EP1614466A2 - System and method to prevent air bubbles in a hybridization chamber - Google Patents

Abstract

A system (1) with hybridizing chambers (5), to hybridize immobilized specimens on carriers (27), having chambers which are defined by the gap between the carrier and a cover (26), and which can be filled with a fluid, is new. The cover seals the chambers against the ambient air. A system (1) with hybridizing chambers (5), to hybridize immobilized specimens on carriers (27), having chambers which are defined by the gap between the carrier and a cover (26), and which can be filled with a fluid, is new. The cover seals the chambers against the ambient air. The system has a pressure assembly to build up the pressures within the chambers by 100 mbar to 1.4 bar over the ambient atmospheric level, to prevent the formation of air bubbles. The pressure is supplied by a pump (8) or a pressure gas bottle, linked to the chambers by pressure lines (10,11) with valves (12).

Description

According to the preamble of independent claim 1, the invention relates to a system with hybridization chambers for hybridizing nucleic acid samples, proteins or tissue sections immobilized on microscope slides. In this case, each hybridization chamber is defined as a substantially gap-shaped, with a liquid substantially fillable space between one of these slides and a lid. Each lid is arranged opposite the slide in such a way that the hybridization chamber is sealed off from the ambient air. Such a system comprises an agitation device with which liquids in the hybridization chambers can be moved relative to the samples immobilized on the object carriers. The invention also relates, according to the preamble of independent claim 14, to a corresponding method for preventing air bubbles in the hybridization chambers of a system for hybridizing nucleic acid samples, proteins or tissue sections immobilized on slides. According to this method, all substantially gap-shaped, between one of these slides and a lid arranged hybridization chambers are filled with a liquid substantially. In this case, the lid is arranged in such a way relative to the slide that the hybridization chamber is sealed off from the ambient air.

The use of DNA samples (DNA = deoxyribose nucleic acid), and especially microarrays of such samples, provides research with an important technique for the simultaneous analysis of thousands of genes. This technique involves the immobilization of DNA probes from many genes on a solid substrate surface, e.g. on a glass slide for a light microscope. The DNA samples are preferably stored in an array of sample spots or "spots", i. arranged in a two-dimensional grid on the substrate and one can later - starting from a certain position within such an array on the origin of the corresponding DNA sample infer. The technique typically involves contacting the DNA sample array with RNA pattern suspensions (RNA = ribose nucleic acid) to detect specific nucleotide sequences in the DNA samples. Typically, pattern suspensions are also used which contain DNA, cDNA and / or proteins or polypeptides.

RNA patterns may be labeled with a so-called "tag" or "label", i. be provided with a molecule which is e.g. emits a fluorescent light having a specific wavelength. Immobilized samples may also include amino acid-containing (e.g., proteins, peptides) or nucleic acid-containing (e.g., cDNA, RNA) probes. Samples added to the immobilized samples may comprise any molecules or chemical compounds that hybridize or otherwise associate with the immobilized samples.

Under good experimental conditions, the RNA patterns hybridize or bind to the immobilized DNA samples and together form hybrid DNA-RNA strands. For each of the immobilized DNA samples and for specific RNA patterns, differences in the hybridization among the DNA samples can be determined by measuring the intensity and wavelength dependency of the fluorescence of each individual microarray element to determine whether the level of gene expression in the DNA sample varies the examined DNA samples. With the use of DNA microarrays comprehensive statements can thus be made about the expression of large quantities of genes and their expression patterns, although only small amounts of biological material must be used.

DNA microarrays have become established as successful tools, and devices for performing DNA hybridization have been continually improved (see, for example, US 6,238,910 or EP 1 260 265 A1 of the Applicant of the current patent application). These documents disclose a device for providing a hybridization space for the hybridization of nucleic acid samples on a slide. These devices are designed to be movable relative to the slide and comprise an annular seal or sealing surface for closing the gap-shaped hybridization space with respect to the ambient air, wherein a surface of this slide is acted upon by the seal or sealing surface. In addition, these devices include lines for feeding or discharging media into the hybridization space or out of the hybridization space as well as a sample feed. An improved temperature control and movement of the liquid with e.g. RNA patterns against DNA samples immobilized on the slide are also disclosed.

It happens on the one hand again and again that air bubbles occur during the filling of liquids or later in the hybridization chamber. On the other hand, attempts have been made (see, for example, US Pat. No. 6,186,659) to use air bubbles specifically as agitating agents in order to achieve more thorough mixing of the reagents in the hybridization chamber. In general, however, air bubbles present in the hybridization medium are undesirable because they disturb the usually very thin liquid film over the immobilized samples. This can lead to an inhomogeneity of the distribution of reagents in the hybridization medium and thus to a falsification of the hybridization results; in the worst case, larger bubbles even displace the hybridization medium from parts of the slides immobilized on the slide.

In addition, numerous methods are known from the prior art to hinder the spontaneous occurrence of air bubbles or the retention of the same in the chamber. For example, a non-parallel arrangement of the slides and lids defining the hybridization chamber has been proposed (see US 5,922,591), or the hybridization media are transported out of the chamber and back into it throughout the hybridization process. The admixing of agents to the hybridization medium, which reduce the surface tension or treating the surfaces of the chamber with water repellent chemical compounds with the aim of hindering the formation of air bubbles is also known.

From US Pat. No. 6,458,526, an arrangement is known which produces "bubble halves 140" protruding into the hybridization space from a solvent-saturated gas. These "bubble halves" are actually dome-shaped interfaces of gas spaces with a defined radius of curvature. These "bubble halves" are located at defined locations in the chamber where they can not interfere with the hybridization of the samples. In a compartment separated from the hybridization space there is a solvent 160 which is contained in the hybridization medium. Over this solvent, a saturated atmosphere 150 is maintained, which is constantly connected to the gas spaces behind the "bubble halves 140" (see Figure 2 in US 6,458,526). Thus, an atmosphere saturated with the solvent is constantly brought to the dome-shaped boundary surfaces and thereby the partial pressure of the solvent present in the hybridization medium is influenced so that any existing air bubbles shrink and are eliminated. This method has the disadvantage that these dome-shaped interfaces must be created and maintained by means of special devices.

The object of the present invention relates to the provision of an alternative system or an alternative method, with which the formation of air bubbles in a hybridization chamber can be prevented in a simple manner.

This object is solved according to a first aspect and the characterizing part of independent claim 1, characterized in that a system described above comprises means for preventing air bubbles in the Hybridisierkammern, which is designed as a pressure means for establishing a chamber pressure in the Hybridisierkammern, wherein This chamber pressure thereby independent of agitation pressure differences over that in the ambient air prevailing, normal atmospheric pressure is that the system comprises at least one spring element, which communicates with the liquid volume used for the hybridization and resiliently counteracts the agitation pressure differences exerted by the agitator on the pressure chamber agitation pressure differences. This object is achieved according to a second aspect and the characterizing part of independent claim 14, characterized in that by means of a pressure device in a system described above with a pressure device of this system in the Hybridisierkammern a chamber pressure is formed, thereby independent of Agitationsdruckunterschieden on the ambient atmospheric pressure prevails that the system comprises a spring member which communicates with the volume of liquid used for hybridization and resiliently counteracts the agitation pressure differential applied by the agitation means to the pressure space. Additional, preferred inventive features will be apparent from the dependent claims, respectively.

