Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Ultrathin-walled multiwell plate for heat block thermocycling
The invention relates to plastic plates for conventional heat block
thermocycling
s of biological samples, particularly to rnultiweil plates. More specifically,
it relates to
ultrathin-walled multiwell plates with an improved heat transfer to small-
volume
samples. Such plates can be used for rapid temperature cycling of multiple,
small-volume
samples (i.e. 1-20 pl) by using heat block thermocyclers with an increased
block
temperature ramping rate (i.e. 4 ° C/second and greater) and standard
heated-lid
~ o technology for sealing the samples.
Temperature cycling of biological samples is a central moment in DNA
amplification by the palymerase chain reaction (PCR:) (Saiki et al., Science,
239, 487-491
[1988]). Much effort is being expended in developing various alternative
reactors and
technologies for rapid temperature cycling of small-volume samples (Kopp et
al., Science
~5 280, 1046-1048 [1998]; Belgrader et al., ].Forensic Science 43, 3i5-319
[1998]; Wittwer
et al., Analytical Biochem., 186, 328-331 [1990] and U.S. Patent No 5,4SS;17S;
Woolley
et al., Analytical Chem., 68, 4081-4086 [(i996]).
One commercially available type of microreactor and thermocycler for rapid
temperature cycling of small samples is a glass capillary tube and a hot-air
thermocycler
zo from Roche Molecular Biochemicals (cat No. 1909 ?.39 and cat No. 20I 1468,
respectively).
The glass capillary tube can hold reaction volumes ranging from 10 to 20 p.l.
The hot-air
thermocycler can hold 32 capillaries and perform 30 - 40 PCR cycles in 20-30
minutes.
However, these rapid DNA amplification technology is connected with various
disadvantages, for example:
25 a) The handling of the individual capillaries is relatively cumbersome.
b) The relatively large glass surface adsorbs components of the standard PCR-
mixtures.
This might inactivate the reaction. Therefore, various carrier molecules, i.e.
proteins
or even DNA, must be added and the concentrations of the components
reoptimized.
c) The cost of the capillary tube, as a disposable PCR container, is high when
compared
3o to the standard 0.2 ml PCR tube.
d) The experimental throughput using this system is limited.
It is surprising that only little research has been conducted to improve the
basic
performance in sample size and speed of the widely used, conventional heat
block
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2
thermocycling of samples contained in plastic tubes or multiwell plates. One
known
improvement of heat block temperature cycling of samples contained in plastic
tubes has
been described by Half et al. (Biotechniques, 10, lOEi-112, j1991] and U.S.
Patent No
5,475,610). They describe a special PCR reaction-compatible one-piece plastic
s microcentrifuge tube, i.e. a thin-walled PCR tube. The tube has a
cylindrically shaped
upper wall section, a relatively thin (i.e. approximately 0.3 rnm) conically-
shaped lower
wall section and a dome-shaped bottom. The samples as small as 20 p,1 are
placed into the
tubes, the tubes are closed by deformable, gas-tight caps and positioned into
similarly
shaped conical wells machined in the body of the heat block. The heated cover
~o compresses each cap and forces each tube down firmly into its own well. The
heated
platen (i:e. heated lid) serves several goals by supplying the appropriate
pressure to the
caps of the tubes: it maintains the conically shaped walls in close thermal
contact with the
body of the block; it prevents the opening of the caps. by increased air
pressure arising in
the tubes at elevated temperatures. In addition, it maintains the parts of the
tubes that
~s project above the top surface of the block at 95° -100° C in
order to prevent water
condensation and sample loss in the course of thermocycling. This made it
possible to
exclude the placing of mineral oil or glycerol into the; wells of the block in
order to
improve the heat transfer to the tubes and the overlaying of the samples by
mineral oil
that prevented evaporation but also served as added tlhermal mass. In
addition, the PCR
zo tubes can be put in a two-piece holder (US patent 5,710,381) of an 8x12, 96-
well
microplate format, which can be used to support the high sample throughput
needs with
any number between 1 and 96 individual reaction tubes.
