 |
Description  |
|
|
FIELD OF THE INVENTION
This invention relates to cuvettes in which reactions are undertaken in
liquids confined within the cuvette, and particularly those reactions
requiring carefully controlled temperatures, limited amount of sample, and
a rapid rate of fluid temperature change.
BACKGROUND OF THE INVENTION
Although this invention is not limited to cuvettes used for nucleic acid
amplification, the background is described in the context of the latter.
Nucleic acid amplification generally proceeds via the following steps
(shown in FIG. 1):
(1) If DNA is to be amplified, a complete DNA double helix is optionally
chemically excised, using an appropriate restriction enzyme(s), to isolate
the region of interest.
(2) A solution of the isolated nucleic acid portion (here, DNA) and
nucleotides is heated to and maintained at 92.degree.-95.degree. C. for a
length of time, e.g., no more than about 10 minutes, to denature the two
nucleic acid strands: i.e., cause them to unwind and separate and form a
template.
(3) The solution is then cooled through a 50.degree.-60.degree. C. zone to
cause a primer nucleic acid strand to anneal or "attach" to each of the
two template strands. To make sure this happens, the solution is held at
an appropriate temperature, such as about 55.degree. C. for about 15
seconds, in an "incubation" zone.
(4) The solution is then heated to and held at about 70.degree. C., to
cause an extension enzyme, preferably a thermally stable enzyme such as a
polymerase isolated from thermus aquaticus, to extend the primer strand
bound to the template strand, by using the nucleotides that are present.
(5) The completed new pair of strands is heated to 92.degree.-95.degree. C.
again, for about 10-15 seconds, to cause this pair to separate.
(6) Steps (3)-(5) are then repeated, a number of times until the
appropriate number of strands are obtained. (See, e.g., U.S. Pat. No.
4,683,202 for further illustration.) The more repetitions, the greater the
number of multiples of the nucleic acid (here, DNA) that is produced.
Preferably the desired concentration of nucleic acid is reached in a
mininum amount of time.
A cuvette is usually used to hold the solution while it passes through the
aforementioned temperature stages. Depending upon the design given to the
cuvette, it can proceed more or less rapidly through the various stages. A
key aspect controlling this is the thermal transfer efficiency of the
cuvette--that is, its ability to transfer heat more or less
instantaneously to or from all of the liquid solution within the cuvette.
The disposition and the thermal resistance of the liquid solution itself
are the major aspects affecting the thermal transfer, since portions of
the liquid solution that are relatively far removed from the heat source
or sink, will take longer to reach the desired temperature.
The crudest and earliest type of cuvette used in the prior art is a test
tube, which has poor thermal transfer efficiency since (a) the walls of
the cuvette by being glass or plastic, do not transfer thermal energy
well, and (b) a cylinder of liquid has relatively poor thermal transfer
throughout the liquid. That is, not only does the liquid have low thermal
conductivity, but also a cylinder of liquid has a low surface to volume
ratio, that is, about 27 in.sup.-1 for a fill of about 100 .mu.l.
Still another problem in DNA amplification is the manner in which the
cuvette allows for ready removal of the liquid after reaction is complete.
A test tube configuration readily permits such removal. However,
modification of the cuvette to provide better thermal transfer efficiency
tends to reduce the liquid transferability.
Recent cuvette or vessel constructions for reaction of liquid are shown in
U.S. Pat. Nos. 4,426,451 issued Jan. 17, 1984 and 3,691,017 issued on
Sept. 12, 1972. In the former, little attempt is made to provide high
thermal transfer efficiency, except that the liquid is distributed as a
thin film that will allow rapid heating, if heat penetrates to the liquid.
However, no mention is made of the cuvette being constructed of metal or
any material that is highly thermally conductive. Furthermore, since the
spacing between top and bottom walls is no greater than 125 microns to
provide a strong capillary effect, removal of liquid from such cuvette
will be difficult. At best, not all the liquid will be removed because of
the strong capillary attraction.
