Compact thermal cycling device and system comprising said thermal cycling device

专利摘要:
The present invention relates to a compact thermal cycling device (1) for heating and cooling at least one housing mount (2) for housing a lower portion of a reagent tube (3), adapted for conducting heat into or out of same; and a thermoelectric heater (6) coupled to said mount (2). Advantageously, the device (1) of the invention further comprises a heating block (8) arranged in a region located above the region of the mount (2), comprising at least one housing port (9) adapted for receiving an upper portion of the tube (3) located above the portion housed by the mount (2), being in thermal contact with said upper portion.
公开号:ES2738665A1
申请号:ES201990017
申请日:2017-08-17
公开日:2020-01-24
发明作者:Fernandez Aitor Ezkerra；Ruiz Javier Berganzo；Lazcano Inigo Aranburu；Eneriz Diana Eneriz；Poza Miguel Roncales
申请人:Brio Apps Alphasip S L；
IPC主号:

专利说明:

[0001]
[0002] COMPACT THERMOCICLING DEVICE V SYSTEM THAT INCLUDES SUCH
[0003]
[0004] FIELD OF THE INVENTION
[0005]
[0006] The present invention is part of the field of experimentation in Molecular, Biochemical and Genomic Biology. More specifically, the invention relates to a thermocycler device used for amplification of DNA sequences, as well as to a system associated with said device.
[0007]
[0008] BACKGROUND OF THE INVENTION
[0009]
[0010] Thermocycler devices, also known as PCR machines (from "Polymerase Chain Reaction") are devices preferably used in the field of Molecular Biology to subject the polymerase enzyme to different temperature cycles, for example, to obtain DNA amplification or for sequencing reactions with the Sanger method.
[0011]
[0012] The most common model of such devices consists of a plurality of wells configured to house reagent tubes, connected to an electrical resistance block that distributes, through a heating plate, a homogeneous temperature to the wells during times that can be programmable , normally with temperature ranges between 4 ° C and 96 ° C.
[0013]
[0014] Since the reactions incubated in the tubes are usually carried out in an aqueous solution, the thermal cyclers usually comprise, in addition, a heating lid (or, in English, "heating lid") arranged to cover the tubes and which is heated from constant form at 104 ° C, so that the condensation of water in the closures of the tubes where the reaction occurs is thus limited, reducing the concentration of solutes to prevent the optimal conditions for the polymerizing enzyme from being modified, thus as to maintain the desired thermodynamics for the pairing of primers (or "primers") that serve as a starting point for DNA replication.
[0015] In the thermal cycler, fast and continuous cycles of temperature change are programmed to initiate the processes of separation and denaturation of the DNA chains at high temperatures, subsequently their banding at low temperatures and, finally, their elongation at high temperatures. This cycle can be repeated multiple times, thus reaching the desired degree of amplification of the DNA fragment.
[0016]
[0017] For several years, several commercial thermal cyclists have also been known, comprising heating means based on Peltier technology, taking advantage of the properties of semiconductors. These materials offer better temperature uniformity, as well as much more steep rise and fall ramps, thus obtaining better results in PCR processes. Also, some market solutions have chosen, in the search to improve the accuracy, accuracy and homogeneity of the temperature, to use metals such as gold, silver and other alloys in the heating blocks of the wells, achieving greater stability and reproducibility of the essays.
[0018]
[0019] However, even in thermocyclers based on heating caps, the problem of condensation is still present and consequently limits the effectiveness of its results. In this sense, the cover that acts as a thermal block in this type of thermal cycler must be folded or slid over the upper part of the tubes, resting on them. This requires a complex construction that limits the access and handling of the pipes, where it is also caused that the heating bulbs (that is, the heaters of the base of the pipes and the heating caps) are excessively far apart, which creates a high level of condensation of the reagents.
[0020]
[0021] As described in the previous paragraphs, it is therefore necessary to have a solution that can be integrated into a thermal cycler that effectively reduces the condensation produced in the samples in which the amplification reactions occur and also allows more direct access to the housing tubes used. The present invention is aimed at solving said need, by means of a novel compact thermocycler device, equipped with a double system of static and dynamic heating of the housing tubes.
