescence of an array with a pressed chip, with white margins showing the background radiation from the displaced sample solution.

The following table shows the figures:

The absolute intensities of signal and background decrease with decreasing gap width, and the difference between the two values remains constant over the measuring range.

The following table shows the data:

Detection of probe signals by suppression of the background fluorescence.

The following table shows the figures:

Detection of probe signals from a DNA array scrubbed from the background.

The following table shows the figures:

Summary of the measurement results for experimental comparison between displacement and exchange of the analyte.

The following table shows the figures:

Reference analysis of PCR in a process unit by gel electrophoresis.

The following table shows the figures:

A schematic representation of a detachable filling unit for filling reaction cartridges with reactive substances or buffers using the following reference symbols: 1Filling unit1.1Mechanical interfaceFilling unit-cartridge2Cartridge2.1Mechanical interfaceFilling unit-cartridge2.2Sealing2.3Reaction chamber2.4Preferred opening for the cannula in the cartridge3Filling channel3.1Fluidic and mechanical interface to sample-laying tools3.2Filling channels4Waste channel with waste container 4.1Ventilation hole4.2Waste channels

The following table shows the data:

Description of the process for filling a reaction cartridge with a modular filling unit.

The following table shows the figures:

A schematic representation of an integrated filling unit for filling reaction cartridges with reactive substances or buffers in the preferred position without penetration of the chamber body seal, using the following symbols: 1Fill-unit-cartridge1.1Mechanical interfaceCartridge-unit-cartridge2Reaction cartridge2.1Mechanical interfaceCartridge-unit-reaction 2.2Sealing2.3Reaction chamber2.4Preferred opening for the cannula in the cartridge housing-housing3Filling channel3.1Fluidic and mechanical interface to the tools to be sampled3.2Filling channels4Waste channel with waste container4.1Fluidic and mechanical interface to the sample-taking units4.2Waste channels5Preference positioning device, here spring

The following table shows the figures:

Description of the process for filling a reaction cartridge with an integrated filling unit.

The following table shows the data:

A schematic representation of an integrated filling unit with an integrated waste receptacle for filling reaction cartridges with reactive substances or buffers in the preferred position without penetration of the chamber body seal, using the following references in addition to the references in Figure 24: 4Waste channel with waste container4.1Ventilation hole

Figure 27: The number of

(a) Filling of the reaction chamber when discharging the excess liquid into a waste tank or channel

The following reference marks shall be used: The test chemical is a chemical that is used to test the reaction.

The following table shows the figures:

Apparatus for processing and detecting reaction cartridges of the invention as described in example 4. 1Reaction cartridge1.1Reaction chamber with micro-array1.2Fluid system interface1.3Sealing of the chamber body1.4Electrical connections for heating system, possibly including temperature sensors1.5Chip1.6Logging system for preferential positioning and guiding of the cannulae1.7Cannulae2Pressure mechanism3Identification system, e.g. barcode or data matrix3.1Identification optics, e.g. barcode or data matrix reader4Detection optics5Fluid connections

The following table shows the data:

Reaction cartridge as described in example 5.

The following table shows the results:

Record the reaction cartridge as described in Example 5 with a thermal imaging camera at 95°C.

Figure 31:

Analysis of the reaction product by Agarosegel electrophoresis as described in Example 5. 1.5: Positive control from the thermocycler2-4: reaction products from cartridges6: 100bp standard

The following is the list of the Member States:

< 110 > CLONDIAG GmbH < 120 > Device and method for detecting molecular interactions < 130 > C8892/MH < 150 > DE 10 2004 022 263.0 The Commission has decided to initiate the procedure provided for in Article 3 (1) of Regulation (EC) No 765/2004. The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following information shall be provided in the form of a summary of the results of the evaluation: The maximum residue levels for the active substance shall be as follows: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following shall be reported for the product concerned: The following are the main categories of products: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following information shall be provided for the purpose of the assessment: The following are the main categories of products: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following information is provided for the purpose of the calculation of the maximum amount of the premium: The Commission shall adopt the implementing acts referred to in Article 17 (2). The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following substances are to be classified as: The following table shows the total number of animals and animals that were subject to the measures laid down in Annex I to Regulation (EC) No 999/2001 and in Annex II to that Regulation: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following information is provided for the purpose of the calculation of the amount of the premium: The following are the main characteristics of the product: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following information is provided for the purpose of the assessment: The following are the main characteristics of the product: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following substances are to be classified as HSV1_UP_NH2 The following are the main characteristics of the product: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following are the main characteristics of the HSV1_UP: The following are the main characteristics of the product: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following information is provided for the purpose of the assessment: The following are the main characteristics of the product: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following substances are to be used: The following shall be indicated in the table: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following information is provided for the purpose of the assessment: The following shall be indicated in the table: The number of employees The following are the types of products: The following are the main characteristics of the product: The following information is provided for the purpose of this Decision: The following are the main characteristics of the product: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following shall be reported in the table: The following are the main characteristics of the product: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following information is provided for the purpose of the calculation of the amount of the premium: The following are the main characteristics of the product: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The test chemical is used to determine the concentration of the test chemical in the test medium. The following shall be added to the list of products: The following are the main categories of products: The following are the types of products: The following are the main characteristics of the product: The following are the main components of the test: The following are the main characteristics of the product: The following are the main factors: The following are the types of products: The following are the main characteristics of the product: The following shall be indicated in the table: The following shall be added:

Claims (11)

1. Device for qualitative and/or quantitative detection of molecular interactions between probe molecules and target molecules in a sample solution, comprising:

   a microarray having probe molecules immobilized on array elements, wherein the microarray (1.5) is arranged on a first surface of the device; and

   a reaction chamber (1.1) which is formed between the first surface having the microarray (1.5) arranged thereon and a second surface,

   wherein laterally limiting compensation zones are provided for the reaction chamber (1.1), which keep the volume within the reaction chamber (1.1) essentially constant upon reduction of the distance between the microarray (1.5) and the second surface such that the sample solution between the microarray (1.5) and the second surface is essentially removed,

   characterized in that the first surface is elastically deformable at least in the zone below the microarray (1.5) such that the microarray (1.5) may be guided relative to the second surface so that the distance between the microarray and the second surface is variable such that the sample solution between the microarray (1.5) and the second surface is essentially removed.
2. Device according to claim 1, further comprising a means (2) for varying the distance between the microarray (1.5) and the second surface in such a way that the sample solution between the microarray and the second surface may be essentially removed.
3. Device according to claim 1 or 2, wherein the distance between the the microarray (1.5) and the second surface is variable in a range of about 0 to about 1 mm.
4. Device according to any of the preceding claims, wherein the device additionally comprises a temperature control unit and/or a temperature regulating unit for controlling and/or regulating the temperature within the reaction chamber (1.1).
5. Device according to any of the preceding claims, wherein the device further comprises a detection system (4), for example a fluorescence optical system like a fluorescence microscope without autofocus, wherein the detection system (4) optionally is connected to a spacer which, when resting upon the second surface, adjusts a distance between the detection system (4) and the second surface.
6. Device according to any of the preceding claims, wherein the second surface is made of an optically transparent material, for example optically transparent plastics.
7. Device according to any of the preceding claims, wherein the first surface is made of elastic plastics.
8. Device according to any of claims 2 to 7, wherein the microarray (1.5) is guidable relative to the second surface by the means (2) for varying the distance and wherein for example the microarray (1.5) is guidable relative to the second surface by the means acting upon the first surface by pressure and/or tension and/or for example the first surface may be set into vibration by the means.
9. Device according to any of the preceding claims, wherein the second surface may be guided relative to the first surface so that the distance between the microarray (1.5) and the second surface is variable, wherein for example the second surface may be guided relative to the first surface by the spacer acting upon the second surface by pressure and/or tension so that the distance between the microarray (1.5) and the second surface is variable.
10. Device according to any of the preceding claims, wherein the probe and/or target molecules are biopolymers selected from the group consisting of nucleic acids, peptides, proteins, antigens, antibodies, carbohydrates and/or analogs thereof, and/or copolymers of the above-mentioned biopolymers.
11. Use of a device according to any of claims 1 to 10 for carrying out microarray-based tests.