WO2007138302A1

WO2007138302A1 - Performance issues in use of vessels for biological applications

Info

Publication number: WO2007138302A1
Application number: PCT/GB2007/001977
Authority: WO
Inventors: David Ward; David Edge; Nelson Nazareth
Original assignee: Bg Research Ltd
Priority date: 2006-05-26
Filing date: 2007-05-29
Publication date: 2007-12-06
Also published as: GB0610432D0; GB2441833A; GB0619128D0; GB2441833B

Abstract

A reaction vessel (10a) for containing a sample during a chemical, biological or biochemical process carried out therein and comprising a heating layer (13) formed of an electrically conductive material whereby an electric current passed therethrough generates heat, and a lining (17) thereto comprising an electrically insulative, thermally conductive material, and apparatus and process employing such vessels.

Description

Field of the invention

The invention relates to an improved reaction vessel and apparatus for heating/cooling/temperature regulation of such vessels and monitoring reactions within such vessels and is applicable with particular advantage to temperature dependant biological or chemical reactions and even more particularly to reactions where enzyme functions in reactions are regulated by temperature such as polymerase chain reactions (PCR). The PCR process is described in detail in US Patents 4683195 and 4683202 owned by Hoffmann - LaRoche, Inc. However, the present invention is not limited in scope to PCR processes.

Typically, but not exclusively as single reaction vessels are used, a large number of reduced volume reactions are carried out simultaneously in one apparatus, with a plurality of reaction vessels being received in a reaction apparatus at one time. They are placed in a tray, with a known footprint format. One such .format is a microtitre plate in which 96 vessels are carried in one 12X8 array. Formats generally have base multiples of 4 vessels (4, 8, 16, 32, 48, 96, 384, and 1536). The present invention is particularly, but not exclusively, concerned with such 4n arrays, where 4n denotes 4 or a multiple thereof. The vessels need not be limited to a microtitre plate format and in addition to single or multiple well formats may for example comprise a recess or specific location on a flat substrate such as a slide or tray, or any other geometry in which a reaction can be carried out.

In order to control and monitor the reactions, the apparatus includes means to monitor the temperature and to control the heating power applied to the reaction vessel contents. Direct thermal measurement may be undertaken using thermopile, pyrometer or contact thermometry based sensing systems.

Many reactions, especially biological reactions such as PCR, can be monitored by optical monitoring using optical interrogation or detection. This optical detection may be colorimetric or fluorometric (including techniques such as time-, resolved fluorescence)

Reaction vessels particularly suited to reduced volume biological reactions and heated by electrically conducting polymers (ECPs) are described in International , Patent Application Publication No PCT/GB97/03187 and a particular such a vessel is described in GB P^*atent specification 2333250. An array of such vessels are introduced into reaction apparatus which includes means to allow heating of the vessels, and means to observe reactions occurring in the vessels. The vessel may be made of an electrically conducting polymer or incorporate electrically conducting polymer such as being surrounded by a sheath of electrically conducting polymer (ECP). The vessel may be accordingly heated by an electrical current flowing through the electrically conducting polymer. The electrical current can be supplied via connection points to the vessel from an electrical supply.

The inventors in respect of the present invention have now discovered however that the close proximity of an electric current to the sample interferes with PCR, making the process quite unreliable.

Summary of the invention

According to a first aspect of the present invention a reaction vessel for containing a sample during a chemical, biological or biochemical process carried out thereon comprises a heating layer formed of an electrically conductive material whereby an electric current passed therethrough generates heat, and a lining thereto comprising an electrically insulative material.

Preferably the lining is also readily thermally conductive.

The lining may for instance be constructed from a polymer which provides a suitable surface for contact with the liquid contents, allows for biological/chemical reactions to take place optimally and provides optimal thermal coupling between the contents and the ECP heating layer. Typically such insulation material may comprise polypropylene, polythene, polyvinylpropylene, glass or other materials such as conformal coatings (e.g. parylene) that can be used in biological/chemical reaction processes. The choice of such material is readily apparent to the skilled addressee of the specification and may, with due regard to any need for rigidity in the reaction vessel, comprise a plastic or other polymeric film. The thickness of this lining may be of the order of 0.25 to 100.0 μm, however thickness is preferably minimised to prevent a thermal lag.

As an example a possible feature may also be to load the liner material with boron nitride or other similar materials that are electrically insulative but thermally conductive or by means of coating the inside of the vessel with for example parylene to provide the electrical insulation while maintaining an optimal thermal conductivity.

The heating layer is preferably constructed from a material allowing the vessel to be heated by application of an electrical voltage differential by direct electrical contact or by induction with resultant heat produced evenly and predictably so as to heat the liquid contents evenly and predictably. Preferably the material provides a low resistance electrical contact. One example of a suitable material is a polypropylene containing (but in no way limited to) carbon fibre, carbon black, carbon flake, Buckminster fullerene tubes or Buckminster fullerene balls. A typical material may have a carbon content of up to 70%, comprising about 10% carbon black and the remainder graphite. Metals and alloys for instance are also suitable materials from which to construct the vessel. It can however be wholly any electrically conducting material, or any electrically conductive material/conductive particles such as carbon, metal within an inert base resin such as polypropylene, polyvinylpropylene or nylon.

Preferably the vessel is a well although a recess in a flat substrate is also possible or performing the reaction on a wholly flat, recessed or tubular substrate. Preferably the base of the well provides an electrical contact surface for the heating layer although where flat substrates with recesses exist, two or more contact points may be employed to ensure optimal heating of the recess.

According to a second aspect of the invention there is provided reaction apparatus in which one or more reaction vessels are received and the reactions therewithin monitored, including one or more vessel receiving stations each for receiving a reaction vessel and for each receiving station a method of thermometry of the reaction vessel. This may comprise contact thermometry or infra-red detection.

