WO2003106032A1

WO2003106032A1 - Hybridization chamber

Info

Publication number: WO2003106032A1
Application number: PCT/EP2003/006253
Authority: WO
Inventors: Ralph Müller; Torsten Gerboth
Original assignee: Axaron Bioscience Ag
Priority date: 2002-06-14
Filing date: 2003-06-13
Publication date: 2003-12-24
Also published as: AU2003238501A8; WO2003106032A8; AU2003238501A1

Abstract

The hybridization chamber has a base (10) with a recess (14) for accommodating a microarray (16). The chamber also comprises a cover (12), which can be placed onto the base (10) thereby resulting in the formation of a hermetically sealed space for the microarray. A covering surface (18) is provided on the cover (12) and is situated at a distance of approximately 20 ?m from the microarray while running parallel thereto. This enables a sample solution (38) to be drawn into the space between the microarray and the covering surface by means of capillary forces and to spread out evenly without requiring any pumps. The hybridization chamber makes it possible to work with minimal sample amounts without any dead space.

Description

hybridization chamber

description

Biochips are increasingly being used for analysis in the field of medicine, genome research or food analysis. The biochips usually consist of a slide on which different types of DNA or RNA oligonucleotides, peptides, proteins, enzymes, etc. are immobilized in separate spots. The spots are arranged in the form of a grid or array, which is why the biochips are also generally referred to as microarrays. With the help of these biochips, for example, hybridization assays, e.g. B. transcription analyzes, usually carried out with the help of appropriately labeled, such as fluorescence-labeled samples. Microarrays are generally suitable for studying affinity reactions.

In order to be able to carry out a hybridization reaction with small volumes of sample solution, a drop of sample solution is occasionally pipetted onto a microarray and a cover glass is then placed on top. The cover glass distributes the sample solution. In addition, the sample solution is distributed well under the cover glass due to capillary forces, which, also due to the capillary forces, strongly adheres to the microarray, which in turn distributes the sample solution well under the cover glass. When applying the cover slip it can easily be used Air bubbles are trapped, especially when the surrounding atmosphere is low in humidity, and especially when neither the cover glass nor the microarray can be completely wetted. Furthermore, this step, which is usually carried out manually, is not very reproducible.

The microarray with sample solution and applied cover glass is then placed in a hybridization chamber. Such a chamber consists of a bottom and a lid. The two parts are sealed against each other by an O-ring. Bottom, O-ring and cover form the walls of a hermetically sealed room that houses the microarray. The word "hybridization chamber" usually designates both the system, consisting of the lid and base, and the hermetically sealed space.

Hybridization chambers are known, inter alia, from US Pat. No. 6,238,910 B1. In the system described there, the hybridization chamber is formed in that an O-ring embedded in the cover presses on the microarray, which is received in the base. A similarly trained

Hybridization chamber can be found in US 2002/0001839 AI. There, a protruding edge of the lid presses on the microarray, thereby forming the hybridization chamber between the microarray and the lid. In US 2002/0048754 AI, the microarray is additionally turned into a microtiter array by placing a grid. In various writings, e.g. B. in US 6,238,910 B1 or WO 01/32934 A2, it is also described how a hybridization chamber is integrated into a system with automatic supply of the required sample liquids (fluidics).

The known hybridization chambers are generally designed as flow-through chambers in which various rinsing steps can also be carried out. In one In the hybridization chamber, a microarray is placed without a cover glass. The sample solution to be examined is then pumped into the hybridization chamber. As a result, a relatively large volume of the liquid used for the hybridization (hybridization volume) is required. This is usually in the range of 200 μl and more, since there are still considerable dead volumes in the hoses and pumps. A sample often has to be diluted for such a system. Experience has shown, however, that this leads to reduced signal strengths and thus reduced sensitivity.

The company Corning offers a hybridization chamber into which the above-mentioned arrangement consisting of a microarray with a sample solution which is distributed by a cover slip placed on it can be inserted. This system has a bottom and a lid. A receptacle for the microarray is provided in the bottom. The bottom also contains a large O-ring that surrounds the entire microarray. The unstructured, flat lid is placed on the

Sealing ring put on. This forms an airtight hybridization chamber between the base, sealing ring and lid, in which the microarray is located.