[0022]
[0023] BRIEF DESCRIPTION OF THE INVENTION
[0024] According to the information set forth in the previous section, an object of the present invention is therefore to provide a device that is capable of significantly reducing the condensation of the samples, in comparison with the known thermal cyclers based on hot lid systems or "heating lid ", where the cover used is folded or slides over the top of the tubes, resting on its ends.
[0025]
[0026] For the purpose described, said object of the invention is carried out by means of a compact thermocycler device for the heating and cooling of at least one housing mount of a lower portion of a reagent tube, wherein said mount is coupled to a thermoelectric heater for the heating / cooling said lower portion of the tube.
[0027]
[0028] Advantageously, the device of the invention additionally comprises a heater block thermally insulated from the mount, disposed in an upper region above it, comprising at least one housing hole adapted to receive an upper portion of the reagent tube and heating said upper portion, above the lower region housed by the mount.
[0029]
[0030] The heating block preferably comprises one or more resistive heating means adapted for thermal contact with the reagent tube when it is housed in the thermal cycler, so that the upper portion of said tubes can be kept at a constant temperature, avoiding condensation of the sample in the walls of the tubes during the different temperature cycles. The combination of the effect of the heater block and the thermoelectric heater makes it possible to reduce the times of obtaining results, as well as to obtain increases and decreases of temperature faster (more pronounced ramps), which is equivalent to a greater efficiency of the reactions, thus improving the results obtained against other thermal cyclers of the state of the art.
[0031]
[0032] In a preferred embodiment of the invention, the thermoelectric heater comprises two Peltier elements, facing and located surrounding the mount. This results in a thermally symmetrical configuration.
[0033] In another preferred embodiment of the invention, the mount comprises a hole for the inspection or optical detection of the reaction that occurs in the tube, as a consequence of the heating and cooling thereof.
[0034]
[0035] In another preferred embodiment of the invention, the resistive heating means are configured to keep the portion of the tube housed by the housing orifice at a constant temperature, preferably equal to or greater than the minimum condensation inhibition temperature of the sample housed in The tube. More preferably, the temperature maintained is substantially 104 ° C. Thus, it is not necessary to fold or slide the device on its upper part, as is the case with most thermocycling devices of the prior art. This allows, therefore, a simpler construction that does not require automatisms or intervention by the user, and that maintains the lateral heating of the tube providing a greater surface free of condensation, as well as a greater precision and uniformity of the temperature in the tube.
[0036]
[0037] In another preferred embodiment of the invention, the mount and the heater block are separated at a distance between 0.5 mm and 5.0 mm.
[0038]
[0039] In another preferred embodiment of the invention, the mount comprises an upper extension or well, which typically has a height between 0.1 mm and 5.0 mm.
[0040]
[0041] This range is suitable to avoid thermal contact between the Peltier heater and the heater block, especially in cases where the tubes are made of low thermal conduction materials, such as plastic materials.
[0042]
[0043] The well can be an integral part of the mount, an extension thereof, or an element in thermal contact with it, and its main function is to extend the heat transfer surface of the thermoelectric heater over the lower portion of the reagent tube . In this way, the well contributes to the heating of the tube above the level of the reagents, thus avoiding thermal losses by the tube wall close to said level and unifying the temperature of the reagents.
[0044]
[0045] In another preferred embodiment of the invention, the device comprises a temperature sensor in thermal contact with the mount, configured to control the power supplied by the thermoelectric heater and the temperature of the tube housed therein. mount. More preferably, the temperature sensor is arranged next to the plane of symmetry of the mount, and / or in the area closest to the reagent housing tube.
[0046]
[0047] In another preferred embodiment of the invention, the thermocycler device of the invention comprises a plurality of reagent tube housing mounts, equipped with their corresponding Peltier heaters, heater block and wells (if used).
[0048]
[0049] This achieves a fully scalable thermal cycler, whose associated technology is not limited by the number of tubes used.
[0050]
[0051] Another object of the invention relates to a system comprising at least one thermocycler device according to any of the embodiments described herein, in combination with one or more reagent tubes housed in one or more housing mounts of said thermocycler.