In the case of infra-red detection each receiving station may have a thermopile sensor. If necessary there may also be a heat guide arranged to collect heat radiated from the surface of the vessel and to guide it onto the sensor. This can avoid having to ensure that the sensor is exactly aligned normal to the surface of an adjacent well. Typically the heat guide is formed of aluminium, copper, or another material with low emissivity and high reflectivity arranged to reflect the heat radiated from the vessel onto the thermopile.

Preferably such thermopile sensors are mounted upon a printed circuit board (PCB) including bores through which the reaction vessels pass. The PCB and the heat guide may be formed with foramens, including bores larger than the local diameter of the vessel, to allow the passage of cooling gas such as air.

This provides an extremely robust, reproducible and non-invasive means of measuring and/or controlling the temperature of individual reaction vessels independently of the other reaction vessels within the reaction vessel matrix. Typically the distance of the thermopile sensor to the vessel is between 0.5mm and 30mm. In the context of microtitre vessels having a maximum diameter of 1 cm, this distance is under 1 cm. Where the location of such a sensor is impossible due to space restrictions a thermal guide such as a glass fibre strand/optical fibre may be used as a waveguide to transport the heat to a remote sensor.

Advantageously the outer layer of the vessel is highly thermally emissive to provide a vessel having as close as possible to black body external surface properties. This is particularly suited to systems where non-contact temperature measurement is required. Where highly thermally emissive materials cannot be used the difference between perfect and actual emissivity may be used to derive the correct temperature of the vessel and the contents thereof.

Where thermally emissive materials are not available or non-contact thermometry is not suitable contact thermometry may be used to derive the temperature of the vessel. Such a contact temperature sensor is preferably sited other than at actively heated or cooled portions of the vessel. A thermally conductive material duct may if desired be employed between the vessel and sensor.

It is usually the case that the vessel is sealed with a cap for the duration of a reaction and such a cap may be translucent or even transparent for at least a part thereof adjacent the sample whereby the progress of the reaction can be monitored. According to features of the invention such a cap may be provided and may be arranged so that the window is heated to slightly above sample temperature and leaves only a minimal, if any, air gap above the sample. This and/or heating the window serves to prevent condensation on the window, enable rapid temperature rise in the sample, and prevent concentration of the sample by evaporation. Advantageously the tube lid has a low thermal mass to allow it to be heated and cooled as quickly as possible.

In an alternative construction the caps are made of a thermally conductive material and heated individually to a thermal profile in a manner to encourage the condensation in the tube to evaporate when optical detection is performed and cooled when optical detection is not required to encourage the condensate to collect on the lid and drip into the vessel. Alternatively the lid may be held at a constant temperature to minimise evaporation that might cause concentration of the reaction within the vessel.

Preferably the vessel is arranged to contain the entire sample in a minimally tapered cylinder, the taper angle being chosen for the optical application and ease of moulding if the vessel is produced by a moulding method. Typically the taper angle is of the order of 1-6° and the thickness of the outer layer is between 0.01 and 1 mm. The taper has the advantage of permitting air above the sample to escape when the cap is being fitted.

In order for the maximum heat transfer to be able to take place as effectively as possible the tube shape should have as large a surface area to volume ratio as possible. The ideal shape would be to have the fluid held between two plates of ECP that would be heated and cooled. However this design does not lend itself to moulding with ECP neither does it lend itself to being locatable in an 96 well MTP format. To circumvent these two problems the design has been limited to an outside diameter of 9mm. inside this footprint the ideal shape is to reduce the diameter of a capillary like tube and extend it's length while maintaining a thin wall thickness. Due to the nature of the filled material that must be used to make an ECP tube, the wall thickness when the reaction vessel is injection moulded is limited to a minimum of 0.1 mm and should be limited to a maximum of 2mm thickness. The ability to transfer heat into and out of the reaction vessel is directly proportional to the wall thickness of the reaction vessel in contact with the heating or cooling medium. Doubling the wall thickness will double the thermal gradient required to transfer the same amount of energy into the reaction vessel. The higher the surface area to volume ratio the better. Preferably the ratio is of surface area to volume is above 3 and preferably above 6.

Preferably the reaction vessel is constructed for use in apparatus with the base of the vessel and an upper edge/portion of the heating layer thereof providing electrical contact areas.

According to another aspect of the invention there is provided a method of manufacture of a reaction vessel, comprising the steps of: a) injection or blow moulding an inner tubular vessel from an electrically insulative and preferably thermally conductive material, the vessel having a base the same thickness or thicker than the walls of the tube in cross section; and b) forming thereon a heating layer of an electrically conducting polymer about the inner tubular vessel.

Moulding of the inner layer first followed by moulding of the outer layer greatly reduces the probability of contamination of the inner layer by the outer layer material, especially when the vessel design is optimised to prevent the outer layer moulding process from damaging the inner surface. Moulding in the reverse order may result in washing of the surface contaminants or material from the outer layer by the inner layer material, which can contaminate the inner layer and hence the liquid contents within the reaction vessel.

According to another aspect of the invention there is provided a method of manufacture of a reaction vessel comprising the steps of: a) injection moulding the reaction vessel to a size and dimension optimal for the process to be used; b) coating of the internal walls of the vessel with an electrically insulative, preferably thermally conductive, layer.

Such layers and application methods include but are not limited to coating of the tube using polyester, polyester-epoxy or fusion bonded epoxy and acrylics, vapour phase deposition of a material such as parylene.

Where contact thermometry is used the tube may have a recess for placement of the temperature sensor to assist in ensuring repeatability between successive vessels.

According to a further feature of the invention there may be provided reaction apparatus for receiving and to allow heating of one or more reaction vessels in associated vessel receiving station(s), including a foraminous upper contact sheet having a plurality of bores, each bore defining the position of a receiving station and a set of foramens for providing air pathways, and a foraminous lower contact sheet having a set of foramens for providing air pathways and having mounted upon it a plurality of electrical contact pads, each pad aligned with a bore of the upper contact sheet, such that a reaction vessel inserted into and contacting a bore of the upper contact sheet will be pushed through to contact an underlying pad on the lower contact sheet to provide means for passing current through the reaction vessel to heat it.

