DE202004012163U1 - System for hybridizing immobilized nucleic acid samples, in sealed hybridizing chambers, has a pressure assembly to build up the chamber pressures over ambient atmospheric levels to prevent the formation of air bubbles - 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

The According to the invention the generic term of the independent Claim 1 a system with hybridization chambers for hybridization from on slides immobilized nucleic acid samples, Proteins or tissue sections. Every hybridization chamber is there as an essentially gap-shaped, with a liquid in the essentially fillable Space defined between one of these slides and a lid. A cover is opposite each other a slide arranged that the hybridization chamber sealed from the ambient air is. Such a system includes a preventive device of air bubbles in the hybridization chambers. The invention relates moreover, according to the generic term of the independent Claim 12, a corresponding method for preventing air bubbles in the hybridization chambers of a system for hybridizing on slides immobilized nucleic acid samples, Proteins or tissue sections. According to this procedure all essentially slit-shaped, between one of these slides and Hybridization chambers with a liquid arranged on a cover essentially filled. The lid is placed opposite the slide in such a way that the hybridization chamber opposite the ambient air is sealed.

The Use of DNA samples (DNA = deoxyribose nucleic acid) and research in particular of microarrays of such samples an important technique for simultaneous or simultaneous analysis of thousands of genes available. This technique involves the immobilization of DNA samples from many genes on a solid substrate surface, such as. on a glass slides for a Light microscope. The DNA samples are preferably in an array of Sample spots or "spots", i.e. in a two-dimensional Grid arranged on the substrate and you can later - starting from a certain position within such an array infer the origin of the corresponding DNA sample. The technology includes typically contacting the DNA sample array with RNA sample suspensions or solutions (RNA = ribose nucleic acid) to detect specific nucleotide sequences in the DNA samples. Typically, sample suspensions are also used, which Contain DNA, cDNA and / or proteins or polypeptides.

RNA samples can with a so-called "tag" or "label", i.e. provided with a molecule which e.g. emits a fluorescent light with a specific wavelength. Immobilized samples can also amino acid-containing (e.g. proteins, peptides) or nucleic acid (e.g. cDNA, RNA) Include samples. Patterns added to the immobilized samples can any molecules or chemical compounds, which with the immobilized Hybridize samples or otherwise connect to them.

Under good experimental conditions hybridize or bind the RNA patterns to the immobilized DNA samples and together form hybrid ones DNA-RNA strands. For every of the immobilized DNA samples and for special RNA patterns differences in hybridization among DNA samples due to the Measurement of the intensity and the wavelength dependency the fluorescence of each individual microarray element and so find out whether the level of gene expression in the examined DNA samples varied. With the use of DNA microarrays you can use the Expression of large amounts of genes and their expression pattern Comprehensive statements are made, although only small amounts of biological material must be used.

DNA microarrays have established themselves as successful tools and the devices for performing DNA hybridization have been continuously improved (cf. e.g. US 6,238,910 or EP 1 260 265 A1 of the applicant of the current patent application). These documents disclose an apparatus 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 specimen slide and comprise an annular seal or sealing surface for closing off the gap-shaped hybridization space from the ambient air, a surface of this specimen slide being acted upon by the seal or sealing surface. In addition, these devices include lines for supplying and discharging media into and out of the hybridization space as well as a sample supply. Improved temperature control and movement of the liquid with, for example, RNA patterns compared to DNA samples immobilized on the slide is also disclosed.

On the one hand, it happens again and again that air bubbles appear when filling liquids or later in the hybridization chamber. On the other hand, an attempt was made (cf. e.g. US 6,186,659 ) To use air bubbles specifically as agitating agents in order to achieve a more thorough mixing of the reagents in the hybridization chamber. In general, however, air bubbles present in the hybridization medium are not wishes because they disrupt the usually very thin liquid film over the immobilized samples. This can lead to an inhomogeneity in the distribution of reagents in the hybridization medium and thus to a falsification of the hybridization results; in the worst case, larger air bubbles even displace the hybridization medium from parts of the samples immobilized on the slide.

Numerous methods are also known from the prior art to hinder the spontaneous occurrence of air bubbles or their remaining in the chamber. For example, a non-parallel arrangement of the specimen slides and lids defining the hybridization chamber has been proposed (cf. US 5,922,591 ), or the hybridization media are conveyed out of and back into the chamber during the entire hybridization process. It is also known to add agents to the hybridizing medium which reduce the surface tension or to treat the surfaces of the chamber with water-repellent chemical compounds with the aim of preventing the formation of air bubbles.

