FIELD OF THE INVENTION
The present invention relates to vessels and apparatus for controlled heating of reagents for example those used in biochemical reactions and to methods for using these.
BACKGROUND OF THE INVENTION
The controlled heating of reaction vessels is often carried out using solid block heaters which are heated and cooled by various methods. Current solid block heaters are heated by electrical elements or thermoelectric devices inter alia. Other reaction vessels may be heated by halogen bulb/turbulent air arrangements. The vessels may be cooled by thermoelectric devices, compressor refrigerator technologies, forced air or cooling fluids. The reaction vessels fit into the block heater with a variety of levels of snugness. Thus, the thermal contact between the block heater and the reaction vessel varies from one design of heater to another. In reactions requiring multiple temperature stages, the temperature of the block heater can be adjusted using a programmable controller for example to allow thermal cycling to be carried out using the heaters.
This type of heater arrangement is particularly useful for reactions requiring thermal cycling, such as DNA amplification methods like the Polymerase Chain Reaction (PCR). PCR is a procedure for generating large quantities of a particular DNA sequence and is based upon DNA's characteristics of base pairing and precise copying of complementary DNA strands. Typical PCR involves a cycling process of three basic steps.
Denaturation: A mixture containing the PCR reagents (including the DNA to be copied, the individual nucleotide bases (A,T,G,C), suitable primers and polymerase enzyme) are heated to a predetermined temperature to separate the two strands of the target DNA.
Annealing: The mixture is then cooled to another predetermined temperature and the primers locate their complementary sequences on the DNA strands and bind to them.
Extension: The mixture is heated again to a further predetermined temperature. The polymerase enzyme (acting as a catalyst) joins the individual nucleotide bases to the end of the primer to form a new strand of DNA which is complementary to the sequence of the target DNA, the two strands being bound together.
A disadvantage of the known block heaters arises from the lag time required to allow the heating block to heat and cool to the temperatures required by the reaction. Thus, the time to complete each reaction cycle is partially determined by the thermal dynamics of the heater in addition to the rate of the reaction. For reactions involving numerous cycles and multiple temperature stages, this lag time significantly affects the time taken to complete the reaction. Thermal cyclers based on such block heaters typically take around 2 hours to complete 30 reaction cycles.
For many applications of the PCR technique it is desirable to complete the sequence of cycles in the minimum possible time. In particular for example where respiratory air or fluids or foods for human and animal stock consumption are suspected of contamination rapid diagnostic methods may save considerable money if not health, even lives.
An alternative thermal cycler contains a number of capillary reaction tubes which are suspended in air. The heating and cooling of the reaction tubes is effected using a halogen lamp and turbulent air from a fan. The thermal dynamics of this system represent a considerable improvement over the traditional block heater design because heated and cooled air is passed across the reaction tubes and the required temperatures are achieved quite rapidly, the fan providing a homogeneous thermal environment and forced cooling. Using this apparatus 30 reaction cycles can be completed in about 15 minutes.
A disadvantage of this thermal cycler is that air cooling and heating are not readily suitable in apparatus which is required to provide different thermal cycling conditions to multiple reactions at the same time, and is certainly not mobile or portable.
SUMMARY OF THE INVENTION
The applicants have developed an efficient system for rapid heating and cooling of reactants which is particularly useful in thermal cycling reactions.
The present invention relates to a reaction vessel comprising an electrically conducting polymer which emits heat when an electric current is passed through it.
Thus in a first aspect, there is provided apparatus for effecting reactions, said apparatus comprising a plurality of reaction vessels for holding reagents, an electrically conducting polymer which emits heat when an electric current is passed through it, and control means for controlling supply of current to the polymer, the polymer being connectable to an electrical supply via the control means.
Further aspects of the invention include specific reaction vessels used in the apparatus as detailed hereinafter as well as methods for carrying out chemical or biochemical reactions.
Electrically conducting polymers are known in the art and may be obtained from Caliente Systems Inc. of Newark, USA. Other examples of such polymers are disclosed for instance in U.S. Pat. No. 5,106,540 and U.S. Pat. No. 5,106,538. Suitable conducting polymers can provide temperatures up to 300° C. and so are well able to be used in PCR processes where the typical range of temperatures is between 30° and 100° C.
