U.S. patent application number 12/671783 was filed with the patent office on 2011-03-17 for reaction vessel comprising electrically conducting polymer as a heating element.
This patent application is currently assigned to ENIGMA DIAGNOSTICS LIMITED. Invention is credited to Richard George Gregory, Graham Gutsell, Ross Peter Jones, Martin Alan Lee, David James Squirrell, Roger James Williamson.
Application Number | 20110065150 12/671783 |
Document ID | / |
Family ID | 38529268 |
Filed Date | 2011-03-17 |
United States Patent
Application |
20110065150 |
Kind Code |
A1 |
Jones; Ross Peter ; et
al. |
March 17, 2011 |
REACTION VESSEL COMPRISING ELECTRICALLY CONDUCTING POLYMER AS A
HEATING ELEMENT
Abstract
A reaction vessel for conducting a chemical or biochemical
reaction, such as a polymerase chain reaction wherein electrically
conducting polymer is arranged to act as a heating element. The
profile of the electrically conductive polymer differs in different
regions of the vessel so as to control thermal gradients. The
profile of the electrically conductive polymer may be arranged to
either increase or reduce the thermal gradient. Reaction systems
comprising combinations of vessels of the invention and apparatus
for heating them, as well as particular reactions vessels are also
described and claimed.
Inventors: |
Jones; Ross Peter;
(Cambridge, GB) ; Williamson; Roger James;
(Hertfordshire, GB) ; Gregory; Richard George;
(Hertfordshire, GB) ; Gutsell; Graham; (Wiltshire,
GB) ; Lee; Martin Alan; (Wiltshire, GB) ;
Squirrell; David James; (Wiltshire, GB) |
Assignee: |
ENIGMA DIAGNOSTICS LIMITED
Wiltshire
GB
|
Family ID: |
38529268 |
Appl. No.: |
12/671783 |
Filed: |
August 1, 2008 |
PCT Filed: |
August 1, 2008 |
PCT NO: |
PCT/GB08/02627 |
371 Date: |
November 30, 2010 |
Current U.S.
Class: |
435/91.2 ;
435/289.1; 435/304.1; 435/305.1 |
Current CPC
Class: |
B01L 2300/1883 20130101;
B01L 3/50851 20130101; G01N 21/0332 20130101; B01L 2300/16
20130101; B01L 7/52 20130101; B01L 2300/1827 20130101; B01L
2300/0887 20130101; B01L 2300/0858 20130101 |
Class at
Publication: |
435/91.2 ;
435/289.1; 435/305.1; 435/304.1 |
International
Class: |
C12P 19/34 20060101
C12P019/34; C12M 1/00 20060101 C12M001/00; C12M 1/22 20060101
C12M001/22; C12M 1/24 20060101 C12M001/24 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 2007 |
GB |
0715170.7 |
Claims
1. A reaction vessel heating system, which comprises electrically
conducting polymer, arranged to act as a heating element for a
reaction vessel, wherein the profile of the electrically conductive
polymer differs in different regions of the vessel so as to control
thermal gradients therealong.
2. A reaction vessel heating system according to claim 1 wherein
the profile of the ECP is arranged to have the effect of reducing
thermal gradients therealong.
3. A reaction vessel heating system according to any of claims 1
and 2 wherein at least one wall of said vessel comprises a highly
thermally conducting layer.
4. A reaction vessel according to claim 3 wherein the highly
thermally conducting layer is a metallic layer.
5. A reaction vessel according to any of claims 3 or 4 further
comprising an inner non metallic layer.
6. A reaction vessel according to any of claims 4 or 5 wherein the
electrically conductive polymer is insulated from the metallic
layer by means of an insulating layer therebetween.
7. A reaction vessel according to claim 6 wherein the insulating
layer is a layer of anodised aluminium, or is a polymer layer.
8. A reaction vessel according to claim 7 wherein the polymer layer
comprises parylene or a derivative thereof.
9. A reaction vessel according to any preceding claim which is an
elongate vessel.
10. A reaction vessel according to claim 9 which comprises a tube
which is sealed at one end, wherein the end is of a transparent
material
11. A reaction vessel according to any one of claims 9-10 which
comprises a capillary vessel or a flattened capillary vessel.
12. A reaction vessel according to any of claims 9-11 wherein the
radial depth of ECP material at the tip of the vessel is less than
the radial depth of ECP material at the centre of the vessel.
13. A reaction vessel according to any of claims 1-12 wherein the
vessel is a thermal cycling vessel.
14. A reaction vessel according to any of claims 1-113 wherein the
vessel is a PCR reaction vessel.
15. A reaction vessel heating system according to claim 1 wherein
the ECP profile is arranged to have the effect of increasing
thermal gradients.
16. A reaction vessel according to claim 15 wherein the vessel is a
culture vessel for the culture of biological materials.
17. A reaction vessel according to claim 16 wherein the vessel is
one of a petri dish, cuvette, chemostat, shake flask, universal
container, bijou.
18. A method for carrying out a chemical or biochemical reaction
which requires at least one heating step, said method comprising
placing chemical or biochemical reagents into a reaction vessel
according to any one of claims 1 to 17 and heating said reagents so
as to bring about a chemical or biochemical reaction.
19. A method according to claim 18 wherein the reaction requires
thermal cycling.
20. A method according to claim 19 wherein the reaction is a
polymerase chain reaction.
21. A method for mixing reagents in a vessel, said method
comprising placing said chemical or biochemical reagents in a
reaction vessel according to any of claims 15-17 and heating the
heating the vessel so as to bring about a temperature gradient to
create convection.
