U.S. patent number RE35,716 [Application Number 08/591,346] was granted by the patent office on 1998-01-20 for temperature control apparatus and method.
This patent grant is currently assigned to Gene Tec Corporation. Invention is credited to Warren R. Jewett, Marilyn J. Stapleton.
United States Patent |
RE35,716 |
Stapleton , et al. |
January 20, 1998 |
Temperature control apparatus and method
Abstract
An apparatus and method for performing automated sample
preparation, DNA amplification and detection, which apparatus has
heat-sinking, flat carriers for holding specimens and reagents,
devices for heating and cooling and maintaining the specimen to or
at any given temperature for any given time periods, and a computer
to generate signals that control said temperatures and times.
Inventors: |
Stapleton; Marilyn J. (Durham,
NC), Jewett; Warren R. (Cary, NC) |
Assignee: |
Gene Tec Corporation (Durham,
NC)
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Family
ID: |
27499469 |
Appl.
No.: |
08/591,346 |
Filed: |
January 25, 1996 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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227348 |
Aug 2, 1988 |
|
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438592 |
Nov 17, 1989 |
5188963 |
|
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|
836348 |
Mar 3, 1992 |
5451500 |
|
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Reissue of: |
855318 |
Mar 23, 1992 |
05281516 |
Jan 25, 1994 |
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Current U.S.
Class: |
435/3; 165/61;
165/86; 435/283.1; 435/286.1; 435/303.1; 435/809 |
Current CPC
Class: |
B01L
7/52 (20130101); G01N 1/31 (20130101); B01L
2400/0406 (20130101); G01N 2035/00138 (20130101) |
Current International
Class: |
B01L
7/00 (20060101); C12M 1/02 (20060101); G01N
1/30 (20060101); G01N 1/31 (20060101); G01N
35/00 (20060101); C12Q 003/00 (); C12M 001/02 ();
C12M 001/38 (); F28F 005/00 () |
Field of
Search: |
;435/3,283.1,286.1,303.1,809 ;165/61,86 ;219/200,201,443 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Tran; Hien
Attorney, Agent or Firm: Barber; Lynn E.
Parent Case Text
This application is a continuation-in-part of copending U.S.
application Ser. No. 07/227,348 filed Aug. 2, 1988, now abandoned,
copending U.S. patent application Ser. No. 07/438,592 filed Nov.
17, 1989, now, U.S. Pat. No. 5,188,963, and copending U.S. patent
application Ser. No. 07/836,348 filed Mar. 3, 1992.Iadd., now U.S.
Pat. No. 5,451,500, .Iaddend.and copending international
application PCT/US90/06768 (01/06768) filed Nov. 16, 1990, the
disclosures of all of which are incorporated herein by reference.
Claims
What is claimed is:
1. An apparatus for providing temperature control to a specimen
carrier, comprising:
(a) one or more specimen carriers, each of said specimen carriers
comprising a compartment for holding a specimen and reaction
fluids;
(b) an enclosure having an interior space and having a site for
positioning said one or more specimen carriers;
(c) a temperature-controlled plate located in said enclosure, said
temperature-controlled plate having a first position in thermal
contact with one side of said one or more specimen carriers placed
at said site and a second position out of said thermal contact;
(d) means for moving said temperature-controlled plate between said
first position and second position;
(e) means for providing laminar air flow between said
temperature-controlled plate and said one or more specimen carriers
when said temperature-controlled plate is in said second
position;
(f) temperature control means capable of adjusting the temperature
of said temperature-controlled plate in response to control
signals; and
(g) computer means connected to said temperature control means and
to said means for providing laminar air flow, wherein when said
temperature-controlled plate is in the first position, the
temperature of the one or more specimen carriers may be brought to
a first temperature through thermal contact with the
temperature-controlled plate and when the temperature-controlled
plate is in the second position, laminar air flow provided between
the temperature-controlled plate and the one or more specimen
carriers may be used to bring the one or more specimen carriers to
a second temperature.
2. An apparatus for providing temperature control to a specimen
carrier according to claim 1, wherein the temperature-controlled
plate is a heating plate.
3. An apparatus for providing temperature control to a specimen
carrier according to claim 1, wherein the means for moving the
temperature-controlled plate between the first and second positions
comprises means for lifting the temperature-controlled plate into
contact with the one or more specimen carriers and for lowering the
temperature-controlled plate away from the one or more specimen
carriers.
4. An apparatus for providing temperature control to a specimen
carrier according to claim 1, wherein said compartment is formed by
a carrier bottom and a cover, said cover movable between an open
and closed position, and used for holding specimens and for
treatment with reaction fluids.
5. An apparatus for providing temperature control to a specimen
carrier according to claim 1, wherein the first temperature is a
temperature which denatures nucleic acid complexes and the second
temperature is chosen to be just below the melting temperature of
primer oligonucleotides.
6. An apparatus for providing temperature control to a specimen
carrier according to claim 1, further comprising sensing means in
thermal contact with the temperature-controlled plate and connected
to said computer means, wherein when a selected temperature is
detected by said sensing means, said computer means causes the
distance between the temperature-controlled plate and said one or
more specimen carriers to change.
7. An apparatus for providing temperature control to a specimen
carrier according to claim 6, wherein when said selected
temperature is detected, laminar air flow between said
temperature-controlled plate and said one or more specimen carriers
is provided by said means for providing laminar air flow.
8. An apparatus for providing temperature control to a specimen
carrier according to claim 1, wherein said means for providing said
laminar air flow comprises a fan directing air through a plenum to
a smaller space between said temperature-controlled plate and said
one or more specimen carriers.
9. An apparatus for providing temperature control to a specimen
carrier according to claim 1, wherein said temperature-control
means is a part of said computer means.
10. A method for providing temperature control to one or more
specimens in one or more specimen carriers in a specimen treatment
process, comprising:
(a) placing one or more specimen carriers in an enclosure having an
interior space and having a site for positioning one or more
specimen carriers, each of the said one or more specimen carriers
comprising a compartment for holding a specimen;
(b) providing:
(i) a temperature-controlled plate located in said enclosure, said
temperature-controlled plate having a first position in thermal
contact with one side of said one or more specimen carriers placed
at said site and a second position out of said thermal contact;
(ii) means for moving said temperature-controlled plate between
said first position and second position;
(iii) means for providing laminar air flow between said
temperature-controlled plate and said one or more specimen carriers
when said temperature-controlled plate is in said second
position;
(iv) temperature control means capable of adjusting the temperature
of said temperature-controlled plate in response to control
signals; and
(v) computer means connected to said temperature control means and
to said means for providing laminar air flow, wherein when said
temperature-controlled plate is in the first position, the
temperature of the one or more specimen carriers may be warmed
through thermal contact the temperature-controlled plate and when
the temperature-controlled plate is in the second position, laminar
air flow provided between the one or more specimen carriers and the
temperature-controlled plate may be used to cool the one or more
specimen carriers;
(c) performing temperature-changing steps in a predetermined
sequence along with one or more specimen treatment steps said
temperature-changing steps including:
(i) changing the temperature of said one or more specimen carriers
to a warmer desired temperature by moving said
temperature-controlled plate to said first position utilizing said
means for moving said temperature-controlled plate between said
first and second positions with respect to said site and providing
control signals to adjust the temperature to said warmer desired
temperature and to maintain the temperature of the
temperature-controlled plate at said warmer desired temperature for
a first predetermined time period; and
(ii) moving said temperature-controlled plate to said second
position utilizing said means for moving the said
temperature-controlled plate between said first and second
positions and one ore more specimen carriers utilizing said means
for providing laminar air flow to adjust the temperature of said
temperature-controlled plate to a cooler desired temperature; and
maintaining said cooler desired temperature for a second
predetermined time period.