An advantage of the invention over a conventional block heater is derived from the fact that polymers which conduct electricity are able to heat rapidly. The heating rate depends upon the precise nature of the polymer, the dimensions of polymer used and the amount of current applied. Preferably the polymer has a high resistivity for example in excess of 1000 ohm.cm. The temperature of the polymer can be readily controlled by controlling the amount of electric current passing through the polymer, allowing it to be held at a desired temperature for the desired amount of time. Furthermore, the rate of transition between temperatures can be readily controlled after calibration, by delivering an appropriate electrical current, for example under the control of a computer programme.
Furthermore as compared to a block heater, rapid cooling can also be assured because of the low thermal mass of the polymer. If desired however, the reaction vessel may be subjected to artificial cooling to further increase the speed of cooling. Suitable cooling methods include forced air cooling, for example by use of fans, immersion in ice or water baths etc.
In addition, the use of polymer as the heating element in a reaction vessel will generally allow the apparatus to take a more compact form than existing block heaters, which is useful when carrying out chemical reactions in field conditions such as in the open air, on a river, on a factory floor or even in a small shop.
Each reaction vessel may take the form of a reagent container such as a glass, plastics or silicon container, with electrically conducting polymer arranged in close proximity to the container. In one embodiment of the vessel, the polymer is provided as a sheath which fits around the reaction vessel, in thermal contact with the vessel. The sheath can either be provided as a shaped cover which is designed to fit snugly around a reaction vessel or it can be provided as a strip of film which can be wrapped around the reaction vessel and secured.
The polymer sheath arrangement means that close thermal contact is achievable between the sheath and the reaction vessel. This ensures that the vessel quickly reaches the desired temperature without the usual lag time arising from the insulating effect of the air layer between the reaction vessel and the heater. Furthermore, a polymer sheath can be used to adapt apparatus using pre-existing reaction vessels. In particular, a strip of flexible polymer film can be wrapped around a reaction vessel of various different sizes and shapes.
Where a sheath is employed it may be advantageous for it to be perforated or in some way reticulated. This may increase the flexibility of the polymer and can permit even readier access by a cooling medium if the polymer is not itself used to effect the cooling.
Alternatively the polymer maybe provided as an integral part of the reaction vessel. The reaction vessel may be made from the polymer by extrusion, injection moulding or similar techniques. Alternatively, the reaction vessel may be manufactured using a composite construction in which a layer of the conducting polymer is interposed between layers of the material from which the vessel is made or in which the internal or external surfaces of the reaction vessel is coated with the polymer, or again in which the vessel is basically made of the polymer coated with a thin laminate of a PCR compatible material. Such vessels may be produced using lamination and/or deposition such as chemical or electrochemical deposition techniques as is conventional in the art
Vessels which comprise the polymer as an integral part may provide particularly compact structures.
If several reaction vessels are required for a particular reaction, any electrical connection points can be positioned so that a single supply can be connected to all the reaction vessels or tubes. The reaction vessels may be provided in an array.
Alternatively, each of or each group of reaction vessels may have its own heating profile set by adjusting the applied current to that vessel or group of vessels. This provides a further and particularly important advantage of reaction vessels with polymer in accordance with the invention over solid block heaters or turbulent air heaters, in that individual vessels can be controlled independently of one another with their own thermal profile. It means that a relatively small apparatus can be employed to carry out a plurality of PCR assays at the same time notwithstanding that each assay requires a different thermal profile i.e. a varying operating temperature and/or dwell times in each stage of a cycle. For example, PCR tests for detecting a fair plurality of organisms in a sample can be carried out simultaneously, notwithstanding that the nucleotide sequence which is characteristic of each organism is amplified at different PCR operating temperatures.
The polymer may suitably be provided in the form of a sheet material or film, for example of from 0.01 mm to 10 mm, such as from 1 to 10 mm, and preferably 0.1 to 0.3 mm thick. By using thin films, the volume of polymer required to cover a particular reaction vessel or surface is minimised. This reduces the time taken for the polymer to heat to the required temperature as the heat produced by passing the current through the polymer does not have to be distributed throughout a large volume of polymer material.