In the case of cuvettes of U.S. Pat. No. 3,691,017, more features suitable
for DNA amplification are provided. For example, by having a spacing of
about 5 mm between major surfaces 23 and 24, it is more readily possible
to remove all of the liquid, there being less capillary attraction left at
such a gross spacing. In addition, a metal layer 38A is provided on the
outside of the cuvette to provide contact with a heating device. However,
this cuvette does not have a low thermal time constant for several
reasons. One reason is that to ensure transparency, the surfaces 23 and 24
are not constructed of metal, but rather of an insulator. As a result, a
long thermal transfer path is needed by extending metal 38A around the
edge of the device and into only a portion 35 of the volume of the
cuvette. This thermal path length is well in excess of 0.5 mm since it is
much more than the thickness of the wall providing the major surface 23 or
24. Indeed, only a portion of the volume of the cuvette is in direct
contact with the metallic thermal-energy transfer element.
Secondly, and more importantly, there is a high thermal resistance in the
cuvette of the '017 patent because of the low fluid surface/volume ratio
provided by cavity geometry, discussed hereinafter in detail.
OTHER RELATED APPLICATIONS
A commonly-owned application entitled "Detection of a Specific Nucleic Acid
Sequence Using A Solid Phase Capture Means" has been cofiled with this
application by Brent A. Burdick et al. That application describes a method
for the detection of a specific nucleic acid sequence in a sample
containing one or more nucleic acids, including the use of
single-stranded, oligonucleotide primers complementary to the sequence, a
labeled probe and known amplication techniques to form primer extension
products of the primers. One of the primers is labeled with a specific
binding moiety which, at some point in the method, is irreversibly
complexed with a receptor thereto. The receptor is covalently bound to a
solid support material, such as polymeric particles.
SUMMARY OF THE INVENTION
We have constructed a cuvette with rapid thermal transfer characteristics,
as measured by its thermal time constant when filled with the liquid of
choice. As such, it is ideally suited for replication of portions of DNA.
More specifically, in accord with one aspect of the invention, there is
provided a thermal cycling cuvette for controlled reaction of components
of a liquid involving cycling through a temperature range of at least
about 35.degree. C. (e.g. between 55.degree. C. and about 90.degree. C.),
the cuvette having at least one liquid-confining chamber defined by two
spaced-apart opposing walls, side walls connecting said two opposing
walls, and means permitting the introduction of liquid into, and the
removal of such liquid from, the chamber. The cuvette is improved in that
the opposing walls and the side walls are dimensioned to provide (a) a
spacing between said opposing walls of no less than 0.5 mm and (b) a
predetermined surface-to-volume ratio of the chamber, at least one of the
opposing walls being provided with a predetermined thermal path length abd
thermal resistance, such that the ratio, path length and thermal
resistance are effective to provide, for pure water contained within the
chamber in contact with the opposing walls, a thermal time constant for
the water that is no greater than about 10 seconds, for a liquid volume of
no greater than about 200 .mu.l.
In accord with another aspect of the invention, the cuvette is improved in
that the permitting means noted in the previous paragraph includes a
liquid access aperture, an air vent, and a movable valve for selectively
sealing off fluid flow between (a) each of the access aperture and air
vent, and (b) the chamber.
In accord with still another aspect of the invention, there is provided a
cuvette for controlled reaction of components of a liquid, the cuvette
having first and second liquid confining chambers for conducting
reactions, each defined by two spaced-apart opposing walls, side walls
connecting respective ones of said two opposing walls, and means
permitting the introduction and removal of the liquid into and from each
of the chambers, each of the chambers providing a major plane of liquid
containment. The cuvette is improved in that the chambers are disposed
such that the major plane of one of the chambers lies disposed above the
major plane of the other chamber; whereby the length and/or width of the
cuvette is reduced compared to a comparable cuvette in which the major
planes are coplanar.