Fig. 1

ein Schema eines zum Ausführen des erfindungsgemässen Verfahrens geeigneten, ersten Geräts bzw. Systems;

Fig. 2

einen der Fig. 1 von EP 1 260 265 A1 entsprechenden senkrechten Längsschnitt durch eine Anordnung mit einer Hybridisierkammer;

Fig. 3

eine der Fig. 2 entsprechende, schematische Ansicht einer Anordnung mit einer Hybridisierkammer, von unten gesehen;

Fig. 4

einen der Fig. 2 entsprechenden, senkrechten Längsschnitt durch eine Anordnung mit einer Hybridisierkammer, Deckel des Systems aufgeklappt;

Fig. 5

einen der Fig. 2 entsprechenden, senkrechten Längsschnitt durch eine Anordnung mit einer Hybridisierkammer, Deckel des Systems geschlossen.

Fig. 6

ein Schema eines zum Ausführen des erfindungsgemässen Verfahrens geeigneten, zweiten Geräts bzw. Systems;

Fig. 7

eine schematische Ansicht einer besonderen, ersten Anordnung eines Deckels mit einer Hybridisierkammer, von unten gesehen;

Fig. 8

eine schematische Ansicht einer besonderen, zweiten Anordnung eines Deckels mit zwei Hybridisierkammern, von unten gesehen.

The system according to the invention and / or the method according to the invention will now be explained in detail with reference to schematic drawings of exemplary embodiments, which do not limit the scope of the invention. Showing:

Fig. 1

a diagram of a first device or system suitable for carrying out the method according to the invention;

Fig. 2

one of Figure 1 of EP 1 260 265 A1 corresponding vertical longitudinal section through an arrangement with a hybridization chamber.

Fig. 3

a corresponding schematic view of an arrangement with a hybridization chamber, seen from below;

Fig. 4

one of Figure 2 corresponding, vertical longitudinal section through an arrangement with a hybridization, lid of the system unfolded;

Fig. 5

one of the Fig. 2 corresponding, vertical longitudinal section through an arrangement with a hybridization chamber, lid of the system closed.

Fig. 6

a diagram of a suitable for carrying out the inventive method, second device or system;

Fig. 7

a schematic view of a particular, first arrangement of a lid with a Hybridisierkammer, seen from below;

Fig. 8

a schematic view of a particular, second arrangement of a lid with two Hybridisierkammern, seen from below.

1 shows a diagram of a system 1 suitable for carrying out the method according to the invention. On the left side of this diagram are a number of vessels 2 for the storage of liquid hybridization media, such as washing liquids (W 3, W 2, W 1), prehybridization buffer ( VP), alcohol cleaning fluid (AR), distilled water (AD), and a container 3 with inert gas (N2). Each of these vessels 2 is preceded by an individual valve 4, via which these media can be supplied to the hybridization chambers 5 (S 1, S 2, S 3, S 4) (shown on the right-hand side). The valve lines 4 comprehensive media lines 6 open into a manifold 7, which in turn opens into a feed pump 8, which is preceded by a vent valve 9. This feed pump 8 draws liquid media from the vessels 2 via the collecting line 7 and pumps them via the distribution line 10 into the inlet lines 11, which open into the hybridization chambers 5 via an inlet valve 12. The hybridizing media leave the Hybridisierkammern 5 via an outlet 13, which each comprise an outlet valve 14 and open into a manifold 15. This manifold 15 in turn opens into a waste line 16, which is closable by means of a waste valve 17. The distribution line 10 and the manifold 15 can communicate with each other via a connecting line 18 and a connecting valve 19. From this connecting line 18 branches off a discharge line 20 with a relief valve 21 and flows into a sump 22 with a loading or vent 23. The sump 22 downstream of a feed pump 24 which is connected via a transition line 25 to the waste line 16.

For better distribution of the Hybridisiermedien in the Hybridisierkammern 5, the system 1 is equipped with an agitation mechanism or with an agitation device 32, as is known from the European patent application EP 1 260 265 A1 of the applicant of the current patent application. The content of this patent application EP 1 260 265 A1 is hereby expressly incorporated by reference, so that this content is part of the present patent application.

FIG. 2 shows a vertical longitudinal section through a hybridization chamber 5 corresponding to FIG. 1 of EP 1 260 265 A1. The cover 26 of this arrangement is movable relative to the slide 27 (in this case pivotable about an axis, so that the hybridization space 5 is replaced by a simple An annular sealing surface 28 serves to close off the hybridization space 5 by acting on a surface 29 of this slide 27. This sealing surface 28 may be a stepped surface of the lid 26 lying flat on the surface 29 of the slide 27; For example, a lip seal may also be used, but an O-ring seal is preferred as the sealing surface 28. The arrangement comprises lines 11, 13 for feeding or discharging media into the hybridization space 5 or out of the hybridization space 5. Such media may include reagents for carrying out the hybridization reaction, such as washings or inert gases (such as nitrogen) for drying the hybridization products on the slides 27 and for blowing out the Hybridisierkammern 5 and the media lines 11,13 be. These supply and discharge lines 11, 13 for hybridization media preferably open into a respective agitation space 30, 30 '. The assembly also includes a sealable pattern feeder 31 through which RNA-containing liquids or other sample liquids can be pipetted by hand. The pattern feeder 31 is preferably closed with a plastic plug (not shown). Alternatively, an automatic or robotized pattern feeder may be provided, as disclosed in different embodiments in EP 1 260 265 A1.

The arrangement comprises a media-separating agitation device 32 for moving liquids against samples of nucleic acids, proteins or tissue sections immobilized on the surface 29 of the slides 27. In the embodiment shown in FIG. 2, the agitation device 32 of the arrangement comprises a membrane 33. This membrane 33 separates a pressure space 34, which can be filled with a pressure fluid (gas or liquid) via a pressure line 35, from an agitation space 30, which flows over a Agitation line 36 is connected to the Hybridisierraum 5. After reaching the thermal equilibrium of the arrangement, adding a certain volume of RNA sample liquid and closing the pattern feed 31, preferably air or another gas (but it could also be a liquid) is introduced into the pressure space 34 via the pressure line 35 or discharged therefrom, so that the membrane 33 bends at the same rhythm and correspondingly reduces or increases the agitation space 30. As a result, the sample liquid is moved in the same rhythm of overpressure or underpressure and relaxation in Hybridisierraum 5 against one or the other end, where preferably at the directed towards the interior of the Hybridisierraums 5 surface 37 of the lid 26 each have a Querströmkanal 38,38 'is located.

On the one hand, these transverse flow channels 38, 38 'facilitate lateral distribution of the RNA molecules contained in the sample solution. This has the effect that the sample liquid or the washing liquids are distributed homogeneously over the entire volume present in the hybridization chamber 5. In addition, the Querströmkanäle 38,38 'serve as a liquid reservoir, so that when the built-in device agitation device 32 generated pendulum movement (filled double arrow) of the sample solution does not dazuführt that parts of the Hybridisierraums 5 are unintentionally dry.

Preferably, a second agitation space 30 ', also provided with a membrane 33', is connected to the hybridization space 5 via a second agitation conduit 36 '. If a pressure surge delivered to the pressure chamber 34 now presses the first diaphragm 33 into the first agitation space 30, this pulse is transmitted via the first agitation conduit 36 to the sample fluid in the hybridization space 5 transfer. The pattern liquid differs somewhat from the second agitation conduit 36 '(and may even partially fill it) and increases the pressure in the second agitation space 30'. As a result, the second membrane 33 'bends upwards and is elastically stretched. As soon as the overpressure in the pressure chamber 34 subsides, both diaphragms 33, 33 'spring back into their rest position and move the sample liquid in the hybridization space 4 in the opposite direction. As a result of this oscillating movement, with the arrangement shown, a sample liquid having a minimum volume (in the range of approximately 100 μl) can be distributed practically homogeneously in the hybridization chamber 5 in less than one minute. Preferably, immediately after the pressure reduction in the pressure chamber 34, a negative pressure in the pressure chamber 34 is generated, so that the backward movement of the sample liquid opposite the preceding pressure surge in the hybridization chamber 5 is enhanced.

