APPARATUS AND METHOD FOR THE THERMAL TREATMENT OF SAMPLES

Abstract

An apparatus for the thermal treatment of samples within sample containers comprises: a sample block a receiving region for the sample containers; a first temperature-control device thermally coupled to the sample block designed to set a temperature of the sample block; a cover plate arranged in the receiving region of the sample block; a second temperature-control device thermally coupled to the cover plate and designed to set a temperature of the cover plate; and a control unit connected to the first and the second temperature-control devices and designed to control the first and the second temperature-control device as to control the temperature of the sample block and the temperature of the cover plate such that the temperatures are coordinated with one another. A method for the thermal treatment of samples contained in sample containers is also disclosed.

Claims

1-18. (canceled)

19. An apparatus for the thermal treatment of samples contained in sample containers, the apparatus comprising: a sample block, including a receiving region adapted to receive the sample containers; a first temperature-control device thermally coupled to the sample block and configured to set a temperature of the sample block; a cover plate configured for the sample containers and dispose in the receiving region of the sample block; a second temperature-control device thermally coupled to the cover plate and configured to set a temperature of the cover plate; and a control unit connected to the first temperature-control device and the second temperature-control device and configured to control the first temperature-control device and the second temperature-control device, wherein the control unit is configured to control the temperature of the sample block and the temperature of the cover plate using the first temperature-control device and the second temperature-control device such that the temperatures of the sample block and the cover plate are coordinated with each other.

20. The apparatus of claim 19, wherein the control unit is configured to control the temperature of the cover plate and the temperature of the sample block such that the temperature of the cover plate is equal to or greater than the temperature of the sample block and such that a difference between the temperature of the cover plate and the temperature of the sample block does not exceed a maximum value.

21. The apparatus of claim 19, wherein the control unit is configured to control the temperature of the cover plate and the temperature of the sample block such that the temperatures of the sample block and the cover plate are coordinated with each other such that the temperature of the cover plate is a function of the temperature of the sample block.

22. The apparatus of claim 19, wherein the control unit comprises a first controller configured to control the temperature of the sample block via the first temperature-control device according to a predetermined setpoint temperature profile.

23. The apparatus of claim 22, wherein the control unit comprises a second controller configured to control the temperature of the cover plate via the second temperature-control device using a reference variable, and wherein the reference variable is a function of one of the following variables: a setpoint temperature of the sample block, the temperature of the sample block and a regulating variable output by the first controller.

24. The apparatus of claim 19, wherein the control unit is configured to control the temperature of the cover plate and the temperature of the sample block such that the temperatures of the sample block and the cover plate are coordinated with each other such that the temperature of the sample block is a function of the temperature of the cover plate.

25. The apparatus of claim 24, wherein the control unit comprises a first controller configured to control the temperature of the sample block via the first temperature-control device, and wherein the control unit comprises a second controller configured to control the temperature of the cover plate via the second temperature-control device according to a predetermined setpoint temperature profile, and wherein the first controller is configured to control the temperature of the sample block using a reference variable that is a function of one of the following variables: a setpoint temperature of the cover plate, the temperature of the cover plate and a regulating variable output by the second controller.

26. The apparatus of claim 19, wherein the cover plate has a planar front surface and is configured to bear against the sample containers disposed in the receiving region of the sample block such that the sample containers are disposed between the sample block and the cover plate.

27. The apparatus of claim 19, wherein the cover plate includes a plurality of openings or windows, which enable optical observation of the sample containers and which are disposed in a central region of the cover plate.

28. The apparatus of claim 27, wherein the second temperature-control device comprises at least one heating element disposed on the cover plate and at least one cooling element, and wherein the at least one cooling element is disposed in a peripheral region surrounding the central region of the cover plate, which includes the plurality of openings or windows.

29. The apparatus of claim 28, wherein the at least one heating element is disposed on or in webs of the cover plate, which extend between the plurality of openings or windows.

30. The apparatus of claim 19, wherein the cover plate comprises a material produced by powder metallurgy containing copper or silver and includes at least one refractory metal.

31. The apparatus of claim 19, wherein the first temperature-control device includes at least one thermoelectric element in contact with the sample block and a heat sink in contact with the at least one thermoelectric element.

32. A method for thermal treatment of samples in sample containers to facilitate DNA amplification, the method comprising: introducing the sample containers into a receiving region of a sample block; closing off the sample containers; covering the sample containers with a cover plate such that the sample containers are disposed between the sample block and the cover plate; controlling a temperature of the sample block via a control unit and a first temperature-control device thermally coupled to the sample block and controlled by the control unit as to set the temperature of the sample block; and controlling a temperature of the cover plate via the control unit and a second temperature-control device thermally coupled to the sample block and controlled by the control unit as to set the temperature of the cover plate, wherein the temperature of the cover plate and the temperature of the sample block are controlled such that the temperatures are coordinated with each other.

