Abstract:
Disclosed are devices for holding a substance library carrier, and which are useful in conducting reactions in which binding between complementary molecules is measured qualitatively or quantitatively. Also disclosed are methods for conducting such reactions in the device.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application is a continuation of PCT/EP02/11313, filed Oct. 9, 2002, which claims priority on the basis of DE 101 49 684.2, filed Oct. 9, 2001. 
     
    
     BACKGROUND OF THE INVENTION  
       [0002]     The invention relates to a modularly built device for the holding of substance library carriers and its use for the qualitative and quantitative detection of certain molecular target molecules.  
         [0003]     Biomedical tests are frequently based on the detection of an interaction between a molecule that is present in a known amount and at a known position (the molecular probe) and an unknown molecule that is to be detected, or unknown molecules that are to be detected (the molecular target molecules). In modern tests, the probes are deposited in form of a substance library onto carriers, the so-called microarrays or chips, so that a sample may be analyzed in parallel with several probes simultaneously (D. J. Lockhart, E. A. Winzeler, Genomics, gene expression and DNA arrays; Nature 2000, 405, 827-836). For the preparation of the microarrays, the probes are typically immobilized in a specified manner on a suitable matrix, as for example the one described in WO 00/12575 (see e.g. U.S. Pat. No. 5,412,087, WO 98/36827) or produced synthetically (see e.g. U.S. Pat. No. 5,143,854).  
         [0004]     The detection of an interaction between the probe and the target molecule occurs as follows:  
         [0005]     The probe or probes are fixed in specified manner on a certain matrix in form of a microarray. The targets are then brought into contact with the probes in a solution and incubated under defined conditions. As a result of the incubation, a specific interaction between the probe and the target takes place. The binding that occurs as a result is distinctly more stable than the binding of target molecules to probes that are not specific to the target molecule. In order to remove target molecules that have not been specifically bound, the system is washed with appropriate solutions or heated.  
         [0006]     Detection of the specific interaction between a target and its probe may occur by a variety of methods normally depending on the nature of the marker which was introduced 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 may be read out fluorescence-optically with high local resolution and, in comparison to other common detection methods (particularly mass-sensitive methods), with very little effort (A. Marshall, J. Hodgson, DNA chips: An array of possibilities, Nature Biotechnology 1998, 16, 27-31; G. Ramsay, DNA Chips: State of the art, Nature Biotechnology 1998, 16, 40-44).  
         [0007]     Depending on the substance library immobilized on the microarray and the chemical nature of the target molecules, interactions between nucleic acids and nucleic acids, between proteins and proteins as well as between nucleic acids and proteins may be analysed using this test principle (for a review see F. Lottspeich, H. Zorbas, 1998,  Bioanalytik,  Spektrum Akademischer Verlag, Heidelberg Berlin).  
         [0008]     Antibody libraries, receptor libraries, peptide libraries, and nucleic acid libraries are possible as substance libraries that may be immobilized on microarrays or chips. The nucleic acid libraries play by far the most important role.  
         [0009]     What is involved here are microarrays, on which desoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules are immobilized. For binding of a target molecule (DNA molecule or RNA molecule) labeled with a fluorescent group to a nucleic acid probe of the microarray, it is necessary that both the target molecule and the probe molecule be present in the form of a single-stranded nucleic acid.  
         [0010]     Only between such molecules can efficient and specific hybridization take place. Single-stranded nucleic acid target molecules and nucleic acid probe molecules are as a rule obtained by heat denaturation and parameters which are to be optimally selected (temperature, ionic strength, concentration of helix-destabilizing molecules) which ensure that only probes with nearly perfect complementary (corresponding to one another) sequences remain paired with the target sequence (A. A. Leitch, T. Schwarzacher, D. Jackson, I. J. Leitch, 1994,  In vitro Hybridisierung,  Spektrum Akademischer Verlag, Heidelberg Berlin Oxford).  
         [0011]     A typical example for the use of microarrays in biological test methods is the detection of microorganisms in samples in biomedical diagnostics. Here, advantage is taken of the fact that the genes for ribosomal RNA (rRNA) are ubiquitously distributed and possess sequence segments that are characteristic for the respective species. These species-characteristic sequences are deposited onto a microarray in form of single-stranded DNA oligonucleotides. The target DNA molecules to be analysed are first isolated from the sample that is to be analysed and provided with fluorescent markers. The labeled target DNA molecules are then incubated with the probes deposited on the microarray in a solution, non-specific interactions are removed by appropriate washing steps and specific interactions are detected by fluorescence-optical analysis. In this way it is possible to detect e.g. several microorganisms in a sample with a single test simultaneously. In this test method, the number of detectable microorganisms in theory only depends on the number of specific probes that have been deposited on the microarray.  
         [0012]     For carrying out these tests in practice, the microarrays or chips are fixed in closed chambers which possess inlets and outlets for exchange of the fluids that are necessary for the washing steps and hybridization steps. Such systems are described e.g. in U.S. Pat. No. 6,287,850 and PCT/EP00/06103.  
         [0013]     The chamber described in U.S. Pat. No. 6,287,850 does not possess aggregates by which the chamber may be heated or cooled. However, this would be desirable for a specific control of the hybridization. Moreover, the assembly of the reaction chamber, which contains the chip, is complicated and involves several gluing steps for the purpose of sealing the chamber, giving rise to the risk of contamination.  
         [0014]     In PCT/EP00/06103, chambers are described that also contain microarrays. These chambers may be heated, but cannot be cooled. Here too, the chamber is assembled in several steps, and gluing steps for sealing the chamber are necessary. The chambers mentioned in the above documents thus allow only limited manipulation of parameters (heating, cooling etc.), the assembly is susceptible to contamination and the peripheral connections that are desirable for an automation of the detection reaction do not exist or can only be achieved with great effort and expense.  