Figure 3 shows a plan view of the arrangement of Figure 2, seen from below. The O-ring seal 28 laterally delimits the hybridization space 5, which has at its opposite ends a respective cross-flow channel 38, 38 ', which are provided as a depression in the surface 37 of the cover 26. The slide 27 (here a glass slide for light microscopy) and the handle panel 55 are shown in dashed lines. Clearly visible is the Abdrückfeder 56, which presses on the handle panel 55 of the slide 27. When opening the Hybridisierraums 5 this Abdrückfeder 56 facilitates the automatic separation of the slide 27 from the lid 26. Also visible is the lines of the inlet line 11, the outlet 13 and the pressure line 35 and the arrangement of agitation rooms 30,30 'and the pattern feeder 31. Die Agitation lines 36,36 'and the pattern feeder 31 open into the Querströmkanälen 38,38'.

All lines 11, 13, 35 for the supply and removal of media preferably open in a common connection plane 57 of the cover 26, which is arranged substantially parallel to the hybridization space 5 and preferably at the same height as the hybridization space 5. The mouth openings of the lines 11,13,35 can, as shown offset from each other or on a transverse to the Device 1 extending line (not shown) may be arranged. Recesses (empty arrows, see Fig. 2) reduce the heat flow from or to the lid 26th

The pressure lines 35, one of which is intended for each of the Hybridisierkammern 5, are shown in dashed lines in Figure 1 and start from a pressure distribution line 39. In this pressure distribution line 39 open a compensation line 40 with a balancing valve 41, a pressure supply line 42 with a pressure relief valve 43 and a vacuum supply line 44 with a vacuum valve 45. The pressure is preferably generated with a low-cost gas (eg air) in a pressure pump 46, in a pressure vessel 47 stored and fed into the pressure supply line 42. The negative pressure is generated in a lower pressure pump 48, stored in a vacuum reservoir 49 and fed into the vacuum supply line 44.

The inert gas container 3 is connected via a gas valve 50 and a gas line 51 to the distribution line 10, which open into the hybridization chambers 5 via inlet lines 11 and one inlet valve 12 each. With inert gas (e.g., with nitrogen gas), depending on the valve positions, all of the hybridization chambers 5 (individually or in groups) may be exhausted via the distribution line 10 and the inlet pipes 11 via the outlet pipes 13 and the manifold 15.

If only the gas valve 50, the connecting valve 19 and the relief valve 21 are opened, the distribution line 10 can be blown into the collecting container 22. If only the gas valve 50, the connection valve 19 and the waste valve 17 are opened, then the distribution line 10 and the manifold 15 can be blown out via the waste line 16.

FIG. 4 shows a vertical longitudinal section corresponding to FIG. 2 through an arrangement with a hybridization chamber 5, wherein the folding frame 54 with the cover 26 of the system 1 inserted therein is unfolded. Preferably, the covers 26 are arranged parallel to each other and in a group of four, because this arrangement just allows a measure of a contact plate 53 of the temperature control thermostat on which a transport frame 52 in the size of a microplate with four parallel to each other slides 27 fits.

Each of these groups of four is assigned to a contact plate 53 connected to a temperature controller. Such a contact plate 53 is thus designed for planar reception of the four slides 27 of a transport frame 52. The frame 52 comprises longitudinal walls, transverse walls and intermediate walls extending substantially parallel to the transverse walls. These walls surround openings which completely penetrate the frame 52, these openings allowing direct contact between the contact plate 53 of the thermostat and the slides 27. Because the slides 27 are held softly in the frame 52 and because the contact plate 53 is formed so that the frame 52 can be slightly lowered toward it, the slides 27 lie directly on the surface of the contact plate 53. Each quad of a processing unit comprises one an axis 58 pivotable and lockable relative to a base plate 59 folding frame 54 with four seats, in each of these seats a lid 26 can be inserted. Each such process unit also comprises a connection plate 60 for sealingly connecting each of an inlet line 11, outlet line 13 and pressure line 35 of the system 1 with the inlet line 11, outlet line 13 and pressure line 35 of a lid 26. As seals for these compounds, preferably O arranged on the system side Rings preferred (not shown).

FIG. 5 shows a vertical longitudinal section corresponding to FIG. 2 through an arrangement with a hybridization chamber 5, wherein the cover 26 of the system inserted into a folding frame 54 is closed. All four hybridization spaces 5 of a group of four defined by a contact plate 53 and such a folding frame 54 are thus associated with the temperature control of a temperature control device. Each group of four procedural unit comprises, as described above, a pivotable about an axis 58 and lockable against a base plate 59 folding frame 54 with four seats, wherein in each of these seats, a lid 26 is inserted. To ensure that the cover 26 can be placed plane-parallel to the slides 27, the folding frame 54 also has a central joint (not shown) with a parallel to the axis 58 mobility. So that the seals 28 close off the hybridization chambers 5 reliably, an additional pressure is exerted on the cover via the snap-on frame 54 26, this can be accomplished via screws, rocker arms or similar known devices (not shown).

The present invention is based on the recognition that by generating an overpressure in the hybridization chambers 5, the spontaneous occurrence of air bubbles during hybridization can be prevented. Here, the chamber pressure should be above the prevailing in the ambient air, normal atmospheric pressure. A chamber pressure which is at least 100 mbar to at most 1.4 bar higher than the ambient pressure is preferred. Even higher pressures in the chamber are possible if counteracted by a sufficiently large contact pressure for sealing the chamber.

In fact, no air bubbles occur under these pressure conditions during hybridization. The underlying mechanism of this phenomenon is not completely clear. However, it is assumed that the increased pressure on the one hand determines or defines the direction of diffusion in the region of the O-ring seal 28, so that no more gas molecules of the ambient air can diffuse into the hybridization chamber 5. On the other hand, due to its pressure dependence, a shift of the phase boundaries in the hybridization medium certainly takes place, so that the spontaneous formation of air bubbles is suppressed. In the context of this invention, therefore, all gas bubbles in the hybridization medium - irrespective of the formation process in the hybridization chamber 5 - are referred to as "air bubbles".

According to the invention, the required overpressure in the hybridization chambers 5 can be achieved from one of the vessels 2 by means of a liquid, for example by means of a hybridization medium pressed by the feed pump 8 into the hybridization chambers 5 (see FIG. If a system 1 comprising this hybridization chamber 5 has an agitation device 32 for moving the hybridization media with respect to the immobilized samples, then this system preferably also comprises a spring element which resiliently counteracts the pressure differences generated by the agitation device 32. Such a spring element may be an elastic tube piece (not shown) in a corresponding inlet or outlet to the hybridization chamber 5; but it can also be a spring loaded Expansion vessel (not shown) may be provided, which is connected via a line to the Hybridisierkammer 5.

Alternatively, inert gas can also be forced out of the container 3 into the distribution line 10 and the inlet lines 11 and the required pressure can be built up via an inlet valve 12 into the hybridization chambers 5 already filled with samples and hybridization media. Preference is given to inert gases such as N ₂ (nitrogen), which do not undergo any chemical interaction or reactions with the hybridization media. It may also be advantageous if the inert gases are not soluble in the Hybridisiermedien. In addition, it is possible to introduce the gas originating from a pressure pump and a pressure vessel (similar to the elements designated by 46 and 47 in FIG. 1) into the distribution line 10. After pressure build-up, all valves can be closed again and hybridization performed. In this case, the gas cushion thus constructed, which is directly related to the liquid volume used for the hybridization, serves as a spring element for resilient counteraction to the agitation pressure differences. If this is necessary, for example because of a minimal but constant pressure drop across the O-ring seals 28, the required chamber pressure may be sporadically corrected or renewed during the many hours of hybridization. Optionally, one or both of the valves 12, 14 (see Fig. 1) may be kept open for this purpose.

