Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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"Heating specimen carriers
The present invention relates to heating and more particularly to the thermal
cycling of specimen carriers.
In many fields specimen carriers in the form of support sheets which may have
a multiplicity of wells or impressed sample sites, are used for various
processes
where small samples are heated or thermally cycled.
A particular example is the Polymerase Chain Reaction method (often referred
to
as PCR) for replicating DNA samples. Such samples require rapid and accurate
thermal cycling, and are typically placed in a multi-well block and cycled
between
several selected temperatures in a pre-set repeated cycle. It is important
that the
temperature of the whole of the sheet or more particularly the temperature in
each
well be as uniform as possible.
The individual samples are normally liquid solutions, typically between 1 l
and
200 1 in volume, contained within individual sample tubes or arrays of sample
tubes that may be part of a monolithic plate. It is desirable to minimise
temperature differentials within the volume of an individual sample during
thermal
processing. The temperature differenfials that may be measured within a liquid
sample increase with increasing rate of change of temperature and may limit
the
maximum rate of change of temperature that may be practically employed.
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Previous methods of heating such specimen carriers have involved the use of
attached heating devices such as wire, strip and film elements and Peltier
effect
thermoelectric devices, or the use of indirect methods where separately heated
fluids are directed into or around the carrier
The previous methods of heating suffer from the disadvantage that heat is
generated in a heater that is separate from the specimen carrier that is
required to
be heated.
The thermal energy must then be transferred from the heater to the carrier
sheet,
which in the case of an attached heater element occurs, through an insulating
barrier and in the case of a fluid transfer mechanism occurs by physically
moving
fluid from the heater to the sheet.
The separation of the heater from the block introduces a time delay or "lag"
in the
temperature control loop. That is to say that the application of power to the
heating elements does not produce an instantaneous or near instantaneous
increase
in the temperature of the block. The presence of a thermal gap or barrier
between
the heater and the block requires the heater to be hotter than the block if
heat
energy is to be transferred from the heater to the block. Therefore, there is
a
further difficulty that cessation of power application to the heater does not
instantaneously stop the block from increasing in temperature.
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The lag in the temperature control loop will increase as the rate of
temperature change of the
block is increased. This can lead to inaccuracies in temperature control and
limit the practical
rates of change of temperature that may be used.
Inaccuracies in terms of thermal uniformity and further lag may be produced
when attached
heating elements are used, as the elements are attached at particular
locations on the block
and the heat produced by the elements must be conducted from those particular
locations to
the bulk of the block. For heat transfer to occur from one part of the block
to another, the first
part of the block must be hotter than the other.
Another problem with attaching a thermal element, particularly a Peltier
effect device, is that
the interface between the block and the thermal device will be subject to
mechanical stresses
due to differences in the thermal expansion coefficients of the materials
involved. Thermal
cycling will lead to cyclic stresses that will tend to compromise the
reliability of the thermal
element and the integrity of the thermal interface.
More recently our PCT application WO 97/26993 published July 31, 1997 has
disclosed a
novel method where the specimen carrier is metallic and direct electrical
resistive heating is
applied to the metallic specimen carrier. The Specification of the aforesaid
PCT application
discloses various features of heating the carrier.
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Now, a problem with heating samples in sample wells of such a carrier is that
agitation or
stirring is sometimes desirable. The present invention aims to solve that
problem.
Accordingly the invention provides a method of heating a specimen carrier in
the form of a
metallic sheet and in which a matrix of sample wells is incorporated in the
sheet,
which method includes applying an alternating current to said sheet to provide
heating of the
samples in the wells, and
a magnet is loosely contained within at least one well and is arranged to be
agitated by the
alternating current so as to provide a stirring action during the heating.
Usually, but not
necessarily always, each well will contain a magnet.
The sheet may be of silver or similar material of high thermal and electrical
conductivity and
will generally have a thin section in the region of 0.3mm thickness, where the
matrix of
sample wells is incorporated in the sheet. The sample wells may incorporate
samples directly
or may carry sample pots or test tubes shaped to closely fit within the wells.
