Note: Descriptions are shown in the official language in which they were submitted.
CA 02464973 2004-05-14
IMPROVEMENTS IN THERMAL CYCLER FOR PCR
FIELD OF THE INVENTION
This invention pertains to the field of computer controlled instruments for
performing the Polymerase Chain Reaction (PCR). More particularly, the
invention
pertains to automated instruments that perform the reaction simultaneously on
many
samples and produce very precise results by using thermal cycling.
BACKGROUND OF THE INVENTION
The background of the invention is substantially as stated in U.S. Patent No.
5,475,610.
To amplify DNA (Deoxyribose Nucliec Acid) using the PCR process, it is
necessary to cycle a specially constituted liquid reaction mixture through
several
different temperature incubation periods. The reaction mixture is comprised of
various components including the DNA to be amplified and at least two primers
sufficiently complementary to the sample DNA to be able to create extension
products
of the DNA being amplified. A key to PCR is the concept of thermal cycling:
alternating steps of melting DNA, annealing short primers to the resulting
single
strands, and extending those primers to make new copies of double-stranded
DNA. In
thermal cycling the PCR reaction mixture is repeatedly cycled from high
temperatures
of around 90°C for melting the DNA, to lower temperatures of
approximately 40°C to
70°C for primer annealing and extension. Generally, it is desirable to
change the
sample temperature to the next temperature in the cycle as rapidly as
possible. The
chemical reaction has an optimum temperature for each of its stages. Thus,
less time
spent at non optimum temperature means a better chemical result is achieved.
Also a
minimum time for holding the reaction mixture at each incubation temperature
is
required after each said incubation temperature is reached. These minimum
incubation times establish the minimum time it takes to complete a cycle. Any
time
in transition between sample incubation temperatures is time added to this
minimum
cycle time. Since the number of cycles is fairly large, this
CA 02464973 2004-05-14
2
additional time unnecessarily Heightens the total time needed to complete the
amplification.
In some previous automated PCR instruments, sample tubes are inserted into
sample wells on a metal block. To perform the PCR process, the temperature of
the metal
block is cycled according to prescribed temperatures and times specified by
the user in a
PCR protocol file. The cycling is controlled by a computer and associated
electronics.
As the metal block changes temperature, the samples in the various tubes
experience
similar changes in temperature. However, in these previous instruments
differences in
sample temperature are generated by non-uniformity of temperature from place
to place
within the sample metal block. Temperature gradients exist within the material
of the
block, causing some samples to have different temperatures than others at
particular times
in the cycle. Further, there are delays in transferring heat from the sample
block to the
sample, and those delays differ across the sample block. These differences in
temperature
and delays in heat transfer cause the yield of the PCR process to differ from
sample vial
to sample vial. To perform the PCR process successfully and eff ciently, anti
to enable
so-called quantitative PCR, these time delays and temperature errors must be
minimized
to the greatest extent possible. The problems of minimizing non-uniformity in
temperature at various points on the sample block, and time required for and
delays in
heat transfer to and from the sample become particularly acute when the size
of the.region
containing samples becomes large as in the standard 8 by I2 microtiter plate.
Another problem with current automated PCR instruments is accurately
predicting
the actual temperature of the reaction mixture during temperature cycling.
Because the
chemical reaction of the mixture has an optimum temperature far each of its
stages,
achieving that actual temperature is critical for good analytical results.
Actual
measurement of the temperature of the mixture in each vial is impractical
because of the
small volume of each vial and the large number of vials.
CA 02464973 2004-05-14
SUMMARY OF THE INVENTION
According to the invention; there is provided an apparatus for performing the
Polymerase Chain Reaction comprising an assembly capable of cycling samples
through
a series of temperature excursions, a heated cover and a computer to control
the process.
The invention further encompasses a sample block with low thermal mass for
rapid temperature excursions. The sample block is preferably manufactured from
silver
for uniform overall heat distribution and has a bottom plate for uniform
lateral heat
distribution. In addition, to further offset heat losses and resulting
temperature gradients
from tl~e center to the edges, a center pin is used as a conducting path to a
heat sink.
The invention also provides a method and apparatus for achieving rapid heating
and cooling using Pettier thermoelectric devices. These devices are precisely
matched to
each other. They are constructed using die cut alumina on one side to minimize
thermal
expansion and contraction. The devices are constructed of bismuth telluride
using
specific dimensions to achieve matched heating and cooling rates. They are
designed
using minimal copper thicknesses and minimal ceramic thicknesses to further
reduce their
heat load characteristics and are asserrzbIed using a specific high
temperature solder in
specified quantities.
The invention is also directed fo a heatsink constructed with a perimeter
trench to
limit heat conduction and losses from its edges. Furthermore, the heatsink has
an
associated variable speed fan to assist in both maintaining a constant
temperature and in
cooling.
The invention is also directed to a clamping mechanism to hold the sample
block
to the heat sink with the thermoelectric devices positioned in between. The
mechanism is
designed to provide evenly distributed pressure with a minimal heat load.. The
design
allows the use of thermal grease as an interface between the sample block, and
the
thermoelectric devices and between the thermoelectric devices and the
heatsink.
