Compact precision thermal cycler with heat accumulator and method for rapid temperature control of samples

Abstract

The invention relates to a compact precision thermocycler with a heat reservoir and a method for the rapid temperature control of samples for accelerated, miniaturized cooling and heating, preferably in medical diagnostics and bioanalytics. The objective is to create a compact precision thermocycler with a heat reservoir and a method for the rapid temperature control of samples, wherein the method enables faster PCR or mRNA analysis, guarantees more precise, accurately defined, and rapidly controllable temperature profiles, is easy to handle, and can be operated energy-efficiently. According to the invention, a first metallic heat reservoir 1 is arranged in the thermocycler, so that it can be set to an optimized temperature by means of a temperature control and separate heating elements, such as Peltier elements 7. Cavity strips 10 are arranged in the first heat reservoir 1.Continuously open sample receiving openings 14 or planar support surfaces are distributed throughout the cavity arrays 10. One, two, or more cooling blocks 2 can be mounted on this heat reservoir 1. The heat reservoir 1 and the cooling block(s) 2 are thermally coupled. A microcomputer 4 is integrated into the supply and control block 3. One, two, or more closed channels or thermally coupled heat pipes 6 are arranged on each cavity array 10. One, two, or more temperature sensors 11 are arranged in or on the heat reservoir 1 or on the cavity arrays 10. All components can be arranged on a two- or three-axis guided xyz stage 12 and the stage adapter 8. In the process, the cavity arrays 10 are force-fitted with reaction chambers containing at least the reaction solution and / or a sample.Temperature control is achieved via thermally connected heat conductors in a multidirectional manner between the sample, Peltier element 7, heat reservoir 1 and an ambient medium, thus transporting energy in an energy-saving and controllable way, so that recuperation takes place during successive heating and cooling cycles.

Description

[0001] The invention relates to a compact, fast, thermal cycler with a heat reservoir and a method for rapid temperature control of samples for accelerated, miniaturized cooling and heating, preferably in medical diagnostics and bioanalytics, particularly in fluorescence microscopy, to thermally initiate, control, or terminate enzymatic reactions and molecular interactions. An example of this is the polymerase chain reaction (PCR), which amplifies nucleic acid segments (ribonucleic acid [RNA], deoxyribonucleic acid [DNA]) and thus serves to detect, among other things, viruses, bacteria, and certain gene segments, messenger RNA, or microRNA.

[0002] Traditionally, a number of different configurations for thermocyclers are known. Ideas for solutions for accelerated, miniaturized cooling and heating have been published time and again, but the desire for even greater speed, precision in temperature profiles, and exact temperature control have not been fully met. To increase the speed of PCR analysis and the associated manufacturing effort, numerous solutions have been developed in the past, some of which have even been protected by patents. For example, there are ULTRA FAST PCR devices that use thin tubes or channels in conjunction with temperature-controlled surfaces for heat transfer. These can be found online, for example, at https: / / www.nextgenpcr.com / .

[0003] Another approach uses nanoparticles to enclose the PCR or mRNA substance and to alternately cool and heat it rapidly. This can be found at https: / / www.eurekalert.orgfpub releases / 2020-12 / ifbs-doh120720.php .

[0004] Furthermore, rapid temperature changes can also be achieved with suitable circulating cold and warm liquids, as proposed in the solution according to EP 000002535427 A2.

[0005] The use of Peltier elements for heating and cooling has established itself as the so-called gold standard, as they allow for the best control of temperature profiles and the precision of temperature control. The use of Peltier elements, which have long been known for their excellent controllability, has been known since 1987 from patent US 4,639,883. Another known solution is the use of discrete Peltier elements, which are connected to the other separate components of the thermocycler using thermal pastes or thermal adhesives in accordance with the state of the art. For example, EP 1 090 141 B1 uses a heating block and separate Peltier elements as well as a heat sink. Various physical effects for heating and cooling have been proposed for conventional thermocyclers, some of which are even used on an industrial scale.Despite the desire for ever shorter amplification times, newer, relatively fast solutions in particular still lack sensitivity and precise quantifiability.