[0052]
[0053] In a preferred embodiment of the system of the invention, one or more tubes comprise a plug or upper closure that penetrates to a height substantially equal to or less than 20 mm above the level of the reagents to be housed in said tubes. More preferably, said height is substantially equal to less than 1 mm.
[0054]
[0055] Definitions of some of the main terms used in the present description, and their scope of interpretation in the light of the invention claimed herein, are given below for illustrative purposes:
[0056]
[0057] A thermocycler device, or PCR machine, must be interpreted as any device capable of subjecting one or more reagent container tubes to different temperature cycles, for example, to obtain DNA amplification or for sequencing reactions with the method of Sanger
[0058]
[0059] A mount is to be interpreted as a housing means of a lower portion or base of a reagent container tube of a thermocycler, said mount being subjected to temperature control by one or more heating means.
[0060] A reagent container tube must be interpreted as any means for housing said reagents whose shape is adapted for housing in a thermocycler mount.
[0061]
[0062] A well must be interpreted as a thermally connected element or part of the mount, and which is arranged at a higher height for the reception of the reagent container tube. The well houses, therefore, a portion of the tube superior to that housed by the mount.
[0063]
[0064] An optical inspection hole is to be interpreted as any access to the frame through which information associated with light transmitted to / from the interior of said frame can be obtained.
[0065]
[0066] A thermoelectric heater must be interpreted as a means of controlling the temperature of the frame through a mechanism based on the thermoelectric effect, in any of its known manifestations.
[0067]
[0068] A Peltier thermoelectric element must be interpreted as a component of the thermoelectric heater based on temperature control by means of the Peltier effect.
[0069]
[0070] A temperature sensor must be interpreted as any mechanism equipped with means for measuring the value of the temperature inside the mount, or of the variations of said value.
[0071]
[0072] A heating block is to be interpreted as any means of resistive heating, intended for the application of heat to an upper portion of the tube housed by the housing orifice
[0073]
[0074] A lower portion of the tube is to be construed as a continuous portion of the tube comprising, respectively, the volume of said tube that houses the reagents subjected to temperature cycles, when said tube is housed in a thermocycler mount.
[0075] An upper portion of the tube is to be interpreted as a continuous portion of the tube that is arranged, when said tube is housed in the mount, at a level higher than that of the lower portion referred to in the preceding paragraph.
[0076]
[0077] A plug or closure of the tube must be interpreted as any upper closure element of said tube, the application of which limits the volume of reagent housing therein.
[0078]
[0079] The term "thermal contact" between two or more elements has to be interpreted, within the scope of the invention, as the intimate contact (understood as a direct contact in the absence of an interface means such as air) between said elements, by means of the which heat transfer between them is allowed.
[0080]
[0081] The term "substantially", applied to the relationship between two or more magnitudes must be interpreted, within the scope of the invention, as equal in a range of variation of ± 1O% over the referred value.
[0082]
[0083] The device of the invention acts on longitudinal configuration tubes intended to be in a vertical position, taking the force of gravity as a reference of this verticality. Thus, relative terms such as upper, lower, above or below, should be interpreted with respect to this vertical direction. The vertical direction of the tube or tubes is established by the configuration of the tube housings configured so that said tube, is its operative position in the thermocycler, adopts the correct position.
[0084]
[0085] The term "comprises" must be interpreted, when applied to the relationship between a principal element with respect to other secondary elements, such as that said principal element includes or contains said secondary elements, but without exclusion of other additional elements.
[0086]
[0087] The expression "consists" must be interpreted, when applied to the relationship between a principal element with respect to other secondary elements, such as including or containing said secondary elements, excluding other additional elements.
[0088]
[0089] DESCRIPTION OF THE FIGURES
[0090] Figure 1 of the present document shows a cross-sectional view of the thermocycler of the invention with a plurality of housed tubes, one of them sectioned and visually accessible, according to a preferred embodiment thereof where the relative position of the dynamic heating block is represented. and of the static heating block, and the separation between them.