The sheet may have an impressed regular array of wells to form a block and a
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basal grid or perforated sheet may be attached to link the tips of the wells
at their
closed ends to form an extremely rigid three-dimensional structure. In some
applications the mechanical stiffness of the block is an important
requirement.
Where a basal grid is used, heating current is also passed through the metal
of the
5 grid. The basal grid is preferably made of the same metal as the block.
While the metallic sheet may be a solid sheet of silver (which may have
cavities
forming wells) an alternative is to use a metallised plastic tray (which may
have
impressed wells), in which deposited metal forms a resistive heating element.
Another alternative is to electro form a thin metal tray (which again may have
impressed wells), and to coat the metal with a bio-compatible polymer.
These measures enable intimate contact to be achieved between the metallic
heating element and the bio-compatible sample receptacles. This gives greatly
improved thermal performance in terms of temperature control and rate of
change
of temperature when the actual temperatures of the reagents in the wells is
measured.
The plastic trays - are conventionally single use disposable items. The
incorporation of the heating element into the plastic trays may increase their
cost,
but the reduction in cycling time for the PCR reaction more than compensates
for
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any increased cost of the disposable item.
The bottom of the composite tray should be unobstructed if fan cooling is
employed. If sub-ambient cooling is required at the end of the PCR cycles,
either
with a composite tray or a block, chilled liquid spray-cooling may be
employed.
The boiling point of the liquid should be below the low point of the PCR cycle
so that liquid does not remain on the metal of the tray or block to impede
heating.
This also allows for the latent heat of evaporation of the liquid to increase
the
cooling effect.
The heating current may be an alternating current supplied by a transformer
system wherein the heating power is controlled by regulating the power
supplied
to the primary winding of the transformer. The sheet to be heated may be made
part of the transformer secondary circuit. The secondary winding may be a
single
or multiple loop of metal that is connected in series with the sheet. By these
means, the high current, low voltage power that is required to heat the highly
conductive sheet may be simply controlled by regulating the high voltage, low
current power supplied to the primary winding of the transformer.
The transformer may comprise a toroidal core having an appropriate mains
primary winding and a single bus bar looped through the core and connected in
series with the metallic sheet to form a single turn secondary circuit.
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Generally the sample wells may be conical in shape. This helps any stirring
action
of each magnet within the respective well.
More specifically, in direct resistance heating using alternating current, an
oscillating magnetic field is produced at each well by the heating current. A
small
bar magnet, (typically 5mm long by 1mm diameter), may be placed in each
sample tube and the heating current will cause oscillating forces to be
applied to
the magnet. The geometry of the conical section of the sample tube will then
constrain the bar to spin about an axis that is not coaxial with, or normal
to, the
axial dimension of the bar. The stirring action is then similar to that which
would
be produced by vigorously stirring each individual tube with a manual stirring
rod.
The magnets may be made of readily available materials, in particular hard
magnetic alloys such as Alnico 4. Rare earth magnets (for example
iron-neodymium-boron or samarium-cobalt) may also be used. To prevent
contamination of the liquid sample, the magnet may be given an inert coating.
Such a coating may be of a bio-compatible polymer such as polypropylene or
polycarbonate, or a noble metal such as gold. A noble metal coating has the
advantage that it adds no significant volume to the magnet when applied in a
coating of sufficient thickness to ensure that the coating is not porous. When
using
gold a 5 m thickness is sufficient to provide a pore-free coating, and adds a
volume of 0.08 1 to the magnet.
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The magnets cost much less than the typical reagent mix to be placed in a
sample
tube, and may therefore be regarded as consumable items. However the magnets
may clearly be easily sorted from the waste reagents for cleaning and re-use.
The magnets may be small. In particular embodiments, for a 100 1 liquid
sample,
a magnet lmm in diameter and 5mm long may be employed. Such a magnet has
a volume of 3.9 l. A 0.5mm diameter by 3mm long magnet may be provided for
use in smaller tubes and would have a volume of 0.58 l. The approximate masses
of these magnet examples would be 31mg and 4.5mg respectively.