[0006] For example, CN111521805 B describes a novel 2019 coronavirus antigen detection reagent and a corresponding manufacturing process as another technical solution. The detection reagent consists of a sample pad, a conjugate pad, an NC membrane, and an absorption pad. The product is simple and convenient to use, has a short detection time, high sensitivity, and is well suited for large-scale screening of primary medical facilities and key outbreak areas of epidemic situations. However, isothermal amplification is less sensitive than traditional quantifiable QPCR diagnostic tests. Another disadvantage of such tests is that the false-negative rate is relatively high and quantitative Ct values ​​are not provided.

[0007] Another rapid test based on isothermal DNA amplification is known from US Pat. No. 7829691 B2. Target-specific hybrid capture (TSHC) is a nucleic acid detection method that is not only rapid and sensitive, but also highly specific and capable of distinguishing highly homologous nucleic acid target sequences. The method generates DNA / RNA hybrids that can be detected using various methods. This isothermal PCR test is less sensitive than the classic quantifiable QPCR diagnostic tests. False-negative results are in the range of 5%.

[0008] Further literature on the state of the art is cited as follows: 1. Rödiger, S, Schierack, P, Böhm, A, Nitschke, J, Berger, E, Frömmel, U, Schmidt, C, Ruhland, M, Schimke, I, Roggenbuck, D, Lehmann, W and Schröder, C (2012) A Highly Versatile Microscope Imaging Technology Platform for the Multiplex RealTime Detection of Biomolecules and Autoimmune Antibodies. Adv Biochem Engin / Biotechnol. DOI: 10.1007 / 10_2011_132 2. Rödiger, S, Lehmann, W, Schröder, C, Schierack, P (2013) Bead technologies. Microparticle systems for nucleic acid diagnostics. BIOspektrum, 153-156. 3. Hanschmann, H, Rödiger, S, Kramer, T, Hanschmann, K, Steidle, M, Fingerle, V, Schmidt, C, Lehmann, W, and Schierack, P (2021) LoopTag FRET Probe System for Multiplex qPCR Detection of Borrelia Species. Life 2021, 11, 1163. https: / / doi.org / 10.3390 / life11111163

[0009] The invention is based on the object of creating a compact precision thermocycler with a heat reservoir and a method for rapid temperature control of samples, i.e. for rapid, miniaturized cooling and heating, wherein the thermocycler has a long service life, the method enables a higher speed of PCR or mRNA analysis, guarantees more precise, precisely definable, quickly controllable temperature profiles, improves the accuracy of the temperature control, the thermocycler requires a small volume, is easy to handle, which can be operated in an energy-efficient manner and which can be used in particular in fluorescence microscopy.

[0010] The problem is solved by the features of the preamble and the characterizing part of the first and fourteenth patent claims. Further advantageous embodiments are described in the dependent, reciprocal subclaims. According to the invention, a first metallic heat reservoir 1, which is generally designed as a plate-shaped support base, is installed and arranged in the novel, compact precision thermocycler for accelerated, miniaturized cooling and heating in medical diagnostics, so that it can be set to an optimized temperature (e.g., 40°C) by means of a temperature controller and separate heating elements, such as Peltier elements 7. One, two, or more cavity strips 10 are arranged in the first heat reservoir 1. In turn, a plurality of continuously open sample receiving openings 14 or planar support surfaces are distributed within the cavity strips 10.At least one, two, or more cooling blocks 2 can be optionally mounted on this heat reservoir 1. The heat reservoir 1 and the cooling block(s) 2 are thermally coupled. At least one microcomputer 4 is integrated into the supply and control block 3. One, two, or more thermally coupled heat pipes 6 are arranged inside each cavity strip 10 or arrangeable heat dissipation strips. The heat reservoir 1 is directly connected to one flat side of the Peltier elements 7. The other flat side of the Peltier elements 7 is thermally directly connected to the cavity strips 10. One, two, or more temperature sensors 11 are arranged in or on the heat reservoir 1 and on the cavity strips 10. The first heat reservoir 1 or further arranged heat reservoirs can be thermally insulated in the direction of a movable xyz object stage 12.With multiple cavity strips 10 arranged, different temperature zones can be formed. The cavity strips 10 of different temperature zones are separated from each other by an insulator 9, which can be strip-shaped. All components are arranged on a guided stage adapter 8 that can be moved along two or three axes.