11. A method of providing temperature control to one or more
specimens in one or more specimen carriers in a specimen treatment
process according to claim 10, and wherein the sequence of the
predetermined sequence of temperature-changing steps and the
specimen treatment steps comprises:
(a) warming the one or more specimen carriers to a temperature just
above a gelling temperature of a selected matrix material before
adding liquid matrix material to the one or more specimen
carriers;
(b) after adding liquid matrix material containing a specimen
having DNA to said one or more specimen carriers, cooling the one
or more specimen carriers to lower than said gelling
temperature;
(c) adding a treatment solution to the one or more specimen
carriers and adjusting the temperature of the one or more specimen
carriers to allow a desired treatment to occur;
(d) heating the one or more specimen carriers to 85.degree. C. and
maintaining g the temperature at about 85.degree. C. until the
gelled matrix material is dehydrated;
(e) denaturing the specimen DNA by saturating the dehydrated matrix
material with distilled water and heating to about 95.degree. C.
for about 3-5 minutes;
(f) heating the one or more specimen carriers to about 72.degree.
C. and maintaining at about 72.degree. C. while adding
amplification reagents;
(g) adding amplification reagents to the one or more specimen
carriers;
(h) initiating amplification thermal cycling after addition of
amplification reagents, adjusting temperature to about 72.degree.
C. and maintaining at about 72.degree. C. for about 2 minutes;
(i) heating the one or more specimen carriers to about 95.degree.
C. and maintaining at about 95.degree. C. for about 20 seconds;
(j) repeating steps (h) and (i) about 24 times;
(k) cooling the one or more specimen carriers to about 72.degree.
C. and maintaining at about 72.degree. C. for about 10 minutes;
and
(l) washing fluids through each specimen earlier to remove unwanted
materials and incubating at an incubation temperature. .Iadd.
12. An apparatus for providing temperature control to one or more
specimens, comprising:
(a) one or more specimen carriers, each of said specimen carriers
comprising a compartment for holding a specimen and reaction fluids
not thicker than 0.5 millimeters;
(b) an enclosure having an interior space and having a site for
positioning said one or more specimen carriers;
(c) a temperature-controlled plate located in said enclosure, said
temperature-controlled plate capable of bringing said one or more
specimen carriers to a first predetermined reaction
temperature;
(d) means for bringing said one or more specimen carriers to a
second predetermined reaction temperature;
(e) temperature control means capable of controlling the
temperature of said temperature-controlled plate and adjusting the
temperature of said one or more specimen carriers, in response to
control signals; and
(f) computer means connected to said temperature control means and
to said means for bringing said one or more specimen carriers to
the second predetermined temperature wherein the amount of time
said one or more specimen carriers is at either predetermined
reaction temperature is repeatedly regulated, and wherein the
temperature-controlled plate has a thermal mass enabling uniform
heat transfer to and away from the specimens and the specimen
carriers, wherein regulation of reaction temperature is
sufficiently rapid to allow an amplification product to be
obtained..Iaddend..Iadd.13. The apparatus for providing temperature
control to one or more specimens according to claim 12, wherein the
specimen carriers comprise microscope slides and a carrier assembly
for receiving the slides as a carrier bottom and providing carrier
edges and top pieces..Iaddend..Iadd.14. An apparatus for providing
temperature control to one or more specimens, comprising:
(a) one or more specimen carriers, each of said specimen carriers
comprising a compartment for holding a specimen and reaction fluids
not thicker than 0.5 millimeters;
(b) an enclosure having an interior space and having a site for
positioning said one or more specimen carriers;
(c) a temperature-controlled plate located in said enclosure, said
temperature-controlled plate capable of bringing said one or more
specimen carriers to a first predetermined reaction
temperature;
.Iadd.(d) means for providing laminar air flow to bring said one or
more specimen carriers to a second predetermined reaction
temperature lower than the first predetermined reaction
temperature;
.Iadd.(e) temperature control means capable of controlling the
temperature of said temperature-controlled plate and adjusting the
temperature of said one or more specimen carriers, in response to
control signals; and
.Iadd.(f) computer means connected to said temperature control
means and to said means for providing laminar air flow to bring
said one or more specimen carriers to a second predetermined
temperature lower than the first predetermined temperature, wherein
the amount of time said one or more specimen carriers is at either
predetermined reaction temperature is repeatedly regulated, and
wherein the temperature-controlled plate has a thermal mass
enabling uniform heat transfer to and away from the specimens and
the specimen carriers, wherein regulation of reaction temperature
is sufficiently rapid to allow an amplification product to be
obtained..Iaddend..Iadd.15. The apparatus for providing temperature
control to one or more specimens according to claim 13, wherein the
specimen carriers comprise microscope slides and a carrier assembly
for receiving the slides as a carrier bottom and providing carrier
edges and top pieces..Iaddend.
Description
BACKGROUND OF THE INVENTION
The present invention relates to the field of automated analyzers
for nucleic acid diagnostics, in particular to temperature control
devices. It is well known in the field of molecular biology that a
reaction is influenced by the temperature at which the reaction is
performed. If the temperature of the reaction varies, the results
could be inconsistent with previous assays or with results of the
calibration reactions. Precise temperature control to provide
heating and cooling cycles is useful in many processes and
particularly useful in gene amplification and detection
processes.
This invention more fully describes an embodiment of the carrier
which incorporates a standard microscope slide as part of the
carrier as described in U.S. Pat. No. 5,188,963 and copending U.S.
patent application Ser. No. 836,348.Iadd., now U.S. Pat. No.
5,451,500.Iaddend.; and PCT US/90/06768. This invention describes a
carrier assembly of multiple carriers which uses a slide as the
main portion of each carrier bottom and frames the edges which fit
around the slide in keeping with the original carrier format. This
invention describes the automated apparatus's temperature control
system and its integration with the carrier for more precise
temperature regulation.
The in situ amplification process (described in U.S. Pat. No.
5,188,963, and copending U.S. patent applications, Ser. No.
227,348.Iadd., now abandoned, having a continuation application
Ser. No. 935,637, now U.S. Pat. No. 5,382,511.Iaddend.; Ser. No.
836,348.Iadd., now U.S. Pat. No. 5,451,500.Iaddend.; and
PCT/90/06768) uses enzymes such as polymerase or ligase, separately
or in combination, to repeatedly generate more copies of a target
nucleic acid sequence by primer extensions to incorporate new
nucleotides or by ligations of adjacent complementary
oligonucleotides, wherein each template generates more copies and
the copies may themselves become templates. By melting
complementary strands of nucleic acids, the original strand and
each new strand synthesized are potential templates for repeated
primer annealing or ligation reactions to make and expand the
number of specific, amplified products. A thermostable polymerase
with reverse transcriptase activity and a thermostable ligase are
now both available and increase the choice of enzymes and
combination of reactions for in situ applications. As stated in
copending U.S. patent application Ser. No. 227,348.Iadd., now
abandoned, having a continuation application Ser. No. 935,637, now
U.S. Pat. No. 5,382,511.Iaddend., if RNA in the specimen is the
target to be amplified, the specimen is treated with reverse
transcriptase to make a nucleic acid complement of the RNA just
prior to amplification. Using a thermostable reverse transcriptase
polymerase such as rTth (Perkin Elmer, Norwalk, Conn.), it may not
be necessary to add another polymerase for rounds of primer
extension amplification. The amplification can either be primer
extensions in one direction for linear amplification, or in
opposing directions, for geometric amplification. The label can
either be incorporated as labeled nucleotides or labeled primers
for one-step detection or labeled probes may be added in a step
following amplification whereby the probes hybridize to the
amplified products for detection.
