WO2012063011A2

WO2012063011A2 - Heating and cooling low volume biological reaction vessels

Info

Publication number: WO2012063011A2
Application number: PCT/GB2011/001565
Authority: WO
Inventors: Nelson Nazareth; Antony Driscoll; David Edge
Original assignee: Bg Research Ltd
Priority date: 2010-11-08
Filing date: 2011-11-08
Publication date: 2012-05-18
Also published as: GB201308226D0; WO2012063011A3; CN103260760B; EP2637789A2; GB2501011A; JP2013544088A; US20150144299A1; CN103260760A; JP5892709B2

Abstract

A vessel cup assembly for receiving a chemical and/or biological reaction process vessel containing reactants and processing the reaction therein and comprising: a reaction vessel receiving portion; a heater portion; and a cooling portion; and wherein the assembly is of integral construction.

Description

Heating and Cooling Low Volume Biological Reaction Vessels

Field of the invention

The invention relates to apparatus for biological or chemical reactions where thermal cycling is employed in the reaction. It is particularly concerned with reactions such as polymerase chain reactions (PCR), although isothermal reactions will also be quite possible.

Background to the invention

The PCR process is described in detail in US Patent specifications 4683195 and 4683202.

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 8 X 1 2 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.

In reactions involving multiple thermal cycles the cooling part of the cycle may be effected using water or non-electrically conducting fluids like 'Fluid XP' or a cooling block, powered by peltier devices, and/or a fan blowing cooled air over the vessei or vessels. Sometimes the cooling is continuously present and the heating part of the cycle is carried out against a background of the constant cooling Thus for example in conventional block thermal cyclers heating is effected using a direct heater eg thermal mats and cooling by either forced air or actively by thermo electric heat pumps. In other thermal cycling apparatus, heating and cooling are effected by shuttling between blown hot air and blown cold air.

There are situations, for example when it is required to identify what may be a dangerous pathogen, in which it is highly desirable to minimise the time taken by such a reaction. Apparatus for minimising the time required in the heating part of the cycle is described in copending UK Patent Application numbers 0609750.5 and 0610432.7. There an electrically conductive polymer is employed as, or as and 0610432.7. There an electrically conductive polymer is employed as, or as part of, the material of the reaction vessel. Cooling is effected using forced cooled or ambient air.

A block based system will inevitably have a relatively high thermal mass while a forced air system will have a low thermal mass. This can militate against rapid heating and cooling.

It is likewise important to achieve heating as quickly as possible, whilst avoiding heat shock or localised boiling. Again, conventional block based systems are limited by the large thermal mass and the insulative properties and geometries of the vessels themselves. Air based systems are similarly limited by the thermal properties of these vessels. Therefore the majority of approaches thus far can only heat the vessels at around 2.5C per second (peak heating rate, when measured at the level of the vessel itself, actual in fluid transitions can be markedly longer)

It is also important however to cater for the fact that a rapid thermal cycling process may impose mechanical considerations and the apparatus or components thereof may have a short life if attention has not been paid to such parameters as material coefficient of expansion and fatigue.

For example, apparatus performing thermal cycling with 96 vessel arrays may be arranged to offer the vessel arrays to apparatus vessel stations for the cycling, and on completion thereof to remove them. As the thermal cycle will be the most efficiently and rapidly effected the more intimate is the contact between each vessel and the heating and cooling systems the more likely is there to be a mechanical fatigue issue for the vessel stations, wrought by constant repeated emplacement and withdrawal of vessels, exacerbated by the thermal cycling. Disintegration of one or more vessel stations may thus occur. The replacement of the components of the vessel receiving station can be a costly exercise.

The present invention provides apparatus wherein thermal cycling in biological or chemical reactions is maintained at a desirable rate whilst at the same time the integrity of each vessel station is maintained for at least an acceptable period of use. A further consideration for rapid detection of DNA species, such as pathogens, is the ability to accurately test for multiple species in a single test within a minimum timeframe. Apparatus capable of independent control and monitoring of each vessel in for example a 96n vessel array would allow multiple tests to be completed simultaneously, thus in itself providing a relatively short timeframe, whilst also conferring the important benefit of greater veracity due to the independent monitoring ability.

Molecular diagnostic tests such as the PCR process and latterly real-time PCR (US Patent No. 6, 171 , 785) have greatly reduced the time to detection of a number of diseases. Current technology is limited by the physics of the process, imposing a lower limit on the time within which the process can be accomplished, whereas an important goal is always to reduce that time. Alternative approaches to the commonly used 96n array (where n is greater than 1 ) have been utilised in order to reduce this cycling time such as air based (US Patent 5,455, 175) thermal cyclers. These have the disadvantage of being unsuited to automation and screening of large numbers of samples simultaneously.

It would therefore be highly advantageous to have a system capable of thermal cycling whether single vessels or multiples including 96n array assays with ramping rates, of both heating and cooling exceeding an average of five degrees Celsius per second 'in liquid' temperatures.

Such a system would then be capable of completing the PCR process in under 20 minutes, for a commonly accepted 3 step, 30 cycle protocol (zero seconds at 95°C, one second at 55°C and five seconds at 72°C - as an example) and is therefore highly suited to rapid detection of pathogens. Additionally it would be highly advantageous for such technology to be applied to any number of vessels and also to be used portably at the point of care. For point of reference standard i 96 vessel thermal cyclers of modern design and construction can cycle at average speeds of up to one and a half degrees Celsius per second in the liquid using standard consumables.

There are myriad interdependent technical problems to be overcome in reducing i process time.

The biological processes described require that each of the arrayed vessels are subjected to exactly the same thermal profile if the resultant data are to be reproducible. A standard 96 vessel block based thermal cycling device is controlled and monitored in at least four positions: if such a system were capable of rapid heating then individual cells in the array could lag thermally behind their neighbours.

It is highly desirable then that there is individual control of each of the 96 vessels and that each is controlled identically. It should be obvious to one skilled in the art that at such speeds even slight differences in control could lead to vastly differing temperature profiles. An accuracy of half a degree Celsius is the accepted norm.

A further disadvantage to existing approaches are the masses that are required to be cycled thermally. Minimisation of the masses involved is a pre-requisite for increasing the thermal cycling rates. Thus, for the advantageous operation in a microtitre context the hardware most closely associated with heating and cooling is preferable of the minimum bulk and weight possible.