Such a hybridization chamber with a microarray, sample solution and cover glass is placed in an immersion bath in order to optimally carry out the hybridization, in which it is heated, for example, at 50 ° C. for twelve hours. The system is then removed from the immersion bath. The lid and base are separated and the microarray is removed. The cover slip is carefully removed. Mechanical damage to the spots and drying of the microarray must be avoided. The microarray is then washed in a buffer bath to remove excess or to remove unbound labeled molecules. Drying follows. The hybridization reaction is then evaluated using a generally optical analysis device.

The various difficulties addressed when carrying out hybridization assays impair the reproducibility of the results and make automation seem desirable.

The object of the invention is to improve the possibilities of automation and reproducibility of hybridization analyzes.

This object is achieved by the inventions with the features of the independent claims. Advantageous developments of the inventions are characterized in the subclaims.

According to the invention, a hybridization chamber or an arrangement for analyzing a sample medium with the aid of a sample carrier, e.g. B. a microarray specified. The sample medium is i. d. Usually be a solution.

The hybridization chamber has a bottom in which the microarray is placed. It also has a lid that can be placed on the floor, thereby forming a closed space in which the microarray is protected against influences from the surrounding atmosphere. The lid has a top surface. The cover surface is designed on the cover (in one piece or mounted) in such a way that it is arranged at a predetermined distance above the microarray and essentially parallel to it, the distance between the microarray and the cover surface being selected such that the sample medium is introduced into the space by capillary forces can be drawn between the microarray and the top surface. A hybridization chamber for microarrays is thus created, in which the volume of the liquid used for the hybridization (hybridization volume) is defined between the surface of the microarray and a top surface of the hybridization chamber arranged closely above the microarray.

If, for example, the sample medium is pipetted onto the microarray through an opening in the top surface, manually or automatically, the sample medium is distributed evenly and without air bubbles over the microarray due to capillary forces that act between the microarray and the top surface. The sample medium spreads in the space between the microarray and the top surface, i.e. over the hybridization volume. This creates a uniform, thin film. The flow of the sample medium is highly reproducible due to the specified distance between the microarray and the top surface. No pumps are required to distribute the sample medium over the microarray.

The distance between the microarray and the top surface also determines the sample volume. This distance can be chosen to be very small by means of suitable measures, as a result of which the sample volume is also very small. A distance of about 20 μm is suitable for aqueous sample solution with a relatively low viscosity and a moderate wettability of the surface of the microarray, which usually consists of glass. Then less than 50μl cover an entire microarray with an area of typically 10 cm ^λ 2.

If the top surface is chosen to be the same size or larger than the microarray, it is possible for the sample medium to be distributed over the entire microarray. It can do the whole Microarray to the edge can be used for hybridization analyzes.

If the cover area is chosen to be smaller than the microarray, the size of the cover area determines the size of that area of the microarray which is wetted by the sample medium and on which hybridization can thus occur. The hybridization area remains limited to the size of the cover area. The latter results from the fact that the sample liquid spreads to the edge of the volume in which the top surface and the microarray are closely spaced. A capillary curvature pressure forms at this edge, which prevents the sample liquid from spreading further. A possible flow of the sample liquid beyond the microarray is prevented.

The size of the required sample volume or the hybridization volume is therefore not determined by any seals or vessel walls that press on the microarray, but rather by the fact that the pipetted

Sample volume is so small that the microarray is only partially covered, or because of the size of the microarray or the top surface. A seal is only used to seal the hybridization chamber as a whole. Usually a seal is placed in the floor. The seal surrounds the surface that is provided for receiving the microarray at a certain distance. When the hybridization chamber is assembled, the lid presses against the seal embedded in the base.

One way of precisely adjusting the distance between the top surface and the microarray is achieved by integrating spacers into the top surface. There is a height of the spacers of for example 20 μm. The spacers are usually arranged in the top surface in such a way that they press on the typically four corners of the microarray. This leaves most of the area of the microarray free for hybridization analysis. A simple form, such

Forming spacers consists in attaching a thin film to the top surface.

