WO2004045772A2

WO2004045772A2 - Reaction vessels and reaction apparatus for use with such vessels

Info

Publication number: WO2004045772A2
Application number: PCT/GB2003/005041
Authority: WO
Inventors: James Howell; Ben Webster; Roderic Fuerst
Original assignee: Bg Research Limited
Priority date: 2002-11-19
Filing date: 2003-11-19
Publication date: 2004-06-03
Also published as: GB2424381A; GB2424199B; GB0609750D0; AU2003286253A1; GB2404882A; GB0226863D0; GB0326878D0; WO2004045772A3; AU2003286253A8; GB0612553D0; GB2424199A; GB2424380A; GB2424381B; GB0612554D0; GB2424380B

Abstract

A reaction vessel (10), typically a microtitre vessel. incorporating heating means (13) for effecting the reaction and having a lid (20) with a window (22) for enabling interrogation of the contents of the vessel (10), the arrangement being such that the heating means (13) also operates to ensure that the window (22) is kept vapour-free. Apparatus within which such reaction vessels are stationed during the reaction has an array of thermopile sensors (52) for measuring vessel temperature.

Description

Improvements in and relating to reaction vessels and reaction apparatus for use with such vessels

Field of the invention

The invention relates to an improved reaction vessel and apparatus for heating 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 such as the polymerase chain reaction (PCR).

The PCR process is described in detail in US Patents 4683195 and 4683202 owned by Hoffmann - LaRoche, Inc.

Typically a large number of reactions are carried out simultaneously in one apparatus, with a plurality of reaction vessels being received in a reaction apparatus at one time. Often the reaction vessels are in the form of a tray, known as a microtitre plate, made up of an array of vessels. . In one standard microtitre plate, 96 vessels are formed in one array. 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. Many reactions, especially biological reactions such as PCR, can be monitored by 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 heated by electrically conducting polymers (ECPs) are described in International Patent Application Publication No PCT/GB97/03187 and a particular such vessel is described in GB Patent 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 surrounded by a sheath of electrically conducting polymer and be accordingly heated by an electrical current flowing through the electrically conducting polymer. The electrical current can be supplied via connection points from an electrical supply. The vessel has a cap which extends into the body of the vessel to form a space of consistent cross section of capillary dimensions. The lid or base of the vessel may be transparent to provide a light guide/window through which the reactions within the vessel can be monitored.

A problem which is apt to occur with such a window arises from a temperature differential between the reaction contents and the inner surface of the light guide or window (which is in contact with the vapour from the reaction contents).

As a result, vapour produced by the hot reaction contents cools on the cooler inner surface of the window and condenses, thus obscuring the contents from the monitor means

A possible arrangement also includes a cap having a channel into which a thermocouple, thermistor, or other contact-based temperature sensor may be inserted to monitor the temperature within a particular reaction vessel, to allow individual temperature control.

A problem that may occur with such a thermometric system is that there can be a large latent period between the application of power to the ECP by the heating control system and the detection of resultant heating by the temperature sensor. The ECP responds quickly to energy input, as a result the ECP can heat excessively during the latent period. This excessive heating may result in irreversible damage to the vessel and/or vessel contents. PCR reactions, for example, are sensitive to inaccuracies in temperature control.

Summary of the invention. According to a first aspect of the invention, there is provided a reaction vessel for containing a chemical or biochemical reaction and incorporating heating means for effecting the reaction 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 vapour-free for the interrogation.

The heating means for effecting the reaction may comprise a layer, preferably an outer layer, which may be composed of an electrically conducting polymer. The lid may have a nose which projects into the vessel, the tip of the nose defining a window through which light may pass for optical monitoring of the vessel contents and the heating means surrounds the vessel and the lid so that the lid window is heated.

The term 'window' is intended to encompass any barrier partially or completely transparent to light and includes light guides.

By extending the outer ECP layer of the vessel to beyond the nose or base of the lid when in position the whole base area of the lid is heated ensuring that there is a minimal temperature differential between the window and the reaction contents, thus minimising the possibility of condensation formation. This allows for more accurate and reproducible optical monitoring of the reactions occurring within the vessel.