Out US 6,458,526 An arrangement is known in which "bubble halves 140" protruding into the hybridization space are produced from a gas saturated with solvent. These "bubble halves" are actually dome-shaped interfaces of gas spaces with a defined radius of curvature. These "bubble halves" are located at defined points in the chamber where they cannot 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. A saturated atmosphere 150 is maintained above this solvent , which is constantly connected to the gas spaces behind the "bubble halves 140" (cf. 2 in US 6,458,526 ). This constantly brings an atmosphere saturated with the solvent to the dome-shaped interfaces, thereby influencing the partial pressure of the solvent present in the hybridizing medium in such a way that any air bubbles that may be present shrink and are eliminated. This method has the disadvantage that these dome-shaped interfaces have to be created and maintained using special devices.

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

This Task is according to the characterizing part of independent claim 1 thereby solved, that a system described in the introduction a printing device for Build up a pressure in the hybridization chamber that over the normal atmospheric conditions prevailing in the ambient air There is pressure. additional, preferred inventive features result from the dependent claims.

The invention System is now based on schematic, not the scope of the invention restrictive Drawings of exemplary embodiments explained in detail. there demonstrate:

1 a schematic of a device or system suitable for carrying out the method according to the invention;

2 one of the 1 of EP 1 260 265 A1 corresponding vertical longitudinal section through an arrangement with a hybridization chamber;

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

4 one of the 2 corresponding, vertical longitudinal section through an arrangement with a hybridization chamber, lid of the system opened;

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

1 shows a diagram of a system suitable for carrying out the method according to the invention 1 , On the left side of this scheme 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 liquid (AR), distilled water (AD), and a container 3 shown with inert gas (N2). Each of these vessels 2 is an individual valve 4 upstream, via which this media connects to the hybridization chambers (shown on the right) 5 (S 1, S 2, S 3, S 4) can be supplied. The the valve 4 comprehensive media lines 6 flow into a manifold 7 which in turn turns into a feed pump 8th leads to the a ventilation valve 9 is connected upstream. This feed pump 8th sucks over the manifold 7 liquid media from the vessels 2 and pumps them over the distribution line 10 into the inlet pipes 11 that have an inlet valve 12 into the hybridization chambers 5 lead. The hybridization media leave the hybridization chambers 5 via an outlet pipe 13 , each with an outlet valve 14 include and in a manifold 15 open out. This manifold 15 in turn leads to a waste line 16 which by means of a waste valve 17 is lockable. The distribution line 10 and the manifold 15 can via a connecting line 18 and a connection valve 19 communicate with each other. From this connecting line 18 branches a relief line 20 with a relief valve 21 and flows into a collection container 22 with a ventilation opening 23 , The collection container 22 a feed pump is connected downstream 24 that have a transition line 25 with the waste line 16 connected is.

For better distribution of the hybridization media in the hybridization chambers 5 is the system 1 with an agitation mechanism or with an agitation device 32 equipped, like this 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 reference is expressly made here, so that this content is considered part of the present patent application.

2 shows one of the 1 of EP 1 260 265 A1 corresponding, vertical longitudinal section through a hybridization chamber 5 , The lid 26 this arrangement is opposite the slide 27 movable (here pivotable about an axis, so that the hybridization space 5 can be opened and closed with a simple movement. An annular sealing surface 28 serves to lock the hybridization room 5 by applying a surface 29 this slide 27 , This sealing surface 28 can have a stepped surface of the lid 26 be what flat on the surface 29 of the slide 27 lies; alternatively, for example, a lip seal can also be used. However, an O-ring seal is preferred as the sealing surface 28 , The arrangement includes lines 11 . 13 for feeding and discharging media into the hybridization room 5 into or out of the hybridization room 5 out. Such media can contain reagents for carrying out the hybridization reaction, such as washing liquids or buffer solutions, but also inert gases (such as nitrogen) for drying the hybridization products on the slides 27 or to blow out the hybridization chambers 5 and the media lines 11 . 13 his. These inlets and outlets 11 . 13 for hybridization media preferably lead to an agitation room 30 . 30 ' , The arrangement also includes a closable sample feeder 31 through which RNA-containing liquids or other sample liquids can be pipetted in by hand. The sample feeder 31 is preferably closed with a plastic plug (not shown). As an alternative to this, an automatic or robotized sample feed can be provided, as is shown in different embodiments in FIG EP 1 260 265 A1 are disclosed.