Another alternative (not shown in the figures) is to use a pressure pump and a pressure vessel (similar to those elements labeled 46 and 47 in FIG. 1) or a gas vessel (such as the N ₂ vessel 3 in FIG. 1). to connect to the manifold 15 and to build up the pressure in the Hybridisierkammern 5 via an opening of the exhaust valves 14. An additional alternative to providing a gas cushion serving as a spring member for resiliently counteracting the agitation pressure differentials is that of a pressure pump and a pressure vessel (similar to the elements indicated at 46 and 47 in Fig. 1) or a gas vessel (such as Figs For example, the N ₂ tank 3 in Fig. 1) to lead gas into one of the lines which open into at least one of the distribution line 10 or manifold 15. Corresponding For example, one or both of the valves 12, 14 (see Fig. 1) may be kept open.

If a system 1 is used with arrangements comprising two agitation membranes 33, 33 '(as described above), the agitator 32 may be used during the preliminary agitation of the hybridization media in the hybridization chambers 5 or even during the hybridization itself. In this case, simply an agitation pressure in the pressure chamber 34 must be generated, which is higher by about 0.5 to 1 bar than the desired chamber pressure of 100 mbar to 1.4 bar above the ambient pressure. The pressure to be used for the agitation moves (depending on the ambient pressure) in the range of approx. 1.6 - 2.4 bar. The second diaphragm 33 'forms in this case a spring element which acts resiliently against this agitation pressure.

• a) Ausspülen von Luftblasen und Flüssigkeitsresten aus der Verteilleitung 10 und der Sammelleitung 15. Zu diesem Zweck werden nur die Ventile 4 (A.D.), 19 und 17 geöffnet und es wird mit der Förderpumpe 8 destilliertes Wasser aus dem Gefäss 2 (A.D.) über das Ventil 4 (A.D.) in die Verteilleitung 10 gepumpt. Das destillierte Wasser strömt durch das Verbindungsventil 19 in die Sammelleitung 15 und von dort über das Abfallventil 17 in die Abfallleitung 16.
  Gleichzeitig werden die Hybridisierkammern 5 aufgebaut. Dies geschieht durch das Auflegen von (vorzugsweise in einem Transportrahmen 52 gehaltenen) Objektträgern 27 mit darauf immobilisierten Proben auf die Kontaktplatte 53 eines Thermostats, durch das Einsetzen von Deckeln 26 in den Klapprahmen 54 des Systems 1 zum Hybridisieren von auf Objektträgern 27 immobilisierten Nukleinsäureproben, Proteinen oder Gewebeschnitten und durch das Verschliessen der Hybridisierkammern 5 mittels Herunterklappen der Deckel 26 auf die Objektträger 24 (vgl. Fig. 4 und 5). Dieses Herunterklappen verbindet die Enden der Einlassleitungen 11, der Auslassleitungen 12 und der Druckleitungen 35 der Agitationseinrichtung 32 jeder einzelnen Hybridisierkammer 5 mit den entsprechenden Leitungsenden des Systems 1. Vorzugsweise werden - entsprechend den vier in einem Transportrahmen 52 aufgenommenen Objektträgern 27 - vier Deckel 26 in einen Klapprahmen 54 eingesetzt.
• b) Füllen und Thermostatisieren der Hybridisierkammern 5 mit Prähybridisier-Puffer. Zu diesem Zweck werden nur die Ventile 4 (V-P), 12, 14 und 17 geöffnet und es wird mit der Förderpumpe 8 Prähybridisier-Puffer aus dem Gefäss 2 (V-P) über das Ventil 4 (V-P) in die Verteilleitung 10 und von dort über die Einlassventile 12 in die Hybridisierkammern 5 gepumpt. Ein Teil des Prähybridisier-Puffers verlässt die Hybridisierkammern 5 über die Auslassventile 14 und die Sammelleitung 15 und gelangt über das Abfallventil 17 in die Abfallleitung 16. Dieser Vorgang verlangt besondere Beachtung, weil hier zum ersten Mal Luftblasen in die Hybridisierkammern 5 eingeschleppt werden können.
• c) Ausblasen der Verteilleitung 10 und der Sammelleitung 15. Zu diesem Zweck werden nur die Ventile 9, 19 und 17 geöffnet und es wird mit der Förderpumpe 8 Luft über das Belüftungsventil 9 angesaugt und über die Verteilleitung 10, das Verbindungsventil 19 in die Sammelleitung 15 und von dort über das Abfallventil 17 in die Abfallleitung 16 gepumpt.
• d) Probenzugabe in die Hybridisierkammern 5. Zu diesem Zweck werden nur die Ventile 14 und 21 geöffnet. Die Proben werden mit einer Pipette über die Musterzuführung 31 im Deckel 26 in die Hybridisierkammern 5 gepresst. Dadurch wird ein entsprechendes Volumen Prähybridisier-Puffer aus den Hybridisierkammern 5 in die Auslassleitung 13 mit den geöffneten Auslassventilen 14 verdrängt. Dies wiederum verdrängt ein entsprechende Volumen Luft aus der Sammelleitung 15. Diese verdrängte Luft strömt durch das geöffnete Entlastungsventil 21 in die Entlastungsleitung 20 und gelangt in den Sammelbehälter 22, den sie über die Entlüftungsöffnung 23 verlässt. Dieser Vorgang verlangt besondere Beachtung, weil hier zum zweiten Mal Luftblasen in die Hybridisierkammern 5 eingeschleppt werden können.
• e) Gleichmässiges Verteilen der Hybridisiermedien in den Hybridisierkammern 5 und gegenüber den auf den Objektträgern 27 immobilisierten Proben. Zu diesem Zweck sind zuerst alle Ventile geschlossen. Dann wird der Kammerdruck wie schon beschrieben erhöht und die Agitationseinrichtung 32 in Betrieb genommen.
  Alle eventuell in den Hybridisierkammern 5 vorhandenen Luftblasen werden bei diesem Druckaufbau eliminiert.
• f) Hybridisieren der Proben während beispielsweise 17 Stunden. Währen der Hybridisierung können die Medien in den Hybridisierkammern 5 konstant oder intermittierend agitiert werden. Dieser Vorgang verlangt besondere Beachtung, weil hier Luftblasen spontan in den Hybridisierkammern 5 entstehen können. Um dies zu verhindern, kann der Kammerdruck während der Hybridisierung korrigierend angepasst und konstant gehalten bzw. gemäss einem individuellen Programm verändert werden. Ein solches Programm kann das Erhöhen und Erniedrigen des Kammerdruckes in bestimmten Druck- und Zeitschritten definieren, so dass ein schonendes Hybridisieren der Proben gewährleistet ist, die eine solch spezielle Behandlung verlangen.
• g) Waschen der hybridisierten Proben mit Puffern. Zu diesem Zweck wird zuerst der Kammerdruck auf Normaldruck reguliert, indem die Auslassventile 14 und das Abfallventil 17 geöffnet werden. Darauf werden Waschflüssigkeiten mittels der Förderpumpe 8 sequentiell und je nach Bedarf aus den Gefässen 2 durch die entsprechend geöffneten Ventile 4 in die Verteilleitung 10 und von da über die geöffneten Einlassventile 12 und die Einlassleitungen 11 in die Hybridisierkammern 5 gepumpt. Die Waschflüssigkeiten und Waschabfälle verlassen die Hybridisierkammern 5 über die Auslassleitungen 13 und die bereits geöffneten Auslassventile 14, werden in der Sammelleitung 15 zusammengeführt und durch das geöffnete Abfallventil 17 in die Abfallleitung 16 abgeführt.
• h) Trocknen der hybridisierten Proben auf den Objektträgern 27. Zu diesem Zweck werden nur die Ventile 12, 14 und 17 geöffnet. Darauf wird das Inertgasventil 50 geöffnet und die Verteilleitung 10, die Einlassleitungen 11, die Hybridisierkammern 5, die Auslassleitungen 13 und die Sammelleitung 15 über das geöffnete Abfallventil 17 und die Abfallleitung 16 mit Inertgas durchgespült bis die Proben trocken sind.
  Anschliessend könne die fertigen Proben entnommen werden.