Reduction in the energy requirements of the thermal cycling process is also a pre-requisite for thermal cycling at rates exceeding 5°C/s in the fluid.

A thermal storage system as demonstrated for example in US12/381 ,953 can reduce the energy requirements for such thermal cycling apparatus. However the apparatus therein described is unable to drive the vessel to a temperature below that allowed by the Dt max of the attached thermoelectric cooler (TEC). Further the heat removal module described in that document of necessity operated at temperatures above the required lowest temperature so that a high cooling rate could not be achieved. Additionally, even in a single piece construction varying thermal diffusion rates across the block are possible as the outside edges may lose more temperature to ambient for example. This could be compensated for by the control electronics, but with greater thermal cycling rates the problem is potentially exacerbated.

The present invention provides apparatus wherein thermal cycling in biological or chemical reactions is performed at cycling rates greatly above those achievable by existing technology and wherein each reaction vessel is controlled independently. Summary of the Invention

According to a first aspect of the present invention there is provided for a chemical and/or biological reaction apparatus a vessel cup assembly for receiving a chemical and/or biological reaction process vessel containing reactants and processing the reaction therein and the vessel cup assembly comprising:

a reaction vessel receiving portion;

a heater portion; and

a cooling portion; and

wherein the assembly is of integral construction.

According to a second aspect of the present invention there is provided a chemical and/or biological reaction apparatus arranged for the reception of a vessel cup assembly as set forth above.

According to a third aspect of the present invention there is provided a chemical and/or biological reaction process utilising the vessel cup assembly and the apparatus set for above.

The vessel cup assembly may be of integral construction. The cooling portion may be constructed as an anchor member to anchor the assembly into a reaction vessel receiving station in a cooler assembly in or for a chemical and/or biological reaction apparatus.

The reaction vessel receiving portion may comprise a cup for receiving snugly a reaction vessel. A snug reception of a reaction vessel into a vessel cup implies both substantial contiguity and separability.

The heater portion may at least partially overlap the reaction vessel and be surrounded by a heater which may be in the form of a wire coil wound thereupon. The coil may be encased in a paint, such as an enamel paint, which can be 'cooked' to stabilise the coil and insulate it exteriorly. Preferably however a glue, such as an ultra-violet curable glue, is employed which also has the property of retaining the wire in place. The wire may be nickel chrome 0.21 mm diameter driven with a voltage of 24 volts and capable of drawing a maximum current of 2.2 amps. The length of the wire in the context of the microtitre vessel having a capillary reaction chamber as herein proposed, is of the order of 36cm. Other reaction vessels will likely have different requirements.

Alternatively the heater may be a sheath of electrically conductive polymer (ecp) (comprising electrically conductive particles such as graphite or metal or similar in an inert plastic carrier such as polypropylene or similar) or of metal film. However, currently available ecp has not been found to be predictable in performance.

The vessel receiving portion may have a constant wall thickness so as to avoid hotspots - temperature variation along the length thereof or therearound. However it has been found that a cup wall which tapers down towards the open end provides an effective control to the flow and even distribution of heat whilst also facilitating installation, snug fitting and removal of a mating reaction vessel. A taper angle of between 2° and 5°, preferably of the order of 4°, has been found suitable. Where for example the cup entry wall thickness is of the order of 0.35 to 0.45, preferably 0.4 mm and the cup base wall thickness is of the order of 0.9 to 1 .1 , preferably 0.95mm, the wall thickness is sufficient at a convenient intermediate station along the length of the cup for the formation of a temperature sensor receiving recess. In this recess can then be located a thermal sensor, such as a thermistor, for thermal control. Measurement Specialties Ltd have supplied a suitable bare thermistor having a length under 3mm and a mean diameter of the order of 0.2mm. To ensure safe anchorage of this thermistor into its recess the tail wire may be wound around the vessel receiving portion a couple of times. The thermistor is preferably located a short distance above the heater so as not to read heater temperature but the temperature of the cup. In this way the thermistor can be arranged or calibrated to operate in a predictive mode, that is it can indicate that the temperature of the reactants will be such and such at a known number of milliseconds later.

A vessel cup assembly as herein set forth has the peculiar advantage that when the heater portion heats up and heat necessarily flows both downwards and upwards, the downward flow acts as a barrier to the operation of the cooler portion whilst the vessel receiving portion is in heating mode.

The sensor (thermistor) should ideally be positioned such that its physical temperature exactly matches that of the reagent fluids, and secondly be constructed such that it imposes a reproducible and calculable thermal lag commensurate with that experienced by the fluid itself.

It has also been found particularly valuable to ensure that there is an air gap between the bottom of the cup and the base of the reaction vessel. This ensures that the reaction vessel is scarcely heated from below, which might be a hot spot. Also, in the manufacture of plastic reaction vessels the base of the vessel is apt to be the least predictable dimensionally so that contact therewith for heat transfer may render the process inconsistent as between one reaction vessel and another. Typically a gap of 2 to 4 mm suffices for this air gap.

It will be appreciated that in the microtitre reaction vessel context the overaii dimensions of a vessel cup assembly in accordance with the invention are of the order of 2 to 4 cm long by 0.4 to 0.7 mm diameter. Effectively the bulk of the vessel cup assembly being quite small heat can be inputted and removed very quickly, that is the ramping rates can be practically as high as the reaction can accept.

In a vessel cup assembly formed for example of machinable aluminium alloy the above dimensions are sufficient for repeated insertion and withdrawal of a reaction vessel. Copper or silver may be used in place of aluminium but the latter is particularly suitable being inexpensive, readily machinable and receptive of anodisation . Anodisation reduces the possibility of shorting across the coil and inhibits corrosion. Where the vessel cup assembly thus comprises a conductive core this latter may constitute the return path for electrical power to the heater.

Preferably the vessel receiving portion and the heater portion comprise a right cylinder, and are formed from a rod, thus facilitating manufacture and the winding of the heating coil where such is used. Advantageously the heater portion has an external screw-thread-like groove for seating the wire. This both facilitates emplacement and retention of the coil and increases the surface areas of the heater portion and the wire which contact each other. Whilst preferably the thread profile is rounded to match the wire profile, a V thread profile has been found acceptable. Insofar as the wire has no insulative coating and the heater portion is insulated, for example by anodisation, then the thread profile will be such that adjacent coils of wire do not touch one another.

