WO2004018105A1

WO2004018105A1 - Thermal engine for a thermocycler with interchangeable sample block

Info

Publication number: WO2004018105A1
Application number: PCT/GB2003/003650
Authority: WO
Inventors: Mark Ebdon; Brian Wilkinson
Original assignee: Quanta Biotech Limited
Priority date: 2002-08-20
Filing date: 2003-08-20
Publication date: 2004-03-04
Also published as: GB0219393D0; AU2003259334A1

Abstract

A thermal engine for a thermocycler, the thermal engine comprising an electrically-controlled heating and cooling element, a heatsink and a heating and cooling stage in thermal contact with the heating and cooling element, the heating and cooling stage having a surface releasably holding an interchangeable sample block having first and second surfaces, the first surface being arranged for receiving samples and the second surface being capable of releasable engagement with the heating and cooling stage of the thermal engine. A thermally conductive gasket is preferably interposed between the heating and cooling stage and the sample block.

Description

THERMAL ENGINE FOR A THERMOCYCLER WITH INTERCHANGEABLE SAMPLE BLOCK

This invention relates to apparatus for the control of chemical reactions. In particular, it concerns automated or semi-automated apparatus and parts therefor for the thermal control of nucleic acid polymerisation reactions.

Many chemical and biochemical reactions performed in vitro require close control of their temperature to ensure predictability and reproducibility of their outcome. This is especially true in the use of the polymerase chain reaction (PCR) for the amplification of polynucleic acid sequences. In PCR, and particularly quantitative PCR (QPCR), it is essential to control the temperatures of samples in order that the intended number of amplifications are carried out accurately in each sample and consistently between samples. The accepted way of achieving such temperature control is to perform the PCR reactions in a thermocycler.

A typical thermocycler comprises a programmable control module having a user interface, together with a thermal engine under the control of the control module. The thermal engine is where samples are placed for thermocycling or incubations requiring high thermal accuracy and uniformity and typically comprises one or more thermoelectric heating and cooling elements in intimate contact both with a heatsink and a fixed sample block. Samples are mounted, in disposable glass or plastics vials, capillaries, microplates, microscope slides or micro arrays, onto or into the sample block and are typically clamped into the sample block by an overhead mechanism provided in the thermal engine. The thermal engine may also contain an overhead heating element to avoid condensation problems in the sample containers.

In EP 1045038 A, a thermocycler for rapid PCR is described, the thermal engine of which employs a small, low-profile, low thermal capacity fixed sample block having an array of spaced-apart sample wells formed in its upper surface. The means by which samples are introduced into the thermal engine consists of an ultra thin-walled multiwell plate whose lower surface follows the profiles of the wells of the plate. The lower surface of the multiwell plate thus keys into the spaced-apart sample wells formed in the upper surface of the sample block. A similar fixed block and plate arrangement is shown in US6153426. US6337435 is concerned with a thermal engine having a number of Peltier effect thermoelectric modules and wire heating elements embedded along the edges of a fixed sample block assembly. The upper surface of the disclosed sample block contains an array of cylindrical wells for receiving a tray of open-top reaction vessels. The outer contours of the reaction vessels conform in shape both to the inner profiles of the reaction vessels and to those of the cylindrical wells. A similar arrangement of fixed sample block and sample reaction vessels is shown in WO98/43740. Further fixed sample block devices are disclosed in US2003/0044969 (published 6th March 2003) and US2002/0030044.

US5939312 describes a miniaturised, integrated multi-chamber thermocycler, the sample block of which is formed from an etched silicone slab. The base of the sample block is fitted with low conductivity projections to isolate the block from its surroundings and a number of heating elements are attached to the base of the block.

In EP 1157744 A, an automated integrated thermocycler is disclosed and has a machined aluminium sample block which has a plurality of wells in its upper surface for receiving sample vials. The sample block is designed to be integrated into the thermal engine of the thermocycler and includes various points of connection to the thermal engine for the supply of coolants and/or heating gases and for permanent fixation to the heating element of the thermal engine, the sample block being specifically designed to have the dimensions of a standard 96-well microtitre plate.

WO03/022440 (published 20th March 2003) describes a multiwelled filter plate and a holder therefor. The holder provides support to the filter during processing of samples following filtration.