Typically, hybridization is performed as follows:

• a) rinsing out of air bubbles and liquid residues from the distribution line 10 and the manifold 15. For this purpose, only the valves 4 (AD), 19 and 17 are opened and it is with the feed pump 8 distilled water from the vessel 2 (AD) on the Valve 4 (AD) is pumped into the distribution line 10. The distilled water flows through the connecting valve 19 into the manifold 15 and from there via the waste valve 17 into the waste line 16.
  At the same time the Hybridisierkammern 5 are constructed. This is done by placing slides (preferably held in a transport frame 52) with samples immobilized thereon onto the contact plate 53 of a thermostat, inserting lids 26 into the snap frame 54 of the system 1 to hybridize nucleic acid samples, proteins immobilized on slides 27 or tissue sections and by closing the Hybridisierkammern 5 by folding down the lid 26 on the slides 24 (see Fig. 4 and 5). This folding down connects the ends of the inlet ducts 11, the outlet ducts 12 and the pressure ducts 35 of the agitation means 32 of each individual hybridizing chamber 5 to the respective duct ends of the system 1. Preferably, four covers 26 are inserted into a snap-in frame 54, corresponding to the four slides 27 received in a transport frame 52.
• b) filling and thermostating the Hybridisierkammern 5 with prehybridization buffer. For this purpose, only the valves 4 (VP), 12, 14 and 17 are opened and it is with the feed pump 8 prehybridization buffer from the vessel 2 (VP) via the valve 4 (VP) in the distribution line 10 and from there via the intake valves 12 are pumped into the Hybridisierkammern 5. A part of the prehybridization buffer leaves the Hybridisierkammern 5 via the outlet valves 14 and the manifold 15 and passes through the waste valve 17 in the waste line 16. This process requires special attention, because here for the first time air bubbles can be introduced into the Hybridisierkammern 5.
• c) blowing out of the distribution line 10 and the manifold 15. For this purpose, only the valves 9, 19 and 17 are opened and it is sucked with the feed pump 8 air through the vent valve 9 and the distribution line 10, the connecting valve 19 into the manifold 15th and pumped from there via the waste valve 17 into the waste line 16.
• d) Sampling in the Hybridisierkammern 5. For this purpose, only the valves 14 and 21 are opened. The samples are pressed with a pipette via the pattern feed 31 in the lid 26 into the hybridization chambers 5. As a result, a corresponding volume of prehybridization buffer is displaced from the hybridization chambers 5 into the outlet line 13 with the outlet valves 14 open. This in turn displaces a corresponding volume of air from the manifold 15. This displaced air flows through the open relief valve 21 in the discharge line 20 and enters the sump 22, which it leaves via the vent opening 23. This process requires special attention, because here for the second time air bubbles can be introduced into the Hybridisierkammern 5.
• e) Uniform distribution of the Hybridisiermedien in the Hybridisierkammern 5 and with respect to the immobilized on the slides 27 samples. For this purpose, first all valves are closed. Then, the chamber pressure is increased as already described and the agitator 32 is put into operation.
  Any air bubbles that may be present in the hybridization chambers 5 are eliminated in this pressure build-up.
• f) hybridizing the samples for, for example, 17 hours. During hybridization, the media in the hybridization chambers 5 can be agitated constantly or intermittently. This process requires special attention because here air bubbles can spontaneously arise in the Hybridisierkammern 5. To prevent this, the chamber pressure during the hybridization can be corrected and kept constant or changed according to an individual program. Such a program may define increasing and decreasing the chamber pressure in certain pressure and time increments so as to ensure gentle hybridization of the samples requiring such special treatment.
• g) washing the hybridized samples with buffers. For this purpose, first, the chamber pressure is regulated to normal pressure by opening the exhaust valves 14 and the waste valve 17. Thereafter, washing liquids are pumped by means of the feed pump 8 sequentially and as needed from the vessels 2 through the correspondingly open valves 4 in the distribution line 10 and from there via the open inlet valves 12 and the inlet lines 11 in the Hybridisierkammern 5. The washing liquids and wastes leave the Hybridisierkammern 5 via the outlet lines 13 and the already open exhaust valves 14 are merged in the manifold 15 and discharged through the open waste valve 17 into the waste line 16.
• h) drying the hybridized samples on the slides 27. For this purpose, only the valves 12, 14 and 17 are opened. Thereafter, the inert gas valve 50 is opened and the distribution line 10, the inlet lines 11, the hybridization chambers 5, the outlet lines 13 and the manifold 15 are flushed through the opened waste valve 17 and the waste line 16 with inert gas until the samples are dry.
  Subsequently, the finished samples can be removed.

As an alternative to process step h) just described, the hybridized samples are dried on the microscope slides 27 by opening only the valves 12, 14 and 21. Then the inert gas valve 50 is opened and the distribution line 10, the inlet lines 11, the Hybridisierkammern 5, the outlet lines 13 and the manifold 15 through the open relief valve 21, the discharge line 20, the sump 22 and the vent 23 are flushed with inert gas until the samples dry are.

An alternative embodiment of the method according to the invention in which the second membrane 33 'of the agitation device 32 can be dispensed with is also preferred. For this purpose, another medium must take over the function of this spring element. This is achieved in that after filling all the samples and media participating in the hybridization into the hybridization chambers 5, all valves are closed (see steps a-d) and then the outlet valves 13 are opened again. Thus, the air-filled volume of the manifold 15 and the directly subsequent part of the waste line 16 is connected to the liquid-filled volumes of the Hybridisierkammern 5. The enclosed between the closed valves 17, 19 and 21 and the sample liquids in the Hybridisierkammern 5 air is elastically compressible and now forms the desired spring element.

Alternatively, step c) can be carried out with an inert gas (eg N ₂ ): In this case, chemical interactions between the gaseous spring element N ₂ and the samples can be excluded.

Physical embodiment

In a first series of experiments, the physical fundamentals were investigated. For this purpose, several 100 slides without samples were processed. The preferably made of an elastomer O-ring seals 28 such as neoprene, silicone, twisted PTFE, polyethylene or Viton all successfully prevented a fluid loss. The basic requirement is of course that the contact pressure on the cover 26, the O-ring 28 and the slide 27 is sufficiently large, ie, significantly higher than the chamber pressure generated. The hybridization chambers 5 used comprised between the cover 26 and the slide 27 an area of 21 × 65 mm. The chamber pressure was increased by 1 bar, ie by 10 ⁵ N / m ² above the normal pressure of the environment. With a current effective area of 13.65 cm ² or 1365 x 10 ^-3 m ² , a force of more than 136.5 N or of approximately 13.9 kp per hybridization chamber 5 had to be used so that they could not spontaneously open. It is also believed that other seal shapes and sealing materials will suffice and prevent fluid leakage if a closing force selected according to the overpressure in the hybridization chamber 5 is applied to the cover 26, seal 28, and slide 27.

While air bubbles were regularly observed in the standard device SN22 during the processing of slides or slides with hydrophobic surfaces, no air bubbles were detected in the prototype according to the invention.