There is also provided a perimeter heater to minimize the thermal non-
uniformity
across the sample block. The perimeter heater is positioned around the sample
block to
counter the heat loss from the edges. Power is applied to the heater in
proportion to the
CA 02464973 2004-05-14
4
sample block temperature with more power applied when the sample block is at
higher
temperatures and less power applied when the sample block is at lower
temperatures.
There is also provided a heated cover, designed to keep the sample tubes
closed
during cycling and to heat the upper portion of the tubes to prevent
condensation. The heated
cover applies pressure on the sample tube cap perimeter to avoid distorting
the cap's optical.
qualities. The cover is self aligning, using a skirt which mates with a sample
tube tray.
The invention is also directed to a method and apparatus for determining an
ideal
temperature ramp rate which is determined so as to take advantage of sample
block
temperature overshoots and undershoots in order to minimize cycle time.
The invention also includes a method and apparatus for characterizing the
thermal
power output from the thermoelectric cooling devices to achieve linear
temperature control
and linear and non-linear temperature ramps.
The invention is further directed to a method for predicting the actual
temperature of
the reaction mixture in the sample vials at any given time during the PCR
protocol.
I S The invention also includes a method and apparatus for utilizing
calibration
diagnostics which compensate for variations in the performance of the
thermoelectric devices
so that all instruments perform identically. The thermal characteristics and
performance of
the assembly, comprised of the sample block, thermoelectric devices and
heatsink, is stored
in an on-board memory device, allowing the assembly to be moved to another
instrument and
behave the same way:
The invention further includes a method and apparatus for measuring the AC
resistance of the thermoelectric devices to provide early indications of
device failures.
According to an aspect of the present invention, there is provided a sample
block for
holding sample vials comprising:
a plurality of sample wells, for receiving sample vials, each well having a
top and
bottom;
an upper support plate connecting the tops of said sample wells; and
a bottom plate connecting the bottom of said sample wells:
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a cross sectional view of a portion of the sample block according
to the invention.
Figure 2 is an enlarged, isometric view of a thermoelectric device constructed
according to
the invention.
CA 02464973 2004-05-14
Figure 2A is a side, elevational view of a thermoelectric device constructed
according to
the invention.
Figure 3 is a cut-away, partial, isometric view of the heatsink according to
the invention.
Figure 4 is an exploded view of an assembly including a sample block,
thermoelectric
5 devices and heatsink.
Figure 5 is an isometric view of the heated cover in accordance with the
invention.
Figure 6 is a chart depicting the Up Ramp (heating rate) vs. Power.
Figure 7 is a chart depicting the Down Ramp (cooling rate) vs. Power.
Figure 8 is a chart for predicting and compensating for temperature overshoots
and
undershoots in accordance with the invention.
Figure 9 is a block diagram of the AC resistance measurement circuit of the
invention.
Figure 10 shows a perimeter heater and its location surrounding the sample
block.
Figure 11 is a detailed view of the perimeter heater of Figure 10.
Figure 12 shows the power applied to the perimeter heater as a function of the
temperature
of the sample block.
Figure 13 shows a thermal model of a sample in a sample vial.
Figure 14 is an illustration of the initial conditions of the thermal model of
Figure 13.
Figure 15 shows the sample block and a seal designed to protect the thermal
electric
devices from the environment.
DETAILED DESCRIPTION OF THE INVENTION
Generally, in the case of PCR, it is desirable to change the sample
temperature
between the required temperatures in the cycle as quickly as possible for
several reasons.
First the chemical reaction has an optimum temperature for each of it's stages
and as such
less time spent at non-optimum temperatures means a better chemical result is
achieved.
Secondly a minimum time is usually required at any given set point which sets
a
minimum cycle time for each protocol and any time spent in transition between
set points
adds to this minimum time. Since the number of cycles is usually quite large,
this
transition time can significantly add to the total time needed to complete the
amplification.
CA 02464973 2004-05-14
6
The absolute temperature that each reaction tube attains during each step of
the
protocol is critical to the yield of product. As the products are frequently
subjected to
quantitation, the product yield from tube to tube must be as uniform as
possible and
therefore both the steady-state and dynamic thermal uniformity must be
excellent across
the block. .
Heat-pumping into and out of the samples is accomplished by using Peltier
thermoelectric devices. These are constructed of pellets of n-type and p-type
bismuth
telluride connected alternately in series. The interconnections between the
pellets is made
with copper which is bonded to a substrate, usually a ceramic (typically
alumina).
The amount of heat-pumping required is dependent on the thermal load and the
ramp rate, that is, the rate at which the temperature is required to change.
The sample
tube geometry and sample volumes are not variables as the sample tubes are
established
as an industry standard, fitting into many other types of instruments such as
centrifuges.
The sample volume- is defined by user need. Therefore the design variables
primarily
affect the sample block, thermoelectric devices, heatsink, fan and the thermal
interface
media between the thermoelectric devices and both the heatsink and the sample
block.
The block geometry must also meet the necessary thermal uniformity
requirements because it is the primary contributor to lateral conduction and
therefore
evens out any variation in thermal uniformity of the thermoelectric coolers
themselves:
The conflicting requirements of rapid ramp rates (indicating low thermal mass)
and high
lateral conduction (indicating a large material mass) are met by concentrating
the bulk of
the block structure in a base plate, and minimizing the thermal mass of the
upper portion
of the block which holds the sample tubes. The optimal material for block
fabrication is
pure silver which has relatively low thermal mass and very good thermal
conduction.