[0091]
[0092] Figures 2a-2b show two views of the mount and the well of the thermocycler of the invention, where some of its main elements are detailed.
[0093]
[0094] Figure 3 shows a section of the reagent tube housed by the device of the invention, wherein said tube is closed by means of a cap.
[0095]
[0096] NUMERICAL REFERENCES USED IN THE FIGURES
[0097]
[0098] (1) Thermocycler device
[0099] (2) Mount to receive a lower portion of a container tube of
[0100] reagents
[0101] (3) Reagent container tube
[0102] (4) Well or upper frame extension
[0103] (5) Optical inspection hole
[0104] (6) Thermoelectric heater
[0105] (6 ', 6 ") Peltier thermoelectric elements
[0106] (7) Temperature sensor
[0107] (8) Heater block
[0108] (9) Housing hole to receive an upper portion of the tube
[0109] (10) Tube cap or closure
[0110]
[0111] DETAILED DESCRIPTION OF THE INVENTION
[0112]
[0113] A detailed description of the invention, referring to a preferred embodiment thereof based on Figures 1-3 of this document, which are provided for illustrative and non-limiting purposes, is set forth below.
[0114]
[0115] As described in previous sections, the thermocycler device (1) object of the present invention (Figure 1) is designed for static combined heating (hot plate mode, or "hot plate") V / o dynamic (thermocycler mode) of unconnected containers or mounts (2), intended to accommodate the reagent tubes (3), where each mount (2) is, optionally, thermally connected or joined in solidarity with a well (4) that allows the heating to be extended on the lower portion of the tubes (3) to a level higher than the volume of the reagents. The heating provided by the wells (4) thus allows the temperature of the sample housed in the tubes (3) to be homogenized, thus minimizing the thermal gradient at the interface between the reagents and the air contained in the tubes (3), and preventing condensation
[0116]
[0117] In more detail, the thermocycler device (1) of the invention comprises, in a compact and preferably monolithic manner, at least the following elements:
[0118]
[0119] - Mount (2): It comprises a preferably metallic structure, through which heat is directed towards the inside or outside of it, causing, respectively, the heating or cooling of the lower region of the tube (3) housed inside , by thermal conduction. The mount (2) additionally comprises a hole (5) (Figures 1 and 2a) for the inspection or optical detection of the reaction that occurs in the tube (3), as a consequence of the heating and cooling thereof. To facilitate said inspection, the mount (2) preferably has a flat bottom base. On the other hand, the upper portion of the frame (2) will be, internally, preferably with a truncated conical section to accommodate the corresponding tube (3).
[0120] - Well (4): It is a housing element of the tube (3) that is thermally connected or is part of the mount (2), and that is arranged at a higher height of it. The well (4) thus houses a portion of the tube (3) superior to that housed by the mount (2), acting as an extension thereof.
[0121] - Thermoelectric heater (6): Preferably, said heater (6) comprises two thermoelectric elements (6 ', 6 ") Peltier, facing and located on each side of the mount (2), so that a greater thermal symmetry is obtained in the application or extraction of heat to the tube (3).
[0122] - Temperature sensor (7) (Figures 2a and 2b): Preferably included in the mount (2) and in thermal contact with it, it allows monitoring the power supplied by the thermoelectric heater (6), thus achieving precise control of the tube temperature (3). - Heater block (8): Said block (8) is arranged in the thermocycler device (1) in an upper region above the mount (2) and the well (4) (if used). In the preferred embodiment, the block (8) is connected to the mount (2), however, both must be separated and thermally insulated. In this same exemplary embodiment, mount (2) and well (4) are the same piece although they can be formed as different pieces thermally communicated. Also, said heater block (8) comprises at least one housing (9) configured to receive a portion of the tube (3) located above the portion housed by the wells (4).
[0123]
[0124] The heater block (8) comprises one or more resistive heating means (for example, "hot plate" type) and a housing orifice (9) for housing the reagent tubes (3), thus being in thermal contact with the same, so that the portion of the tube (3) received by said holes (9) can be maintained at a constant temperature, preventing condensation of the sample on the walls of said tube (3) during the PCR reactions.