[0011] According to the invention, in the compact precision thermocycler, in addition to the first heat reservoir 1, a second heat reservoir 5 or additional heat reservoirs can preferably be arranged in order to adapt the thermocycles to a required sample-specific, as fast as possible, temperature control.

[0012] Advantageously, the solution according to the invention can be mounted on two- or three-axis movable xyz stage 12 or stage adapter 8 of commercially available fluorescence microscopes or an automatic pipetting device coupled with a fluorescence microscope with viewing direction 13.

[0013] Furthermore, the first heat reservoir 1 and / or the cavity strip 10 consists of fast heat-conducting materials such as silver, copper or aluminum and the second heat reservoir 5 consists of non-metals with phase transition (PCM material), which has a high heat storage capacity and is designed with an enlarged surface.

[0014] In the compact precision ThermoCycler, the continuously open sample receiving openings 14 in the cavity bar(s) 10 are conical in shape. Transparent, thermostable reaction vessels with a planar bottom can be inserted into them with a force fit, or transparent, planar, thermostable slides or biochips can be placed on the planar support surface. This enables the analysis of a wide variety of samples using different types of sample containers.

[0015] In a special embodiment, a heatable weighting plate can be arranged above the cavity strip(s) 10 in order to counteract the formation of condensate.

[0016] In the compact precision ThermoCycler, the Peltier element(s) 7 consist of ceramic or metal plates, which are connected by soldering, gluing, or pressing to the cavity strip(s) 10 and, on the other hand, to the heat reservoir 1, ensuring rapid and low-loss heat flow. Advantageously, additional insulation 9 is arranged between the different temperature zones.

[0017] It is possible that heat conductors, heat pipes 6, heat foils, thermal pastes, heat spreaders and / or vapor chambers are embedded inside the cavity strip 10, the heat reservoir 1, or arrangeable heat dissipation strips.

[0018] In the compact precision ThermoCycler according to the invention, Peltier elements 7 can be arranged on both sides of the cavity strips 10 if required in order to improve the heat input or heat dissipation.

[0019] Advantageously, in the compact precision thermocycler, one or more cavity strips 10 can form a temperature zone, which are distributed in strips and separated from each other by thermal insulators 9. Several heat pipes 6, heat spreaders, and / or vapor chambers can be embedded inside a cavity strip 10 to accelerate the heat transfer to the heat reservoir 1 and the propagation rate of the heat flows during heating and cooling.

[0020] Compact precision ThermoCyclers can operate in a device configuration with an automated inverted or upright light or fluorescence microscope, whereby the device configuration consists of a compact precision ThermoCycler with arranged microscope adapter, a light or fluorescence microscopic optics, a camera, a two- or three-axis movable, motorized xyz stage 12 and a connected and connected control and evaluation software in order to automatically temperature control samples in coordination with the fully automated measurement and evaluation process.

[0021] Compact precision thermocyclers can operate in a device configuration with an automated inverted or upright light or fluorescence microscope, with an upstream station for sample loading into the cavities (sample receiving openings 14), a transport device (tray, conveyor belt) for the targeted movement and positioning of one, or two or more compact precision thermocyclers, and a second station for analysis equipped with at least one microscope and an evaluation device, such as a camera.

[0022] In the method according to the invention for the rapid and precise temperature control of samples in reaction chambers for conducting and evaluating polymerase chain reactions (PCR tests) using a compact precision thermocycler, the cavity strips 10 are force-fitted with reaction chambers containing at least the reaction solution and / or a sample. Temperature control is achieved via thermally connected heat conductors multidirectionally between the sample, cavity strip 10, Peltier element 7, heat reservoir 1, and a possibly second heat reservoir 5 in an energy-saving and controllable manner, so that recuperation occurs during successive heating and cooling cycles. This makes it possible for heat to be released passively or actively to an ambient medium, such as the ambient air, at an accelerated rate as needed. In principle, however, analysis can also be performed under a different protective medium.With the method according to the invention, reaction solutions, reaction gels, reaction solids or reaction gases are rapidly tempered qualitatively and quantitatively thermocyclically, isothermally or by a temperature gradient in order to carry out chemical and / or enzymatic reactions, crystallizations, molecular interactions or conformational changes, the growth of cells, tissues or.