Silver also lends itself well to electroforming. Fn practice the optimal block
geometry has
a light clcctroformcd upper portion to hold the sample tubes fxed to a
relatively thick
base plate which provides lateral conduction. The thermal mass of the block is
concentrated in the base plate where the material contributes the most to
thermal
uniformity. The electroforrned portion of the block has a minimum thickness
which is
defined by two parameters: first, the material cannot be so thin as to make it
too delicate
CA 02464973 2004-05-14
7
far normal handling; second, the wall thickness is required to conduct heat
out of the
upper regions of the sample tube. Circulation in the sample itself is achieved
by
convection inside the tube and sample temperature is relatively uniform along
the height
of the tube, but good thermal conductivity between the tube walls and the base
plate
increases the effective surface area available for conduction of heat between
the sample
and the base plate. The base plate thickness has a minimum value defined by
lateral
conduction requirements which is a function of the thermal uniformity of the
thermoelectric coolers and structural rigidity.
Another contributor to the thermal mass is the alumina ceramic layers which
form
part of the structure of the thermoelectric cooler itself. There are two
alumina layers in
the construction of the thermoelectric cooler, one on the sample block side
and another on
the heatsink side. The thickness of the layers should be minimized as much as
possible,
in this case the practical limit of thinness for the alumina thickness is
defined by the
manufacturing reduirements of thermoelectric cooler fabrication. This
particular layer of
ceramic could in principal be replaced by a different layer altogether such as
a thin sheet
of Kapton which would reduce the thermal mass even more, but at the present
time
although coolers are available with this structure, reliability is unproven.
It is anticipated
that once the technology has been developed further, then a cooler of such a
design may
be preferred. 1-lowever, the thin alumina layers also contribute to system
reliability.
The copper conductors within the cooler are a significant thermal load and are
not
overlooked in the design of the system. The thickness of the copper traces is
defined by
the requirement of carrying current through the device. Once the current is
known the
required copper thickness can be calculated.
Samtslc Block
Figure 1 shows a cross sectional view of a portion of the sample block 3b
which
typically lzas 96 wells 20, each for receiving a sample vial. The sample block
is
constructed of silver and comprises an upper support plate 21 and the sample
wells 20
electroformed as one piece fastened to abase plate 22. The base plate 22
provides lateral
conduction to compensate for any difference in the thermal power output across
the
CA 02464973 2004-05-14
surface of each individual thermoelectric device and for differences from one
thermoelectric device to another.
There are always boundary losses in any thermal system. In a rectangular
configuration there is more heat loss in the corners. One solution is to use a
round
sample block, but the microtiter tray format that is in common usage is
rectangular
and this must be used to retain compatibility with other existing equipment.
Once the.
edge effects have been eliminated using all standard means, such as insulation
etc.,
there remains a tendency for the center of the sample block to be warmer than
the
corners. Typically it is this temperature difference that defines the thermal
uniformity
of the sample block. In accordance with the invention, the center temperature
is
reduced by providing a small thermal connection from the center of the sample
block
to the heat sink. By using a pin 24 which acts as a "heat leak" in the center
of the
sample block, the temperature gradient across the sample block can be reduced
to an
acceptable level. The amount of conduction required is quite small and a l.Smm
diameter stainless steel pin has been found to be sufficient. Moreover, a pin
made of
the polymer ULTEMTM, manufactured by General Electric may also be used. As
more fully described below, the pin also serves to help position and lock into
place
components of the assembly illustrated in Figure 4.
Peltier Thermoelectric Devices (TED'S)
Thermal uniformity of the sample block is critical to PCR performance. One
of the most significant factors affecting the uniformity is variations in the
thermoelectric device performance between devices. The most difficult point at
which to achieve good uniformity is during a constant temperature cycle far
from
ambient. In practice this i.s a constant temperature cycle at approximately
95°C. The
thermoelectric devices are matched under these conditions to make a set of
devices
for each heatsink assembly which individually produce the same temperature fox
a
given input current. The thermoelectric devices are matched to within
0.2°C in any
given set, this value being derived from the maximum discrepancy that can be
rectified by the lateral conduction of the sample block baseplate.
CA 02464973 2004-05-14
9
Figure 2A shows a side view of a typical Peltier thermoelectric device 60. The
device is composed of bismuth telluride pellets 30, sandwiched between two
alumna
layers 26. The pellets are electrically connected by solder joints 28 to
copper traces 29
plated onto the alumina layers. One alumina layer has an extension 31 to
facilitate
S electrical connections. The thickness of the extended areas is reduced to
decrease the
thermal load of the device.
Figure 2 shows an isometric view of a typical Peltier thermoelectric device.
The
alumina layer 26 that forms the outer wall of the thermoelectric device,
expands and
contracts during temperature cycling at a different rate than the sample block
19. The
motion of the alumina is transmitted directly to the solder 28 connecting the
internal
bismuth telluride pellets 30. This motion can be reduced dramatically by
cutting the
alumina into small pieces 32 called die so that the field of expansion is
small. The
minimum size of the die is defined by the size of the copper traces required
to carry
current through the thermoelectric device and the requirements that the device
retain some
strength for handling.