Description
[0001] The present invention relates to reaction vessels useful in
chemical and biochemical reactions which are required to undergo
controlled heating and/or cooling, in particular, vessels which are
required to undergo thermal cycling, where a sequence of different
temperatures are required.
[0002] A particular example of such a reaction are a number of
nucleic acid amplification methods, in particular the polymerase
chain reaction (PCR). As is well known, in this reaction,
exponential amplification of nucleic acids is achieved by cycling
the sample containing or suspected of containing the target nucleic
acid through an iterative sequence of different temperatures in the
presence of specifically designed primer sequences and polymerase
enzymes able to extend those primer sequences. These temperatures
represent the temperatures necessary for nucleic acid denaturation
(and generally requires temperatures of about 95.degree. C.),
primer annealing (at a lower temperature for example at about
55.degree. C.) and primer extension (which may require and
intermediate temperature for example of about 74.degree. C.).
[0003] There is frequently a need to obtain the results of a PCR
reaction quickly, for example in cases of environmental
contamination which may be the result of hostile activity. However,
even in a clinical or diagnostic situation, the production of quick
results can be helpful, in particular where patient compliance or
return can be problematic.
[0004] Clearly, for fast PCR, the sample must be rapidly heated and
cooled. This is facilitated by making the sample small to reduce
its thermal mass and by minimising the distances over which heat
must be transferred. The same considerations must be applied to the
container of the sample.
[0005] Thus, a number of examples of apparatus designed to carry
out PCR reactions utilize reaction vessels which comprise a
capillary tube format (ie long and thin WO 2005/019836) or as a
planar structure (flat and thin) (WO2006024879), the content of
which are incorporated herein by reference.
[0006] A variety of heating systems are utilized in order to
achieve rapid PCR. These include for example fluid based systems in
which hot fluid such as air is fed to the container of the sample
for heating purposes, and non-heated fluid is supplied to effect
cooling (see for example U.S. Pat. No. 6,787,338 and WO2007/054747,
the content of which is incorporated herein by reference).
[0007] In an alternative type of apparatus, electrically conducting
polymer (ECP) is used as both the heating element and in some cases
also the container (see WO 98/24548, the content of which is also
incorporated herein by reference).
[0008] The ECP acts as a resistive heater and so it is required to
be connected to an electrical supply by way of electrical
connections. As an inevitable consequence of reducing the thermal
mass of the sample and facilitating heat transfer into and out of
it, the means of connecting and locating the ECP can have
significant thermal effects upon it.
[0009] A particular problem is the formation of temperature
gradients as heat can be conducted both out from and in to the
extremities of the ECP tube, through the electrical connections, as
it is heated and cooled, respectively.
[0010] This problem has been addressed in some instances by
examining the electrical connections themselves and in particular,
the mountings for the electrodes. These must be electrically
insulating and are preferably also thermally insulating. However,
the property of thermal insulation is in itself insufficient.
[0011] Electrical connectors (or electrodes) that are thermally
insulating heat and cool slowly which has the effect of making them
important contributors to the formation of longitudinal temperature
gradients during rapid thermal cycling. The mountings must also
therefore have a low thermal mass as well as being thermal
insulators. This may be achieved by placing insulating materials
that have been structured to reduce their thermal mass whilst
retaining the physical integrity needed to support the electrodes.
Such structuring, in its essence, requires the inclusion of air
gaps in the mountings. This may be achieved by using foam
materials, such a honeycomb or reticulated foam to form a mount for
the electrode. The mounts are suitable in the form of a pillared or
corrugated mount for the electrical connector (as described for
example in WO2005/0011834, WO2004/045772 and copending British
Patent Application No. 0623910.7).
[0012] In PCR, it would be ideal to have all parts of the sample at
the same controlled temperature all of the time. This is extremely
difficult in a system that is being rapidly heated and cooled. In
the capillary format, both radial and longitudinal temperature
gradients are formed.
[0013] The applicants have found however that the profile of the
ECP can be adjusted to control the thermal gradient
[0014] A first aspect of the present invention provides a reaction
vessel heating system, which comprises electrically conducting
polymer, arranged to act as a heating element for a reaction
vessel, wherein the profile of the electrically conductive polymer
differs in different regions of the vessel so as to control thermal
gradients therealong.
[0015] The ECP profile may be arranged to have the effect of
reducing thermal gradients. This is particularly suitable for
reaction vessels used for thermal cycling reactions, for example
PCR.
[0016] In one embodiment, the profile of the ECP is itself adjusted
to vary its radial thickness so that the resistance heating is
distributed unevenly in a way so as to reduce gradients. This may
be done empirically, in relation to any particular vessel type, by
determining the gradient profile which occurs and adjusting the
thickness of the ECP in various regions of the vessel accordingly.
Typically, the profile will be tapered, being narrower at the
bottom of the vessel than the top. Instead of varying the radial
thickness, the area of contact between the vessel and ECP may be
varied.
[0017] The thermal gradients may be further reduced by providing
the vessel with at least one wall which comprises a highly
thermally conducting layer. The highly thermally conducting layer
may comprise a metallic layer, for example aluminium. The
electrically conductive polymer is insulated from the metallic
layer by means of an insulating layer therebetween, for example a
layer of anodised aluminium, or is a polymer layer, such as
parylene or a derivative thereof. Preferably the vessel further
comprises an inner non metallic layer to improve
biocompatability.
[0018] The reaction vessel may be an elongate vessel. In a
preferred embodiment, the reaction vessel comprises a tube which is
sealed at one end, wherein the end is of a transparent material.
The reaction vessel may comprise a capillary vessel or a flattened
capillary vessel.
[0019] In another embodiment, the ECP profile is arranged to have
the effect of increasing thermal gradients. A thermal gradient is
thus created along the vessel.