Another way to set the distance between the top surface and the microarray can be achieved by adjusting the distance between the top and bottom using adjusting screws. Furthermore, in that suitable spacers are integrated in the base or cover, which act directly between the cover and the base and not on the microarray. Various lids can be preformed in such a way that they can be spaced apart at specific distances, e.g. B. 20, 30 or 40 microns are set.

A further possibility of precisely adjusting the distance between the top surface and the microarray can be achieved by countersinking holes in the lid in an area outside the top surface, usually four pieces in the corners of a rectangle that is larger than the microarray. The counterbores are designed so that they leave only a thin layer of the cover adjacent to the hybridization chamber. In addition, a thread is cut in the counterbores. It can then be a screw, e.g. B. a grub screw, are inserted into each counterbore, in such a way that it presses on the thin remaining layer and deforms it elastically. When viewed from the hybridization chamber, the cover then has a convex deformation. This can act on suitable abutments in the bottom of the hybridization chamber, as a result of which the desired distance between the top surface and the microarray can be set. The lid is advantageously a plastic block or injection molded part made of plastic that has been reworked by milling. It is important that the lid is sufficiently stiff so that it does not deform over the length of the microarray. For this purpose, the lid typically has a thickness of at least 10 mm, as a rule 12 mm, with a length of approximately 10 cm predetermined by the shape of microarrays which are regularly formed on specimen slides.

Many possibilities are conceivable as material for the lid, for example different plastics, such as PMMA, silicone, Teflon, or glass, etc. Teflon types, polysulphones or PEEK (polyether ether ketone) are also possible. However, polycarbonate (PC) is particularly suitable. Polycarbonates are linear polycondensates and therefore show thermoplastic behavior. Compared to other thermoplastics, polycarbonates have good mechanical and thermal properties. As a result, polycarbonate is well suited for classic machining as well as for the injection molding process. The continuous use temperature for PC is 130 ° C. In addition, polycarbonate does not store dyes, in contrast to other plastics that store hydrophobic and hydrophilic dyes. Polycarbonate is mechanically stable, chemically essentially inert, can be cleaned, does not absorb water and does not swell.

Polycarbonate is also transparent. The wetting of the microarray during the application of a sample can thus be observed with the naked eye. The hybridization can be optically detected online, through the lid.

If the lid and base are made of inexpensive plastic, the hybridization chamber can also be used as a consumable. In such a case, the hybridization chamber can also be used as packaging for transporting microarrays are used. The packaging or hybridization chamber can be used immediately after transport for an assay to be carried out without the microarray having to be removed from its packaging. This reduces the risk of contamination.

The lid has an inlet opening in the area above the microarray through which the sample medium can be pipetted onto the microarray. This inlet opening can be arranged centrally above the microarray or at the edge of the microarray. The inlet opening is closed, for example, by a plug or a septum, e.g. B. by a pocket septum. A septum is an elastic lip. A capillary for pipetting the sample medium can be pushed through the lip. If the capillary is pulled back, the elastic lip closes again airtight.

With such a system it is possible to work completely dead volume free. For this purpose, a capillary is used in the so-called "humming bird" mode ("humming bird" = hummingbird). H. the capillary is brought up to a liquid sample medium. Part of the sample is drawn into the capillary. The capillary is then passed through the septum. The sample volume sucked into the capillary can then be pipetted onto the microarray without any significant residues remaining in the capillary. The capillary is then removed through the septum. The sample volume pipetted onto the microarray is distributed over the microarray due to the capillary forces. The system is therefore able to work very economically and to lose practically nothing from a sample. The sample is used optimally.

The cover can have an individual, preferably machine-readable code. This enables the Regularly check the quality of the analysis results obtained with the help of the respective lid. If it can be seen that a certain lid repeatedly gives bad results, this lid can be excluded from further use. Such a code can e.g. B. in the form of a bar code on the lid.

It is also conceivable to integrate a so-called electronic phone card chip (smart card) into the cover. This chip can contain information about properties of the cover, for example about the size and / or position of the hybridization area or the distance between the cover area and the microarray.

Such a chip can also be used as protection against imitation of covers. To do this, a

Identification algorithm for the lid partly in the chip and partly externally, stored in a computer.