33. The method of claim 32, wherein the temperature of the cover plate and the temperature of the sample block are controlled such that the temperatures are coordinated with each other such that the temperature of the cover plate is equal to or greater than the temperature of the sample block and such that a difference between the temperature of the cover plate and the temperature of the sample block does not exceed a maximum value.

34. The method of claim 32, wherein the temperature of the sample block is controlled according to a predetermined setpoint temperature profile, and wherein the temperature of the cover plate is controlled using a reference variable which is a function of one of the following variables: a setpoint temperature of the sample block, the temperature of the sample block, and a regulating variable, which is output to the first temperature-control device by the control unit for setting the temperature of the sample block.

35. The method of claim 32, wherein the temperature of the cover plate is controlled according to a predetermined setpoint temperature profile, and wherein the temperature of the sample block is controlled using a reference variable that is a function of one of the following variables: a setpoint temperature of the cover plate, the temperature of the cover plate, and a regulating variable, which is output to the second temperature-control device by the control unit for setting the temperature of the cover plate.

36. An arrangement for thermal treatment of samples in sample containers to facilitate DNA amplification, the arrangement comprising: an apparatus according to claim 19; and a plurality of sample containers containing the samples to be thermally treated, wherein the sample containers are combined in a microtiter plate, and wherein the microtiter plate is disposed in the receiving region of the sample block and covered by the cover plate.

Description

[0047] In the following, the invention is explained on the basis of the exemplary embodiments shown in the figures. The same reference signs indicate the same parts of the apparatus shown. Shown are:

[0048] FIG. 1 a diagram of the time temperature profile of a sample block, a cover plate and a sample contained in a microtiter plate and arranged between the sample block and the cover plate in a conventional thermocycler during a PCR cycle;

[0049] FIG. 2 a schematic sectional view of an apparatus for the thermal treatment of samples;

[0050] FIG. 3 a schematic representation of a top view of the apparatus according to FIG. 2; and

[0051] FIG. 4 a schematic representation of the temperature profile over time of the sample block, of the cover plate and of a sample in the apparatus according to FIGS. 2 and 3.

[0052] As described at the outset, conventional thermocyclers comprise a sample block which is intended and is designed for receiving sample containers with samples to be thermally treated in sample containers. In order to carry out PCR methods, especially also in real-time PCR applications with simultaneous analysis of the sample, the sample containers and a cover sealing the sample containers, for example a sealing film, are covered with a cover plate which is heated in order to avoid condensate formation on the cover. Microtiter plates with sample containers for small sample volumes accordingly also have a low total height. The temperatures occurring in such an arrangement on the sample block, the cover plate and in the samples in a conventional thermocycler with a temperature control of the sample block and the cover plate according to the prior art are illustrated in the diagram shown in FIG. 1.

[0053] The temperature of the sample block (solid line) is controlled in accordance with a predetermined temperature profile for a PCR cycle: In a first time period, the sample block is heated to 95° C. (denaturation phase), the sample block is then cooled to 50° C. (primer hybridization phase) and maintained at this temperature for a second time period. The sample block is then heated again to a temperature of 72° C. (amplification phase). Following this third time period, the sample block is heated again to 95° C. and a new cycle begins. The temperature of the heatable cover plate (dashed line) is kept constant at 95° C. The dotted line represents the profile of the sample temperature. It can be seen that the temperature of the samples does not precisely follow the profile of the temperature of the sample block due to the influence of the heated cover plate on the temperature of the samples. It is particularly disadvantageous that the samples do not reach the final temperature of 50° C. desired for the primer hybridization phase but remain at an actual final temperature of approximately 60° C.

[0054] In FIG. 2, an example of an improved apparatus for the temperature control of samples according to the invention is shown schematically in a sectional view. The apparatus has a sample block 4 on the underside of which temperature-control elements 5 are arranged which are in thermal contact with the sample block 4 in order to heat or cool it. The temperature-control elements 5 may be designed, for example, as Peltier elements. On their rear side facing away from the sample block 4, the temperature-control elements 5 are in contact with a heat sink 6. In the present example, the heat sink 6 comprises a plurality of cooling elements for dissipating heat away from the temperature-control elements 5. The temperature-control elements 5 are thus sandwiched between the sample block 4 and the heat sink 6. The sample block 4 is formed from a material with high thermal conductivity, for example from silver or from aluminum. This makes it possible to set the temperature of the sample block 4 quickly by means of the temperature-control elements 5.

[0055] On its front side, which is opposite the rear side in contact with the temperature-control elements 5, the sample block 4 has a receiving region for sample containers 9. In the present example, the sample containers 9 are combined in a microtiter plate 3. The microtiter plate 3 used here is a microtiter plate for small volumes. The receiving region of the sample block 4 correspondingly has a surface with recesses for receiving the sample containers 9, which bears against the rear side of the microtiter plate 3.

