EP0836884A2 - System for carrying out thermal reaction processes without contamination - Google Patents

Abstract

System to apply a heat reaction to liquids has ≥ 1 vessel, with ≥ 1 inner fluid-tight closure. At least 1 outer closure can give a pressure seal to the vessels. Also claimed is an operation to carry the liquid through a pipette unit, which can be given inner and outer seals. The outer seals are removed from the vessel(s), and the pipette is inserted through the inner seal(s). The liquid is extracted from the vessel(s) into the pipette unit, or a liquid is discharged from the pipette system into the vessel(s).

Description

The invention relates to a system for the contamination-free processing of thermal Reaction processes and a method for carrying out liquid transport processes into and out of vessels.

The method according to the invention can be used in analytical methods which can be falsified by the entry or removal of interfering substances. Especially for carrying out molecular diagnostic analyzes with sample preparation (Sample preparation) and amplification (e.g. PCR according to EP-B-0 201 184) are due the reaction problem requires closed reaction vessels. At a However, these vessels need to be performed automatically several times be opened and closed. Furthermore, temperatures of 4 - 95 ° C applied, the increased requirements for the security of the vessels put.

WO 95/11083 describes a reaction vessel with a closed one, especially for PCR Described septum, which is, however, only for the automatic removal from a reaction vessel, but not suitable for automatic loading. For loading the The container with the sample must be opened completely manually. In addition, this embodiment is only conditional for the use of disposable pipette tips can be used because a considerable force is required to pierce the septum the usual plastic pipette tips cannot apply. In addition, leaves desired lid counter heating due to the thermal instability of the septum only apply conditionally. Both effects run in parallel, i. H. the thinner the septum, the lighter penetrable, the higher the thermolability.

EP-A-0 642 828 describes a system with the same components, which are the same Disadvantages.

WO 83/01912 describes a vessel for the determination of hemoglobin with the aid of potassium cyanide described, which has a first plug with a channel and a lid Prevents the escape of dissolved potassium cyanide. This vessel is for implementation not thought of and suitable for temperature treatments.

WO 94/05425 describes a lid for a container which contains a valve which can be opened by pressing on the lid so that liquid that is in the Lid is in the container can flow.

US-A-4,362,698 describes a closure for sample vessels, which consists of two sub-elements exists, which, however, neither close pressure-tight, nor without their destruction Function can be separated from the sample vessel and from each other.

DE-A-44 12 286 describes a system with a lid, reaction vessel and lid handler, which enables such a process to be fully automated. However, it has the disadvantage that an additional device component as a lid handler is needed. This also applies to the embodiment described in DE 29505707. Farther these embodiments are through the separate lid opening mechanism slowly.

US-5,282,543 describes a capping of reaction tubes, with the miss are arranged as a single sealing element in a composite. This arrangement is for direct, automatic pipetting into the reaction vessels is unsuitable.

The object of the present invention was to provide a system in which the Liquid transport can be carried out using conventional plastic pipette tips can, and which nevertheless enables such a tight closure of the vessels that of the Liquid inside the vessels does not spill out during heating can.

Fig. 1

Ablaufschema eines Pipettiervorgangs mit den erfindungsgemäßen Elementen

Fig. 2

Reaktionsgefäß mit den Dichtelementen

Fig. 3

Reaktionsgefäß mit Dichtelementen und eingeführter Pipettenspitze

Fig. 4

Anordnung in einem Heizblock mit Deckelgegenheizblock

Fig. 5

Device zum Arretieren mehrerer Reaktionsgefäße und beweglichem Dichtelement 2

Fig. 6

Anordnung des Device in Abb. 5 in einem Herzblock mit Deckelgegenheizblock

Fig. 7

Formen der Öffnungen im Dichtelement 1

Fig. 8

Ausführungsform für Verbindung von Dichtelement 1 mit Reaktionsgefäß

Fig. 9

Obenaufsicht einer Vorrichtung enthaltend 16 Reaktionsgefäße

Fig. 10

Querschnitt/Seitenansicht einer Vorrichtung gemäß Abb. 9 verbunden über Kunststoffstegen

Fig. 11

Oberaufsicht eines Mehrfachdichtelements für die Vorrichtung gemäß Abb. 9

Fig. 12

Querschnitt/Seitenansicht eines Mehrfachdichtelements

Fig. 13

Seitenansicht eines in einen Automaten eingelegten Mehrfachelements gemäß Fig. 5

The core of the present invention is a two-component seal on a reaction vessel, a first, inner sealing element sealing the vessel in a liquid-tight but not pressure-tight manner, and a removable second, outer sealing element closing the vessel in a pressure-tight manner.