A method of forming the vessel cup assembly may accordingly comprise: taking a metal, preferably aluminium rod of appropriate dimensions;

drilling at one end a vessel receiving portion as described herein;

forming a screw thread on a designated heater portion thereof;

forming a temperature sensor recess toward the base of the vessel receiving portion;

anodising the rod;

placing a glue on the heater portion

winding a heater wire on the heater portion; and

curing the glue

The glue may be applied as a spot at the beginning and end of the wire. A heat insulative glue or jacket may be applied to the coil assembly and cured subsequently.

The manufacturing process may be assisted by retaining a spigot below the designated cooler portion of the assembly by which spigot the rod may be held for the drilling, turning, milling if required, gluing and wire winding and curing steps, the spigot then being removed. If the cooling portion is to project into the coolant then the spigot may constitute the projecting member or, if fins are to be formed on the projecting member the spigot may be below the projecting member for subsequent removal.

In the preferred embodiment of the assembly which has a stop flange between the heater and the cooler portions the manufacturing process includes the step of turning a rod of the outside diameter of the stop flange down at the cup receptor, heater, and cooler portions.

The heat transfer portion may accordingly comprise a pin which will protrude into a cooling liquid channel in the cooling assembly. The pin may be perforated or ribbed so as to maximise the heat transfer surface thereof. If the pin has a transverse hole for the passage of cooling fluid then the vessel cup assembly may incorporate an indicator, if not a keyway, wherewith to ensure alignment. The intrusion of the pin into the cooling fluid passage, in conjunction with the liquid flow rate is advisedly such that, where several such pins intrude in series array the liquid temperature is substantially the same at each.

The cooler portion is preferably formed to be an interference and sealing fit in the intended cooling assembly and may accordingly incorporate the above mentioned annular anchor stop for ensuring correct insertion thereof into a cooling assembly. The arrangement is accordingly preferably such that the vessel cup assembly may be pressed into a cooling assembly, perhaps employing a mandrel shaped to engage the interior base of the vessel receiving portion. The interference fit of course assists heat transfer by conduction. For this purpose it is particularly advantageous if the cooling portion and the cooling assembly are formed from the same material, preferably machinable aluminium. In this way expansion and contraction due to heat should not affect the fit, which preferably remains continuous around and beneath the cooling portion, in other words with the cooling portion not contacting the cooling fluid except insofar as a hole may be formed in the cooling assembly to allow air to bleed from between the cooling portion and the cooling assembly. However the air bleed hole may be blind and formed either upwards in the cooling portion or downwards in the cooling assembly.

According to a further feature of the invention the cooling assembly may comprise a matrix having therein a channel adapted for the flow of a coolant liquid. The matrix may be formed from subassemblies arranged to mate at a half channel plane. Usually the channel will be labyrinthine and serpentine.

For the context of a cooling assembly whose cooling liquid contacts the cooling portion the fitment of the vessel station it is even more important for the assembly of cooling portion to cooling assembly to be in a sealed manner. It is also advantageous that replaceable sealing devices such as O-rings and sealing gels are avoided thus minimising cost and complexity and so that their emplacement cannot be forgotten. Although the use of similar material is preferred for the cooling portion, and hence the vessel cup assembly, and the cooling assembly an alternative is to use a harder material for one than the other. The cooling assembly may be accordingly constructed from polypropylene, resin or other such flexible material and is preferably, in the case of a 96 well array, a single block. It should be noted that the pin assembly need not intrude into the liquid channel, the passage of the fluid through the block itself being able to remove the excess thermal energy at the required rate.

Fortunately it is usually the case that the lower temperature required in biological or chemical reactions involving thermocycling is higher than ambient. Often it is anyway necessary that the lower temperature is as precisely controlled as the upper temperature. Accordingly apparatus for effecting such reactions and incorporating a cooling assembly according to the invention may also have a heater for heating the coolant to the desired temperature. This has the added advantage of preventing condensation from forming on the exterior of the module.

Cooling of these assemblies may therefore be provided by a liquid coolant, which in the preferred embodiment is a non-charge carrying fluid such as Fluid XP. In order that cooling remain constant the fluid may be arranged into a circuit incorporating a radiator whereby excess heat can be vented to atmosphere. In order to minimise thermal losses this radiator may be controlled so that only the required amount of heat is removed and then this fluid is returned to a reservoir.

The temperature of the fluid entering the block is typically arranged, via a heat exchanger, to be of the order of 20 to 40°C preferably room temperature, ie around 25°C. The temperature can in fact also be any below the lowest in the cycle; however a higher temperature reduces the energy requirements generally. In the 96 vessel array embodiment of the invention there may be a manifold via which the heat exchanger feeds eight coolant tubes arranged to pass twelve vessel cup assemblies or twelve coolant tubes arranged to pass eight vessel cup assemblies.

It will be appreciated that in the apparatus above set forth thermal cycling is performed via means of increasing current to the heating coil wrapped intimately around the vessel receiving portion and that this is against the constant cooling of the liquid. When cooling is required the power is removed from the heater and the rod will very rapidly drop to the temperature of the coolant fluid. The temperature sensor inserted immediately below the reaction chamber is used to control how much current is required to be supplied to the coil. It is an important feature of the construction of a preferred embodiment of the invention that heat from the heater spreads both downwards and upwards at given and controlled rates with the result that a thermal blanket is formed above the cooling portion whilst heat is permeating uniformly upward into the vessel receiving portion and thence into the reaction vessel.

It will also be appreciated that an important advantage of the invention may lie in the removability and replaceability of the vessel cup assembly in relation to the cooling assembly. To that end an insertion tool may be provided for driving a vessel cup assembly home in the cooling assembly. A failed assembly may be removed with a suitable gripping device such as pliers or, more likely, by drilling through the failed vessel cup assembly.

The cooling assembly may incorporate a manifold or mount for contacts such as those for electrical supply to the heater and for the thermal sensor. The manifold or mount may conveniently comprise a printed circuit board (PCB).

The apparatus may be of a type employing a single station - for a single reaction vessel. Alternatively the apparatus may be of the type employing a multiplicity of stations. In particular the apparatus may be arranged to receive in stations a standard array of 96, or an integer multiple thereof, microtitre reaction vessels in a rectangular array, usually comprising 12 x 8 such stations. Further, the apparatus may be a thermocycling apparatus for performing, for example, polymerase chain reactions (PCR).