Although in theory the outer layer may comprise an open ended tube surrounding the elongate surfaces of the inner layer preferably the outer layer itself is in the form of an elongate vessel so that the base of the inner layer is also covered by electrically conducted polymer to form a contact area.

The inner layer is preferably 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 material may comprise polypropylene, polythene, polyvinylpropylene, glass or other materials that can be used in biological/chemical reaction processes. The choice of such material is readily apparent to the skilled addressee of the specification

The outer layer preferably constructed from a material allowing the vessel to be heated by application of an electrical voltage differential 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 carbon fibre and carbon black. It can however be any electrically conducting material with conductive particles such as carbon, metal, within an inert base resin such as polypropylene, polyvinylpropylene or nylon.

Advantageously the outer layer is black to provide a vessel having black body external surface properties, which is a good radiating source. This is particularly suited to systems where non-contact temperature measurement is required (for instance in accordance with the fourth aspect of the invention).

Preferably the vessel is designed to contain the entire body of heated liquid 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 1-2° and the thickness of the outer layer is between 0.1 and 1 mm.

In vessels according to the invention the liquid heating region is apt to be the region of most stringent manufacturing tolerance and is carefully calibrated and compensated for the variation in the vessel radius to give even heating without hot spots. This means that the liquid at each depth in the vessel is then subjected to conditions as close as possible to those at any other depth. The vessel has advantageously the smallest radius practical given the taper angle, to reduce to a minimum the maximum distance from any point in the vessel contents to the nearest heated internal surface, hence reducing temperature lag between the outer layer and vessel contents while heating or cooling. The maximum radius of the working portion of the vessel may accordingly be 1 to 4mm.

Preferably the vessel is heated at the same power per unit area at all depths, to produce even heating by means of the method of 'Tube Radius Compensation'. The outer layer is arranged so as to provide an even heating power per unit area of contact with the inner layer across the interface between the outer and inner layers. This in turn will produce an even heating of the liquid contents, providing optimal heating characteristics. This characteristic is important to the success of processes like PCR. In order to achieve this over a varying radius formed by the outer surface of the inner layer the thickness of the outer layer may be varied so as to precisely maintain a given cross sectional area of the outer layer, perpendicular to the direction of flow of electrical current through the outer layer.

Preferably the reaction vessel is constructed for use in apparatus in accordance with a fourth aspect of the invention (set out below) with the base of the vessel and an upper region thereof providing electrical contact areas. In this case the outer layer is constructed so as to integrate two contact areas, allowing a low resistance reliable contact to be formed from a pair of external contacts to the outer layer. These areas are advantageously arranged to reduce heating at the contact point, due to contact resistance, to a minimum, and to allow for electrical contact to be made to both areas by pressing the vessel vertically downwards into a suitably constructed contact pack. In this way an array of vessels can also be easily and simultaneously connected to an array of contacts, including but not limited to a microtitre plate application.

Preferably the base of the vessel forms the lower contact region. The outer layer at the base of the vessel may thus be thicker than the heating regions to reduce possible heating at contact points to a minimum, and to provide for robust contact. Preferably the upper contact region is located above the heating regions, consisting of a thicker horizontal ridge in the outer layer. This allows for an upper electrical contact to press vertically against the contact region for assured contact with minimum heating at the contact point.

The vessel may include sections of different radius, in which case the vessel may include at least one transition area where the inner and outer layers are thicker. One application for this is to provide a larger radius section for insertion of the lid. To avoid unpredictable heating in transitional areas and to facilitate manufacture by moulding (where this process is used) the transition areas may be given a thicker inner and outer layer. The thicker inner layer allows margin for some erosion of the inner layer during the moulding process. This margin prevents contamination as a result of the transfer of material from the outer layer to the inner surface of the vessel. A thicker outer layer in the transitional regions ensures that the current density (current per unit cross-sectional area) flowing through the region is lower than in heating regions, producing less heating and avoiding hot spots.

According to a second aspect of the invention there is provided a method of manufacture of a reaction vessel, comprising the steps of: a) injection moulding an inner tubular vessel having a base thicker than the walls of the tube in cross section; and b) injection moulding an outer layer of an electrically conducting polymer about the inner tubular vessel, with the outer layer also having a base thicker than its walls.