[0020] A vessel with a thermal gradient may be used for the culture
of biological materials, with different regions along the thermal
gradient being optimal for different biological material. The
vessel may comprise a petri dish, cuvette, chemostat, shake flask,
universal container, bijou.
[0021] The profile may be adapted by changing the radial thickness
of ECP. Alternatively, the area of contact between the ECP and the
vessel may be varied.
[0022] A second aspect of the present invention provides a method
for carrying out a chemical or biochemical reaction which requires
at least one heating step, said method comprising placing chemical
or biochemical reagents into a reaction vessel according to the
present invention and heating said reagents so as to bring about a
chemical or biochemical reaction.
[0023] Preferably the reaction requires thermal cycling. More
preferably the reaction is a polymerase chain reaction.
[0024] A third aspect of the present invention provides a method
for mixing reagents in a vessel, said method comprising placing
said chemical or biochemical reagents in a reaction vessel
according to the present invention and heating the heating the
vessel so as to bring about a temperature gradient to thereby
create convection.
[0025] Such modifications may be included in vessels which include
or utilise ECP resistance heaters, irrespective of the presence or
otherwise of the highly thermally conducting layer, and such
vessels form yet a further aspect of the invention.
[0026] According to a fourth of the present invention there is
provided a reaction vessel for conducting a chemical or biochemical
reaction, wherein at least one wall of said vessel comprises a
layer of a highly thermally conducting material, (in particular a
metallic layer) and an inner non-metallic layer.
[0027] For the avoidance of doubt, the term "layer" as used herein
refers to any essentially laminar arrangement of material,
including both self-supporting layers as well as coatings. Layers
or coatings which are not self supporting, will generally conform
to the shape of the relevant substrate, and so for example may in
practice may be any shape, including in particular tubular.
Similarly, self-supporting layers may take whatever form is
particularly convenient in relation to the context in which they
are used.
[0028] Reaction vessels of the invention have good thermal
conductivity as a result of the presence of the highly thermally
conducting layer of the wall, and therefore can be used in
reactions where temperature control, or in particular temperature
cycling with good temperature uniformity is important. Therefore,
they may be particularly useful in reactions such as nucleic acid
amplification reactions which involve thermal cycling such as the
polymerase chain reaction (PCR). The good thermal conductivity
means that significant temperature gradients through the vessel do
not form, or are rapidly evened out if they do occur, so that the
temperature profile along and across the sample is made flatter
(more homogeneous).
[0029] Generally, metal walled vessels have not be used hitherto in
reaction vessels because they are generally chemically reactive in
particular with biological molecules such as nucleic acids and
proteins, and so the metal interferes with reagents in the vessel
and so disrupts the reaction. However, the applicants have found
that this problem can be overcome by the provision of an inner
non-metallic layer, in contact with the thermally conducting layer
such as the metallic layer so as to effectively form a
composite.
[0030] The vessel is suitably an elongate vessel, with the layered
wall forming at least one of the long walls so as to increase the
surface area of the metal containing wall which is in the proximity
of a reagent in the vessel. In particular, the vessel is a
capillary vessel or a flattened capillary vessel, where the length
is selected to accommodate the volume of the sample and inner
diameters are small. In particular, the inner diameter of a
capillary tube is in the range of from 0.2 to 2 mm. The thickness
of the wall is generally from about 0.1 to about 1.5 mm.
[0031] Examples of such vessels are described for example in
WO2004/054715, U.S. Pat. No. 6,015,534 and WO 2005/019836, the
content of which is incorporated herein by reference.
[0032] Such vessels effectively comprise a single radial side wall
and this suitably comprises a metallic layer over substantially
all, and preferably all its area.
[0033] Where flattened tubes are used, they may be of a shape
described in WO2006024879, the content of which is incorporated
herein by reference. Specifically, such vessels have a width:depth
ratio of about 2:1 or more, for example, of 3:1 or more. Typically
the width of the vessels may be of the order of 1 mm or less for
example 0.8 mm or less, whereas the depth is generally 0.5 mm or
less, and suitably less than 0.3 mm. The vessels may be
tapered.
[0034] In these cases, at least one side wall comprises a metallic
layer, and preferably all side walls comprise a metallic layer. The
lower wall may also have this construction, although in many
instances, it is preferred that the lower wall, which forms the
base of the vessel is of a transparent material such as glass or a
transparent polymer so that the contents of the reaction vessel can
be optically monitored during the reaction. This is particularly
helpful in the case of the use of the so-called "real-time" PCR
reactions where optical signals, in particular fluorescent signals
from signalling reagents added to the PCR reaction, produce a
variable signal as the reaction progresses, so that the progress of
the reaction can be monitored. Such monitoring gives rise to the
option of quantifying the amount of target nucleic acid within the
initial sample, so providing further information which may be of
use, for example in diagnostics, in determining the seriousness of
a particular condition.
[0035] Suitable non-metallic materials for the inner non-metallic
layers may include polymeric materials or glass or even a
passivated layer created through anodisation of a metal, or similar
process, or combinations of these. In particular, however, the
inner non-metallic layer is a polymer or glass or combination of
these.
[0036] In a particular embodiment, the inner non-metallic layer is
a glass layer, since glass is generally well recognised as being
compatible with many biochemical and chemical reactions including
the polymerase chain reaction.
[0037] However, polymeric materials such as polyurethane,
polyethylene, polypropylene, or polycarbonates, as well as
silicones which are compatible with the sample and with the
particular reaction being carried out within the reaction
vessel.