In accord with yet another aspect of the invention, there is provided a
method of amplifying a nucleic acid sequence comprising the steps of
treating a mixture of a separated strand of nucleic acid with a molar
excess of an oligonucleotide primer at a temperature effective to attach
the primer to the strand, heating the bound primer and strand in the
presence of an extension enzyme to extend the primer to form a
complementary strand, heating the bound complementary strand and the
strand of nucleic acid at a temperature that causes them to separate into
two template strands, and then repeating each of the previous steps on the
template strands to produce many numbers of amplified strands. The method
is improved in that it further includes the steps of:
adding to the mixture a detection probe and a capture probe each comprising
a complementary sequence of nucleotides that bond to an oligonucleotide
sequence of choice and either a signal moiety capable of reacting to
produce a detectable signal, or a capture moiety that will immobilize a
strand bonded to the probe, respectively;
reacting the probes with the amplified strands to bind the probes to the
amplified strands;
transferring a mixture containing the bound probes and strands to a
separation medium;
and separating strands bearing capture probes on them from those lacking
such probes, by passing the mixture through a filter containing a
substance that binds with the capture moiety but which allows passage of
strands lacking the capture moiety.
Thus, it is an advantageous feature of the invention that rapid thermal
energy transfer occurs in and out of the cuvette and its liquid contents,
permitting the liquid to undergo multiple temperature changes as needed
for the desired reaction. As a result, a required number of repetitive
cycles can be achieved in less time than has been heretofore possible.
It is a related advantageous feature of the invention that a cuvette is
provided which allows rapid thermal processing of DNA strands through a
multizoned cycle requiring many heat-and-cool repetitions.
It is still another related advantageous feature of the invention that such
a cuvette and its liquid has a reduced thermal time constant, requiring
very little delay just to heat up or cool down the liquid.
It is another advantageous feature of the invention that such a cuvette
also permits relatively easy withdrawal of the complete liquid contents,
once the reactions are complete.
Another advantageous feature of the invention is the provision of such a
cuvette in which the contents can be readily sealed from exposure to the
atmosphere.
Still another advantageous feature of the invention is the provision of
such a cuvette with two chambers and with a minimum of length and width.
Other advantageous features will become apparent upon reference to the
following detailed description of the preferred embodiments, when read in
light of the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph plotting typical temperature changes for a cuvette in
which DNA replication is occurring;
FIG. 2 is an isometric view of a cuvette prepared in accordance with the
invention;
FIG. 3 is a plan view of the cuvette of FIG. 2;
FIG. 4 is a section view taken generally along the line IV--IV of FIG. 3;
FIG. 5 is an enlarged, sectioned fragment taken from the encircled portion
of the cuvette, labeled "V";
FIG. 6 is a plot of an actual time-temperature response of such a cuvette,
to obtain the thermal time constant thereof;
FIG. 7 is a plot of the heating response curve that such a cuvette will
have, in light of the data of FIG. 6;
FIGS. 8 and 9 are sectional views similar to that of FIG. 4, but
illustrating two different embodiments;
FIG. 10 is a fragmentary sectional view similar to FIG. 4, illustrating the
cooperation of both a pipette and a seal, with the cuvette.
FIG. 11 is a fragmentary plan view of an alternate embodiment, particularly
of the valving mechanism;
FIG. 12 is a section view taken generally along the line XII--XII of FIG.
11;
FIG. 13 is a fragmentary plan view similar to that of FIG. 11, but with the
valve rotated about 45.degree.;
FIG. 14 is a section view similar to FIG. 12, but illustrating the valve
rotated as noted for FIG. 13, and taken along the line XIV--XIV of FIG.
13;
FIG. 15 is a section view similar to that of FIG. 12, but of still another
embodiment;
FIG. 16 is a plan view of another embodiment of the invention partially
broken away;
FIG. 17 is a section view taken generally along the line XVII--XVII of FIG.
16;
FIG. 18 is a section view similar to that of FIG. 17, but illustrating yet
another embodiment;
FIG. 19 is a schematic illustration of an incubating apparatus that is
useful in cycling the cuvette through its DNA processing temperatures;
FIG. 20 is a section view similar to FIG. 17, of still another embodiment;
and
FIG. 21 is a section view taken generally along the line of XXI--XXI of
FIG. 20.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention is described hereinafter preferably for temperature cycling
over a range of at least 35.degree. C., as is particularly useful in
replicating DNA strands. In addition, it is also useful for any kind of
reaction of liquid components and reagents that requires repetitive
heating and cooling of the cuvette within the reaction is conducted.