In certain embodiments, a magnet is placed in each of the wells to be
agitated. In
standard practice the shape of the individual wells is conical and the magnet
length is chosen such that the long axis of the bar magnet is constrained to
be
within a range of between 5 and 30 degrees of the axis of the well. Such
orientation ensures that the agitation magnet will spin eccentrically and will
not
jam in the well. The diameter of the magnet should be as small as is
practical, in
order to minimise the volume of the magnet. The passage of the alternating
heating current through the block gives rise to an alternating magnetic field
circling the block in a plane normal to the direction of current flow. The
alternating magnetic field causes aliernating forces to be applied to the bar
magnets as they try to align themselves with the magnetic field. The conical
shape
of the wells constrains the movement of the magnets, which then spin
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eccentrically in each well.
The effect of the eccentric spinning of the magnets is to vigorously stir the
liquid
sample in each of the wells to which a magnet has been introduced. The
stirring
effect almost completely eliminates any of the temperature differentials that
may
be observed in a static sample during thermal cycling.
Preferably, the bottom of the sheet, even if a basal grid is attached, has an
open
structure with a large surface area. Such a surface is ideal for forced-air
cooling.
Moreover, preferably there are no attached elements to impede free and full
contact between the metal of the sheet and moving air.
Ducting of the air may be provided to encourage even cooling effects over the
extent of the sheet. To allow for controlled cooling rates, the air movement
may
be under proportional control. The control response time of a device that
imparts
movement to air, for instance a mechanical element such as a fan, is slow
compared to the fast electronic control response of the heating system. The
heating
system may therefore be used together with the fan to control the temperature
changes of the sheet during cooling.
The secondary winding in series with the sheet may have more than one loop
through the core of the transformer.
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The power supply means and control for the heating current may be a high
frequency AC power supply permitting a reduction in the amount of material in
the transformer core.
5 The thermal uniformity of the sheet will be dependent on the heating power
dissipation at any point in the sheet being matched to the thermal
characteristics
of that point. For instance, a point around the centre of the sheet will be
surrounded by temperature controlled metal, whereas a point at the edge of the
sheet or block will have temperature controlled metal on one side and ambient
air
10 on the other. The geometry of the sheet may be adjusted with the aim of
achieving
thermal uniformity. In general practice the geometry of sample sites or wells
of
a sheet or block will be a standardised regular array. The industry standard
arrays
consist of 48, 96 or 384 wells in a 110 X 75mm rectangular plate or block.
These
layouts are arbitrary and larger arrays of 768 and 1536 wells are appearing.
Typically, the geometric factors that may be varied comprise the thickness of
the
metal from which the sheet is formed, and if a basal grid is used, the
geometry of
the webs in the plane of the grid.
Embodiments of the invention will now be described by way of example with
reference to the accompanying diagrammatic drawings in which:
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Figure I is a side elevation of a heating apparatus;
Figure 2 is a plan view of the apparatus of Figure;
Figure 3 is a side view of sample tubes incorporating magnets and located in
wells
of a sheet of the heating apparatus of Figure 1;
Figure 4 is a top plan view showing the magnet location, and
Figure 5A to 5C shows a perspective, plan and side view of the block specimen
carrier of the apparatus shown in Figure 1.
A metallic sheet specimen carrier in the form of a multi-well block (1)
measuring
110mm x 75mm and having 96 wells (2) disposed in a grid layout is formed in
silver nominally 0.3mm thick. This is attached to bus bars (3) of substantial
cross-sectional area. The bus bars loop once through a transformer (toroidal
or
square), core (4). The core (4) has a primary winding (5) appropriate for the
mains voltage employed. The bus bars (3) also act as a structural member
supporting the block (1). The transformer primary current is controlled using
a
triac device (6). 'The triac device receives current from an AC source and is
controlled by a temperature control circuit (7) which uses at least one fine
wire
thermocouple (8) soldered to a central underside region of the block to sense
the
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temperature of the block. The temperature control circuitry may be operated
manually or by a personal computer (9). More specifically, the heating power
may
be controlled by proportional phase angle triggering of the triac (6) in
response to
signals from the thermocouples (8) combined with programmed temperature / time
information entered to describe the required thermal behaviour of the
apparatus.
