EP3386637B1 - Temperature-control device having a reaction vessel - Google Patents

Description

A major analysis technique for nucleic acids is the polymerase chain reaction, commonly referred to as PCR. The nucleic acids are amplified by incubating them in a special reagent mix at defined temperatures until a finished PCR product is created. Today, this is routinely carried out in special temperature control devices, so-called thermal cyclers, which normally incubate a larger number of samples (e.g. 24, 48 or 96) in a reaction vessel consisting of a tube closed on one side and a cap. These thermal cyclers are usually relatively large as table-top units and are not designed for the analysis of individual samples.

To analyze the finished PCR product, it must be transferred to an appropriate analyzer. For this purpose, the caps are removed from the reaction vessels and then the PCR product is picked up with a pipette and transferred. Alternatively, the reaction vessels can also be sealed with foils and the reagent mix can be covered with overlay media (oil, liquid wax, chill-out wax, etc.). To transfer the reagent mix or the finished PCR product, the foils and, if necessary, the overlay media are pierced with a pipette tip, a cannula or the like. A buffer can also be added in this way. The resulting mix is often heated again after pipetting on.

For pipetting up or down, the reaction vessels are removed from the thermal cycler and, if necessary, they are inserted again into the thermal cycler.

In order to prevent the sample from condensing on the cap of the reaction vessel during the PCR, current thermal cyclers are equipped with a lid heater. So z. B. from the DE 92 05 824 U1 a temperature control device, here a metal block thermostat, known, with a cover which completely covers the sample container inserted into a metal block. The cover can either indirectly through good thermal contact with the metal block or directly over the Cover existing heating elements to be heated. Avoiding condensation on the lids of the sample containers is specified as the decisive effect for the heating. The disadvantage of indirect heating is that the temperature profile of the cover is dependent on the heating of the metal block. So that the lid of the sample container can actually be heated to the same temperature as the sample in the sample container, a great deal of insulation is required to avoid any heat loss on the way to the lid. In practice, the temperature of the lid will always be at least slightly below the temperature of the metal block, so that condensation cannot be avoided 100%. Regardless of whether the cover is heated directly or indirectly, the sample containers are positioned in the temperature control device in such a way that the heated cover is pressed directly onto the cover of the sample container in order to heat it up to a corresponding temperature via heat conduction.

With a temperature control device, here a thermal cycle device for performing the PCR as described in the WO 98/43740 A2 is set out to solve the problem that samples which are each arranged in a reaction vessel in a sample block are heated differently. Among other things, it is proposed to arrange a peripheral heating device around the sample block. The peripheral heater is not connected to the sample block. It should only warm up the air in the immediate vicinity to the temperature of the sample block if possible. In addition, the device has a heated lid. With the heated lid, pressure is exerted on the caps of the reaction vessels, so that the reaction vessels remain tightly closed on the one hand and on the other have good thermal contact with the sample block, via which the bottom of the reaction vessel and thus the sample is heated. The heating plate integrated in the lid is controlled so that it always reaches a temperature above the sample temperature in order to ensure that the sample does not condense on the cap of the reaction vessel. The heating plate has recesses in which, if necessary, curvatures of the caps can be positioned, on which no direct pressure is then exerted in order to avoid their deformation. With such a design of the closure caps, in addition to the function of closing the reagent vessel, an optical function can also be assigned to them, so that a finished PCR product without the Cap is removed or replaced, an optical reader can be supplied and the sample is optically accessible through the respective recess.

In the EP 0 706 649 B1 discloses a temperature control device with which the course of the nucleic acid amplification can be optically monitored. This temperature control device includes a reaction chamber consisting of a tube closed on one side, in which the optically tracked reaction mixture is located, and a cap. In order to avoid condensation of the reaction mixture on walls of the reaction chamber which are in the optical path of the device, these walls are heated. In the exemplary embodiment shown, the cap represents the part of the wall that lies in the optical path. It is heated indirectly via a thermally conductive circuit board (printed circuit board) with a heating element, without the circuit board or the heating element limiting the optical path.

In addition to a parallel incubation and implementation of the PCR of increasingly more samples, such as those from the aforementioned WO 98/43740 A2 Known temperature control device can be carried out, various applications have also become established in recent years, in which the PCR is only carried out on a single sample.

For this purpose, using a temperature control device, in particular a thermal cycler, which is designed to accommodate a large number of reaction vessels, is inappropriate because of the disproportionately high purchase costs and the space and energy requirements.