Orientations such as "up", "down", "above" and "below" are used with
respect to the cuvette as it is preferably used.
Turning first to FIGS. 2-4, a cuvette 30 constructed in accord with the
invention comprises a liquid-confining chamber 32 defined by two opposing
walls 34 and 36, FIG. 4, spaced apart a distance t.sub.1. Such spacing is
achieved by side walls 38 and 40, FIG. 3, that join at opposite ends 42
and 44 of chamber 32. Most preferably, the shape of side walls 38 and 40
is one of a gradual concavity, so that they diverge at end 42 at an angle
alpha of about 90.degree., and at a point halfway between ends 42 and 44,
start to reconverge again at an angle of about 90.degree.. Distance
t.sub.1, FIG. 4, is selected such that such distance, when considered in
light of the shape of sidewalls 38 and 40, minimizes the quantity of
liquid that is retained in the cuvette upon removal of liquid. That is, it
is well-known that capillary spacings resist liquid removal. Therefore,
the thickness of the liquid between closely-spaced walls 34 and 36 is
preferably a non-capillary spacing, namely a spacing .gtoreq.0.5 mm and
most preferably, 0.5 to 2.5 mm. More precisely, given a particular shape
to the liquid container, the ability of the container to empty against
such adverse factors as capillary attraction, can be expressed as a
capillary number C. The equation is N.sub.ca =.mu..V/.gamma.. In this
equation, .mu. is viscosity, and for water solutions, this is about 0.01
centipose. .gamma. is surface tension, which for water solutions is about
70 dynes/cm. V is velocity in cm/sec, which is determined by the total
volume to be emptied, divided by the flow-through area of the exit, and
divided by the acceptable time of emptying. Therefore, the velocity factor
requires detailed consideration.
The volume to be emptied varies from 200 to 100 .mu.l. The acceptable times
are from 1 to 10 secs. Thus, V ranges between 200/A.1 as a maximum and
100/A.10 as a minimum. For purposes of the invention, A is assumed to be
about 5.2.times.10.sup.-3 cm.sup.2, the flow-through area of exit 60.
Thus, V ranges between about 1.92 cm/sec and about 38.5 cm/sec. In turn,
then, the capillary number for this cuvette is between about 0.0003 and
about 0.006. However, this must be coupled with a non-capillary spacing,
since only that spacing allows such a rate of emptying to occur.
Walls 34 and 36 provide the major surfaces in contact with the liquid. As
such, their surface area is selected such that, when considered in light
of the thickness of spacing t.sub.1, the surface-to-volume ratio for
chamber 32 is optimized for a high rate of thermal energy transfer. A
highly preferred example is the exposed surface area of 2.4 cm.sup.2 (0.37
in.sup.2) for each of walls 34 and 36, with the surface from the side
walls providing a contact area of about 0.36 cm.sup.2. Most preferably,
therefore, the surface-to-volume ratio is between about 65 in.sup.-1 and
about 130 in.sup.-1 for a fill volume of between 200 and 100 .mu.l,
respectively.
Such a large fluid surface-to-volume ratio provides an advantage apart from
a rapid thermal energy transfer. It also means that, for a given volume, a
much larger surface area is provided for coating reagents. This is
particularly important for reagents that have to be coated in separate
locations on the surface to prevent premature mixing, that is, mixing
prior to injection of liquid within the chamber. Also, a more effective
dissolution occurs for those reagents when liquid is introduced.
Optionally, therefore, one or more reagent layers 50 can be applied to the
interior surface of wall 36, FIG. 4, in a form that will allow the one or
more layers to enter into a reaction with liquid sample inserted into
chamber 32. Thus "a reagent" is a substance that will interact physically
or chemically with the liquid sample. Such reagent layer can be applied in
conventional ways, such as by spraying and drying. Such reagents can
include a polymerase enzyme, salts, buffers and stabilizers. The coated
layer may also include primer-pairs and dinucleotides necessary for
replication.
A liquid access aperture 60 is formed in wall 36 adjacent end 42. The
aperture has an upper portion 62 and a lower portion 64 that connects the
upper portion with chamber 32. Preferably at least portion 62 is conical
in shape, the slope of which allows a large number of different conical
pipette designs P, FIG. 2, to mate therewith.