Cooling of the block is by means of a fan (10) mounted under the block,
passing
ambient air over the protruding well forms (2), the air being directed by the
enclosure in which the block is mounted. The fan is controlled by the same
temperature control circuitry that drives the heater triac. Although not shown
in
detail, the airflow is guided to give even cooling of the block (1) by means
of
multiple shaped air inlets on the top, sides and bottom of the apparatus
enclosure.
The fan extracts air from the inside of the enclosure. The negative pressure
within
the case is varied proportionally by proportionally controlling the fan speed.
It will be appreciated that the rear surface of the block (1) has a large
surface area
which is ideally suited to the dissipation of heat.
The measured performance of the example apparatus gives rates of change of
temperature in excess of 6 degrees per second and over/under shoots of less
than
0.25 degrees within the typical PCR working range of 50-100 degrees. The
thermal uniformity of the block is such that within 10 seconds of any
temperature
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transition, even at rates of change of temperature in excess of 6 degrees
Celsius
per second, the range of temperatures that may be measured in wells around the
block does not vary more than +/- 0.5 degrees from the mean temperature.
The block (1) of the present embodiment will have an electrical resistance of
around 0.00015 Ohms. To obtain the levels of heating desired, a current in the
order of 1600A is supplied to the block. The order of this required current is
easily calculable on the basis of the size of the block and the innate
properties of
silver. The current in the primary winding (5) might be up to around 3A at
240V
or 7A at 110V. Thus even though high current is supplied across the block (1),
the
voltage across the block remains low, say 0.25V. Further, the block (1) and
bus
bars (3) are isolated from mains power and may be connected to ground to
enhance safety further.
The described example uses a silver block with cavities, but metallised
plastic tray
inserts, or electro formed thin metal trays, as previously described, may also
be
used.
The system as described has several important advantages.
1.1 The block is heated directly with no requirement for heat transfer
from an attached heat source. This is very efficient and taken together with
the
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very low specific heat capacity of silver allows very rapid temperature
changes.
1.2 Direct heating means that there is no thermal lag at all. Temperature
control functions are immediate so that the block may be cycled in temperature
with little or no over or undershoot. Temperature control is therefore
inherently
precise.
1.3 Since there are no obstructions or thermal barriers attached to the
block, simple forced-air cooling of the back of the block provides rapid and
controllable cooling.
1.4 The fine wire thermocouple is soldered directly to the block so as to
provide close temperature measurement and control. Any other temperature
measurement device may be used as long as it does not introduce significant
sensor lag.
1.5 The temperature distribution around the surface of the block is
dependent on the evenness of heating and the thermal conductivity of the
block.
The thermal conductivity of silver is very high, and the distribution of heat
energy
around the block is -dependent upon the distribution of the heating current.
This
may be regulated by varying the geometry of the multi-well block. The
variation
in geometry will typically be achieved by spatial variation in the thickness
of the
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block (1) such that, (for instance), the minimum metal thickness (of about
0.25mm), may be found at the middle of the block surface and the maximum
metal thickness (of about 0.4mm), may be found along the edges of the block
(1)
parallel to the longer axis. The variations in metal thickness are used to
maintain
5 thermal uniformity across the area of the block during thermal cycling by
compensating for the differing thermal environments experienced by different
points in the block (1).
The variations in metal thickness are produced whilst manufacturing the block
by
10 electroforming. During the electroforming process the distribution of the
electrodepositing current is modulated such that the depositing current is
higher
in areas where a greater thickness of metal is required.
The overall geometry of the block is standardised to accept liquid samples of
15 20-100 l contained in either individual 200 1 sample tubes or arrays of
samples
contained in a 96 well microplate.
The large currents required may be easily produced and controlled since the
block
becomes part of a heavy secondary circuit of the transformer. The cross-
sectional
area of the winding- bars is made considerably larger than the cross-sectional
area
of the block so that significant heat generation only occurs in the block. The
current can be easily controlled in the primary winding (where the current is
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small), using thyristors, triacs or other devices. Alternatively, the primary
winding
may be driven by a high frequency, switch mode, controllable power supply.