Fig. 1

Flow diagram of a pipetting process with the elements according to the invention

Fig. 2

Reaction vessel with the sealing elements

Fig. 3

Reaction tube with sealing elements and inserted pipette tip

Fig. 4

Arrangement in a heating block with cover counter heating block

Fig. 5

Device for locking several reaction vessels and a movable sealing element 2

Fig. 6

Arrangement of the device in Fig. 5 in a heart block with lid counter heating block

Fig. 7

Forming the openings in the sealing element 1

Fig. 8

Embodiment for connection of sealing element 1 with reaction vessel

Fig. 9

Top view of a device containing 16 reaction vessels

Fig. 10

Cross-section / side view of a device according to Fig. 9 connected via plastic webs

Fig. 11

Top view of a multiple sealing element for the device according to Fig. 9

Fig. 12

Cross section / side view of a multiple sealing element

Fig. 13

Side view of a multiple element inserted into an automatic machine according to FIG. 5

The invention relates to a system for processing thermal reaction processes in liquids containing a variety of vessels, a variety of first Closures for individual of these vessels and one or more second closures can seal a large number of vessels at the same time. Also subject of the Invention is a method of transporting a liquid into or out of a vessel Vessel, which is closed with a first and a second sealing element, into or out with the help of a pipetting unit.

A reaction vessel in the sense of the invention means a vessel (1) in which a liquid can be stored. It can therefore be closed or locked. However, the vessel has at least one opening, which is connected to the first and second closure can be closed. In addition, the vessel preferably has no opening that cannot be closed or closed in a liquid-tight and pressure-tight manner. The In addition, the vessel is preferably thermostable, particularly with regard to shape and Inertness towards the liquid to be stored, especially at temperatures up to at 110 ° C. Preferred materials for reaction vessels are preferably thermoformable Plastics such as polystyrene, polyethylene or polypropylene. You can use the Injection molding technology can be manufactured in a simple manner. They are especially for single use. A particularly suitable material for vessels is polypropylene, as it tolerates temperatures up to 110 ° C without deformation. The reaction vessel preferably has a particularly thin wall thickness, preferably between 0.15 and 0.45 mm, particularly preferably approx. 0.3 mm, and preferably has a conical shape that follows is tapered at the bottom. The vessels preferably have a volume of between 0.05 and 50 ml, preferably 0.2 to 2 ml. The external shape of the vessel is usually determined by the The fact that it is a temperature control unit, e.g. B. a Kitzeblock, in the form as good as possible is adjusted. This improves the heat transfer from the temperature control unit through the Wall of the vessel into the interior of the vessel and, if applicable, the one located therein Liquid. Such reaction vessels are commercially available.

Several reaction vessels can also be connected to one another. So it was, for example proved to be expedient, 16 vessels with curved webs in two rows of 8 positions are connected. The vessels are preferably connected to one another in such a way that they are incorporated into conventional thermal cyclers, e.g. B. for implementation a PCR, fit. Several of these joined vessels can be juxtaposed are treated, e.g. B. in 96-well microtitration plate format. If in the following is spoken of a large number of vessels, it is preferably 4 to 96, particularly preferably 8 to 16 vessels.