[0125]
[0126] Additionally, the combination of the effect of the heater block (8) and the thermoelectric heater (6) results in a shorter time to obtain results, as well as faster increases and decreases in temperature (more pronounced ramps), which is equivalent to a greater efficiency of the reactions, thus improving the results obtained against other thermocyclers of the prior art.
[0127]
[0128] Preferably, the static heater block (8), located in the upper part of the tube (3), heats it by its lateral regions, at a temperature of substantially 104 ° C, so that it is not necessary to lower it or slide it over the upper part of the tubes (3), as is the case with most thermocycler devices of the prior art. This allows, therefore, a simpler construction that does not require automatisms or intervention by the user, and that maintains the lateral heating of the tube (3) providing a greater surface free of condensation (that is, without condensation), as well as greater precision and uniformity of the temperature in said tube (3).
[0129]
[0130] In a preferred embodiment of the invention, to provide greater independence to the dynamic and static heating regimens of the thermocycler device (1), the mount (2) and the heater block (8) are separated, so that they are thermally isolated from each other. Preferably, its separation range varies between 0.5 mm and 5.0 mm. Said separation is considered adequate since the tube material, usually plastic, is a bad thermal conductor and, therefore, there will be no thermal contact between the heater ( 6) thermoelectric and heater block (8).
[0131] For its part, the configuration of the mount (2) according to the present invention is optimized to maximize thermal symmetry and minimize thermal inertia. This leads to the reduction of the energy input necessary to reach the target temperature in the entire frame, which contributes to the improvement of the efficiency of the thermal cycler (1).
[0132]
[0133] In this sense, in a preferred embodiment of the invention, the shape of the mount (2) is designed such that the mass, determining thermal inertia, decreases as the distance to the main thermal focus of the thermal cycler device (1) increases. said thermal focus being the thermoelectric heater (6). This favors the transmission of heat to or from the thermoelectric heater (6) since it is where the dimensions of the frame section (2) is maximum, and the reduction of possible temperature gradients, due to the progressive distance of the upper regions of the tube (3). Likewise, the lower mass in the central area in contact with the tube (3) minimizes the thermal inertia of the mount (2). Since this arrangement is symmetrical, the temperature uniformity is maximum in the tube portion inserted in the mount (2).
[0134]
[0135] In embodiments of the invention incorporating a well (4) in the mount (2), said well (4) preferably has a height between 0.1 mm and 5.0 mm, which allows heating by above the level of the reagents, avoiding losses by the tube wall close to said level and unifying the temperature of the reagents.
[0136]
[0137] In another embodiment of the invention, the temperature sensor (7) is arranged next to the plane of symmetry of the mount (2), that is, in its potentially cooler zone. This allows to ensure that the minimum temperature in the tube (3) is at a level as close as possible to the target temperature for each reaction. Alternatively or in addition, in another embodiment of the invention, the temperature sensor (7) is located in the area in contact with the tube (3). This allows to ensure that the minimum temperature in the tube is as close as possible to the target temperature for each reaction.
[0138]
[0139] The combination of a mounting shape (2) that provides maximum thermal symmetry and minimum thermal inertia, together with the location of the temperature sensor (7) in the plane of symmetry and / or in the area closest to the tube (3), allows to optimize the qualities of the thermal cycler device (1), namely, uniformity and heating / cooling speed, while maintaining a minimized energy consumption.
[0140] The approach of the invention is scalable for application to an arbitrary number of tubes (3), so that the thermal cycler device (1) has a modular and replicable structure, which allows its use both in a single tube (3) and in large scale reactions
[0141]
[0142] This implies, in addition, another difference with respect to the devices of the state of the art designed for a plurality of tubes (3), based on this or other heating / cooling methods (such as oil, air, etc.). In particular, the reduction of the typical designs to a single mount, results in a frame of uniform section in its entire length, less efficient than the one proposed by the present invention. In particular, reducing the distribution of heating / cooling elements to a single mount, results in an asymmetric distribution of them around the tube, which translates into a lower uniformity than that described herein.
