U.S. patent application number 13/261654 was filed with the patent office on 2015-05-28 for apparatus including a vessel cup assembly for heating and cooling low volume biological reaction vessels and methods associated therewith.
The applicant listed for this patent is Antony Drtscoll, David Edge, Nelson Nazareth. Invention is credited to Antony Drtscoll, David Edge, Nelson Nazareth.
Application Number | 20150144299 13/261654 |
Document ID | / |
Family ID | 45349226 |
Filed Date | 2015-05-28 |
United States Patent
Application |
20150144299 |
Kind Code |
A1 |
Nazareth; Nelson ; et
al. |
May 28, 2015 |
APPARATUS INCLUDING A VESSEL CUP ASSEMBLY FOR HEATING AND COOLING
LOW VOLUME BIOLOGICAL REACTION VESSELS AND METHODS ASSOCIATED
THEREWITH
Abstract
A vessel cup assembly 98 for receiving a chemical and/or
biological reaction process vessel 200 containing reactants and
processing the reaction therein includes a reaction vessel
receiving portion 100, a heater portion 101, and a cooling portion
102, wherein the assembly is of integral construction.
Inventors: |
Nazareth; Nelson; (Upper
Dean, GB) ; Edge; David; (Warlingham, GB) ;
Drtscoll; Antony; (Langford, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nazareth; Nelson
Edge; David
Drtscoll; Antony |
Upper Dean
Warlingham
Langford |
|
GB
GB
GB |
|
|
Family ID: |
45349226 |
Appl. No.: |
13/261654 |
Filed: |
November 8, 2011 |
PCT Filed: |
November 8, 2011 |
PCT NO: |
PCT/GB2011/001565 |
371 Date: |
February 3, 2015 |
Current U.S.
Class: |
165/61 ;
62/3.6 |
Current CPC
Class: |
B01L 2300/1894 20130101;
B65D 81/38 20130101; F25B 21/02 20130101; B01L 2300/0627 20130101;
B01L 2300/1822 20130101; B01L 2300/0832 20130101; B01L 9/06
20130101; B01L 2300/1827 20130101; B01L 7/52 20130101 |
Class at
Publication: |
165/61 ;
62/3.6 |
International
Class: |
F25B 21/02 20060101
F25B021/02; B65D 81/38 20060101 B65D081/38 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 8, 2010 |
GB |
1018794.6 |
Jan 14, 2011 |
GB |
1100625.1 |
Claims
1-75. (canceled)
76. 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.
77. 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.
78. A vessel cup assembly as claimed in claim 2 and wherein the cup
wall thickness tapers down towards the open end thereof.
79. A vessel cup assembly as claimed in claim 3 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.
80. A vessel cup assembly as claimed in claim 2 and wherein there
is arranged to be an air gap between the bottom of the cup and the
base of the reaction vessel.
81. A vessel cup assembly as claimed in claim 1 and wherein the
heater portion has a heater in the form of a wire coil wound upon
the heater portion.
82. A vessel cup assembly as claimed in claim 6 and wherein the
coil is encased in a thermally insulative material.
83. A vessel cup assembly as claimed in claim 6 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.
84. A vessel cup assembly as claimed in claim 6 and wherein a glue,
such as an ultra-violet curable glue, is employed to retain the
wire in place.
85. A vessel cup assembly as claimed in claim 6 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.
86. A vessel cup assembly as claimed in 6 and wherein the heater
portion has an external screw-thread-like groove for seating the
wire.
87. A vessel cup assembly as claimed in claim 1 and wherein the
cooling portion comprises a pin arranged for protruding into a
cooling liquid duct in a reaction apparatus cooling assembly.
88. A vessel cup assembly as claimed in claim 1 and wherein the
cooling portion is arranged for attachment to a TEC or Peltier
cell.
89. A vessel cup assembly as claimed in claim 13 and wherein the
cooling portion is of frusto-conical shape, with a broad base
arranged for being soldered to the face of the TEC.
90. A vessel cup assembly as claimed in claim 1 and comprising a
metal rod.
91. A vessel cup assembly as claimed in claim 1 and incorporating a
thermal sensor.
92. A vessel cup assembly as claimed in claim 1 and arranged to
receive a microtitre reaction vessel having overall dimensions of
the order of 2 cm long and 0.7 cm maximum diameter with a reaction
portion which tapers down from about 0.45 cm to about 0.3 cm and a
funnel entry portion for accepting a transparent lid.
93. A vessel cup assembly as claimed in claim 1 and having overall
dimensions of the order of 2 to 4 cm long by 0.4 to 0.7 mm
diameter.