         [0015]     In many tests in biomedical diagnostics the problem arises that before the actual test method takes place, the target molecules first have to be present in sufficient quantities and, for this reason, often have to be amplified from the sample. The amplification of DNA molecules takes place through polymerase chain reaction (PCR). For the amplification of RNA, the RNA molecules have to be converted into the corresponding complementary DNA (cDNA) by means of reverse transcription. This cDNA may then also be amplified by PCR. PCR is a standard laboratory method (J. Sambrook, E. F. Fritsch, T. Maniatis, 1989,  Molecular Cloning: A laboratory manual,  2 nd  edition, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press).  
         [0016]     The amplification of DNA by PCR is relatively fast, allows a high sample throughput in low batch volumes by means of miniaturized methods, and is work-efficient by means of automation. Characterization of nucleic acids solely by means of amplification is however not possible. Rather, it is necessary to use analysis methods such as nucleic acid sequence determinations or electrophoretic separation and isolation methods for the characterization of the PCR products after amplification.  
         [0017]     From documents U.S. Pat. No. 5,716,842, DE 195 19 015A1, and WO 94/05414, as well as U.S. Pat. No. 5,498,392, several miniaturizable or miniaturized methods and devices (thermocyclers) for carrying out of the PCR are known. The integration of a microarray or the integration of a DNA chip is not dealt with in these documents.  
         [0018]     In documents U.S. Pat. No. 5,716,842, WO 91/16966, WO 92/13967, miniaturizable or miniaturized thermocyclers are described that function according to the principle that the fluid sample is continuously pumped over three temperature zones. These continuously working thermocyclers also do not provide for integration of a DNA chip.  
         [0019]     The disadvantage of all of the above-mentioned solutions is that online detection only provides information on whether and possibly how much nucleic acid has been amplified. A better characterization of the amplification products is not possible.  
         [0020]     The U.S. patent specification No. 5,856,174 discloses a system, by which it is possible to pump back and forth between three miniaturized chambers. In one chamber of this system the PCR takes place, in the next chamber a processing/purification reaction is carried out, and in the third chamber the reaction products are detected, e.g. on a DNA chip. The miniaturized PCR vessel is a standard vessel of the kind sufficiently described in the literature (S. Poser, T. Schulz, U. Dillner, V. Baier, J. M. Kohler, D. Schimkat, G. Mayer, A. Siebert, Chip element for fast thermocycling, Sensors and Actuators A, 1997, 62672-675). The disadvantage of this arrangement is that a system of pressure-driven fluidics, which is complicated, liable to malfunction and complex to regulate, has to be set up in order to transport the fluid sample from the PCR chamber to the detection chamber. In addition, separating amplification and detection leads to an increase in the overall analysis time.  
         [0021]     In PCT/EP00/06103 a unit is described, with which PCR and nucleic acid hybridization may be carried out on a DNA chip as a single chamber reaction in a sample chamber with integrated heating system. However, this unit has the disadvantage that it cannot be cooled, that a complicated quadropol system is necessary for mixing the samples, and that the assembly of this unit requires the gluing of parts. Moreover, this unit, which is a disposable product, is uneconomical because of its high manufacturing costs.  
         [0022]     In R. C. Anderson, X. Su, G. J. Bogdan, J. Fenton (A miniature integrated device for automated multistep genetic assays, Nucleic Acids Research, 2000, Vol. 28, No. 12), a finger-thick cartridge is described in which DNA purification steps, PCR, an enzymatic processing and hybridization may be carried out on a DNA chip, the different reactions occurring in different reaction chambers. By means of a complex compressed air-driven unit, the sample solution is pumped from reaction chamber to reaction chamber in order to carry out the different steps of the process. The disadvantages of this system are that a great amount of effort goes into the construction of a pressure-driven fluidics, there is a tendency to malfunction and there are no integrated heating and cooling aggregates. Moreover, only the PCR and the enzymatic processing may be automated.  
         [0023]     Considering the mentioned state of the art, it becomes clear that there is a great need for devices that enable the fully automated carrying out of microarray-based detection tests. In particular, there is a need for devices for the carrying out of microarray-based tests in which the control of parameters such as temperature regulation, such as cooling and flow control is fully automated. Furthermore, there is a need for devices for the carrying out of microarray-based tests which allow the in-situ synthesis, e.g. by PCR, of the components required for the test such as the target molecules and the use of the amplification products directly in the test system without manual processing. There is generally a need for devices for the carrying out of microarray-based tests that are characterized by simple construction, easy manageability, avoidance of sources of contamination, reproducibility of the tests, and low manufacturing costs.  
       SUMMARY OF THE INVENTION  
       [0024]     Hence, it is an object of the present invention to provide a device that allows for the fully automated and parameter-regulated carrying out of microarray-based tests. It is a further object of the present invention to provide a device that is characterized by simple construction, easy manageability and therefore cost-effective manufacturing. Further, it is an object of the present invention to provide a device that allows for the simultaneous performance of PCR and microarray-based tests in a single chamber system by avoidance of sources of contamination. It is a further object of the present invention to provide a manual filling station for the loading of the sample chamber that contains the microarray.  
         [0025]     These and other objects of the present invention are solved by providing the subject matter described in the patent claims. Preferred embodiments are defined in the sub-claims. In this connection, the objects are solved according to the invention as follows: by pressing together a layer composite  400 ,  300 ,  200  by means of two holding elements ( 101 ,  102 ) that are fixable with one another a device  1  is created that comprises an optically translucent chamber  500  which, in turn has a detection surface having a substance library  201  and in which microarray-based tests as well as reactions such as PCR may be carried out.  
         [0026]     This kind of construction and the nature of the components of the layer composite, which are a base element  400 , an intermediate element  300 , and a lid element  200 , are responsible for advantageous effect of the created chamber  500 , hereinafter also referred to as sample or reaction chamber. The entire device  1  is hereinafter also referred to as cartridge  1 .  