[0143]
[0144] Additionally, in other embodiments of the invention the thermocycler device (1) may comprise the use of a plug (10) or closure (Figure 3) in its tubes (3) that penetrates, preferably in the form of a plunger, substantially up to an equal height. less than 20 mm above the level of reagents to be housed in said tubes (3). More preferably, said height is substantially equal to less than 1 mm.
[0145]
[0146] The use of the plug (10) in the tubes (3) further improves the temperature uniformity, reducing the evaporation of the reagents and their condensation. All this further increases the efficiency of the PCR reactions, as a result of which, when penetrating into the tube (3), the plug (10) is heated longitudinally by the static heater block (8) to the proximity of the reagents, thus contributing to the reduction of heat losses and condensation. The plug (10) also reduces the volume of air inside said tube (3), whereby the saturation pressure corresponding to the reaction temperature is reached by transforming less liquid reagent into steam. This minimizes the change in reagent concentrations, assuming an additional advantage over other prior art thermal cyclers.

权利要求:
Claims (1)
[0001]
1. - Compact thermocycler device (1) for heating and cooling of at least one mount (2), said device (1) comprising at least one mount (2) for housing a lower portion of a reagent tube (3) provided to accommodate a sample, wherein said mount (2) is coupled to a thermoelectric heater (6) for heating / cooling said lower portion of the tube (3); and where the device (1) is characterized in that it additionally comprises a heater block (8) thermally insulated from the mount (2) and disposed in an upper region above it, comprising at least one housing hole (9 ) adapted to receive an upper portion of the reagent tube (3) and heat said upper portion above the lower region housed by the mount (2);
- where the mount (2) and the heater block (8) are separated at a distance between 0.5 mm and 5.0 mm;
- where the heater block (8) comprises one or more resistive heating means, configured to keep the portion of the tube (3) housed by the housing orifice (9) at a constant temperature at a value equal to or greater than the minimum temperature of condensation inhibition of the sample housed in the tube (3).
where the mount (2) comprises a hole (5) for the inspection or optical detection of the reactions produced in the tube (3) as a result of heating and cooling thereof.
2. - Device (1) according to the preceding claim, wherein the mount (2) comprises a well (4) for accommodating at least part of the lower portion of the tube (3), wherein said well (4) is an integral part of the mount (2), an extension thereof, or an element in thermal contact with it, arranged in an upper region of the mount (2) and adapted to extend the heat transfer surface of the thermoelectric heater (6) in said lower portion of the tube (3).).
3. - Device (1) according to the preceding claim, wherein the well (4) has a height between 0.1 mm and 5.0 mm.
4. - Device (1) according to any of the preceding claims, wherein the thermoelectric heater (6) comprises two thermoelectric elements (6 ', 6 ") Peltier, facing and located on each side of the mount (2).
6. - Device (1) according to the preceding claim, wherein the temperature of the heater block (8) is substantially 104 ° C.
7. - Device (1) according to the preceding claim, wherein the mount (2) comprises a temperature sensor (7) in thermal contact with said mount (2) to control the power supplied by the thermoelectric heater (6) and the temperature of the tube (3) of reagents.
8. - Device (1) according to the preceding claim, wherein the temperature sensor (7) is arranged on the plane of symmetry of the mount (2), and / or in an area in contact with the tube (3).
9. - Device (1) according to any of the preceding claims, comprising a plurality of mounts (2) for housing reagent tubes (3), equipped with their corresponding thermoelectric heaters (6) V block (8) heater.
10. - System comprising at least one thermocycler device (1) according to any of the preceding claims, resulting from the combination with one or more reagent tubes (3) housed in one or more frames (2) comprised in said device (1 ).
11. - System according to the preceding claim, wherein one or more tubes (3) comprise a plug (10) or upper closure that penetrates substantially at a height equal to or less than 20 mm above the level of the reagents to be housed in said tubes (3 ).
12. - System according to the preceding claim, wherein the height is substantially equal to or less than 1 mm.

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EP16382401|2016-08-23|

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PCT/EP2017/070817|WO2018036895A1|2016-08-23|2017-08-17|Compact thermal cycling device and system comprising said thermal cycling device|

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