EP2255880B1 - System and method to prevent air bubbles in a hybridization chamber - Google Patents

Description

The invention relates, according to the preamble of independent claim 1, to a method of preventing air bubbles in the hybridization chambers of a system for hybridizing nucleic acid samples, proteins or tissue sections immobilized on slides. This method comprises providing a substantially gap-shaped hybridization chamber between a slide and a lid, wherein the lid is arranged opposite the slide such that the hybridization chamber is sealed from the ambient air. Furthermore, this method comprises substantially filling the provided hybridization chamber with a liquid.

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 samples from many genes on a solid substrate surface, such as on a glass slide for a light microscope. The DNA samples are preferably arranged in an array of sample spots or "spots", ie in a two-dimensional grid on the substrate, and it is later possible to deduce the origin of the corresponding DNA sample from a specific position within such an array. The technique typically involves contacting the DNA sample array with RNA pattern suspensions (RNA = ribose nucleic acid) with specific nucleotide sequences in them Detect DNA samples. Typically, pattern suspensions are also used which contain DNA, cDNA and / or proteins or polypeptides. RNA patterns can be provided with a so-called "tag" or "label", ie a molecule which, for example, emits a fluorescent light with a specific wavelength. Immobilized samples may also include amino acid-containing (eg, proteins, peptides) or nucleic acid-containing (eg, cDNA, RNA) samples. 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 established themselves as successful tools and devices for performing DNA hybridization have been continually improved (see, eg US 6,238,910 or EP 1 260 265 A1 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 a movement of the liquid with eg RNA patterns towards DNA samples immobilized on the slide is 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, an attempt was made (cf., eg US 6,186,659 ), Targeted use of air bubbles as an agitating agent in order to achieve a thorough mixing of the reagents in the hybridization. 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 (cf. US 5,922,591 ), or the hybridization media are transported out of the chamber and back into it throughout the hybridization process. The admixture of agents to the hybridization medium which reduce surface tension or treat the surfaces of the chamber with water repellent chemical compounds with the aim of hindering the formation of air bubbles is also known.

Out US 6,458,526 For example, 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 becomes a saturated Atmosphere 150 maintained, which is constantly connected to the gas spaces behind the "bubble halves 140" (see. Fig. 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 is to provide an alternative method by which the formation of air bubbles in a hybridization chamber can be prevented in a simple manner.

1. (a) das Bereitstellen einer im Wesentlichen spaltförmigen Hybridisierkammer zwischen einem Objektträger und einem Deckel, wobei der Deckel derart gegenüber dem Objektträger angeordnet wird, dass die Hybridisierkammer gegenüber der Umgebungsluft abgedichtet ist; und
2. (b) das im wesentlichen Füllen der gemäss Schritt (a) bereitgestellten Hybridisierkammer mit einer Flüssigkeit.

This object is achieved according to the features of independent claim 1 by proposing a method for preventing air bubbles in a substantially gap-shaped hybridization chamber of a system for hybridizing nucleic acid samples, proteins or tissue sections immobilized on slides, the method comprising:

1. (a) providing a substantially gap-shaped hybridization chamber between a slide and a lid, wherein the lid is disposed opposite the slide so that the hybridization chamber is sealed from the ambient air; and
2. (b) substantially filling the hybridization chamber provided according to step (a) with a liquid.

• (c) das Ausbilden eines Kammer-Drucks in der Hybridisierkammer mit einer Druckeinrichtung dieses Systems;
• (d) das Aufrechterhalten dieses Kammer-Drucks während eines Hybridisiervorgangs auf einem Wert, der mindestens 100 mbar bis maximal um 1.4 bar über dem in der Umgebungsluft herrschenden, normalen atmosphärischen Druck liegt,
• (e) ein stossweises Aufbauen eines Agitationsdrucks in einem Druckraum (34), wobei der Agitationsdruck um 0.5 bis 1 bar höher ist als der Kammerdruck, wobei dieser Druckraum (34) im Deckel (26) angeordnet ist und durch eine Membran (33) von einem Agitationsraum (30) getrennt ist, und wobei der Agitationsraum (30) über eine Agitationsleitung (36) mit der Hybridisierkammer (5) verbunden ist; und
• (f) ein federndes Entgegenwirken von durch die Agitationseinrichtung (32) auf den Druckraum (34) ausgeübten Agitationsdruckunterschieden mit einem Federelement, das mit dem für die Hybridisierung verwendeten Flüssigkeitsvolumen in Verbindung steht, und das als zweite, im Deckel (26) angeordnete Membran (33') oder als ein mit einem Gas gefülltes Volumen einer Sammelleitung (15) oder einer Auslassleitung (13) ausgebildet ist.