An internal closure (sealing element 2) for this vessel has the function of being unwanted Prevent liquids from escaping from the opening of the vessel. The is preferred inner closure made of an elastic sealing material, e.g. B. latex or silicone rubber. The hardness of the material is preferably 20 to 100 shore, preferably 30 to 60 shore. Elastosil LR 3003/40 (Wacker Chemie GmbH, FRG) used, which has an elasticity of 40 shore. The inner closure has a thickness of 0.1 to 20 mm, particularly preferably 0.5 to 1.2 mm. This is to be understood that this thickness refers to the thinnest point on the outside. So z. B. for Total stiffening of the closure or to adapt the sealing element to the opening of the vessel (e.g. as a sealing ring 15) greater thicknesses are possible on the belt. To make things easier the introduction of a pipetting unit, e.g. B. a plastic pipette tip is inside Closure provided a further dilution or an opening (4). This opening can have different geometric shapes (see FIGS. 7a-7d). So z. B. a Slit with a length of 1 to 15 mm, preferably 1 to 10 mm, can be provided. It is also possible to provide the opening as a cross slot or in a star shape. It's important, that the opening is closed so that no liquid can escape to the outside by itself. Such slots can for example be cut or cut with knives in the first closure to be punched. If desired, you can also use it during the casting process Recesses are kept clear.

The inner stopper is a stopper that is separate for each container can be put on (single lock). However, a multiple lock is appropriate the number of reaction vessels possible. Fixing the multiple lock (2) is preferably carried out via a fitting element on the reaction vessel and / or the closure but via sealing rings (15) of the closure, which protrudes into the opening of the vessel (1). The inner closure preferably remains on the vessel and closes the vessel during the entire treatment process and is preferably not removed afterwards. The inner closure preferably covers the entire opening and is particularly preferred also the upper edges of the vessel, but can also be partially inserted into the vessel be. The inner closure is preferably not pressure-tight.

An outer closure (sealing element 3) is intended to serve this purpose, whether intentionally or not to seal any leaks in the inner seal from the outside. He therefore covers in all cases all openings in the inner closure, for example as Slit were already preformed or were created (using the pipette tip). This External closure can in principle consist of any materials if they are only firm Allow enough to be pressed onto the vessel opening and / or the inner closure. Prefers are therefore again elastic materials for the outer closure, as described above for the first closure were described. Because the outer closure is not for provision or the opening must be suitable, preferably not from the pipetting unit must be penetrated, larger limits are possible with regard to the thickness, especially upwards. However, thicknesses of more than 20 mm are then unsuitable, if a separate lid heater is to be provided. Preferred thicknesses are between 0.1 and 10 mm. The lid heater, which the evaporation may change inner closure to precipitate condensation, can in the outer Closure should be integrated. However, the lid heater can also be used in an independent Component be housed. It is particularly preferred that the outer closure as a thin, removable layer of elastic sealing material, e.g. B. a mat on one heated component is provided, which in its entirety on the Reaction vessel is pressed with the first closure. The outer closure is preferably suitable for the closure of a large number of vessels simultaneously, e.g. B. of 4 to 96, preferably 8 to 16, of such vessels (corresponding to the number of vessels or internal closures). A feature of a particularly preferred outer closure is that it can be completely separated from the inner closure. This happens when a liquid with the help of a pipetting unit through the inner closure into the vessel to be transported in or out of the vessel. For this process the Removed the outer stopper from the vessel and the inner stopper so that the pipetting needle when passing through the opening of the inner closure, not through the outer closure must pass through.

Liquid-tight in the sense of the invention means in particular that liquids are not without the action of additional forces, such as pressure (e.g. by heating) or conscious generation or widen an opening.

Pressure-tight means that the closure pressures up to 2.5 bar, preferably up to 1.2 bar, endures without gas or liquid from enclosed space z. B. the vessel allow.

The invention also relates to a method for transporting a liquid With the help of a pipetting unit into or out of a vessel, which is connected to a first and second closure is closed, comprising the steps of removing an outer Sealing element (3) from the vessel and an inner sealing element (2), introduction of Pipette into the vessel through the inner sealing element (2) and absorb the liquid into the pipettes from the vessel or dispensing the liquid from the pipetting unit the container.