         [0027]     The construction of the cartridge  1  and the reaction chamber  500  according to the invention allows the production of cartridges  1  and reaction chambers  500  in which the substance library carrier may be replaced without much effort. Another advantage of this construction principle is that the created reaction chamber  500  is hermetically sealed without having to be glued, which saves several working steps in comparison with the usual assembly of the reaction chamber  500  according to the state of the art. Hence, the device according to the invention as such is better; in addition, potential contamination of the sample space due to the gluing steps is prevented. These and other advantageous properties of the created reaction chamber  500  are based on the nature of the base, intermediate and lid elements forming the reaction chamber. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0028]      FIG. 1  is a representation of the assembled cartridge  1  consisting of two holding devices  101  and  102 , media connection side  120  with data matrix  600 , antireflection structure  109 , and snap-fit  106  at the media connection side. Furthermore, alignment pin holes  108  are illustrated.  
         [0029]      FIG. 2  is a representation of the individual components of the cartridge  1  in an exploded drawing. In the upper part of the picture, a holding device  102 , in the middle the core unit  500  consisting of base element  400 , intermediate element  300 , and lid element  200 , in the lower part of the picture, the second holding device  101  is seen.  
         [0030]      FIG. 3  is a representation of the cartridge  1  attached to the connector  1000 .  
         [0031]      FIG. 4  is a representation of the connector  1000  with labeling of the cartridge terminal strip  
         [0032]      FIG. 5  is an overview of the opening of the reaction space  301  by cannulae  1201  by means of a slide.  
         [0033]      FIG. 6  is a detailed representation of the opening of the reaction space  301  by cannulae  1201  that puncture the sealing septum  300  and form an inlet  1202  and an outlet  1203  in the reaction space  301 . The cannulae are positioned toward the sample chamber by the locating lug  1103  on the slide  1100  and the needle guide  113  in the cartridge  1 .  
         [0034]      FIG. 7  is a representation of the filling station  2000  with installed injection syringe  2401 , put on lid  2200 , and installed cartridge  1 . The filling station  2000  stands on the filling station base  2300  and is held by magnets.  
         [0035]      FIG. 8  is representation of the body  2100  of the filling station with installed injection syringe  2401  with cannula  2402  and ventilation cannula  2403 .  
         [0036]      FIG. 9  is an image of an electrophoresis gel. The mass reference-DNA (one fragment every 100 base pairs) was loaded on lane A, the PCR products that were produced in a conventional thermocycler on lanes B and D, and the PCR products that were produced in the cartridge on lanes C and E. Genomic DNA of Corynebacterium glutamicum was amplified. The amplification product has a length of approx. 500 base pairs. It becomes apparent from  FIG. 9 , that the cartridge  1  may be used for the amplification of DNA by means of PCR and that it is as equally efficient as a conventional thermocycler.  
         [0037]      FIG. 10  is an analysis of the PCR reaction carried out in example 2. Intensive hybridization signals are only detected at the spots that were occupied by the P 1 -sequence perfectly complementary to the labeled strand of the PCR. 
     
    
     DETAILED DESCRIPTION  
       [0038]     According to the invention, the designation lid element  200  refers to a carrier element  202  that has a substance library  201  on a detection surface and is optically translucent and non-fluorescent, at least within the area of the detection surface. Detection surface means the area of the carrier element  202  on which the substance library  201  is immobilized. In such a preferred embodiment of the invention, the substance library  201  is directly deposited on the lid element  200 .  
         [0039]     In other preferred embodiments, the substance library  201  is deposited on an optically translucent, non-fluorescent chip which, in turn, is fixedly connected to the carrier element  202  that is, at least in the detection area defined by the chip, optically translucent and non-fluorescent, the dimensions of the chip being smaller than the dimensions of the carrier element  202 . In this case, the chip carrying the substance library  201  and the carrier element  202  together forms the lid element  200 .  
         [0040]     The carrier element  202  consists of optically translucent, non-fluorescent materials. The materials are preferably glass, Borofloat 33 (Schott), quartz glass, monocrystalline CaF 2  (Schott), monocrystalline silicon, phenyl methyl methacrylate, and/or polycarbonate.  
         [0041]     If the substance library is not directly deposited on the carrier element  202  but on a chip, the chip also consists of optically translucent, non-fluorescent materials. Preferred materials are glass, Borofloat 33 (available from Schott), quartz glass, monocrystalline CaF 2  (available from Schott), monocrystalline silicon, phenyl methyl methacrylate, and/or polycarbonate.  
         [0042]     The substance library may also be located on the base element  400 . It is obvious to a person skilled in the art that in this case the lid element  200  consists of a carrier element  202  having an area whose position, size and shape is defined by the position, size and shape of the substance library  201  on the base element  400 . If the analysis of the target-probe interaction is carried out using optical detection methods, the lid element  200  is in this case optically translucent and non-fluorescent at least in this area.  
         [0043]     The substance libraries  201  may be protein substance libraries, peptide substance libraries, and nucleic acid substance libraries. The protein substance libraries may in particular be antibody libraries, receptor molecule libraries, and membrane protein libraries. The peptide libraries may in particular be receptor ligand libraries, they may be pharmacologically-active peptide libraries and peptide hormone libraries. Nucleic acid substance libraries may in particular be DNA molecule libraries and RNA molecule libraries. In case of DNA molecule libraries, in particular ribosomal DNA sequences of microorganisms may be deposited on the lid element  200 . In addition, they may be nucleic acid substance libraries for SNP analysis. Furthermore, they may be protein substance libraries or nucleic acid substance libraries which allow a so-called “expression profiling”. They may also be combinatorial substance libraries.  
         [0044]     In this case, the substance libraries  201  are deposited on the carrier element  202  in such a way that they are in contact with the sample space of the resulting chamber. The lid element  200  of the created reaction chamber is thus characterized according to the invention in that it has a detection surface with a substance library on its underneath side and is optically translucent at least in the detection area.  
         [0045]     An advantageous embodiment of the invention is that the resulting reaction chamber  500  is sealed and aqueous samples may be heated to temperatures of up to 100° C. over the course of hours without leakage of liquid or evaporation of the samples occurring.  
