lm rovements in and relating to reaction vessels and reaction apparatus
for use with such vessels _________ 2424199
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 polymerase chain reactions (PCR).
The PCR process is described in detail in US Patents 4683195 and 4683202 owned by Hoffmann - La Roche, Inc. Typically 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. 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 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 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 electricalfy 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 monitqred.
A problem which is apt to occur with such a window arises from a teirperature 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 fight 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 cylinder1 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 angie is of the order of 1-2 and the thickness of the outer layer is between 0.1 and 1mm.
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, polyvinyipropylene 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 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 1cm, this distance is under 1cm.
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 450 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 therniopile 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 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 minimum 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 -25 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.
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 photorriuftiplier 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 5lOnm 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 a.ncFijlk oI mirror is_arragedtobesh_as1j. jive effient radiation collection w,tt hiinimumscanninJuqçj] 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 eachvessel 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, andr removes ffects related to variation in signal between the acquisition 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.
Brief DescriDtion 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; and, Figure 3 is a schematic view of the steps of the method and apparatus for monitoring the optical effects of the reaction.
DescriDtion of the Preferred Embodiment A reaction vessel 10 for receiving reagents in a reaction cavity lOa has a lid 20 for sealing the vessel, the vessel 10 comprising an inner tubular layer 11 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 1-, Li 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 accuate 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 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 radiusthan 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 lOs 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. f.
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 II, 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 lOa 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 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 1 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. 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 pontamination 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. 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 61. In use a reaction vessel 10 is pushed through bore 51 in upper contact sheet 49 until base of oiter layer 91 comes into contact with lower contact pad 59, and upper contact ridge 61 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 c;ontrolled 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.
The optical monitoring system for the reaction apparatus is illustrated in figure 3.
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 71, scanning apparatus 79 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 diffraction grating 73 to a multi-anode photomultiplier tube assembly 75 operating in a photon counting mode. The foraminous mirror 93 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 photomuftiplier tube assembly 75 here comprises a multianode 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 71 is a diode pumped solid state laser (DPSS Laser) which is smaller and lighter thar conventional gas lasers typically used in optical monitoring systems. - The scanning apparatus comprises one or more platar rotatable mirrors, for clarity only one such mirror 79 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 93 which reflects the majority of the emitted radiation through lens 81 which fouses the radiation upon diffraction grating 73.
A Fresnel lens 83 is interposed between the rotatable mirrors, e.g. mirror 79, and the receiving stations 67 to ensure that the light entering each reaction vessel 69 is substantially vertical. 18 -