The arrangement comprises a media-separating agitation device 32 for moving liquids opposite on the surface 29 the slide 27 immobilized samples of nucleic acids, proteins or tissue sections. In the in 2 The embodiment shown comprises the agitation device 32 the arrangement a membrane 33 , This membrane 33 separates a pressure room 34 that via a pressure line 35 is designed to be filled with a pressurized fluid (gas or liquid) from an agitation room 30 who has an agitation leadership 36 with the hybridization room 5 connected is. After reaching the thermal equilibrium of the arrangement, adding a certain volume of RNA sample liquid and closing the sample feeder 31 is via the pressure line 35 preferably air or another gas (but it could also be a liquid) intermittently into the pressure chamber 34 brought or drained from it so that the membrane 33 bends in the same rhythm and accordingly the agitation room 30 reduced or enlarged. As a result, the sample liquid becomes in the same rhythm of the positive or negative pressure and relaxation in the hybridization room 5 against one or the other end, where preferably against the inside of the hybridization space 5 facing surface 37 of the lid 26 one cross-flow channel each 38 . 38 ' located.

These cross-flow channels 38 . 38 ' on the one hand facilitate the cross-distribution of the RNA molecules contained in the sample solution. This has the effect that the sample liquid or the washing liquids are homogeneous over the whole in the hybridization space 5 present volumes are distributed. The cross-flow channels also serve 38 . 38 ' also as a liquid reservoir, so that in the agitation device built into the device 32 generated pendulum movement (filled double arrow) of the sample solution does not result in parts of the hybridization space 5 unintentionally lie dry.

Preferably there is a second one, also with a membrane 33 ' provided agitation room 30 ' via a second agitation line 36 ' with the hybridization room 5 connected. If now on the pressure room 34 pressure surge emitted the first membrane 33 in the first agitation room 30 presses, this impulse is sent through the first agitation line 36 to the sample liquid in the hybridization room 5 transfer. The sample liquid gives way slightly to the second agitation line 36 ' off (can even partially fill it) and increases the pressure in the second agitation room 30 ' , This causes the second membrane to bend 33 ' upwards and is stretched elastically. As soon as the overpressure in the pressure chamber 34 subsides, both membranes spring 33 . 33 ' back to their rest position and move the sample liquid in the hybridization room 4 in the opposite direction. By means of this pendulum movement, a sample liquid with a minimum volume (in the range of approx. 100 μl) can be practically homogeneous in the hybridization space in less than a minute with the arrangement shown 5 be distributed. Preferably immediately after the pressure reduction in the pressure chamber 34 a negative pressure in the pressure chamber 34 generated so that the backward movement of the sample liquid in the hybridization space opposite to the previous pressure surge 5 is reinforced.

3 shows a plan view of the arrangement of 2 , seen from below. The O-ring seal 28 laterally limits the hybridization space 5 , each of which has a cross-flow channel at its opposite ends 38 . 38 ' has, which as a depression in the surface 37 of the lid 26 are provided. The slide 27 (here a glass slide for light microscopy) and its handle field 55 are shown in dashed lines. The pull-off spring is also clearly visible 56 which on the grip field 55 of the slide 27 suppressed. When opening the hybridization room 5 makes this spring easier 56 the automatic separation of the slide 27 from the lid 26 , The lines of the inlet pipe are also visible 11 , the outlet pipe 13 and the pressure line 35 as well as the arrangement of the agitation rooms 30 . 30 ' and the sample feeder 31 , The agitation leaders 36 . 36 ' and the sample feeder 31 flow into the cross-flow channels 38 . 38 ' ,

All lines 11 . 13 . 35 for feeding or discharging media preferably lead to a common connection level 57 of the lid 26 , which are essentially parallel to the hybridization space 5 and preferably at the same level as the hybridization room 5 is arranged. The mouth openings of the lines 11 . 13 . 35 can be offset from one another as shown or on a cross to the front direction 1 extending line (not shown). Cutouts (empty arrows, cf. 2 ) reduce the heat flow from or to the lid 26 ,

The pressure lines 35 , of which for each of the hybridization chambers 5 one is determined are in 1 shown in dashed lines and go from a pressure distribution line 39 out. In this pressure distribution line 39 open a compensating line 40 with a compensating valve 41 , an overpressure supply line 42 with a pressure relief valve 43 and a vacuum supply line 44 with a vacuum valve 45 , The positive pressure is preferably with an inexpensive gas (eg air) in a positive pressure pump 46 generated in a pressure vessel 47 stored and in the overpressure supply line 42 fed. The vacuum is in a vacuum pump 48 generated in a vacuum container 49 stored and in the vacuum supply line 44 fed.