In DE 4022792 the inventors describe a plate with cylindrically shaped walls
of
the wells and spherically shaped bottoms thereof. ThE; individual wells of the
plate were
zs formed by melting a polycarbonate sheet in the range of 0.27-0.5 mm by a
stream of hot
air. This technology leads to relatively thin walls in the range of 0.08-
0.2mm. The
biological samples were placed into the wells, covered with polycarbonate film
(0. i mm)
and the individual wells were thermosealed by a special press. Upon sealing
the plate was
placed on the thermoblock and fixed by screws. Though theoretically the heat
transfer to
3o the samples is improved, however, the way of positioning the plate on the
block and the
cylindrical and spherical geometry of the well prevent a close thermal contact
with the
heating block. During thermocyling, due to the large thermal expansion, the
plate fixed by
CA 02348564 2001-04-25
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screws becomes deformed and the close thermal contact is not maintained
anymore.
Therefore, by using the above technology rapid cycling reactions cannot be
performed.
The other known improvement of heat block thermocycling is described in PCT
patent application WO 98/43740. It concerns a neat block thenmocycler with an
increased
ramping rate, i.e. 4° Clsecond). The thermocycler ca~;~ hold 96 PCR
tubes (each of a
volume of 0.2 ml) or 96-well PCR plates. Theoretica,lIy, the thermocycler can
perform 30
PCR cycles in 20-30 minutes, provided that only a fe;w seconds are spent to
reach the
temperature equilibrium between the heat block and the samples.
However, as described in U.S. Patent No 5,508,197, even if the temperature of
the
~o heat-transfer media; i.e. water, is changed almost instantaneously, it
takes approximately
15 seconds to reach equilibrium between water and t:he 15-20 p.l samples in
the standard
PCR plates. This means that for 30 PCR cycles approximately 20 minutes are
spent to
reach the equilibrium between heat-transfer media ar.~d the 15-20 ul samples
in the plates.
In comparison, the above mentioned heat block cycler (WD 98/43740) operating
~s at a ramping rate of 4° C/second, needs for the heat-block
temperature transitions during
30 PCR cycles 10 minutes only. This shows that the :major limiting factor for
rapid
temperature cycling of small samples in platic PCR tubes or PCR plates is the
low
efficiency of the heat transfer through the walls of conventional PCR tubes or
plates,
respectively.
zo The present invention concerns plastic multiwell plates for performing heat
block
thermocycling of multiple samples. More specifically, it concerns ultrathin-
walled
multiwell plates with an improved heat transfer to small samples. Ultrathin-
walled
multiwell plates are suited for rapid, oil-free, heat block temperature
cycling of small-
volume samples (i.e. approximately 1-20 p,l), wherea:> the Lower limit is
given by the
zs reliability of the conventional pipetting systems.
Figure 1 illustrates an example of a multiwell plate according to the
invention.
Figure 2 illustrates the positioning of the plate in the )block of the thermal
cycler.
One aspect of the present invention concerns the considerably decreased
thickness
(i.e. approximately 7.5-15 fold) of the well walls when compared to known thin-
walled
3o PCR tubes (U.S. Patent No 5,475,610.). This can be reached, for example, by
means of
thermoforming the plates out of thin thermoplastic films. Such thermoplastic
films are,
for example, polyolefin films, such as metallocene-catalyzed polyolefin films
and/or
copolymer films. Usually, the multiwell plate is vacuumformed out of cast,
unoriented
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4
polypropylene fzlm, polypropylene-polyethylene copolymer films or metallocene-
catalyzed polypropylene films. The film is formed into a negative ("female")
mould
comprising a plurality of spaced-apart, sonically shaped wells which are
machined in the
body of a mould in the shape of rectangular- or square-array. The thickness of
the film
s for vacuumforming sonically shaped wells is chosen according to the standard
rule used
for thermoforming, i.e. thickness of the film = well draw ratio x thickness of
the wall of
the formed well.
