WO2018036895A1

WO2018036895A1 - Compact thermal cycling device and system comprising said thermal cycling device

Info

Publication number: WO2018036895A1
Application number: PCT/EP2017/070817
Authority: WO
Inventors: Aitor EZKERRA FERNÁNDEZ; Javier Berganzo Ruiz; Íñigo Aranburu Lazcano; Diana ENÉRIZ ENÉRIZ; Miguel RONCALÉS POZA
Original assignee: Laboratorios Alpha San Ignacio Pharma, S.L.
Priority date: 2016-08-23
Filing date: 2017-08-17
Publication date: 2018-03-01
Also published as: TW201809284A; ES2738665A1; DE112017004226T5

Abstract

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.

Description

COMPACT THERMAL CYCLING DEVICE AND SYSTEM COMPRISING SAID

THERMAL CYCLING DEVICE

Field of the Invention

The present invention is comprised in the experimental field in molecular biology, biochemistry and genomics. More specifically, the invention relates to a thermal cycling device used for DNA sequence amplification, as well as to a system associated with said device.

Background of the Invention

Thermal cycling devices, also known as PCR (Polymerase Chain Reaction) machines are apparatus preferably used in the field of molecular biology to subject the polymerase enzyme to different temperature cycles, for example for performing DNA amplification or for sequencing reactions using Sanger's method. The most common model of said devices consists of a plurality of wells configured for housing reagent tubes, connected to an electrically resistant block distributing, through a heating plate, a homogenous temperature to the wells for programmable times, usually with temperature ranges comprised between 4°C and 96°C.

Given that the incubated reactions in the tubes are usually performed in an aqueous solution, thermal cyclers usually further comprise a heating lid arranged for covering the tubes and heated constantly at 104°C, such that condensation of water in the closures of the tubes where the reaction takes place is thereby limited, reducing the concentration of solutes to prevent the optimal conditions for the polymerizing enzyme from change, and to maintain the desired thermodynamics for the pairing of primers used as a starting point for DNA replication.

In the thermal cycler, fast and continuous temperature change cycles are programmed to start the processes of separating and denaturing DNA strands at high temperatures, and then the annealing thereof at low temperatures, and finally the extension thereof at high temperatures. This cycle can be repeated multiple times, thereby achieving the desired degree of amplification of the DNA fragment.

Various commercial thermal cyclers comprising heating means based on Peltier technology which use the properties of semiconductors have also been known for some years now. These materials offer greater temperature uniformity, as well as much steeper upward and downward temperature ramps, thereby obtaining better results in PCR processes. Likewise, in the search to improve temperature precision, accuracy and homogeneity, some solutions on the market have chosen to use metals such as gold, silver and other alloys in the well heating blocks, achieving greater assay stability and reproducibility.

Nevertheless, even in thermal cyclers based on heating lids, the problem with condensation still remains and, accordingly, limits the effectiveness of their results. In this sense, the lid acting as a heating block in thermal cyclers of this type must be lowered onto or slid over the upper portion of the tubes, resting on said tubes. This requires a complex construction that limits access to and handling of the tubes, where it furthermore causes the focal heating points (i.e., heaters of the base of the tubes and the heating lids) to be located too far away, which creates a high level of reagent condensation .

As described in the preceding paragraphs, it is therefore necessary to provide a solution that can be integrated in thermal cyclers which efficiently reduces the condensation taking place in the samples in which amplification reactions take place, and which also allows more direct access to the housing tubes used. The present invention seeks to meet said need by means of a novel, compact thermal cycling device provided with a dual static and dynamic heating system for heating the housing tubes.

Brief Description of the Invention

According to the information proposed in the preceding section, one object of the present invention is to provide a device that is capable of considerably reducing condensation of the samples, compared with known thermal cyclers based on heating lid systems, where the lid used is lowered onto or slides over the upper portion of the tubes, resting on their ends .

For the purpose described above, said object of the invention is achieved by means of a compact thermal cycling device for heating and cooling at least one housing mount for housing a lower portion of a reagent tube, where said mount is coupled to a thermoelectric heater for heating/cooling said lower portion of the tube.

Advantageously, the device of the invention additionally comprises a heating block thermally isolated from the mount, arranged in an upper region above same, comprising at least one housing port adapted for receiving an upper portion of the reagent tube and heating said upper portion above the lower region housed by the mount.