According to a further feature of the invention there may be provided spring contacts instead of or in addition to a foraminous contact sheet. The spring contact may be made from beryllium copper, copper, phosphor bronze or other such metallic or electrically conducting material. The spring contacts will allow the electrical contact to be made with the vessel without causing catastrophic failure of the vessel by for example mechanical abrasion. The upper contact sheet can be constructed in various ways. For example, a flexible compressible sheet may be covered with a conducting layer to form a surface which forms around the vessel contact areas and affords a good electrical contact, which is maintained even at low contact pressure. The flexible material may be silicone foam, neoprene, sheet rubber or various foams. In addition, the flexible/compressible nature of this sheet allows for vessel misalignment to be tolerated. The lower contact pads can be constructed from pieces of the same type of flexible sheet to afford the same benefits.

The conductive surface of the contact sheets may be formed by woven metal fabrics including nickel copper alloy woven with rip stop nylon. The choice of other suitable materials is apparent to the skilled addressee.

In addition, the outer layer of the vessel may be shaped, or treated with a coating or preparation to decrease the resistance of contacts to the vessel surface. An example of such a coating is a silver or aluminium paint such as electrolube silver conductive paint

The apparatus is applicable with particular advantage to reaction vessels in accordance with the first aspect of the invention.

Preferably the reaction apparatus includes means to apply an independent and distinct voltage to each individual or plurality of reaction vessel stations across its pairs of contacts. Conveniently this is achieved by including a circuit upon the lower contact sheet allowing the voltage applied to the lower conductive pads to be controlled independently. In this case typically the upper contact sheet is coupled to one voltage only. This may alternatively however be achieved by having the lower contacts at a common voltage and the upper contacts controlled individually. By such arrangements no wires need be connected to the reaction vessels to achieve heating and a repeatable low resistance electrical connection can be attained at two contacts on each vessel in a manner tolerant of misalignment and minimum contact pressure.

In one embodiment the upper contact assembly consists of a flexible contact sheet bonded to a substrate using electrically conductive adhesive. An example of a suitable adhesive includes a silver loaded epoxy. The contact pads of the lower PCB may cover at least some areas of the lower surface of the contact sheet to provide an electrical contact with the conductive adhesive. Another example of the contact mounting method is to use contact retention pieces which may be metal and may readily be electrically and physically bonded to the lower PCB or substrate, by a process such as soldering, and which then accept the flexible contact pads.

Other examples of contact arrangements include flexible wire forms, such as helical springs, rings and fingers to achieve the upper and lower contacts, spikes or clamps.

Preferably the reaction vessels include a flange of larger cross section, the flange being part of the outer layer of the vessel such that the flange bears against the outer layer of the upper flexible contact sheet to provide a contact area ridge.

The apparatus may include means to provide cooling via airflow. Airflow perpendicular to the plane of the array of vessels provides effective cooling for the reaction vessels. Foramens in the contact apparatus enable such airflow to be readily achieved. Cooling may additionally or alternatively be provided via liquid or phase change coolant, preferably brought into close proximity to the reaction vessels to optimise heat transfer and dissipation.

According to a feature of the invention the apparatus may have a location to which a microtitre plate bearing reaction vessels may be offered and a movable platform carrying the lower contact plate and the thermopile and heat guide array, the arrangement being such that upon initiation the platform is moved upwards against a fixed foraminous lid/surface such that the contact sheets are compressed against the contact positions of the vessel.

This feature of the invention can be advantageously combined with the third aspect of the invention since the PCB carrying the thermopiles can be located between the upper contact sheet and the lower contact sheet. In such case the thermopile PCB is foraminous with a series of bores through which reaction vessels may pass and a series of foramens for providing air pathways or, preferably, substantially the only bores are those through which the vessels pass but the leave a space around said vessels whereby cooling air is directed over the vessel surface. The foramens may be holes of varying size to allow "tuning" of airflow across each vessel. The airflow may be a fast flowing "jacket" of air near the surface of each vessel providing for rapid cooling. The fast flow rate improves cooling rate, and if the air flows parallel to the surface of the vessel it reduces heating effects on the neighbouring thermopile sensors. In addition the jacket can be used to produce uniform predictable cooling over the entire surface of the vessel.

Alternatively turbulence could be deliberately introduced into the airflow since this may aid cooling in other systems, for example where the thermopile sensors are more distant from the vessel.

The means for providing the airflow may comprise a fan capable of maintaining good airflow at high pressure. This characteristic is suited to forcing air through a convoluted path.

A further feature of this invention is to use cooled air to perform the cooling of the vessels. The cooling air might be generated by using Peltier Thermo electric coolers to cool the air or a Refrigeration system to cool the air. Cooling speed is in part dictated by the delta temperature between the internal volume of the vessel (sample location) and the external volume of the vessel (cooling air). The greater this delta temperature the greater the cooling speed of the system will become.

According to a further feature of the invention a chilled block may be placed in contact with the reaction vessel. Instead of using the forced air method of cooling the heat would be absorbed by the block and carried to a heat exchanger elsewhere. The material from which the cooling block is created e.g. copper, aluminium or silver is such that it allows a high thermal conductivity of the heat from the vessel into a heat exchanger for removal elsewhere. The use of the chilled block over the forced air cooling has the potential to provide greater cooling speeds as the thermal conductivity of the block (which is a further deciding factor in cooling speed) will be greater than air. The block can be chilled by passing coolant liquid through channels therein and/or Peltier pumps or refrigeration systems

According to yet another feature of the invention there may be provided an optical monitoring system for a reaction apparatus, where the reaction apparatus defines a plurality of receiving stations, each such station receiving a reaction vessel in which a reaction may take place.