[0023] Microorganisms, fluorescence measurement processes, phase transitions, dissolution processes, detachment processes, hybridizations or dehybridizations, antibody / antigen binding or dissociations can be thermally initiated, controlled, or terminated. The compact precision thermocycler according to the invention enables reaction solutions to be thermally initiated, appropriately controlled, or terminated with process precision in order to perform polymerase chain reactions, ligase chain reactions, isothermal nucleic acid amplifications, and melting curve analyses of nucleic acids and proteins.The integrated microcomputer 4 contains control software with comprehensive algorithms which controls the process in such a way that the heat can be transferred from the heat reservoir 1 via the heat conduction system and from the Peltier element 7 into the sample with very little loss and quickly and can be dissipated back into the heat reservoir 1 or the respective arranged heat reservoirs in order to realize fast thermal cycles in the sample in the most energy-efficient way possible.

[0024] Advantageously, in the method according to the invention, the heat reservoirs 1 and 5 are set to a temperature between the upper temperature level (denaturation at 94-96° C) and the room temperature (at 25° C) and the small temperature differences to be bridged are controlled by means of Peltier elements 7.

[0025] The advantage of the solution according to the invention lies in the fact that the heat loss occurring during operation of the Peltier elements 7 is not dissipated through intensive cooling during each cycle, as was previously the case, but is temporarily stored directly in the ThermoCycler. This is because it is complex and expensive to repeatedly dissipate large amounts of heat (cooling) and subsequently reinsert it (heating). These heat flows to and from the heat reservoir 1, combined with direct temporary storage, significantly reduce the energy required for operation, resulting in a recuperation effect. In addition to energy savings, the entire arrangement can also be significantly reduced in size and made very compact. Furthermore, the disclosed invention reduces the thermal material load on all components, particularly the Peltier elements 7.In addition to the benefits of energy and cost savings, the service life is also extended when the compact precision ThermoCycler is integrated into a microscopic measuring instrument enclosed in a housing, which is known to be sensitive to temperature fluctuations. The higher the energy input into the measuring instrument, the more complex the active cooling of the measuring instrument becomes, resulting in additional costs during construction and operation. In routine laboratories, the negative impacts on the working environment caused by waste heat and fan noise are well-known disturbances.

[0026] By reducing the temperature differences that the Peltier elements must overcome, their efficiency increases. Heat reservoir 1 can be easily adjusted to an optimized temperature. The optimal temperature (e.g., 40°C) is determined by the duration of the heating and cooling cycles and the required temperature differences, taking into account minimal heat transfer. The effect of recuperation is particularly effective with optimal temperature control, i.e., up to the extent limited by the efficiency of the Peltier elements 7 during cooling.

[0027] A further significant advantage is that the power loss peaks that occur during cooling can be quickly dissipated into a metallic (for example made of silver, copper, or aluminum) heat reservoir 1. In the effort to achieve even faster temperature changes, a further increase in heat storage capacity can be achieved by using a second heat reservoir 5 made of PCM (phase change material). The metallic heat reservoir 1 quickly absorbs, in particular, short-term power peaks, while the second PCM heat reservoir 5 temporarily stores relatively larger amounts of heat. This results in an overall better power-to-mass ratio, since the PCM material can absorb and release significantly more heat energy than metallic versions in the relevant temperature range.

[0028] The PCM material can be selected according to the optimal required heat reservoir temperature. To dissipate sufficient energy from the first heat reservoir 1 despite the slower heat storage in the second heat reservoir 5, the contact surface between the two reservoirs is increased by extending metal pins or fins of the first heat reservoir 1 deep into the PCM. The metallic first heat reservoir 1 significantly determines the speed and effectiveness of the overall arrangement.