At opposite end 44, an air vent 70 is provided, in a manner similar to that
described in U.S. Pat. No. 4,426,451. Most preferably, air vent 70 extends
into a passageway 72, FIG. 3, that is routed back to a point adjacent end
42, where it terminates in opening 74 adjacent access aperture 60.
To allow a single closure device to seal both the access aperture 60 and
opening 74 of the air vent, both of these are surrounded by a raised,
cylindrical boss 80. Any conventional closure mechanism is useful with
boss 80, for example, a stopper 82, FIG. 10. Such stopper can have
external threads 84 for engaging mating internal threads, not shown, on
the boss, or it can be constructed for a force fit within the boss 80.
Most preferably, except for wall 34 described below, the walls of the
device are formed from less wettable materials, such as polycarbonate
plastics.
In accord with one aspect of the invention, the wall 34 opposite to wall 36
is constructed with a predetermined thermal path length and thermal
resistance that will provide a high rate of thermal energy transfer. Most
preferably, such path length (t.sub.2 in FIG. 5) is no greater than about
0.3 mm, and the thermal resistance is no greater than about 0.01.degree.
C./watt. These properties are readily achieved by constructing wall 34 out
of a metal such as aluminum that is about 0.15 mm thick. Such aluminum has
a thermal resistance R, calculated as thickness .chi..1/(thermal
conductivity K.surface area A), which is about 0.003.degree. C./watt.
(These values can be contrasted for ordinary glass of the same thickness,
which has a thermal resistance of about 0.24.degree. C./watt.)
Wall 34 can be secured to sidewalls 38 and 40 by any suitable means. One
such means is a layer 90, FIG. 5, of a primer, which comprises for example
a conventional high temperature acrylic adhesive, followed by layer 92 of
conventional polyester adhesive. In some instances, layers 90 and 92 need
not extend over the surface area of wall 34, as such would increase the
thermal resistance of wall 34, and possibly interfere with reactions
described within chamber 32. On the other hand, it will be apparent that
some reactions can equally be adversely affected by metal ions present and
for these reactions, the adhesive coats the entire surface area of wall
34.
A cuvette constructed as described above for FIGS. 2-4, has been found to
produce a thermal time constant tau (.tau.) for its contained liquid that
is no greater than about 10 seconds. Most preferred are those in which
.tau. is of the order of 3-8 seconds. That is, FIG. 6, when such a
cuvette, filled with water, is heated along the exterior of wall 34, and
its temperature is measured at point Y, FIG. 4, a thermal response curve
is generated from 28.degree. C. to a final temperature of 103.9.degree. C.
The time it takes for the liquid therein to reach a temperature of
76.degree. C. (63% of the difference (103.9-28)) is the approximate value
of tau (.tau.). This derives (approximately) from the well-known thermal
response equation:
Temperature T (t)=Final Temperature+(Initial Temperature-Final
Temperature).e.sup.-t/.tau. (1)
Thus, if the time interval t in question equals tau, then e.sup.-t/.tau.
=e.sup.-1 .perspectiveto.0.37. In such a case, T (t) (at t=tau) is the
temperature which is equal to the final temperature, plus 63% of (Initial
Temperature-Final Temperature). (The step-wise shape of the data in FIG. 6
is an artifact of the recorder.)
Therefore, tau for the liquid of that cuvette is about 3.5 seconds
(assuming that the response curve of FIG. 6 obeys the equation (1) above,
which it does to a sufficiently close approximation).
If the adhesive of layers 90 and 92 does extend over all the surface of
wall 34, then tau can be increased to as much as 7 or 8 sec.
With a value of tau equal to about 3.5 sec, the thermal response curve of
the same cuvette can be predicted under the conditions it will be
subjected in accordance with the protocol of FIG. 1. FIG. 7 is such a
response curve, generally for only the last segment of FIG. 1 marked "X",
where the stepped portions "A" are the oven or incubator temperatures, and
the curve "B" is the temperature of the liquid contents, for tau
.perspectiveto.3 seconds. (However, in this example, the incubation
temperature was selected to be 37.degree. C. instead of 55.degree. C.).