Typically such standard arrays have the vessels on rectangular centres 9mm apart. Clearly this imposes restraints upon the construction of devices in accordance with the present invention, and it is a feature thereof that such construction is entirely realizable.

The reaction vessel as such for the 96n array context is typically a microtitre vessel of by now somewhat standard construction. In this construction the vessel, having overall dimensions of the order of 2cm long and 0.7cm maximum diameter, has a reaction portion which tapers down from about 0.45cm to about 0.3cm and a funnel entry portion for accepting a transparent lid. It is usually the case that the vessel is sealed with a cap for the duration of a reaction and such a cap may be translucent or even transparent for at least a part thereof adjacent the sample whereby the progress of the reaction can be monitored by an optical system external to the reaction vessel.

The reaction vessel may be formed of a plastic loaded with carbon particles for thermal conductivity. By using such a vessel in a vessel cup assembly in accordance with the invention the target of completion of PCR minutes in less than 20 minutes is achievable.

An alternative reaction vessel may be formed simply of polypropylene, indeed moulded as a 12 X 8 well block. This is considerably cheaper of manufacture than the individual, carbon loaded well above described and a PCR process conducted with it will be somewhat longer than the 20 minutes. This of course implies a bank of 96 vessel cup assemblies. It will be appreciated that these assemblies will be rooted in a cooling assembly; also that this can be the case for individually formed reaction vessels as above described. These latter are usually mounted in a preformed tray.

In an alternative embodiment of the invention the vessel cup assembly cooling portion may be arranged for attachment to a TEC or Peltier cell. In order to effect rapid loss of heat into a TEC the cooling portion may be of frusto-conical shape, with a broad base which can be soldered to the face of the TEC. A low temperature indium based solder is preferred which will be adhered to a TEC face in a vapour phase oven to achieve an even temperature distribution across the base.

A particular advantage of this arrangement is that the TEC can be rendered inert during the heating part of the cycle so that very little heat goes other than into the cup. However aluminium, whether or not anodised, is not currently susceptible of soldering, so the cooling portion may comprise a pedestal of a metal such as copper attached to the aluminium, for example by swaging, thereafter to the TEC by soldering. An alternative is to have a mechanical frame to hold the vessels onto the TECs or to screw the copper to the aluminium.

Thus each of the vessels may have its own individual heat removal module, with masses and construction optimised to further reduce energy requirements of thermal cycling. These modules may be entirely independent of each other but arranged so that several may be ganged for an identical reaction process as required. Discrete elements can also constitute an array of up to either 4 or to 16 vessels, this being a two by two or four by four array. This arrangement has the further advantage that elements can be removed individually for routine maintenance purposes. A single vessel cycling unit may use an 8mm by 8mm TEC device, the 4 and 16 vessels using devices with dimensions of 17mm by 17mm and 35mm by 35mm respectively.

While use of a TEC in this way can greatly increase the cooling rates achievable by this system it may concurrently reduce the ability of the system to heat rapidly This problem can be overcome by the provision of a second heating circuit connected in series with the peltier elements allowing additional heating to be supplied to the system as and when required. The additional heating circuit may be printed onto the top of the TEC device or indeed sandwiched between 2 ceramic plates acting as the top surface of the TEC.

Rapid thermal cycling of these vessels can place significant stress upon the individual components in the system, the TECs in particular may be prone to failure. Materials possessing a high modulus of elasticity may be particularly useful to minimise the effects of thermally induced mechanical stress. The top and bottom surfaces of the TEC may accordingly be attached using Indium based solders or other such materials possessing high thermal conductivity and a high modulus of elasticity. Also shown to work is soldering one side of the device with an indium based solder while attaching the other with a thermally conductive silicone "glue". However the preferred construction method is an indium based solder to both surfaces of the TEC with the assembly constructed in a vapour phase oven using a gallium based liquid whose temperature can be controlled to reach the liquidus point of the solder. To facilitate this TEC devices should preferably be "metallised" by pre-tinning to provide a surface capable of keying the indium based solder to.

In order to protect the assembly further the entire assembly or any of its component parts can be covered in a conformal coating, preferably parylene in order to prevent failure due to atmospheric conditions, in particular condensation of water which could cause shorts in the TEC pillars and or corrosion/cracks during thermal changes.

In order to achieve thermal cycling speeds over 5°C/s it is necessary to incorporate TECs with sufficient heat pumping power. The optimal temperature for the heat removal module to be controlled to has been found to be 35-45°C, this gave the best balance of additional cooling power while minimising the impact on the heating capability.

As to the transfer of heat itself from the vessel cup assembly to the liquid reactants good component mating is important. The mould for the reaction vessels might therefore be highly polished as well as the interior surface of the reaction vessel receiving portion. A ramp rate in excess of 5°C/s can be achieved with a reaction vessel wall thickness below 0.3mm, bearing in mind construction techniques known in the art such as plastic moulding, and constructed of a material possessing thermal conductivity of minimally 1 W/MK. In the preferred embodiment the vessels are formed of highly loaded polypropylene containing 60% carbon w/v and with a wall thickness of 0.2mm. Additionally, in order to minimise thermal gradients vertically in a tubular vessel the entirety of the reactant fluid column is preferably encapsulated in the vessel holder portion. The cups themselves may be gold plated for optimum performance. This and the elasticity provided by Indium solder reduces failure rates of the component parts of the cycling system. A further level of control can be provided by the addition of an independently controlled heated compression ring at the top of the apparatus, this provides two functions. Firstly, it ensures that the vessel is compressed into the tapered fitting ensuring thermal contact and secondly it provides means to add additional heat, whether to ensure no condensation can form on the lid of the tube or to prevent thermal gradients forming when larger volumes such as over 100μΙ are used in the apparatus

An additional diode sensor may be built into the centre of each TEC to facilitate temperature measurement and control.

Preferably the apparatus employs electronic control circuits capable of individually addressing each of the vessels and controlling them at the desired ramping to achieve temperature accuracies of less than +/- 0.5, possibly even 0.2°C. The control gear can incorporate diodes in its circuitry arranged so as to provide feedback on the thermal performance of the TEC devices, for example to determine if a failure has occurred and assess whether minimum requirements for heating/cooling power are being met by every TEC device in the array.