[0029] Furthermore, the heat propagation rate can be significantly accelerated by a system of interconnected heat conductors using heat pipes 6, heat spreaders, heat foils, or vapor chambers, and metallic structural elements such as cavity strips, metallic Peltier elements, and solder joints. The complete structure of the ThermoCycler includes the guide for the movable stage adapter 8, fastening elements and insulation between the temperature zones 9, and several cavity strips 10. Furthermore, thermal insulation can be arranged in the direction of the xyz stage 12 to prevent, for example, a microscope from heating up.

[0030] The compact design and small dimensions allow for easy use in a wide variety of commercially available microscopes. In the method according to the invention, the combination is preferably carried out with automated microscopes, so that the temperature control and the measurement process can be carried out fully automatically, software-controlled, and coordinated. In addition to the preferred microscopic evaluation of the sample, it is also possible to equip the compact precision ThermoCycler itself with appropriate measurement technology based on optical or electrical sensors. This measurement technology can be fully integrated, but is preferably connected via the microscope adapter as an independent reader module.

[0031] The combination of rapid thermocyclic temperature control of reaction chambers with microscopic optical analysis, and in particular multicolor fluorescence microscopy, allows for a range of investigations of microscopic structures while simultaneously controlling the temperature of the reaction chamber and thus also of the microstructures, which were previously impossible or at least not optimally possible. This particularly applies to fluorescence-coupled, thermocyclic, or isothermal reactions such as multiplex real-time PCR or LAMP on biochips and bead assays, for example, for the detection of various microorganisms or molecular tumor markers. Such multiplex detection methods can also be combined with melting curve analyses to measure the binding strength of analytes, such as the avidity of antibodies, or to analyze single nucleotide polymorphisms (SNPs) in genes.To conduct melting curve analyses, step gradients or linear gradients are used with simultaneous stepwise or continuous reaction evaluation. The disclosed method of coupling eversensic or, preferably, inverse multicolor fluorescence microscopy with thermal reaction control allows cells, tissues, or biomolecules to be examined individually or in combination in multiplex systems, down to single-molecule resolution, i.e., the optical resolution of nanostructures. It is also possible to use the microscope optics in such a way that the aforementioned reactions can optionally be measured summatively without optical resolution of the micro- and nanostructures in the reaction chamber.Alternatively, it is possible to design the evaluation similarly to the state of the art in the simplest case by a combination of LEDs, optical filters and photodiodes using a simple fluorescence optics and to apply and evaluate it in combination with the solution according to the invention and the associated method according to the invention for the rapid tempering of samples, e.g. PCR tests or isothermal amplification reactions, without imaging and image analysis methods.

[0032] The concept of imaging analysis of thermocyclically controlled reactions was first described by Lehmann et al. (EP2167964) and implemented as a functional model by Rödiger et al. (2012) using a single-cavity cycler. However, for modern molecular biology laboratories, the analysis of individual samples is not sufficient for routine use. Rödiger et al. (2013) were able to demonstrate that it was possible to construct and build a ThermoCycler for a cavity bar with 8 cavities, with considerable effort in heat dissipation through external heat sinks. However, this type of thermal management precluded the further integration of at least three cavity bars, each comprising 8 cavities, into a ThermoCycler.Thanks to the inventive design of the compact precision thermocycler and the disclosed thermal management method for thermocyclers, the integration of a sufficient number of microscopically viewable cavity strips 10 into a thermocycler can now be implemented for the first time without exceeding the standard dimensions of a microscope's microplate specimen holder. By using four cavity strips 10, each with 3 x 8 cavities, the 96-well format commonly used in routine laboratories is achieved. Each of these cavity strips 10 can be individually temperature-controlled in the form of a temperature zone.