Whilst the materials used for the inner tubular vessel formed in accordance with this second aspect of the invention may be any injection mouldable polymer, preferably they comprise one of a polypropylene, polythene, polyvinylpropylene, or other materials suitable for use in biological and/or chemical reaction processes. Preferably the outer layer is a polypropylene containing carbon fibre and carbon black. It can be any electrically conducting material with conductive particles such as carbon, metal, within an inert base resin such as polypropylene, polyvinylpropylene or nylon. In the case where carbon fibres are used, preferably these are milled or otherwise processed to produce an optimal fibre length. Use of shorter fibre length facilitates moulding by reducing the tendency of the fibres to block the moulding apparatus.

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.

The thicker inner and outer layers ensure that if erosion of the inner layer occurs during outer layer moulding, the inner layer will still be sufficient to prevent contamination of the liquid contents. Erosion of the inner layer can be worse at the base of the reaction vessel, near the injection point.

Preferably the vessel includes sections of different radius as proposed in relation to the first aspect of the invention. In this case, preferably the radius change region has a thicker cross section both at the inner and outer layer. This allows for some erosion of the inner vessel as the outer layer material flows around the increased radius of inner layer material. Where the outer layer includes as one of its components, carbon fibres, the thicker cross section also reduces possible breakage/ shearing of the carbon fibres. Since the final layer thickness is hard to predict, the thicker outer layer ensures that minimal heating will occur. This protects against the possibility of the outer layer becoming thinner in this region than in the region surrounding the main body of the inner vessel forming the liquid heating region, which could produce an undesirably high temperature. According to a third aspect of the invention there is provided reaction apparatus in which a plurality of reaction vessels are received and the reactions within monitored, including a plurality of vessel receiving stations, each for receiving a reaction vessel and for each receiving station a sensor, preferably a thermopile sensor, arranged spaced from the station so that when a vessel is at the station the sensor measures the temperature of the vessel, but is not in contact with the vessel.

This provides an extremely robust reproducible, 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. With the method being non-invasive there is no risk of biological cross contamination such as could occur with temperature sensors placed within vessels and exposed to vessel contents. Also with no need for mechanical contact for heat transfer between the vessel and the thermopile there is a reduced potential for variability in the temperature measurement. The lack of mechanical contact reduces wear and degradation of the temperature monitoring apparatus, or the vessels.

In a plate layout the space is very limited. As a result the thermopiles are preferably equidistant from surrounding vessels to minimise the chance of physical contact. In a layout with more space available, it is preferable to place the thermopiles sufficiently far away from the vessels that they will not be significantly heated by warm air around the vessels. This stabilises the reading environment and reduces reading variation. The maximum distance can be determined by the sensitivity of the thermopile and its field of view. The further away the thermopile is placed, the less radiation is received from the vessel.

Typically therefore 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. Preferably the reaction vessels are in accordance with the first aspect of the invention and include an outer layer which provides black body external surface properties which facilitates the non-contact temperature measurement.

Typically the reaction apparatus receives a standard array of reaction vessels in a microtitre plate, usually 96 vessels, or integer multiples thereof, wherein the reaction vessels may be arranged in an array which may be square/rectangular or other geometric shape that allows for optimum conditions to be met. In this case, preferably the plurality of the thermopile sensors of the apparatus are also arranged in an array with their axes aligned, so that when the vessels are in their stations, the thermopile sensor for that station is pointed at the outer surface of the corresponding vessel, but not in direct physical contact with any vessel. Preferably the axis of the centre of the field of view of each sensor is inclined at 45° to the major axis of the microtitre plate and lies in a horizontal plane. This allows the thermopile sensors themselves to fit into the spaces between the vessels in the array with a minimum of interference to airflow over the surface of the vessels. Preferably such thermopile sensors are mounted upon a printed circuit board including bores through which the reaction vessels pass.