The inert gas container 3 is about a gas valve 50 and a gas pipe 51 with the distribution line 10 connected, which via inlet lines 11 and one inlet valve each 12 into the hybridization chambers 5 lead. With inert gas (eg with nitrogen gas) all hybridization chambers can, depending on the valve positions 5 (individually or in groups) via the distribution line 10 and the inlet pipes 11 via the outlet lines 13 and the manifold 15 be blown out.

Will only be the gas valve 50 , the connection valve 19 and the relief valve 21 opened, so the distribution line 10 in the collection container 22 be blown out. Will only be the gas valve 50 , the connection valve 19 and the waste valve 17 opened, so the distribution line 10 and the manifold 15 via the waste line 16 be blown out.

4 shows one of the 2 corresponding, vertical longitudinal section through an arrangement with a hybridization chamber 5 , with the snap frame 54 with the lid inserted in it 26 of the system 1 is opened. The lids are preferred 26 arranged parallel to each other and in a group of four, because this arrangement is just a measure of a contact plate 53 of the temperature control thermostat on which a transport frame 52 the size of a microplate with four slides arranged parallel to each other 27 fits. Each of these groups of four is a contact plate connected to a temperature control device 53 assigned. Such a contact plate 53 is thus for the flat recording of the four slides 27 of a transport frame 52 educated. The frame 52 comprises longitudinal walls, transverse walls and intermediate walls running essentially parallel to the transverse walls. These walls surround openings that frame the frame 52 penetrate completely, these openings making direct contact between the contact plate 53 the thermostat and the slides 27 enable. Because the slides 27 as part of 52 are held gently springy and because of the contact plate 53 is designed so that the frame 52 you ge slides can be lowered compared to the slide 27 directly on the surface of the contact plate 53 , Each group of four of a processing unit comprises one around an axis 58 swiveling and opposite a base plate 59 lockable snap frame 54 with four seats, with a lid in each of these seats 26 is insertable. Each such process unit also includes a connection plate 60 for sealingly connecting one inlet line each 11 , Outlet pipe 13 and pressure line 35 of the system 1 with the inlet pipe 11 , Outlet pipe 13 and pressure line 35 a lid 26 , O-rings arranged on the system side are preferred as seals for these connections (not shown).

5 shows one of the 2 corresponding, vertical longitudinal section through an arrangement with a hybridization chamber 5 , with the in a snap frame 54 inserted lid 26 the system is closed. All four hybridization rooms 5 one through a contact plate 53 and such a snap frame 54 Defined groups of four are thus assigned to the temperature control of a temperature control device. As described above, each group of four of a process unit comprises one about an axis 58 swiveling and opposite a base plate 59 lockable snap frame 54 with four seats, with a lid in each of these seats 26 is insertable. To make sure the lid 26 parallel to the slides 27 can be placed, the snap frame shows 54 also a central joint (not shown) with one to the axis 58 parallel mobility. So that the seals 28 the hybridization rooms 5 lock reliably, is via the snap frame 54 an additional pressure on the lid 26 exercised, this can be accomplished via screws, rocker arms, or similar known devices (not shown).

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

In fact, under these pressure conditions, there are no more air bubbles during the hybridization. The functional mechanism underlying this phenomenon has not been fully elucidated. However, it is assumed that the increased pressure on the one hand is the direction of diffusion in the area of the O-ring seal 28 determined or defined so that no gas molecules of the ambient air into the hybridization chamber 5 can diffuse. On the other hand, due to their pressure dependency, the phase boundaries in the hybridizing medium are also shifted, so that the spontaneous formation of air bubbles is suppressed. In connection with this invention, therefore, all gas bubbles in the hybridization medium - regardless of the process of formation in the hybridization chamber 5 - referred to as "air bubbles".

According to the invention, the required overpressure in the hybridization chambers 5 by means of a liquid, such as, for example, one from the feed pump 8th into the hybridization chambers 5 pressed hybridization medium from one of the vessels 2 can be achieved (cf. 1 ). If this hybridization chamber 5 comprehensive system 1 an agitation facility 32 for moving the hybridization media with respect to the immobilized samples, this system preferably also includes a spring element which passes through the agitation device 32 generated pressure differences resiliently counteracts. Such a spring element can be an elastic piece of pipe (not shown) in a corresponding feed or discharge to the hybridization chamber 5 his; however, a spring-loaded expansion vessel (not shown) can also be provided, which is connected to the hybridization chamber via a line 5 connected is.