[0038] Such inner layers will generally be rigid and supporting
structures, which may be formed by processes such as injection or
extrusion moulding and the like. These may then be coated with a
metallic layer, or they may be extruded or formed directly onto the
metallic layer.
[0039] However, if necessary or required a thin layer for example
of polymeric material may deposited on the metallic layer for
example using techniques such as vapour deposition, liquid phase
deposition or plasma polymerisation to provide a relatively thin
layer which may itself constitute the inner non-metallic layer.
Alternatively, such a thin layer may be applied to a different
inner non-metallic layer as described above to form a composite
structure.
[0040] A particularly suitable polymeric layer of this type is
formed of parylene or derivatives thereof. Parylene is a generic
name applied to polyxylylene as for example as described in U.S.
Pat. No. 3,343,754, the content of which is incorporated herein by
reference.
[0041] Compounds of this type can be represented by the general
formula (I)
##STR00001##
where is R is a substituent group, m is 0 or an integer of from 1
to 3 and n is sufficient for the compound to be a polymer.
[0042] Where m is greater than 1, each R group may be the same or
different.
[0043] In one embodiment, m is 0.
[0044] Suitable substituent groups R include but are not limited to
R.sup.1, OR.sup.1, SR.sup.1, OC(O)R.sup.1, C(O)OR.sup.1, hydroxyl,
halogen, nitro, nitrile, amine, carboxy or mercapto and where
R.sup.1 is any hydrocarbon group and where R.sup.1 may be
optionally substituted by one or more groups selected from
hydroxyl, halogen, nitro, nitrile, amine or mercapto.
[0045] Suitable hydrocarbon groups include alkyl groups such as
straight or branched chain C.sub.1-10alkyl groups, alkenyl groups
such as straight or branched C.sub.2-10alkenyl groups, alkynyl
groups such as straight or branched C.sub.2-10alkynyl groups, aryl
groups such as phenyl or napthyl, aralkyl groups such as aryl
(C.sub.1-10) alkyl for instance benzyl, C.sub.3-10cycloalkyl,
C.sub.3-10cycloalkyl (C.sub.1-10) alkyl, wherein any aryl or
cycloalkyl groups may be optionally substituted with other
hydrocarbon groups and in particular alkyl, alkenyl or alkynyl
groups as described above.
[0046] Particular examples of groups R include alkyls such as
methyl, ethyl, propyl, butyl or hexyl, which may be optionally
substituted with hydroxy, halo or nitrile such as hydroxymethyl or
hydroxyethyl, alkenyls such as vinyl, aryls in particular phenyl or
napthyl which may be optionally substituted by halo or alkyl groups
such as halophenyl or C.sub.1-4alkylphenyl, alkoxy groups such as
methoxy, ethoxy, propoxy, carboxy, carbomethoxy, carboethoxy,
acetyl, propionyl or butyryl.
[0047] In particular, R is selected from halogen (particularly
chlorine or bromine), methyl, trifluoromethyl ethyl, propyl, butyl,
hexyl, phenyl, C.sub.1-4alkylphenyl, naphthyl, cyclohexyl and
benzyl.
[0048] Examples of such polymers are sold as "Parylene". Particular
variety of parylene which may be obtained include Parylene N (where
m is 0), Parylene C (where m is 1 and R is chloro), Parylene F
(where m is 1 and R is trifluoromethyl) and Parylene D (where m is
2 and each R is chloro).
[0049] Parylene is a particularly convenient polymeric material for
providing an internal coating for the metallic surface, as it may
be readily applied using a vapour deposition process. In this
process a solid dimer of formula (II)
##STR00002##
where R and m are as defined above, is placed into a suitable
vaporisation chamber in solid form. When the chamber and heated
under reduced pressure, for example to temperatures of about
150.degree. C. at low pressure, for example of about 1 mmHg, the
diner vapourises. The vapour is then transferred into a pyrolysis
chamber where the temperature is much higher, for example at about
650.degree. C. and the pressure is for example of 0.5 mmHg,
Pyrolysis occurs so as to cause the formation of a reactive
monomeric species of formula (III).
##STR00003##
[0050] If this is allowed to pass into a further chamber containing
the item to be coated which is at ambient temperature, but also at
low pressure, for example of 0.1 mmHg, polymerisation of the
species (III) occurs on the surface of the object, so that a
coating of the polymer of formula (I) above is produced.
[0051] The species (III) condenses on the surface in a
polycrystalline fashion, providing a coating that is conformal and
pinhole free. This is important to ensure that any sample within
the reaction vessel is isolated from the metallic layer.
[0052] Compared to liquid processes, the effects of gravity and
surface tension are negligible--so there is no bridging, thin-out,
pinholes, puddling, run-off or sagging. And, since the process
takes place at room temperature, there is no thermal or mechanical
stress on the object.
[0053] Parylene is physically stable and chemically inert within
its usable temperature range, which includes the temperatures at
which PCR reactions are conducted. Parylene also provides excellent
protection from moisture, corrosive vapours, solvents, airborne
contaminants and other hostile environments.
[0054] It is widely used in the electronics industry to coat and
protect electronic components. However, the applicants are the
first to find that parylene is compatible with chemical or
biochemical reactions and in particular with the PCR reaction, and
the use of parylene for coating reaction vessels and in particular
PCR reaction vessels is described and claimed in the applicants
copending patent application of even date.
[0055] In the vessel of the apparatus, the metallic layer
effectively forms a thermal shunt, conducting heat rapidly from one
part of the reaction vessel to another. Thus it minimises the
build-up of thermal gradients in the vessel and therefore in the
sample during the reaction, which is important in ensuring that the
reaction proceeds efficiently and well.