[0056] The microtiter plate 3 is covered by a sealing film 10 which tightly closes the individual sample containers 9 which are designed as wells in the microtiter plate. The microtiter plate 3 with the sealing film 10 is covered by a cover plate 2. The cover plate 2 is thermally coupled to a temperature-control device comprising one or more heating elements 1. In the present example, a single, flat heating element 1 is present. The heating element(s) 1 can, for example, be designed as resistance heating elements.

[0057] FIG. 3 shows a view of the apparatus schematically from above. This representation shows that the heating element 1 has a plurality of openings 12 through which optical measurements, e.g., fluorescence measurements, on samples contained in the sample containers 9 of the microtiter plate 3 is possible. The cover plate 2 may be made of a material that is transparent to the measuring radiation. Alternatively and advantageously, it may be made of a material which is not transparent to the measuring radiation and has low thermal capacity in addition to good thermal conductivity. Suitable materials are refractory metals and refractory metal alloys, e.g., materials produced by powder metallurgy containing copper or silver and comprising at least one refractory metal. Refractory metals are usually understood to be high-melting, non-precious metals of subgroups 4, 5, and 6 of the periodic table of the elements, e.g., titanium, zirconium, hafnium, vanadium, niobium, tantalum, chromium, molybdenum and tungsten, as well as alloys containing one or more of said metals. Advantageous for use as the material of the cover plate 2 are such refractory metals or alloys which have thermal conductivity in the range between 160 and 400 W/m.Math.K and heat capacity between 150 and 300 J/kg.Math.K. For example, a tungsten-copper sintered material having thermal conductivity between 160 and 230 W/m.Math.K and heat capacity between 200 and 250 J/kg.Math.K may be considered. In this case, the cover plate 2 has openings aligned with the openings 12 of the heating element 1, through which measuring radiation can be detected from the sample containers of the microtiter plate 3. The sealing film 10 comprises of a polymer that is transparent to the measuring radiation.

[0058] In addition to the heating element 1, the temperature-control device for the cover plate 2 also comprises a plurality of cooling elements 11. In the present example, the cooling elements 11 are arranged on the periphery of the cover plate 2. They may, for example, be Peltier elements, heat pipes or fluid coolers, e.g., water or air coolers.

[0059] The apparatus for the thermal treatment of the samples contained in the microtiter plate 3 further comprises a control unit 13, which is electrically connected to the temperature control elements 5 for setting the temperature of the sample block 4 and to the heating element 1 and the cooling elements 11 for setting the temperature of the cover plate 2. The control unit 13 may comprise an electrical or electronic control circuit, especially with a microcontroller with software for running a data memory containing a temperature control. The control unit 13 is designed to control the temperature of the cover plate 2 and the temperature of the sample block 4 so that said temperatures are coordinated with one another. In the present example, the control unit 13 comprises two controllers, namely a first controller 7 for controlling the temperature of the sample block 4 and a second controller 8 for controlling the temperature of the cover plate.

[0060] The first controller 7 is designed to determine a regulating variable on the basis of a reference variable which represents the setpoint temperature profile of the PCR cycle and a control variable representing the current actual temperature of the sample block 4 and to output to the temperature-control elements 5 in order to set the temperature of the sample block 4 to the value of the reference variable. The control variable may be determined, for example, by a temperature sensor detecting the current actual temperature of the sample block 4 (not shown in FIGS. 2 and 3). The first controller 7 is further designed to output a signal representing the actual temperature of the sample block 4 to the second controller 8. The signal may be, for example, the reference variable of the first controller 7, the control variable of the first controller 7 or the regulating variable output by the first controller 7 or a signal derived from one of said variables.

[0061] The second controller 8 is designed to determine a regulating variable on the basis of a reference variable derived from the signal received by the controller 7 and representing the actual temperature of the sample block 4 and a control variable representing the current actual temperature of the cover plate 2 and to output it to the heating element 1 and/or to the cooling elements 11 in order to set the temperature of the cover plate 2 to the value of the reference variable. The control variable may be, for example, a temperature of the cover plate 2 detected by a temperature sensor (not shown in FIGS. 2 and 3). The reference variable for the second controller 8 may be determined based on the signal provided by the first controller 7 in such a way that it corresponds to a setpoint temperature of the cover plate 2 that is equal to the actual temperature of the sample block 4 or is higher than the actual temperature of the sample block 4 by a few Kelvin, for example, 2 to 10 K.