Nucleic acid amplification had been limited to solution reactions
wherein the nucleic acid is released from cells or tissue. In U.S.
Pat. No. 5,188,963 and copending U.S. patent application Ser. Nos.
227,348.Iadd., now abandoned, having a continuation application
Ser. No. 935,637, now U.S. Pat. No. 5,382,511 .Iaddend.and
836,348.Iadd., now U.S. Pat. No. 5,451,500.Iaddend., a process to
amplify nucleic acid targets within cells was described and a
method for embedding the cellular specimens in a matrix was
described to immobilize and stabilize the cells during
amplification and detection. A number of examples for using in situ
amplification are given in U.S. Pat. No. 5,188,963. A
photomicrograph of cells which had amplified and labeled DNA was
included in Ser. No. 836,348.Iadd., now U.S. Pat. No. 5,451,500
.Iaddend.to show that the amplified fragments are retained in
individual cells and such cells can be enumerated under microscopic
observation.
The process requires at least one denaturing or high temperature
stage, and one primer annealing or low temperature stage in each
cycle. To achieve the desired results, the embedded cell specimens
are heated to nucleic acid denaturation temperature and temperature
control commences before reagent addition. Since the specificity of
nucleic acid hybridization is influenced by temperature, uniform
and accurate temperature for all specimens is maintained throughout
the reaction. The time required for the specimen to be brought to
the reaction temperatures can be a large percentage of the time
allowed for the biochemical processes to be performed; therefore,
means to cycle temperature rapidly and reliably are desirable.
There are various techniques and devices for adjusting temperature
of reagents and specimens thereafter controlling the reaction
temperature. For example, it is known to use individual reaction
heating coils around individual reaction vessels. While a
circulating air or water bath can control temperature of a large
number of reactions simultaneously, the rate at which heat
transfers from such a bath to a reaction vessel is substantially
proportional to the difference between the temperature of the
vessel and the temperature of the bath, to the heat capacity of the
fluid, and to the efficiency of the contact therebetween. (See, for
example, U.S. Pat. No. 5,038,852 where circulating fluid reservoirs
or Peltier heat pumps are described for heating and cooling a
reaction mix.) The specific heat of air is so small that it becomes
very difficult to control the temperature of reaction vessels
accurately in circulating air. While water has a superior specific
heat compared to air, it must be moved rapidly about the reaction
vessels to maintain narrow temperature tolerances and, unfavorably,
the water supports microbial growth. In addition to fluid baths, it
is also commonly known to install reaction vessels in thermal
contact with a temperature controlled body or mass having good
thermal conductivity and a specific heat as high as practical. For
example, a plurality of reaction vessels may be located within an
aluminum or copper body.
The aforementioned in situ amplification for cellular analyses,
which requires precise temperature regulation, creates a need for
an improved apparatus which adjusts and controls the temperature of
the cellular specimens An apparatus designed for rapid temperature
cycling necessitates reducing thermal loads to increase the rate at
which heat transfers occur. The carriers used in this invention are
thin, flat reaction vessels whose bottom piece transfers and
spreads the heat quickly to the ultra-thin specimen within. Using
the word "thin" herein for carrier means that the carrier bottom
that conducts heat to the specimen is preferably not thicker than 1
millimeter. Using the word "ultra-thin" herein for specimen means
that a rehydrated matrix and specimen is preferably not thicker
than 0.5 millimeter. Because the specimen is ultra-thin and
represents a significantly greater surface area to volume ratio
than what would be found in a conical tube, the specimen
temperature more closely matches the temperature of the bottom
piece. For e.g., the surface area-to-volume ratio of 100
microliters in a conical tube is 132:1; whereas, the surface
area-to-volume ratio in a flat carrier (with a 2 cm.times.2 cm
matrix and specimen holding area) holding an equivalent 100
microliter sample is 830:1, or more than six times greater. For
example, a conical microfuge tube filled to a depth of 1 centimeter
at a maximum width of 0.62 centimeters has a surface area of 1.32
cm.sup.2 and a volume of 0.1 cubic centimeter (100 microliters). A
carrier with a sample 2 cm.times.2 cm.times.0.025 cm also has
volume of 0.1 cubic centimeter, but has a surface area of 8:3
cm.sup.2.
When glass slides are inserted in a carrier assembly as separate
carrier bottoms, each glass slide becomes part of the heat flow
transfer to and from a specimen. A specimen in the thin,
aforementioned configuration has greater surface contact with the
slide (carrier bottom), thereby reflecting quicker temperature
changes with respect to the flat carrier bottom, than a
specimen-containing solution with respect to the aforementioned
conical tube. Using glass in the bottom carrier piece, or a
material with comparable heat conductivity characteristics, also
improves the heat transfer capability of the carrier format over
standard microfuge tubes made of polypropylene. A flat
configuration of the matrix and specimen holding area on a carrier
enables convenient microscopic analysis of molecular targets within
the individual cells immobilized throughout the specimen.
Discrimination between binding specificity of different nucleic
acid primers and probes to target molecules is affected by
temperature. Minor sequence variations in nucleic acid base
composition may be detected within individual cells either by
labeling newly-incorporated nucleotides from specific
oligonucleotides and/or amplifying the target sequence and then
hybridizing a labeled probe to the amplification products. These
sequence variations may be used in DNA-based diagnostics to
identify infectious disease, genetic disease, cancer or
identity-testing. Precise temperature control is required to use
genetic sequence information most fully and produce exquisitely
accurate results.
The object of the invention is to provide an apparatus and method
of accurately controlling the temperature of simultaneous
biochemical reactions, bringing all the individual reactions to a
desired temperature, holding the reactions to the specified
temperature for a period of time, cooling the reactions to a
desired temperature, and holding the reactions at the specified
temperature for a period of time. Further objects, features and
advantages of the invention will become apparent from a
consideration of the following description, taken in conjunction
with the accompanying drawing figures.
SUMMARY OF THE INVENTION
The present invention overcomes the limitations and drawbacks
described above and provides a device for reaction vessels and an
apparatus which rapidly brings cellular specimens to a higher or
lower predetermined reaction temperature. The apparatus is suitable
for cycling and controlling the temperature of a plurality of
reaction vessels and can be readily adapted for use in an automated
analyzer of DNA diagnostics.
In accordance with the present invention, an apparatus for
providing a controlled temperature environment for a plurality of
specimens includes an assembly for receiving specimen carriers, a
heating plate with means to raise and lower its position relative
to the plane of the specimens and means to create a laminar air
flow between the specimens and the heating plate to cool the
heating plate and specimens rapidly. A heating element in thermal
contact with the heating plate heats the heating plate therein to a
predetermined reaction temperature. The apparatus also includes
sensing means in thermal contact with the heating plate for sensing
the temperature of the apparatus and controlling the heater to
reach and maintain the specimen at the predetermined
temperature.
In one embodiment disclosed herein the apparatus is generally
rectangular in shape with specimen carriers in rows and includes a
plurality of platforms on the heating plate extending upwardly
therefrom, all in thermal contact with the specimen carriers when
the heating plate is in the raised position. When the heating plate
is lowered, a plenum is coextensive with the space between the
specimen carriers and the heating plate, providing a channel for a
laminar air flow to quickly cool the specimens and heating plate.