The method according to the invention is characterized in that it additionally comprises:

• (c) forming a chamber pressure in the hybridization chamber with a pressure device of that system;
• (d) maintaining said chamber pressure during a hybridization operation at a value which is at least 100 mbar and at most 1.4 bar above the normal atmospheric pressure prevailing in the ambient air,
• (E) a jerky build up of a Agitationsdrucks in a pressure chamber (34), wherein the agitation pressure by 0.5 to 1 bar is higher than the chamber pressure, said pressure chamber (34) in the lid (26) is arranged and by a membrane (33) of an agitation space (30) is separated, and wherein the agitation space (30) via an agitation line (36) is connected to the hybridization chamber (5); and
• (f) a resilient counteraction of agitation pressure differences exerted by the agitation device (32) on the pressure chamber (34) with a spring element which communicates with the volume of liquid used for the hybridization, and as a second membrane (26) arranged in the cover (26). 33 ') or as a gas-filled volume of a manifold (15) or an outlet conduit (13) is formed.

Additional, preferred inventive features emerge from the dependent claims.

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 aufge- klappt;

Fig. 5

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

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 method according to the invention and a system for carrying it out 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 the Fig. 1 from EP 1 260 265 A1 corresponding vertical longitudinal section through an arrangement with a hybridization chamber;

Fig. 3

one of the Fig. 2 corresponding schematic view of an arrangement with a hybridization chamber, seen from below;

Fig. 4

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

Fig. 5

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

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.

FIG. 1 1 shows a diagram of a system 1 suitable for carrying out the method according to the invention. On the left side of this scheme are a number of containers 2 for the storage of liquid hybridising 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) shown. Each of these vessels 2 is preceded by an individual valve 4, via which these media the (on the right side shown) Hybridisierkammern 5 (S 1, S 2, S 3, S 4) can be supplied. 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 hybridization 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 connection line 18 branches off a discharge line 20 with a relief valve 21 and opens into a collecting container 22 with a loading or vent 23. The collection container 22 is followed by 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 these from the European patent application EP 1 260 265 A1 the applicant of the current patent application is known. On the content of this patent application EP 1 260 265 A1 is hereby incorporated by reference, so that this content is considered part of the present patent application.

FIG. 2 shows one of the Fig. 1 from EP 1 260 265 A1 The cover 26 of this arrangement is movable relative to the slide 27 (here pivotable about an axis, so that the hybridization space 5 can be opened and closed by a simple movement.) An annular sealing surface 28 serves to close off This sealing surface 28 may be a stepped surface of the lid 26 lying flat on the surface 29 of the slide 27, alternatively, for example, a lip seal may be used Ring seal as a sealing surface 28. The arrangement includes cables 11,13 for feeding or discharging media in the hybridization 5 in or out of the hybridization 5 out. Such media may be reagents for carrying out the hybridization reaction, such as wash liquors or buffer solutions, but also inert gases (such as nitrogen) for drying the hybridization products on the slides 27 or for blowing out the hybridization chambers 5 and the media lines 11,13. 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 in different embodiments EP 1 260 265 A1 are disclosed.

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 in FIG. 2 In the embodiment shown, the agitation device 32 of the arrangement comprises a membrane 33. This membrane 33 separates a pressure space 34, which is filled with a pressurized fluid (gas or liquid) via a pressure line 35, from an agitation space 30 which communicates with the hybridization space via an agitation conduit 36 5 is connected. 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 transverse flow channels 38, 38 'also serve as a liquid reservoir, so that the pendulum movement (filled double arrow) generated by the agitator device 32 built into the device does not lead to the sample solution causing parts of the hybridization chamber 5 to be 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. 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.

FIG. 3 shows a plan view of the arrangement of FIG. 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 Hybridisierungsraums 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. The agitation 36, 36 'and the pattern feed 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 conduits 11, 13, 35 may be arranged as shown offset relative to one another or on a line (not shown) running transversely to the device 1. Recesses (empty arrows, cf. Fig. 2 ) reduce heat flow from or to the lid 26.