The additional step of opening the thermocycler to remove or insert the reaction vessel after or before pipetting up or down further increases the disproportion, since after each opening and closing of the thermocycler, a disproportionately large volume of space has to be reset to a defined temperature .

From the publication US 2008 / 0254532A1 is known a temperature control device for a symmetrical chemical reaction chamber with an inner volume for receiving a sample, which has a closable inlet. The The reaction chamber sits in a carrier housing with a thin, elastic material wall that has heating elements that emit heat to the peripheral surface of the reaction chamber.

Another temperature control device for carrying out a PCR can be found in the publication US 6,558,947 B1 forth. It consists of a large number of sleeves into which the reaction vessels can be inserted and which can be heated individually over their circumferential surfaces.

From the publication US 2008/0057544 A1 a temperature control device with a divisible heating block is known, which is also used to heat reaction vessels via their surrounding area.

Out EP 642 831 A1 a device for the automatic execution of temperature cycles in a large number of test tubes is known. The device has a thermoblock for heating the test tubes and a cover that can be heated with a resistance heater to cover the test tubes. Openings are provided in the lid, which are arranged coaxially to the sample tubes and through which a pipetting needle can be inserted into the sample tubes.

The publication G 92 05 824 U1 discloses a metal block thermostat with a metal block in which a large number of sample vessels can be inserted and a cover which can be removed from the metal block. The cover can be heated independently of the metal block or can be heated by thermal contact with the metal block.

It is the object of the invention to find a temperature control device with only one reaction vessel in which pipetting up and down of a sample is possible without removing the reaction vessel from the temperature control device.

The object is achieved by a temperature control device with a reaction vessel, with a thermally insulated interior in which the reaction vessel is accommodated, and with a cover which covers the interior, the reaction vessel consisting of a hollow body which is symmetrical about an axis of symmetry and closed on one side with a base and a cap. There is a heatable heating block in the interior. It has an inner surface which is adapted to the bottom of the reaction vessel and is in contact with it, to heat a sample in the reaction vessel. The lid is made of a heat-conducting material, the temperature control device having a heat-insulating cover, from which the lid is covered, and the lid abutting the cap. The thermal conductivity of the heat-insulating cover is necessarily lower than that of the heat-conducting material of the cover. It is essential to the invention that a heatable heating element is present which surrounds the hollow body and on the latter

Shape is adjusted. The radiator is connected to the lid via a heat-conducting contact area, so that the radiator heats the hollow body directly and the cap indirectly via the lid. The lid has a hole through which a cannula, through the cap, can be inserted into the hollow body.

The contact area is advantageously formed by a conical jacket-shaped radiator outer surface formed on the radiator and a conical jacket-shaped lid outer surface formed on the cover.

The outer surface of the radiator is favorably arranged on the inside, ie facing the hollow body.

It is advantageous if the radiator is formed from at least two radiator shells arranged symmetrically to the axis of symmetry and the radiator shells are mounted in the interior via at least one spring element, so that the radiator shells are applied to the hollow body with a restoring force of the at least one spring element.

The radiator shells are advantageously held together by at least one elastic ring.

It is also advantageous if the cover is formed from two cover shells which are spring-loaded to one another.

The invention is described below using an exemplary embodiment and drawings.

Fig. 1a

eine Temperiervorrichtung im Längsschnitt und

Fig. 1

b eine Temperiervorrichtung nach Fig. 1a im Querschnitt.

Show:

Fig. 1a

a temperature control device in longitudinal section and

Fig. 1

b a temperature control device Fig. 1a in cross section.

Each temperature control device according to the invention, shown for example in the 1a and 1b , is specially designed for a reaction vessel 1, that is, it is individually adapted to the geometric shape and dimension of the reaction vessel 1, which is why it is not universal in connection with a reaction vessel of any geometric Shape and dimension can be used. Accordingly, the features of the temperature control device can also be described only in connection with a reaction vessel 1.

The reaction vessel 1 consists of a hollow body 1.1 which is symmetrical about an axis of symmetry 1.0 and is closed on one side by a base 1.2 and a cap 1.3.

The temperature control device is not limited to the application for the PCR and cell suspensions, reagent mixes, e.g. B. consisting of reactants and catalysts, as well as finished PCR products, in liquid form, to be understood.

The temperature control device has a thermally insulated interior 2, closed by a lid 3, in which there is a heatable heating block 7, which has an inner surface 7.1 which is adapted to the bottom 1.2 of the reaction vessel 1. The reaction vessel 1 is arranged standing on the heating block 7 such that its bottom 1.2 is in contact with the inner surface 7.1 in order to heat a sample 8 located in the reaction vessel 1. The cover 3 is made of a heat-conducting material, is covered by a heat-insulating cover 9 and is in contact with the cap 1.3.