As a comparative example, the cuvette described in U.S. Pat. No. 3,691,017
has the following properties. Its overall thickness (col. 8, lines 42-44)
is 5/16" or 0.31". The space occupied by the liquid has a thickness of 5
mm (line 44, col. 8). The window is 1/2" by 1/4" (line 45), so that the
volume of the liquid of a unit area of one square inch, is estimated to be
about 0.197 in .sup.3, and its surface area contacting the cuvette is
estimated to be about 2.79 in .sup.2. This produces at best a
surface/volume ratio of only about 14.2 in .sup.-1. A sphere of the same
volume has a S/V ratio of 8.3 in .sup.-1, indicating that the cuvette is
only slightly better in its S/V ratio than the worst possible
configuration (the sphere). In addition, the heating element is a foil of
aluminum that has to extend into the cavity from the outside, thus
producing a thermal path length well in excess of 1 mm. All of this
clearly indicates that the thermal time constant tau of water in such a
cuvette is substantially greater than 10 sec.
It is not necessary that the cuvette be assembled using adhesive. A
non-adhesive embodiment is illustrated in FIG. 8, wherein parts similar to
those used in the previous embodiment bear the same reference numeral to
which is appended the letter "a". Thus, cuvette 30a comprises two opposed
walls 34a and 36a which, with side walls (of which only 40a is shown),
define a chamber 32a. A liquid access aperture 60a and an air vent 70a are
provided, also as in the previous embodiment, and wall 34a has the same
properties as discussed above. However, in this embodiment there are no
adhesive layers between wall 34a and side wall 40a at any point. Instead,
around the entire edge of chamber 32a, there is a gasket seal 100 of an
elastomer, or other suitable gasket material, between wall 34a and the
side wall. In addition, bumps of plastic 110 extend from the side walls
through appropriate openings in the wall 34a. Bumps 110 are then upset by
heat or pressure to rivet wall 34a in place. Optionally, gasket seals 100
can also extend around such bumps.
When the access aperture and the air vent are sealed, and the cuvette is
heated, pressure builds within chamber 32 and 32a. It is preferred,
therefore that a part of the cuvette, other than the thermally conductive
wall, become deformed to accommodate the pressure increase, in order to
maintain intimate contact between reaction vessel wall 34 and the
incubator. Still further, deformation of some other wall reduces the
strain on the seal that holds the thermally conductive wall in place. Such
an embodiment is shown in FIG. 9, as described and claimed in the commonly
owned application Ser. No. 123,752, filed Nov. 23, 1987, entitled "Cuvette
with Non-Flexing Thermally Conductive Wall" filed by Helfer et al. Parts
similar to those previously described bear the same reference numeral, to
which the distinguishing suffix "b" is appended.
Thus, cuvette 30b comprises opposed walls 34b and 36b defining, with side
walls 40b (only one shown), a chamber 32b having a spacing t.sub.1. These
and the access aperture 60b and air vent 70b are constructed as in either
of the previous embodiments. However, to insure that wall 34b does not
deform under pressure, wall 36b is constructed to have a flexure strength
that is less than that of wall 34b. Specifically, this is preferably done
as follows: if wall 34b comprises aluminum that is about 0.15 mm thick,
then its flexure strength K at the center of flexure is determinable,
based on the following:
Deflection X is determined by the well-known equation
X=.alpha.Pa.sup.2 /Et.sup.3 (2)
where P=total applied load, E=plate modulus of elasticity, t=plate
thickness, a=the length of one side of the plate (here, the flexing wall),
and .alpha. is an emperical coefficient (usually equal to about 0.015).
Rearranging,
P/X=Et.sup.3 /.alpha.a.sup.2 (3)
Because P/X is analogous to F/X which equals K, then
K.perspectiveto.Et.sup.3 /.alpha.a.sup.2 (4)
This allows K to be calculated to be about 6.11.times.10.sup.6 dynes/mm.