In a further embodiment the TEC uses a larger base ceramic arranged for mounting 2, 4, 8, 16 or more individual die sets thereupon, each TEC die set having its own isolated upper ceramic plate separate from its adjacent TEC. The TECs thus form an easily locatable group that can be mounted more accurately than individual TECs. Each TEC keeps its own individual thermal profile.

Wires for connections can be either threaded through holes in the lower ceramic base plate or through vias formed using PCB through hole plating techniques. This enables compression and solder fixing to a sub assembly that provides both electrical and thermal connection. If a requirement for additional heating exists then these TEC devices may similarly be modified by constructing a TEC using an additional top ceramic tile incorporating a heating element. The two top tiles can then be bonded together in a sand wedge so that the heating element is protected between the ceramic sand wedge. The heating element can be etched or silk screen printed on to the surface of the ceramic. Moreover this larger TEC device can incorporate an additional top ceramic tile incorporating a temperature measurement element, the two top tiles forming a sand wedge that encapsulates a temperature measurement element such as a thermistor or diode junction. This can provide temperature feedback on the operation of the TEC itself including indicating failure.

Where a heat transfer module (HRM) is employed it is preferably individual to a unique reaction vessel or at most to a small group thereof, such as an array of four. In this way, particularly in the 96n vessel context, cooling of a particular vessel or group of vessels occurs in parallel with that of another vessel or group and is consequently somewhat more consistent and perhaps quicker than if a larger group were cooled in series by a single heat reduction module.

A suitable heat transfer module may comprise a jacket having cooling liquid (water) inlet and outlet, and a core fitting within the jacket, the core having a rugose, ridged or finned surface to a cooling liquid chamber therearound. The jacket may resemble a waffle, having a plurality of cooling liquid inlets, one per vessel or small group of vessels, and one or more liquid outlet manifolds. The core may resemble a bolt with a cylindrical shank and, for example, a square head, the shank sealably locatable and removable from the jacket and having the rugose, ridged or finned surface formed thereon and the head comprising a base for a single reaction vessel or group of perhaps four such vessels. The core may be made of a highly thermal conductive metal such as copper or aluminium and the fins or the like may be the more pronounced toward the end thereof distal from the head thereby to assist in drawing heat away from the head.

The core may have a bore therein, perhaps axial, for electrical conduits and may further carry at the shank distal end a printed circuit board (pcb) carrying various electrical command, control and communications elements.

Typically the core in the 96n microtitre vessel context has a head 2cm square and a total length not exceeding about 3cm.

A peitier cell (TEC) may be mounted upon the core head and arranged to be initiated cyclically to assist in the removal of heat from the reaction vessel during a cooling part of a cycle and, with polarity perhaps reversed, to resist the downward flow of heat in a heating part of the cycle. The peitier cell may, particularly in the 96n microtitre vessel context, comprise a group of four discrete cells or perhaps a single cell having a common base plate and one operative upper plate per vessel, ie. perhaps four operative upper plates. Typically in that context the peltier cell may measure 8mm square per reaction vessel. The peltier cell may further be formed with one or more holes in the base plate thereof to enable the electrical conduits thereto to pass directly downward rather than out at edges thereto. This can be particularly useful if the peltier cell is mounted upon a pcb in turn mounted upon a core head as the conduits can be soldered directly to the pcb.

The depth of the peltier cell may be relatively unimportant, though the deeper it is the more readily it resists delamination due to thermal expansion and contraction. Typically however the depth may be of the order of 2 - 3 mm, with the plates having a thickness of the order of 0.3 - 0.5 mm .

A heater plate may be interposed between the core head and the heater cup, or form part of the heater cup, above the peltier cell when the latter is employed. The heater plate may comprise a ceramic sheet. A heater element in the form of an electrical conduit may take a serpentine path between a like serpentine path of adhesive holding the heater plate in situ. The heater plate may be unique to one vessel or group of, for example, four vessels.

A temperature sensor may be disposed centrally below the heater plate. Whilst sensors are available which are very thin, eg less than 2mm, even 1 mm, a recess or hole in the heater plate may allow location of the sensor. The sensor may be connected to the pcb below the core shank. In an alternative construction the sensor is mounted in the heater cup or on the heater plate just below the vessel, where there is, as mentioned above, preferably a hot spot reduction void.

The pcb may incorporate an H bridge for controlling the direction of current to the peltier cell and means for controlling the heat cycle, including reference to the sensor.

Where the peltier cell is mounted on a pcb there may be a void in the pcb, or between pcb's allowing the more rapid transfer of heat to the core. Alternatively the pcb may incorporate voids containing thermally conductive material. The attachment of the various elements one to another may be effected using indium based solders, this constituting a highly thermally conductive but mechanically flexible medium.

In another embodiment of the invention the vessel receiving portion may comprise a tray, perhaps substantially flat, and perhaps rectangular, with low side walls. It may thus be adapted to receive a slide or a capsule carrying the reactants.

In yet another embodiment of the invention the vessel receiving portion may define a slot adapted to receive a reaction chamber formed on part of a slide, the slide being perhaps of credit card dimensions and, perhaps, structure. Such a slide may incorporate enclosed channels wherein reactants are propelled from one station to another to undergo successive stages in a biological, chemical or bio-chemical process.

A particular advantage of the vessel cup assembly according to the present invention is that it, or an array thereof, can readily be incorporated into apparatus for carrying out a chemical, biological or biochemical process. In a preferred construction the apparatus comprises a platform upon which the vessel cup array may be installed, the platform then raised to a process station and lowered therefrom upon completion of the process. The platform may incorporate the cooling station or part of the cooling system where the vessel cup assembly also comprises part of a cooling system, in particular an attached pettier cell or TEC.

Brief Description of the Drawings

An embodiment of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: -

Figure 1 is a schematic side elevation of first embodiment of a vessel cup assembly in accordance with the invention;

Figure 2 is a section on ll-ll in figure 1 but showing a reaction vessel in place. Figure 3 is a schematic side view of a second embodiment of a vessel cup assembly in accordance with this invention;

Figure 4 is a section on IV-IV in figure 3;

Figure 5 is a side elevation of a further vessel cup assembly embodiment;

Figure 6 is a section on VI-VI in figure 5;

Figure 7 is a side view of a vessel cup assembly adapted for reception of a reaction vessel in the form of a flat faced capsule or slide;

Figure 8 is a section on VIII-VIII in figure 7;

Figure 9a is an exploded isometric view of a peltier cell;

Figure 9b is an exploded side view of the cell of figure 9a;

Figure 9c is a section on JX-IX in figure 9b; and

Figure 9d is a plan view of the cell shown in figure 9b.