It is understood that other embodiments are equally feasible such
that, for example, the specimens could be arranged annularly in an
apparatus having an annular heating plate and carrier assembly. In
yet another embodiment the heating plate and the carrier assembly
may be arranged more vertically than horizontally so that a closed
position and an open position (for the distance between the heating
plate and carrier assembly) is more descriptive than a raised
position or a lower position for either the heating plate or the
carrier assembly. While the heating plate moves in the embodiment
described herein and in FIGS. 1-6, it is equally possible that the
heating plate is fixed and the carrier assembly moves either to
contact the heating plate or create a space for the laminar air
flow.
The preferred specimen carriers are thin and flat wherein the
biochemical reactions are performed in a thin aqueous film or
matrix rather than in standard tube or cuvette-type containers. The
preferred specimen carrier and the fluid delivery system are
further described in U.S. Pat. No. 5,188,963. The thin, flat
specimen carriers are best suited for in situ DNA amplifications
and detections which integrate specimen collection, preparation and
gene detection in one reaction vessel. In the instances where the
specimen to be analyzed is put on a standard glass slide for the
convenience of microscopic observation, a carrier assembly holding
the slides supplies carrier edges and top pieces, and said carrier
assembly incorporates other features of a supporting carrier rack
such as providing the collecting trough. The glass slide is
inserted in the carrier assembly which is then placed in the
apparatus for processing just as carriers are positioned in racks
described in U.S. Pat. No. 5,188,963.
To accomplish precise heating and cooling, the present invention
utilizes a specimen carrier assembly with openings through which a
surface of each specimen carrier is in communication with a heating
plate. Heating elements, sandwiched within or beneath the heating
plate, heat the heating plate and transfer heat quickly and
uniformly to the specimen carriers. Means to move the heating plate
away from the specimen carriers break communication between the
specimen carriers and the heating plate, and cooling commences
immediately. A fan directs a laminar air flow in a channel between
the surface of the heating plate facing the carrier and the surface
of the specimen carrier facing the heating plate. The laminar air
flow serves as a medium for the transfer of heat away from both the
heating plate and the specimen carriers for rapid and uniform
cooling.
The laminar flow cooling system of the invention cools thin, flat
specimen containers. Said containers could resemble thin-walled
cuvettes or tubes having a thin specimen holding area. The
difference which defines laminar cooling is that air between the
specimen holders and the heat source is compressed into a rapidly
moving stream to cool objects on both sides of the air flow quickly
and representative temperatures of both the heating plate and the
specimen containers are monitored and adjustments are made in the
air flow rate to bring each toward the temperature of the other.
The apparatus of this invention provides for control of the
temperature of specimens in the carrier and control of the distance
between the heating plate and the carrier and control of the
laminar air flow cooling. The slides' matrix and specimen holding
areas are aligned with the raised heating platforms. The distance
between each slide and the corresponding heating platform is
uniformly adjusted and may be changed during heating and cooling.
Other specimen containers having a thin specimen holding area and
made of thin pieces to transfer heat efficiently and which use a
laminar air stream for rapid cooling, as described herein, are
within the scope of this invention.
Other aspects and features of the invention will be more fully
apparent from the following disclosure and appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view of the apparatus of the invention
showing one embodiment of a heating plate and lifting mechanism and
a cut away view of a carrier assembly holding standard microscope
slides. Slides are positioned in four carriers showing how said
slides contact the heating plate. Two of the carrier top pieces, or
covers, are shown in a closed position.
FIG. 2 is an perspective view of a carrier assembly holding glass
microscope slides and one slide in a partially inserted position. A
dotted line shows one of the covers going from an open to a closed
position.
FIG. 3 is an enlarged view showing the path of the laminar air flow
between the carrier's slide bottom and the platforms on the heating
plate when a measured distance separates the heating plate from the
carrier slide bottom.
FIG. 4 is an enlarged view showing the path of the laminar air flow
between the carrier's slide bottom and the platforms on the heating
plate when a minute distance separates the heating plate from the
carrier slide bottom.
FIG. 5 is a cross-sectional view of a carrier and heating plate
taken along line 5--5 in FIG. 3 showing an individual raised
heating platform on the heating plate in alignment with a slide
positioned in the carrier, carrier edges which define the matrix
and specimen holding area between the top (cover) and bottom pieces
of the carrier, the bottom carrier edge enveloping the slide and
the position of the cover and the retainer.
FIG. 6 is a perspective view of a retainer which closes over the
carrier, keeping retainer ribs in alignment with the carrier
sections between specimens so that carrier edges seal fluids by
making contact with the inserted slides. Also shown is the
mechanism for opening and closing the carrier top pieces, or
covers, over the specimen and matrix holding area.
FIG. 7 is a general block diagram of the temperature cycling
apparatus.
FIG. 8 is a logic flow diagram to show the steps in temperature
control.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
THEREOF
The invention broadly comprises an apparatus for heating and
cooling multiple specimens within carriers in a carrier assembly.
The specimen holding areas of the carriers are thin so as to spread
the specimen for cellular analysis and allow for rapid temperature
change. Individual specimen carriers are held in a carrier assembly
for molecular processing at precise temperatures. With reference to
FIG. 1, a temperature control apparatus 10 in accordance with the
present invention includes a heating plate 12, a lifting mechanism
14 and heating element 16. The heating plate 12 is preferably
formed of a heat conductive material such as aluminum alloy or
copper. Heating plate designs were referred to in copending U.S.
patent application Ser. No. 836,348.Iadd., now U.S. Pat. No.
5,451,500.Iaddend.. The heating plate surfaces closest to the
carrier assembly may have protruding sections in a pattern that
permits intimate contact with the carrier bottoms and specific
means of heating, such as insulated resistive heating wire
elements, may be incorporated in specific locations in the carrier
assembly or disposed within the heating plate 12 by milling
cavities in the hating plate 12 to direct heat to specific areas of
the carrier bottom. Heating elements are fixed within such cavities
by means known in art, for example, laser welding, to enclose the
heaters.
The heating plate 12 embodied herein has raised platforms 18
integrally formed with the heating plate 12, for example, by
machining or die-cast injection molding, or the platforms may be
separately formed and bonded to the heating plate by soldering,
brazing or with a suitable heat-conductive epoxy compound. If the
platforms 18 are formed separately with separate heating elements
positioned with the individual platforms 18, the heating plate 12
may be formed of aluminum, or an aluminum frame with as little
thermal mass as possible, and the platforms 18 formed from copper.
In all cases the heating plate surface meeting the carrier assembly
32 must be shaped so that intimate contact is achieved overall for
optimal distribution of heat to the carrier bottom. In the
preferred embodiment disclosed herein, the heating element is an
insulated thermofoil material (Minco Products, Inc., Minneapolis,
Minn.) having a total resistance in the range of about 6-16 ohms
and being adapted to dissipate approximately 12 watts of power per
square inch when 24 volts DC is applied thereto.
In the preferred embodiment disclosed herein, the temperature
sensors 20A and 20B (FIG. 7) comprise thermistors, or
thermocouples, bonded to, or embedded in, the heating plate 12 and
a representative carrier, respectively. The temperature sensor 20
may have a nominal resistance of approximately 10,000 ohms at
25.degree. C. Electrical connections for both the heating elements
16 and temperature sensors 20 are provided by means of
feed-throughs.