The heating block preferably comprises one or more resistive heating means adapted for the thermal contact thereof with the reagent tube when said tube is housed in the thermal cycler, such that the upper portion of said tubes can be kept at a constant temperature, preventing condensation of the sample on the walls of the tubes throughout the different temperature cycles. The combination of the effect of the heating block and of the thermoelectric heater allows reducing times for obtaining results, as well as obtaining quicker increases and decreases in temperature (steeper ramps), which is the equivalent to greater reaction efficiency, thereby improving the results obtained compared with other thermal cyclers of the state of the art.

In a preferred embodiment of the invention, the thermoelectric heater comprises two Peltier elements located opposite one another and surrounding the mount. A thermally symmetrical configuration is thereby achieved.

In another preferred embodiment of the invention, the mount comprises a port for the optical detection or inspection of the reaction taking place in the tube as a result of the heating and cooling thereof.

In another preferred embodiment of the invention, the resistive heating means are configured for keeping the portion of the tube housed by the housing port at a constant temperature, having a value preferably equal to or greater than the minimum condensation inhibition temperature of the sample housed in the tube. More preferably, the temperature is kept substantially at 104°C. It is therefore not necessary to lower the device onto or slide it over the upper portion thereof, as occurs with most thermal cycling devices of the state of the art. It therefore allows a simpler construction that does not require automatons or the user intervention, and that maintains lateral heating of the tube providing a larger condensation-free surface, as well as greater temperature precision and uniformity in the tube.

In another preferred embodiment of the invention, the mount and the heating block are separated from one another by a distance comprised between 0.5 mm and 5.0 mm.

In another preferred embodiment of the invention, the mount comprises an upper prolongation or well which typically has a height comprised between 0.1 mm and 5.0 mm. Said range is suitable for preventing thermal contact between the Peltier heater and the heating block, particularly in cases in which the tubes are made of materials with low heat conductivity, such as plastic materials.

The well can be an integral part of the mount, a prolongation thereof, or an element in thermal contact with it, and its main function is to enlarge the heat transfer surface of the thermoelectric heater in the lower portion of the reagent tube. The well therefore contributes to heating the tube above the level of the reagents, thereby preventing heat losses through the wall of the tube close to said level and making the temperature of the reagents uniform.

In another preferred embodiment of the invention, the device comprises a temperature sensor in thermal contact with the mount, configured for controlling the power supplied by the thermoelectric heater and the temperature of the tube housed in said 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 housing tube for housing the reagents.

In another preferred embodiment of the invention, the thermal cycling device of the invention comprises a plurality of housing mounts for housing reagent tubes, provided with their corresponding Peltier heaters, heating block and wells (if used) . A fully scalable thermal cycler is thus obtained, the associated technology of which is not limited by the number of tubes used is thereby obtained.

Another object of the invention relates to a system comprising at least one thermal cycling 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 thermal cycler.

In a preferred embodiment of the system of the invention, one or more tubes comprise a stopper or upper closure entering until 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 or less than 1 mm .

Definitions of some of the main terms used in the present description and of its scope of interpretation in light of the invention herein claimed are provided below for illustrative purposes :

A thermal cycling device, or PCR machine, must be interpreted as any device suitable for subjecting one or more tubes containing reagents to different temperature cycles, for example for performing DNA amplification or for sequencing reactions with the Sanger's method.

A mount must be interpreted as a means for housing a lower portion or base of a tube containing reagents of a thermal cycler, said mount being subjected to temperature control by one or more heating means.

A tube containing reagents must be interpreted as any means for housing said reagents the shape of which is adapted for being housed in a mount of the thermal cycler. A well must be interpreted as a thermally connected element or an element that is part of the mount, and arranged at a height above said mount for receiving the tube containing reagents. The well therefore houses a portion of the tube located above the portion housed by the mount.

An optical inspection port must be interpreted as any access to the mount by means of which information associated with light transmitted to/from the inside of said mount can be obtained .

A thermoelectric heater must be interpreted as a means for controlling the temperature of the mount through a mechanism based on any of the known manifestations of the thermoelectric effect .

A thermoelectric Peltier element must be interpreted as component of the thermoelectric heater based on controlling temperature by means of the Peltier effect.