Preferably the thermopile sensor comprises a thermopile encapsulated in a 'can' with a window in the can allowing a specific wavelength range of radiation particularly infra-red radiation to enter the can window from the measured reaction vessel only, causing the thermopile to -produce a signal. The signal from the thermopile is read via a pair of electrical contacts. This provides a reading related to the amount of infrared radiation being absorbed by the thermopile. Preferably each thermopile sensor package also includes an internal embedded temperature sensor, which allows measurement of the local temperature of the thermopile and encapsulation, via further electrical contacts. This provides for compensation of the reading to produce an accurate final temperature. The thermopile sensor could alternatively have an external temperature sensor, say externally bonded to the can. In some cases the thermopiles could have their temperatures regulated in some way that they did not require individual thermistors.

Preferably each thermopile sensor has a field of view restricted to a portion of the outer surface of the vessel whose temperature is to be measured. This may be provided by an aperture, preferably made from a material which is a good IR reflector, including but not limited to copper or aluminium, shaped with a window allowing access of infra red radiation to the thermopile sensor only from the desired region of the specific reaction vessel whose temperature is being measured by the sensor. The size of the aperture depends upon the size and position of the sensor and vessel but it is preferably arranged so that its field of view covers only the heated part of the vessel and specifically blocks the view of everything else, particularly other vessels at different temperatures. The aperture may be separated from the thermopile or in mechanical contact with the device, for example being fixed onto the thermopile can as a cap.

According to a fourth aspect of the invention there is provided reaction apparatus for receiving and to allow heating of a plurality of reaction vessels in a plurality of vessel receiving stations, 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.

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 silicon 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.

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 a independent and distinct voltage to each reaction vessel station across its pair of contacts. Conveniently this is achieved by including a circuit upon the lower contact sheet allowing the voltage to be applied to the 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 low contact pressure. Preferably 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 preferably includes means to provide cooling via airflow. Airflow perpendicular to the plane of the array of vessels provides effective cooling for the reaction vessels. The foramens in the contact apparatus enable such airflow to be readily achieved.

The fourth aspect 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.

In a typical reaction apparatus operating upon a 96n well array the reaction vessels are either part of a microtitre plate or are fitted thereto and the plate is offered to the apparatus after the wells have been charged with the reactants and sealed with their caps. According to a feature of the invention there may be provided, for use in particular with reaction apparatus cooled by air up-draft, a microtitre plate incorporating stations for receiving reaction vessels in accordance with the invention and having foramens surrounding the receiving stations, whereby cooling air can in use be ducted up therethrough adjacent the reaction vessels.

Advantageously such a foraminous microtitre plate is arranged, in relation to the size of the reaction vessels, to grip the latter once these have been offered into their respective receiving stations. If desired microtitre plates according to this feature of the invention may be arranged to provide one or even both of the electrical contacts to the reaction vessels. However, as these microtitre plates may well be disposable items, not least for reasons of maintaining effectiveness and minimising contamination, it is preferred that they are not thus encumbered.

According to a fifth aspect of the invention there is 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, 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 multi-channel 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 51 Onm 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 mirror is arranged to be such as to achieve efficient radiation collection with maximum 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 (and same temperature) 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 due to the acquisition of different wavelengths at different times and/or temperatures.

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.

Brief Description of the Drawings

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

Figure 1 is a section through part of the reaction apparatus holding a reaction vessel;

Figure 2 is a schematic plan view of the reaction apparatus illustrating a plurality of reaction vessels with their temperature sensor and control means; and Figure 3 is a schematic view of the steps of the method and apparatus for monitoring the optical effects of the reaction. Description of the Preferred Embodiment

In Figure 1 can be seen a section of a reaction vessel 10 for receiving reagents in a reaction cavity 10a and having a lid 20 for sealing the vessel. The vessel 10 comprises an inner tubular layer 1 1 having a base 12 and an open top through which reagents are introduced and an outer layer 13 of electrically conducting polymer. 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 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 1 1 and 13 are formed by two shot injection moulding with the two layers of different polymers. The inner layer is polypropylene that provides an optimal surface for contact with the reaction contents to be expected in many biological reactions and also provides good thermal coupling between the outer layer 13 and the contents. The outer layer is polypropylene containing carbon fibre and carbon black, 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 length for the manufacturing process for the vessels. The outer layer 13 varies in thickness so that the heat applied to the contents is even.