The pressure lines 35, of which one is intended for each of the hybridization chambers 5, are shown in FIG FIG. 1 shown by dashed lines 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 (eg with nitrogen gas), depending on the valve positions, all the hybridization chambers 5 (individually or in groups) can be blown out via the distribution line 10 and the inlet lines 11 via the outlet lines 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. Become only the gas valve 50, the connecting valve 19 and the waste valve 17 are opened, the distribution line 10 and the manifold 15 can be blown out via the waste line 16.

FIG. 4 shows one of the Fig. 2 corresponding, vertical longitudinal section through an arrangement with a hybridization chamber 5, wherein the folding frame 54 is unfolded with the lid 26 inserted therein of the system 1. 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 one of the Fig. 2 corresponding vertical longitudinal section through an arrangement with a Hybridisierkammer 5, wherein the inserted into a snap frame 54 cover 26 of the system is closed. All four hybridization rooms 5 of a defined by a contact plate 53 and such a folding frame 54 group of four 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. In order for the seals 28 to reliably seal the hybridization spaces 5, additional pressure is exerted on the covers 26 via the snap-on frame 54; 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 Hybridisierkammern 5 by means of a liquid, such as by means of one of the feed pump 8 in the Hybridisierkammern 5 pressed Hybridisiermedium be reached from one of the vessels 2 (see. Fig. 1 ). 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 provided a spring-loaded expansion vessel (not shown), which is connected via a line to the hybridization chamber 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, there is the possibility of a pressure pump and a pressure vessel (similar to the elements designated 46 and 47 in Fig. 1 ) gas into the distribution line 10 to initiate. 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 (cf. Fig. 1 ) are kept open.

Another alternative (not shown in the figures) is to use a pressure pump and a pressure vessel (similar to the elements labeled 46 and 47 in FIG Fig. 1 ) or a gas container (such as the N ₂ container 3 in Fig. 1 ) to connect to the manifold 15 and the pressure in the Hybridisierkammern 5 to build on 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 those shown at 46 and 47 in FIG Fig. 1 ) or from a gas container (such as the N ₂ container 3 in Fig. 1 ) to lead gas in one of the lines which open into at least one of the distribution line 10 or manifold 15. Accordingly, one or both of the valves 12, 14 (cf. Fig. 1 ) are 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. 0.6 - 2.4 bar. The second diaphragm 33 'forms in this case a spring element which acts resiliently against this agitation pressure.

1. 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.
2. 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.
3. 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.
4. 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 entsprechendes 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.
5. 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 Kammer-druck wie schon beschrieben erhöht und die Agitationseinrichtung 32 in Betrieb genommen.
   Alle eventuell in den Hybridisierkammern 5 vorhandenen Luftblasen werden bei diesem Druckaufbau eliminiert.
6. 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.
7. 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.
8. 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:

1. 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) on the contact plate with specimens immobilized thereon 53 of a thermostat, by the insertion of lids 26 in the snap frame 54 of the system 1 for hybridizing nucleic acid samples immobilized on slides 27, proteins or tissue sections and by closing the Hybridisierkammern 5 by folding down the lid 26 on the slides 24 (see. 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 agitator 32 of each individual hybridization chamber 5 to the respective duct ends of the system 1. Preferably, four lids 26 are formed into one according to the four slides 27 received in a transport frame 52 Folding frame 54 used.
2. 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.
3. 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.
4. 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 from the hybridization chambers 5 is introduced into the outlet line 13 with the outlet valves open 14 displaced. This in turn displaces a corresponding volume of air from the manifold 15. This displaced air flows through the open relief valve 21 into the relief 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.
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 agitation device 32 is put into operation.
   Any air bubbles that may be present in the hybridization chambers 5 are eliminated in this pressure build-up.
6. 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.
7. 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 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 pumped into 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.
8. 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 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 ad) 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. It concerns a standard device of the applicant (Tecan HS 400, serial number 22, briefly SN22 called), which at normal pressure was operated 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 operated at about 0.5 bar, that in PT3 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 kit is designed to validate hybridization systems and is designed to pre-define the hybridization temperatures and to deliver the "OK" signals only if the intended temperatures and test parameters related to washing, agitation and drying are maintained accurately. The samples immobilized on the slides 27 are these are oligonucleotides whose sensitivity to temperature differences is known.