Using thin alumina layers in the thermoelectric device (of the order of 0.508
mm) not only reduces the thermal Load but also means that for a given required
heat
pumping rate the temperature that the ends of the pellet reaches is reduced
due to the
increase in thermal conductivity k. This enhances reliability by reducing the
thermal
stress on the solder joint.
Generally in PCR the reaction temperatures are above ambient and in the range
35
to 96°C. Tn the most important cases the block is heated or cooled
between two above
ambient temperatures where the flow of heat due to conduction is from the
block to the
heat sink. The key to optimizing the system cycle time, given an optimized
block
configuration, is to balance the boost to the ramp rate when cooling provided
by the
conduction, against the boost provided to the heating ramp rate by the Joule
effect of
resistance heating.
If the cross-section of the bismuth telluride pellets in a given
thermoelectric device
were considered constant, the heating ramp rate would be increased by
increasing the
height of the pellet. This is because the conduction path through the
thermoelectric
CA 02464973 2004-05-14
device would be made longer thereby decreasing k. This also has the effect of
reducing the
current required to maintain a given block temperature in the steady state.
During the
down ramp, i.e. cooling the block, the decreased k means that the conduction
contribution
will be reduced and so the down ramp rate will be reduced.
5 Conversely, if the height of the bismuth telluride pellet were to be
decreased for a
given cross-section, then k would be increased. This would increase the
current required
to maintain an elevated temperature in the steady state and would increase the
cooling
ramp rate. Heating ramp rates would be reduced as a larger portion of the heat
in the
block would be conducted directly to the heat sink. Decreasing.the bismuth
telluride
10 pellet height also increases the holding power required for a given
temperature due to the
losses through the thermoelectric devices and reduces the thermal load,
increasing the
maximum possible ramp rate for given power. Therefore the optimized
thermoelectric
device can be derived by adjusting the height of the Bismuth Telluride pellets
until the
heating rate matches the cooling rate.
The ratio I:A for the pellets also defines the resistance of the device i.e.
R=nr(hlA)
where n is the number of pellets, r is the resistivity of the Bismuth
Telluride being used, h
is the height of the pellet and A is the cross-sectional area.
The resistance must be measured as an AC resistance because of the Seebeck
effect. Because the geometry defines the resistance of the device, another
design
boundary is encountered in that the device must use a cost effective current
to voltage
ratio because too high a current requirement pushes up the cost of the
amplifier. The
balanced solution for tl~e silver electroformed block described above is
Pellet height = 1 .27mm
Pellet cross-sectional area = 5.95mm2
CA 02464973 2004-05-14
I1
If the thermal cycler was to be used as part of another instrument, e.g.
integrated with detection technology, then it may be more convenient to use a
different current source which would lead to a modified thermoelectric device
geometry. The current source in the present embodiment consists of a class D
type
switch-mode power amplifier with a current sending resistor in series with the
device
and ground.
Because the thermoelectric devices are soldered together, excess solder can
wick up the side of the bismuth telluride pellets. Where this occurs, k is
increased
which results in a local cold spot, also called a mild spot. These cold spots
are
reduced in number and severity by application of the minimum amount of solder
during the assembly process of the thermoelectric device. For the same reason,
it is
also necessary to ensure that the solder used to attach the connecting wires
to the
thermoelectric device does not contact the pellet
High temperature solder has been shown to not only have improved high
temperature performance but it is also generally more resistant to failure by
stress
reversals and hence is most appropriate in this application. T'he solder used
in this
invention may be of the type as described in U.S. Patent 5,441,576.
Heatsink
Figure 3 shows the heatsink 34 assembled with the thermoelectric devices 39
and the sample block 36. A locating frame 41 is positioned around the
thermoelectric
devices to align them with the sample block and the heatsink to ensure
temperature
uniformity across the sample block. The frame is composed of Ultem or other
suitable
material and has tabs 43 at its corners to facilitate handling. The heatsink
34 has a
generally planer base 35 and fins 37 extending from base 35. The thermal mass
of the
heat sink is considerably larger than the thermal mass of the sample block and
samples
combined. The sample block and samples together have a thermal mass of
approximately
100 joules/°K and that of the heat sink is approximately 900
joules/°K. This means that the
sample block clearly changes temperature much faster than the heat sink for a
given
amount of heat pumped. In addition the heat sink temperature is controlled
with a
variable speed fan as shown in Figure 9). The temperature of the heat sink is
measured by
CA 02464973 2004-05-14
12
a thermistor 38 placed in a recess 40 within the heatsink and the fan speed is
varied to
hold the heat sink at approximately 45°C which is well within the
normal PCR cycling
temperature range, where maintaining a stable heat sink temperature improves
the
repeatability of system performance. When the block temperature is set to a
value below
ambient then the heat sink is set to the coolest achievable temperature to
reduce system
power consumption and optimize block thermal uniformity. This is accomplished
simply
operating the fan at full speed.
The heat sink temperature measurement is also used by the thermoelectric
device
control algorithm described below in linearizing the thermal output power from
the
IO thermoelectric devices.
The heatsink temperature uniformity is reflected in the uniformity of the
block
temperature. Typically the heatsink is warmer in the middle than it is at the
edges and this
adds to other effects that lead to the corners of the block being the coldest.