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 to the open neck of the vessel 7. 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 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 1 1 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 1 1 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 1 1 , with the outer layer 13 also having a base thicker than its wallsMoulding of the inner vessel 11 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 outer layer also contains thicker sections in these regions, to give a low current density when heating, and hence a low heating power, avoiding undesirable heating effects.

The inner surface of the upper region 16 of the inner vessel 1 1 includes a flange 19 which interfits with a corresponding flange 23 on the exterior surface of lid 20 to provide a snap fit.

Figure 1 also illustrates a single reaction vessel receiving station 30 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 30, 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 dimensioned 96-well microtitre plate (MTP). Such a microtitre plate of standard dimensions comprises a plate 40 having reaction vessel receiving stations 41 and cooling air flow foramens 42. interspersed between each station 41. The stations 41 are arranged for gripping the reaction vessels fitted therein sufficiently tightly to prevent relative movement thereof during reactions. The plate 40 has side members, not shewn, which project therefrom and provide both means for maintaining the plates 40 substantially rigid and for locating the plate accurately within the reaction apparatus.

The spatial relationship between the plate 40 carrying a reaction vessel 10 and the reaction apparatus is illustrated in figure 1 , where the microtitre plate 40 is shewn above various elements of the reaction apparatus which have functions in relation to the reaction vessels 10.

The temperature sensing arrangement is illustrated in figure 2. This shows a foraminous pcb 50 having receiving stations 51 and temperature sensing units 52, for each receiving station 51 a temperature sensor unit 52 arranged spaced from the station. 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 52 and the vessel 10 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 52 comprises a thermopile 53 encapsulated in a 'can' 54 with a window 55 through which a specific frequency range of radiation, from a specific field of view, may enter the can 54 to cause the thermopile to produce a signal which is then passed to temperature control means (not shewn) via the pcb 50. Here the radiation passes through an Infra red filter (not shown) before reaching the thermopile.

The sensor unit 52 also includes (not shown) a local temperature sensor to provide an accurate final temperature. The can 54 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 60 having a plurality of bores defining the position of a receiving station 30 and a set of foramens 61 for providing air pathways, and a foraminous lower contact sheet 70 having a set of foramens 71 for providing air pathways and having mounted upon it a plurality of electrical contact pads 72, each pad 72 aligned such that a reaction vessel 10 inserted into a station 30 will contact an underlying pad 72 for passing current through the reaction vessel 10 to heat it.

The upper contact sheet 60 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 61. In use a reaction vessel 10 is pushed through bore 30 in the upper contact sheet 60 until base of outer layer 13 comes into contact with lower contact pad 72, and upper contact ridge 13c sits against and in contact with upper contact sheet 60.

The foramens 61 and 71 provide ventilation and therefore ducted cooling of the reaction vessels. Likewise, it will be noted that the foramen providing the station 51 in the pcb 50 is somewhat larger than the vessel 10 and is accordingly arranged to ensure that cooling air passes close to the vessel 10 at its base and in the region of the reaction cavity 10a.

The voltage difference applied across each vessel can be controlled independently. In this case the voltages applied to the lower contact pads 72 are controlled independently whilst the upper contact sheet 60 is coupled to one voltage only.

The optical monitoring system for the reaction apparatus is illustrated in figure 3. The system comprises at least one light source 80, scanning apparatus 81 , 82 for directing the light to the reaction vessels 10 in the receiving stations 30, and for receiving radiation emitted by the reaction vessels 10 and directing the radiation via a lens 83 and diffraction grating 84 to a multi-anode photomultiplier tube assembly 85 operating in a photon counting mode. The mirror 81 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 84, since at this point the emitted light beam is of much greater diameter than the foramen.

The multi-anode photomultiplier tube assembly 85 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 84 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 80 is a diode pumped solid state laser (DPSS Laser) which is smaller and lighter than conventional gas lasers typically used in optical monitoring systems.

There is a plurality of mirrors 82, or which for clarity only one is illustrated The mirrors 82 are motor driven to rotate and are controlled by means (not shewn) to direct the light from the laser to any receiving station 30. Radiation emitted is returned to the foraminous mirror 81 which reflects the majority of the emitted radiation through lens 83 which focuses the radiation upon diffraction grating 84.