1. 1. Erstes Waschen bei 43 °C bzw. 61 °C, Dauer 30 sec;
2. 2. Injektion von 105 µl der Oligonukleotidlösung bei 43 °C bzw. 61 °C in jede Kammer;
3. 3. Hybridisierung bei 43 °C bzw. 61 °C; bei starker Agitation und während 60 min;
4. 4. Waschschritt 1 bei 23 °C (Kanal 1) während 30 sec; Absaugen während 30 sec; 2 Wiederholungen;
5. 5. Waschschritt 2 bei 23 °C (Kanal 2) während 30 sec; Absaugen während 30 sec; 2 Wiederholungen;
6. 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:The executed program for each test run is in detail:
a) Preparation of "HybCheck Wash Buffers 1 and 2 It. Manufacturer data;
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. 1. First wash at 43 ° C or 61 ° C, duration 30 sec;
2. 2. Injection of 105 μl of the oligonucleotide solution at 43 ° C or 61 ° C into each chamber;
3. 3. hybridization at 43 ° C or 61 ° C; with strong agitation and during 60 min;
4. 4th washing step 1 at 23 ° C (channel 1) for 30 sec; Suction for 30 sec; 2 repetitions;
5. 5. wash step 2 at 23 ° C (channel 2) for 30 sec; Suction for 30 sec; 2 repetitions;
6. 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. 1. 6 sub arrays (6x9 spots) on a microscope slide (Microslide)
2. 2. Perfect Match (PM) at 43 ° C: 24 spots per slide (4 per sub array)
3. 3. Mismatch 1 (MM1) at 43 ° C: 24 spots per slide (4 per sub array)
4. 4. Mismatch 2 (MM2) at 43 ° C: 24 spots per slide (4 per sub array)
5. 5. Negative Controls: 36 spots per slide (6 per sub array)
6. 6. Of all PM's 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. 7. Discrimination PM: MM1 (1: 5) as well as Discrimination PM: MM2 (1:20) calculated
8. 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. 1. 6 sub arrays (6x9 spots) on a microscope slide (Microslide)
2. 2. Perfect Match (PM) at 61 ° C: 24 spots per slide (4 per sub array)
3. 3. Mismatch 1 (MM1) at 61 ° C: 24 spots per slide (4 per sub array)
4. 4. Mismatch 2 (MM2) at 61 ° C: 24 spots per slide (4 per sub array)
5. 5. Negative controls: 36 spots per slide (6 per sub array)
6. 6. Of all PM's 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. 7. Discrimination PM: MM1 (1: 5) as well as Discrimination PM: MM2 (1:20) calculated
8. 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 FIG. 6 shows a schematic of a suitable for carrying out the inventive method, second device or system. This system 1 'differs from that in FIG. 1 shown system 1 essentially by the fact that each lid 26 two Hybridisierkammern 5A, 5B are assigned. 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 an agitation room 30 provided with a membrane 33. This agitation room 30 is connected in each case with an agitation line 36 to the hybridization chamber 5A, 5B, the membrane 33 separating 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 supplied in series, ie synchronously. 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 at FIG. 1 described, fed via the exhaust 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 mentioned in connection with FIG FIG. 1 described). 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 above 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 ruling, 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.

• Zuerst wird jede Hybridisierkammer 5A,5B mit Waschpuffer gefüllt. Dabei werden die Einlassleitungen 11A,11B sowie die Auslässe 13A,13B und die Auslassleitung 13 ebenfalls mit Waschpuffer gefüllt. Nach dem Einpipetieren der Musterflüssigkeiten werden die Musterzuführungen 31 und die Einlassventile 12A,12B wieder geschlossen. Die Auslassventile 14 bleiben geöffnet. Werden nun die Blasventile 63 geöffnet, so dringt Gas aus einem Kompressor oder einem Druckspeicher über die individuellen Blasleitungen 64 in die Auslassleitungen 13 ein und verdrängt den Waschpuffer aus diesen Auslassleitungen 13. Dieses Gas bildet dann das Gaskissen in der Auslassleitung 13. Durch deren vergrösserten Durchmesser, weist das Gaskissen das notwendige Volumen auf, um erfolgreich als Federelement dienen zu können. Durch die beim Agitieren auftretenden Druckspitzen wird das Gas in den Auslassleitungen entsprechend komprimiert und federt beim Nachlassen des Agitationsdruckes zurück. Dadurch entsteht die gewünschte Pendelbewegung der Medien im den Hybridisierkammern 5. In dieser eben beschriebenen Ausführungsform kann das Federelement als Teil des Agitationsmechanismus betrachtet werden; tatsächlich erübrigt sich hier das Anbringen einer zweiten Membran 33'. Ist allerdings der Querschnitt der Auslassleitung 13 zu klein, um den ganzen für die Federelement-Wirkung benötigten Raum zur Verfügung zu stellen, so kann ergänzend doch eine zweite Membran 33' (vgl. z.B. Fig. 1) vorgesehen werden.