A temperature sensor must be interpreted as any mechanism provided with means for measuring the value of the temperature inside the mount, or for measuring the variations of said value.

A heating block must be interpreted as any resistive heating means intended for applying heat to an upper portion of the tube housed by the housing port.

A lower portion of the tube must be interpreted as a continuous portion of the tube comprising, respectively, the volume of said tube housing the reagents subjected to temperature cycles when said tube is housed in a mount of the thermal cycler.

An upper portion of the tube must be interpreted as a continuous portion of the tube which is arranged, when said tube is housed in the mount, at a higher level than the lower portion referred to in the preceding paragraph.

A stopper or closure of the tube must be interpreted as any upper closure element of said tube, the application of which limits the volume of reagents that can be housed therein.

The expression "thermal contact" between two or more elements must be interpreted in the scope of the invention as the close contact (this being understood as direct contact in the absence of an interfacing medium such as air) between said elements, whereby heat transfer between them is allowed.

The expression "substantially" applied to the relationship between two or more magnitudes must be interpreted in the scope of the invention as being equal to or comprised in a range with a ±10% variation with respect to the value in question.

The device of the invention acts on longitudinal tubes intended for being in a vertical position, the force of gravity being taken as a reference for this verticality. Therefore, relative terms such as upper, lower, above or below must be interpreted with respect to this vertical direction. The vertical direction of the tube or tubes is established by the configuration of the housing areas of the tube configured so that said tube adopts the correct position in its operating position in the thermal cycling device.

When the expression "comprises" is applied to the relationship between a main element and other secondary elements, it must be interpreted that it includes or contains said secondary elements, but without excluding other additional elements .

When the expression "consists" is applied to the relationship between a main element and other secondary elements, it must be interpreted that it includes or contains said secondary elements, excluding other additional elements. Description of the Drawings

Figure 1 of this document shows a cross-section view of the thermal cycler of the invention with a plurality of tubes housed therein, one of them being sectioned and visually accessible, according to a preferred embodiment thereof where the relative position of the dynamic heating block and of the static heating block, and the separation between both, are depicted.

Figures 2a-2b show two views of the mount and the well of the thermal cycler of the invention, where some of the main elements thereof are shown in detail.

Figure 3 shows a section of the reagent tube that can be housed by the device of the invention, where said tube is closed by means of a stopper.

Reference Numbers used in the Drawings

Detailed Description of the Invention

A detailed description of the invention in reference to a preferred embodiment thereof based on Figures 1-3 herein, provided for non-limiting and illustrative purposes, is provided below .

As described in preceding sections, the thermal cycling device (1) object of the present invention (Figure 1) is conceived for the combined static heating (hot plate mode) and/or dynamic heating (thermal cycler mode) of non-communicated containers or mounts (2) intended for housing reagent tubes (3), where each mount (2) is optionally thermally connected or joined in an integral manner to a well (4) which allows extending heating in the lower portion of the tubes (3) to a higher level than that occupied by the volume of the reagents. The heating provided by the wells (4) therefore allows homogenizing the temperature of the sample housed in the tubes (3), thereby minimizing the thermal gradient in the interface between the reagents and air contained in the tubes (3) , and preventing condensation .

In further detail, the compact and preferably monolithic thermal cycling device (1) of the invention comprises at least the following elements :

- Mount (2) : This comprises a preferably metallic structure through which heat is conducted into or out of same, bringing about, respectively, the heating or cooling of the lower region of the tube (3) housed therein due to thermal conduction. The mount (2) additionally comprises a port (5) (Figures 1 and 2a) for the optical detection or inspection of the reaction taking place in the tube (3) as a result of the heating and cooling thereof. To make said inspection easier, the mount (2) preferably has a flat lower base. In turn, the upper portion of the mount (2) will internally preferably have a frustoconical section for housing the corresponding tube (3) .

- Well (4) : This is an element for housing the tube (3) which is thermally connected to or is part of the mount (2), and is arranged at a height greater than said mount (2) . The well (4) therefore houses a portion of the tube (3) located above the portion housed by the mount (2), acting as a prolongation thereof .