The heating plate 12 has screws 22 and posts 24 connecting it to
the lifting mechanism 14. The posts 24 are preferably made of non
heat-conductive material and are attached to the heating plate with
non-metal screws 22. The lifting mechanism consists of means to
raise and lower the heating plate and may be accomplished by any
number of possible assemblies such as a combination of squeeze
clamp solenoids and levers, or an electric gear motor and cam
action. A further lifting mechanism may comprise a stepper motor,
switch and double helix, whereby the heating plate is raised and
lowered by moving one centrally-located post up and down. Four arms
from the center post to each corner of the heating plate support
the heating plate, and movement of the double helix raises and
lowers the center post. Spring-like action, executed from below the
heating plate by means know in the art such as gaskets between the
arms and the heating plate "float" the heating plate so that the
heating plate surface is aligned with respect to the carrier
assembly when the heating plate is raised.
In the preferred embodiment of the lifting mechanism disclosed
herein, four tubular solenoids 26 move four lever 28 arms at the
same time to lift the four corners of the heating plate 12 and
position its top surface with raised platforms 18 in immediate
contact with the carriers 30. Referring to FIG. 1, two pairs of
arrows show the direction each squeeze clamp moves in order to lift
the opposite corner of the heating plate. One of the open arrows
shows the direction one solenoid clamp 26 retracts to lift the
opposite corner of the heating plate in the direction indicated by
the other open arrow when lever arms 28 rotate around a pivot rod
29. The stippled pair of arrows demonstrates a similar action for
the other solenoid clamp and heating plate corner.
With continued reference to FIGS. 1 and 2, the specimen carriers 30
are held by a carrier assembly 32. The carrier assembly 32
positions the specimen carriers 30 containing the material to be
assayed which may be solid or liquid tissue specimens embedded or
immobilized in a matrix material on the carrier bottom piece 33. In
the embodiment shown the carrier assembly 32 has a plurality of
rectangular openings 34 formed therethrough adapted to receive
standard microscope slides, each of which said microscope slide 35
becomes part of the carrier bottom 33. The openings 34 are aligned
with respect to the platforms 18 on the heating plate 12 so that
each specimen carrier bottom 33 makes thermal contact with the
respective platform 18 when the heating plate 12 is in the raised
position. The preferred specimen carriers 30 are made of glass or a
heat-resistant plastic material and are more fully described in
copending patent application Ser. No. 438,592.Iadd., now U.S. Pat.
No. 5,188,963.Iaddend..
Referring to FIGS. 3-6, a retainer 38, comprised of ribs 40 and
fastened by means of hinges 42 to the apparatus, presses against
the carriers 30 between the slide openings when said retainer is
closed by a spring-loaded closure 43, insuring thermal contact with
the platforms 18 when the heating plate 12 is in the proper
position. The retainer ribs 40 define spaces 44 adapted to allow
the top piece or cover 45 of the specimen carriers 30 to be opened
and closed. The cover actuator 46 grippingly moves over the
retainer ribs 40 to open and close the carrier. The spaces 44 also
provide a path through the retainer 38 for the delivery of fluid
reagents to the specimens as described in U.S. Pat. No. 5,188,963
and copending applications Ser. Nos. 227,348.Iadd., now abandoned,
having a continuation application Ser. No. 935,637, now U.S. Pat.
No. 5,382,511 .Iaddend.and 836,348.Iadd., now U.S. Pat. No.
5,451,500.Iaddend.. As is well known in the art, the material to be
assayed may comprise a mixture of suitable reagents and a patient
specimen or control or calibration sample.
Returning to FIG. 3, a fan 48 connects to a plenum 50 coextensive
with the laminar flow air space 52, which occurs when the heating
plate 12 is retracted a distance of 2-10 millimeters from the
carrier. A baffle (not shown) made as known in the art is
configured in such a way within the plenum 50 so as to even out the
rate of air flow entering the laminar air flow space at all carrier
positions 30. The carrier bottoms 33, and microscope slides 35
which are inserted at carrier positions 30, situated in the carrier
assembly 32 form the upper boundary of the laminar flow space 52
and the heating plate forms the lower boundary of the laminar flow
space 52.
In FIG. 4 the distance is reduced between the heating plate and
carrier to show the heating plate touching the carrier bottom
piece. As disclosed in copending application Ser. No.
836,348.Iadd., now U.S. Pat. No. 5,451,500.Iaddend., included
herein by reference, actual temperature data representing the
heating plate and the carrier were recorded using a prototype
device. The carrier held 25 mm.times.75 mm glass slides and the
distance between said heating plate and slide was constant during
cycling. The importance of the data is that heat convected from the
heating plate via the air cushion overcame differences in starting
temperatures at different cycles to bring the slide closer to the
desired higher temperature setpoint, but lower temperature
setpoints varied from one cycle to the next. The data suggest that
adjusting the distance between the heating plate and the carrier is
as least as important, or even more important, in maintaining
consistent lower setpoint temperatures versus higher ones. The data
also demonstrate that temperature cycling control is possible
without intimate contact between the heating plate and the carrier
with each in a fixed position, the fixed position affects the slope
of the heating/cooling curve, and preferably the distance (which
may be 0-2 cm, or somewhere in between at a particular point in the
temperature cycling) changes during the programmed temperature
cycle.
The instant invention improves temperature regulation by
controlling a laminar air flow between the heating plate and the
carrier. The invention further provides mechanisms and computer
means to control changing the distance between heating plate and
carrier. Referring to FIG. 4, the elevational view of one
embodiment of a lifting mechanism 14 further shows the positional
change of the solenoid-operated 26 lever arms 28 as the lever arms
rotate to lift the heating plate 12 at each of the four posts 24.
The dotted line shows where the heating plate meets a slide 35
within the carrier assembly 32. Arrows show the direction of
rotational movement of the lever arms around the pivot rod 29 to
lift the heating plate 12.
The cross-section view of the carrier assembly in FIG. 5 is located
in FIG. 3 by the line marked 5--5. The heating plate 12 is shown in
a retracted portion relative to the carrier, as in FIG. 3, to
illustrate the laminar air flow space 52. The platforms 18 on the
heating plate 12 are shown aligned with the slide 35 and carrier
bottom 33.
The preferred thickness of the carrier bottom is 1 millimeter or
less. The carrier assembly 32 is made of a heat-resistant material
and may formed as one plastic piece by compression or injection
molding processes to hold slides 35. Alternatively, the carrier may
be made by arranging separate sections, which sections are cut from
long extruded plastic into appropriate lengths, whose cross-section
is shown as extrusion piece 54 in FIG. 5, and which are placed at
intervals to accommodates the slides and joined with cross pieces
by means known in the art such as laser welding.
The cross-section view in FIG. 5 also illustrates the sequence of
parts through the carrier assembly and heating plate starting with
retainer ribs 40, the carrier top piece 45, or covers, in a closed
position, the position of carrier edges 56 forming matrix and
specimen holding spaces 58, the slides 35, carrier bottoms 33, the
laminar air flow spaces 52, the heating platforms 18 elevated above
the main part of the heating plate 12, the heating plate 12 in a
retracted position and the hater 16.
The apparatus is controlled by a microcomputer or CPU
(microprocessor) 60 with memory 62 as shown in FIG. 7. The user
enters a heating/cooling profile into the computer via a keyboard
64 or touch pad in response to queries on the menu display 66. A
profile comprises a time to heat to setpoint temperature SP.sub.h
(ramp), time T.sub.h to reside at setpoint temperature (soak), a
selected time to decrease temperature to a lower setpoint
temperature SP.sub.l /(ramp) and time T.sub.l to reside at lower
setpoint temperature (soak). Generally two or three different soak
temperatures are selected by the user and default ramp rates are
preset, but may be overridden if ramp time is also designated by
the user. A temperature, SP.sub.h, is preferably within the range
of from about 60.degree. C. to 95.degree. C. A temperature,
SP.sub.l, is preferably within the range of from about 35.degree.