         [0046]     These advantageous effects are achieved through the sealing material properties of the intermediate element  300 . In addition, the intermediate element is elastic and repeatedly puncturable with cannulae, wherein the cannulae are extractable and after the extraction of the cannulae a leakage of liquid from the intermediate element does not occur. The intermediate element preferably consists of polydimethylsiloxane (available under the names Sylgard 184 or 182), natural rubber, butadiene rubber, chloroprene rubber, nitrile-butadiene rubber, butyl rubber, isoprene-styrene rubber, polynorbornene rubber, ethylene-propylene rubber, fluor rubber (available under the names Biton, Tecnolflon, Fluorel, Daiel), perfluor rubber (available under the name Klarez), methyl-phenyl-silicon rubber, methyl-vinyl-silicon rubber, methyl-fluor-silicon rubber, fluor-silicon rubber, polysulfide rubber, urethane rubber, polyester or polyether prepolymers on the basis of 4,4′-methylene di(phenylisothiocyanate) or toluene diisocyanate (available under the names Adipren, Elastothane, Genthane, Urepan, Vibrathan).  
         [0047]     Within the scope of the invention, the intermediate element  300  is also referred to as septum or sealing septum. The intermediate element  300  is characterized in that it has an enclosed recess  301 . Due to this recess  301  that defines the volume of the reaction space (provided by  301 ) of the reaction chamber  500 , both the geometry and the volume of the reaction space may be varied. The reaction space  301  is hereinafter also referred to as sample space or chamber space.  
         [0048]     Preferably, a reaction space  301  with volumes between 5 and 100 μL, preferably between 10 and 50 μL or between 15 and 40 μL, between 15 and 35 μL or between 15 and 25 μL is formed by the sealing septum  300 .  
         [0049]     Depending on the geometry of the recess  301  in the sealing septum  300 , substance libraries  201  may be deposited on the lid element  200  in various geometric arrangements, the geometry of the arrangement of the substance library  201  only depending on the geometry of the recess  301  of the sealing septum  300 .  
         [0050]     The advantageous design of the sealing septum  300  allows an air bubble-free filling of the reaction space  301 . The geometry of the reaction space that is defined by the sealing septum  300  is preferably in the shape of a D; another advantageous shape is that of a new moon, a sickle or a tangerine segment.  
         [0051]     An advantageous embodiment of the invention is that the geometric shape of the reaction space is defined by the sealing septum  300  and, as a result, may be changed without altering the entire device  1  and may be customized to individual problems.  
         [0052]     A further advantageous embodiment of the invention is that the enclosure of the reaction chamber  500  achieved by the sealing septum  300  on the one hand increases the storage life of the chip and, on the other hand, reduces the danger of contamination during the analysis. A further advantageous embodiment of the invention is that depending upon the design of the sealing septum  300 , substance libraries  201  with different outer geometric dimensions may be deposited on the lid element  200  and used.  
         [0053]     A further advantageous embodiment of the invention is that the sealing septum  300  has a recess of any desired geometry  301 . The sealing septum  300  preferably consists of a sealing, elastic material so that the sample chamber may be loaded by repeated puncturing of the sealing septum from the side, the sealing septum being sealed in such a way that there is no liquid leakage after the extraction of the cannulae with which the sample chambers are loaded. The sealing septum  300  in this case preferably consists of materials such as polydimethylsiloxane (available under the names Sylgard 184 or 182), of natural rubber, butadiene rubber, chloroprene rubber, nitrile-butadiene rubber, butyl rubber, isoprene-styrene rubber, polynorbornene rubber, ethylene-propylene rubber, fluor rubber (available under the names Biton, Tecnolflon, Fluorel, Daiel), perfluor rubber (available under the name Klarez), methyl-phenyl-silicon rubber, methyl-vinyl-silicon rubber, methyl-fluor-silicon rubber, fluor-silicon rubber, polysulfide rubber, urethane rubber, polyester or polyether prepolymers on the basis of 4,4′-methylene di(phenylisothiocyanate) or toluene diisocyanate (available under the names Adipren, Elastothane, Genthane, Urepan, Vibrathan).  
         [0054]     The base element of the sample chamber  400  is preferably designed in such a way that it possesses an integrated heater/sensor substrate. The heating/sensor substrate is generally a temperature-regulating heating system. Such heating/sensor substrates are described in PCT/EP00/06103.  
         [0055]     The base element  400  in this case is at least optically translucent and non-fluorescent. in the sample space area defined by the recess of the sealing septum  300  or in the area defined by the detection surface of the lid element. Preferably, it consists of materials such as Borofloat 33 (available from Schott), quartz glass, monocrystalline CaF 2  (available from Schott), and/or monocrystalline silicon. Through the heater/sensor substrate integrated in the base element  400 , the temperature may be adjusted to ±1° C. within a range from 0° C. to 100° C., preferably from 0° C. to 95° C. and/or from 50° C. to 95° C.  
         [0056]     In one embodiment of the invention, the substance library  201  is situated on the base element  400 . The base element  400  then also has to be optically translucent and non-fluorescent within the area whose size, shape, and position are defined by the detection surface of the substance library  201 . If the substance library is situated on a chip and if this chip is affixed to the base element  400 , the chip has to be optically transparent and translucent.  
         [0057]     In a special embodiment of the invention, the substance library  201  is situated directly on the base element  400 . In this case, the base element  400  may have an electrode structure which does not allow the detection of interactions of the target molecules with the probe molecules of the substance library by optical detection methods, but rather by electronically measurable variables such as impedance, conductivity, potential, capacity etc. (U.S. Pat. Nos. 6,013,166, 628,590, 6,245,508, 5,965,452, Tu et al. (2000) Electrophoresis, 21). In this case, neither the base element  400  nor the lid element  200  has to be optically transparent and non-fluorescent in the areas defined by the substance library  201 , although they may be.  
         [0058]     The layered reaction chamber  500  consisting of the base element  400 , the intermediate element  300 , and the lid element  200 , is also referred to as core unit  500 . For the fixation and alignment of the core unit, the base element  400 , the intermediate element  300 , and the lid element  200  are placed on top of one another in recesses  117  that are mounted in the holding elements  101  and  102  that may be engaged with one another.  