Alternatively, inert gas can also be drawn from the container 3 into the distribution line 10 and the inlet pipes 11 pressed and via an inlet valve each 12 the pressure required in the hybridization chambers already filled with samples and hybridization media 5 being constructed. Inert gases such as N ₂ (nitrogen) which do not undergo any chemical interaction or reactions with the hybridization media are preferred. It can also be advantageous if the inert gases are not soluble in the hybridization media. There is also the option of a pressure pump and a pressure vessel (similar to those with 46 and 47 designated elements in 1 ) originating gas in the distribution line 10 initiate. After the pressure builds up, all valves can be closed again and the hybridization can be carried out. The gas cushion constructed in this way, which is directly connected to the volume of liquid used for the hybridization, serves as a spring element for resiliently counteracting the agitation pressure differences. If this is the case, for example because of a minimal but constant pressure loss via the O-ring seals 28 , should the required chamber pressure be corrected sporadically or renewed or kept constant during the hybridization, which usually lasts many hours. One or both of the valves can optionally be used for this purpose 12 . 14 (see. 1 ) are kept open.

Another alternative (not shown in the figures) consists of a pressure pump and a pressure vessel (similar to the one with 46 and 47 designated elements in 1 ) or a gas container (such as the N ₂ container 3 in 1 ) with the manifold 15 to connect and the pressure in the hybridization chambers 5 by opening the exhaust valves 14 build. An additional alternative to providing a gas cushion, which serves as a spring element for resiliently counteracting the agitation pressure differences, is that of a pressure pump and a pressure vessel (similar to the one with 46 and 47 designated elements in 1 ) or from a gas container (such as the N ₂ container 3 in 1 ) lead gas into one of the lines, which in at least one of the distribution line 10 or manifold 15 lead. Accordingly, one or both of the valves 12 . 14 (see. 1 ) are kept open.

Becomes a system 1 used with arrangements which (as described above) have two agitation membranes 33 . 33 ' may include, during the preparatory agitation of the hybridization media in the hybridization chambers 5 or during the hybridization itself, the agitation device 32 be used. It simply has to be an agitation pressure in the pressure room 34 generated, which is about 0.5 to 1 bar higher than the desired chamber pressure of 100 mbar to 1.4 bar above the ambient pressure. The pressure to be used for the agitation is (depending on the ambient pressure) in the range of approx. 1.6 - 2.4 bar. The second membrane 33 ' in this case forms a spring element which counteracts this agitation pressure.

typically, hybridization is carried out as follows:

• a) Rinsing out 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 opened and it is with the feed pump 8th distilled water from the vessel 2 (AD) via the valve 4 (AD) into the distribution line 10 pumped. The distilled water flows through the connection valve 19 into the manifold 15 and from there through the waste valve 17 into the waste line 16 , At the same time, the hybridization chambers 5 built up. This is done by placing (preferably in a transport frame 52 held) slides 27 with samples immobilized on the contact plate 53 a thermostat by inserting lids 26 in the snap frame 54 of the system 1 for hybridizing slides 27 immobilized nucleic acid samples, proteins or tissue sections and by closing the hybridization chambers 5 by folding down the lid 26 on the slides 24 (see. 4 and 5 ). This flip down connects the ends of the inlet lines 11 , the exhaust pipes 12 and the pressure lines 35 the agitation facility 32 every single hybridization chamber 5 with the corresponding line ends of the system 1 , Preferably - according to the four in a transport frame 52 recorded slides 27 - four lids 26 in a snap frame 54 used.
• b) filling and thermostatting of the hybridization chambers 5 with prehybridization buffer. For this purpose only the valves 4 (VP) 12 . 14 and 17 opened and it is with the feed pump 8th Prehybridization buffer from the vessel 2 (VP) via the valve 4 (VP) in the distribution line 10 and from there through the intake valves 12 into the hybridization chambers 5 pumped. Part of the prehybridization buffer leaves the hybridization chambers 5 via the exhaust valves 14 and the manifold 15 and gets through the waste valve 17 into the waste line 16 , This process requires special attention because this is the first time that air bubbles have entered the hybridization chambers 5 can be introduced.
• c) blowing out the distribution line 10 and the manifold 15 , For this purpose only the valves 9 . 19 and 17 opened and it is with the feed pump 8th Air through the ventilation valve 9 sucked in and over the distribution line 10 , the connection valve 19 into the manifold 15 and from there through the waste valve 17 into the waste line 16 pumped.
• d) Sample addition into the hybridization chambers 5 , For this purpose only the valves 14 and 21 open. The samples are pipetted over the sample feeder 31 in the lid 26 into the hybridization chambers 5 pressed. As a result, a corresponding volume of prehybridization buffer is obtained from the hybridization chambers 5 into the outlet pipe 13 with the exhaust valves open 14 repressed. 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 discharge line 20 and gets into the collection container 22 that they have through the vent 23 leaves. This process requires special attention because it is the second time that air bubbles have entered the hybridization chambers 5 can be introduced.
• e) Even distribution of the hybridization media in the hybridization chambers 5 and versus those on the slides 27 immobilized samples. For this purpose, all valves are closed first. Then the chamber pressure is increased as already described and the agitation device 32 put into operation. All possibly in the hybridization chambers 5 existing air bubbles are eliminated with this pressure build-up.
• f) Hybridizing the samples for 17 hours, for example. During hybridization, the Media in the hybridization chambers 5 be agitated constantly or intermittently. This process requires special attention because here air bubbles spontaneously in the hybridization chambers 5 can arise. To prevent this, the chamber pressure can be corrected during the hybridization and kept constant or changed according to an individual program. Such a program can define the increase and decrease of the chamber pressure in certain pressure and time steps, so that a gentle hybridization of the samples is guaranteed, which require such special treatment.
• g) washing the hybridized samples with buffers. For this purpose the chamber pressure is first regulated to normal pressure by the exhaust valves 14 and the waste valve 17 be opened. Washing liquids are then pumped on 8th sequentially and as required from the vessels 2 through the correspondingly opened valves 4 into the distribution line 10 and from there through the open intake valves 12 and the inlet pipes 11 into the hybridization chambers 5 pumped. The washing liquids and washing waste leave the hybridization chambers 5 via the outlet lines 13 and the exhaust valves already open 14 , are in the manifold 15 merged and through the open waste valve 17 into the waste line 16 dissipated.
• h) drying the hybridized samples on the slides 27 , For this purpose only the valves 12 . 14 and 17 open. Then the inert gas valve 50 opened and the distribution line 10 who have favourited Inlet Pipes 11 , the hybridization chambers 5 , the exhaust pipes 13 and the manifold 15 via the open waste valve 17 and the waste line 16 flushed with inert gas until the samples are dry. The finished samples can then be taken.

As an alternative to the method step h) just described, the hybridized samples are dried on the slides 27 by just the valves 12 . 14 and 21 be opened. Then the inert gas valve 50 opened and the distribution line 10 who have favourited Inlet Pipes 11 , the hybridization chambers 5 , the exhaust pipes 13 and the manifold 15 via the open relief valve 21 who have favourited Discharge Line 20 , the collection container 22 and the vent 23 flushed with inert gas until the samples are dry.

An alternative embodiment of the method according to the invention, in which the second membrane is applied, is also preferred 33 ' the agitation facility 32 can be dispensed with. For this purpose, another medium must take over the function of this spring element. This is achieved in that after all the samples and media participating in the hybridization have been filled into the hybridization chambers 5 all valves closed (see steps a-d) and then the exhaust valves 13 be opened again. This will make the volume of the manifold filled with air 15 as well as the directly connected part of the waste line 16 with the volumes of the hybridization chambers filled with liquid 5 connected. The between the closed valves 17 . 19 and 21 and the sample liquids in the hybridization chambers 5 trapped 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 basics were examined. Several 100 slides were processed without samples. The O-ring seals, which are preferably made of an elastomer 28 made of eg neoprene, silicone, twisted PTFE, polyethylene or Viton all successfully prevented fluid loss. The basic requirement is, of course, that the contact pressure on the lid 26 , the o-ring 28 and the slide 27 sufficiently large, ie significantly higher than the generated chamber pressure. The hybridization chambers used 5 included between the lid 26 and the slide 27 an area of 21 × 65 mm. The chamber pressure was raised 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 1,365 × 10 ^-3 m ² , a force of more than 136.5 N or approx. 13.9 kp per hybridization chamber was required 5 be used so that they could not open spontaneously. It is assumed that other types of seals and sealing materials are sufficient and prevent leakage, if one corresponds to the excess pressure in the hybridization chamber 5 selected closing force on the lid 26 who have favourited Seal 28 and the slide 27 is exercised.

While at standard equipment SN22 during the processing of slides or slides with hydrophobic surfaces regularly Air bubbles were observed in the prototype according to the invention no air bubbles found.