[0033] The device according to the invention is intended to be Fig. 1 and 2 their structure and mode of operation are explained in more detail. Fig. 1shows the exploded view of a compact precision ThermoCycler with a heat reservoir 1. The cooling block 2, which is advantageously designed as a unit consisting of a square aluminum profile with an integrated fan, serves to dissipate excess heat. To ensure that the first heat reservoir 1 quickly reaches the optimal operating temperature, the first heat reservoir 1 is connected to a demand heater 15.

[0034] If greater heat storage capacity and thus greater heat recuperation (heat recovery) is desired, one or more heat reservoirs made of PCM material 5 can be connected downstream of the fast-acting metallic heat reservoir 1. The combination of a first heat reservoir 1 and at least one second heat reservoir 5 results in a highly effective, complex heat reservoir that combines the properties of high volume and weight efficiency, high thermal energy storage capacity, and rapid heat power absorption. The inherently relatively slow charging and discharging rate of the PCM heat storage device 5 can be significantly increased by enlarging the contact surface between the two reservoirs. This effect is advantageously achieved by arranging metal pins or fins on the first heat reservoir 1, which are designed to extend deep into the PCM heat reservoir 5.At the same time, this eliminates an undesirable heat transfer surface compared to a more obvious separate design of the heat reservoirs.

[0035] The cavity strips 10 contain conical openings (cavities) for receiving the sample vessels, such as Nucleolink modules (Nunc). The angle of the cones (e.g., 17.2°) is selected so that the sample vessels are held in the cavity strip 10 in a self-locking and force-fitting manner by friction during cyclic temperature changes. This ensures good, stable heat transfer between the samples via the sample vessels and the cavity strips 10.

[0036] For cooling and heating, Peltier elements 7 are used, in accordance with the gold standard of classical PCR technology. The characteristic curve of the temperature in the Fig. 2demonstrates the outstanding precision of the temperature control achieved without any significant overshoots in the temperature curves. To shorten the temperature transition times, additional heat pipes 6 are embedded inside the cavity strips 10. This effectively increases the speed of the temperature transitions by an order of magnitude.

[0037] The Peltier elements 7 positioned at both ends of the heat pipes 6 can, if necessary, significantly increase the heat transfer capacity of the heat pipes 6 by up to four times compared to the use of a single-sided tempered heat pipe 6. This is done by introducing a heat flow into the cavity strips 10 from both sides and additionally halving the heat flow path.

[0038] For precise control of the temperature profiles, several temperature sensors 11 are arranged, which are suitably attached to a temperature zone at least in each cavity strip 10 and to the heat reservoir 1. The energy supply to the Peltier elements 7 is controlled by an attached energy supply and control block 3. Advantageously, the corresponding algorithms are implemented in the form of digital control by means of a microcomputer 4 integrated into the energy supply and control block 3.

[0039] Due to its compact, very small design, the ThermoCycler according to the invention can be mounted directly on an xyz stage 12 of a conventional fluorescence microscope (in Figure 1(only indicated schematically). With a feasible weight of the inventive ThermoCycler of only approximately 1.5 kg, state-of-the-art xyz stages of standard microscopes with high acceleration rates and thus very short travel times can be used.

[0040] Fig. 1shows the preferred viewing direction of a microscope 13, i.e. the viewing direction of the microscope 13 is directed from below onto the bottom of the sample vessels and thus through these onto the samples. This viewing direction offers the advantage that imaging particles of the samples (e.g. beads during the evaluation of bead assays, see Rödiger et al. 2012, Rödiger et al. 2017, Hanschmann et al. 2021), which collect and settle at the bottom of the sample containers, thus generally keeping the distance of the particles to the microscope constant within a narrow range. This fulfills an essential prerequisite for quantitative, high-quality PCR analysis by providing sharp, high-contrast images in real time, from which the data for the evaluation of, for example, PCR or melting curve analyses is ultimately extracted. The optical arrangement described here primarily refers to inverted microscopes.In principle, however, the inventive solution can also be used with upright microscopes if this is advantageous, for example, for in situ studies on living cells. Imaging can also be omitted entirely or optionally during analysis.