[0056] Thus vessels comprising a highly thermally conducting
material most suitably comprise a material which has a thermal
conductivity in excess 15 W/mK. Materials having this property will
generally be metallic in nature, but certain polymers, in
particular those known as "cool polymers" or polymers containing
thermally conducting ceramics such as boron nitride as well as
diamond, may have the desired level of thermal conductivity. In
particular however, the highly thermally conducting material is
metallic, which may be of any suitable metal or metal alloy
including aluminium, iron, steel such as stainless steel, copper,
lead, tin or silver. In particular, the metallic layer comprises
aluminium.
[0057] The reaction vessel in accordance with the invention, may be
heated by any suitable heating means, and as a result of the
presence good thermal conductivity of the walls of the vessel due
to the presence of the highly thermally conducting layer, the heat
will be readily transferred to the vessel contents.
[0058] Thus the vessels are suitable for use in a wide range of
apparatus in particular thermal cycling equipment. These may be
heatable and/or coolable using a number of different technologies,
including the use of fluid heaters and coolers such as air heaters
and coolers in particular those heated by halogen bulbs, as
described for example in U.S. Pat. No. 6,787,338 and WO2007/054747,
the content of which is incorporated by reference, as well as in
vessels using ECP as resistive heating elements, for example as
described in WO 98/24548 and WO 2005/019836 as will be discussed
further below. The vessels may also be used in more conventional
devices such as solid block heaters that are heated by electrical
elements. For cooling the apparatus may incorporate thermoelectric
devices, compressor refrigerator technologies, forced air or
cooling fluids as necessary. However, where the vessels of the
invention comprise a metallic layer, this means that they may also
be capable of being heated using for example induction methods.
Apparatus used to heat vessels of the invention in this way will
have the facility to heat the vessel by electromagnetic induction,
for example by using a high-frequency alternating current (AC) to
induce eddy currents within the metal. Resistance of the metal to
these currents leads to Joule heating of the metal. Heat is also
generated by magnetic hysteresis losses. For use in induction
heating apparatus, it will be clear that the metallic layer within
the vessel should be of a suitable material to allow it to be
heated in this way, and so for example iron metallic layers may be
preferred to say stainless steel or copper.
[0059] Reaction systems comprising combinations of reaction vessels
as described above, and apparatus which is able to accommodate said
reaction vessel, and which comprises a heating system adapted to
controllably heat and cool said vessel, in particular using any of
the methods discussed above, form a further aspect of the
invention.
[0060] When the reaction vessels of the invention are utilised in
combination with resistive heating elements, such as ECP, it is
necessary to ensure that where the highly thermally conducting
layer is also electrically conducting, such as a metallic layer,
this is electrically insulated from the resistive heater in order
to prevent short circuits etc. The applicants have found that it is
possible to passivate the surface of a metal layer so that it is
electrically isolated from the ECP, but still in good thermal
contact. For example in the case of an aluminium metallic layer,
anodisation of any surface of the aluminium layer which is to be in
contact with the resistive heater such as the ECP provides such
insulation.
[0061] Alternatively, a parylene layer, preparable as described
above may be applied to the surface of the highly thermally
conducting layer such as the metallic layer which contacts the ECP
so as to provide an effective electrically insulating layer. Such
layers have the benefit that they do not significantly add to the
size or thermal mass of the vessel.
[0062] The ECP is suitably arranged as a sheath or coating arranged
outside the highly thermally conducting layer and the electrically
insulating layer thereof, as described in WO 98/24548. By keeping
the elements of the reaction vessel small, in particular as thin
layers, the thermal mass of the vessel remains low, and so fast
heating and cooling can take place as is required for rapid
PCR.
[0063] Thus in a particularly preferred embodiment, a wall of the
reaction vessel, and suitably the entire side walls of the vessel
comprise an inner non-metallic layer, for example of glass or a
polymeric material. This is covered by a metallic layer as
described above, which is itself covered by an electrically
insulating layer, for example a layer of anodised aluminium or
parylene or a derivative thereof as described above. Outside of
this layer is suitably provided a layer of electrically conducting
polymer. Such vessels are generally intended to be disposable after
a single use.
[0064] The electrically conducting polymer needs to be connected to
an electrical supply, and so electrical connections, which may be
integral with the vessel, are suitably provided at each end of the
ECP.
[0065] The ECP elements used in the vessels as resistive heating
elements can be manufactured by various processes, but most a
convenient process involves injection moulding of the polymer.
However, in the process of injection moulding, the material tends
to form an outer polymer-rich skin that may creates at least a
partial electrically insulating barrier to any external means of
making electrical contact.
[0066] In such cases, the applicants have found that it is helpful
to break through the insulating skin and make electrical contact
with the bulk material in order to increase the efficiency of the
heating system.
[0067] Thus, in a particular embodiment, the reaction vessel as
described above, is connectable to an electricity supply by means
of barbed electrical contacts which pierce the surface of the
electrically conducting polymer. These are suitably integral with
the vessel.
[0068] Such barbed connectors may take various forms depending upon
the particular configuration of the reaction vessel itself. In
particular, where the vessel is of a generally tubular
configuration, suitable barbed connectors may take the form of
annular metal rings with inwardly projecting barbs or the like,
similar in design to "Starlock Washers".
[0069] The inwardly projecting barbs will cut through the
insulating skin to make electrical contact with the bulk conducting
material, as well as hold the ring in position. The outer portion
will present a metallic surface for electrical interconnection, and
so apparatus intended to accommodate the vessels will be configured
appropriately.
[0070] Furthermore, the barbs provide effectively a scalloped edge
which helps to reduce the size and therefore the thermal mass of
the connectors and also, reduces the contact area with the ECP.