C. to 60.degree. C.
The CPU programs comprise instructions to enter and store user
profiles and interfaces with a temperature control circuit 68 which
contains programming for the lifting control 70, heating control 72
and fan, or laminar air flow, control 74 as diagramed in FIG. 7.
The temperature control circuit 68 contains a proportional or a
proportional-integral-derivative (PID) algorithm for hating and
cooling control. Proportioning may be accomplished either by
varying the ratio of "on" time to "off" time, or, preferably with
proportional analog outputs as known in the art which decrease the
average power being supplied either to the heater or the fan as the
temperature approaches setpoint. PID control combines the
proportional mode with an automatic reset function (integrating the
deviation signal with respect to time) and rate action (summing the
integral and deviation signal to shift the proportional band). The
1990/91 Temperature Handbook by Omega Engineering, Inc. (Stamford,
Conn.) contains explanations of the various control modes in the
"Introduction to Temperature Controllers" on pages P-5 to P-10.
Such microprocessor control systems are well known in the art and
need not be further described herein. Control functions required
for automatic temperature control particular to apparatus of the
invention are more fully explained herein for each step in the
logic flow diagram in FIG. 8.
The process starts with a command to the CPU 60 from the user to
begin temperature control in Step 82 of FIG. 8. A user-defined
temperature profile is selected from the computer's memory or
entered from the keyboard to begin operation. After checking that
the retainer closure 43 is in a closed position, the heating plate
moves to make physical contact with the slides. The lifting control
70 in this embodiment activates four solenoid-operated lever arms,
SOL-1, SOL-2, SOL-3 and SOL-4 in FIG. 7 to position the heating
plate in contact with the carrier bottom 33.
The CPU monitors the temperature of the heating plate and, upon
receiving the run command, issues the proper command signal to
begin heating in Step 84. Upon receiving the proper command, the
CPU retrieves the first setpoint data and issues a proper signal to
cause heating for a user-defined temperature profile at a default
rate and starts the clock. The heater heats the heating plate to
the high temperature equal to a user-defined level, which is
referred to as temperature variable SP.sub.h. The heater begins
heating at full voltage and heats to the desired setpoint in the
shortest time possible unless the user defines the time period for
reaching setpoint temperature. Heat transfers from the heating
plate by conduction to the carriers until the desired carrier
temperature is reached and during the incubation period to maintain
the temperature. The lifting mechanism remains activated until a
set point temperature is retrieved that is lower than the previous
one.
In Step 86 the CPU reads the temperature of heating plate as the
temperature sensor 20-A (FIG. 7) develops a signal as known in the
art that is proportional to the temperature of the heating plate 12
and such a signal is converted to a signal for the digital
temperature control circuit 68. The CPU monitors the temperature of
the heating plate and issues the proper command signal to cause the
heater to heat the heating plate until the desired temperature is
reached, and then issues the proper commands to the temperature
control apparatus to cause the desired temperature to be
maintained. Using either the proportional or the
proportional-integral-derivative (PID) algorithm, the CPU computes
a set point as a target temperature, continuously monitors the
temperature of the plate and compares it as it approaches the set
point on the user-defined temperature profile. An error signal is
generated by comparison of the actual temperature to the calculated
set points in the algorithm. The temperature control circuit 68
generates a signal that is proportional to the error voltage
applied thereto and the rate of change of such error voltage. The
resulting signal from the temperature control circuit 68 generates
a modulated output proportional to the signal applied thereto. The
output is in turn applied to the heating element 16. The voltage to
the heater is controlled by the temperature control circuit and may
be turned on and off and the rate of heating may be tuned by
adjusting the voltage.
When a specimen carrier slide 35, which has a temperature lower
than the selected reaction temperature, is added to the carrier
assembly 32, or, when a carrier 30 that is already installed in the
carrier assembly 32, is filled or washed with a fluid, e.g. as in
U.S. Pat. No. 5,188,963, that is lower than the temperature of the
heating plate 12, heat from the heating plate 12 flows to the
specimen carrier 30 through the thermally conductive platform 18.
In response to the heat flow, localized cooling of the platform 18
in the immediate area of the specimen carrier 30 draws heat from
the heating plate 12. As this process continues, the temperature
control circuit 68 with the temperature sensor 20-A and the heating
element 16 operate as described above to maintain the reaction
temperature of the heating plate 12 at the predetermined
temperature. Heat flow in the opposite direction occurs if the
carrier assembly has a temperature higher than the heating plate,
and said heat is absorbed by the larger thermal mass of the heating
plate, such that adjustments are made in the heater control.
In Step 88 the CPU keeps track of the elapsed time at particular
temperatures to implement desired incubation periods. At least one
temperature sensor 20-B (FIG. 7) is placed in a specimen carrier 30
and used to develop a signal. The temperature is monitored via
sensor 20-B and the CPU determines whether the carrier is at the
correct process temperature. During ramp periods the carrier
temperature may lag behind in ramping to a higher setpoint
temperature or said carrier temperature may move ahead in ramping
to a lower temperature at any given moment in time. For this reason
it is important that elapsed time for incubation start when the
carrier bottom, not the heating plate, attain soak temperature. The
microcomputer control system may start counting the incubation
period when temperature sensor 20-B, representing the temperature
of the slide 35 or carrier bottoms 33, reaches the predetermined
temperature. To implement timing of the incubation period, the
computer restarts a clock and times the elapsed time from when the
temperature sensor 20-B equals the temperature, SP.sub.h. The
incubation time variable is generally set by the user according to
the requirements of a desired biochemical process.
In Step 90 the CPU compares the elapsed time that the slides are at
the desired incubation temperature, SP.sub.h, with the selected
incubation time, T.sub.h. If the actual time is less than the
selected time, the program continues to maintain temperature and
compare the elapsed time.
When the elapsed time that the slides are at temperature, SP.sub.h,
equals the desired incubation time as determined in Step 90, the
CPU sends the proper command in Step 92 to the heating and cooling
apparatus to cause the heating plate to be cooled toward a low
temperature, SP.sub.l, set by the user.
In some profiles the next setpoint temperature after moving to a
higher temperature may be an even higher temperature. In this case
the program reenters at Step 84.
Control of laminar flow cooling is integrated into the temperature
control circuit 68 as follows. When the next desired temperature of
the heating plate 12 is lower than the present heating plate
temperature, the temperature control circuit 68 develops a signal
to the lifting control 70 to deactivate the solenoids, causing
retraction of the heating plate 12. A simultaneous signal to the
air flow control 74 activates the fan 48. Air enters the fan 48 and
is pressurized into the plenum 50 into a laminar air flow through
the laminar flow air space 52. Air flows through the laminar air
flow space 52 between the platforms 18 and specimen carrier bottoms
33, removing heat from he specimen carriers 30 and the heating
plate 12. The laminar air flow space 52 is thin enough and the air
flow pressurized enough by compressing it into a thin space that
air turbulence is kept to a minimum.
The program returns to the proportional or PID algorithm in Step 86
to execute heating/cooling control towards a lower setpoint in a
similar way in which control was executed towards a higher
setpoint, but involving different output control signals. The
transmission of commands by the CPU activate a laminar air flow to
cool the heating plate and the carriers simultaneously. The
temperature of the heating plate 12 and the slide 35 or carrier
bottom 33 are monitored by the CPU and an error signal is generated
by comparison or the actual temperature to the calculated set
points in the proportional or the proportional-integral-derivative
algorithm to control the temperature of the heating plate.