[0041] For temperature-controlled biochip examinations, possibly in combination with amplification, the biochips can be placed on the upper support surface of the cavity bars 10. Depending on the scaling, up to 96 observation points can then be used through the openings in the cavity bars 10. Further configuration options for this approach include the placement of suitable slides and the arrangement of biochips and slides on the underside of the cavity bars 10 using a simple, suitable pressure or suction option.

[0042] A typical temperature profile achieved with the compact precision ThermoCycler and using the method according to the invention for PCR cycles is shown Fig. 2 The three temperature phases of the PCR cycle are approached very quickly in the solution according to the invention and are thus kept very stable: 1. Denaturation at 94-96°C: At this high temperature, the complementary double strands are separated along the hydrogen bonds, i.e., denatured, so that they exist as single strands. 2. Hybridization in 50-70°C: The mirror image copy of the characteristic section of the DNA sequence to be detected attaches to the single strands by forming hydrogen bonds between the complementary nucleic bases. 3. Polymerization at70-74°C: At this temperature, a DNA polymerase, for example, optimally synthesizes the new strand by linking the complementary binding nucleotides.

[0043] Fig. 2 shows in the temperature range from 28° C to 32° C (lower solid line) the only very slight fluctuations in the temperature of the heat reservoir 1 with a compact precision thermocycler according to the invention as a result of the absorption or release of heat energy during heating or cooling. Reference symbol list for the compact precision ThermoCycler

[0044] 1First heat reservoir, metallic 2Cooling block 3Power supply and control block 4Integrated microcomputer 5Second heat reservoir made of PCM material 6Heat pipe 7Peltier element 8Stage adapter 9Thermal insulator between temperature zones 10Cavity bar 11Temperature sensor 12Movable xyz stage 13Viewing direction of a microscope 14Sample opening (cavity) 15Demand heating for the first heat reservoir

Claims

1. Compact precision ThermoCycler with heat storage and at least one Peltier element (7) arranged and connected thereto and a supply and control block (3), characterized by that one or two or more heat reservoirs are arranged, these heat reservoirs are designed to be coupled to one another, thata first metallic heat reservoir (1) is designed as a support base for the thermocycler, one, two or more cavity strips (10) are connected and arranged with the first heat reservoir (1) via Peltier elements (7), a plurality of continuously open sample receiving openings (14) and / or planar support surfaces are arranged in the cavity strips (10), at least one, two or more cooling blocks (2) are arranged on this first heat reservoir (1), the first heat reservoir (1) and the further heat reservoirs and the cooling block(s) (2) are thermally coupled, at least one microcomputer (4) is integrated into the supply and control block (3), one, two or more thermally coupled heat pipes (6) are arranged on or inside each cavity strip (10), the first heat reservoir (1) or further heat pipes are thermally insulated in the direction of a movable xyz object stage (12) are trained,one, two or more temperature sensors (11) are arranged in or on the heat reservoir (1) and in or on the cavity strips (10), the cavity strips (10) of one temperature zone are separated from cavity strips (10) of other temperature zones by an insulator (9), and all parts can be placed on a two- or three-axis movable guided xyz object stage (12) or object stage adapter (8).

2. Compact precision thermocycler according to claim 1, characterized by that two heat reservoirs (1 and 5) are arranged, which are designed to be coupled to one another, thata first metallic heat reservoir (1) is designed as a support base for the thermocycler, one, two or more cavity strips (10) are connected and arranged with the first heat reservoir (1) via Peltier elements (7), a plurality of continuously open sample receiving openings (14) or planar support surfaces are arranged in the cavity strips (10), at least one, two or more cooling blocks (2) are arranged on this first heat reservoir (1), the first heat reservoir (1) and the second heat reservoir (5) and the cooling block(s) (2) are thermally coupled, at least one microcomputer (4) is integrated into the supply and control block (3), one, two or more thermally coupled heat pipes (6) are embedded on or inside each cavity strip (10), the heat reservoirs (1 and 5) are thermally are insulated, in or on the heat reservoir (1) and in or on the cavity strips (10) each,two or more temperature sensors (11) are arranged, the cavity strips (10) of one temperature zone are separated from the cavity strips (10) of other temperature zones by an insulator (9), and all parts can be arranged on a two- or three-axis movable guided xyz object stage (12) and the object stage adapter (8).