Description of the Preferred Embodiments

Illustrated in figures 1 and 2 is a vessel cup assembly having, in order from top to bottom a vessel receiving portion 100, a heater portion 101 and a cooling portion 102. There is an annular anchor stop 103 between the cooling portion 102 and the heater portion 101 .

The vessel receiving portion 00 is in the form of a cup for receiving snugly a reaction vessel. The wall of the cup wall tapers down towards the open upper end with a taper angle of about 4°. The cup entry wall thickness is about 0.4 mm and the cup base wall thickness is about 0.95mm. At the base of the vessel receiving portion 100 an allowance is made for a gap 100a between the base and the bottom of the reaction vessel. The gap 100a will be of the order of 2 to 4 mm deep. At a convenient intermediate station along the length of the cup 100 is a temperature sensor receiving recess 104. In this recess is located a thermistor 105. Measurement Specialties Ltd have supplied a suitable bare thermistor having a length under 3mm and a mean diameter of the order of 0.2mm. To ensure safe anchorage of this thermistor 105 into its recess 104 the thermistor tail wire is wound around the vessel receiving portion 1 00 a couple of times. The thermistor 105 is located a short distance above the heater so as not to read heater temperature but the temperature of the cup. The thermistor is arranged to operate in a predictive mode.

The heater portion 101 has an external spiral screw-thread-like groove 101 a of V profile for seating a heater wire coil 106 (shown in figure 5). The wire is nickel chrome 0.21 mm diameter arranged to be driven with a voltage of 24 volts and capable of drawing a maximum current of 2.2 amps. The length of the wire in this micro-titre vessel context is 36 cm. An ultra-violet curable glue is employed in retaining the wire 1 06 in place. The groove 1 01 a is formed so that when the rod is anodised adjacent wire coils do not contact one another.

The cooling portion 1 02 is constructed as an anchor member to anchor the assembly into a reaction vessel receiving station in a cooler assembly in or for a chemical and/or biological reaction apparatus. The cooling portion 1 02 is formed in relation to the cooler assembly so as to be contiguous therewith both at periphery and at base . A 1 .0mm blind hole at the base of the cooling assembly cooling portion receiving well serves to contain air compressed during fitment of one to the other. The annular anchor stop 1 03 controls the depth of the cooling portion to visibly assure that the cooling portion is home.

Figure 2 demonstrates the fitting of a reaction vessel 200 in the cup 100. The vessel is a microtitre reaction vessel comprising a reaction chamber 201 and a funnel portion 202. The reaction chamber portion is a snug fit in the vessel receiving portion 100, leaving the gap 100a between them at the bottom of the reaction vessel. The funnel portion 202 receives a lid of a transparent material.

In some versions of the embodiment illustrated in Figures 1 and 2 a further probe or pip, perhaps finned, may extend from the base of the cooling portion 102 and project into coolant, the cooling assembly being arranged, of course, for such projection to occur. However such a probe or pip may serve as a spigot whilst winding the wire coil, after which it may be removed. The vessel cup assembly shown in figures 1 and 2 is formed by turning from a rod of aluminium alloy which is anodized, after machining, for electrical insulation purposes. It is accordingly of integral construction.

The embodiment of the invention illustrated in Figures 3 and 4 has a vessel receiving portion 300 and a heater portion 301 similar to those of the embodiment described with respect to figures 1 and 2. However the cooling portion 302 is of frustoconical form with the base broader than the apex so as to maximize the surface of contact with cooling apparatus, in this case a peltier cell 303, to the upper plate 304 of which it is attached. At least the base of the cooling portion (the shoe 305) is formed from copper the more readily to attach it with solder. The copper shoe 305 is swaged to the cooling portion 302. The vessel cup assembly illustrated in figures 3 and 4 is otherwise formed from machinable aluminium rod.

Attached to the lower plate 306 of the peltier cell 303 is a secondary cooling block 307. As with the cooling portion 102 illustrated in figures 1 and 2 the cooling block 307 is constructed to fit tightly into a heat reduction module which, in this instance, will stabilise the temperature of the lower plate 306.

The vessel cup assembly illustrated in figures 5 and 6 is similar to that illustrated in figures 3 and 4 except that the cooling block 307 has formed thereon a probe 308. The probe 308 is formed with fins 309 thereon adapted for projection into a coolant liquid duct in a heat reduction module (HRM).

Indium based solder is used for the attachment of the shoe 305 to the peltier cell upper plate 304 and of the lower plate 306 to the secondary cooling block 307.

The vessel cup assembly illustrated in figures 7 and 8 is similar to those illustrated in figures 3 and 4 except that the vessel receiving portion 700 is frustoconical in form. The portion 700 has a shallow tray 701 formed therein and adapted to receive a reaction vessel in the form of a flat capsule or slide.

The peltier cell (TEC) shown in figures 9a - 9d, can be the cell 303 shown in figures 3 to 6. It comprises the basic peltier cell 900 with a ceramic heater plate 901 and a serpentine heater element 902 sandwiched between the plate 901 and the cell 900 itself. For use with microtitre reaction vessels the vessel cup assembly illustrated in figures 1 to 4 has an overall length of 26 mm, a cup external diameter (vessel receiving portion 100, 300) of 5.0 mm and a cooling portion 102 of 6.0 mm diameter. If a pip remains with the cooling portion 102 its dimensions are typically 4.0 mm long and 3.0 mm diameter.

In the case particularly of the embodiment illustrated in figures 1 and 2, the vessel cup assembly is but one of an array of 8 x 12 = 96 thereof emplaced in an individual receiving station in a heat reduction module (HRM). The receiving stations are in square array on 1 .0 cm centres. The heat reduction module is a machinable aluminium block incorporating a labyrinth of channels for coolant liquid. It is formed as separate blocks mating at half channel depth. The heat reduction module is mounted in a reaction apparatus which houses also a reservoir of coolant liquid and means for controlling the temperature thereof. Entry and exit manifolds of the heat reduction module are connected to this reservoir.