94. A vessel cup assembly for receiving a chemical and/or
biological reaction process vessel containing reactants and
processing the reaction therein and comprising a metal rod
incorporating: a reaction vessel receiving portion comprising a cup
adapted to receive snugly a microtitre reaction vessel and wherein
there is arranged to be an air gap between the bottom of the cup
and the base of the reaction vessel; a heater portion having an
external screw-thread-like groove seating a wire coil encased in an
insulative material arranged to hold the coil in the groove; a
cooling portion; and a thermal sensor; and wherein the assembly is
of integral construction.
95. Apparatus comprising a vessel cup assembly as claimed in claim
1 and incorporating a cooling assembly.
96. Apparatus as claimed in claim 20 and wherein the cooling
assembly comprises a block having therein a channel adapted for the
flow of a coolant liquid and a heater for heating the coolant to
the desired temperature.
97. Apparatus as claimed in claim 20 and wherein the cooling
assembly incorporates a manifold or mount for electrical
contacts.
98. Apparatus as claimed in claim 20 and arranged to receive in
stations a standard array of 96, or an integer multiple thereof,
microtitre reaction vessels in a rectangular array.
99. Apparatus as claimed in claim 20 and wherein the cooling
assembly comprises at least one peltier cell (TEC).
100. Apparatus as claimed in claim 20 and employing electronic
control circuits capable of individually addressing each of the
vessels and controlling them at the desired ramping.
101. Apparatus as claimed in claim 20 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.
102. Apparatus as claimed in claim 20 and incorporating an optical
reading facility arranged for monitoring the reaction in the or
each reaction vessel.
103. A method of forming a vessel cup assembly as claimed in claim
1 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; 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; curing the glue; and emplacing a temperature sensor
in the recess therefor.
104. A method of performing a thermal biological, chemical or
biochemical reaction and employing a vessel cup assembly as claimed
in claim 1.
105. A method as claimed in claim 29 and employing apparatus as
claimed in claim 20.
106. A method as claimed in claim 29 and wherein the reaction is a
polymerase chain reaction.
Description
FIELD OF THE INVENTION
[0001] 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
[0002] The PCR process is described in detail in U.S. Pat. Nos.
4,683,195 and 4,683,202.
[0003] 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.times.12
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.
[0004] 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 vessel 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.
[0005] 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 part of, the material of the reaction vessel. Cooling is
effected using forced cooled or ambient air.
[0006] 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.
[0007] 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 2.5 C per second (peak
heating rate, when measured at the level of the vessel itself,
actual in fluid transitions can be markedly longer)
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] Molecular diagnostic tests such as the PCR process and
latterly real-time PCR (U.S. Pat. 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 (U.S. Pat. No. 5,455,175) thermal
cyclers. These have the disadvantage of being unsuited to
automation and screening of large numbers of samples
simultaneously.
[0013] 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.
[0014] 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.degree. C., one second at
55.degree. C. and five seconds at 72.degree. 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 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.
[0015] There are myriad interdependent technical problems to be
overcome in reducing process time.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] Reduction in the energy requirements of the thermal cycling
process is also a pre-requisite for thermal cycling at rates
exceeding 5.degree. C./s in the fluid.
[0020] A thermal storage system as demonstrated for example in U.S.
Ser. No. 12/381,953 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.
[0021] 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
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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 36 cm.
Other reaction vessels will likely have different requirements.
[0028] 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.
[0029] 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.degree. and 5.degree.,
preferably of the order of 4.degree., 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 0.95 mm, 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 3 mm
and a mean diameter of the order of 0.2 mm. 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] It will be appreciated that in the microtitre reaction
vessel context the overall 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] The temperature of the fluid entering the block is typically
arranged, via a heat exchanger, to be of the order of 20 to
40.degree. C. preferably room temperature, ie around 25.degree. 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.
[0048] 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.
[0049] 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.
[0050] 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).
[0051] 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.times.8 such stations. Further, the apparatus may be a
thermocycling apparatus for performing, for example, polymerase
chain reactions (PCR).
[0052] Typically such standard arrays have the vessels on
rectangular centres 9 mm 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.
[0053] 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 2 cm long and 0.7 cm maximum diameter,
has a reaction portion which tapers down from about 0.45 cm to
about 0.3 cm 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.
[0054] 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.
[0055] An alternative reaction vessel may be formed simply of
polypropylene, indeed moulded as a 12.times.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 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.
[0056] 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.
[0057] 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.