This has the further advantage of further minimising heat exchange
between the electrical connectors and the ECP, so further assisting
in reducing unwanted thermal gradient formation.
[0071] The connectors and particularly the barbs thereof, are
suitably constructed of a material which have high mechanical
strength, so that the barbs can be sharpened to enhance the
penetration ability. Whilst many metals are able to fulfil this
function, a particularly suitable material for the connectors has
been found to be stainless steel. This not only has the required
mechanical strength and electrical conductivity, but also, it has a
high corrosion resistance, at 16 W/mK, a surprisingly low thermal
conductivity compared to many other metals (cf Copper @ 399 W/mK,
Aluminium @ 237 W/mK). By using this material, and by designing the
connectors so that their area is as small as possible, helps to
reduce unwanted thermal effects at the electrodes.
[0072] Connections of this type are cost effective to manufacture,
which will be a particular advantage in cases where the proposed
PCR vessel is to be a high volume disposable item.
[0073] They may, in fact, be used in connection with any reaction
vessel or device which includes ECP as a resistive heater, and such
reaction vessels and devices form a further aspect of the
invention.
[0074] As discussed briefly above, the apparatus into which the
vessel is accommodated for use suitably is provided with mounts of
a material which is both an electrical and thermal insulator (i.e
the material absorbs and releases heat slowly) and also have a low
thermal mass. This can be achieved by making the electrode mounts
"air-like" ie formed as foam, skeletal or honeycomb structures.
However they must be able to physically support and position the
electrodes, and therefore they must have a degree of rigidity,
firmness and/or resilience.
[0075] Thus in a particular embodiment of the apparatus for
thermally cycling the contents of the reaction vessel as described
above comprises a solid foam material arranged in direct contact
with at least one of said electrical contacts.
[0076] The solid foam material acts as an insulator, preventing
heat flows and particularly heat loss through the electrical
contacts. As a result, a more uniform temperature can be maintained
within the reaction vessel. By utilising specifically a solid foam
in preference to a conventional solid insulator, such as a solid
plastics insulator, the performance of the apparatus is
significantly enhanced. Thermal gradients set up within the
reaction vessel can be further reduced.
[0077] It is believed that this is due to the fact that foam-like
materials have a lower thermal mass than solids. Therefore, in
addition to providing insulation preventing the flow of heat into
and out of the reaction vessel through the electrical contact, they
do not significantly hinder the heating and cooling process.
[0078] In contrast, solid insulators with significant thermal mass
were found to heat up and cool down slowly (rather than not at
all), thereby acting as sources or sinks of heat depending on their
temperature relative to the sample. This was found to be
disadvantageous in this context.
[0079] Suitable solid foam materials include metal, glass, carbon,
polymer, ceramic foams or composites made of several of these.
[0080] Particular examples of such foam materials are polymeric
foams such as polyurethane or polystyrene foams.
[0081] In a particular embodiment, the foam material is a ceramic
foam. Ceramic foams generally comprise inorganic, non-metallic
materials (such as metal oxides, silicides, nitrides, carbide or
borides) with a crystalline structure, which have usually been
processed at a high temperature at some time during their
manufacture.
[0082] Many such foams are now commercially available. They have
been developed mainly for the aerospace industry where their
utility as insulators is a result of their light weight.
[0083] Solid foams generally comprise solids which have many gas
bubbles trapped within them. They may be rigid or pliable in
nature, but are preferably rigid so as to support the electrical
contacts. Where they are pliable, the foam material suitably has a
good "shape memory".
[0084] Various forms and types of solid foam materials are known.
Some are known as "refractory" foams. They are made by various
methods depending upon the nature of the material used. For
instance, solid foams comprising polymers may be readily prepared
including foaming agents into the preparation process, as is well
understood in the art. Syntactic foams and self-foamed materials
such as foam glass may be prepared in a similar way.
[0085] Other solid foams may utilise a foamed polymer as the basic
starting material and materials are essentially coated onto these
or onto carbonaceous skeletons formed by pyrolysis of the polymer
foam. For example, ceramic foams can be produced by coating a
polymer foam or a carbon skeleton derived from it, with an
appropriate binder and ceramic phases, and then sintering at
elevated temperatures. Metallic foams may be formed by
electrolytically depositing the metal onto a polymer foam, utilizes
an electrodeless process for the deposition of a metal onto the
polymer foam precursor via electrolytic deposition.
[0086] Suitably the solid foam material is of a material, which has
no fluorescent or phosphorescent properties, even when illuminated
with a light source. This means that it may not interfere with the
fluorescent signalling or labelling systems that are frequently
utilised for detecting the products of an amplification reaction.
Such systems may be used to detect the product of amplification
either at the end point of the reaction, or, increasingly, in
"real-time" as the reaction progresses. These systems, which
include the well known "Taqman.TM." system as well as other systems
such as those described for example in Homogeneous fluorescent
chemistries for real-time PCR. Lee, M. A., Squirrell, D. J.,
Leslie, D. L. and Brown, T. in Real-time PCR: an essential guide,
J. Logan, K. Edwards & N. Saunders eds., Horizon Scientific
Press, Wymondham, p. 31-70, 2004, the content of which is
incorporated herein by reference. For instance, generic methods
utilise DNA intercalating dyes that exhibit increased fluorescence
when bound to double stranded DNA species. Fluorescence increase
due to a rise in the bulk concentration of DNA during
amplifications can be used to measure reaction progress and to
determine the target molecule copy number. Furthermore, by
monitoring fluorescence with a controlled change of temperature,
DNA melting curves can be generated, for example, at the end of PCR
thermal cycling.
[0087] However, when the material has a degree of fluorescence,
this may be obviated by dying, coating or inking the foam before
use.