Liquids that can be transported are, for example, sample liquids or derived liquids in which the presence of ingredients is proven should be, e.g. B. serum, buffy coat, plasma and liquids derived therefrom, liquids, are the result of a nucleic acid amplification process, but also liquids, the reagents for the detection, preparation or amplification of ingredients of sample liquids.

A pipetting unit is understood to mean a device or a device into which Liquid can be absorbed and released again. This is preferred by sucking in and expelling the liquid into a receiving area of the pipetting unit. In principle, this can also be done with reusable receiving units, such as metal needles, be. For the purposes of the invention, however, the use of plastic pipette tips is particularly preferred (5), which are used only once for liquid transport. This are usually made of materials such. B. polypropylene, which is a mechanical load only endure very limited. Furthermore, they are not very pointed, so that an independent Puncturing conventional lids with them is not possible. For this reason in the sense of the invention it prefers to whom the inner closure is pre-punched Opening, e.g. B. in the form of a slot.

The insertion of the pipetting unit into the vessel through the inner closure is preferred made automatically, e.g. B. with the help of a commercially available pipetting machine. In principle, however, is also the manual processing of the introduction of the pipetting unit possible, e.g. B. with a pipette. Here, the opening in the inner closure, for. B. the slots, expanded so far that the pipetting unit through the closure inside the Vessel can penetrate.

As a further step of the method according to the invention, either one is already in the Contained liquid in the pipetting unit or one in the Liquid contained in the pipetting unit is dispensed into the vessel. For example, it is possible Reagents for performing a reaction in the vessel to a sample liquid to admit. However, it is also possible to use a sample liquid that has already been processed withdraw certain amount and add it to the pipetting unit.

The system according to the invention has particular advantages in processes in which a liquid to be heated in the vessel. The vessel is preferred during heating steps closed by both the inner and the outer closure, so that practical neither liquid nor gas can escape from the vessel. For this the outer Closure with a certain force depending on the number of reaction vessels held down. This force is 0.1 to 10 N per reaction vessel, preferably 1 to 5 N. This pressure is preferred by vertically pressing the closure, e.g. B. with a Bracket (12), such as a metal plate, which can also contain the lid heater, generated. For this purpose, it is preferred to hold the bracket as uniformly and vertically as possible in the direction of To move the vascular occlusion. In the case of the use of multiple devices the simultaneous closure of all vessels under the same pressure conditions is preferred. In this case, the outer closure prevents gaseous liquid components from escaping (e.g. water vapor or aerosols) into the environment outside the Vessel, against the gas pressure. Procedure in which the liquid is inside the vessel The amplification methods are for example heated and cooled several times of nucleic acids, e.g. B. the polymerase chain reaction. But also simple denaturations and hybridization reactions can include heating steps.

With the help of the system according to the invention, several preferred embodiments of Procedure feasible. In a first execution phase, a sample liquid pipetted through the inner closure into the vessel, which then serves as a reaction vessel. Likewise, those required to carry out the desired reaction (s) Reagents either presented or pipetted into the sample liquid. During these pipetting steps, the outer closure is open, i.e. H. the sealing element (3) removed. Before carrying out heating or / and cooling steps, the outer closure closed. After completion of the temperature steps and preferred after re-setting a gas pressure in the vessel that corresponds to the external pressure the outer closure can be removed. If desired or necessary, will be further pipetting and / or temperature steps carried out, analogously to that described above Action. Should z. B. after a temperature step, liquid is removed from the vessel are, the outer closure is opened after pressure equalization, the pipetting unit inserted through the inner closure and the desired amount of liquid from the Contained in the pipetting unit. Then the pipetting unit is replaced by the internal closure removed and the liquid transported as desired, z. B. in a measuring cell (for analytical methods) or in another reaction vessel.

The process according to the invention preferably contains in a further step after completion each liquid transport through the inner closure the reclosure of the Vessel with the outer closure. After that, further temperature treatment reactions can be carried out be performed.

In Fig. 1 is the preferred method according to the invention for the transport of liquids shown schematically. A vessel (1) is closed with the help of an inner closure (2), an opening (4), for. B. in the form of a slot. When inserting the pipette tip (5) the outer closure (3) is separated from the inner closure. After introducing the Pipetting unit can absorb liquid into it or dispense it out of it will.