It is essential to the invention that there is a heatable radiator 4 which surrounds the hollow body 1.1 and is adapted to its shape and which is connected to the lid 3 via a heat-conducting contact area 5, so that the heater 4 directly connects the hollow body 1.1 and the cap 1.3 over the lid 3 indirectly heated.

With a temperature control device according to the invention, which is a thermal cycler in connection with the PCR, it is possible to insert a cannula, a detection probe, a mixing stick, a pipette tip or the like through the lid 3 of the temperature control device and through the cap 1.3 of the reaction vessel 1 and so on e.g. B. pipette off or on, detect or mix the sample without opening the temperature control device. Since the cover 3 remains closed, the defined room climate is also maintained in the temperature control device. The removal and, if necessary, reinstallation in the Temperature control device is not required. In addition, with a temperature control device according to the invention, it is possible to pipette off without additional means, which will be explained later. The cap 1.3 advantageously has a screw cap and is filled with a septum.

In order to be able to carry out a pipetting up and down with the temperature control device closed, it is essential to the invention that the lid 3 has a hole 3.3. Providing such a hole 3.3 in the cover 3 is possible in an uncomplicated manner, since the cover 3 according to the invention has no heating, but is heated by heat conduction via the heating element 4, with which it is in contact via the contact area 5 when the temperature control device is closed. The lid 3 is therefore not heated by active heating, but passively via heat conduction. Since the cover 3 bears on the cap 1.3 at least over the area of the hole 3.3, the interior 2 of the temperature control device is closed despite the hole 3.3.

The contact area 5 is formed by an outer surface 4.1 of the heater formed on the radiator 4 and an outer surface 3.1 formed on the cover 3. In the simplest form, the outer surface 4.1 of the radiator and the outer surface 3.1 of the cover are arranged in a ring and in a radial plane to the axis of symmetry 1.0. In order to make the contact area 5 larger for better heat conduction, the outer surface 4.1 of the radiator and the outer surface 3.1 of the cover may be conical. The larger the cone angle is selected, the larger the contact area 5 becomes, with otherwise the same dimensions of the temperature control device. For a simple closing of the interior 2 of the temperature control device with the cover 3, the outer surface 4.1 of the radiator can be arranged inside the outer surface 3.1 of the cover.

The radiator 4 not only has the task of heating the lid 3, but is also intended to heat the hollow body 1.1 of the reaction vessel 1 and thus the gas volume of the reaction vessel 1, which is inevitably located above the sample 8. In this way, condensation on the inner wall of the hollow body 1.1 is primarily avoided. Furthermore, the pressure of the gas volume can be changed by changing the temperature, which is often used as a so-called thermopneumatic effect in fluidics. A manipulation (pipetting off, Aliquoting, mixing) of the sample is also possible without a pump [ Keller, M .; Focke, M .; Strohmeier, O .; Reith, P .; Roth, G .; Mark, D .; Zengerle, R .; von Stetten, F .: "Centrifugal-thermopneumatic aliquoting on the LabDisk and application for DNA-based detection of various bacteria"; in: Microsystem Technology Congress 2013, Aachen, October 14-16, 2013, pp. 31 - 34 ].

When a cannula is inserted into the sample 8, it is pushed into the cannula while expanding the gas volume. For this purpose, the cannula must be closed at the outer first end during insertion until the inner second end is in contact with the sample 8. If the first end is then opened, the gas volume can expand in that the sample 8 rises in the cannula until a normal pressure is established in the gas volume or the sample 8 is completely absorbed. The heater 4 enclosing the hollow body 1.1 thus not only enables the use of a passively heated cover 3 but also a quasi-passive removal of the sample 8.

The radiator 4 can be a tubular body or advantageously consist of at least two radiator shells 4.2 arranged symmetrically with respect to the axis of symmetry 1.0, with exactly two radiator shells 4.2 in the form of half-shells being advantageous. While a tubular heating element 4 is arranged in a fixed position within the interior 2 with respect to the axis of symmetry 1.0, in one embodiment of the heating element 4 as two element shells 4.2 the two element shells 4.2 with respect to the axis of symmetry 1.0 are mounted radially elastically via at least one spring element 6 in the interior 2 and are supported via at least one elastic ring 11 held together.