[0088] The solid foam material is arranged in contact with at least
one and preferably both of the electrical contacts. Suitably
sufficient foam material is arranged so that in use, it effectively
isolates the electrical contacts from environmental effects. This
will generally be achieved by arranging the solid foam material in
contact with at least the remote edges of the electrical
contacts.
[0089] If desired, a solid insulator material may also be provided
and arranged to contact the solid foam material.
[0090] Suitable solid insulator materials are well known in the
art, and include polymeric or fibrous materials. In particular the
solid insulator is a polymeric insulator such as an acetal
homopolymer resin such as Delrin.TM.,
acrylonitrile-butadiene-styrene terpolymer (ABS) or
polytertrafluoroethylene (PTFE).
[0091] The solid insulator may be arranged to contact a substantial
portion, for example at least one side of the solid foam material.
This additional insulation will protect the solid foam material
itself from changes in environmental temperature and so enhance the
overall reliability of the system. Suitably the additional solid
insulator is provided so as to effectively isolate the reaction
vessel from the external environment when in use. The precise
arrangement of the solid insulator therefore will vary depending
upon the nature of the reaction vessel and the manner in which it
is used.
[0092] As mentioned above, example of mounts which include
foam-like materials are described for example in WO2005/0011834,
WO2004/045772, where the foam materials have a degree of resilience
or compliance to allow them to hold the connectors firmly, or
co-pending British Patent Application No. 0623910.7, where firm
foam-like materials may be used, which are sprung-loaded to ensure
that a sufficiently firm hold on the connectors is achieved.
[0093] Reaction vessels as described above and reaction systems
comprising them can be used in chemical and biochemical reactions
as required. Thus, in a further aspect, the invention provides a
method for carrying out a chemical or biochemical reaction which
requires at least one heating step, said method comprising placing
chemical or biochemical reagents into a reaction vessel as
described above, and heating said reagents so as to bring about
said chemical or biochemical reaction. In particular, the vessel is
positioned into apparatus specifically designed to hold it, and to
heat and/or cool it as required. In particular, the apparatus
comprises a thermal cycler as described above, and the reaction
requires thermal cycling, in particular is a polymerase chain
reaction.
[0094] The invention will now be particularly described by way of
example with reference to the accompanying diagrammatic drawings in
which:
[0095] FIG. 1 shows a section through a reaction vessel according
to the invention;
[0096] FIG. 2 shows an enlarged view of the portion of the reaction
vessel shown in claim 1, which incorporates the elements of the
invention;
[0097] FIG. 3 is a perspective end view of the reaction vessel of
FIG. 1; and
[0098] FIG. 4 shows an electrical connection used in the embodiment
of FIG. 3; and
[0099] FIG. 5 is a schematic diagram showing a vessel as shown in
FIG. 1 in position in a thermal cycling apparatus.
[0100] FIG. 6 illustrates a sample vessel coated with ECP, showing
tapering of the ECP layer;
[0101] FIG. 7 is a graph illustrating the internal temperatures of
a sample vessel at a thermopile setting of 43.degree. C.;
[0102] FIG. 8 is a graph illustrating the internal temperatures of
a sample vessel at a thermopile setting of 63.degree. C.;
[0103] FIG. 9 is a graph showing the effect of tapering the ECP at
different thermopile settings; and
[0104] FIG. 10 is a graph showing the different temperatures
recorded on a tapered section of ECP.
[0105] The reaction vessel shown in FIG. 1 is of the same general
type as those described in for example WO2005/019836, which is
intended for use in an apparatus for conducting a PCR reaction.
[0106] The vessel comprises a plastics body (1) with an upper
sample receiving portion (2) with a relatively wide mouth so that
reagents can, with ease, be added. In the illustrated embodiment,
the upper portion includes projecting flanges (6) which are able to
interact with a lifting arm in an apparatus such as that described
in WO 2005/019836 so as to allow the vessel to be moved in an
apparatus adapted to carry out reactions automatically.
[0107] At the lower end of the sample receiving portion (2), the
vessel terminates in a capillary tube (3) which is sealed at the
lower end by a transparent seal (4), so as to form an elongate thin
reaction vessel, which can contain relatively small sample (7)
within the capillary section.
[0108] An aluminium coating layer (5) surrounds the capillary tube
(3) and is in close thermal contact with it. This coating layer (5)
acts as a thermal shunt, which is able to rapidly dissipate thermal
gradients which build up along the tube (3) and thus within a
sample (7) which is subject to heating and/or cooling. The outer
surface of the aluminium coating layer (5) has been anodised so as
to produce an insulating layer thereof. In an alternative
embodiment however, the outer surface of the aluminium coating
layer (5) is coated with parylene to provide electrical
insulation.
[0109] An ECP layer (8) completely encases the aluminium coating
layer (5) as well as the sides of the lower seal (4) and the base
of the upper portion (2). Is it provided with upper and lower
ridges (9, 10) respectively which can accommodate upper and lower
annular electrical connectors (11, 12) respectively. Each
electrical connector (11,12) is provided with a number of inwardly
projecting barbs (13) (FIGS. 3 and 4) which are able to pierce the
surface of the ECP to ensure that electrical contact is made with
the body of the ECP.
[0110] In use, this particular vessel can be loaded with sample and
PCR reagents, as described in WO 2005/019836. A prepared sample (7)
to which has been added all the reagents necessary for carrying a
PCR reaction is placed in the upper portion (2) and if necessary a
cap (not shown) is placed over open end. The entire vessel is then
then centrifuged to force the sample (7) into the capillary tube
(3) section of the vessel.