The outer closure is also open for this. After removing the pipette tip, it will The vessel is closed with the help of the outer sealing element (3) and is used for temperature treatment to disposal.

Fig. 2 shows a reaction vessel, as it can be used in Fig. 1, in an enlarged view. In addition to the elements described in Fig. 1, a sealing ring (15) can also be seen, the Is part of the inner closure (2).

Fig. 3 shows an enlarged view of the state when a pipette tip (5) through the inner Closure (2) is inserted into the vessel (1). The cover (3) is removed.

The system according to the invention can preferably be arranged in an arrangement of several Insert the connected reaction vessels (1) (Fig. 4), with the sealing element (3) then serves as a "mat" for a number of reaction vessels. The sealing elements (2) with individual openings (4) can also form a connected unit. Orders in 96-well microtitration plate format are preferred.

The sealing element (3) is preferred, as shown in Fig. 4, as an easily replaceable element the cover counter heating block (7). For this purpose, brackets (8) for the Mat provided. The heating element (7) with the mat can be hinged (Pivot point 9) can be folded onto the vessels (1).

5 shows a variant of the embodiment. Here is a holder (12) for the reaction vessels shaped so that the sealing element (3) simultaneously via a hinge (11) and a frame (10) is integrated and can be opened accordingly, so that a Pipetting, as shown in Fig. 1, can be done.

Fig. 6 shows the arrangement of the variant of Fig. 5 in a heating block (6) with a cover counter heating block (7) and pivot point (9).

Fig. 7 shows some geometric variants of the opening (4) for insertion according to the invention a pipette tip. 7a and 7b are slits, 7c and 7d are also small Recesses included in the material.

The sealing elements are preferably made of silicone, the reaction vessel made of polypropylene.

These materials are known for their poor bonding properties. Fig. 8 shows a possibility by forming widenings (14) of the band of the reaction vessels ("Ears") with corresponding holes (13) the silicone seal (2 / not shown) rivet. For sealing, the sealing ring (15 / not shown) is also used, the protrudes into the reaction vessel (see Fig. 2).

9 and 10 show reaction vessels for carrying out a PCR. It is a matter of 16 reaction vessels connected by curved bars in 2 rows with 8 positions each are. The webs are shaped so that a higher temperature load does not Deformation results. The reaction vessels have widenings (ears) (14, see Fig. 8) for welding to the sealing element (2) (see Fig. 11).

In Fig. 11 a mat is shown as a sealing element (2). It contains the individual Vessels associated positions slots (4). The sealing element can be widened (13) welded onto the vessel plate (top).

In Fig. 12, a sealing element (2) is shown in section. You can see them in the vessels protruding sealing rings (15) and the slots (4) (in longitudinal section).

FIG. 13 shows a multiple device according to FIG. 5 in the opened state. It is possible, e.g. B. using a barcode to attach an identification of the device on the flap.

The advantages of the present invention are that in addition to those commonly available Modules, e.g. B. a pipetting unit, no special automation modules necessary are so that z. B. Direct applicability in machines such as Elecsys 1010 or in TECAN pipetting stations for the application of molecular diagnostic determinations. When used as a seal for PCR cups, all tubes are pipetted from The sample and PCR mix are sealed. An aerosol entry is therefore not possible. this applies also for all further processing steps. This is a significant simplification of the manual PCR pipetting, as there is no longer any need to open the lid. Use as Reaction vessel in sample preparation processes is advantageously possible. An accessibility for pipetting with steel needles or disposable pipette tips. Microtitration plate formats are possible and inexpensive to manufacture. In contrast to State of the art, at least one of the closures is self-closing. A huge advantage of the system according to the invention is that a low-contamination multiple removal of Liquid from the vessel itself is possible after PCR.

The following examples are intended to explain the invention further:

example 1

In this example, the materials for a system containing elements according to the 6, 9, 10, 11 and 12 are described.