The hybridization chamber is typically divided into one

Slide the mounting frame in which the lid and base are pressed together in the press fit. This recording frame is preferably heatable and coolable. The temperature of the hybridization chamber can be adjusted by suitable heating or cooling elements, e.g. B. a thermostat or a Peltier

Element, which can also be integrated directly into the floor. It is also possible to place the hybridization chamber in a suitable holder, which in turn is compatible with commercially available PCR machines (PCR = polymerase chain reaction). The latter can then be used

Temperature control of the hybridization chamber can be used. It is also possible to subject the hybridization chamber to temperature cycles or to run a PCR in it. Furthermore, the hybridization chamber can be integrated into a system that is designed to automatically fill the chamber. Such a system can also be designed to carry out washing, rinsing and drying steps automatically. Such a system can have an xyz robot for automatically picking up sample medium and dispensing the sample medium onto the microarray after the septum has passed.

It is also possible to integrate the hybridization chamber into a system in which the microarrays - with the cover removed - are first set up spot by spot. The lid is then placed on top and the microarray that has just been created is used for hybridization analyzes.

It is also conceivable to integrate stirring or agitation agents, such as an ultrasound source in the lid, top surface or bottom. Pressure waves can also be conducted into the chamber. Finally, it is also possible to move the sample medium on the microarray by mechanical pressure on the lid.

The arrangement according to the invention can also be used to process a plurality of microarrays in parallel

Have hybridization chambers. Or it can have a large hybridization chamber with a correspondingly large base with a plurality of cutouts for receiving a plurality of microarrays under a correspondingly large cover. This is particularly useful in combination with handling by a robot.

The application of a thin film of a sample medium to a sample carrier, for example a microarray, achieved by the arrangement according to the invention can generally be described as a method become. According to the method according to the invention, a cover surface is first positioned above the sample carrier and essentially parallel to it at a predetermined distance, as a result of which a gap is formed between the sample carrier and the cover surface. The distance between the microarray and the top surface is chosen such that the sample medium can be drawn into the space between the microarray and the top surface by capillary forces. The sample medium is then applied at a location where the gap formed between the sample carrier and the top surface is exposed, for example at the lower end of one

Inlet opening in the top surface on the sample holder. The sample medium is then drawn into the space between the microarray and the top surface by capillary forces and spreads out in the form of a thin film on the sample carrier.

The invention is explained below with reference to

Embodiments explained, which are shown schematically in the figures. The same reference numbers in the individual figures denote the same elements. In detail shows:

1 is a sectional view of the hybridization chamber; and FIG. 2 three views of the cover.

Fig. 1 shows the hybridization chamber. It consists of a base 10 and a cover 12. FIG. 2 shows three views of the cover 12.

The bottom 10 has a recess 14 into which a microarray 16 is inserted. The recess 14 is shallower than the height of the microarray 16. Furthermore, the base 10 has an annular groove 20 for receiving an O-ring 22. The O- Ring 22 seals the space around microarray 16 when base 10 and lid 12 are pressed together.

The contact pressure required for sealing between base 10 and cover 12 is achieved in that the hybridization chamber composed of cover 10 and base 12 is pushed into a receiving frame (not shown). The mounting frame is designed in such a way that the

Hybridization chamber is clamped in it. The receiving frame exerts forces on the lid and the bottom of the hybridization chamber in such a way that they are pressed against one another. The O-ring in the bottom of the hybridization chamber provides the elastic restoring force required for this.

The lid 12 is made by machining

Molded solid polycarbonate. It has an increased cover area 18 compared to the microarray 16. The cover surface 18 has the function of a cover glass and reduces the distance between the cover 12 and the microarray 16 to approximately 20 μm. In order to fix the base 10 and cover 12 or the microarray 16 and cover surface 18 to one another, the base 10 has at least two upstanding registers 24 which engage in corresponding recesses 26 in the cover 12.

The exact adjustment of the distance between the microarray 16 and the top surface 18 is ensured with the aid of the countersunk bores 28 milled into the cover 12. The counterbores 28 end in projections 30 which are formed in the surface of the cover 12 facing the microarray 16. Four projections 30 are formed in the corners of a rectangle, which is larger than the microarray 16, in the cover 12. The counterbores 28 are designed such that they leave only a thin layer 32 of the cover 12 adjacent to the hybridization chamber. In addition, a thread is cut in the counterbores 28. A set screw 34 is in each counterbore 28 introduced, in such a way that it presses on the thin remaining layer 32 and deforms it elastically. When viewed from the hybridization chamber, the cover 12 has a convex deformation. With the aid of this convex deformation, the desired distance of approximately 20 μm between the top surface 18 and the microarray 16 is set by changing the position of the grub screws 34.