Thermoelectric heater (6) : Said heater (6) preferably comprises two thermoelectric Peltier elements (6', 6'') located opposite one another and on each side of the mount (2) such that greater thermal symmetry is obtained when applying heat to or removing heat from the tube (3) .

- Temperature sensor (7) (Figures 2a and 2b) : Preferably comprised in the mount (2) and in thermal contact with same, it allows monitoring the power supplied by the thermoelectric heater (6), thereby achieving precise control over the temperature of the tube (3) .

- Heating block (8) : Said block (8) is arranged in the thermal cycling device (1) in an upper region above the mount (2) and the well (4) (if used) . In the preferred embodiment, the block (8) is joined to the mount (2), but nevertheless they must both be thermally separated and isolated from one another. In this same embodiment, the mount (2) and well (4) are one and the same part, though they can be formed as different parts thermally communicated with one another. Said heating block (8) also comprises at least one housing element (9) configured for receiving a portion of the tube (3) located above the portion housed by the wells (4) .

The heating block (8) comprises one or more resistive heating means (for example "hot plate" type means) and a housing port (9) for housing the reagent tubes (3), therefore being in thermal contact therewith, such that the portion of the tube (3) received by said ports (9) can be kept at a constant temperature, preventing condensation of the sample on the walls of said tube (3) during PCR reactions. Additionally, the combination of the effect of the heating block (8) and of the thermoelectric heater (6) results in a shorter time for obtaining results, as well as in quicker increases and decreases in temperature (steeper ramps), which is the equivalent to greater reaction efficiency, thereby improving the results obtained compared with other thermal cyclers of the state of the art .

Preferably, the static heating block (8) located in the upper portion of the tube (3) heats said tube (3) in its lateral regions substantially at a temperature of 104°C, such that said block (8) does not have to be lowered onto or slid over the upper portion of the tubes (3), as occurs with most thermal cycling devices of the state of the art. This therefore allows a simpler construction that does not require automatons or user intervention, and that maintains lateral heating of the tube (3) providing a larger condensation-free surface (i.e., without condensation) , as well as greater temperature precision and uniformity in said tube (3) .

In a preferred embodiment of the invention, to provide greater independence to the dynamic and static heating regimens of the thermal cycling device (1), the mount (2) and heating block (8) are separated such that they are thermally isolated from one another. Their range of separation preferably ranges between 0.5 mm and 5.0 mm. Said separation is considered suitable since the material of the tube, which is usually plastic, is a poor heat conductor and, therefore, there will be no thermal contact between the thermoelectric heater (6) and the heating block (8) .

In turn, the configuration of the mount (2), according to the present invention, is optimized in order to maximize thermal symmetry and minimize thermal inertia. This brings about a reduction in energy input required for reaching the target temperature in the entire mount, which contributes to improving the efficiency of the thermal cycler (1) .

In this sense, in a preferred embodiment of the invention the shape of the mount (2) is designed such that the mass, which is a determining factor of thermal inertia, decreases when the distance to the main focal heating point of the thermal cycling device (1) increases, with said focal heating point being the thermoelectric heater (6) . This thereby favors heat transfer to or from the thermoelectric heater (6), given that this is where the section dimensions of the mount (2) are maximum, and the reduction of the possible temperature gradients due to the progressively greater distances between 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) . Given that this arrangement is symmetrical, temperature uniformity is maximum in the tube portion inserted in the mount (2) .

In the embodiments of the invention incorporating a well (4) in the mount (2), said well (4) preferably has a height comprised between 0.1 mm and 5.0 mm, which allows performing heating above the level of the reagents, preventing losses through the wall of the tube close to said level and making the temperature of the reagents uniform.

In another embodiment of the invention, the temperature sensor (7) is arranged next to the plane of symmetry of the mount (2), i.e., in the potentially coldest area thereof. This allows assuring that the minimum temperature in the tube (3) is at a level that is as close as possible to the target temperature for each reaction. Alternatively, or complementarily, in another embodiment of the invention, the temperature sensor (7) is located in the area in contact with the tube (3) . This allows assuring that the minimum temperature in the tube is as close as possible to the target temperature for each reaction.

The combination of a shape of the mount (2) which 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 optimizing qualities of the thermal cycling device (1), namely, heating/cooling uniformity and rate, at the same time keeping energy consumption at a minimum.