The optical monitoring system may comprise at least one radiation source. Also provided is a scanning apparatus for directing radiation to vessels in the receiving stations, and for directing radiation emitted by the reaction vessel contents into photometric apparatus. The photometric apparatus directs received radiation to a diffraction grating or equivalent technology, and thence to a photomultiplier tube assembly, preferably operating in photon counting mode.

The photomultiplier tube assembly may comprise a series of single channel multi- anode photomultiplier tubes but preferably the assembly comprises a multichannel multi-anode photomultiplier tube (MAPMT).

Radiation emitted by the vessel contents is dispersed over the pixels of the MAPMT by use of a diffraction grating such that the range of wavelengths of radiation impinging upon a photocathode of the multi-anode photomultiplier tube correlates with the position of the photocathode in the MAPMT.

In one embodiment the MAPMT is a 32 pixel linear array over which radiation from around 510-720 nm is dispersed. Thus the optical monitoring system provides for the use of a broad range of fluorophores emitting radiation at wavelengths between about 510nm and about 720nm without the need to change filter sets as required in other instrumentation.

The use of the PMT and operating it in photon counting mode provides for sensitive detection of radiation facilitating the measurements of low levels of incident fluorescence associated with high sampling frequencies. Measurements using a PMT operating in photon counting mode are less affected by changes in the electromagnetic environment, than if the PMT is operated in analogue mode.

The optical monitoring means is preferably an integral part of the reaction apparatus.

Preferably the light source is a single light source, typically a laser. Preferably the laser is a diode pumped solid-state laser (DPSSL) in contrast to the gas lasers used in conventional reaction apparatus and optical monitoring systems. Preferably means are provided for monitoring the reactions within a plurality of tubes, by directing radiation from a single excitation source to the tubes, and collecting the resultant radiation from the tubes to be measured by a single photometric system. This means may comprise one or more rotatable mirrors, where the configuration of mirrors can be controlled to direct light to and from any specific tube. An array of two mirrors is preferred. The size and bulk of the mirror is arranged to be such as to achieve efficient radiation collection with minimum scanning frequency.

The use of a single excitation source and a single photometric system in the same way for all vessels under observation reduces the possibility of variability being introduced into measurements due to the differences between multiple detectors or sources of excitation. In addition it facilitates the cost-effective use of high quality components in excitation and detection sub systems. This is particularly suited for use with high quality photomultiplier systems

The acquisition of a full spectrum from each vessel at each sampling point facilitates the concurrent use of multiple different fluorophores in the array of reaction vessels in the apparatus (including use of multiple different fluorophores within a single vessel) as required by some fluorometric applications. This spectrum may also be acquired in a single operation reading all channels of the MAPMT concurrently, in contrast to systems where readings at different wavelengths must be acquired consecutively, for example by use of a filter wheel or other means. This affords higher sampling rates, and removes effects related to variation in signal between the acquisitions of different wavelengths.

A Fresnel lens may be used in the path of the laser. A Fresnel lens is light, cost- effective and very compact compared to a standard lens of the same diameter and optical properties. The Fresnel lens ensures that the radiation from the excitation source is always directed substantially vertically when it enters each vessel. The rotating mirrors cause the beam to be reflected at an angle, such that it hits the Fresnel lens at a point above the vessel to be illuminated, the Fresnel lens refracts the beam from this point to enter the vessel vertically. The resultant emitted radiation from the vessel is refracted from vertical travel to the correct angle to return to the rotating mirrors and hence to the photometric system. A plurality of light sources may be used as the excitation source to illuminate the sample with a variation of radiation spectra. The excitation sources may be a plurality of individually attenuated LASERs, a plurality of Light Emitting Diodes, a Light Emitting Diode (LED) capable of generating a variety of spectra (RGB LED's) or multiple incandescent or fluorescent lamps.

Software and/or physical filters may be used to remove incident light from the detected sample spectra and also to remove emissions resultant from excitation from one source from those resultant from another source and in this way allow non source-specific emissions to be subtracted and experimentally link fluorophores in the reaction to specific light sources as discussed above. This allows the apparatus to excite at a number of individual wavelengths simultaneously while removing the necessity to change filters using a filter wheel. Where single excitation sources are used a physical filter may be used to remove the excitation spectra from the detected sample spectra. Filter Wheels are generally regarded as slow devices capable of performing several colour changes per second. The use of the software filtering allows up to 1500 samples per second to be filtered. As to detection, CCD, a photomultiplier tube or an avalanche photo diode array are among the possibilities.

Brief Description of the Drawings

A reaction vessel in accordance and apparatus for heating such vessels and monitoring reactions within such vessels in accordance with the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: -

Figure 1a is a section through part of a reaction vessel with an inert electrically insulative lining;

Figure 1 b is a section through part of a two-part {2 shot) reaction vessel with an inert electrically insulative inner part surrounded by an outer electrically conductive part which forms the heating element;

Figure 2 is a schematic plan view of the reaction apparatus illustrating a plurality of reaction vessels;

Figure 3 is a schematic cross section of a thermopile and heat guide array; Figure 4 is a schematic view of a first apparatus for monitoring the optical effects of the reaction;

Figure 5 is a schematic view of a contact temperature sensor, its location in the system and the Beryllium copper contacts which may be used;

Figure 6 is a schematic view of a plurality of light sources used to excite the reaction; and

Figure 7 is a schematic cross section of a block-cooled embodiment of the invention.