To fill in the sample solution, the cover 12 has a cutout 36 arranged above the microarray 16, which in FIG

Direction towards the microarray 16 funnel-shaped. The cut-out 36 serves as an inlet opening for a capillary which contains the sample solution 38.

In order to avoid loss due to evaporation of the sample solution 38, an exchangeable septum 40 is let into the inlet opening 36 and its position is fixed by two essentially cylindrical plastic parts 42, 44. The two cylindrical plastic parts 42, 44 are screwed into the inlet opening 36.

If one pierces through the septum 40 with a capillary or pipette to apply the sample solution 38 to the microarray 16, the rubber lips of the septum 40 are pressed apart. The lips close again when the capillary or pipette is pulled out.

Typically, a capillary with a diameter of e.g. B. 1/16 inch used. The septum 40 is pierced with the capillary until the capillary touches the microarray 16 or is only a small distance from the microarray 16. The sample solution 38 is then conveyed out of the capillary into the opening 50 or onto the microarray 16 by a slight overpressure. The capillary is then passed through the septum 40 removed back. The septum 40 closes again hermetically due to its elastic restoring forces.

The sample solution 38 is then initially in the form of a drop on the microarray 16. The sample transport into the

Reaction space between the microarray 16 and the top surface 18 is further carried out via capillary forces. Are the surfaces of the microarray 16 and the top surface 18 z. B. wetted with water vapor, the sample liquid spreads quickly in the thin capillary gap between microarray 16 and top surface 18.

As a result of the funnel-shaped design of the inlet opening 36, its diameter widens very rapidly with increasing distance from the microarray 16. As a result, there are large gaps between the capillary and the walls of the inlet opening 36 upwards. This prevents the sample solution 38 due to Capillary forces are drawn into the area between the capillary and the wall of the inlet opening 36 in the cover 12. This prevents sample loss.

If the sample solution 38 has been distributed uniformly over the microarray 16, the hybridization chamber is tempered together with the receiving frame. This is typically done for 12 hours at 40-60 ° C to allow optimal hybridization. Preferably, the temperature control

Hybridization chamber either uses a water bath or a Peltier-controlled temperature control unit - similar to a PCR system - into which the hybridization chamber is inserted.

At these temperatures, the sample solution 38 can evaporate at the open ends of the hybridization volume between the microarray 16 and the top surface 18. To prevent this, a reservoir 46 is provided in the bottom 10, into which water is poured. The water filled in creates a saturated water vapor atmosphere in the closed room of the Hybridization chamber and thus prevents evaporation of the sample solution 38. An enlargement of the reservoir 46 can be achieved in that channels are formed below the microarray 16, which can hold a larger amount of water.

After the hybridization has proceeded as completely as possible, it is necessary for a series of hybridization assays to remove the sample solution 38 from the microarray 16 by rinsing.

For this purpose, the cover 12 has closable through bores 48, preferably for suction, through which liquids for flushing processes or nitrogen for drying can be fed into the hybridization chamber. If the completely assembled hybridization chamber is inserted into the receiving frame, then there are suitable connections in the system of the liquid and gas supply directly above the openings of the through bores 48, so that the liquids or gases can get directly into the through bores 48.

In general, the microarray 16 does not need to be removed from the hybridization chamber in order to carry out the rinsing and drying. It will be the after hybridization

Hybridization chamber with the microarray 16 therein removed from the water bath. The inlet opening 36 and / or the through holes 48 are opened.

A washing solution, water or buffer is usually used for rinsing. These can enter the chamber through the inlet opening 36 and / or the through bores 48 and can be pumped or drawn through the chamber. The wash solution is preferably injected into the chamber through the inlet opening 36 using a syringe. In order to carry out the washing or rinsing steps, the distance between the microarray and the top surface can be increased temporarily. For drying, nitrogen or dry compressed air is pressed through the hybridization chamber at about 5 bar for a few seconds, which removes all moisture. In addition to compressed air, a vacuum can also be used. It is also conceivable that compressed air is pressed through the septum 40 into the chamber, which escapes again through the through bores 48.