Usually and also advantageously, the hollow body 1.1 of the reaction vessel 1 is a conical jacket with a small cone angle, so that when the reaction vessel 1 is introduced into the temperature control device and thus between the radiator shells 4.2, the latter are increasingly pressed apart, with an increasingly large restoring force on the radiator shells 4.2 acts, which ensures that the radiator shells 4.2 nestle against the hollow body 1.1. This restoring force is determined by the elasticity and the spring characteristic of the at least one elastic ring 11, for example a rubber ring and the at least one spring element 6 influenced.

In the case of the design of the radiator 4 as two radiator shells 4.2, the cover 3 can advantageously also be made from two cover shells 3.2, advantageously in the form of half-shells. The lid outer surface 3.1 and the radiator outer surface 4.1, as in FIG Fig. 1a shown, cone-shaped. When the lid 3 is closed, the lid shells 3.2 are then spread apart and the lid outer surfaces 3.1 are pressed against the outer heater surfaces 4.1 by restoring forces. In the in Fig. 1a In the embodiment shown, the restoring forces are generated by a spiral spring 10 enclosing the cover shells 3.2.

If the cover 3 is made from one piece, then the cover outer surface 3.1 remains firmly positioned with respect to the symmetry axis 1.0, while the position of the radiator outer surface 4.1 shifts radially to the symmetry axis 1.0.

If the outer surface 3.1 of the cover and the outer surface 4.1 of the radiator are designed as ring surfaces, only the contact area 5 is thereby reduced. In the case of a cone-shaped design of the outer cover surface 3.1 and the outer surface of the radiator 4.1, the outer surface 3.1 of the cover is placed on the outer surface 4.1 of the radiator at a different height in the axial direction of the axis of symmetry 1.0, depending on how far apart the radiator shells 4.2 are, which is why the cover 3 in the cover 9 is mounted axially sprung.

Reference list

1

Reaction vessel

1.0

Axis of symmetry

1.1

Hollow body

1.2

ground

1.3

cap

2nd

inner space

3rd

cover

3.1

Lid outer surface

3.2

Lid shell

3.3

hole

4th

radiator

4.1

Radiator outer surface

4.2

Radiator shell

5

Contact area

6

Spring element

7

Heating block

7.1

Inner surface

8th

sample

9

cover

10th

Coil spring

11

elastic ring

Claims (6)

1. Temperature-control device with a reaction vessel (1), with a thermally insulated interior area (2) in which the reaction vessel (1) is accommodated, and with a cover (3), which covers the interior area (2), wherein the reaction vessel (1) consists of a hollow body (1.1) and a cap (1.3), said hollow body being closed on one side with a floor (1.2) and being symmetrical in relation to a symmetrical axis (1.0), and wherein a heating block (7) is provided in the interior area (2), said heating block having an interior surface (7.1), which is adapted to the floor (1.2) of the reaction vessel (1) and is in contact with said vessel in order to heat a sample (8) located in the reaction vessel (1), wherein the cover (3) is made from a thermally conductive material, and wherein the temperature-control device has a thermally insulating enclosure (9) by which the cover (3) is covered, and wherein the cover (3) rests on the cap (3.1), characterized in that a heatable heater (4) that surrounds the hollow body (1.1) and is adapted to its shape is provided, wherein the heater is in contact with the cover (3) via a thermally conductive contact zone (5) in such a way that the heater (4) directly heats the hollow body (1.1) and indirectly heats the cap (1.3) via the cover (3), and wherein the cover (3) has a hole (3.3) via which a tube can be introduced into the hollow body (1.1) through the cap (1.3).
2. Temperature-control device as claimed in Claim 1, characterized in that the contact zone (5) is formed by a heater outer surface (4.1) in the shape of a lateral surface of a cone formed on the heater (4), and a cover outer surface (3.1) in the shape of a lateral surface of a cone formed on the cover (3).
3. Temperature-control device as claimed in Claim 2, characterized in that the heater outer surface (4.1) is arranged on the interior.
4. Temperature-control device as claimed in one of the previous claims, characterized in that the heater (4) at least consists of two heater shells (4.2) symmetrically arranged in relation to the symmetrical axis (1.0), and the heater shells (4.2) are mounted in the interior area (2) via at least one spring element (6) in such a way that the heater shells (4.2) are applied on the hollow body (1.1) with a restoring force of the at least one spring element (6).
5. Temperature-control device as claimed in Claim 4, characterized in that the heater shells (4.2) are kept together via at least an elastic ring (11).
6. Temperature-control device as claimed in Claim 5, characterized in that the cover (3) consists of two cover shells (3.2), which are mounted in an elastic manner in relation to one another.

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