3. Compact precision thermocycler according to claim 1, characterized by that a second heat reservoir (5) or further heat reservoirs are arranged next to the first heat reservoir (1).

4. Compact precision thermocycler according to claim 1, characterized by that the two- or three-axis movable xyz object table (12) is designed as a table of a fluorescence microscope or an automatic pipetting machine with a fluorescence microscope.

5. Compact precision thermocycler according to claim 1, characterized by thatthe first heat reservoir (1) and / or the cavity strip (10) consists of rapidly heat-conducting materials such as silver, copper or aluminum and the second heat reservoir (5) consists of non-metals with phase transition (PCM material), has a high heat storage capacity and is designed with an enlarged surface.

6. Compact precision thermocycler according to claim 1, characterized by that the continuously open sample receiving openings (14) in the cavity strip(s) (10) are conical, into which transparent, thermostable reaction vessels with a planar bottom can be inserted in a force-fitting manner, or transparent, planar, thermostable microscope slides or biochips can be placed on the planar support surface.

7. Compact precision thermocycler according to claim 1, characterized by that a heatable weighting plate is arranged above the cavity strip(s) (10).

8. Compact precision thermocycler according to claim 1, characterized by that the Peltier element(s) (7) consist of ceramic or metal plates which are connected by soldering, gluing or pressing to the cavity strip(s) (10) and, on the other hand, to the heat reservoir (1) in a force- and heat-locking manner to ensure a rapid and low-loss heat flow.

9. Compact precision thermocycler according to claim 1, characterized by that Heat conductors, heat pipes (6), heat foils, thermal pastes, heat spreaders and / or vapor chambers are embedded inside the cavity strip (10), the heat reservoir (1) and arrangeable heat dissipation strips.

10. Compact precision thermocycler according to claim 8, characterized by that Peltier elements (7) are arranged on both sides of the cavity strip (10).

11. Compact precision thermocycler according to claim 1, characterized by thatone or more cavity strips (10) form temperature zones which are distributed in strips and separated from one another by thermal insulators (9), one or more heat pipes (6), heat spreaders and / or vapor chambers are arranged on or inside the cavity strip (10) and these are directly coupled to the heat reservoir (1), wherein the heat transfer to the heat reservoir (1) and the propagation speed of the heat flows are accelerated during heating and cooling.

12. Compact precision thermocycler according to claim 1 in a device configuration with an automated inverted or upright light or fluorescence microscope, characterized by thatthe device configuration consists of a compact precision ThermoCycler with arranged microscope adapter, a light or fluorescence microscopic optics, a camera, a two- or three-axis movable, motorized xyz stage (12) and a connected and connected control and evaluation software in order to automatically temper samples in coordination with the fully automatic measurement and evaluation process.

13. Compact precision thermocycler according to claim 1 in a device configuration with an automated inverted or upright light or fluorescence microscope according to claim 9, characterized by thatan upstream station for loading samples into the cavities (sample receiving openings 14), a transport device (tray, conveyor belt) for the targeted movement and positioning of one or two or more compact precision thermocyclers is arranged and a second station is equipped with at least one microscope and an evaluation device, such as a camera, for analysis.

14. A method for the rapid and precise temperature control of samples in reaction chambers for carrying out and evaluating polymerase chain reactions (PCR tests) using a compact precision thermocycler according to claim 1, characterized by thatthe cavity strips (10) are force-fitted with reaction spaces containing at least the reaction solution and / or a sample, the temperature control is transported multidirectionally between the sample, Peltier element (7), heat reservoir (1) and an ambient medium in an energy-saving, controllable manner via thermally connected heat conductors, and that Recuperation occurs during successive heating and cooling cycles.

15. Method according to claim 14, characterized by that Heat reservoirs (1, 5) are set to a temperature between the upper temperature level (denaturation at 94-96° C) and the room temperature (at 25° C) and thus smaller temperature differences have to be bridged by the Peltier elements (7).

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