The HRM forms part of a platform arranged in reaction apparatus to be available for the reception of an array of reaction vessels containing reactants and to which their lids have been fitted, the platform also having an associated sprung lift device. The apparatus is thus constructed to raise the platform and press the reaction vessels via the lids thereof against a pressure plate to maintain the reaction vessels in even snug contact with the vessel cup assemblies during the reaction process. The pressure plate is perforated adjacent the centre of the lids to assist visibility of the reactants to apparatus optical equipment.

In a reaction apparatus for carrying out PCR simultaneously on samples in each of the reaction vessels 200, there is a holder within which the vessels are held in their array and charged with reactants. The vessel lids are then emplaced. and the holder is offered to the heat reduction module (HRM).

The HRM is mounted on an elevator toward the top of which is a heater plate, foraminous coincidental with the reaction vessel lids. Thus, when the elevator hoists the loaded HRM to a reaction station the compression plate ensures that the reaction vessels are in snug contact with the cup receiving portions of their corresponding vessel cup assembly. Optical interrogation apparatus is sited above the compression plate. The reaction can then commence. In the case of carrying out PCR, the reactants in the vessels are brought to the upper temperature for denaturing the DNA in the reactants, held briefly at that temperature, cooled to the intermediate, annealing temperature and held briefly there, then cooled to the lower temperature where denatured DNA extends and held briefly there, this cycle being repeated several times until a change of colour is detected in the reactants.

Claims

1 . A vessel cup assembly for receiving a chemical and/or biological reaction process vessel containing reactants and processing the reaction therein and comprising:

a reaction vessel receiving portion;

a heater portion; and

a cooling portion; and

wherein the assembly is of integral construction.

2. A vessel cup assembly as claimed in claim 1 and wherein the vessel receiving portion comprises a cup adapted to receive snugly a reaction vessel.

3. A vessel cup assembly as claimed in claim 2 and wherein the cup has a constant wall thickness.

4. A vessel cup assembly as claimed in claim 2 and wherein the cup wall thickness tapers down towards the open end thereof.

5. A vessel cup assembly as claimed in claim 4 and wherein the cup wall thickness has a taper angle of between 2° and 5°.

6. A vessel cup assembly as claimed in claim 4 or claim 5 and wherein the taper angle is of the order of 4°.

7. A vessel cup assembly as claimed in any one of claims 4 to 6 and wherein the cup wall thickness is of the order of 0.35 to 0.45 mm. at the rim thereof and of the order of 0.9 to 1 . 1 mm at the base thereof.

8. A vessel cup assembly as claimed in any one of claims 2 to 8 and wherein there is arranged to be an air gap between the bottom of the cup and the base of the reaction vessel.

9. A vessel cup assembly as claimed in claim 8 and wherein the gap is 2 to 4 mm deep.

10. A vessel cup assembly as claimed in claim 1 and wherein the vessel receiving portion comprises a tray with low side wails.

1 1 . A vessel cup assembly as claimed in claim 10 and wherein the tray is adapted to receive a slide or a capsule carrying reactants.

12. A vessel cup assembly as claimed in claim 1 and wherein the vessel receiving portion defines a slot adapted to receive a reaction chamber formed on part of a slide.

13. A vessel cup assembly as claimed in claim 12 and wherein the slot is adapted to receive a reaction chamber contained in a slide of credit card dimensions.

14. A vessel cup assembly as claimed in any one of the preceding claims and wherein the heater portion has a heater in the form of a wire coil wound upon the heater portion.

15. A vessel cup assembly as claimed in claim 14 and wherein the coil is encased in a thermally insulative material.

16. A vessel cup assembly as claimed in claim 15 and wherein the coil is encased in a paint, such as an enamel paint, which can be 'cooked' to stabilise the coil and insulate it exteriorly.

17. A vessel cup assembly as claimed in claim 14 or claim 15 and wherein a glue, such as an ultra-violet curable glue, is employed to retain the wire in place.

18. A vessel cup assembly as claimed in any one of claims 14 to 17 and wherein the wire is nickel chrome 0.21 mm diameter driven with a voltage of 24 volts and capable of drawing a maximum current of 2.2 amps.

19. A vessel cup assembly as claimed in any one of claims 14 to 18 and wherein the length of the wire is of the order of 36 cm.

20. A vessel cup assembly as claimed in any one of claims 14 to 19 and wherein the heater portion has an external screw-thread-like groove for seating the wire.

21 . A vessel cup assembly as claimed in any one of the preceding claims and wherein the cooling portion is formed to be an interference and sealing fit in a reaction apparatus cooling assembly.

22. A vessel cup assembly as claimed in claim 21 and wherein the cooling portion incorporates a rib or a groove transverse thereto to assist in maintaining anchor and seal.

23. A vessel cup assembly as claimed in any one of the preceding claims and wherein the cooling portion comprises a pin arranged for protruding into a cooling liquid duct in a reaction apparatus cooling assembly.

24. A vessel cup assembly as claimed in claim 23 and wherein the pin is perforated or ribbed so as to maximise the heat transfer surface thereof.

25. A vessel cup assembly as claimed in any one of claims 1 to 20 and wherein the cooling portion is arranged for attachment to a TEC or Peltier cell.

26. A vessel cup assembly as claimed in claim 25 and wherein the cooling portion is of frusto-conical shape, with a broad base which can be soldered to the face of the TEC.

27. A vessel cup assembly as claimed in claim 26 and wherein the solder is a low temperature indium based solder.

28. A vessel cup assembly as claimed in claim 27 and wherein the cooling portion is adhered to a TEC face in a vapour oven.

29. A vessel cup assembly as claimed in any one of claims 25 to 28 and wherein the cooling portion comprise a pedestal of copper.

30. A vessel cup assembly as claimed in any one of the preceding claims and comprising a metal rod.

31 . A vessel cup assembly as claimed in claim 30 and wherein the rod is formed of aluminium.

32. A vessel cup assembly as claimed in claim 31 and wherein the rod is anodised.

33. A vessel cup assembly as claimed in any one of the preceding claims and having a niche for the reception of a thermal sensor.

34. A vessel cup assembly as claimed in any one of the preceding claims and incorporating a thermal sensor.

35. A vessel cup assembly as claimed in any one of the preceding claims and arranged to receive a microtitre reaction vessel having overall dimensions of the order of 2cm long and 0.7cm maximum diameter with a reaction portion which tapers down from about 0.45cm to about 0.3cm and a funnel entry portion for accepting a transparent lid.