US6341490 discloses an improved heat transfer apparatus having a number of upstanding pins sited in the upper surface of a fixed sample block. A similar principle is employed in US6238913, wherein a fluid-heated apparatus for use with deep-welled microplates is described. The apparatus is stand-alone and does not have interchangeable components, other than the microplates. The ability to cycle different shapes and sizes of reaction vials or liquid volumes through the thermocycler is extremely important. In general, current thermocycler technology only allows this to be done by removing the entire thermal engine and replacing it with another thermal engine having a different format sample block. This has been done for some time, the perceived advantage lying in the fact that the thermal engines are usually separate units to the thermocycler control module and, since the thermal engine is the part most likely to fail with use, this allows thermal engine replacement without having to replace the control module and/or shutdown the throughput of reactions for significant periods. However, there are significant disadvantages to the current regime. The thermal engine is generally a very heavy unit since it contains, amongst other components, the heatsink and, importantly, the thermal engine also typically contains high cost thermoelectric Peltier heating and cooling devices. These factors mean that, for each sample block format chosen by a user, a separate thermal engine must be purchased at significant cost. Thermal engines which are not in use for a given set of reactions must also be stored carefully away or take up valuable bench space.

Thus, it is an object of the present invention to provide thermal engines, sample blocks and thermocyclers which reduce the problems identified above in relation to current apparatus.

Accordingly, one aspect of the present invention provides a thermal engine for a thermocycler, the thermal engine comprising an electrically-controlled heating and cooling element, a heatsink and a heating and cooling stage in thermal contact with the heating and cooling element, the heating and cooling stage having a surface releasably holding an interchangeable sample block having first and second surfaces, the first surface being arranged for receiving samples and the second surface being capable of releasable engagement with the heating and cooling stage of the thermal engine.

The thermal engine according to the invention has the advantage that it can be used with a plurality of interchangeable sample blocks of appropriate dimensions and being capable of releasable engagement. Thus, a range of sample block formats can be used with the same thermal engine. The efficiency of heat transfer between the block and the stage is enhanced when, in preferred embodiments, the second surface of the sample block has a complementary profile to that of the surface of the heating and cooling stage. The profile of the surface of the heating and cooling stage is not related to any specific sample block format. The sample block can be removed and replaced with an alternative sample block without disassembly of the remaining components of the thermal engine and, in particularly preferred embodiments, no external tools are required to enable the engagement and disengagement of the sample block with the heating and cooling stage.

Preferably, the heating and cooling stage has a substantially flat surface, although other profiles are not excluded. The heating and cooling stage may comprise a distinct component of the thermal engine or may form part of the heating and cooling element.

Since an interface between two components can provide undesirable thermal conduction properties, particularly in the case of a metal to metal interface, it is preferred that a thermally-conductive gasket is interposed between the heating and cooling stage and the sample block. The thermally conductive gasket comprises a compliant, thermally conductive material and may comprise at least one material selected from phase change materials (for example, T725 or T766 from Chromerics Inc.), preferably less than 1 mm thick phase charge materials, carbon fibre-impregnated foamed plastics materials, silicone-impregnated foamed plastics materials, metallic strips, conductive adhesive (e.g AR clad™ 8043 or AR clad™ 8044 from Adhesives Research, Inc)-coated metallic strips, thermally conductive greases (e.g Microfaze A4, a dry film thermal grease available from AOS Thermal Compounds of Eatontown, New Jersey), thermally conductive grease-coated mica, polyimide or ceramics, and combinations thereof. Combination gaskets are preferred and examples of such combinations include a thermally conductive paste surrounded by metal foil, thermally-conductive grease surrounded by metal foil, or a laminate of elastomeric binder (e.g silicon rubber)/thermally conductive filler applied to a carrier firm (e.g fibreglass, polyimide, polyester or aluminium film), such as that sold by Berquist as Sil-Pad. The use of the thermally-conductive gasket between the sample block and the heating and cooling stage avoids the creation of microscopic air gaps due to minute imperfections in the complementarity of the respective surfaces. Such air gaps would generally rely on convection to transfer heat across the interface; such transfer is relatively inefficient. The thermally-conductive gasket bridges the gap, preferably 'wetting out' on each surface, and thus eliminates air gaps, providing improved thermal transfer to and from the sample block. The gasket provides a good thermal transfer with low thermal resistance and a higher level of consistency from one sample block to the next.