Biological embodiment

To carry out an example of a hybridization performed according to the invention under elevated pressure, two systems were used simultaneously and independently of each other. This is a standard device of the applicant (Tecan HS 400, serial number 22, abbreviated SN22), which was operated at atmospheric pressure and a prototype according to the invention (abbreviated to PT3), which was operated at a chamber pressure increased by 0.8 to 1.0 bar , Both devices were equipped with a corresponding agitation mechanism, as it was basically described above. This agitation device 32 in SN22 was at about 0.5 bar, the one in PT3 operated at an agitation pressure of about 1.5 bar. In both devices, the observance of the exact temperature was checked in advance; they were (apart from the mentioned, test-related differences) operated with exactly the same parameters (eg temperatures). This made it possible to draw direct conclusions about the causes from the results obtained.

The hybridization procedure (buffer preparation, pattern injection, program definition on the hybridization systems used) was carried out according to the technical instructions of Alopex (ALOPEX GmbH, Fritz-Hornschuch-Str. 9, D-95326 Kulmbach, Federal Republic of Germany). In each case two slides 27 were inserted into the hybridization systems SN22 and PT3 and processed at 43 ° C. or at 61 ° C. From the following processed samples the results are discussed: 43 ° C 43 ° C HS 400 SN22 HS 400 PT3 Slide # 29, # 31 Slide # 24, # 27 61 ° C 61 ° C HS 400 SN22 HS 400 PT3 Slide # 33, # 34 Slide # 35, # 37 A test kit "HybCheck" from Alopex was used with the kit lot number: 040524). This test kit is designed to validate the hybridization systems and is designed to pre-define the hybridization temperatures and to deliver the "OK" signals only if the intended temperatures and the relevant test parameters for washing, agitation and drying are strictly adhered to. The samples immobilized on the slides 27 are oligonucleotides whose sensitivity to temperature differences is known.

• 1. Erstes Waschen bei 43 °C bzw. 61 °C, Dauer 30 sec;
• 2. Injektion von 105 µl der Oligonukleotidlösung bei 43 °C bzw. 61 °C in jede Kammer;
• 3. Hybridisierung bei 43 °C bzw. 61 °C; bei starker Agitation und während 60 min;
• 4. Waschschritt 1 bei 23 °C (Kanal 1) während 30 sec; Absaugen während 30 sec; 2 Wiederholungen;
• 5. Waschschritt 2 bei 23 °C (Kanal 2) während 30 sec; Absaugen während 30 sec; 2 Wiederholungen;
• 6. Trocknen der objektträger während 3 min bei 23 °C;

f) Nach Beendigung der Hybridisierung wurden beide Objektträger herausgenommen und in einen Laser Scanner (Tecan LS400) eingelegt;
g) Messeinstellung des Laser Scanners: 1. Scan Mode: einzelne Wellenlänge 2. Laserwellenlänge: 543 nm (grün) 3. Filterwellenlänge: 590 nm 4. Gain: 165 5. Autofokus Modus: HS Autofokus, Ebene 1 6. Scan Resolution: 10 µm 7. Pinhole: klein 8. Oversampling Faktor: 1
h) Nach Messung im Laser Scanner wurden die erhaltenen Rohdaten ausgewertet wie folgt:
Hybridisierung bei 43 °C:

• 1. 6 Sub Arrays (6x9 Spots) auf einem Objektträger (Microslide)
• 2. Perfect Match (PM) bei 43 °C: 24 Spots pro Slide (4 pro Sub Array)
• 3. Mismatch 1 (MM1) bei 43 °C: 24 Spots pro Slide (4 pro Sub Array)
• 4. Mismatch 2 (MM2) bei 43 °C: 24 Spots pro Slide (4 pro Sub Array)
• 5. Negative Controls: 36 Spots pro Slide (6 pro Sub Array)
• 6. Von allen PM's und MM1's pro Microslide (24 Einzelwerte) wurde der Mittelwert, die Standartabweichung und der CV berechnet. Dabei gilt: CV = (Standartabweichung/Mittelwert) x 100
• 7. Diskriminierung PM:MM1 (1:5) sowie Diskriminierung PM:MM2 (1:20) berechnet
• 8. Die CV-Wert und die Diskriminierungs-Werte als "OK" (falls CV über ein ganzes Slide < 18%) oder "failed" angegeben.

Hybridisierung bei 61 °C:

• 1. 6 Sub Arrays (6x9 Spots) auf einem Objektträger (Microslide)
• 2. Perfect Match (PM) bei 61°C: 24 Spots pro Slide (4 pro Sub Array)
• 3. Mismatch 1 (MM1) bei 61°C: 24 Spots pro Slide (4 pro Sub Array)
• 4. Mismatch 2 (MM2) bei 61°C: 24 Spots pro Slide (4 pro Sub Array)
• 5. Negativkontrollen: 36 Spots pro Slide (6 pro Sub Array)
• 6. Von allen PM's und MM1's pro Microslide (24 Einzelwerte) wurde der Mittelwert, die Standartabweichung und der CV berechnet. Dabei gilt: CV = (Standartabweichung/Mittelwert) x 100
• 7. Diskriminierung PM:MM1 (1:5) sowie Diskriminierung PM:MM2 (1:20) berechnet
• 8. Die CV-Werte und die Diskriminierungs-Werte als "OK" (falls CV über ein ganzes Slide < 18%) oder "failed" angegeben.

The executed program for each test run is in detail:
a) Preparation of the "HybCheck Wash Buffers 1 and 2 according to the manufacturer's instructions;
b) dissolving lyophilized, fluorescently labeled oligonucleotides in 250 μl HybCheck buffer for hybridization preheated to a hybridization temperature (43 ° C and 61 ° C respectively);
c) placing 2 slides (each with 6 sub-arrays) in positions 1 and 2 in the hybridization system, using the large hybridization chambers (63.5 mm x 20 mm);
d) closing the hybridization chambers and starting the hybridization program;
e) Hybridization program:

• 1. First wash at 43 ° C or 61 ° C, duration 30 sec;
• 2. Injection of 105 μl of the oligonucleotide solution at 43 ° C or 61 ° C into each chamber;
• 3. hybridization at 43 ° C or 61 ° C; with strong agitation and during 60 min;
• 4th washing step 1 at 23 ° C (channel 1) for 30 sec; Suction for 30 sec; 2 repetitions;
• 5. wash step 2 at 23 ° C (channel 2) for 30 sec; Suction for 30 sec; 2 repetitions;
• 6. drying the slides during 3 min at 23 ° C;

f) After hybridization was complete, both slides were removed and placed in a laser scanner (Tecan LS400);
g) Measurement setting of the laser scanner: 1. Scan Mode: single wavelength 2. laser wavelength: 543 nm (green) 3. Filter wavelength: 590 nm 4. Gain: 165 5. Autofocus mode: HS autofocus, level 1 6. Scan Resolution: 10 μm 7. Pinhole: small 8. Oversampling factor: 1
h) After measurement in the laser scanner, the obtained raw data were evaluated as follows:
Hybridization at 43 ° C:

• 1. 6 sub arrays (6x9 spots) on a microscope slide (Microslide)
• 2. Perfect Match (PM) at 43 ° C: 24 spots per slide (4 per sub array)
• 3. Mismatch 1 (MM1) at 43 ° C: 24 spots per slide (4 per sub array)
• 4. Mismatch 2 (MM2) at 43 ° C: 24 spots per slide (4 per sub array)
• 5. Negative Controls: 36 spots per slide (6 per sub array)
• 6. Of all PMs and MM1's per Microslide (24 individual values) the mean, the standard deviation and the CV were calculated. Where: CV = (standard deviation / mean) x 100
• 7. Discrimination PM: MM1 (1: 5) as well as Discrimination PM: MM2 (1:20) calculated
• 8. The CV value and the discrimination values are indicated as "OK" (if CV over an entire slide <18%) or "failed".