The overall procedure of washing, rinsing and drying preferably takes place immediately after the hybridization, without removing the sample holder. This keeps the sample carrier free from contamination.

After the hybridization assay has been completed, the microarray 16 is evaluated. To do this, it is removed from the hybridization chamber and placed in a biochip reader.

The microarray 16 is i. d. Usually only used once. Lid 12 and septum 40, however, are rinsed and used again.

Alternatively, the entire hybridization chamber, consisting of base 10, cover 12 and closure of inlet opening 36 including inserted sample holder 16, can be designed as a disposable or consumable.

Such a hybridization chamber as a disposable could also be used to transport the sample carrier 16. It would therefore have the function of a container for receiving and transporting the sample carrier 16. The sample carrier 16 would be delivered inserted into the hybridization chamber, the hybridization chamber used for this being a disposable. Finally, it is also conceivable that the hybridization chamber designed as a disposable is designed in such a way that a sample carrier 16 is no longer required. Raised areas can be integrated in the bottom 10 of the hybridization chamber, which reflect the dimensions of the sample carrier 16 and take over its function. The advantage of using a sample carrier 16 made of glass, however, is that it has more suitable surfaces for spotting and thus for assembling the arrays than the plastics which are generally used for the hybridization chamber. There are a variety of functional coatings for sample carriers made of glass, from which you can choose.

reference numeral

Bottom cover recess for receiving the microarray 16 microarray, sample holder top surface annular groove for receiving the O-ring 22 O-ring upright register with the upstanding register 24 corresponding recesses countersunk bores projections thin layer grub screw inlet opening sample solution, sample medium septum first plastic part to fix the position of the septum 40 second plastic part for fixing the position of the septum 40 solvent reservoir through holes for supplying the hybridization chamber with media opening of the inlet 36

Claims

claims

1. Arrangement for analyzing a sample medium (38) with the aid of a sample carrier (16) a) with a bottom (10) which can accommodate the sample carrier (16); b) with a lid (12) which can be placed on the floor (10), whereby a closed space is formed in which the sample holder (16) can be; characterized in that c) that the cover (12) has a cover surface (18); d) that the cover surface (18) is formed on the cover (12) such that it is arranged at a predetermined distance above the sample carrier (16) and essentially parallel to it, e) the distance between the sample carrier (16) and the cover surface (18) is selected such that the sample medium

(38) can be drawn into the space between the sample holder and the top surface by capillary forces.

2. Arrangement according to the preceding claim, characterized in that counterbores (28) are formed in the cover (12); and that the counterbores (28) are designed such that they leave a thin layer (32) of the cover (12) in the direction of the sample carrier (16).

3. Arrangement according to one of the preceding claims, characterized in that the cover (12) has a ratio of its thickness to its length of at least one to ten.

4. Arrangement according to one of the preceding claims, characterized in that the cover (12) is formed from polycarbonate.

5. Arrangement according to one of the preceding claims, characterized in that the cover (12) has an inlet opening (36) in the region above the sample carrier (16); and that the inlet opening (36) is closed by a septum (40).

6. Arrangement according to one of the preceding claims, characterized in that the cover (12) is characterized by an individual code.

7. Arrangement according to one of the preceding claims, characterized by a device for tempering the sample carrier (16).

8. Arrangement according to one of the preceding claims, characterized by a device for automatic handling of the sample medium (38) and for performing washing steps and / or rinsing steps and / or drying steps.

9. A method for applying a thin film of a sample medium (38) to a sample carrier (16) with the following steps: a) above the sample carrier (16) and essentially parallel to it, at a predetermined distance Positioned top surface (18), whereby a gap is formed between the sample carrier and the top surface; b) the distance between the sample carrier (16) and the top surface (18) is chosen such that the sample medium (38) can be drawn into the space between the sample carrier and the top surface by capillary forces; and c) the sample medium (38) is applied at a location at which the gap formed between the sample carrier (16) and the cover surface (18) is exposed, so that the sample medium is transferred into the space between by capillary forces

Sample holder and top surface can be pulled and can spread out in the form of a thin film on the sample holder.