Description of the Preferred Embodiment

In figures 1 a and 1 b a reaction vessel 10 for receiving reagents has a reaction cavity 10a and a lid 20 (cf figure 3) for sealing the vessel. In figure 1 b the vessel 10 is a two-shot tube comprising an inner tubular layer 11 having a base 12 and an open top through which reagents are introduced and an outer, heating layer 13 of electrically conducting polymer. Figure 1a represents a single-shot tube with an inert electrically insulative lining 14. As shewn in figure 3, the lid 20 has a nose 21 which projects into the body of the vessel 10, the tip of the nose defining a window 22 through which light may pass for optical monitoring of the vessel contents. The nose of the lid 20 is such that the air gap between the reaction sample in the reaction cavity 10a and the lid is of minimal volume when a sample of standard volume is introduced so as to enable a rapid temperature rise in the sample and less heating of the air gap, with a tendency to minimise condensation formed on the window and to prevent concentration of the sample by evaporation. The outer layer 13 extends from the base 12 of the inner tubular vessel 11 to beyond the level of the outer surface of the lid window 22 such that the lid window 22 is heated. This minimises the possibility of condensation formation, and thus allows for accurate and reproducible optical monitoring of the reactions occurring within the vessel.

The inner and outer layers 11 and 13 are formed by two shot injection moulding with the two layers of different polymers. The inner layer is polypropylene loaded with boron nitride. This renders the inner layer both highly electrically insulative and highly thermally conductive and provides an optimal surface for contact with the reaction contents to be expected in many biological reactions. The outer layer is polypropylene containing carbon flake which heats on application of a voltage differential with the heat produced evenly and predictably. In this case the carbon fibres are milled carbon fibres so that the fibres are of optimal size for the manufacturing process of the vessels. The outer layer 13 varies in thickness so that the heat applied to the contents is even where the cross-sectional area of the vessel varies.

The inner vessel 11 comprises three regions of different radius, namely a lower region 14 in the base region of least radius, a mid region 15 of slightly larger radius and an upper region 16 of greatest radius in the region from the mouth of the vessel to the open neck thereof.

A shoulder 17 formed between lower region 14 and mid region 15 provides a seat upon which the lid 20 sits in position. A shoulder 18 between mid region 15 and upper region 16 supports a contact ridge 13c/18c for providing an electrical connection to the vessel.

The outer layer 13 extends about the lower region 14 and mid region 15 of the inner vessel and thus itself comprises a lower region 13a of smaller radius than upper region 13b of larger radius.

The inner layer 11 is heated at the same power per unit area at all depths in regions 14 and 15 to produce even heating of the reaction cavity 10a and window 22 with the thickness of the outer layer being varied to precisely maintain the cross sectional area of the outer layer perpendicular to the direction of flow of electrical current through the outer layer. Both the inner vessel and outer layer have a thicker cross section of in each of the transitional areas formed at shoulders 17 and 18, and at the base 12 of the vessel.

The method of manufacture of the reaction vessel comprises the steps of injection moulding an inner tubular vessel 11 having a base 12 thicker than the walls of the vessel in cross section, and then injection moulding an outer layer 13 of an electrically conducting polymer about the inner tubular vessel 11 , with the outer layer 13 also having a base thicker than its walls.

Moulding of the inner vessel 1 1 first, followed by moulding of the outer layer 13, reduces the possibility of contamination of the inner surface of vessel 11 by the outer layer material.

The moulding of the outer layer may cause erosion of the inner layer. To allow for this, the inner layer is provided with thicker regions where erosion of the inner layer is expected to be worst, specifically around the base 12 of the vessel, and at the shoulder 17 where the outer layer moulding flows around the inner layer. This ensures that the outer layer material never penetrates the inner layer, hence avoiding contamination of the reaction cavity 10a by outer layer materials.

The inner surface of the upper region 16 of the inner vessel 11 includes a flange 19 which interfits with a corresponding flange 23 on the exterior surface of lid 20 to provide a snap fit.

In this embodiment Figure 2 also illustrates a single reaction vessel receiving station 33 in a reaction apparatus in which a plurality of reaction vessels 10 are received and the reactions within monitored. It has an array of 96 receiving stations 33, four of which are illustrated in figure 2.

It has been found possible to mount temperature measurement, contact and cooling means in the 9mm x 9mm square space allowed for each vessel within a standard 96-well MTP (microtitre plate). Accordingly there is for each receiving station a temperature sensor unit 35 arranged spaced from the station 33 so that when a vessel is at the station 33 the unit 35 is not in contact with the vessel but measures the temperature of the vessel. Thus each reaction vessel 10 has its temperature monitored independently of the other reaction vessels within the reaction vessel matrix. There is no contact between the sensor unit 35 and the vessel and thus there is accurate and consistent temperature measurement, with no risk of cross contamination between different vessels due to transfer of vessel contents via the sensor, for example to subsequent received vessels.

The sensor unit 35 comprises a thermopile 37 encapsulated in a 'can' 39 with a window 41 through which a specific frequency range of radiation, from a specific field of view, may enter the can 39 to cause the thermopile to produce a signal. Here the radiation passes through an infra red filter (not shown) before reaching the thermopile. As illustrated in figure 3, the signals are read via electrical contacts 45, which couple the sensor 35 to a printed circuit board 47.

The sensor unit 35 also includes (not shown) a local temperature sensor to provide an accurate final temperature. The can 39 is provided with an aluminium aperture which blocks access of infra red radiation to the thermopile from any objects other than the specific reaction vessel that is being measured by the sensor.

The reaction apparatus includes a foraminous upper contact sheet 49 having a plurality of bores defining the position of a receiving station 33 and a set of foramens 53 for providing air pathways, and a foraminous lower contact sheet 55 having a set of foramens 57 for providing air pathways and having mounted upon it a plurality of electrical contact pads 59, each pad 59 aligned such that a reaction vessel 10 inserted into a station 33 will contact an underlying pad 59 for passing current through the reaction vessel to heat it.

The upper contact sheet 49 comprises a flexible compressible sheet covered with a conducting layer, in this case a conducting fabric. This sheet is adhered by silver loaded epoxy electrically conductive adhesive to a substrate, in this case a foraminous steel sheet.