Periodically, an error signal based upon the comparison between the
computed slope of the user-defined temperature profile and that of
the new set point is generated from calculation of the slope and
the elapsed time. The error signal is converted to the proper
control signal to control the lowering of temperature to a lower
setpoint.
The speed of the fan is controlled by inputs from temperature
sensors 20A (representation of heating plate temperature) and 20B
(representation of slide carrier temperature) to the proportional
controlling algorithm. Changing the speed of the fan increases or
decreases the airflow so that the rate of cooling is within bounds
of the user-defined time or the default rate set by the program.
The adjustments in airflow compensate for fluctuations in the
temperature of intake air and internal heat build-up within the
apparatus. When sensor 20-B reaches the lower setpoint temperature,
the clock starts counting the elapsed time set for the incubation
period, T.sub.l. If the error calculated by the CPU between sensor
20-A and sensor 20-B indicates that sensor 20-A is lower than
sensor 20-B when the setpoint temperature is reached, the heater is
activated; if sensor 20-A is higher than sensor 20-B, the airflow
continues to cool the plate after sensor 20-B reaches the low
setpoint temperature. Comparing thermal loss rates detected by
sensors 20-A and 20-B during the cooling phase and making the
aforementioned adjustments work toward equilibrating the
temperature of the heating plate and the carrier just as the lower
setpoint temperature is reached. At the point when the heating
plate and slide carriers are both very near the low setpoint
temperature, a control signal activates the lifting mechanism to
restore contact between the heating plate and the carrier and
another signal to the fan control deactivates the fan. Maintaining
a stable temperature for an incubation time period operates
similarly for high and low setpoint temperatures. Heat loss to the
surrounding environment requires activating the heater control to
keep heating plate at low setpoint temperature.
The controller algorithm is also programmed to change fan speed
when a differential temperature between sensor 20-A and 20-B is
greater than a predetermined amount. Ideally, the heating plate and
the carrier are designed to have balanced thermal load and heat
loss characteristics. Variable airflow works to fine tune cooling
so that when cooling is achieved in less than the user-defined ramp
time, or a predetermined default time, or the temperature
differential between sensors 20-A and 20-B is greater than a
predetermined amount, a decrease in airflow allows more efficient
convective transfer of heat from the heating plate through the air
cushion to the carrier, or vice versa, and works toward achieving
thermal equilibration before low setpoint is reached.
Step 88 following a cooling phase toward a lower setpoint
temperature is the same as one that follows moving to a higher
setpoint temperature. The CPU measures the elapsed time from the
time the slides temperature reaches the SP.sub.l of the heating
plate.
Step 90 again compares elapsed time to the user-defined low
temperature incubation time, T.sub.l. As soon as the elapsed time
equals the desired low-temperature incubation time, T.sub.l, Step
92 involves the CPU retrieving the next setpoint temperature in the
profile and continues until all setpoint temperatures have been
executed.
When one profile is completed the CPU counts the number of times
the profile has been run and compares the number to a user-defined
variable in memory. The number of times a profile is to be run is
the cycle count. In Step 94 the CPU compares the cycle count to the
user set variable. If the cycle count does not match the desired
number of cycles, processing returns to Step 84. If the cycle count
equals the set variable for the desired number of cycles,
processing proceeds to Step 96.
After the desired number of cycles has been performed, Step 96
determines whether the user wishes to run another temperature
profile stored in another "file" or database. Every temperature
profile entered by the user has a link data field in which there is
stored the profile identification of the next file or temperature
profile to be run, if any. The contents of this field are read. If
the field finds a profile number in the link field, then processing
returns to Step 84 and restarts by retrieving the first setpoint
temperature in the new profile and continues processing through
Step 96 again to achieve each setpoint temperature in the profile
for the set number of cycles.
In Step 98 the contents of the data link field are read and if the
user has made no further entry to the link field, signals to lifter
and fan controls work to cool the heating plate until no further
reduction in temperature occurs. When the heating plate temperature
as sensed by temperature sensor 20-A is not lowered for a preset
time period, an "end" message is displayed and the control
functions shut off the temperature control apparatus.
Automated DNA analyzers containing the temperature control
apparatus may or may not utilize temperature control during other
functions. In many instances controlled temperature is desirable to
achieve consistent clinical results and the invention herein may be
used to replace other temperature control systems for a more
precise temperature control. The localized heating provided to each
specimen carrier 30 on the apparatus 10 is very rapid and precise,
particularly in comparison to other air and water bath
techniques.
The unique heating and cooling system combines the conductive
heating via the heating plate 12, providing means to distance the
heating plate 12 from the specimen carriers 30, and convection
cooling via the laminar air flow. A primary difference between the
apparatus of the invention and other kinds of moving air systems,
which are used to remove heat from the reaction vessels or heat
sinks, is that the thermal load of the system has been reduced to a
heating plate 12 of just sufficient mass to spread heat evenly and
the specimen carriers 30 themselves become part of the heat sinking
system. Since the temperature at which heat transfers from the
reaction vessel to the laminar air flow is substantially
proportional to the difference between the temperature of the
vessel and the temperature of the air, cooling the specimen carrier
from high temperatures of 95.degree. C. with air at 25.degree. C.
or lower is rapid at temperature ranges between 55.degree. C. and
95.degree. C. The heat transfer capacity of the fluid air is
increased by increasing its flow rate to supply air of lower
temperature to the laminar flow space 52. The efficiency of the
contact between the air flow and the surfaces to be cooled is
increased by pressurizing the air flow into a thin laminar pathway,
thereby, reducing air turbulence.
Studies using glass slides, 1 mm.times.25 mm.times.75 mm, as
specimen carriers 30 demonstrated in situ DNA amplification as
shown in copending patent application U.S. Ser. No. 227, 348.Iadd.,
now abandoned, having a continuation application Ser. No. 953,637,
now U.S. Pat. No. 5,382,511.Iaddend.. The studies made evident that
spacing the specimen carrier a distance as short as 2 mm from the
heating plate 12 permitted more rapid cooling of the glass slide
and the air cushion in this space further increased the rate of
cooling over cooling that could be achieved when the slide was in
contact with the heating plate. Temperature data results for
heating specimen carriers was included in the specification of
copending patent Ser. No. 836,348.Iadd., now U.S. Pat. No.
5,451,500.Iaddend.. The instant invention provides a means for
moving air through the air cushion, said air flow directed between
the carrier and the heating plate and describing the invention
herein comprising a temperature control apparatus for DNA-based
detections in cellular diagnostic tests and the essential
controlling program logic to attain precise temperature
control.
The use of DNA amplification cycling temperatures for annealing and
denaturation are both above ambient air temperatures, making
refrigeration or Peltier-cooling of the specimen carriers
unnecessary in automated clinical DNA analyzers. However, a means
of refrigerating or Peltiercooling air may be employed to prechill
the air entering the fan and plenum, thereby augmenting the speed
of cooling by increasing the temperature differential between the
specimens and the air used to cool them.
Other modifications of the above-described embodiments of the
invention as used by those of skill in the mechanical and
electrical arts and related disciplines are intended to be within
the scope of the invention.