         [0059]     By pressing the two holding elements together, the core unit consisting of the base, intermediate and lid element is sealingly compressed. For that purpose, the recesses of the holding elements  101  and  102  that are fixable with one another in a preferred embodiment contain barbs (Widerlager/Widerhaken)  118 , pushing the sealing septum  300  to the side. In order not to hinder this process, the sealing septum  300  is constructed in such a way that it possesses expansion joints  302 . In order to ensure a secure positioning of the sealing septum  300  in the holding elements  101  and  102  despite the expansion joints  302 , the sealing septum has alignment ears  304  at its corners.  
         [0060]     In another embodiment, lid element  200 , intermediate element  300 , and base element  400  may be conglutinated with one another.  
         [0061]     In order to inject the thus created hermetically sealed reaction space  301  free of dead volume, at least two cannulae fixed at a defined distance are, at a precise position, inserted from the side into the sealing septum  300 . The needles then penetrate into the reaction space  301  at the corners of the flat side of the D-shaped recess or at the corner of the new moon-shaped recess. In this way, the reaction space  301 , and thus the reaction chamber  500 , have an inlet and an outlet. The sample is now injected into the reaction chamber  500  with a syringe. In the same manner, the chamber may be emptied or the sample fluid may be exchanged with e.g. a rinsing buffer.  
         [0062]     The shape of the reaction space  301  is designed fluidically in such a way that bubble-free filling with sample fluid is possible with high reproducibility. After filling, the needles are extracted so that the insertion holes in the elastic sealing septum  300  close up and the sample remains pressure-tight and hermetically sealed in the reaction chamber  500 . The entire sample fluid is situated in the reaction space  301  and not in feeding or exit channels—which is the reason why the cartridge  1  works free of dead volume.  
         [0063]     Through the hermetic sealing of the reaction chamber  500 , the substance library  201 , which is sensitive to mechanical stress, is protected against damage. Another advantage according to the invention is that in case of a detection of substances that are highly sensitive to contamination, the reaction space  301  may be filled with protective substances such as protective liquids and protective gases until analysis takes place. Such protective gases are preferably argon, nitrogen, and inert gases. If the surface-bound substance library consists of e.g. RNase sensitive RNA molecules, the sample chamber  500  may be protected with RNase inhibitors (e.g. DEPC-water) until analysis takes place. During the filling with the sample material, the protective substance is removed through simple displacement.  
         [0064]     Furthermore, standard substances may be provided in the reaction space  301  during the manufacturing process. For example, a mixture of nucleotides, primers, polymerase and PCR buffer would be suitable for PCR.  
         [0065]     An advantageous embodiment of the invention allows for the cooling of the core unit  500 . For that purpose, the holding elements  101  and  102  may contain cooling channels  105  that may be operated with different cooling media. The cooling agents are preferably fluorohydrocarbons, R 134 , ammonia, volatile hydrocarbons such as propane, cooled air, cooled gases such as liquid or gaseous CO 2 . This advantageous embodiment allows in particular a precise performance of the PCR.  
         [0066]     By using e.g. CO 2  or R 134 , the sample in the cartridge may be thermostat regulated to temperatures distinctly below room temperature, a mode of operation that is particularly desirable for the hybridization reaction. In an advantageous embodiment of the invention, cooling media are blown onto the core unit  500  via cooling agent inlets  112  and  117  and the cooling agent outlet  116  that are appropriately integrated into the holding elements  101  and  102 . By using e.g. CO 2  or another cooling agent R 134 , the core unit may be cooled to temperatures below room temperature. By means of the heater/sensor substrate  400 , a temperature below room temperature may be regulated with a precision of ±1° C. The available operating range of the cartridge is then −30° C. to 100° C., preferably 0° C. to 95° C. and/or 50° C. to 95° C.  
         [0067]     One advantageous embodiment of the invention allows for the two holding elements  101  and  102  that are fixable with one another to represent two half shells engaging with one another. The holding elements fixed with one another or the half shells fixed with one another together with the core unit  500  forms a device that is also referred to as cartridge  1 .  
         [0068]     According to the invention, the cartridge may be assembled from one side by simply positioning the different components that are the upper holding element  101 , the lid element  200 , the intermediate element  300 , the base element  400 , and the bottom holding element  102 , on top of each other.  
         [0069]     The cartridge is advantageously designed in such a way that all media connections (for cooling media  105  and heating system contacts  403 ) are located on one side  120 .  
         [0070]     The cartridge is advantageously designed in such a way that it also has a recess  103  on the side where the media connections  120  are located, via which recess  103  the reaction chamber  500  may be loaded by piercing with at least two cannulae from the side into the sealing septum  300 .  
         [0071]     In an advantageous embodiment of the invention, the recess  103  is designed in such a way that a locating lug  1103  may be inserted into the recess in such a way that it fits snugly. In order to inject the sample into the reaction chamber  500 , in an advantageous embodiment of the invention, at least two cannulae that are fixed with a defined spacing may be inserted into the sealing septum  300  from the side and positioned exactly by means of a locating lug  1103  which may be inserted into the recess  103  of the cartridge  1  and two locating holes in the cartridge (needle guide  113 ). The sample is injected into the reaction space  301  of the sample chamber  500 , the sample chamber  500  may likewise be emptied via the two cannulae or the fluid sample may be exchanged e.g. with a rinsing buffer.  
         [0072]     In an advantageous embodiment of the invention, the cartridge  1  is designed in such a way that it has snap closures  106  on the side of the recess  103  and the media connections  120 . Because of this, it is possible to attach or connect the cartridge  1  to any connector of a defined construction type (e.g.  1000 ) and to immediately have all media connections available. The connector  1000  corresponding to this advantageous embodiment of the invention has a slide  1100  that carries the insertion cannulae  1201  together with locating lug  1103 . The slide  1100  is pressed into the cartridge  1  by a servomotor in order to open the sample chamber  500  for filling or for rinsing. The sample chamber  500  is closed again by pulling out the slide  1100 .  