In use, the polymer component of the reaction vessel is arranged such that an electric current can be generated within the polymer. This can either be achieved by providing the polymer with connection points for connection to an electrical supply or by inducing an electric current within the polymer, for example by exposing the polymer to suitable electrical or magnetic fields.
The close thermal contact between the polymer and the reagents or reagent container which may be established in the reaction vessels of the invention reduces or eliminates the insulating effect of the air layer between the heating element and the reaction vessel.
The vessel may comprise a flat support plate such as a two-dimensional array in particular a chip such as a silicon wafer chip; or a slide, in particular a microscope slide, on which reagents may be supported. The plate may be made from the polymer or the polymer may be provided as an integral part of the plate, either as a coating on one side of the plate or as a polymer layer within a composite construction as previously described. Where appropriate, and particularly when the plate is a chip, the polymer may be deposited and/or etched in the preferred format on the chip using for example printed circuit board (PCB) technology.
Where the reaction vessel comprises a slide or chip, the apparatus may comprise the slide or chip, an electrical supply, means for connecting the electrical supply to the slide or chip or for inducing an electrical current in the polymer and a means for controlling the current passing through the polymer layer in the slide or chip.
Vessels of this type may be particularly useful for carrying out in-situ PCR for example on tissue samples.
These vessels are novel. Thus in a further aspect the invention provided a reaction vessel comprising a slide or a chip and an electrically conducting polymer which emits heat when an electric current is passed through it, said polymer being arranged to heat reactants on said slide or chip.
Other suitable reaction vessels are tubes and cuvettes, which are known in the art.
In a preferred embodiment of the invention, the vessel comprises a capillary tube. The heat transfer from a capillary tube to reagents contained within it is more rapid than that achieved using conventional reagent vessels as the surface area to volume ratio of the reagents in the capillary tube is larger than in a conventional reagent vessel. Furthermore, the volume of samples used in these reactions is frequently very small, of the order of microliters or less and small volume vessels are thus essential.
Also, the invention provides apparatus for carrying out reaction at controlled temperatures, which apparatus comprises a reaction vessel comprising a slide or chip and a means for controlling the supply of electric current to the electricity conducting polymer so as to control the temperature thereof.
Capillary tube reaction vessels are usually filled by allowing the sample to be drawn into the tube under capillary action. The ends of the tube are then sealed. In the case of a glass tube, which is the usual form, sealing is typically effected thermally.
This thermal sealing method has a major disadvantage in being liable to degrade the sample. Also however, a glass tube of what might well be less than 2 mm outside diameter and about 4 cm length, is very fragile. There are capillary reaction vessels which have one end presealed. These may be filled by employing centrifuge or vacuum techniques. These are however time consuming and besides entail a risk of retained air and contamination from air.
It is not uncommon for a newly opened box to contain four or five broken tubes in a bank of 96 such vessels, which is one popular quantity for use in biochemical thermocycling apparatus. Further breakages are very likely to occur during filling and mounting and even in use, not least because heating and cooling is typically effected using turbulent hot and cold air.
There exist also reaction vessels formed from plastics material and vessels which are not capillary in form. Such vessels typically have a maximum internal diameter of 5 to 10 mm and are conical or paraboloid tapering down to the base. These are relatively easily filled and are provided with caps which seal thereto. They are relatively unbreakable but have the disadvantage that the required temperatures may not be accurately attained or consistently attained throughout the sample or with each cycle. Because of the low surface area to volume ratio, heat transfer is poor in conventional tubes.
Reaction vessels in which a cap for a reaction vessel projects into the vessel in order to reduce the volume thereof are described for example in EF-A-245994 and U.S. Pat. No. 4,578,588.
In a particularly preferred embodiment, the reaction vessel used in the present invention comprises a container, a cap member, and an electrically conducting polymer which is arranged so as to heat reagents in the reaction vessel when current is supplied to said polymer, the cap member being formed so as to project into the container to reduce the capacity thereof and to create a space therebetween of substantially consistent proportions.
Thus in a further aspect the invention provides a reaction vessel comprising a container, a cap member, and an electrically conducting polymer which is arranged so as to heat reagents in the reaction vessel when current is supplied to said polymer, the cap member being formed so as to project into the container to reduce the capacity thereof and to create a space therebetween of substantially consistent proportions.