The flat, thin configuration of the specimen carrier 30 is a
departure from the commonly used centrifuge tube or cuvette. A
specimen carrier assembly 32 also has a considerably different
configuration than found in support racks or blocks designed for
tubes or cuvettes. In tube and cuvette-type vessels the dynamics of
the biochemical within the contained solution are subject to
molecular distribution in the solution. The flat specimen carrier
makes use of a supporting matrix and thin-film fluid dynamics for
molecular processing. The flat specimen carrier assemblies 32,
adapted to use a standard microscope slide 35 as part of the
carrier bottom 33, are caused to press against the microscope slide
35 by the retainer 38 to insure good thermal contact between the
slide 35 and the heating plate. Together the heating control 72,
fan control 74, and lifting control 70 comprise a temperature
control circuit 68 in a CPU to meet the demands of DNA
amplification temperature cycling for clinical DNA analyzers.
The preferred way of doing the method is described as follows. The
method utilizes standard microscope slides, either blank slides or
ones with cellular specimens on them. The slides are inserted into
a carrier assembly and the carrier assembly is loaded into the
slide temperature control apparatus. A temperature profile to warm
the slides to a temperature just above the gelling temperature of
the matrix material insures even spreading in the matrix and
specimen holding area before gelation. Plain liquid agarose, or
liquid agarose mixed with cell suspensions, is added to the slide,
filling the matrix and specimen holding area of the slide under the
cover. For example, 5 ul of a cell culture solution (10.sup.6
cells/ml) may be mixed with 500 ul of 1% agarose (Molecular Biology
Grade Agarose, IBI, New Haven, Conn.). Another temperature profile
allows the matrix temperature to drop below its gelling
temperature, forming a gel matrix embedding the specimen. After gel
matrices have formed, slide covers are opened with the actuator on
the retainer.
A freshly-prepared specimen treatment solution consisting of 1
mg/ml Pronase (Life Technologies, Rockville, Md.) in 0.01 M Tris.
Cl pH 7.8, 0.001 M EDTA, and 0.1% Triton X-100 (v/v) is added to
the matrix surface in excess and incubated at 37.degree. C. for
5-15 minutes. If RNA is the molecular target, RNAase inhibitors as
known in the art would be included in the treatment solution. The
mixture is rinsed from the matrix by three 500 ul washes of
dH.sub.2 O over 15 minutes. The matrices are then dried to the
upper surface of the slides by ramping to and maintaining a
85.degree. C. temperature. The in situ sample preparation method
unmasks DNA within the matrix-embedded specimen and permits the
nucleic acid of the specimen to be used as a template for
transcription.
Specimen DNA is denatured by saturating the matrix with 500 ul
dH.sub.2 O and using a temperature profile that heats the carrier
to 95.degree. C. and maintains 95.degree. C. for 3-5 min. Adding
dH.sub.2 O drop by drop as needed keeps the matrices from
completely drying out during denaturation. The target nucleic acids
within the nucleoid of the specimen's cells or within virions are
available for primer hybridization and polymerase activity. The
genetic material is capable of acting as a template for
transcription of DNA or reverse transcription of mRNA within the
treated cells using an exogenous polymerase.
A temperature profile heats slides to a primer annealing
temperature of, for example, 72.degree. C. and maintains
temperature while amplification reagents are added. Each
partially-dehydrated matrix is rehydrated with 100 ul of the
nucleotide/primer mix, 5 Units of Taq DNA Polymerase (Boehringer
Mannhelm, Indianapolis, Ind.). In the one-step method at least one
of the nucleotides is modified in order to detect incorporation.
The nucleotide mixture for example may contain 140 uM each dATP,
dGTP and dCTP, 70 uM dTTP and 70 uM Digoxgenin-11-dUTP (Boehringer
Mannheim) in buffer (10 mM Tris. Cl, 50 mM KCl, 1.5 mM MgCl.sub.2).
The primers are specific for the target sequence, for example,
cultured cells of CaSki or SiHa which contain integrated copies of
human papillomavirus type 16 (HPV-16), are detected by using one or
more specific primers at 1 uM each for type-specific regions in the
E5, E6 or E7 gene sequences of HPV-16.
A temperature profile for amplification thermal cycling consists
of, for example, ramping to and maintaining 72.degree. C. for 2
minutes and ramping to and maintaining 95.degree. C. for 20 seconds
for 25 cycles, and ending with a profile of 10 min @ 72.degree. C.
Polymerase activity utilizes the target template within the
nucleoid of cells each time the previous extension product melts
under temperature denaturation and permits another primer molecule
to bind and initiate polymerization. Adding a strip of rubber
cement at the cover's edge will keep matrix from drying out, or
dH.sub.2 O may be added drop by drop at the cover's edge as needed
to replenish volume which may be decreased by evaporation.
After amplification, matrices are rinsed by filling the fluid
receiving area with dH.sub.2 O or detection buffer to start fluid
flow through the carrier. The matrices are rinsed by first adding
dH.sub.2 O to one edge of the cover slide, and then 1 ml of TMN (40
MM Tris. Cl, pH 7.8, 6 mM MgCl.sub.2, 5 mM NaCl), so that the
fluids passed slowly through the diffusion layer between each
matrix and its cover. Two ul of anti-Digoxgenin monoclonal antibody
from the GENIUS.TM. Detection System (Boehringer Mannheim) in 225
ul TMN are added to each matrix and incubated for 30 min at
37.degree. C. Two ml of suitable alkaline phosphatase substrate
buffer is added slowly for 15 minutes to rinse unbound antibody
conjugate away before adding alkaline phosphotase subtrates, NBT
(4.4 ul) and BCIP (3.3 ul) in 500 ul of the AP substrate buffer to
the matrices. The reaction is incubated with a temperature profile
of 1-2 hours at 35.degree. C. and stopped by the addition of 2 ml
dH.sub.2 O through the diffusion layer. The matrices are stained,
still within the carrier, for 15 minutes in nuclear fast red to
counterstain cellular structures. The carrier assembly is removed
from the apparatus and the slides are removed from the carrier
assembly and placed on the microscope stage. The cells are observed
within the matrices under the microscope and both negative cells
without the amplification label and positively-identified cells are
visible. The amplified product is observed to remain within the
target cells and enable such cells to be enumerated. Cells having
copies of the HPV genome integrated into their genome may be
identified by this method. The flat matrix format maintains
cellular and tissue structures so they may be visible after
processing. The use of polymerase activity to amplify in situ
increases detection sensitivity, permits unambiguous signal
detection and enables the genetic entity to be tied to specific
locations in the specimen.
Detection systems utilizing biotin, digoxgenin, fluorescence,
antibodies or enzymes or combinations of these produce unambiguous
signals within cells embedded in an agarose matrix. If primers or
nucleotides are not labeled during amplification; labeled
oligonucleotides may be hybridized to amplification products for a
30-minute incubation, followed by rinsing twice at minute intervals
and once with slide at stringent temperature without zone spreading
of signal from target cells. Standard aqueous hybridization buffers
without formamide and standard SSC washes of 500 ul each may be
used as known in the art. Labeled cells are visualized with
standard light or fluorescent microscopy, depending upon which
label is used.
In summary, the present invention provides an apparatus for
performing automated sample preparation, DNA amplification and
detection, which apparatus has heat-sinking, flat carriers for
specimens and reagents, means from heating and cooling and
maintaining the specimen to or at any given temperature for a given
time period, and a computer means to generate signals that control
said temperatures and times.
While the invention has been described in detail with respect to
specific illustrative examples and embodiments, it will be apparent
that numerous other variations, modifications and embodiments are
possible, and accordingly all such variations, modifications and
embodiments are to be regarded as being within the scope of the
invention. Such variations include, but are not limited to the
detection of proteins or other cellular components using known
detection methods and reagents.
* * * * *