For wall 36b to have a flexure strength less than that, it need only
comprise a layer of polyethylene or polypropylene that is about 0.3 mm
thick (thickness t.sub.3 that is twice that of the aluminum wall 34b), to
have a flexure strength of about 8.3.times.10.sup.5 dynes/mm, calculated
in the same manner. In such a construction, wall 36b will dome upwardly as
pressure is generated within chamber 32b, leaving wall 34b lying planar
against the heating element (shown in phantom as "E").
In use, any of the above-described embodiments is filled to about point 44,
FIG. 3, which provides a fill of about 90%, with a liquid containing the
desired sample for reaction, for example, a solution of a DNA sequence
that is to be amplified. The device is then inserted into an appropriate
incubator and cycled through the necessary stages for the reaction.
In accord with another aspect of the invention, a movable valve is disposed
to selectively open and close access to the chamber within the cuvette.
Parts similar to those previously described bear the same reference
numeral to which the distinguishing letter "c" has been appended.
Thus, in FIGS. 11-14, end 42c of chamber 32c has both the liquid access
aperture 60c and the air vent passageway 72c located adjacent to each
other, within raised boss 80c, FIG. 11. Upper portion 62c is conically
shaped, as in previous embodiments, to allow a mating engagement of
pipette P, FIG. 12.
However, in this embodiment a valve is provided, comprising a rotatable
portion 120 mounted within a mating conically shaped aperture 122, FIG.
12. Rotatable portion 120 has an exterior, conically shaped surface 123.
The valve has an axis of rotation 124, preferably concentrically disposed
within aperture 60c. A handle 126 permits manual or automated rotation of
the valve. Both the access aperture 60c, as well as air vent opening 74c,
are located in the movable valve portion 120, FIGS. 11 and 13.
To selectively connect aperture 60c and opening 74c to chamber 32c, two
paths are formed in rotatable portion 120. Path 130 extends from aperture
60c to the exterior surfaces 123, FIG. 12, where it is open over an angle
theta of that surface, FIG. 11. Path 132 extends, FIG. 14, from aperture
74c to an exit 134 on surface 123. The heights of the paths 130 and 132,
relative to the chamber 32c and passageway 72c, respectively, are such as
to allow the paths to be fluidly connected to the chamber or passageway,
when rotatable portion 120 is rotated to the position shown in FIG. 13. In
addition, because of the angle theta of access provided by path 130,
portion 120 can be rotated also to a position (not shown) wherein only the
access aperture 60c is in fluid communication with chamber 32c, if
desired.
A tail portion 140 holds rotatable portion 120 vertically in place, and
rides in a slot 144, FIG. 12.
The access and air vent valve need not be rotatable. Translatable valves
are also useful. Parts similar to those previously described bear the same
reference numeral to which the distinguishing suffix "d" is appended.
Thus, in FIG. 15, valve portion 120d is disposed at end 42d so as to
incorporate both the access aperture 60d and air vent opening 74d.
However, unlike the previous embodiment, portion 120d is vertically,
translatably mounted, with tail portion 140d riding vertically in a slot
144d that permits no rotation. A spring 150 biases portion 120d so that,
normally, it is raised to misalign paths 130d and 134d with respect to
chamber 32d and vent passageway 72d, respectively, thus sealing off
chamber 32d from the outside environment. To access the chamber, the user
need only press down on portion 120d, such as by engaging access aperture
60d with a pipette P, and push against spring 150.
Vent opening 74d is, in this embodiment, a groove formed in exterior
surface 123d, which allows venting of passageway 72d when portion 120d is
pushed down a distance sufficient to fluidly connect chamber 32d also with
aperture 60d.
In certain reactions, it is desirable that the liquid be moved to a second
chamber, after an appropriate residence time in the first chamber. For
example, DNA probe assays require a single, additional, new thermal cycle
with which to bond DNA probes to the amplified target sequence. Subsequent
to the DNA amplification temperature cycling discussed above, the
DNA-bearing broth is brought into contact with DNA probes specific to the
amplified target sequence. Such probes can be coated onto the walls of a
reaction vessel, and more specifically, on the walls of a second, DNA
probe bearing chamber, as shown in FIGS. 16-17. Parts similar to those
previously described, bear the same reference numeral to which the
distinguishing suffix "e" is appended.