[0111] In an alternative embodiment however, the sample and the
reagents may be loaded directly into the capillary tube (3) using a
specifically designed fine tipped pipettor, and with accompanying
close control of pipettor removal, as described and claimed in a
copending British patent application of the applicants of even date
to the present application.
[0112] The vessel is then suitably positioned in an apparatus (FIG.
5) able to accommodate it such that the connectors (11, 12) are
connectable to an electrical supply but seat on a pair of
supports.
[0113] A ring of a solid foam insulator material (14) (a
polyurethane engineering foam) is provided in contact with the
lower edge portion of the upper electrical contact (11). The lower
electrical contact (12) is held on a shaped support (15), also of
the solid foam insulator material.
[0114] The solid foam insulator material is arranged to minimise
heat transfer from the electrical contacts, but does not interfere
with the contact to the electrical supply (not shown).
[0115] A ring (16) of solid insulator material such as Delrin is
provided adjacent the ring of solid foam insulator material (14) in
contact with it. The ring (16) effectively surrounds the rest of
the upper electrical contact (11) but is not in direct contact with
it. As a result, it acts as an insulator from environmental effects
coming from above, but does not act as either a heat sink or heat
source in relation to the electrical contact itself.
[0116] Similarly, the shaped support (15) of a solid foam insulator
material is itself supported on a ring (17) of solid insulator
material such as Delrin so as to provide similar protection for the
lower electrical contact (5). The solid insulator rings (16, 17)
are provided with conduits for electrical connection and
spring-mounted in housing (18).
[0117] In use, the solid insulator rings (16, 17) combined with the
apparatus in which the vessel is held define a chamber for the tube
(2) that is effectively isolated from the environment. However, the
electrical contacts themselves are in contact with the solid foam
material of low thermal mass.
[0118] When arranged in this way, the apparatus could be utilised
in a polymerase chain reaction in a far more effective and reliable
manner, as compared to devices which had no or alternative
arrangements of insulator material.
[0119] The connectors (11,12) are then connected to the electrical
supply, which is controlled, suitably automatically, to pass
current through the ECP layer (5) so it rapidly progresses through
a series of heating and cooling cycles, ensuring that the sample is
subjected to similar cycling conditions. This will allow the sample
(7) to be subjected to a PCR reaction.
[0120] Where a real-time monitoring system is included in the
sample (7), the progress of the PCR can be monitored through the
seal (4) using conventional methods.
[0121] As a result, rapid PCR can be achieved. The presence of
thermal gradients within the vessel (1) and therefore the sample
(7) is reduced by the measures taken, including in particular the
presence of the aluminium coating layer (5) which acts as a thermal
shunt. Thus reliable and reproducible results may be achieved.
[0122] During thermal cycling of the sample vessel in the PCR
process, temperature gradients can arise along the length of the
vessel. These temperature gradients typically arise due to the
influence of the electrical connections, the temperature being
higher in the region adjacent to the electrical connections during
heating due to conduction. The applicants have discovered that the
thermal gradients can be minimised by tapering the ECP coating of
the sample vessel.
[0123] FIG. 6 illustrates the sample vessel of FIGS. 1 and 2. As
previously described the sample vessel has a lower portion
comprising a capillary which is coated in ECP. The capillary is
11.6 mm and FIG. 6 shows the radial thickness of the ECP at three
positions along its length. The ECP tapers from an area of 3.12 mm
at the top, 2.78 mm in the middle and 2.51 mm at the bottom.
[0124] FIGS. 7 and 8 show experimental data measuring the internal
temperature of three sample vessels, having different radial
thicknesses of ECP at the tip. The three sample vessels had uniform
thickness of ECP along the length, apart from the tip 27, which had
different adjustment to the tip diameter.
[0125] FIG. 7 illustrates the mean, tip and centre internal
capillary temperatures at a thermopile setting of 43.degree. C.
[0126] FIG. 8 illustrates the mean, tip and centre internal
capillary temperatures at a thermopile setting of 63.degree. C.
[0127] Both sets of results show that when the tip diameter was
reduced, causing a tapering of the ECP, the different between the
temperature at the tip and centre was also reduced.
[0128] FIG. 9 shows the effect of tapering the ECP on the
temperature gradient inside the capillary, showing results for
thermopile settings at both 43.degree. C. and 63.degree. C. It can
be seen that reducing the tip diameter significantly reduces the
temperature difference between the tip and the centre and that the
effect is increased for higher temperatures.
[0129] This effect of minimising temperature gradients by altering
the ECP profile can be further enhanced by the use of a layer of
highly thermally conducting material, for example aluminium as
described in the previous embodiments.
[0130] The applicants have discovered that the profile of ECP can
be adjusted to increase the thermal gradient. FIG. 9 shows that the
temperature gradient inside the capillary is increased when the tip
diameter is increased.
[0131] A temperature gradient caused by profiling ECP can be
created as described in the following example.
EXAMPLE
[0132] A 1 mm thick sheet of ECP (carbon black filled polyethylene)
was cut into the shape of an elongated equilateral triangle with
the tip cut off, having dimension of 90 mm long, 20 mm wide at the
base and 4 mm wide at the tip. This was connected to a laboratory
power supply using crocodile clips that were attached at either
end.
[0133] A ruler was used to measure 10 mm steps (from the narrow end
to the wide end) starting at 10 mm from the narrow end and
finishing at 70 mm from the narrow end. The ECP was placed on a
foam block and temperature at each step was measured by placing a
thermocouple probe on the surface with a small pad of foam on the
top.
[0134] The temperature measurements are shown in FIG. 10. As can be
seen, a temperature gradient is formed between the higher
temperature at the narrow end and the lower temperature at the
wider end.
* * * * *