In this way, the insertion of the cap member into the vicinity of the sample results in an increase in the surface area to volume ratio of the sample, so that the required temperature can be consistently and rapidly attained throughout the reagent mass. In addition the reaction vessel which is easy to fill.
The expression “substantially consistent proportions” used herein means that the space, which will form the reagent volume is of substantially similar cross section throughout. This means that externally applied factors such as heating or cooling means, will be effective throughout the entire volume of the reagent in a substantially consistent manner.
As before, the reaction vessel of this embodiment may take the form of a reagent container such as a glass or plastics container, with electrically conducting polymer arranged in close proximity to the container. In one embodiment of the vessel, the polymer is provided as a sheath which fits around the reaction vessel, in thermal contact with the vessel. The sheath can either be provided as a shaped cover which is designed to fit snugly around a reaction vessel or it can be provided as a strip of film which can be wrapped around the reaction vessel and secured.
In a preferred arrangement, the polymer is provided as an integral part of the reaction vessel, and in this case, it may either be as part of the container or the cap member. The container and/or cap member may be made from the polymer by extrusion, injection moulding or similar techniques Alternatively, the container or cap member may be manufactured using a composite construction in which a layer of the conducting polymer is interposed between layers of the material, such as plastics or glass, from which the container or cap member is made. In a further alternative, the internal or external surfaces of the container and/or cap member are coated with the polymer. Alternatively, the container or cap member is basically made of the polymer coated with a thin laminate of a PCR compatible material. Such vessels may be produced using lamination and/or deposition such as chemical or electrochemical deposition techniques as is conventional in the art.
Reaction vessels and apparatus of the invention can be used in a variety of situations where chemical or biochemical reactions are required to be carried out. Thus the invention further provides a method of carrying out a reaction such as a chemical or biochemical reaction which method comprises heating reagents in apparatus or in a reaction vessel as described above.
In particular the invention provides a method of carrying out a chemical or biochemical reaction which requires multiple temperature stages; said method comprising placing reagents required for said reaction in a reaction vessel which comprises an electrically conducting polymer which emits heat when an electric current is passed through it, supplying current to said polymer so as to heat reagents to a first desired temperature; and thereafter adjusting the current so as to produce the subsequent temperatures stages required for the reaction.
As well as amplification reactions such as PCR reactions already mentioned above, the vessels and apparatus of the invention can be used for the purposes of nucleic acid sequencing and in enzyme kinetic studies wherein are studied the activity of enzymes at various temperatures, likewise other reactions, especially those involving enzymic activity, where precise temperatures need to be maintained. The reaction vessels of the invention allow precise temperatures to be reached and maintained for suitable time periods, and then changed rapidly as desired, even in mobile or portable apparatus in accordance with some embodiments of the invention.
For PCR reactions, the temperature conditions required to achieve denaturation, annealing and extension respectively and the time required to effect these stages will vary depending upon various factors as is understood in the art. Examples of such factors include the nature and length of the nucleotide being amplified, the nature of the primers used and the enzymes employed. The optimum conditions may be determined in each case by the person skilled in the art. Typical denaturation temperatures are of the order of 95° C., typical annealing temperatures are of the order of 55° C. and extension temperatures of 72° C. are generally of the correct order. When utilising the reaction vessels and apparatus of the invention, these temperatures can rapidly be attained and the rate of transition between temperatures readily controlled.
Generic DNA intercalating dyes and strand specific gene probe assays, eg TaqmanŽ assays as described in U.S. Pat. No. 5,538,848 and Total Internal Reflection Fluorescence (TIRF) assays such as those described in WO93/06241 can of course be employed with many embodiments of the invention. In such assays, a signal from the sample such as a fluorescent signal or an evanescent signal is detected using a fluorescence monitoring device. When this type of process is undertaken, the fluorescence monitoring device must be arranged such that it is able to detect signal emanating from the sample. In some instances, it may be helpful if at least a part of the vessel, for example an end where the vessel is a tube of the invention may be optically clear so that measurements can be made through it. Alternatively the vessel can be provided with means of conveying a signal from the sample to the monitoring device, for example, an optic fibre or an evanescent wave guide.
The fluorescence monitoring device may be set to read a fluorescent signal at one or more wavelengths depending upon the nature of the signally system being used.