Biological embodiment

To carry out an example of a hybridization carried out according to the invention under increased pressure, two systems were used in parallel and independently of one another. It is a standard device of the applicant (Tecan HS 400, serial number 22 ; abbreviated to SN22), which was operated at normal 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 described in principle above. This agitation facility 32 in SN22 was operated with approx. 0.5 bar, the one in PT3 with an agitation pressure of approx. 1.5 bar. In both devices, the exact temperature was checked beforehand; apart from the test-related differences mentioned, they were 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, sample injection, program definition on the hybridization systems used) was carried out according to the technical instructions from Alopex (ALOPEX GmbH, Fritz-Hornschuch-Strasse 9, D-95326 Kulmbach, Federal Republic of Germany). Two slides each were placed in the hybridization systems SN22 and PT3 27 used and processed at 43 ° C or at 61 ° C. The results of the following processed samples are discussed: 43 ° C 43 ° C HS 400 SN22 HS 400 PT3 Slides # 29, # 31 Slides # 24, # 27 61 ° C 61 ° C HS 400 SN22 HS 400 PT3 Slides # 33, # 34 Slides # 35, # 37

A test kit "HybCheck" from Alopex with the kit lot number: 040524) was used. This test kit is used to check the hybridization systems and is designed in such a way that the hybridization temperatures are predefined and that the "OK" signals are only met if the intended ones are strictly observed Temperatures and the relevant test parameters in relation to washing, agitation and drying are given. With those on the slides 27 immobilized samples are oligonucleotides whose sensitivity to temperature differences is known.

The executed Program for every test run is in detail:

• a) Preparation of the "HybCheck Wash Buffer 1 and 2 according to manufacturer's instructions;
• b) Loosen of lyophilized, fluorescence-labeled oligonucleotides in 250 μl to one Hybridization temperature (43 ° C or 61 ° C) preheated HybCheck buffer for the hybridization;
• c) Insert 2 slides each (with 6 sub arrays each) at positions 1 and 2 in the hybridization system, use of the large ones Hybridization chambers (63.5 mm × 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. Inject 105 μl of the oligonucleotide solution 43 ° C or 61 ° C in every chamber; 3. Hybridization at 43 ° C or 61 ° C; with strong agitation and while 60 min; 4. Wash step 1 at 23 ° C (channel 1) for 30 sec; Suction during 30 sec; 2 repetitions; 5. Washing step 2 at 23 ° C (channel 2) during 30 sec; Suction during 30 sec; 2 repetitions; 6. Dry the slides during 3 min at 23 ° C;
• f) After completion of the hybridization, both slides were removed and placed in a laser scanner (Tecan LS400);
• g) Measurement setting of the laser scanner: 1. Scan mode: single wavelength Second Laser wavelength: 543 nm (green) Third Filter wavelength: 590 nm 4. Gain: 165 5.Autofocus mode: NS autofocus, level 1 6. Scan resolution: 10 μm 7. Pinhole: small 8th. Oversampling factor: 1
• h) After measurement in the laser scanner, the raw data obtained evaluated as follows:

Hybridization at 43 ° C:

• 1. 6 sub arrays (6 × 9 spots) on a 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 PM's and MM1's per microslide (24 individual values) became the mean, the standard deviation and the CV calculated. The following applies: CV = (standard deviation / mean) × 100
• 7. Discrimination PM: MM1 (1: 5) and discrimination PM: MM2 (1:20) calculated
• 8. The CV value and the discrimination values as "OK" (if CV over a whole slide <18%) or "failed" is specified.

Hybridization at 61 ° C:

• 1. 6 sub arrays (6 × 9 spots) on a 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 PM's and MM1's per microslide (24 individual values) became the mean, the standard deviation and the CV calculated. The following applies: CV = (standard deviation / mean) × 100
• 7. Discrimination PM: MM1 (1: 5) and discrimination PM: MM2 (1:20) calculated
• 8. The CV values and the discrimination values as "Ok" (if CV over a whole slide <18%) or "failed" is specified.

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 The results shown not only prove that both devices used a lot deliver useful results. Rather, they are in the two devices SN22 and PT3 achieved results (ordered by temperature) so similar, that an influence of the heightened Pressure on the hybridization can be excluded.

The invention Preventing air bubbles is not limited to use in hybridization chambers. Also other devices can be designed so that the occurrence of unwanted air bubbles in them can be prevented. Such devices or instruments can e.g. come from the field of microfluidics technology, such as “Lab on a chip "systems.