         [0073]     Computer-controlled external devices such as pumps and valves for the filling of the cartridge  1  may be connected via the connector  1000 . The computer-controlled thermo-regulator with the cartridge  1  is also connected via the connector  1000  to the contacts of the heating element  403 . In addition, the computer-controlled cooling agent supply may be connected via the connector  1000  to the medium connection  105  of the cartridge  1  provided for this purpose.  
         [0074]     The connector  1000  is preferably constructed flat and small in order to be able to install it in different devices as universally as possible and has suitable mounting holes  1002  and alignment pin holes  1001  for making screw connections and fixations.  
         [0075]     In addition, the cooling channel  1300  is made of insulation material in order to protect the cooling media from heating up. If required, the cooling agent is pressed into the cooling channel  105  through a cooling agent tube  1204  which is connected to the insulated cooling channel  1300  via a cooling agent connection  1205 .  
         [0076]     The connector  1000  possesses an electric cable  1206  and an electric connection  1207  for supplying the cartridge  1  with electricity.  
         [0077]     An advantageous embodiment of the invention allows for the fully-automated operation of the cartridge  1  via the connector  1000 .  
         [0078]     The slide  1100  is flexibly supported by a sliding linear bearing (Gleitlinearlager)  1003  so that the locating lug  1103  may be inserted into the recess  103  of the cartridge  1  provided for this purpose. The cannulae are connected with filling tubes  1202  through which different solutions may be pumped into the sample chamber  500 . In order to be able to drive the slide electromechanically, it has a slide rod  1210  that is provided with a thread  1211  to attach the drive. The slide rod  1210  is provided with a mechanical damping  1212  to compensate for jerky motions of the electromechanical drive. Thus, economically priced drives may be used.  
         [0079]     Moreover, several of these connectors  1000  may be set up in parallel so that the simultaneous analysis of several samples in different cartridges is possible.  
         [0080]     In an advantageous embodiment of the invention, the cartridge is designed in such a way that it possesses alignment pinholes  108  by means of which it can be positioned in a DNA reader so that adjustment of the image field or the focal point becomes unnecessary.  
         [0081]     In an advantageous embodiment of the invention, the holding elements  101  and  102  that are fixable with one another are two interlocking half shells that are held together by simple pressing against each other by means of press fit  115 .  
         [0082]     In other advantageous embodiments of the invention, the two holding elements  101  and  102  of the cartridge are screwed together.  
         [0083]     In another advantageous embodiment of the invention, the two holding elements  101  and  102  that are fixable with one another are designed in such a way that they may be conveniently manufactured from plastic by injection molding and are thus inexpensive. The materials used in the manufacture of the holding elements are preferably polycarbonate plastics (available e.g. under the name Makrolon), polystyrenes that may contain glass fibers as aggregate, plexiglass which may be colored or uncolored, or SPS GF30 (available from the company Schulatec).  
         [0084]     In an advantageous embodiment of the invention the cartridge  1  is designed in such a way that it may be discarded after one use. For this reason, any form of cleaning after it has been used becomes unnecessary.  
         [0085]     The two holding elements  101  and  102  of the cartridge  1  are constructed according to the invention in such a way that they have viewing windows above and below the core unit  500  so that the optical translucency of the reaction chamber is guaranteed.  
         [0086]     In an advantageous embodiment of the invention, the cartridge is constructed in such a way that on the side of the holding element  101  that is turned to the read-out optics there is an antireflection structure  109  that suppresses scattered light. This structure preferably has the shape of parallel, narrow grooves (riffling). Other shapes usable as reflection structure are knobs, roughening, and pyramid arrangements. Due to this advantageous property of the cartridge  1 , a multitude of optical methods (dark field, incident light, oblique light and transmitted light-fluorescence measurement, confocal fluorescence measurement, luminescence measurement, phosphorescence measurement, absorption measurement) may be utilized for the analysis of the interaction investigated in the sample chamber.  
         [0087]     In another advantageous embodiment of the invention, each cartridge  1  is individually identified via a data matrix  600 . For that purpose, a dataset is stored in a database while the cartridge is being assembled that in addition to the parameters for the heater/sensor substrate  400  contains information as to how the installed substance library  201  has to be analyzed and how the sample in the cartridge  1  has to be handled for a successful diagnosis. This dataset gets a number that is added on the cartridge in the form of a data matrix  600 . The number registered in the data matrix  600  accesses the dataset created during assembly. On the basis of the protocols found there, the sample is processed fully automatically. All the user has left to do is to inject the sample and to make a note of the analysis results.  
         [0088]     In another advantageous embodiment of the invention, the sample chamber  500  may be loaded manually by means of a manual filling station  2000  that is subsequently also referred to as injector  2000 . This device assumes the function of the connector only for the injection of the sample, and like it has integrated quick-closure connectors  2106 .  
         [0089]     In particular, it possesses cavities for the charging and venting of the sample chamber  500  of the cartridge  1  as well as cavities for receiving the cartridge  1 . Preferably, these can be cavities for receiving a disposable syringe  2401  filled with sample fluid with attached cannula  2402  and a single ventilation cannula  2403 , so that during the attachment of the cartridge  1 , the cannulae enter contemporaneously into the reaction space  301  of the sample chamber  500 . The sample may then be injected into the sample chamber  500  of the cartridge  1 , the ventilation of the sample chamber  500  taking place via the ventilation cannula  2403 . After loading the sample chamber  500 , the cartridge  1  is removed from the injector  2000  and attached to the connector  1000  for further processing. Since the syringe  2401  and the cannulae  2402  and  2403  are disposable articles, contamination of the sample space with undesired substances is avoided.  
         [0090]     The manual filling station  2000  consists of two parts, a lid  2200  and a body  2100 , which possess the appropriate recesses for the fixation of the device according to the invention  1 , a filling unit, and a ventilation unit. The filling unit is preferably a syringe with cannula  2401  and  2402  and the ventilation unit is a cannula  2403 . Lid  2200  and body  2100  may be affixed to one another by means of any device. This fixation preferably occurs by means of magnets.  