Thus, cuvette 30e comprises chamber 32e disposed between two opposing walls
34e and 36e, FIG. 17, having major surfaces, and side walls 38e and 40e,
FIG. 16, all of which are generally similar to previous embodiments, with
an access aperture 60e, air vent 70e and air vent opening 74e. However,
unlike the previous embodiments, metallic wall 34e is above, rather than
below, chamber 32e, FIG. 17, so that heat is applied downwardly from above
cuvette 30e. (A filter membrane, not shown, can be optionally positioned
at 70e.)
This rearrangement of walls 34e and 36e is due to the fact that a second
chamber 160 is provided, disposed underneath chamber 32e. That is, each of
the two chambers provides a major plane of liquid containment, and
preferably, has a generally planar configuration due to the opposing walls
of primary surface area being also generally planar and respectively
coplanar. Thus, the major containment plane of chamber 32e is disposed
above the major containment plane of chamber 160.
As is apparent in FIG. 17, chamber 160 has an opposing, primary surface
wall that is in fact wall 36e. The opposite wall 164 is the wall that
provides the thermal energy transfer, by being constructed substantially
the same as wall 34e. Side walls 166 and 168, FIG. 16, shape the chamber
160 as in the case of chamber 32e, except that chamber 160 is rotated
about 45.degree. with respect to chamber 32e, so that its air vent,
described below, does not conflict with the lower portion 64e of access
aperture 60e.
An air vent and passageway 170 is provided for chamber 160, FIG. 16. It is
constructed similar to the air vent passageway of the air vent of
previously embodiments. Alternatively, not shown, air vent opening 74e can
be shaped and positioned to allow direct removal of liquid from chamber
160, without requiring it to be withdrawn back through chamber 32e as
shown in FIG. 16.
In use, liquid in chamber 32e is forced to flow into chamber 160, as per
the arrows in FIG. 17, by injecting either air pressure into chamber 32e
through the access aperture, or, e.g., solutions optionally containing
reagents for further reaction in the cuvette, or by pulling a vacuum on
chamber 160.
Alternatively, the two chambers can overlie each other with side walls that
are exactly aligned, rather than offset by 45.degree.. This embodiment is
shown in FIG. 18, wherein parts similar to those previously described bear
the same reference numeral to which the distinguishing letter "f" is
appended.
Thus, cuvette 30f comprises upper and lower chambers 32f and 160f, sharing
a common wall 36f that is not a thermal transfer wall. Air vent 70f feeds
liquid from chamber 32f to 160f, and walls 34f and 164f provide the
necessary rapid transfer to thermal energy. Unlike the previous
embodiment, however, sidewalls (40f of chamber 32f being shown) are
aligned with the sidewalls of chamber 160f (wall 166f being shown). This
is rendered possible by rotating boss 80f 90.degree. so as to provide
access aperture 60f that is totally within the major plane of liquid
confined by chamber 32f. In such a cuvette, air vent 170f and its opening
74f are located within the major plane of liquid confined within chamber
160f.
In each of the two embodiments of FIGS. 16-18, the advantage achieved is
that the length or width of the cuvette is not extended appreciably beyond
that of the cuvette of FIG. 2, as it would be if the two major planes of
liquid confinement were coplanar. Only the vertical thickness is slightly
increased to permit the two chambers to be vertically stacked to provide a
three-dimensional flow.
Yet another useful configuration is one shown in FIGS. 20 and 21, in which
a third liquid-confining chamber is fluidly connected to said second
chamber, with a liquid-permeable membrane isolating and separating the
third chamber from the second chamber. Parts similar to those previously
described bear the same reference numeral, to which the distinguishing
suffix "g" is appended.
Thus, cuvette 30g comprises a first chamber 32g having opposing walls 34g
and 36g (of which wall 34g is metallic), sidewalls 38g and 40g (FIG. 21)
and access aperture 60g. Chamber 32g leads via passageway or air vent 70g
to the second chamber 160g, disposed in a major plane of configuration
that is parallel to that plane of configuration occupied by chamber 32g,
as in the embodiment of FIG. 16. However, in this embodiment, chamber 160g
is above chamber 32g, rather than below it. Chamber 160g is | | |