For example, vacuumforming wells with a draw ratio of two and an average
thickness of the walls of 30 microns results-in a film thickness of 60
microns. The
~o average optimum wall thickness was found to be 20-~40 microns. The
thickness of the
well is reduced 7.5-15 fold when compared to the wall thickness of the
formerly
improved PCR tube desribed in U.S. Patent No 5,475,610. Using the Fourier
equation for
heat transfer and the equation for temperature transfer through solid
substances, it can be
shown that heat transfer through one square millimeter of the surface of the
well of the
~s plate is increased 7.5-15 fold and the time of temperature transfer through
the wall is
decreased 56-225 fold when compared to the said PC'R tube. This drastic
decrease in time
can be explained by the fact that the time needed for ahe transfer of
temperature front is
proportional to the square power of distance. It can be easily calculated that
the time of
the temperature transfer through the ultrathin walls of the mufti-well plate
is in the range
Zo of milliseconds, whereas for the said PCR tube (LT.S. Patent No 5,475,610)
it is in the
range of seconds. This explains the well known fact that thin (20-40 microns)
plastic
films are poor thermo insulators.
The thickness of the walls of the formed well:. is gradually reduced to the
bottoms
of the wells due to vacuumforming of the wells into a~ negative mould. This
geometry of
zs the walls of the wells provides several advantages:
~ The relatively thick upper parts of the walls of the wells cause additional
rigidity of the
whole multiwell plate.
~ During heating of the heat block of the thermocycl.er, a vertical
temperature gradient is
formed in the sample, due to the gradient of the wf;ll-wall thickness. This
vertical
3o temperature gradient causes intensive convective mixing of the sample in
sonically
shaped wells and increases the heat transfer through the sample. In
comparison, this
convective mixing of the sample is much less eff dent in conventional PCR
plates/tubes with a uniform wall thickness.
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Another aspect of the invention concerns the height of the wells of the
rnultiwell
plate. The height of the conically shaped wells is equal to the height of the
similarly
shaped sample wells machined in the body of the heat block. Thus, this
geometry of the
wells (2) enables the positioning of the plate (1) on i:he heat block (4) as
shown in Figure
s 2. As shown (Figure 2), in contrast to the conventional PCR plates, the
walls of the wells
(2) of the mufti-well plate (1) do not project above tile top surface of the
block {4). The
type of positioning provides several advantages: Thc; pressure caused by the
screw (12) to
the lid (10) {heating element (11)) can be increased i.n order to obtain
efficient sealing of
the samples (9) sealed, for example, by, a silicon mat: (13). In this case the
pressure is
~o actually directed to those parts of the multiwell plate; (1) which are
supported by the top
surface of the heat block (4) (or by parts of the top surface surrounding
individual wells
depending on the geometry of the heat block) and not to the thin walls of the
wells of the
plate as it is the case for the PCR tubes or conventional PCR plates. This
advantage
makes it possibe to increase the sealing pressure of t;he heated lid (10)
several fold when
~5 compared to the conventionally used pressure of 30-50 g per well without
cracking the
conically shaped walls of the wells (2).
The extremely thin walls of the wells, i.e. 20-~40 microns, are highly
flexible~as
the multiwell plates are thermoformed out of highly elastic films (or sheets
depending on
the draw ratio). The walls of the wells are highly resistant against stress
cracking, due to
zo their flexibility and elasticity. As the wells of the plate, positioned on
the heat block, are
tightly sealed at room temperature, the air pressure in the wells will
increase at elevated
temperatures. The increased air pressure causes a deformation of the wails of
wells and
brings them in tight thermal contact with the surface of the walls of the
individual sample
wells machined in the body of the heat block. Standard PCR plates {having
relatively
z5 thick and rigid walls of.the wells) require that the co~ucally shaped walls
of the wells have
to match perfectly with the shape of the wells machined in the body of the
heat block to
guarantee a close thermal contact (see for example U.S. Patent No 5,475,610).
This
requirement is not as critical for the ultrathin walled multiwell plates of
the invention, due
to flexibility and elasticity of the walls of the wells. Using this advantage,
special shapes
30 of both, the walls of the wells of the plate and the wells of the heat
block can be
differently designed. These differently designed wells can promote an even
closer thermal
contact after positioning the plate into the heat block.
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6
Another aspect of the invention concerns the frame of the multiwell plates. As
the
plates can be formed of very thin films (depending on the. draw ratio of the
well; supra)
the flexibility of, for example, standard-format plates, i.e. 96-well PCR (8;5
x 12,5 cm)
plates, is such that handling is not easily possible anymore. Therefore,
depending on the
s geometry of the plate, a supporting frame might be needed, for example for
industry
standard formats, i.e. 96-, I92-, 384-well PCR plates. This frame can support,
for
example in case of small plates, the edges of the plate, or individual wells
of the plate, or
groups of wells. For handling with robots, for example, the frame can be
injection molded
in the form of the standard skirted microplates containing the array of holes
in the top
vo surface of the frame matching the array of wells of the ultrathin multiwell
plate. The plate
can be attached to the frame by for example heat bonding. However, for small
format
plates including the frame can be formed as a single piece by using specially
designed
moulds.