The upper section of the outer layer 13 overlies the shoulder 18 between the mid and upper sections of the inner vessel 11 to form an upper contact ridge 13c. In use a reaction vessel 10 is pushed through bore 51 in upper contact sheet 49 until base of outer layer 13 comes into contact with lower contact pad 59, and upper contact ridge 13c sits against and in contact with upper contact sheet 49. The foramens 53 and 57 provide ventilation and therefore effective cooling of the reaction vessels.

The voltage difference applied across each vessel can be controlled independently. In this case the voltages applied to the lower contact pads 59 are controlled independently whilst the upper contact sheet 49 is coupled to one voltage only.

The PCB 47 upon which the thermopile sensors 35 are mounted also includes bores 63 aligned with bores 51 through which vessels may pass.

An optical monitoring system for the reaction apparatus is illustrated in figure 4. Within the reaction apparatus is defined a plurality of receiving stations 67, each for receiving a reaction vessel 69 in which a reaction may take place. The system comprises at least one light source 60, scanning apparatus 61 for directing the light to the reaction vessels 69 in the receiving stations 67, and for receiving radiation emitted by the reaction vessels and directing the radiation via a foraminous mirror 64, a collimating lens 65, and a diffraction grating 73 to a multi-anode photomultiplier tube assembly 75 operating in a photon counting mode. The foraminous mirror 64 contains a foramen at 45 degrees to the plane of the mirror, permitting laser light to pass through it to the vessels. The majority of diverging emitted light from the vessels is reflected to the diffraction grating 73, since at this point the emitted light beam is of much greater diameter than the foramen.

The multi-anode photomultiplier tube assembly 75 here comprises a multi-anode photomultiplier tube (MAPMT) with a 32 pixel array over which radiation from around 510 to 720 nm is dispersed. Radiation emitted by the reaction vessel contents is dispersed over the pixels of the MAPMT by the diffraction grating 73 such that the wavelength range of the radiation impinging on a photocathode of the MAPMT correlates with the position of the photocathode in the MAPMT

The light source 60 is a diode pumped solid state laser (DPSS Laser) which is smaller and lighter than conventional gas lasers typically used in optical monitoring systems.

The scanning apparatus comprises one or more planar rotatable mirrors, for clarity only one such mirror 61 is illustrated. These are motor driven and controlled by means which are omitted from the drawings for clarity. The system of mirrors can be configured to direct the light from the laser to any receiving station 67. Radiation emitted is returned to foraminous mirror 64 which reflects the majority of the emitted radiation through lens 65 which focuses the radiation upon diffraction grating 73.

A Fresnel lens 83 is interposed between the rotatable mirrors, e.g. mirror 61 , and the receiving stations 67 to ensure that the light entering each reaction vessel 69 is substantially vertical.

In a further embodiment using contact thermometry the tube 91 of Figure 5 has a "doughnut" recess at the base thereof to envelop the temperature sensor 96. The temperature sensor is surface mounted to a PCB 95. Flexible thermally conductive material 97 (such as silicone rubber) is used to fill the space between the sensor and tube and ensure a good thermal contact. The beryllium copper spring ring 93 is also surface mounted onto the PCB and is arranged such that the electrical contact position 94 is at a height which ensures that there is no localised heating of the base of the tube and likewise that the base of the tube is not held at a temperature which is too low. The top of the spring contact may be tapered to guide the tube into position.

In a yet further embodiment using a plurality of light sources, Figure 6 shows a plurality of light sources 100, comprising either LEDs, lasers, multiple incandescent or fluorescent lamps. These can also be replaced with a single RGB LED to provide a plurality excitation spectra. The emitted light is converged by a lens assembly 101 to a resultant incident light 102, and directed by a scanning apparatus 61 (figure 4) to the reaction vessel 69 (figure 4).

Either singly or combined the resultant excitation spectrum can be used to excite various combinations of fluorophores for detection that would otherwise not be possible. For example dyes excited at high wavelengths.

Emission patterns can be determined for the different sources for different dyes. Using software a source and dye can be experimentally linked in terms that the emissions (for a dye that are associated with unwanted sources for other dyes) can be subtracted from the emissions resulting from the desired source. Noise from various sources can also be subtracted in this way. The embodiment illustrated in figure 7 shows a vessel 201 comprising an inner electrically insulative and thermally conductive layer and an outer layer of electrically conductive polymer, as per figures 1 and 3. The vessel 201 comprises a reaction chamber portion 201a, a shank portion 201 b and a funnel portion 201 c. A lid 202, having a transparent base 202a, is a snap fit in the shank and funnel portions of the vessel 201. The vessel and lid are accordingly as above described the vessel being of microtitre proportions. There is a recess 201 d at the base of the vessel 201.

The receiving station for the vessel comprises a PCB 203, a cooling block 204 having coolant channels 205, and a contact sheet 206. Mounted on the PCB 203 is an electrical contact clip 207 surrounding a thermosensor 208. A thermally conductive flexible cushion 209 sits on the thermosensor 208.

Thus the electrical supply circuit comprises the PCB 203 with the clip 207 contacting the conductive heating layer of the vessel 201 at the side of the base of the reaction chamber 201 a, and the contact sheet, made of a material having a degree of elasticity, contacting the shoulder between the shank portion 201 b and the funnel portion 201 c of the vessel 201. This latter contact, located as it is towards the top of the shank, means that the shank, surrounding the lid window 202a, is caused to heat the lid 202 and keep the window condensation free.

The coolant channels 205 convey coolant liquid between the block 204 and a heat exchanger, not shewn. In operation the coolant is maintained at a temperature somewhat above a specified normal room temperature, for example 22° to 25°C, but below the lowest temperature of the thermocycling programme, while the electrical circuit is arranged to cycle the reaction chamber temperature between specified upper and lower temperatures.