The surface of the heating and cooling stage may be provided with a sealing gasket towards its perimeter. This perimetric sealing gasket may be made of foamed plastics material and acts to prevent fluid ingress from any spillages and/or condensation of the contents of a sample block into the region of the heating and cooling stage. The material of the perimetric sealing gasket is preferably thermally insulating in order to minimise thermal losses at the periphery of a sample block engaged with the heating and cooling stage.

The sample block may be held to or released from the thermal engine by means of a clamping mechanism. The clamping mechanism may be electrically actuated. An electrically actuated clamping mechanism is advantageous in that it tends to facilitate the automation of reactions carried out in the thermal engine of the present invention, the sample blocks then possibly being loaded or unloaded from the thermal engine by robotic means. Alternatively, or in addition, the clamping mechanism may comprise one or more manually-operated clamping members situated adjacent the heating and cooling stage. Such clamping members may comprise rotatable cam members positioned, for example, at opposite sides or corners of the heating and cooling stage for engaging with the sample block. Spring-loaded, possibly lever-operated, lugs can also be built into the thermal engine to engage with a corresponding peripheral lip provided on the sample block. Clamping may also be provided by means of a lid for the thermal engine and which releasably engages with the thermal engine so as to clamp the sample block to the heating and cooling stage when the lid is closed. A further alternative or additional clamping mechanism involves the use of a series of electromagnets positioned adjacent the heating and cooling stage and which attract ferrous components attached either to the sample block itself or to a peripheral skirt provided around the sample block.

The provision of a clamping mechanism in the thermal engine helps to ensure as intimate a contact as possible between the complementary surfaces of the sample block and heating and cooling stage. When a thermally-conductive gasket is interposed between those two surfaces, the clamping mechanism can be expected to provide a degree of compressive deformation of the thermally-conductive gasket to enhance its contact with those two surfaces and thus improve thermal transfer between them.

Preferably, the thermal engine has means for identifying the specification of a sample block by means of one or more unique identifiers on the sample block.

Since the thermal engine of the present invention is capable of use with a variety of sample blocks of potentially different mass, thermal properties and sample holding, a mechanism for the communication of the sample block type to a control module controlling the thermal engine is important. This can, of course, be achieved by manual input of the block type but, to avoid error, an automatic identification is advantageous. Such identification can be achieved, for example, by a unique series of projections for each different type of sample block, the projections interacting with an array of optical sensors or micro-switches positioned proximal to the heating and cooling stage and connected either directly to the input to the heating and cooling element or to the control module. As an alternative to projections on the sample block, reflective areas (to interact with reflective optical switches provided in the thermal engine), magnets (to interact with reed switches in the thermal engine) barcodes or metal electrical contacts could be provided on the sample block.

Preferably, the second surface of the sample block is non-complementary in shape to the first surface. It is preferred that the first and second surfaces of the sample block are oppositely facing.

The sample blocks employed in the thermal engine of the present invention have the advantage, as mentioned above, that they can be used interchangeably. This avoids the necessity for a fixed sample block in the thermal engine. Thus, different sample block formats can be used, i.e. to allow the loading of different sizes and numbers of vials or liquid samples, with the same heating and cooling stage and thermal engine. The second, heating and cooling stage-engaging surfaces of the sample blocks are not dependent in their profiles on the sample-loading format and the blocks are thus fully interchangeable by means of releasable engagement to the heating and cooling stage. Complementarity between the surface of the heating and cooling stage and the second surface of the sample block improves the efficiency and uniformity of thermal transfer to the samples.

The sample block may be at least partially metallic. The first surface may have a plurality of wells. The first surface is preferably suitable for receiving sample vials of 100 μL to 2,000 μL volume or for receiving a multiwelled comsumable, such as a consumable having a multiple of 96 wells in number, having well volumes of 2μl to 200 μl. The metallic sample block may comprise aluminium, silver, a silver alloy or copper. Preferably, the copper is oxygen-free.