The polypropylene-based plastics are PCR-compatible and therefore widely used
~s for injection molding of PCR tubes and/or multiwell plates. In addition,
they are resistant
to stress cracking and have a reduced water vapor sorption when compared to
other
plastics (e.g. polycarbonate). Such plates can be thermoformed in both,
standard industry
formats, i.e. 96-, 192- and 384-well PCR plates for large scale applications,
supported by
robots and small foot-print formats to match small foot-print thermocyclers,
i.e. "personal
zo thermocyclers".
The following example serves to illustrate the invention but should not be
construed as a
limitation thereof.
2s Example:
Fig.l illustrates a 36-well ultrathin walled multiwell plate according to the
invention. The
plate was designed for rapid temperature cycling of saunples ranging from 0.5-
4 p.l using a
small foot-print pettier-driven heat block thermocycler supplied with a "wine-
press" type
heated lid (Fig. 2). The volume of the wells is 16 p,l and the distance
between the wells is
30 4.5 mm, i.e. industry standard for high sample density 384-well PCR plates.
The diameter
of the openings of the wells is 3.8 mm and the height ~of the wells is 3 mm.
The average
thickness of the walls of the wells is 30 p,m. The frame (3) was cut out of a
polypropylene
sheet of a thickness of 0.5 mm and heat bonded to the plate (1). The area of
the plate (1)
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7
is 30 x 30 mm. As shown in Figure 1, the handling of the plate (1) containing
the multiple
wells (2) is facilitated, by a rigid 0.5-1 mm thick plastic frame (3) which is
heat bonded to
the plate. As shown in Figure 2, the frame (3) is noi; in direct thermal
contact with the
block (4) during thermocycling because the inner contour (5) of the frame (3)
matches the
s outer contour (6) of the heat block (4) of the thermocycler (7 =
thermoelectric heat pump
and 8 = air-forced heat sink).
The ultrathin walled multiwell plate according to the invention (Fig. l } was
experimentally tested for the amplification of a 455=base pairs long fragment
of human
~o papilloma virus DNA. The sample volume was 3 pl. For various PCR reactions,
the
average romping rate of the thermo cycler was varied from 4° C to
8° C per second. The
samples (i.e. standard PCR-mixtures without any carrier molecules) were
transferred into
the wells of the plate by means of conventional pipelaing equipment. The plate
was
covered by standard sealing film (Microseal A; MJ-Research, USA); transferred
into the
~s heatblock of the thermocycier and tightly sealed by the heated lid as shown
in Fig. 2.
Upon sealing, a number of 30 PCR cycles was performed in 15-25 minutes
depending on
the romping rate of the thermo cycler. The PCR product was analyzed by
conventional
agarose electrophoresis. The 455-base pairs long DNA fragment was amplified
with a
high specificity at the indicated romping rates (supra).
2o Plates according to the invention with well volumes of 35 pl were
successfully
tested for temperature cycling of samples of a volume of 20 ~I. Thereby, 30
PCR cycles
were performed in 20-30 minutes at a romping rate of 6° C per second.
Surprisingly,
although the average thickness of the walls was 20 rn~icrons and the volume of
the wells
was 35 pl, samples of a volume of as few as O.S p,l can be easily amplified
without
zs reducing the PCR efficiency.
In conclusion, the ultrathin walled multiwell plates according to the
invention,
allow a simple and rapid loading of multiple samples by conventional pipettes,
rapid
sealing of all samples by using conventional sealing i~lms and rapid DNA
amplification
30 (15-30 minutes for 30 cycles) with an improved spec:ifcity typical for
rapid cycling
(Wittwer et al., Analytical Biochem:, 186, 328-331 [1990]) using appropriate
heat block
thermocyclers (i.e. romping rate in the range of 4° C to 8° C
per second).