In a modification to the construction shewn in figure 7, the block 204 is recessed to carry a thermopile located to read the temperature of the surface of the reaction chamber and the base of the vessel 201 is arranged to impinge in an associated elastic material of the PCB 203 thus providing electrical contact.

Claims

1. A reaction vessel for containing a sample during a chemical, biological or biochemical process carried out thereon and comprising a heating layer formed of an electrically conductive material whereby an electric current passed therethrough generates heat, and a lining thereto comprising an electrically insulative material.

2. A vessel as claimed in claim 1 and wherein the lining is also thermally conductive.

3. A vessel as claimed in claim 1 or claim 2 and wherein the lining is formed from any one of polypropylene, polythene, polyvinylpropylene, or glass, conformal coatings, parylene, polyester, polyester epoxy or acrylic. .

4. A vessel as claim in any one of claims 1 to 3 and wherein the lining is loaded with boron nitride.

5. A vessel as claimed in any one of the preceding claims and wherein the heating layer is constructed from plastic containing carbon fibre, carbon black, carbon flake, Buckminster fullerene tubes (carbon nanotubes, or bucky tubes), Buckminster fullerene balls (bucky balls), or a conductive metal flake or metal.

6. A vessel as claimed in any one of the preceding claims and which is a well or a recess or a specific location on a flat substrate.

7. A vessel as claimed in claims 1 to 6 and wherein there are flanking contacts providing an electrical contact surface for the heating layer.

8. A vessel as claimed in any one of claims 1 to 7 and which is a microtitre vessel.

9. A vessel as claimed in any one of the preceding claims and having a reaction chamber portion and a lid retaining portion, wherein the reaction chamber portion has a maximum diameter of 2.0 to 4.0mm and a wall thickness of 0.5 to 1.0mm, the vessel tapering outwards from the base thereof upwards by 1-6°.

10. A vessel as claimed in claim 9 and having a recess at the base thereof.

11. A vessel as claimed in claim 10 and wherein the base is adapted to receive an electrical contact.

12. A vessel as claimed in claim 10 and wherein the base is adapted to receive a temperature sensor.

13. A vessel as claimed in any one of claims 9 to 12 and having a shank portion and an upper portion, the shank portion and the upper portion being arranged to receive a lid, there being a first shoulder between the reaction chamber portion and the shank portion and a second shoulder between the shank portion and the upper portion, the first shoulder being arranged for seating a lid and the second shoulder being arranged for the provision of an upper electrical contact, whereby heating the tube also heats a lower part of the lid.

14. A vessel as claimed in any one of claims 9 to 13 and wherein the reaction chamber has an internal aspect ratio of 4:1 , and is just greater than capillary dimensions to an aqueous liquid.

15. A vessel as claimed in any one of the preceding claims and having a lid, the lid having a window whereby the contents of the vessel can be interrogated, the arrangement being such that the heating means also operates to ensure that the window is kept condensation-free for the interrogation.

16. A vessel as claimed in claim 15 and wherein the lid vessel combination is arranged such that, with a sample of standard volume in the vessel, the lid window contacts the sample.

17. A vessel as claimed in claim 15 or claim 16 and wherein the lid has a low thermal mass.

19. A vessel as claimed in any one of claims 15 to 17 and wherein the lid is arranged to be directly and individually heated.

20. A vessel as claimed in any one of claims 15 to 19 and wherein the lid is arranged to be held at a constant temperature.

21. A vessel as claimed in any one of the preceding claims and whereof the outer surface behaves as a black body heat source or has a high thermal emissivity.

22. Apparatus for carrying out a chemical or biochemical reaction and having a plurality of reaction vessel receiving stations adapted to receive a reaction vessel as claimed in any one of the preceding claims and, for each station, a sensor arranged to measure the temperature of an adjacent vessel.

23. Apparatus as claimed in claim 22 and wherein the sensors are arranged in a circuit for controlling vessel temperature.

24. Apparatus as claimed in claim 23 and arranged to apply an independent and distinct voltage to individual vessels or groups of vessels.

25. Apparatus as claimed in any one of claims 22 to 24 and having compensator temperature sensor means for measuring the sensor environment temperature and correcting the sensor output.

26. Apparatus as claimed in claim 25 and wherein the compensator temperature sensor means comprises a sensor located on each sensor.

27. Apparatus as claimed in any one of claims 22 to 26 and wherein the temperature sensor is a thermopile sensor.

28. Apparatus as claimed in claim 27 and having a heat guide to reflect the heat radiated from the vessel onto the thermopile.

29. Apparatus as claimed in any one of claims 22 to 26 and wherein the sensor is a contact sensor.

30. Apparatus as claimed in any one of claims 22 to 29 and having a printed circuit board (PCB) with bores through which the reaction vessels pass, the sensors being mounted on the PCB.

31. Apparatus as claimed in claim 30 and wherein the PCB is formed with foramens, including bores larger than the local diameter of the vessel, to allow the passage of cooling gas such as air.

32. Apparatus as claimed in claim 30 or claim 31 and having a foraminous upper contact sheet having a plurality of bores providing said receiving stations and a plurality of bores providing air pathways, and a foraminous lower contact sheet having mounted thereon a plurality of electrical contact pads, each pad aligned with a receiving station bore in the upper contact sheet, and having a plurality of bores providing air pathways, the arrangement being such that when a vessel is inserted into a receiving station bore in the upper contact sheet the base of the vessel will contact a pad on the lower contact sheet.

33. Apparatus as claimed in claim 32 and wherein one or both of the upper and lower contact sheet areas comprise a fϋexible compressible sheet covered with a conducting layer.

34. Apparatus as claimed in claim 33 and wherein the flexible material comprises one or more of silicon or other flexible plastics foam, neoprene, and sheet or foam rubber.