Alternatively, the sample block may comprise a thermally-conductive, biochemically inert material. Such a material is preferably a plastics material. The first surface of such a sample block may have a plurality of wells. The first surface of the sample block made of thermally-conductive, biochemically inert material is preferably suitable for receiving sample vials of 100 μL to 2,000 μL volume or for receiving a multiwelled consumable, such as a consumable having a multiple of 96 wells in number, having well volumes of 2 μl to 200 μl. Alternatively or additionally, when the first surface of such a sample block has a plurality of wells, such wells may be suitable for receiving liquid samples of 1-1,000 μL volume. This latter embodiment has the advantage that liquid samples of very low volume may be loaded directly into the sample block with no need for the use of sample vials or other additional consumables. The mount may also be made by an injection moulding process, resulting in a low cost component with a low thermal mass and thinner walls than could otherwise be achieved. If elastomeric materials are used, a compliant block may be produced which shows improved thermal conductance with the heating and cooling stage by virtue of an enhanced intimacy of contact.

The thermally-conductive, biochemically inert material may be selected from: liquid crystalline polymers, such as Cool Poly RS372™; thermally conductive polypropylene, such as Cool Poly RS032™; thermally conductive polycarbonate, such as Cool Poly RB019™; and thinly cast standard grade polypropylenes and polycarbonates. The Cool Poly™ polymers are products of Cool Polymers, Inc. of Warwick, Rhode Island. The thinly cast standard grade polypropylenes and polycarbonates are preferably less than 1 mm thick, more preferably less than 0.7 mm thick.

Regardless of the type of material used to construct the sample block, it is preferred that the region of the first surface where samples are received, and possibly the whole sample block, is non-porous.

As a further alternative, the sample block may have an at least two part construction, a first part providing the first surface and being composed of a thermally-conductive, biochemically inert material and a second part providing the second surface and being metallic.

It is preferred that the second surface of the sample block is substantially flat. A flat-surfaced block is easier to manufacture.

Preferably, the sample block has a peripheral sealing gasket which is preferably made of foamed plastics material. The peripheral sealing gasket prevents the ingress of fluid, for example from spillages and/or condensation, into the region of the heating and cooling stage when the block is engaged with the thermal engine. The material of the peripheral sealing gasket is preferably thermally insulating in order to minimise thermal loses at the periphery of the sample block.

In another aspect, the invention provides a thermocycler having a thermal engine as described above.

The thermocycler of the present invention provides great versatility to the user in that it only requires one thermal engine to enable a wide range of different sample blocks to be employed. In addition, it is extremely well adapted for automation. Given that the only part that needs to be replaced to conduct a different format of reaction is the sample block, rather than the entire thermal engine, the machinery required for the automated process is simpler, lighter, less cumbersome and more compact than that used hitherto. The ability to automatically identify the sample block format and releasably engage the sample block with the thermal engine are also important factors in the suitability of the thermocycler of the present invention for an automated process. In embodiments employing a sample block made of a thermally-conductive, biochemically inert material and having wells for receiving 1 to 1,000 μL liquid samples, automation is further simplified compared to prior art instrumentation since no sophisticated mechanisms for the removal of reagent containers from the sample block are required.

In a further aspect, the invention provides a kit of parts for a thermal engine as described above, the kit comprising: an electrically-controlled heating and cooling element, a heatsink and a heating and cooling stage in thermal contact with the heating and cooling element, the heating and cooling stage having a surface for releasably holding an interchangeable sample block; and one or more interchangeable sample blocks having first and second surfaces, the first surface being arranged for receiving samples and the second surface being capable of releasable engagement with the heating and cooling stage.

The kit of parts described above preferably includes a thermally conductive gasket for interposition between the heating and cooling stage and the sample block.

In yet another aspect, the invention provides a kit of parts for use in the assembly of a thermal engine as described above, the kit comprising: an electrically-controlled heating and cooling element, a heatsink and a heating and cooling stage in thermal contact with the heating and cooling element, the heating and cooling stage having a surface for releasably holding an interchangeable sample block; and a thermally conductive gasket for interposition between the heating and cooling stage and the sample block.