In the present example, polypropylene (Novolen 1100 UCX with an additive 11 NU 10 in a concentration of 3.5%) used as material for the reaction vessels. The Additive that up to max. 10% can be added, improves heat conduction, so that the reaction material is heated particularly quickly in a thermoblock can be done. This is due to the particularly thin wall thickness of the reaction vessel 0.3 mm, and the conical shape supports.

The reaction vessels have a volume of 0.25 ml.

The sealing element (2) preferably consists of elastic silicone rubber with a thickness of 1 mm, Elastosil LR 3003/40 (Wacker Chemie GmbH), which has an elasticity of 40 shore (DIN 53505).

In this case, there is a slot from to facilitate removal or pipetting 4 mm provided.

The sealing element (3) consists of the same material, also with a thickness of 1 mm.

Example 2 Sample preparation

An analog reaction vessel with a higher volume e.g. B. 1.5 ml and the invention System can also be used for sample preparation in nucleic acid analysis will. According to a protocol by B. Vogelstein and D. Gillespie (Proceedings of Natl. Acad. Sci. USA, February 1979, vol. 76, no. 2, pp 615 -619) are for the isolation of Nucleic acid from agarose gels according to Method II, various tempering steps at 25 ° C and 37 ° C necessary. The invention can be used to reduce the risk of contamination Use system preferably for automation of the protocol. In R. Boom et al. (J. of Clinical Microbiology, March 1990, pp. 495-503) and with P.M. Lizardi et al (Analytical Chemistry Vol. 98, pp 116-122 (1979)) use comparable protocols Incubations at 56 ° C or 50 ° C described with the inventive system reduced risk of contamination can be carried out. Here, the Pipetting steps using an x, y, z pipetting robot (TECAN, Canberra Packard) be carried out automatically. After removing the second sealing element (3) and a fixed placement of the reaction vessels on the working surface of the pipetting device Contamination-free pipetting can be carried out without additional device-side elements. It is particularly advantageous that all vessels are closed by means of a sealing element (2) and thus no aerosols can get into the reaction vessels.

Reference list

(1)

Reaction vessel

(2)

Sealing element 1

(3)

Sealing element 2

(4)

Opening in the sealing element 1

(5)

Pipette tip

(6)

Heating block

(7)

Lid heater block

(8th)

Bracket for sealing element 2

(9)

Pivot for lid counter heating block to open

(10)

Frame for holding sealing element 2

(11)

Open hinge

(12)

Frame for locking several reaction vessels

(13)

Widening for welding sealing element 1

(14)

Rivet point for welding sealing element 1 to reaction vessel

(15)

Sealing ring on sealing element 2

Claims (9)

System for the thermal treatment of liquids containing one or more vessels, one or more internal liquid-tight closures for these vessels, one or more external closures, which can seal one or more vessels pressure-tight. A method for transporting a liquid with the aid of a pipetting unit into or out of a vessel, which can be closed with an inner liquid-tight and outer pressure-tight closure, comprising the steps: optionally removing the outer closure from the vessel and the inner closure, Inserting the pipetting unit into the vessel through the inner closure, Withdrawing a liquid from the vessel into the pipetting unit or dispensing a liquid from the pipetting unit into the vessel. A method according to claim 2, characterized in that the pipetting unit Plastic pipette tip is or contains. Method according to claim 2 or 3, characterized in that the second closure has a preformed liquid-tight penetration point for the pipetting unit. A method according to claim 2 to 4, characterized in that the liquid in Vessel is heated before or after taking or dispensing liquid in the vessel. A method according to claim 5, characterized in that the outer closure is pressed against the vessel during heating, so that the closure is gastight is. A method according to claim 2, characterized in that the pipetting unit after Completion of the liquid transport through the inner closure from the vessel removed and the vessel is sealed gas-tight with the inner closure. A method according to claim 6, characterized in that the outer closure is heated with the help of a lid heater while the liquid is being heated. System for the thermal treatment of liquids containing a plurality of vessels connected to one another to form a single device, a first closure element with which this large number of vessels can be closed together, a physically separable second closure element, with which the first closure element is covered and the vessels are closed.

Images