35. Apparatus as claimed in claim 33 or claim 34 and wherein the conducting layer comprises a woven metal fabric including nickel copper alloy with rip stop nylon.

36. Apparatus as claimed in any one of claims 33 to 34 and wherein the conducting layer includes one or more of conducting springs, rings, fingers, spikes and clamps.

37. Apparatus as claimed in any one of claims 33 to 36 and wherein the lower contact sheet includes a circuit for applying an independent voltage at each vessel station or at distinct groups of vessel stations.

38. Apparatus as claimed in any one of claims 33 to 37 and wherein the lower contact sheet comprises a printed circuit board (PCB).

39. Apparatus as claimed in any one of claims 22 to 38 and having air cooling and fan means whereby cooled air is used to cool the or each vessel.

40. Apparatus as in claim 39 and wherein the air cooling means is a Peltier thermo electric cooler or a refrigeration system

41. Apparatus as claimed in any one of claims 22 to 29 and having a thermally conductive block surrounding the vessel whereby the heat may be removed from the vessel to a remote location for dissipation.

42. Apparatus as claimed in claim 41 and wherein the cooling block is formed of copper.

43. Apparatus as claimed in claim 41 and wherein the cooling block is formed of aluminium.

44. Apparatus as claimed in claim 43 and wherein the aluminium is anodised.

45. Apparatus as claimed in any one of claims 41 to 44 and having a heat exchanger associated with the thermally conductive block.

46. Apparatus as claimed in any one of claims 41 to 45 and wherein the block is formed with channels for the conveyance of a cooling liquid.

47. Apparatus as claimed in any one of claims 22 to 46 and having a location to which a microtitre plate bearing at least one reaction vessel may be offered and a movable platform carrying the lower contact plate and the thermopile and heat guide array, the arrangement being such that upon initiation the platform is moved upwards until the lower contact sheet is compressed against the base of the at least one vessel.

48. Apparatus as claimed in any one of claims 22 to 47 and having optical monitoring means comprising at least one radiation source, a scanning apparatus for directing radiation into vessels in the receiving stations and for directing radiation emitted by the reaction vessel contents into a photometric apparatus, the photometric apparatus being arranged to direct received radiation to a diffraction grating and hence to a photomultiplier tube assembly.

49. Apparatus as claimed in claim 48 and wherein the photomultiplier tube assembly is arranged to operate in photon counting mode.

50. Apparatus as claimed in claim 49 and wherein the photomultiplier tube assembly is arranged to operate in current mode.

51. Apparatus as claimed in any one of claims 48 to 50 and wherein the photomultiplier tube assembly comprises a multichannel multi-anode photomultiplier tube (MAPMT).

52. Apparatus as claimed in claim 51 and wherein the MAPMT is a 32 pixel linear array over which radiation from around 510-720 nm is dispersed.

53. Apparatus as claimed in any one of claims 48 to 51 and wherein the radiation source is a single light source.

54. Apparatus as claimed in claim 53 and wherein the light source is a laser.

55. Apparatus as claimed in claim 54 and wherein the laser is a diode pumped solid-state laser.

56. Apparatus as claimed in any one of claims 48 to 56 and including at least one rotatable mirror controllable to direct light to and from any specific reaction vessel.

57. Apparatus as claimed in any one of claims 48 to 56 and incorporating a Fresnel lens in the path of the radiation source and arranged to direct light substantially vertically into each reaction vessel.

58. Apparatus as claimed in any one of claims 22 to 58 and arranged to accept a 96n reaction vessel array, where n is an integer.

59. Apparatus as claimed in any one of claims 22 to 47 and having reaction monitoring means comprising means for cycling between a plurality of light sources in order to illuminate the samples with a full spectrum of light or parts thereof and software filter means to distinguish returned emission spectra for different incident lights and fluorophores present in the reaction.

60. Apparatus as claimed in claim 59 and wherein the reaction monitoring means comprises means for selecting one or a plurality of excitation sources.

61. Apparatus as claimed in claim 59 or claim 60 and wherein the reaction monitoring means is arranged to either excite at multiple wavelengths concurrently or individually.

62. Apparatus as claimed in any one of claims 59 to 61 and wherein the reaction monitoring means is arranged to mechanically filter an individual or plurality of excitation sources from the detected sample spectra.

63 Apparatus as claimed in any one of claims 59 to 62 and wherein the reaction monitoring means incorporates software or firmware for filtering an individual or plurality of non-specific emissions from the detected sample spectra.

64. Apparatus as claimed in any one of claims 59 to 63 and wherein the reaction monitoring means are arranged to select a specific wavelength or group of wavelengths.

65. Apparatus as claimed in any one of claims 59 to 64 and wherein the light sources include LASER based sources.

66. Apparatus as claimed in claim 65 and wherein a plurality of LASERs is employed attenuated differently to cover the full excitation spectra.

67. Apparatus as claimed in any one of claims 59 to 64 and wherein the light sources include light emitting diodes (LEDs).

68. Apparatus as claimed in any one of claims 59 to 67 and having detection means comprising a CCD, a photomultiplier tube or an avalanche photo diode array.

69. A method of manufacture of a reaction vessel, comprising the steps of: a) injection or blow moulding an inner tubular vessel from an electrically insulative conductive material, the vessel having a base the same thickness or thicker than the walls of the tube in cross section; and b) forming thereon a heating layer comprising an electrically conducting polymer about the inner tubular vessel.

70. A method of manufacture of a reaction vessel comprising the steps of: a) injection moulding the reaction vessel from an electrically conductive polymer; b) coating of the internal walls of the vessel with an electrically insulative layer.

71. A method as claimed in claim 69 or claim 70 and wherein the electrically insulative material is also thermally insulative.

72. A method as claimed in claim 71 and wherein the electrically and thermally insulative material comprises polyester, polyester-epoxy or fusion bonded epoxy and acrylics, vapour phase deposition of a material such as parylene.