36. A vessel cup assembly as claimed in claim 35 and wherein the reaction vessel has a wall thickness wall thickness below 0.3mm .

37. A vessel cup assembly as claimed in claim 35 or claim 36 and wherein the microtitre reaction vessel is formed of a material possessing thermal conductivity of minimally 1 W/MK.

38. A vessel cup assembly as claimed in any one of claims 35 to 37 and arranged to receive a reaction vessel formed of a plastic loaded with electrically conductive particles such as carbon.

39. A vessel cup assembly as claimed in any one of claims 35 to 38 and wherein the vessels are formed of highly loaded polypropylene containing 60% carbon w/v.

40. A vessel cup assembly as claimed in any one of the preceding claims and having overall dimensions of the order of 2 to 4 cm long by 0.4 to 0.7 mm diameter.

41 . An array of vessel cup assemblies, each assembly being as claimed in any one of claims 1 to 35 and arranged to receive a reaction vessel array in the form a 12 X 8 well block.

42 An array as claimed in claim 41 and wherein the block is formed from polypropylene.

43. Biological, chemical or bio-chemical reaction apparatus adapted to receive a vessel cup assembly as claimed in any one of the preceding claims

44. Apparatus as claimed in claim 43 and having a cooling assembly adapted to receive a vessel cup assembly as claimed in any one of claims 1 to 40

45. Apparatus as claimed in claim 43 or claim 44 and wherein the cooling assembly comprises a block having therein a channel adapted for the flow of a coolant liquid.

46. Apparatus as claimed in claim 44 and wherein the cooling assembly has a heater for heating the coolant to the desired temperature.

47. Apparatus as claimed in any one of claims 44 to 46 and wherein the cooling assembly incorporates a radiator whereby excess heat can be vented to atmosphere.

48. Apparatus as claimed in any one of claims 44 to 47 and wherein the cooling assembly incorporates a manifold or mount for electrical contacts.

49. Apparatus as claimed in claim 48 and wherein the manifold or mount comprises a printed circuit board (PC ).

50. Apparatus as claimed in any one of claims 42 to 49 and having a single station for a vessel cup assembly.

51 . Apparatus as claimed in any one of claims 42 to 50 and arranged to receive in stations a standard array of 96, or an integer multiple thereof, microtitre reaction vessels in a rectangular array.

52. Apparatus as claimed in claim 51 and wherein the array comprises 12 x 8 stations.

53. Apparatus as claimed in any one of claims 42 to 52 and wherein the cooling assembly comprises at least one peltier cell.

54. Apparatus as claimed in claim 53 and wherein a diode sensor is built into the centre of each TEC.

55. Apparatus as claimed in claim 53 or claim 54 and wherein the TEC is arranged for mounting 2, 4, 8, 16 or more individual die sets thereupon, each TEC die set having its own isolated upper ceramic plate separate from its adjacent TEC.

56. Apparatus as claimed in any one of claims 53 to 55 and where the TEC has an additional top ceramic tile incorporating a heating element.

57. Apparatus as claimed in claim 56 and wherein the heating element is etched or silk screen printed on to the surface of the ceramic.

58. Apparatus as claimed in claim 56 or claim 57 and wherein the TEC device has an additional top ceramic tile incorporating a temperature measurement element.

59. Apparatus as claimed in any one of claims 42 to 58 and wherein the cooling assembly is individual to a unique vessel cup assembly or to a small group thereof, such as an array of four.

60. Apparatus as claimed in any one of claims 42 to 59 and employing electronic contro) circuits capable of individually addressing each of the vessels and controlling them at the desired ramping.

61. Apparatus as claimed in any one of claims 42 to 60 and wherein the cooling assembly comprises a platform in which the vessel cup assembly or assemblies are fitted and into which the reaction vessel array charged with reactants and lid or lids fitted are placed, the platform then being arranged to be offered up to a work station comprising a plate against which the reaction vessel lids are pressed, the plate being perforated for visual access to the reactants by apparatus optical equipment.

62. Apparatus as claimed in any one of claims 42 to 61 and arranged for conducting a thermocycling process or an isothermal reaction or temperature modulated biochemical, or chemical reaction

63. Apparatus as claimed in claim 62 and adapted for carrying out a polymerase chain reaction (PCR).

64. Apparatus as claimed in any one of claims 42 to 63 and comprising a platform upon which the vessel cup array may be installed, the platform being arranged for being raised to a process station and lowered therefrom upon completion of the process.

65. Apparatus as claimed in claim 64 and wherein the platform incorporates the cooling station or part of the cooling system where the vessel cup assembly also comprises part of a cooling system, in particular an attached pettier cell or TEC.

66. Apparatus as claimed in any one of claims 42 to 65 and incorporating an optical reading facility arranged for monitoring the reaction in the ??or each reaction vessel.

67. A method of forming a vessel cup assembly as claimed in any one of claims 1 to 40 and comprising:

taking a metal, preferably aluminium rod of appropriate dimensions;

drilling at one end a vessel receiving portion as described herein;

forming a screw thread on a designated heater portion thereof;

anodising the rod;

placing a glue on the heater portion;

winding a heater wire on the heater portion; and

curing the glue.

68. A method as claimed in claim 67 and comprising retaining a spigot below the designated cooler portion of the assembly by which spigot the rod may be held for the drilling , turning , milling if required, gluing and wire winding and curing steps, the spigot then being removed.

69. A method of assembling a vessel cup assembly as claimed in any one of claims 1 to 40 to a cooling assembly, comprising pressing a cooling portion into the cooling assembly.

70. A method as claimed in claim 69 and employing a mandrel shaped to engage the interior base of the vessel receiving portion.

71. A method of performing a thermal biological, chemical or biochemical reaction and employing a vessel cup assembly as claimed in any one of claims 1 to 41.

72. A method as claimed in claim 71 and employing apparatus as claimed in any one of claims 42 to 61 .

73. A method as claimed in claim 71 or claim 72 and wherein the reaction is a polymerase chain reaction (PCR).

74. A vessel cup assembly as claimed in any one of claims 1 to 41 and substantially as hereinbefore described with reference to the accompanying drawings.

75. Apparatus as claimed in any one of claims 42 to 61 and substantially as hereinbefore illustrated in the accompanying drawings.