This
allows the same degree of control of the current induced in the secondary
winding
incorporating the block, but the high frequency allows the use of a more
compact
core in the transformer.
Referring now to Figures 3 and 4, a novel stirring arrangement is shown. A
sample carrier (1)(which is equivalent to the block (1) described above) has
conical cavities (12) carrying 200 1 sample tubes (13). Then, within each tube
is
loosely carried a magnet (14).
Each is a small bar magnet, (typically 5mm long by 1mm diameter), which is
placed in each sample tube and the heating current is then able to cause
oscillating
forces to be applied to the magnet. The geometry of the conical section of the
sample tube will then constrain the bar to spin about an axis that is not
coaxial
with, or normal to, the axial dimension of the bar. The stirring action is
then
similar to that which would be produced by vigorously stirring each individual
tube with a manual stirring rod.
The magnets can be made of readily available materials such as Alnico 4 and
coated with non-reactive materials such as polypropylene or PTFE or nobel
metals
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such as gold, for example a 5 m layer of acid hard gold plating may be used.
The
magnets cost much less than the typical reagent mix to be placed in a sample
tube,
and may therefore be regarded as consumable items. However the magnets may
clearly be easily sorted from the waste reagents for cleaning and re-use.
The magnets are small, lmm diameter by 5mm long which gives a volume of
3.92 l for use in a 200 l sample tube. A 0.5mm diameter by 3mm long magnet
for use in smaller tubes has a volume of 0.58 1. The approximate masses of
these
magnets are 31mg and 4.5mg respectively.
The action of the agitation magnets not only removes measurable temperature
differentials from the 100 1 liquid samples used, but also increases the
overall rate
of heat transfer from the block to the sample. Thus the programmed
temperature/time profile is more accurately reproduced in the thermal
processing
experienced by the liquid sample.
Figures 5A to 5C show the sample carrier sheet (block) (1) of Figures 1, 2 and
4
in more detail. As desribed above this metallic specimen carrier is in the
form of
a multi-well block (1). This block (1) measures 110mm X 75mm and has an 8 X
12 array of standardised conical wells 12mm deep and is formed in silver
having
an average metal thickness of 0.33mm. An attached basal grid may also be
provided which ties together to exterior bottoms (101) of the wells.
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It will be seen that the wells in the sheet (1) have a significant depth and
thus
include side walls (102) and have an overall generally frustoconical shape.
The
wells are arranged to accept and surround a significant portion of any sample
tubes positioned in the wells. This can help in the efficient transfer of heat
into
and/or out of samples. A large surface area of tube is in contact with the
sheet (1).
Furthermore, in cooling it will be noted that this large area of tube is in
direct
contact with a portion of the sheet, ie the exterior or underside of the
wells, over
which ambient air is fed.
Similar considerations also apply if samples are placed directly in the sheet
rather
than in a sample tube.
It has been found that mains frequency currents eg 50Hz provide a good
stirring
effect.
The fact that the rear of the carrier sheet is exposed can lead to various
other
advantages, in particular other apparatus may be located behind the sheet
and/or
access to the rear of the sheet is easy to obtain. In a particular
alternative, a
method and apparatus for realtime analysis or monitoring of reactions
occurring
in the sample sites during heating and/or stirring can be provided. This may
be
implemented by providing a optical probe in each sample site or well,
typically
this probe will be the tip of a optical fibre which is located in an aperture
towards
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the base of the well. The fibre in each well will lead away from the rear (or
underside) of the sheet to suitable transmitter, receiver and analysis
equipment.
The monitoring will typically make use of the fact that the fluorescing
characteristics of the reagents change as the reaction progresses. Thus an
exciting
frequency of light will be fed from the transmitter along the fibres to each
well.
This exciting frequency will cause fluorescence in the reagents and the
emitted
light will travel back along the fibres to the receiver and analysis equipment
where
the fluorescence or changes in fluorescence will be analysed to give an
indication
of the state of the reaction.