         [0091]     In principle, the device according to the invention  1  may be used for all test methods that are based on the specific interaction of a target molecule with a probe that is fixed on a microarray. These interactions may be protein-protein interactions, protein-nucleic acid interactions, and nucleic acid-nucleic acid interactions.  
         [0092]     The device according to the invention  1  is preferably used for testing procedures that are investigations of the interaction between proteins or peptides with an antibody library that is fixed on a chip. In another preferred application, the device according to the invention is used for microarray-based studies of interactions between a target nucleic acid and a nucleic acid probe. In an especially preferred use, the device according to the invention is used for the detection of microorganisms in clinical samples.  
         [0093]     In another especially preferred embodiment, the device according to the invention  1  is used for the detection of the presence of DNA sequences. From the detection of certain DNA sequences, for example the presence of pathogens and their resistances against therapeutic agents may be deduced. Especially preferred is also the use of the device  1  for the determination of the genetic state of cells or organisms such as the detection of mutations that lead to hereditary diseases (e.g. mucoviscidosis, phenylketonuria, infertility etc.) or the detection of polymorphisms. A field of application that is also preferred is the detection of genetic differences that lead to the identification of individuals (e.g. applications in the field of forensics, for paternity tests, among other things).  
         [0094]     The detection of the interaction between probes and targets may generally occur through common methods such as optical analysis using fluorescent markers such as Cy3, Texas Red etc., through radioactive markers, or also through chemical reactions such as silver-precipitation (WO 98/04740).  
         [0095]     Especially preferred is also the use of the device for the detection of the physiological state of cells e.g. by expression profiling. In this case, linear amplification is preferred, for example by linear PCR amplification.  
         [0096]     Especially preferred is also the use of the device in combination with other amplification methods that require a cyclic temperature regimen in order to be carried out, such as the ligase chain reaction (LCR) or ligase detection reaction (LDR), in particular when this is combined with an array-based analysis.  
         [0097]     The device according to the invention allows the fully automated, temperature-controlled and flow-controlled performance of test methods based on microarrays. The device according to the invention further allows the simultaneous performance of microarray-based test methods and a PCR without reprocessing of the intermediates.  
         [0098]     The device according to the invention will preferably be used to simultaneously amplify nucleic acids by PCR and to analyze the products of the PCR by a microarray-based test in which nucleic acids are utilized as probes.  
         [0099]     Reactions such as the ligase chain reaction (LCR) and/or the ligase detection reaction (LDR) may also be carried out in the device according to the invention  1 .  
         [0100]     The construction of the device  1  according to the invention allows the cost-effective manufacture of a disposable cartridge that may be used to carry out microarray-based test methods.  
         [0101]     The following examples are intended to illustrate the invention without limiting it in any way.  
       EXAMPLE 1  
     PCR  
       [0102]     A cartridge  1  was assembled according to  FIG. 2 . At the same time all technical data concerning this cartridge as well as protocols were stored in a database. The cartridge was assigned an individual number that allows the automatic linking of the cartridge  1  with the corresponding dataset in the database. The number was printed as data matrix  600  and glued on the cartridge  1 .  
         [0103]     A PCR mixture consisting of 1 μL of genomic DNA of  Corynebacterium glutamicum  (5 pg/μL), 1 μL primer (AGA GTT TGA TCC TGG CTC AG) (10 pg/μL), 1 μL primer (TAC CGT CAC CAT AAG GCT TCG TCC CTA) (10 pg/μL), 1 μL MgCl 2  (25 mM), 5 μL PCR buffer, 1 μL 50 fold dNTP (10 mM per base), 0.5 μL Taq-polymerase (5 units/pL), and 39.5 μL water was drawn in an injection syringe  2401  with cannula  2402 .  
         [0104]     Injection syringe and ventilation cannula  2401 ,  2402 , and  2403  were placed into the body  2100  of the injector  2000 , the lid  2200  was put on and the cartridge  1  was slid into the cartridge pocket  2001 . 20 μL of the PCR mixture were then injected into the sample chamber  500  and the cartridge  1  was removed from the injector  2000 .  
         [0105]     The remainder of the PCR mixture was put into reaction tubes for the amplification in a conventional thermocycler and amplified.  
         [0106]     The cartridge  1  was attached to the connector  1000  and the individual cartridge number encoded in the data matrix  600  was read out and automatically transmitted to the operating program. With this number, the technical data as well as protocols required for the PCR were read out from the database. The technical data for the temperature regulation were transmitted to the temperature controller. The slide  1100  was not slid into the cartridge  1  so that the reaction space  301  remained closed. The PCR was carried out in the cartridge using the following temperature protocol: initial denaturation for 240 s at 45° C. 30 elongation cycles with 60 s at 95° C., 60 s at 58° C. and 150 s at 72° C. each as well as a final extension of 420 s at 72° C.  
         [0107]     To speed up the cooling times, compressed air was pumped as cooling medium into the cartridge through the cooling agent tube and the insulated cooling channel. After the PCR, the slide  1100  was slid into the cartridge  1  in order to open the sample chamber  500  and the PCR sample was pumped out. The sample was loaded on an electrophoretic gel in order to obtain proof of the functionality of the cartridge. The success of the experiment in comparison with the results in a conventional thermocycler is documented in  FIG. 9 .  
       EXAMPLE 2  
     Hybridization and PCR  
       [0108]     A cartridge  1  was assembled according to  FIG. 2 . At the same time all technical data concerning this cartridge as well as protocols were stored in a database. The cartridge  1  was assigned an individual number that allows the automatic linking of the cartridge  1  with the corresponding dataset in the database. The number was printed as data matrix  600  and glued on the cartridge  1 .  