A trench 44 is
cut into the heat sink outside the perimeter of the thermoelectric device area
to limit the
conduction of heat and decreases edge losses from the area bounded by the
trench.
Thermal Interface and Clamping Mechanism
Thermoelectric device manufacturers recommend that thermoelectric devices be
held under pressure to improve life-expectancy. (The pressure recommended is
often
def ned by the thermal interface media selected.) The pressure that is
recommended varies
from manufacturer to manufacturer but is in the range of 30 to I00 psi for
cycling
applications.
There are many thermal interface media available in sheet form which can be
used
to act as a compliant layer on each side of the thermoelectric devices, but it
has been
demonstrated that thermal grease gives far superior thermal performance for
this
application. Unlike other compliant sheets which have been shown to require 30
psi or
more even under optimal conditions. Thermal grease does not require high
pressure to
ensure that good thermal contact has been made. Also thermal grease acts as an
effective
lubricant between the expanding and contracting silver block and the
thermoelectric
CA 02464973 2004-05-14
13
device surface, enhancing life-expectancy. Thermalcote II thermal grease
manufactured by
Thermalloy, Inc. may be used.
Because the silver block is relatively flexible and soft it cannot transmit
lateral
clamping pressure very effectively. However, because the thermal interface
media is
S thermal grease, the clamping force required is low.
Figure 4 shows an exploded view of the assembly with the preferred embodiment
of the clamping mechanism. Bach clamp 46 is made up of a series of fingers 48
extending from a spine 49. The fingers 48 are sized, shaped and spaced so as
to fit
between the wells 20 of the sample block 36 and thus apply pressure at a
corresponding
series of points on the base plate 22 of the sample block 36. The open
honeycomb
structure of the electroformed sample wells allows the fingers to be inserted
some
distance into the block, thereby applying the pressure more evenly than~an
edge clamping
scheme would. These fingers apply pressure at a series of local points to
minimize the
contact area between the mass of the clamp and the sample block so that the
clamp does
not add significantly to the thermal load. The clamps are molded from a glass
filled
plastic which has the necessary rigidity for this application. The pressure is
applied by
deforming the fingers with respect to mounting posts SO which may be separate
clamp
structures, but are preferably integrally formed with the clamps 46. The
clamps 46 are
held flush to the surface of the heat sink with a series of screws 52
extending through
corresponding hole 53 in clamps 46 and then into threaded holes 55 in heatsink
34. This
scheme eliminates the necessity to set the pressure with adjustment screws as
the clamps
can simply be tightened down by standard torqueing techniques.
The resulting even pressure distribution ensures that the full area of the
thermoelectric devices is in good thermal contact with the block and the
heatsink reducing
local thermal stresses on the thermoelectric devices.
Figure 4 shows other important features of the invention. A printed circuit
board
82 a memory device 96 for storing data and surrounds the thermoelectric
devices and
provides electrical connections. Alignment pins 84 are seated in holes 86 in
the heatsink
and protrudes through alignment holes 88 to align the printed circuit board
with the
heatsink. The locating frame 41 is positioned around the thermoelectric
devices and has
CA 02464973 2004-05-14
14
a cross beam 90 with a through hold 92. Pin 24 (shown in Figure 1) fits into a
hole (not
shown) in the sample block, extends through hold 92 in the locating frame and
further
extends into hole 94 in the heatsink.
Perimeter Heater
In order to bring the temperature uniformity across the sample block to
approximately ~0.2°C, a perimeter heater is positioned around the
sample block to
eliminate heat losses from its edges. Preferably, the heater is a film type,
having low
mass with inside dimensions slightly larger than the sample block. Figure 10
shows the
perimeter heater 74 and its approximate location surrounding the sample:block
36. The
heater is not fastened in place, it is simply positioned in the ai.r around
the perimeter of
the sample block in order to warm the air in the immediate vicinity.
Figure 1 I shows a detailed view of the perimeter heater 74. The heater is
rectangular as determined by the dimensions of the sample block and is
manufactured so
that it has separate power densities in specific areas to reflect the varying
amounts of heat
loss around the perimeter of the block. Matching lower power density regions
76 (0.73
W/in'-) are located in the center portions of the short sides of the rectangle
and matching
higher power density regions 78 (I .3 W/in2) are located in the longer sides,
extending into
the shorter sides.
As shown in Figure 12, the power applied to the perimeter heater,is regulated
to
correspond to the temperature of the sample block, with more power applied to
the heater
at higher block temperatures and less applied at Iower block temperatures.
Heated Cover:
Figure 5 shows the heated cover 57. The heated cover applies pressure to the
sample vial caps to ensure that they remain tightly closed when the sample is
heated.
Further, pressure transferred to the vials assures good thermal contact with
the
sample block. The cover is heated under computer control to a temperature
above that of
the sample to ensure that the liquid does not condense onto the tube cap and
instead
remains in the bottom of the tube where thermal cycling occurs. This is
described in
CA 02464973 2004-05-14
United States Patent No. 5,475,610, mentioned above. The heated platen 54 in
the present
invention does not press on the dome of the cap but instead presses on the cap
perimeter.
The platen has a surface shaped in this manner so that optical caps are not
distorted by the
application of pressure. Thus, tubes that have been cycled can be directly
transferred to
an optical reader without the need to change the cap.