In a related aspect, the invention provides a thermal engine for a thermocycler, the thermal engine comprising an electrically-controlled heating and cooling element, a heatsink and a heating and cooling stage in thermal contact with the heating and cooling element, the heating and cooling stage having a surface for releasably holding an interchangeable sample block. In yet another related aspect, the invention provides a sample block for interchangeable use in a thermal engine as described above. The invention also provides a kit of parts comprising such a sample block and a thermally conductive gasket for interposition between the heating and cooling stage of the thermal engine and the sample block.

The parts and the kits of parts according to the present invention allow the user to maximise the interchangeability of sample blocks in the thermal engine.

The invention will now be described in more detail by way of example only and with reference to the drawings, of which:

Figure 1 shows a cross-section through the interior of a thermal engine according to the present invention; and

Figure 2 shows, in perspective, a simplified representation of a sample block according to the present invention and composed of a biochemically inert, thermally-conductive material.

The thermal engine, generally indicated 11, has a number of Peltier heating and cooling devices 12 sandwiched between a fan-forced heatsink 13 and a machined aluminium interface plate 14 providing a heating and cooling stage. The interface plate 14 also clamps the Peltier devices 12 into position and gives a degree of protection thereto from sample spillage and physical damage during use. Onto the interface plate 14 is placed a thermally-conductive gasket 15, comprising a thermally-conductive grease sandwiched and contained by aluminium foil, and, onto the gasket 15, a machined aluminium sample block 16. The lower surface of the sample block 16 is flat, as is the upper surface of the interface plate 14. In use, sample vials 17 are placed into the sample block 16. The thermal engine shown also has an overhead heated plate 18 to inhibit condensation of sample solvents during reactions and the inaccuracies in temperature control associated therewith. A housing 19 is provided around the heated plate 18 to minimise temperature variations due to airflow around sample tubes. A lid-mounted clamping mechanism consisting of a compression spring 20 is used to ensure that the sample vials 17 are sealed securely in the sample block 16.

In use, the Peltier heating and cooling devices 12 are caused to undergo a rise in temperature by an electrical input from a control module (not shown). The heat of the Peltier devices 12 is transferred to the interface plate 14 and to the heatsink 13. Intimate contact between the Peltier devices 12 and the plate 14 and heatsink 13 is maintained by the use of thermal grease or phase-change gaskets. The heat transferred to the interface plate 14 is transferred, via the gasket 15, to the sample block 16 and hence to the sample vial 17 and its contents. During a cooling phase of a cycle, the heatsink 13 and the Peltier devices 12 disperse heat from the rest of the components in the thermal path.

To change the sample arrangement or volume, the sample block 16 is simply removed from the gasket 15 and interface plate 14 and an alternative sample block, preferably having the same lower surface profile, in this case flat, is put onto the then vacant gasket 15 and interface plate 14.

In Figure 2, the sample block, generally indicated 21, has a flat lower surface 22. On the upper surface 23 there are provided a plurality of low volume wells 24 of the order of 100 μL volume. For simplicity, only a small number of wells 24 are shown in Figure 2; in practice, a 96-well (8 x 12) arrangement would be typical. In use, liquid samples are placed directly into the wells 24. The sample block 21 is constructed from a thermally conductive polypropylene (Cool Poly RS032™), a material which has both high thermal conductivity and low biochemical reactivity, and hence the liquid samples may be thermocycled and reacted in the block 21 without needing to place the samples in vials or other additional consumables beforehand.

Claims

1. A thermal engine for a thermocycler, the thermal engine comprising an electrically-controlled heating and cooling element, a heatsink and a heating and cooling stage in thermal contact with the heating and cooling element, the heating and cooling stage having a surface releasably holding an interchangeable sample block having first and second surfaces, the first surface being arranged for receiving samples and the second surface being capable of releasable engagement with the heating and cooling stage of the thermal engine.

2. A thermal engine according to claim 1 in which the second surface of the sample block has a complementary profile to that of the surface of the heating and cooling stage.

3. A thermal engine according to claim 1 or claim 2 in which the heating and cooling stage has a substantially flat surface.

4. A thermal engine according to any of claims 1 to 3 in which a thermally conductive gasket is inteφosed between the heating and cooling stage and the sample block.