Hybridization at 61 ° C:

• 1. 6 sub arrays (6x9 spots) on a microscope slide (Microslide)
• 2. Perfect Match (PM) at 61 ° C: 24 spots per slide (4 per sub array)
• 3. Mismatch 1 (MM1) at 61 ° C: 24 spots per slide (4 per sub array)
• 4. Mismatch 2 (MM2) at 61 ° C: 24 spots per slide (4 per sub array)
• 5. Negative controls: 36 spots per slide (6 per sub array)
• 6. Of all PMs and MM1's per Microslide (24 individual values) the mean, the standard deviation and the CV were calculated. Where: CV = (standard deviation / mean) x 100
• 7. Discrimination PM: MM1 (1: 5) as well as Discrimination PM: MM2 (1:20) calculated
• 8. The CV values and the discrimination values are indicated as "OK" (if CV over an entire slide <18%) or "failed".

The following results were achieved:

43 ° C / HS 400 SN22

CV Perfect Match OK CV Mismatch 1 OK Discrimination Perfect Match and Mismatch 1 OK Discrimination Perfect Match and Mismatch 2 OK spot quality OK negative controls OK No gradient was observed

43 ° C / HS 400 PT3

CV Perfect Match OK CV Mismatch 1 OK Discrimination Perfect Match and Mismatch 1 OK Discrimination Perfect Match and Mismatch 2 OK spot quality OK negative controls OK No gradient was observed

61 ° C / HS 400 SN22

CV Perfect Match OK CV Mismatch 1 OK Discrimination Perfect Match and Mismatch 1 OK Discrimination Perfect Match and Mismatch 2 OK spot quality # 33 failed, # 34 OK Negative Controls OK No gradient was observed

61 ° C / HS 400 PT3

CV Perfect Match OK CV Mismatch 1 OK Discrimination Perfect Match and Mismatch 1 OK Discrimination Perfect Match and Mismatch 2 OK spot quality failed negative controls OK No gradient was observed

The results show not only that both devices used give very useful results. Rather, the results obtained in the two devices SN22 and PT3 (ordered by temperature) are so similar to each other that an influence of the increased pressure on the hybridization can be excluded.

The inventive method is not limited to the use in hybridization. It can also be used in other devices or used to prevent the occurrence of unwanted air bubbles. Such devices or instruments may e.g. from the field of microfluidics technology, such as "lab on a chip" systems.

FIG. 6 shows a diagram of a second device or system suitable for carrying out the method according to the invention. This system 1 'differs from the system 1 shown in FIG. 1 essentially in that each lid 26 is assigned two hybridizing chambers 5A, 5B. These two hybridization chambers 5A, 5B can be arranged over a single slide with, for example, two identical DNA microarrays. However, it is also possible to use two smaller slides 27, each of which together with one cover 26 only defines one hybridization chamber 5 (not shown). Each Hybridisierkammer 5A, 5B is supplied by the distribution line 10 via a respective individual inlet valve 12A, 12B and one individual inlet line 11A, 11B with liquids, such as washing buffer or Hybridisiermedien. Thus, all Hybridisierkammern either self-sufficient, ie independently or jointly, ie operated simultaneously.

In order to be able to move the liquids present in each of the two hybridization chambers 5A, 5B under a cover 26 relative to the samples immobilized on the slides, each cover 26 of this particularly preferred system 1 'has its own hybridization chamber 5A, 5B each with its own This agitation space 30 is in each case connected to an agitation line 36 with the hybridization chamber 5A, 5B, wherein the membrane 33 separates the agitation space 30 from a pressure space 34. These pressure chambers 34 are filled via the pressure line 35 with a pressurized fluid and sporadically, for example, acted upon pulsating with the agitation pressure. In Fig. 6, the membranes 33 of the hybridization chambers 5A, 5B are charged in series, i. synchronous. This has the advantage of a simplified construction, in which after the pressure relief valve 43, no further valves or control elements in the pressure distribution line 39 or in the leading to the pressure chambers 34 pressure lines 35 must be provided. However, it can also be provided that each diaphragm 33 is pressurized with a separate pressure line 35 (parallel) (not shown).

The outlets 13A, 13B of a lid 26 open into the outlet 13 of the same and are, as already described in Figure 1, supplied via the outlet valve 14 of the manifold 15. According to a preferred embodiment of this system 1 ', the outlet duct 13 has an enlarged diameter and thus provides the space in which a gas cushion serving as a spring element can be accommodated. This spring element is - directly via a continuous fluid line, or indirectly (separated by a membrane) - in communication with the volume of liquid used for the hybridization. This spring element resiliently counteracts the agitation pressure differences exerted by the agitation device 32 on the pressure chamber 34 (similar to what has already been described in connection with FIG. 1). This ensures that the chamber pressure, which is built up by means of a pressure device for preventing air bubbles in the Hybridisierkammern 5, regardless of Agitationsdruckunterschieden over the prevailing in the ambient air, normal atmospheric Pressure can be maintained. As spring elements can also serve membranes which are acted upon directly by the media in the Hybridisierkammern 5, or which are separated by a liquid pad or a gas cushion from these media. A spring element - no matter how it is formed - is preferably elastic or compressible or stretchable and compensates for the Agitationsdruckunterschiede in the Hybridisierkammern 5, so that the chamber pressure over a certain time, for example during the entire hybridization, so far above that in the ambient air prevailing, normal atmospheric pressure is that in the Hybridisiermedium no gas bubbles can form.

In addition to the system 1 described therein, the preferred system 1 'presented here comprises a blow line 62, which via a blow valve 63 preferably leads straight to the point where the outlet 13B opens into the outlet line 13.

This blow line 62 is operated as follows:
First, each hybridization chamber 5A, 5B is filled with wash buffer. In this case, the inlet lines 11A, 11B and the outlets 13A, 13B and the outlet 13 are also filled with washing buffer. After the sample liquids are pipetted in, the pattern feeders 31 and the inlet valves 12A, 12B are closed again. The exhaust valves 14 remain open. If the blow valves 63 are now opened, gas from a compressor or a pressure accumulator flows via the individual blow lines 64 into the outlet lines 13 and displaces the wash buffer from these outlet lines 13. This gas then forms the gas cushion in the outlet line 13 , The gas cushion has the necessary volume to successfully serve as a spring element. Due to the pressure peaks occurring during agitation, the gas in the outlet lines is compressed accordingly and springs back when the agitation pressure decreases. This results in the desired pendulum movement of the media in the Hybridisierkammern 5. In this embodiment just described, the spring element can be regarded as part of the Agitationsmechanismus; in fact, it is not necessary here to attach a second membrane 33 '. However, if the cross section of the outlet 13 is too small, In order to make available the entire space required for the spring element effect, a second membrane 33 '(cf., for example, FIG. 1) can additionally be provided.

Figure 7 shows a schematic view of a particular, first arrangement of a lid 26 with a hybridization chamber 5, seen from below. The essential difference from the embodiment already shown in FIG. 3 lies here in the outlet conduit 13 with the enlarged diameter, whereby the space for a gas cushion serving as a spring element is made available. The individual outlet 13 'of the hybridization chamber 5 opens into this outlet line 13 in the vicinity of its end. The individual blow line 64 assigned to this hybridization chamber 5 also discharges in the same region. All media lines each have a connection opening which is located in the common connection plane 57 of the Cover 26 is located. These five openings are preferably arranged on a line, but they can also be arranged arbitrarily. All other parts correspond to the embodiment of Fig. 3. In this embodiment, a second membrane 33 'is provided, which closes a second agitation space 30'. The second diaphragm 33 'in this case serves to support the spring element arranged as a gas cushion in the outlet line 13. The second agitation space 30 'is here designed as a closed space and contains a gas cushion, which is formed on the membrane 33' compressible and re-expandable. If the outlet line 13 provides a sufficiently large gas cushion, the second diaphragm 33 'and the second agitation space 30' can be dispensed with (see FIG.