Claims (16)

System ( 1 ) with hybridization chambers ( 5 ) to hybridize on slides ( 27 ) immobilized nucleic acid samples, proteins or tissue sections, each hybridization chamber ( 5 ) as a substantially gap-shaped space, which can be essentially filled with a liquid, between one of these slides ( 27 ) and a lid ( 26 ) is defined, and the cover ( 26 ) compared to the slide ( 27 ) that the hybridization chamber ( 5 ) is sealed from the ambient air, whereby the system ( 1 ) a device for preventing air bubbles in the hybridization chambers ( 5 ), characterized in that this device for preventing air bubbles in the hybridization chambers ( 5 ) as a pressure device for building up a chamber pressure in the hybridization chambers ( 5 ) is formed, this chamber pressure being above the normal atmospheric pressure prevailing in the ambient air. 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 is a pressure source ( 8th . 3 ) which, via pressurizable lines ( 9 . 51 ) with the hybridization chambers ( 5 ) is connectable. System ( 1 ) according to claim 3, characterized in that the pressure source is a feed pump ( 8th ) or a pressurized gas bottle ( 3 ) includes. System ( 1 ) according to claim 3 or 4, characterized in that the pressurizable lines ( 9 . 51 ) a connection valve ( 19 ), a distribution line ( 10 ), a manifold ( 15 ) and for each of the hybridization chambers ( 5 ) an inlet valve ( 12 ) and an outlet valve ( 14 ) include. System ( 1 ) according to claim 5, characterized in that the pressurizable lines ( 9 . 51 ) also a waste line ( 16 ) with a waste valve ( 17 ) and a relief line ( 20 ) with a relief valve ( 21 ) includes. System ( 1 ) according to one of the preceding claims, characterized in that it is an agitation device ( 32 ) with which liquids in the hybridization chambers ( 5 ) compared to those on the slides ( 27 ) immobilized samples can be moved. System ( 1 ) according to claim 7, characterized in that the agitation device ( 32 ) a pressure pump ( 46 ) and a pressure tank ( 47 ), which each have a pressure chamber ( 34 ) a lid ( 26 ) can be connected, the cover ( 26 ) also a membrane ( 33 ) which covers the pressure chamber ( 34 ) from an agitation room ( 30 ) separates. System ( 1 ) according to claim 7 or 8, characterized in that the agitation device ( 32 ) a vacuum pump ( 48 ) and a vacuum tank ( 49 ), which each have a pressure chamber ( 34 ) a lid ( 26 ) can be connected, the cover ( 26 ) also a membrane ( 33 ) which covers the pressure chamber ( 34 ) from an agitation room ( 30 ) separates. System ( 1 ) according to one of claims 7 to 9, characterized in that the agitation device ( 32 ) comprises a spring element, which one on the pressure chamber ( 34 ) counteracts overpressure or underpressure. System ( 1 ) according to claim 10, characterized in that the spring element as in the lid ( 26 ) arranged second membrane ( 33 ' ) or as in the manifold ( 15 ) between the waste valve ( 17 ), the connection valve ( 19 ) and the relief valve ( 21 ) arranged air space is formed. System ( 1 ) according to one of the preceding claims, characterized in that it is designed to generate the required chamber pressure by means of a liquid or by means of a gas. System ( 1 ) according to claim 12, characterized in that the liquid for generating the chamber pressure from a feed pump ( 8th ) in a distribution line ( 10 ) and via inlet valves ( 11 ) against the liquid in the hybridization chambers ( 5 ) is pressed hybridization medium. System ( 1 ) according to claim 12, characterized in that the gas for generating the chamber pressure from a feed pump ( 8th ) via a ventilation valve ( 9 ) sucked air or in a container ( 3 ) is stored inert gas, this gas via a distribution line ( 10 ) and via inlet valves ( 11 ) against the liquid in the hybridization chambers ( 5 ) is pressed. System ( 1 ) according to one of claims 12 to 14, characterized in that there is a pressure chamber ( 34 ) includes the lid ( 26 ) is arranged and with a membrane ( 33 ) opposite an agitation room ( 30 ) in which an agitation pressure can be built up that is 0.5 to 1 bar higher than the desired chamber pressure System ( 1 ) according to claim 15, characterized in that there is a second, in the lid ( 26 ) arranged membrane ( 33 ' ) or a volume of a manifold filled with a gas ( 15 ) and a waste line directly connected to it ( 16 ) between closed valves ( 17 . 19 . 21 ) lie and form a spring element which counteracts this agitation pressure.