5. A thermal engine according to claim 4 in which the thermally-conductive gasket comprises at least one material selected from phase change materials, carbon fibre-impregnated foamed plastics materials, silicone-impregnated foamed plastics materials, metallic strips, conductive adhesive-coated metallic strips, thermally-conductive greases, thermally-conductive grease-coated mica, polyimide or ceramics, and combinations thereof.

6. A thermal engine according to claim 5 in which the thermally-conductive gasket comprises a thermally-conductive grease or paste surrounded by metal foil.

7. A thermal engine according to any of claims 1 to 5 in which the sample block is held or released by means of a clamping mechanism.

8. A thermal engine according to claim 7 in which the clamping mechanism is electrically-actuated.

9. A thermal engine according to claim 7 or claim 8 in which the clamping mechanism comprises one or more manually-operated clamping members situated adjacent the heating and cooling stage.

10. A thermal engine according to any of claims 1 to 9 having means for identifying the specification of a sample block by means of one or more unique identifiers on the sample block.

11. A thermal engine according to any preceding claim in which the second surface of the sample block is non-complementary in shape to the first surface.

12. A thermal engine according to any of claims 1 to 11 in which the sample block is metallic.

13. A thermal engine according to claim 12 in which the sample block comprises aluminium, silver, a silver alloy or copper.

14. A thermal engine according to any of claims 1 to 11 in which the sample block comprises a thermally-conductive, biochemically inert material.

15. A thermal engine according to claim 14 which the thermally-conductive, biochemically inert material is selected from liquid crystalline polymers, thermally conductive polypropylene, thermally conductive polycarbonate and thinly cast standard grade polypropylenes and polycarbonates.

16. A thermal engine according to any preceding claim in which the first surface of the sample block has a plurality of wells.

17. A thermal engine according to claim 16 in which the first surface of the sample block is suitable for receiving sample vials of 100 μl to 2000 μl volume or for receiving a multiwelled consumable.

18. A thermal engine according to claim 14 or claim 15 in which the first surface of the sample block has a plurality of wells for receiving liquid samples of lμl to 1,000 μl volume.

19. A thermal engine according to any preceding claim in which the sample block has an at least two part construction, a first part providing the first surface and being composed of a thermally-conductive, biochemically inert material and a second part providing the second surface and being metallic.

20. A thermal engine according to any preceding claim in which the second surface of the sample block is substantially flat.

21. A thermal engine according to any preceding claim in which the sample block has a peripheral sealing gasket.

22. A thermal engine according to claim 21 in which the peripheral sealing gasket is made of foamed plastics material.

23. A thermal engine according to any preceding claim in which the heating and cooling stage forms part of the heating and cooling element.

24. A thermocycler having a thermal engine according to any preceding claim.

25. A kit of parts for a thermal engine according to claim 1, the kit comprising: an electrically-controlled heating and cooling element, a heatsink and a heating and cooling stage in thermal contact with the heating and cooling element, the heating and cooling stage having a surface for releasably holding an interchangeable sample block; and one or more interchangeable sample blocks having first and second surfaces, the first surface being arranged for receiving samples and the second surface being capable of releasable engagement with the heating and cooling stage.

26. A kit of parts according to claim 25 including a thermally conductive gasket for interposition between the heating and cooling stage and the sample block.

27. A kit of parts for use in the assembly of a thermal engine according to claim 1, the kit comprising: an electrically-controlled heating and cooling element, a heatsink and a heating and cooling stage in thermal contact with the heating and cooling element, the heating and cooling stage having a surface for releasably holding an interchangeable sample block; and a thermally conductive gasket for interposition between the heating and cooling stage and the sample block.

28. A thermal engine for a thermocycler, the thermal engine comprising an electrically-controlled heating and cooling element, a heatsink and a heating and cooling stage in thermal contact with the heating and cooling element, the heating and cooling stage having a surface for releasably holding an interchangeable sample block.

29. A sample block for interchangeable use in a thermal engine according to any of claims 1 to 23.

30. A kit of parts comprising a sample block according to claim 29 and a thermally conductive gasket for interposition between the heating and cooling stage of the thermal engine and the sample block.