Figure 8 shows a schematic view of a particular, second arrangement of a lid 26 with two Hybridisierkammern 5A, 5B, seen from below. Like the embodiment shown in FIG. 7, here the cover 26 has an outlet duct 13 with the enlarged diameter, whereby the space for a gas cushion serving as a spring element is made available. In this case, the provided cavity is sufficient to guarantee the effect as a spring element, therefore, both hybridization chambers 5A, 5B lying behind one another have no second membrane 33 'and no second agitation space 30'. The two individual outlets 13A, 13B open into this outlet line 13, wherein an orifice near the end and the other mouth is located somewhere in a central region of the outlet duct 13. The agitation in the Hybridisierkammern 5A, 5B is operated via a common Agitationsleitung 35.

Alternatively, an individual agitation line may each lead to the agitation chambers 30 of the two hybridization chambers 5A, 5B (not shown). In this alternative case, the pressure line 35 could branch within the lid, so that only one common connection for the individual pressure lines on the common connection plane 57 of the lid 26 would be necessary. However, it could also be provided to provide the two hybridization chambers 5A, 5B each with an individual and independent agitation device 32. This would be accomplished with an additional pressure line 42 'and pressure distribution line 39' and with an individual agitation line 35 'which can be filled per hybridization chamber 5A, 5B via an additional connection in the common connection plane 57 of the cover 26 (not shown).

By analogy, more than two, e.g. three hybridization chambers 5 per cover 26 are arranged. The number of Hybridisierkammern 5 per cover 26 is limited only by the practicality of the arrangement of the necessary supply and discharge lines, seals and Agitationsvorrichtungen. Optionally, a single slide 27 may have a plurality of regions preferably provided with a nucleotide array, so that a plurality of hybridization chambers 5 per cover 26 and slide 27 may be operated with a divided cover 26 (see for example Fig. 8). However, smaller slides 27 'may also be provided which essentially define just a single, smaller hybridization chamber 5 so that the plurality of hybridization chambers 5 per cover 26, but on a plurality of slides 27, can be operated.

Claims (20)

A system (1) comprising hybridization chambers (5) for hybridizing nucleic acid samples, proteins or tissue sections immobilized on slides (27), each hybridization chamber (5) being defined as a substantially gap-like, liquid-substantially fillable space between one of said slides (27). and a lid (26), wherein the lid (26) is disposed opposite to the slide (27), that the hybridization chamber (5) is sealed from the ambient air, and wherein the system (1) comprises an agitator (32) in which liquids can be moved in the hybridization chambers (5) with respect to the samples immobilized on the slides (27), characterized in that the system comprises means for preventing air bubbles in the hybridization chambers (5) serving as pressure means for constructing a Chamber pressure in the Hybridisierkammern (5) is formed, whereby this chamber pressure thereby una Depending on agitation pressure differences above the normal atmospheric pressure prevailing in the ambient air, the system (1) comprises at least one spring element, which communicates with the liquid volume used for the hybridization and resiliently flows through the agitation device (32) onto the pressure chamber ( 34) counteracts applied Agitationsdruckunterschieden. System (1) according to claim 1, characterized in that the pressure device is designed to build up a chamber pressure in a range between 100 mbar to 1.4 bar above the normal atmospheric pressure in the ambient air. System (1) according to claim 1 or 2, characterized in that the pressure device comprises a pressure source (8,3) which can be connected via pressurizable lines (9,51) with the Hybridisierkammern (5). System (1) according to claim 3, characterized in that the pressure source comprises a feed pump (8) or a compressed gas cylinder (3). System (1) according to claim 3 or 4, characterized in that the pressurizable lines (9,51) a connecting valve (19), a distribution line (10), a manifold (15) and for each of the Hybridisierkammern (5) a Include inlet valve (12) and an outlet valve (14). System (1) according to claim 5, characterized in that the pressurizable lines (9, 51) further comprise a waste line (16) with a waste valve (17) and a discharge line (20) with a discharge valve (21). System (1) according to one of the preceding claims, characterized in that the agitation device (32) comprises a positive pressure pump (46) and an overpressure container (47), which are each connectable to a pressure chamber (34) of a lid (26), wherein the Cover (26) also comprises a membrane (33) which separates the pressure chamber (34) from an agitation space (30). System (1) according to claim 7, characterized in that the agitation device (32) comprises a vacuum pump (48) and a vacuum container (49) which are each connectable to a pressure chamber (34) of a lid (26), wherein the lid ( 26) further comprises a membrane (33) which separates the pressure space (34) from an agitation space (30). System (1) according to one of the preceding claims, characterized in that the spring element is designed as a cover (26) arranged in the second membrane (33 '). System (1) according to any one of claims 1 to 8, characterized in that the spring element is a gas cushion associated with a hybridization chamber (5) during the hybridization process. System (1) according to claim 10, characterized in that the spring element as in the outlet (13) in the lid (26) arranged, in connection with the Hybridisierkammer (5) standing gas cushion is formed. System (1) according to claim 10, characterized in that the spring element as in the manifold (15) between the waste valve (17), the connecting valve (19) and the relief valve (21) arranged air space is formed. System (1) according to any one of the preceding claims, characterized in that it comprises lids (26) defining two or more hybridising chambers (5A, 5B). A method for preventing air bubbles in the hybridization chambers (5) of a system (1) for hybridizing nucleic acid samples, proteins or tissue slices immobilized on slides (27), according to which each substantially slit-like, between one of these slides (27) and a lid (26 ) is filled with a liquid substantially, wherein the lid (26) is disposed opposite to the slide (27), that the Hybridisierkammer (5) is sealed from the ambient air, and wherein the system (1) a Agitation device (32), with which liquids in the hybridization chambers (5) can be moved relative to the specimens immobilized on the slides (27), characterized in that a pressure device of this system (1) in the Hybridisierkammern (5) a chamber Pressure is formed, thereby independent of Agitationsdruckunterschieden over that in the ambient air At the normal atmospheric pressure prevailing, the system (1) comprises a spring element, which communicates with the volume of liquid used for the hybridization and resiliently counteracts the Agitationsdruckunterschieden exerted by the agitator (32) on the pressure chamber (34). A method according to claim 14, characterized in that a chamber pressure is generated which is at least 100 mbar to a maximum of 1.4 bar higher than the normal ambient pressure. A method according to claim 14 or 15, characterized in that the chamber pressure is generated by means of a liquid or by means of a gas. A method according to claim 16, characterized in that the liquid for generating the chamber pressure is one of a feed pump (8) in a distribution line (10) and via inlet valves (12) against the liquid in the Hybridisierkammern (5) pressed Hybridisiermedium. A method according to claim 16, characterized in that the gas for generating the chamber pressure from a feed pump (8) via a vent valve (9) sucked air or in a container (3) stored inert gas, said gas via a distribution line ( 10) and is pressed via inlet valves (11) against the liquid in the Hybridisierkammern (5). A method according to any one of claims 14 to 18, characterized in that in an in the cover (26) arranged and opposite a agitation space (30) with a membrane (33) separated pressure chamber (34) an agitation pressure is built up, which by 0.5 to 1 bar higher than the desired chamber pressure A method according to any one of claims 14 to 19, characterized in that a second, in the cover (26) arranged membrane (33 '), or filled with a gas volume of a manifold (15) or an outlet conduit (13) are used as a spring element , which counteracts these agitation pressure differences.

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