The proposed invention is scalable for being applied to a random number of tubes (3) , such that the thermal cycling device

(1) has a modular and replicable structure, allowing use thereof both in a single tube (3) and in large scale reactions. This additionally entails another difference with respect to devices of the state of the art conceived for a plurality of tubes (3) based on this heating/cooling method or other heating/cooling methods (such as oil, air, etc.) . Reducing conventional designs to a single mount in particular gives rise to a mount having a uniform section along its entire length, which is less efficient than the one proposed by the present invention. Reducing the distribution of heating/cooling elements to a single mount in particular gives rise to an asymmetrical distribution thereof around the tube, which translates into less uniformity than what is described herein.

Additionally, in other embodiments of the invention the thermal cycling device (1) can comprise the use of a stopper

(10) or closure (Figure 3) in the tubes (3) that enters, preferably in the form of a plunger, until a height substantially equal to or less than 20 mm above the level of the reagents to be housed in said tubes (3) . More preferably, said height is substantially equal to or less than 1 mm.

The use of the stopper (10) in the tubes (3) improves temperature uniformity even more, reducing evaporation of the reagents and condensation thereof. All this additionally increases the efficiency of PCR reactions as a result of the fact that as the stopper (10) enters the tube (3) , said stopper (10) is longitudinally heated by the static heating block (8) until the area close to the reagents, thereby contributing to the reduction of heat losses and condensation. The stopper (10) furthermore reduces the volume of air inside said tube (3) , so the saturation pressure corresponding to the reaction temperature is reached, transforming less liquid reagent into vapor. This minimizes the change in reagent concentrations, which is an additional advantage with respect to other thermal cyclers of the state of the art.

Claims

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), wherein said mount (2) is coupled to a thermoelectric heater (6) for heating/cooling said lower portion of the tube (3); and wherein the device (1) is characterized in that it additionally comprises a heating block (8) thermally isolated from the mount (2) and arranged in an upper region above same, comprising at least one housing port (9) adapted for receiving an upper portion of the reagent tube (3) and heating said upper portion above the lower region housed by the mount (2);

wherein the mount (2) and the heating block (8) are separated from one another by a thermally isolated distance comprised between 0.5 mm and 5.0 mm; and wherein the heating block (8) comprises one or more resistive heating means configured for keeping the portion of the tube (3) housed by the housing port (9) at a constant temperature at a value equal to or greater than the minimum condensation inhibition temperature of the sample housed in the tube (3) .

The device (1) according to the preceding claim, wherein the mount (2) comprises a well (4) for housing at least part of the lower portion of the tube (3), wherein said well (4) is an integral part of the mount (2), a prolongation thereof, or an element in thermal contact with it, arranged in an upper region of the mount (2) and adapted for enlarging the heat transfer surface of the thermoelectric heater (6) in said lower portion of the tube (3) above the level of the reagents to be housed in said tube (3) .

3. The device (1) according to the preceding claim, wherein the well (4) has a height comprised between 0.1 mm and 5.0 mm.

The device (1) according to any of the preceding claims, wherein the thermoelectric heater (6) comprises two thermoelectric Peltier elements (6', 6'') located opposite one another and on each side of the mount (2) .

The device (1) according to any of the preceding claims, wherein the mount (2) comprises a port (5) for the optical detection or inspection of the reactions taking place in the tube (3) as a result of the heating and cooling thereof.

The device (1) according to the preceding claims, wherein the temperature of the heating block (8) is substantially 104°C.

The device (1) according to the preceding claims, wherein the mount (2) comprises a temperature sensor (7) in thermal contact with said mount (2) for controlling the power supplied by the thermoelectric heater (6) and the temperature of the reagent tube (3) .

The device (1) according to the preceding claims, 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. The device (1) according to any of the preceding claims, comprising a plurality of mounts (2) for housing reagent tubes (3), provided with their corresponding thermoelectric heaters (6) and heating block (8) .

10. A system comprising at least one thermal cycling 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 mounts (2) comprised in said device (1) .

11. The system according to the preceding claim, wherein one or more tubes (3) comprise an upper closure or stopper (10) entering until a height substantially equal to or less than 20 mm above the level of the reagents to be housed in said tubes (3) .

The system according to the preceding claim, wherein the height is substantially equal to or less than 1 mm.