[0062] An example of a method for the thermal treatment of samples in sample containers with the aim of DNA amplification is described below. In the exemplary embodiment described here, the method is carried out by means of the apparatus shown in FIGS. 2 and 3. First, the sample-containing sample containers 9, which are combined in the microtiter plate 3, are arranged in the receiving region of the sample block 4. The sample containers 9 are previously or subsequently closed, for example by means of a sealing film 10 which is placed or glued onto the microtiter plate 3. The sample containers 9 are then covered with the cover plate 2. The microtiter plate 3 is then sandwiched between the sample block 4 and the cover plate 2. The temperature of the cover plate 2 and the temperature of the sample block 4 are controlled so that said temperatures are coordinated with one another by means of the control unit 13 such that the samples pass through a desired temperature profile, for example a temperature profile serving DNA amplification by means of PCR. For this purpose, the control unit 13 may control the temperature of the cover plate 2 and of the sample block so that said temperatures are coordinated with one another such that the temperature of the cover plate 2 is equal to or greater than the temperature of the sample block 4 and such that the difference between the temperature of the cover plate 2 and the temperature of the sample block does not exceed a maximum value. In the present exemplary embodiment, this maximum value may be a few Kelvin, e.g., 2 to 10 K.

[0063] In the present exemplary embodiment, the temperature of the cover plate 2 is controlled as a function of the temperature of the sample block 4. By means of the first controller 7 of the control unit 13, the temperature of the sample block 4 is controlled for the thermal treatment of the samples, in the present example namely according to a profile predetermined for the PCR cycles. For this purpose, the first controller 7 controls the temperature-control elements 5 thermally coupled to the sample block 4 using a reference variable corresponding to the desired setpoint temperature profile of the PCR cycles and a control variable detected by means of a temperature sensor and representing the current actual temperature of the sample block 4. The controller 7 determines a regulating variable from the reference and control variables and outputs it to the temperature-control elements 5 in order to set the temperature of the sample block 4 to the value of the reference variable.

[0064] The first controller also outputs a signal representing the actual temperature of the sample block 4 to the second controller 8, which signal is, for example, the reference variable of the first controller 7, the control variable of the first controller 7 or the regulating variable output by the first controller 7 or a signal derived from one of said variables. The second controller 8 determines its reference variable from the signal, for example in the simplest case by adding an amount representing a desired temperature difference between the sample block 4 and the cover plate 2 to the value of the signal. The reference variable thus determined corresponds to a setpoint temperature of the cover plate 2 which is higher than the current actual temperature of the sample block 4 by the desired temperature difference. The second controller controls the temperature of the cover plate 2 based on the determined reference variable and a control variable representing the current actual temperature of the cover plate 2, which control variable can be determined, for example, by means of a temperature sensor. For this purpose, it can output a regulating variable to the heating element 1 and/or to the cooling elements 11 in order to set the temperature of the cover plate 2 to the value of the reference variable.

[0065] The profiles of the temperatures of the sample block 4, of the cover plate 2, and of a sample contained in the microtiter plate 3 in the course of the described method are shown in a schematic representation in FIG. 4. The temperature of the sample block 4 (solid line) follows the predetermined temperature profile for a PCR cycle. It substantially corresponds to the temperature profile of the sample block with the denaturation phase, the primer hybridization phase, and the amplification phase, as shown in FIG. 1. In contrast to the embodiment shown in FIG. 1 according to prior art, the temperature of the cover plate 2 of the apparatus shown in FIGS. 2 and 3 does not constantly remain at a high value. Rather, by means of the described control, which is a function of the temperature of the sample block 4, the temperature of the cover plate 2 (dashed line) is always adapted to the temperature profile of the sample block 4 so that a temperature difference between the cover plate 2 and the sample block 4 does not exceed a maximum value of 10 K. From FIG. 4 it can be seen that, as a result of this temperature control of the cover plate 2, the temperature of the sample (dotted line) substantially follows the temperature profile of the sample block 4 with significantly improved speed and precision. It should be noted, especially, that during the second time period of the cycle, i.e., during the primer hybridization phase, the final temperature of the sample reaches the value predetermined by the sample block 4 after a short cooling phase.

[0066] The invention comprises a plurality of variants and variations of the exemplary embodiment described herein. For example, it is also possible for a temperature sensor that detects the current actual temperature of the sample block to output a signal representing the actual temperature of the sample block to the second controller of the control unit in order to derive the reference variable of the second controller.

[0067] In a further variation of the exemplary embodiment of an apparatus and a method detailed here for the thermal treatment of samples, it is possible for a first controller connected to the temperature-control elements for the sample block to control the temperature of the sample block as a function of the temperature of the cover plate controlled by a second controller. In this case, a reference variable representing the course of the PCR cycles is specified to the second controller, while the first controller receives from the second controller a signal representing the actual temperature of the cover plate, on the basis of which the reference variable of the first controller is determined for the temperature control of the sample block.

[0068] A plurality of other variants is conceivable without departing from the inventive idea.