Because the heated platen has recesses 56 in it to clear the cap domes, there
is a
need to align the plate to the tube positions before applying pressure to
avoid damage to
the tubes. This is accomplished by use of a "skirt" 58 around the perimeter of
the platen
which aligns to the microtiter tray before the plate touches the tube caps. -
The cover has a
sliding mechanism similar to that used on the PYRIS Differential Scanning
Calorimeter
by the Perkin Elmer Corporation allowing the cover to slide back to allow
sample vials to
be inserted into the sample block and forward to cover the sample block and
move down
engage the vials.
Determining the Ideal Ramp Rate:
The optimized ramp rate has been empirically determined to be
4°Clsec. Any
system which has a higher block ramp rate than this cannot fully utilize the
benefits
temperature of overshoots and consequently achieves an insignificant reduction
in cycle
time.
Figure 6 is a chart depicting the Up Ramp (heating rate) vs. Power and Figure
7 is
a chart depicting the Down Ramp (cooling rate) vs. Power.
When heating the block to a temperature above ambient, the Joule heating and
the
Seebeck heat pumping both act to heat the sample block against conduction.
When
cooling the block between two temperatures above ambient, the Seebeck heat
pumping
and coriduction act against the Joule heating. During cooling, significant
power is
required to hold the block temperature steady against the flow of heat out of
the block by
conduction. Therefore even with zero power applied, the block will cool at a
significant
rate. As the current is increased; the Seebeck effect increases the cooling
obtained.
However as the current is increased further the joule effect, which is
proportional to the
square of the current, quickly starts to take over acting against the Seebeck
cooling,
CA 02464973 2004-05-14
16
Therefore a point is reached where applying additional power acts against the
required
effect of cooling. In the heating mode these two effects act together against
conduction
and no ceiling is reached. In practice the heating power vs. input current is
approximately
linear. This is why the design criteria centers around meeting the cooling
rate
requirements; the heating rate can always be achieved by the application of
more power.
Characterizing the Output of the TED's
The following equation describes the total heat flow from the cold side of a
thermoelectric
cooler.
0 = %a* R(taVg)* IZ + t~ *S(~a"g~* I -(k(tavg)*(tc'th~+Qc )
where
t~ - cold side temperature of cooler
th - hot side temperature of cooler
ta,,g - average of t~ and th
R(t) - electrical resistance of cooler as a function
of temperature
S(t) - Seebeck coefficient of the cooler as a function
of temperature
K(t) Conductance of cooler as function of temperature
-
I - electrical current applied to cooler
- total heat flow from the cold side of the cooler
Given a desired heat flow, Q~, and the hot and cold side temperatures, tc and
th, the
equation is solved for I, the current required to produce Q~, The solution of
this equation
is used for three purposes:
1) To achieve linear temperature transitions or ramps.
For linear temperature transitions, constant thermal power is required. To
maintain
constant thermal power when temperatures tc and th are changing, it is
necessary to
solve for I in equation I periodically. The result is the current then applied
to the
CA 02464973 2004-05-14
I7
coolers. To compensate for errors a proportional integral derivative (PID}
control loop is
applied where:
Error input to PID = Set point Rate - Actual Rate
and Output from the PID is interpreted as percent Q
2) To achieve a linear PID temperature set point control algorithm over the
desired temperature range:
Input to the P1D control is the error signal t~ - Set point.
Output from the PID control is interpreted as a % of Qm~.
Equation I is used to-determine the current value, I, which will result in the
% of
Qm;,~ output by the PID control, under the current temperature conditions.
3) To achieve non-linear temperature transitions or ramps where temperature
transitions are defined by the derivative of temperature with respect to time,
dT/dt, as a
function of block temperature.
This function is approximated by a table containing Block temperature .T,
dTfdt
data points in 5 C increments for cooling and by a linear equation for
heating. The small
effect of sample mass on dT/dt profiles, although measurable, is ignored.
Knowing the
total thermal mass, MCP (joules/°K), involved during temperature
transitions, the amount
of thermal power, Q (joules/sec), required to achieve the desired rate
profile, dT/dt
(°K/sec), is given at.any temperature by the following equation:
Q = MCp * dTldt
The solution to equation 1 is used to determine the current value, I, which
will
result in the desired Q under the current temperature conditions. This process
is repeated
periodically during temperature transitions.
Controlling_overshoot and undershoot
There is a practical limit to the ramp rates and the resulting cycle times
that can be
achieved. The sample has a time constant with respect to the block temperature
that is a
function of the sample tube and tube geometry which, because the tube is an
industry
CA 02464973 2004-05-14
I8
standard, cannot be reduced. This means that even if the sample tube wall
temperature is
changed as a step function e.g. by immersion in a water bath, the sample will
have a finite
ramp time as the sample temperature exponentially approaches the set point.
This can be
compensated for by dynamically causing the block to overshoot the programmed
temperature in a controlled manner. This means that the block temperature is
driven
beyond the set point and back again as a means of minimizing the time taken
for the
sample to reach the set point. As the possible ramp-rates increase, the
overshoot required
to minimize the time for the.sarnple to reach the set point gets larger and a
practical limit
is soon reached. This occurs because although the average sample temperature
does not
overshoot the set point, the boundary liquid layer in the tube does overshoot
to some
extent. When cooling to the priming temperature, too great an overshoot can
result in
non-specirc priming. Therefore the best advantage is to be gained in a system
which
utilizes this maximum ramp rate combined with optimized overshoots that are
symmetrical on both up and down ramps.