A Fresnel lens 86 is interposed between the rotatable mirrors, e.g. mirror 81 , and the receiving stations 30 to ensure that the light entering each reaction vessel 10 is substantially vertical.

Claims

1.A vessel for containing a chemical or biochemical reaction and incorporating heating means for effecting the reaction 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.

2. A vessel as claimed in claim 1 and which is a microtitre vessel.

3. A vessel as claimed in claim 1 or claim 2 and wherein the heating means comprises an electrically conductive coating on the vessel.

4. A vessel as claimed in claim 3 and wherein the coating is a polymer.

5. A vessel as claimed in claim 3 and wherein the coating is an ink.

6. A vessel as claimed in any one of the preceding claims and formed of an electrically insulative plastics material

7. A vessel as claimed in any one of the preceding claims and whereof the outer surface is black.

8. A vessel as claimed in any one of the preceding claims and which is a composite, comprising an inner electrically non-conductive polymer layer, and an outer electrically conductive layer.

9. A vessel as claimed in claim 8 and having a base and sides and wherein both layers on the base are thicker than those on the sides.

10. A vessel as claimed in claim 8 or claim 9 and having an inner layer composed of a thermoplastic polymer material and an outer layer composed of a thermoplastic polymer material with conductive filler.

1 1. A vessel as claimed in claim 10 and wherein the inner layer comprises polypropylene.

12. A vessel as claimed in claim 10 or claim 11 and wherein the outer layer comprises polypropylene filled with carbon powder and/or carbon fibre.

13. A vessel as claimed in any one of claims 8 to 12 and wherein the two layers have thickness varying with the diameter of the vessel to keep constant the total cross-sectional area of the outer layer at any given height.

14. A vessel as claimed in any one of the preceding claims and which is substantially cylindrical and tapered, having an upper region of greater diameter than the lower.

15. A vessel as claimed in any one of the preceding claims and having contact areas for electrical connection.

16. A vessel as claimed in any one of the preceding claims and having an electrical contact area at a base thereof and another approximate an upper rim hereof.

17. A vessel as claimed in claim 16 and having a substantially horizontal upper ridge carrying the upper electrical contact.

18. A vessel as claimed in any one of the preceding claims and wherein the lid projects into the vessel, whereby the window is contained within the vessel.

19. A vessel as claimed in any one of the preceding claims and having at least one discontinuity or step in the side thereof.

20. A vessel as claimed in any one of the preceding claims and wherein the lid is optically clear and arranged to permit interrogation of the contents using fluorescence, bioluminescence, phosphorescence and/or chemiluminescence.

21. A method of fabricating a vessel and comprising the steps of: a. injection moulding an inner tubular vessel having a base thicker than the sides thereof; and b. injection moulding an outer layer of an electrically conducting polymer about the inner vessel, the outer layer also having a base thicker than the walls thereof.

22. Apparatus for carrying out a chemical or biochemical reaction and arranged to accept at least one vessel as claimed in any one of the preceding claims.

23. Apparatus as claimed in claim 22 and arranged to apply an independent and possibly distinct voltage to individual vessels or groups of vessels at any given time.

24. Apparatus for carrying out a chemical or biochemical reaction and having a plurality of reaction vessel receiving stations adapted to receive a reaction vessel and, for each station, a thermopile sensor so spaced from the station as to measure the temperature of an adjacent vessel.

25. Apparatus as claimed in claim 24 and wherein the thermopile sensors are arranged in a circuit for controlling vessel temperature.

26. Apparatus as claimed in claim 24 or claim 25 and wherein the thermopile sensors are between 0.5mm and 30mm from the associated vessel.

27. Apparatus as claimed in claim 26 and wherein the sensors are of the order of 4mm from the associated vessel.

28. Apparatus as claimed in any one of claims 24 to 27 and wherein the vessel receiving stations include a plate in which said stations are arranged and the axis of the centre of the field of view of each thermopile sensor is at 45° to a major axis of the plate and lies in a horizontal plane.