[0058] 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 8 mm by 8 mm TEC device, the 4 and 16
vessels using devices with dimensions of 17 mm by 17 mm and 35 mm
by 35 mm respectively.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] In order to achieve thermal cycling speeds over 5.degree.
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.degree. C., this
gave the best balance of additional cooling power while minimising
the impact on the heating capability.
[0063] 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.degree. C./s
can be achieved with a reaction vessel wall thickness below 0.3 mm,
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 0.2 mm.
[0064] 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
1000 are used in the apparatus
[0065] An additional diode sensor may be built into the centre of
each TEC to facilitate temperature measurement and control.
[0066] 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.degree. 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.
[0067] 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
TEC's. Each TEC keeps its own individual thermal profile.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] Typically the core in the 96n microtitre vessel context has
a head 2 cm square and a total length not exceeding about 3 cm.
[0073] A peltier 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 peltier 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 8 mm 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.
[0074] 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.
[0075] 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.
[0076] A temperature sensor may be disposed centrally below the
heater plate. Whilst sensors are available which are very thin, eg
less than 2 mm, 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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 peltier cell or TEC.
BRIEF DESCRIPTION OF THE DRAWINGS
[0083] An embodiment of the invention will now be described, by way
of example only, with reference to the accompanying drawings, in
which:
[0084] FIG. 1 is a schematic side elevation of first embodiment of
a vessel cup assembly in accordance with the invention;
[0085] FIG. 2 is a section on II-II in FIG. 1 but showing a
reaction vessel in place.
[0086] FIG. 3 is a schematic side view of a second embodiment of a
vessel cup assembly in accordance with this invention;
[0087] FIG. 4 is a section on IV-IV in FIG. 3;
[0088] FIG. 5 is a side elevation of a further vessel cup assembly
embodiment;
[0089] FIG. 6 is a section on VI-VI in FIG. 5;
[0090] FIG. 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;
[0091] FIG. 8 is a section on VIII-VIII in FIG. 7;
[0092] FIG. 9a is an exploded isometric view of a peltier cell;
[0093] FIG. 9b is an exploded side view of the cell of FIG. 9a;
[0094] FIG. 9c is a section on IX-IX in FIG. 9b; and
[0095] FIG. 9d is a plan view of the cell shown in FIG. 9b.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0096] Illustrated in FIGS. 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.
[0097] The vessel receiving portion 100 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.degree.. The cup entry wall thickness is about 0.4 mm and the cup
base wall thickness is about 0.95 mm. 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.
[0098] 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 3 mm
and a mean diameter of the order of 0.2 mm. To ensure safe
anchorage of this thermistor 105 into its recess 104 the thermistor
tail wire is wound around the vessel receiving portion 100 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.
[0099] The heater portion 101 has an external spiral
screw-thread-like groove 101a of V profile for seating a heater
wire coil 106 (shown in FIG. 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 106 in place. The
groove 101a is formed so that when the rod is anodised adjacent
wire coils do not contact one another.
[0100] The cooling portion 102 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 102 is formed in relation to the
cooler assembly so as to be contiguous therewith both at periphery
and at base. A 1.0 mm 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 103 controls the depth of the cooling portion to visibly
assure that the cooling portion is home.
[0101] FIG. 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.
[0102] In some versions of the embodiment illustrated in FIGS. 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.
[0103] The vessel cup assembly shown in FIGS. 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.
[0104] The embodiment of the invention illustrated in FIGS. 3 and 4
has a vessel receiving portion 300 and a heater portion 301 similar
to those of the embodiment described with respect to FIGS. 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
FIGS. 3 and 4 is otherwise formed from machinable aluminium
rod.
[0105] 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 FIGS. 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.
[0106] The vessel cup assembly illustrated in FIGS. 5 and 6 is
similar to that illustrated in FIGS. 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).
[0107] 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.
[0108] The vessel cup assembly illustrated in FIGS. 7 and 8 is
similar to those illustrated in FIGS. 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.
[0109] The peltier cell (TEC) shown in FIGS. 9a-9d, can be the cell
303 shown in FIGS. 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.
[0110] For use with microtitre reaction vessels the vessel cup
assembly illustrated in FIGS. 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.
[0111] In the case particularly of the embodiment illustrated in
FIGS. 1 and 2, the vessel cup assembly is but one of an array of
8.times.12=96 thereof emplaced in an individual receiving station
in a heat reduction module
[0112] (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.
[0113] 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.
[0114] 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).
[0115] 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.
[0116] 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.
* * * * *