         [0109]     The lid element  200  built into in the cartridge, which lid element is also referred to as array, carried a substance library consisting of 4 different probes and that was arranged on the surface in the pattern of a chessboard. Each element of the array had a size of 256×256 μm. Each probe was arranged redundantly, i.e. each on 16 spots. Due to the surface structure, the individual spots have a raster scan. The DNA library consisted of the sequence P 1  (complementary to the PCR fragment) and three different deletion variants:  
                                       P1: 5′ CCTCTGCAGACTACTATTAC 3′                           P1 del9_11: 5′ CCTCTGCAATACTATTAC 3′                       P1 del10_12: 5′ CCTCTGCAGCACTATTAC 3′                       P1del9_10_11_12: 5′ CCTCTGCAACTATTAC 3′          
 
         [0110]     PCR mixture comprising consisting of the following components was drawn into an injection syringe  2401 : 5 μL Advantage 2  PCR buffer (Clontech, Palo Alto, USA), 1 μL dNTP Mix 20 mM, 1 μL Taq-polymerase (Advantage 2 , Clontech, Palo Alto, USA), 1 μL Primer P 1  (10 pmol/μL) (5′ CCTCTGCAGACTACTATTAC 3′) (MWG, Ebersberg, Germany), 1 μL Primer P 2  (10 pmol/μL), coupled with the fluorescent dye Cyanine  3  at the 5′-end (5′ CCTGAATTCTTGCTGTGACG 3′) (MWG, Ebersberg, Germany), 1 μl Template 106-mer PCR product (1 ng/μL) with the sequence 5′ CCTCTGCAGACTACTATTACATAATACGACTCACTATAGGGATCTGCACGTATACTTCTATAGTGTCACCTAAATAGGCAGTCTGTCGTCACAGCAAGAATTCAGG3′, 40 μL deionized water.  
         [0111]     Injection syringe and ventilation cannula  2401 ,  2402 , and  2403  were placed into the body  2100  of the injector  2000 , the lid  2200  was put on and the cartridge  1  was slid into the cartridge pocket  2001 . 20 μL of the PCR mixture were then injected into the sample chamber  500  and the cartridge  1  was removed from the injector  2000 .  
         [0112]     The cartridge  1  was contacted via the connector  1000 , the individual cartridge number encoded in the data matrix  600  was read out and automatically transmitted to the control program. With this number, the technical data as well as protocols required for the PCR were read out from the database. The technical data for the temperature regulation were transmitted to the temperature controller. The slide  1100  was not slid in so that the reaction space  301  of the cartridge  1  remained closed. The PCR was carried out in the cartridge using the following temperature protocol:  
         [0113]     Initial denaturation 120 s at 95° C., 30 elongation cycles with 35 s at 95° C., 40 s at 42° C. and 40 s at 72° C. each, final extension of 240 s at 72° C.  
         [0114]     To speed up the cooling times, compressed air was pumped into the cartridge  1  as cooling medium through the cooling agent tube and the insulated cooling channel.  
         [0115]     After the PCR, the PCR product was hybridized to the surface-bound DNA library on the DNA array. For that purpose, the cartridge was heated to 95° C. for 5 min. and then incubated for 1 h at 30° C. A rinsing process followed in order to remove non-specifically bound DNA from the surface and unbound fluorophores from the cartridge. For that purpose, the slide  1100  was slid into the recess  103  of the cartridge  1  and the PCR sample in the sample chamber  500  was replaced by continuous pumping of 500 μL of washing buffer  1  (2×SSC, 0.2% SDS) (flow rate approx. 0.1 mL per minute), the cartridge  1  being kept at 30° C. The cartridge was then rinsed with 500 μL washing buffer  2  (2×SSC) with the same flow rate and temperature. At the end of the rinsing process, the sample chamber  500  remained filled with washing buffer  2 . The fluidic connections were removed from the sample chamber  500  by moving the slide  1100 .  
         [0116]     The hybridization signals were detected in washing buffer  2  (2×SSC) under a Zeiss fluorescence microscope (Zeiss, Jena, Germany). The excitation occurred in the incident light with a white light source and a set of filters suitable for cyanine  3 . The signals were recorded with a CCD camera (PCO-Sensicam, Kehlheim, Germany). The exposure time was 5000 ms ( FIG. 10 ).  
         [heading-0117]     Reference Number List  
         [none]    
       
           1  cartridge  
           101  upper holding element  
           102  lower holding element  
           103  recess for the incorporation of a lug/slide  
           104  viewing window  
           105  medium connection for cooling liquids  
           106  snap-fit  
           108  alignment pin holes  
           403  contacts for heating element  
           120  media connection side  
           109  antireflection structure  
           600  data matrix  
           117  cooling channel outlet  
           118  barb (Widerlager/Widerhaken)  
           400  base element with integrated heater/sensor substrate  
           401  carrier of the integrated heater/sensor substrate  
           402  optically translucent recess  
           403  contacts for heater/sensor substrate  
           404  contacts for heater/sensor substrate  
           300  sealing, elastic, repeatedly puncturable intermediate element  
           301  recess enclosed in  300 , defines reaction space  
           302  expansion joints  
           304  alignment ears  
           200  lid element  
           202  lid carrier element  
           201  substance library or chip carrying substance library  
           500  core unit or chamber or reaction chamber or sample chamber  
           105  cooling channel  
           109  snap-fit  
           110  snap-fit  
           111  recess for heating/sensor substrate contacts  403   
           112  cooling agent inlet  
           116  cooling agent outlet  
           117  cooling agent inlet  
           115  press fit  
           1000  connector  
           1100  slide  
           1103  locating lug  
           1001  alignment pin holes  
           1002  mounting holes  
           1006  electric cable  
           1207  electrical connection  
           1202  filling tubes  
           1210  slide rod  
           1211  thread  
           1212  damping  
           1204  cooling agent tube  
           1205  cooling agent connection  
           1300  cooling channel  
           1201  insertion cannulae  
           2000  manual filling station  
           2106  quick-closure connector  
           2401  disposable syringe  
           2404  cannula  
           2403  ventilation cannula  
           2402  tip of a cannula  
           2403  tip of a cannula  
           2100  manual filling station body  
           2200  manual filling station lid  
           2101  recessed grip  
           2102  recessed grip