29. Apparatus as claimed in claim 28 and wherein the plate is a printed circuit board providing electrical relationship with the sensors.

30. Apparatus as claimed in any one of claims 24 to 29 and wherein each thermopile sensor comprises a thermopile encapsulated in a can, the can having a window arranged to allow only infrared radiation therethrough.

31. Apparatus as claimed in any one of claims 24 to 30 and having compensator temperature sensor means for measuring the thermopile environment temperature and correcting the thermopile sensor output.

32. Apparatus as claimed in claim 31 and wherein the compensator temperature sensor means comprises a sensor located on or in each thermopile sensor.

33. Apparatus as claimed in any one of claims 24 to 32 and wherein each thermopile sensor has a mask defining an aperture whereby the thermopile sensor only receives radiation from its associated reaction vessel.

34. Apparatus as claimed in any one of claims 24 to 33 and arranged to accept vessels as claimed in any one of claims 1 to 20.

35. Apparatus for receiving and to allow heating of a plurality of reaction vessels in a plurality of vessel receiving stations, including 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.

36. Apparatus as claimed in claim 35 and wherein one or both of the upper and lower contact sheet areas comprise a flexible compressible sheet covered with a conducting layer.

37. Apparatus as claimed in claim 36 and wherein the flexible material comprises one or more of silicon or other flexible plastics foam, neoprene, and sheet or foam rubber.

38. Apparatus as claimed in claim 36 or claim 37 and wherein the conducting layer comprises a woven metal fabric including nickel copper alloy with rip stop nylon.

39. Apparatus as claimed in any one of claims 36 to 38 and wherein the conducting layer includes one or more of conducting springs, rings, fingers, spikes and clamps.

40. Apparatus as claimed in any one of claims 35 to 39 and wherein the lower contact sheet includes a circuit for applying independent and possibly distinct voltage at each vessel station or at distinct groups of vessel stations at any given time.

41. Apparatus as claimed in claim 40 and wherein the lower contact sheet comprises a printed circuit board (PCB).

42. Apparatus as claimed in 35 and wherein the upper contact sheet comprises a flexible contact sheet bonded to a rigid substrate with an electrically conductive adhesive.

43. Apparatus as claimed in any one of claims 35 to 42 and including features as claimed in any one of claims 24 to 34.

44. Apparatus as claimed in claim 43 and wherein the thermopiles are mounted on a foraminous thermopile plate intermediate the upper and lower contact sheets, the foramens including holes for both receiving reaction vessels and guiding airflow therethrough.

45. Apparatus for receiving and to allow heating of a plurality of reaction vessels in a plurality of vessel receiving stations 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..

46. Apparatus as claimed in claim 45 and wherein the photomultiplier tube assembly is arranged to operate in photon counting mode.

47. Apparatus as claimed in claim 45 and wherein the photomultiplier tube assembly is arranged to operate in current mode.

48. Apparatus as claimed in any one of claims 45 to 47 and wherein the photomultiplier tube assembly comprises a multichannel multi-anode photomultiplier tube (MAPMT).

49. Apparatus as claimed in claim 48 and wherein the MAPMT is a 32 pixel linear array over which radiation from around 510-720 nm is dispersed.

50. Apparatus as claimed in any one of claims 45 to 49 and wherein the radiation source is a single light source.

51. Apparatus as claimed in claim 50 and wherein the light source is a laser.

52. Apparatus as claimed in claim 51 and wherein the laser is a diode pumped solid-state laser.

53. Apparatus as claimed in any one of claims 45 to 52 and including at least one rotatable mirror controllable to direct light to and from any specific reaction vessel.

54. Apparatus as claimed in any one of claims 45 to 53 and incorporating a Fresnel lens in the path of the radiation source and arranged to direct light substantially vertically into each reaction vessel.

55. Apparatus as claimed in any one of claims 45 to 54 and incorporating features as claimed in any, one of claims 24 to 44.

56. Apparatus as claimed in any one of 24 to 55 and arranged to accept a 96n reaction vessel array, where ή is an integer.

57. Apparatus as claimed in any one of claims 22 to 56 and having a ^"fan arranged to maintain an airflow therethrough.