Figure 8 is a chart far predicting and compensating for temperature overshoots
and undershoots. In order to drive the block temperature beyond the set point
and back
again in a controlled fashion the system first measures the block temperature,
Tbn+1 and
then solves the following equations:
Ts"+, = Ts~ + (Tbn+, - Ts") * 0.174/RC
Tsf" _ (Tb" - Ts~ - mRC)( 1-a ~'"''~~) + mtrn + Tsn
where Tb is the measured block temperature, Ts is the calculated sample
temperature, Tsf
is the final calculated sample terhperature if the block is ramped down at
time tn , R is the
thermal resistance between the sample block and the sample, C is the thermal
capacitance
of the sample, m is the slope of a line defined by the points 'rb and Tsf and
tr is the time
for the sample block to return to the set point if the system caused if to
ramp toward the
set point at the same rate it is was ramping away.
If the resulting Tsfn is within a particular error window around the set point
then
the system causes the sample block to ramp hack to the set point at the same
rate it was
CA 02464973 2004-05-14
19
ramping away. If the resulting Tsfn is outside the particular error window
then the system
causes the sample block to continue to ramp away from the set point at the
same rate.
While ramping back toward the set point the same proportional integral
derivative (PID)
control loop described above is applied.
Determining Sample Temperature
The temperature of a sample in a sample vial is determined by using the model
illustrated in Figure 13 where:
TBIk is the measured baseplate temperature;
TSmp is the calculated sample temperature; y
TPlastic is the calculated plastic temperature;
TCvr is the measured cover temperature;
RI is the thermal resistance of the plastic vial between the block and sample
mixture;
C1 is the thermal capacitance ofthe sample mixture;
R2 and R3 represent the thermal resistance of air in parallel with the plastic
vial between
the sample mixture and the cover; and
C2 is the thermal capacitance of the plastic vial between the sample mixture
and the
cover.
The model above is solved for TSmp(t) and TPlastic(t) given that: TBlk = mt +
TBlkO, TCvr = K and initial conditions are non-zero. Taking initial conditions
and the
slope of TBIk to be the only variables, as illustrated in Figure 14, the
equations are
refactored giving equations for 'Tsrnp and TPIastic.
Given the following relationships:
g1 = I/ItI;
g2 = 1182; .
g3 = 1 /R3 ;
a = (g 1 + g2)/C 1;
b= g2/Cl;
f=g2/C2;
CA 02464973 2004-05-14
g = {g2 + g~) / C2;
alpha = -(-g/2 -alt - (sqrt(g*g - 2*g*a + a*a +4*f*b))/2); and
beta = -(-gl2 -alt + (sqrt(g*g - 2*g*a + a*a +4*f*b))/2),
the coefficients for the sample temperature equation become:
coefl = (g3/C2)*(-b/(beta*(alpha-beta))*exp(-beta*T) + bl(alpha*beta) +
(b/(alpha*(alpha-beta)))*exp(-alpha*T))
coef2 = (b/(alpha-beta))*exp(-beta*T) - (bl(alpha-beta)}*exp(-alpha*T)
coef3 = (gl/Cl)*(gl(alpha*beta) + (-alpha+g)*exp(-alpha*T}!(alpha*(alpha-
beta)) +
(beta-g)~'exp(-beta*T)I(beta*{alpha-beta))}
coef4 = (gl/C1)*((g-beta)*exp(-beta*T)/(pow(beta,2)*(alpha-beta)) -
g/(beta*pow{alpha,2)) + (1+ T*g)/(aipha*beta) + (-g+alpha)*exp(-
alpha*T)/(pow(aIpha,2)*(alpha-beta)) - gl(alpha*pow(beta,2))}
coef5 = (-g+alpha)*exp{-alpha*T')/(alpha-beta) + (g beta)*exp(-beta*T)/(alpha-
beta)
and the coefficients for the plastic vial temperature equation become:
coef6 = (g3/C2) * ((beta - a)*exp{-beta*T)l(beta*{alpha-beta)) +
a/(alpha*beta) + (-
alpha+a) * exp{-alpha*T')/{alpha* (alpha-beta))}
coef7 = {-beta+a)*exp(-beta*T)/(alpha-beta) + (alpha-a)*exp(-alpha*T)/(alpha-
beta)
coef8 = (gllC1 ) * (f*exp(-beta* T)l(pow(beta,2)*(alpha-beta)) -
(/(beta*pow(alpha,2)) -
f* exp(-alpha*T}/(pow(aIpha,2)*{alpha-beta)) + T*f/(alpha*beta) -
.f/(alpha*pow(beta,2)))
coef~ = (gl/C1) * (-f*exp(-beta*T)I(beta*(alpha-beta)) + (/(alpha*beta) +
f*exp(-
aIpha*T)/(algha*(alpha-beta)))
coefl0 = f*exp{-beta*T)/(alpha-beta) - f'''exp(-alpha*T)I(alpha-beta)
and
slope = (TBtk-TBlkO)lT where T is the sampling period ~?.174sec)
Utilizing the model in Figure I 3 then, .