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 '. If, however, the cross-section of the outlet line 13 is too small to make the entire space required for the spring element effect available, then a second membrane 33 '(cf., for example, FIG Fig. 1 ).

FIG. 7 shows a schematic view of a particular, first arrangement of a lid 26 with a hybridization chamber 5, seen from below. The main difference to the in FIG. 3 already shown embodiment is here in the outlet 13 with the enlarged diameter, whereby the space is provided for serving as a spring element gas cushion. 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 membrane 33 'and the second agitation space 30' can be dispensed with (cf. Fig. 8 ).

FIG. 8 shows a schematic view of a particular, second arrangement of a lid 26 with two hybridization chambers 5A, 5B, seen from below. Like the in FIG. 7 In the embodiment shown 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 provided. 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 duct 13, one mouth near the end and the other mouth lying 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 cover 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).

Analogously, more than two, for example, three hybridization chambers 5 per cover 26 could be 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 with a divided cover 26 (cf. Fig. 8 ) several hybridization chambers 5 per cover 26 and slide 27 can be operated. 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 (5)

1. Method to prevent air bubbles in a substantially slot-shaped hybridization chamber (5) of a system (1) for hybridizing nucleic acid specimens, proteins or tissue sections immobilized on specimen slides (27), wherein the method comprises:

   (a) providing a substantially slot-shaped hybridization chamber (5) between a specimen slide (27) and a cover (26), wherein the cover (26) is arranged with respect to the specimen slide (27) in such a way that the hybridization chamber (5) is sealed against ambient air; and

   (b) substantial filling of the hybridization chamber (5) provided according to step (a) with a liquid;
   characterized in that the method further comprises:

   (c) formation of a chamber pressure in the hybridization chamber (5) with a pressure device of said system (1);

   (d) maintaining said chamber pressure during a hybridization process to a value which lies at least 100 mbar to a maximum of 1.4 bar above the normal atmospheric pressure prevailing in ambient air;

   (e) pulsating build-up of an agitation pressure in a pressure chamber (34), wherein the agitation pressure is higher by 0.5 to 1 bar than the chamber pressure, wherein said pressure chamber (34) is arranged in the cover (26) and is separated by a membrane (33) from an agitation chamber (30), and wherein the agitation chamber (30) is connected via an agitation line (36) to the hybridization chamber (5); and

   (f) resilient counteracting of agitation pressure differences caused by the agitation device (32) on the pressure chamber (34) by a spring element which is in connection with the liquid volume used for hybridization and which is formed as the second membrane (33') arranged in the cover (26) or as a gas-filled volume of a collecting line (25) or an outlet line (13).
2. Method according to claim 1, characterized in that the chamber pressure is generated by means of a liquid or by means of a gas.
3. Method according to claim 2, characterized in that the liquid for generating the chamber pressure is a hybridization medium which is pressed by a feed pump (8) into a distribution line (10) and via inlet valves (12) against the liquid in the hybridization chamber (5).
4. Method according to claim 2, characterized in that the gas for generating the chamber pressure is air aspirated by a feed pump (8) via a venting valve (19) or an inert gas stored in a container (3), wherein said gas is pressed via a distribution line (10) and via inlet valves (11) against the liquid in the hybridization chamber (5).
5. Method according to claim 1, characterized in that the chamber pressure is held irrespective of the agitation pressure differences by 0.1 bar to a maximum of 1.4 bar above the normal atmospheric pressure prevailing in ambient air.

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