TSmp = coefI *TCvrO + coef2*TPlastic0 + coef3*TBlkO + coef4*slope +
coef5*TSmpO
CA 02464973 2004-05-14
21
TPlastic = coef6*TCvrO + coef7*TPlastic0 + coef8*slope + coef3*TBllcO +
coefl0*TSmpO
The coefficients are recalculated at the beginning of each PCR protocol to
account
for the currently selected sample volume. TSmp and TPlastic are recalculated
for every
iteration of the control task.
To determine the sample block set point, TBIkSP, during a constant temperature
cycle, Tblk is determined using the equation for TSmp.
TblkO = (Tsmp - coefl*TCvrO - coef2*TPlastic0 - coef4*slope -
coef5*TSmpO)/coef3
When maintaining a constant temperature the slope = 0 and Tsmp = TsmpO
TSmpSP (sample temperature set point) and:
TBIkSP = (TSmpSP - coefl*TCvr - coef2*TPlastic - coef5*TSmpSP)/coef3
The equation for TBIkSP is solved on every pass of the control loop to update
the
sample block set point to account for changes in temperature of the plastic
and cover.
Calibration Diagnostics:
The control software includes calibration diagnostics which permit variation
in
the performance of thermoelectric coolers from instrument to instrument to be
compensated for so that all instruments perform identically. The sample block,
thermoelectric devices and heatsink are assembled together and clamped using
the
clamping mechanism described above. The assembly is then ramped through a
series of
known temperature profales during which its actual performance is compared to
the
specified performance. Adjustments are made to the power supplied to the
thermoelectric
devices and the process is repeated until actual performance matches the
specification. The
thermal characteristics obtained during this characterization process are then
stored in a
CA 02464973 2004-05-14
22
memory device residing on the assembly. This allows the block assembly to be
moved
from instrument to instrument and still perform within specifications.
AC Resistance Measurement:
The typical failure mode for the thermoelectric devices is an increase in
resistance caused by a fatigue failure in a solder j oint. This results in an
increase in the
temperature of that joint which stresses the joint further, rapidly leading to
catastrophic
failure. It has been determined empirically that devices that exhibit an
increase in AC
resistance of approximately 5% after about 20,000 to 50,000 temperature cycles
will
shortly fail. The AC resistance of the thermoelectric devices are monitored by
the
instmment to detect imminent failures before the device in question causes a
thermal
uniformity problem.
This embodiment automates the actual measurement using a feedback control
system and eliminates the need to remove the thermoelectric device from the
unit. The
control system compensates for the temperature difference between the two
surfaces of
the thermoelectric device caused by the heat sink attached to one side and the
sample
block attached to the other. The control system causes the thermoelectric
device to
equalize its two surface temperatures and then the AC resistance measurement
is made.
The micro-controller performs a polynomial calculation at the referenced time
of the AC
measurement to compensate for ambient temperature error.
Figure 9 shows the sample block 36, a layer of thermoelectric device 60 and
heatsink 34 interfaced with the system microcontroller 62 and bipolar power
amplifier 64.
The temperature sensor already present in the heatsink 38 and an additional
temperature
sensor attached to the sample block 36 with a clip (not shown) formed of music
wire are
utilized to determine the temperature differential of the surfaces of the
thermoelectric
device.
The bipolar power amplifier supplies current in two directions to the device.
Current in one direction heats the sample block and current in the other
direction cools
the sample block. The bipolar power amplifier also has signal conditioning
capability to
measure the AC voltage and AC current supplied to the thermoelectric device. A
band
CA 02464973 2004-05-14
23
pass filter 68 is incorporated into the signal conditioning to separate an AC
measurement
signal from the steady state signal that produces a null condition for the
temperature
difference across the thermoelectric device.
The micro-controller incorporates the necessary capability to process the
measurement information and perform the feedback in real time. It also stores
the time
history of the AC resistance and the number of temperature cycles of the
thermoelectric
device and displays the information to the operator on the display 70. The AC
measurement is normally done during initial turn on. However, it can be
activated when
self diagnostics are invoked by the operator using the keypad 72. An analog to
digital
and digital to analog converter along with signal conditioning for the
temperature sensors
and AC resistance measurement is also integrated into the micro-controller in
order for it
to perform its digital signal processing.
Sealing the Thermoelectric Device Area from the lJnvironment.
The thermoelectric devices are protected from moisture in the environment by
seals and the chamber is kept dry with the use of a drying agent such as
silica gel. The
seal connects from the silver electroform to the surrounding support and as
such adds to
the edge losses from the block. These losses are minimized by the use of a low
thermal
conductivity pressure seal 98 and by the use of the perimeter heater described
above. 'The
seal 98 has a cross-section generally in the shape of a parallelogram with
several tabs 100
spaced about the lower surface of seal 9$ for holding seal 98 to the edge of
the sample
block as shown in Figure 15.
The seal 98 is installed by first applying RTV rubber (not shown) around the
perimeter 110 of the upper portion of the sample block. The seal 98 is then
placed on the
RTV rubber. More RTV rubber is applied to the perimeter 120 of the seal and
then a
cover (not shown) is installed which contacts the RTV rubber = seal
combination. The
cover has a skirt which also contacts a gasket (not shown) on the printed
circuit board to
effect a more effective seal.
